WO2014016358A1

WO2014016358A1 - Inhibition of hiv-1 infection by inhibition of binding of cpsf to viral capsid

Info

Publication number: WO2014016358A1
Application number: PCT/EP2013/065665
Authority: WO
Inventors: Leo James
Original assignee: Medical Research Council
Priority date: 2012-07-24
Filing date: 2013-07-24
Publication date: 2014-01-30
Also published as: GB201213087D0

Abstract

The invention describes a method for identifying a compound capable of inhibiting infection by HIV, comprising contacting a HIV CA polypeptide with a CPSF6 polypeptide in the presence of the compound, and determining the influence of the compound on the binding of HIV CA to CPSF6, by measuring the interaction of CPSF6 with HIV CA at one or more of residues 53, 56-57, 66-67, 70, 73-74, 105, 7, 109 and 130 thereof.

Description

INHIBITION OF HIV-1 INFECTION BY INHIBITION OF BINDING OF CPSF TO VIRAL CAPSID

The present invention relates to the inhibition of HIV-1 cellular infection by disruption of the interaction between the HIV-1 capsid protein (CA) and the RNA processing factor CPSF6, and to anti-HIV agents which target this interaction.

The HIV-1 virus enters target cells encapsulated in a fullerene capsid cone composed of capsid (CA) protein. The role of the capsid in early events of the HIV-1 replication cycle is not known, nor is it clear exactly how long the capsid remains associated with the infectious particle, or where the capsid disassembles. Early biochemical studies led to the view that the capsid is an inert shell, required purely during cellular entry, whereupon it then rapidly falls apart ('uncoats') to permit reverse transcription [1 , 2]. However, recent data suggest that uncoating may occur later than previously thought, either during transport of the reverse transcribing virus to the nucleus, or once the reverse transcribing virus has docked at the nuclear pore [3-5]. This accommodates the possibility that the capsid may facilitate transit of the core towards the nucleus by interacting with the cell's cytoskeletal transport system [5]. An intact capsid would also be expected to protect the viral genome from host pattern recognition as it undergoes reverse transcription, in addition to maintaining a high stoichiometry of reverse transcriptase enzyme to viral template, which is necessary for overcoming the rate limiting steps in reverse transcription [4, 6].

Like all lentiviruses, HIV-1 is able to infect non-dividing cells, which requires the exploitation of active host cell nuclear import processes [7]. CA mutations have been identified that specifically affect nuclear entry in non-dividing cells [8], suggesting a link between CA and nuclear import. However, the role of capsid in anything other than a structural context is contradicted by that fact that, apart from the Cyclophilin A (CypA)-binding loop, there are no other known interfaces on CA through which it can interact with host cell cofactors. Consequently, CA mutations outside of the CypA- binding loop are currently believed to exert their effect by altering capsid stability. For example, it has been proposed that CA mutations that decrease the ability of HIV-1 to enter the nucleus (Q63A Q67A) or infect non-dividing cells (T54A N57A) do so by causing the capsid to uncoat faster or slower than wild type [8, 9]. Despite having no known interfaces on CA, viral cofactors have been identified whose dependencies appear to map to CA. Recent genome-wide screens have implicated a number of nuclear import components as HIV-1 cofactors, including the nuclear pore protein RanBP2 (also called NUP358) and the karyopherin TNP03 [12, 13]. There is evidence that the requirement for RanBP2 may map to CA [14], [15], [16]. Intriguingly, the determinant for TNP03 requirement also maps to CA [17] and CA mutation N74D affects the sensitivity of HIV-1 to TNP03 knockdown [14].

Mutation N74D arose spontaneously during passage of HIV-1 in cells expressing CPSF6-358, an artificially truncated version of cleavage and polyadenylation specific factor 6 (CPSF6, also known as CF Im) that perturbs HIV-1 nuclear entry [14].

CPSF6 is a pre-mRNA processing protein that dynamically shuttles between the nucleus and the cytoplasm [18] and contains a C-terminal nuclear-targeting arginine/serine-rich (RS-) domain [19, 20] of the type bound by TNP03 [21 , 22]. CPSF6 lacking this RS-domain is no longer confined to the nucleus but is found in addition in the cytoplasm [20]. CPSF6-358 (which is truncated at position 358 and therefore lacks the C-terminal RS-domain) was also found to be cytoplasmic and restricted HIV-1 before nuclear entry [14]. It is therefore significant that the HIV-1 CA N74D mutation not only allows escape from CPSF6-358 restriction but also results in the loss of viral dependence on TNP03. Lee et al (36) demonstrated that removal of the C-terminal 58 residues of CPSF6 in CPSF6-358 impairs specific binding of CPSF6 to HIV-1 CA, and identified residues 313-327 of CPSF6 as being key in the interaction between the two proteins. However, they provided no information concerning the nature of the interaction of the HIV CA protein with CPSF6.

Summary of the Invention

Many CA mutations that give clear infectivity defects are located on an exposed surface in the CA hexamer structure (see Figure 1 ; [10, 1 1]). We therefore hypothesised that these mutations might not directly affect capsid stability.

We show herein that CPSF6 binds specifically to a novel protein -protein interface on the N- terminal domain of HIV-1 CA. We show that this interface is structurally and functionally conserved across diverse lentiviruses and is accessible in the context of an intact CA hexamer, suggesting that CPSF6 may interact with incoming capsid during the early post-entry stages before uncoating.

In a first aspect, therefore, there is provided a method for identifying a compound capable of inhibiting infection by HIV, comprising contacting a HIV CA polypeptide with a CPSF6 polypeptide in the presence of the compound, and determining the influence of the compound on the binding of HIV CA to CPSF6 by measuring the interaction of CPSF6 with HIV CA at one or more of residues 53, 56-57, 66-67, 70, 73-74, 105, 107, 109 and 130 thereof.

Structure-guided mutagenesis of this interface reveals that CA residues that mediate CPSF6 binding also mediate dependence on TNP03 and RanBP2. Moreover, addition of an ectopic nuclear localization signal (NLS) to CPSF6-358 recovers its nuclear localization and restores HIV-1 infectivity, suggesting that the functional outcome of CPSF6 interaction with HIV-1 depends on whether CPSF6 is trafficking into the nucleus or remaining in the cytosol. Together, our data reveals that HIV-1 CA possesses a previously undescribed, conserved protein -protein interface that dictates cofactor dependence. Furthermore, it suggests that HIV-1 uses CA to interact directly with host cofactors and exploit cellular nuclear import pathways.

In a preferred aspect, the CPSF6 polypeptide comprises residues 313-327 of CPSF6. As shown herein, these residues define the binding interface with CA. In one embodiment, the CPSF6 polypeptide preferably comprises at least residues V314, L315 and F321 . These residues interact directly with a channel formed in the structure of CA, fitting to pockets in the centre of the channel. The CA polypeptide comprises the structure of the intact CA polypeptide as defined by residues 53, 56-57, 66-67, 70, 73-74, 105, 107, 109 and 130. These residues have been shown to define the surface of HIV CA which interacts with CPSF6.

Accordingly, the CA polypeptide preferably comprises residues 53-130 of HIV CA. In one embodiment, modelling is used to assess the interaction of putative HIV drugs with the interface formed between HIV CA and CPSF6 as described herein.

In a further embodiment of the present invention, there is provided a method for identifying a compound or compounds capable, directly or indirectly, of modulating the interaction of HIV CA and CPSF6 and thereby the infectivity of HIV, comprising the steps of :

(a) incubating a CA polypeptide with the compound or compounds to be

assessed ; and

(b) identifying those compounds which bind to the CA polypeptide in the region defined by residues 53, 56-57, 66-67, 70, 73-74, 105, 107, 109 and 130. As demonstrated below, the forgoing regions of CA and CPSF6 are required for direct interaction of the two proteins. Moreover, the interaction of HIV CA with CPSF6 is shown to be involved in nuclear entry of HIV, though indirect interaction with nuclear entry cofactors such as TP03. Accordingly, compounds which modulate the interaction of CA and CPSF6 have the potential to modulate the infectivity of HIV.

Moreover, the invention provides methods for producing polypeptides capable of modulating the interaction between CPSF6 and HIV CA, including expressing nucleic acid sequences encoding them, methods of modulating the interaction between CPSF6 and HIV CA in cells in vivo, and methods of treating conditions associated with HIV infection.

In a further embodiment, the invention concerns a method for modulating the interaction between CPSF6 and HIV CA. In a preferred embodiment, the invention provides a method for identifying a lead compound for a pharmaceutical, comprising : incubating a compound or compounds to be tested with a CPSF6 polypeptide and a HIV CA polypeptide, for example as defined above, under conditions in which, but for the presence of the compound or compounds to be tested, the interaction between CPSF6 and HIV CA induces a measurable chemical or biological effect;

determining the ability of the HIV CA polypeptide to interact, directly or indirectly, with the CPSF-6 polypeptide to induce the measurable chemical or biological effect in the presence of the compound or compounds to be tested ; and

selecting those compounds which modulate the interaction between CPSF6 and HIV CA.

In a related aspect, the invention provides a method for identifying a compound which regulates the interaction between CPSF6 and HIV CA.

In a preferred embodiment of the foregoing aspects, the method encompasses the use of a CPSF6 polypeptide, preferably a recombinant polypeptide, that includes an amino acid sequence having at least 75% identity with the amino acid sequence of residues 313-327 of wild-type CPSF6. Preferably, the CPSF6 sequence is the sequence set forth in SEQ ID No 1 . In a preferred aspect, the method encompasses the use of an HIV CA polypeptide, preferably a recombinant polypeptide, that includes an amino acid sequence having at least 75% identity with residues 53-130 of the amino acid sequence of wild-type HIV CA. Preferably, the HIV CA sequence is the sequence set forth in SEQ ID No 2.

Binding between HIV CA and CPSF6 can be determined in a number of ways. In one embodiment, a direct binding assay, such as a two-hybrid assay, can be used. In other embodiments, a HIV nuclear infection assay can be used, in which the ability of HIV to enter the nucleus of cells is assayed. Preferably, the cells are non-dividing cells, which can be infected by lentiviruses. In various embodiments described herein, viruses related to HIV, such as SIV or FIV, or pseudotyped HIV, may be used instead of HIV.

Moreover, indirect interactions can be assayed. For example, assays can be configured to detect the effect of a compound or compounds on the indirect interaction between nuclear entry cofactors, such as TP03 and RanBP2, with HIV CA. This interaction is mediated via the direct interaction between HIV CA and CPSF6.

In the foregoing aspects and embodiments, the method includes the use of a test compound that is, for example, protein based, carbohydrate based, lipid based, nucleic acid based, natural organic based, synthetically derived organic based, or antibody based.

In another preferred embodiment, the invention provides a compound identified according to the method of the foregoing aspects, and preferably, such a compound is suitable for treating a condition associated with HIV infection.

In another aspect, the invention provides a method for treating a condition associated with an HIV infection in a subject in need thereof by modulating the interaction of HIV CA and CPSF6, by administering a pharmaceutical composition capable of modulating interaction of HIV CA and CPSF6 in an amount sufficient to modulate the nuclear infection by HIV.

In a related aspect, the invention provides a method for treating a condition associated with HIV infection in a subject by, administering a composition capable of modulating the interaction of HIV CA and CPSF6 in a therapeutically-effective amount sufficient to modulate the HIV mediated condition in the recipient subject. In further embodiments, the invention provides a compound which modulates the interaction of HIV CA and CPSF6 for use in modulating an HIV mediated condition.

In embodiments of the present invention, the condition associated with HIV is HIV- AIDS.

In a second aspect, the invention relates to a method for developing an anti-HIV drug comprising the steps of (a) identifying one or more compounds which demonstrate anti-HIV infection activity; (b) screening said compounds and selecting one or more compounds which affect the interaction of HIV CA and CPSF6; (c) determining the structure of the compound and using structure-guided mutagenesis to prepare variants of the compound with improved activity.

Advantageously, the forgoing aspect of the invention makes use of a crystal structure of a complex of CPSF6 and HIV CA.

According to a third aspect, there is provided a drug cocktail comprising two or more drugs for use in the treatment or prevention of an HIV infection, wherein at least one of said drugs in indicated for the disruption of the interaction between an HIV CA polypeptide and a nuclear entry cofactor.

Preferably, the interaction between an HIV CA polypeptide and a nuclear entry cofactor is indirect, and is mediated via an interaction between HIV CA and CPSF6. For example, the nuclear entry factor may be RanBP2 or TP03.

In accordance with the third aspect of the invention, the drug cocktail may further comprise one or more anti-HIV drugs selected from the group consisting of efavirenz, emtricitabine, tenofovir, disoproxil fumarate, rilpivirine, lamivudine, zidovudine, emtricitabine, azidothymidine( AZT), nevirapine, amprenavir, tipranavir, indinavir, saquinavir mesylate, lopinavir, ritonavir, Fosamprenavir Calcium, darunavir, atazanavir sulfate, nelfinavir mesylate, raltegravir, maraviroc and enfuvirtide.

Drugs capable of disrupting of the interaction between the HIV CA polypeptide and a nuclear entry cofactor can be selected according to the preceding aspects of the invention. In on example, the drug is PF-3450074. Brief Description of the Figures

Figure 1. Exposed CA mutations affecting HIV-1 infectivity

Location of CA mutations that are solvent-exposed in the hexameric CA structure and which are found to result in decreased HIV-1 infectivity [10]. CA mutations are labeled and represented as red spheres. The CA hexamer structure was derived from pdb: 3H47 [1 1 ] by generating symmetry-related copies in PyMOL. Left: view looking down onto the hexamer, right: view from the side, with CypA-binding loops at the top.

Figure 2. CPSF6 binds diverse lentiviral capsids

Isothermal titration calorimetry (ITC) of CPSF6313-327 against CAN domains of lentiviral capsids. CPSF6313-327 binding to HIV-1 is specific (A), being abolished by CA mutation N74D (B). CPSF6313-327 also binds to diverse lentiviral capsids, HIV- 2, FIV and SIVmac (C). The stoichiometry (N), affinity (Kd), enthalpy (ΔΗ) and entropy (AS) of interaction are shown.

Figure 3. Crystal structure of HIV-1 CAN in complex with CPSF6313-327

(A) Crystal structure of the HIV-1 CAN:CPSF6313-327 complex. HIV-1 CAN is shown as cartoon representation and CPSF6 as sticks. Secondary structure elements in

HIV-1 CAN are labeled. The electron density for the CypA-binding loop was poor, so this region is represented by a dashed line. (B) Close-up view of the HIV-1

CAN:CPSF6313-327 interface, showing HIV-1 CAN as surface representation. The three CPSF6 residues that fill the centre of the channel in HIV-1 are indicated. (C) The CPSF6- binding interface on HIV-1 CAN. Residues involved in binding to

CPSF6313-327 are labeled and shown in green. (D) Intramolecular interactions in CPSF6313-327. Residues involved in intramolecular hydrogen bonding interactions are labeled and the interactions shown as dashed lines. The sequence of

CPSF6313-327 is shown for reference. (E) Stereo figure showing overview of the HIV-1 CAN:CPSF6313-327 interaction. The N- and C-termini of CPSF6313-327 (labeled) project out of the binding channel in HIV-1 CAN.

Figure 4. Interactions in the HIV-1 CAN :CPSF6313-327 complex

(A-D) Detailed views of the HIV-1 CAN:CPSF6313-327 interface. HIV-1 CAN is shown as grey cartoon, HIV-1 residues that bind CPSF6 are in green and

CPSF6313-327 in yellow. Water molecules involved in water-mediated interactions in the complex are shown as cyan spheres. (A) Overview of the HIV-1 CAN:CPSF6313- 327 interface, showing all interacting residues and intermolecular hydrogen-bonding interactions. Views of the close-ups shown in (B), (C) and (D) are indicated. (B-D) HIV-1 CAN ohelices are labeled to aid orientation. Interacting residues are labeled (CPSF6 in normal font; HIV-1 in italics). (E-F) CPSF6 residue F321 is critical for interaction with HIV-1 CAN. (E) HeLa cells expressing empty vector (white bar), HA- tagged CPSF6-358 (black bar) or CPSF6-358 bearing mutation F321 N (striped bar) were infected with GFP-encoding VSV-G pseudotyped HIV-1 vector. F321 N abolished restriction by CPSF6-358, confirming the importance of this residue in the HIV-1 CAN:CPSF6 interaction. (F) Western blot to show CPSF6-358 and CPSF6-358 F321 N expression levels, with actin as loading control.

Figure 5. The CPSF6-binding interface is accessible and highly conserved in HIV-1 virions.

(A) Model of CPSF6313-327 binding to the HIV-1 CA hexamer. The hexamer structure was derived from pdb: 3H47 [1 1] by generating symmetry-related copies in PyMOL. The model was composed by superposition of the HIV-1 CAN chains from the HIV-1 CAN:CPSF6313-327 complex structure on the pdb: 3H47 hexamer using secondary structure matching. HIV-1 CA helices are shown as cylinders and

CPSF6313-327 as spheres. Left: top view of hexamer, right: side view of hexamer. N-terminal and C- terminal CA domains (NTD and CTD) are labeled. (B) Close-up of boxed region in (A). CAs are shown as cartoon representation and CPSF6313-327 as sticks. The region between CTD positions 175 and 188 was disordered in the hexamer structure and so is represented by a dashed line. (C) Close-up of same view as in (B), showing exposed CA mutations which are found to result in decreased HIV-1 infectivity [10] (see Figure 1). CA mutations are labeled and shown as red spheres. Note the similarity between the location of the CPSF6-binding interface in

(B) and the mutations in (C). (D) Model of CPSF6313-327 binding at a hexamer- hexamer interface. Neighbouring hexamers were derived from pdb: 3H47 by generating extended symmetry-related copies in PyMOL. HIV-1 CAN:CPSF6313-327 was superposed on the hexamer using secondary structure matching. The model shows that CPSF6313-327 binding is likely to be accommodated at neighbouring CTD-mediated hexamer-hexamer junctions. (E) The CPSF6-binding interface is highly conserved within HIV-1 viruses. The ConSurf Server [33, 34] was used to map -100 unique HIV-1 CAN sequences onto the HIV-1 CAN:CPSF6313-327 structure. HIV-1 CAN is shown as surface representation and CPSF6313-327 as yellow sticks. The level of conservation at each position in CAN is shown by the colour, with dark blue being the most conserved and red being the least conserved. Residues at the CPSF6-binding interface are among the most highly conserved of all, suggesting that this is a functionally important interface. The figure was generated using the PyMOL script output by ConSurf, with conservation grades replacing the B-factor column. Figure 6. The CPSF6-binding interface determines HIV-1 nuclear entry requirements

(A) ITC of CPSF6313-327 against mutant HIV-1 CANs. All mutations at the CPSF6- binding interface resulted in reduced affinity to CPSF6313-327. The stoichiometry (N), affinity (Kd), enthalpy (ΔΗ) and entropy (AS) of interaction are shown. (B) Titres of VSV-G pseudotyped GFP-encoding HIV-1 vectors bearing wild type or mutant CA on HeLa cells expressing empty vector (EV), CPSF6-358, control knockdown cells (shC) and cells depleted for TNP03 (shTNP03) or RanBP2 (shRanBP2). The data are representative of two independent experiments, each using three different virus doses. Mutation of HIV-1 CAN residues involved in binding to CPSF6 resulted in the loss of dependence on TNP03 and RanBP2, suggesting a link between CPSF6 binding and normal nuclear import of HIV-1. (C) Western blot to show knockdown of TNP03 and RanBP2, with cyclophilin A (CypA) as loading control.

Figure 7. Drug PF-3450074 binds to part of the CPSF6-binding interface in HIV- 1 CA

(A) Superposition of HIV-1 CAN: PF-3450074 structure (pdb: 2XDE) [26] on HIV-1 CAN:CPSF6313-327 using secondary structure matching of the HIV-1 CAN domains. Drug PF-3450074 is shown in cyan, CPSF6313-327 in yellow. The three CPSF6 residues that fill the centre of the channel in HIV-1 are indicated. One of the phenyl rings in PF-3450074 superposes almost exactly with the phenyl ring of F321 in CPSF6313-327. (B) Crystal structure of HIV-1 CAN:PF-3450074 (pdb: 2XDE) showing residues involved in binding to CPSF6 (green sticks). (C) ITC of PF- 3450074 against wild type and mutant HIV-1 CANs. Figure 8. Addition of ectopic NLS rescues CPSF6-358 nuclear localization and HIV-1 infection

(A) Typical confocal microscopy images of HeLa cells expressing HA-tagged full- length CPSF6 (CPSF6-FL), a monoclonal cell line of CPSF6-358 (CPSF6-358.4) and CPSF6-358 with an additional C- terminal SV40 NLS (CPSF6-358-NLS). Addition of the SV40 NLS onto CPSF6-358 rescued CPSF6 nuclear localization. Scale bars, 10 μηη. (B) Titres of VSV-G pseudotyped GFP-encoding HIV-1 vector on HeLa cells expressing empty vector (EV), or the indicated CPSF6 constructs. Addition of the SV40 NLS onto CPSF6-358 rescued HIV-1 infection. (C) HA-tagged CPSF6 expression levels as determined by western blot, with actin as loading control.

Detailed Description of the Invention

Unless otherwise stated, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. Methods, devices, and materials suitable for such uses are now described. All publications cited herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing the methodologies, reagents, and tools reported in the publications that might be used in connection with the invention.

The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology and pharmacology, known to those of skill of the art. Such techniques are explained fully in the literature. See, e. g. , Gennaro, A. R., ed. (1990) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; Hardman, J. G., Limbird, L. E., and Gilman, A. G., eds. (2001 ) The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill Co.; Colowick, S. et al., eds., Methods In

Enzymology, Academic Press, Inc.; Weir, D. M. , and Blackwell, C. C, eds. (1986) Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications; Maniatis, T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Vols. I-III, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al., eds. (1999) Short Protocols in Molecular Biology, 4th edition, John Wiley & Sons; Ream et al., eds. (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press; Newton, C. R., and Graham, A., eds. (1997) PCR (Introduction to Biotechniques Series), 2nd ed., Springer Verlag.

The capsid of HIV-1 has historically been thought of as a packaging device to carry viral protein and nucleic acid into the cell. Mutations in CA that alter infectivity are typically thought to do so by altering capsid stability and assembly [10, 28, 29]. The discovery of a conserved protein -protein interface on HIV-1 CA is therefore important, because it suggests that CA has more than a purely structural role, and accommodates a mechanism whereby CA interacts with host proteins to facilitate virus infection. In particular, it implies that variation in CA sequence may impact on infectivity by modulating these interactions. There is increasing evidence that CA determines susceptibility to restriction factors such as TRIM5alpha and CPSF6-358, and dependency on nuclear import proteins such as TNP03, RanBP2 and NUP153 [16, 17, 30]. Our data support the hypothesis that at least some CA mutations alter these susceptibilities and dependencies by modifying direct interactions rather than causing a change in uncoating kinetics. Interestingly, some CA mutations have concomitant effects on multiple host proteins. For instance, N74D escapes CPSF6- 358 restriction whilst simultaneously relieving dependence on nuclear transport factors such as TNP03 and a functional nuclear pore. On the basis of structure- guided mutagenesis of the CPSF6-binding interface we have identified five more CA mutations that have the same pleiotropic effects as N74D (N57A, M66F, Q67A, K70A and T107A). These findings support the hypothesis of flexible nuclear import pathways, or functional redundancy, proposed by Lee ei a/ [14]. Redundancy in HIV- 1 infection is an attractive explanation as it allows otherwise puzzling data to be rationalized. It is also conceptually appealing as it provides a mechanism for viral escape from host immunity and rapid zoonosis. The seeming interdependency of multiple host factors on a single capsid mutant also suggests that some of these host factors operate in a single pathway, which the virus utilizes for efficient infection. Given the host factors involved, this single pathway most likely involves nuclear import of the virus.

A compelling model for the role of the CPSF6 interface in nuclear entry of HIV-1 is that it recruits CPSF6 to achieve active transport into the nucleus. Under this model, CPSF6 in turn recruits karyopherins such as TNP03, which can shuttle through the nuclear pore. In support of this model, TNP03 is known to bind RS-domains of the type found in CPSF6 [21 , 22]. Moreover, the redundancy that has been noted in the use of nuclear entry cofactors by HIV-1 could be explained if the RS-domain of CPSF6 is recognised by several karyopherins. Likewise, other RS-domain containing proteins may use the CPSF6 interface. Most importantly, if CPSF6 is an HIV-1 co- factor it would explain why mutation N74D, and the additional mutations we have described, result in concomitant loss of both CPSF6 interaction and dependence on TNP03 and RanBP2. Furthermore, it may also resolve seemingly conflicting data that report different viral targets for TNP03 requirement and interaction: the requirement for TNP03 maps to CA [17], but TNP03 has been found to bind integrase (IN) and not CA [31]. Recruitment of TNP03 to CA-bound CPSF6 could explain why CA determines TNP03 requirement, while also accommodating a role for IN as the direct viral binding partner of TNP03. Although, it is also possible that upon formation of a ternary complex some direct interactions between TNP03 and CA are made. Further evidence in support of CPSF6 as a co-factor is that addition of an ectopic NLS to CPSF6- 358 restores both nuclear localization and HIV-1 infectivity. However, we cannot rule out that this might be due to a reduction in the concentration of cytosolic CPSF6-358 that would otherwise prevent functional interaction of endogenous CPSF6 with CA. Nevertheless, it seems an unlikely coincidence that nuclear shuttling CPSF6 happens to bind to a conserved interface in HIV-1 capsid in which point mutations alter dependence on nuclear entry cofactors.

Irrespective of the role of CPSF6 itself, the conservation, location and phenotypic dominance of the CPSF6 interface provides compelling evidence that this binding site plays a key role in HIV-1 infection. The importance of the CPSF6-binding interface in HIV-1 infection is further reinforced by the striking similarity between the location of the CPSF6-binding interface and the binding site for the recently described drug, PF-3450074, which inhibits HIV-1 replication (Figure 7A) [26]. The CPSF6- binding interface on HIV-1 CA possesses several important attributes that make it an ideal antiviral drug target, in that it is highly conserved, functionally important and druggable. Since PF-3450074 occupies only a subset of the entire CPSF6-binding site, it is unlikely to be as effective a drug as one that inhibits the entire interface. Our complexed HIV-1 CAN:CPSF6313-327 crystal structure provides a molecular delineation of the CPSF6 interface that may be useful in the

development of antiviral therapeutics.

HIV, as referred to herein, is a member of the genus Lentivirus, part of the family of Retroviridae. Two types of HIV have been characterized: HIV-1 and HIV-2. HIV-1 is the virus that was initially discovered and termed both LAV and HTLV-III. It is more virulent, more infective, and is the cause of the majority of HIV infections globally. References to HIV herein are preferably references to HIV-1. The sequences of many isolates of HIV-1 are freely available in public databases.

The HIV CA protein, also known as p24, is derived from the GAG gene and forms the viral capsid. Sequences of many isolates of the GAG gene, and p24, are available from public databases. The preferred HIV CA N-terminal sequence is set forth in SEQ ID No. 2. CPSF6 refers to human cleavage and polyadenylation factor 6, as described by Li et al., Cell Res. 21 (7), 1039-1051 (201 1 ). The preferred sequence of human CPSF6 is set forth in SEQ ID No 1. A "compound" which influence the interaction of CPFS6 and HIV CA may be of almost any general description, including low molecular weight compounds, organic compounds which may be linear, cyclic, polycyclic or a combination thereof, peptides, polypeptides including antibodies, or proteins. In general, as used herein, "peptides", "polypeptides" and "proteins" are considered equivalent.

"Modulation" includes inhibition and potentiation, or enhancement, of an activity. Preferably, the activity is measurable, as is its inhibition or enhancement; and modulation refers to the effecting of a measurable change in said modulation. In some embodiments, the modulation represents an increase or decrease of 10%, 20%, 30%, 40%, 50%, 100% or more of the activity. Increases in activity can be in excess of 100%, 200%, 300% or more.

The terms "direct" and "indirect", as applied herein, refer to interactions between entitles which either require, or do not require, an intermediary. An "indirect" action proceeds through an intermediary; for instance, an interaction between HIV CA and TP03 proceeds via the intermediary CPSF6; there is not believed to be any direct binding of HIV CA to TP03. However, the interaction between HIV CA and CPSF6 has been shown to be direct. Measuring the interaction between CPSF6 and HIV CA residues 53-130 refers to any assay which is configured to detect this interaction, as opposed to any other interactions which may occur outside of the HIV CA region identified herein. Assays can be implemented as any type of biochemical assay; for example, competition assays can be defined using reagents which specifically bind with HIV CA within the region 53-130. Such reagents can be antibodies, peptides or any other specific binding reagent.

Isolated protein domains comprising residues 53-130 of HIV CA may also be used, in any desired binding assay, to detect protein interactions which target this region of HIV CA. If isolated domains are used, it should be ensured that the three- dimensional structure of the domains which is present in the full-length HIV CA is maintained in the isolated domains. For example, the N-terminal domain of HIV CA may be used, since the N-terminal and C-terminal domains of HIV CA are mostly alpha-helical and fold independently.

In a further embodiment, all or part of the region defined by residues 53-130 of HIV CA can be modeled by polypeptides which have been designed to have the same three-dimensional structure. In particular, smaller parts of the N-terminal domain can be replaced in binding assays by small polypeptides which have the same, or a similar, structure. Rational design of peptide mimics is known in the art; see, for example, Methods in Molecular Biology Volume 993 2013: In Silico Models for Drug Discovery.

Sequence homology (or identity) may moreover be determined using any suitable homology algorithm, using for example default parameters. Advantageously, the BLAST algorithm is employed, with parameters set to default values. The BLAST algorithm is described in detail at http ://www. nchi. nih. gov/BLAST/blast help, html, which is incorporated herein by reference. The search parameters are defined as follows, and are advantageously set to the defined default parameters.

Advantageously, homology of nucleic acid sequences can be assessed using a suitable algorithm, such as BLAST. Preferred levels of homology, when assessed by BLAST, equate to sequences which match with an EXPECT value of at least about 7, preferably at least about 9 and most preferably 10 or more. The default threshold for EXPECT in BLAST searching is usually 10. BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx ; these programs ascribe significance to their findings using the statistical methods of Karlin and Altschul (see http://www.ncbi.nih.gov/BLAST/blast~help.html) with a few enhancements. The BLAST programs were tailored for sequence similarity searching, for example to identify homologues to a query sequence. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al. (1994) Nature Genetics 6 : 1 19-129.

In one embodiment, sequence homology refers to percentage sequence identity, which can be assessed without the aid of an algorithm. Preparation of a HIV CA or CPSF6 polypeptide

The invention encompasses the production of HIV CA or CPSF6 polypeptides for use in the assays as described herein. Preferably, HIV CA or CPSF6 polypeptides are produced by recombinant DNA technology, by means of which a nucleic acid encoding a HIV CA or CPSF6 polypeptide can be incorporated into a vector for further manipulation. As used herein, vector (or plasmid) refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles are well within the skill of the artisan. Many vectors are available, and selection of appropriate vector will depend on the intended use of the vector, i. e. whether it is to be used for DNA amplification or for DNA expression, the size of the DNA to be inserted into the vector, and the host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification of DNA or expression of DNA) and the host cell for which it is compatible. The vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence and a signal sequence.

Both expression and cloning vectors generally contain nucleic acid sequence that enable the vector to replicate in one or more selected host cells. Typically in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses.

The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2p plasmid origin is suitable for yeast, and various viral origins (e. g. SV 40, polyoma, adenovirus) are useful for cloning vectors in mammalian cells.

Generally, the origin of replication component is not needed for mammalian expression vectors unless these are used in mammalian cells competent for high level DNA replication, such as COS cells. Advantageously, an expression and cloning vector may contain a selection gene also referred to as selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e. g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media.

As to a selective gene marker appropriate for yeast, any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker gene. Suitable markers for yeast are, for example, those conferring resistance to antibiotics G418, hygromycin or bleomycin, or provide for prototrophy in an auxotrophic yeast mutant, for example the URA3, LEU2, LYS2, TRP1 , or HIS3 gene.

Since the replication of vectors is conveniently done in E. coli, an E. coli genetic marker and an E. coli origin of replication are advantageously included. These can be obtained from E. coli plasmids, such as pBR322, Bluescript® vector or a pUC plasmid, e.g. pUC 18 or pUC 19, which contain both E. coli replication origin and E. coli genetic marker conferring resistance to antibiotics, such as ampicillin. Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up HIV CA or CPSF6 nucleic acid, such as dihydrofolate reductase (DHFR, methotrexate resistance), thymidine kinase, or genes conferring resistance to G418 or hygromycin. The mammalian cell transformants are placed under selection pressure which only those transformants which have taken up and are expressing the marker are uniquely adapted to survive. In the case of a DHFR or glutamine synthase (GS) marker, selection pressure can be imposed by culturing the transformants under conditions in which the pressure is progressively increased, thereby leading to amplification (at its chromosomal integration site) of both the selection gene and the linked DNA that encodes HIV CA or CPSF6. Amplification is the process by which genes in greater demand for the production of a protein critical for growth, together with closely associated genes which may encode a desired protein, are reiterated in tandem within the

chromosomes of recombinant cells. Increased quantities of desired protein are usually synthesized from thus amplified DNA.

Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to HIV CA or CPSF6 nucleic acid. Promoters suitable for use with prokaryotic hosts include, for example, the, sslactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Their nucleotide sequences have been published, thereby enabling the skilled worker operably to ligate them to DNA encoding CPSF6 or HIV CA, using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems will also generally contain a Shine-Delgamo sequence operably linked to the DNA encoding HIV CA or CPSF6.

Preferred expression vectors are bacterial expression vectors which comprise a promoter of a bacteriophage such as phagex or T7 which is capable of functioning in the bacteria. In one of the most widely used expression systems, the nucleic acid encoding the fusion protein may be transcribed from the vector by T7 RNA polymerase (Studier et al, Methods in Enzymol. 185 ; 60-89,1990). In the E. coli BL21 (DE3) host strain, used in conjunction with pET vectors, the T7 RNA

polymerase is produced from the lambda lysogen DE3 in the host bacterium, and its expression is under the control of the IPTG inducible lac UV5 promoter. This system has been employed successfully for over production of many proteins. Alternatively the polymerase gene may be introduced on a lambda phage by infection with an int- phage such as the CE6 phage which is commercially available (Novagen, Madison, USA), other vectors include vectors containing the lambda PL promoter such as PLEX (Invitrogen, NL), vectors containing the trc promoters such as

pTrcHisXpressTm (Invitrogen) or pTrc99 (Pharmacia Biotech, SE), or vectors containing the tac promoter such as pKK223-3 (Pharmacia Biotech) or PMAL (new England Biolabs, MA, USA).

Moreover, the HIV CA or CPSF6 gene according to the invention preferably includes a secretion sequence in order to facilitate secretion of the polypeptide from bacterial hosts, such that it will be produced as a soluble native peptide rather than in an inclusion body.

The peptide may be recovered from the bacterial periplasmic space, or the culture medium, as appropriate.

Suitable promoting sequences for use with yeast hosts may be regulated or constitutive and are preferably derived from a highly expressed yeast gene, especially a Saccharomyces cerevisiae gene. Thus, the promoter of the TRP 1 gene, the ADHI or ADHII gene, the acid phosphatase (PH05) gene, a promoter of the yeast mating pheromone genes coding for the a- or a-factor or a promoter derived from a gene encoding a glycolytic enzyme such as the promoter of the enolase,

glyceraldel_yde-3phosphate dehydrogenase (GAP), 3-phospho glycerate kinase (PGK), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase, 3phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase or glucokinase genes, the S. cerevisiae GAL 4 gene, the S. pombe nmt 1 gene or a promoter from the TATA binding protein (TBP) gene can be used. Furthermore, it is possible to use hybrid promoters comprising upstream activation sequences (UAS) of one yeast gene and downstream promoter elements including a functional TATA box of another yeast gene, for example a hybrid promoter including the UAS (s) of the yeast PH05 gene and downstream promoter elements including a functional TATA box of the yeast GAP gene (PH05- GAP hybrid promoter). A suitable constitutive PH05 promoter is e. g. a shortened acid phosphatase PH05 promoter devoid of the upstream regulatory elements (UAS) such as the PH05 (-173) promoter element starting at nucleotide -173 and ending at nucleotide -9 of the PH05 gene.

Transcription of a DNA encoding HIV CA or CPSF6 by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are relatively orientation and position independent. Many enhancer sequences are known from mammalian genes (e. g. elastase and globin). However, typically one will employ an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270) and the CMV early promoter enhancer. The enhancer may be spliced into the vector at a position 5' or 3' to HIV CA or CPSF6 DNA, but is preferably located at a site 5' from the promoter.

Eukaryotic expression vectors will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5'and 3'untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding HIV CA or CPSF6.

Construction of vectors according to the invention employs conventional ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a known fashion. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing HIV CA or CPSF6 expression and function are known to those skilled in the art. Gene presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), or in situ hybridization, using an appropriately labeled probe which may be based on a sequence provided herein. Those skilled in the art will readily envisage how these methods may be modified, if desired. HIV CA and CPSF6 are drug development targets

According to the present invention, HIV CA or CPSF6 polypeptides are used as targets to identify compounds, for example lead compounds for pharmaceuticals, which are capable of modulating the infectivity of HIV by modulating its interaction with nuclear transport factors.

Accordingly, the invention relates to an assay and provides a method for identifying a compound or compounds capable, directly or indirectly, of modulating the infectivity of HIV, comprising the steps of :

(a) incubating HIV CA or CPSF6 polypeptides with the compound or compounds to be assessed ; and

(b) identifying those compounds which influence the binding of HIV CA to CPSF6. HIV CA or CPSF6 binding compounds

According to a first embodiment of this aspect invention, the assay is configured to detect polypeptides which bind directly to the HIV CA or CPSF6 polypeptides.

Binding to HIV CA or CPSF6 polypeptides may be assessed by any technique known to those skilled in the art.

Examples of suitable assays include the two hybrid assay system, which

measures interactions in vivo, affinity chromatography assays, for example involving binding to polypeptides immobilized on a column, fluorescence assays in which binding of the compound (s) and HIV CA or CPSF6 polypeptides is associated with a change in fluorescence of one or both partners in a binding pair, and the like.

Preferred are assays performed in vivo in cells, such as the two-hybrid assay. In a preferred aspect of this embodiment, the invention provides a method for identifying a lead compound for a pharmaceutical useful in the treatment of disease involving HIV infection, comprising incubating a compound or compounds to be tested with a HIV CA or CPSF6 polypeptides, under conditions in which, but for the presence of the compound or compounds to be tested, HIV CA associates with CPSF6 with a reference affinity;

determining the binding affinity of HIV CA for CPSF6 in the presence of the compound or compounds to be tested ; and

selecting those compounds which modulate the binding affinity of HIV CA for CPSF6 with respect to the reference binding affinity.

Preferably, therefore, the assay according to the invention is calibrated in absence of the compound or compounds to be tested, or in the presence of a reference compound whose activity in interacting with HIV CA or CPSF6 polypeptides is known or is otherwise desirable as a reference value. For example, in a two-hybrid system, a reference value may be obtained in the absence of any compound. Addition of a compound or compounds which increase the binding affinity of HIV CA for CPSF6 increases the readout from the assay above the reference level, whilst addition of a compound or compounds which decrease this affinity results in a decrease of the assay readout below the reference level.

Compounds which modulate the functional CA-CPSF6 interaction In a second embodiment, the invention may be configured to detect functional interactions between a compound or compounds and HIV CA or CPSF6

polypeptides. Such interactions can affect the ability of HIV CA to interact with nuclear transport factors such as TP03 or RanBP2, and therefore HIV infectivity. Assays which detect modulation of the functional interaction between HIV CA and CPSF6 polypeptides are preferably cell-based assays. For example, they may be based on infection assays using cultured cells which are exposed to HIV virions in the presence or absence of the test compound(s). In preferred embodiments, a nucleic acid encoding a HIV CA or CPSF6 polypeptide is ligated into a vector, and introduced into suitable host cells to produce transformed cell lines that express the HIV CA or CPSF6 polypeptides. The resulting cell lines can then be produced for reproducible qualitative and/or quantitative analysis of the effect (s) of potential compounds affecting HIV CA or CPSF6 polypeptides function. Thus HIV CA or CPSF6 polypeptide-expressing cells may be employed for the

identification of compounds, particularly low molecular weight compounds, which modulate the interaction between HIV CA and CPSF6 polypeptides. Thus host cells expressing HIV CA or CPSF6 polypeptides are useful for drug screening and it is a further object of the present invention to provide a method for identifying compounds which modulate the activity of HIV CA or CPSF6, said method comprising exposing cells containing heterologous DNA encoding HIV CA or CPSF6 polypeptides, wherein said cells produce functional HIV CA or CPSF6, to at least one compound or mixture of compounds or signal whose ability to modulate the interaction of said HIV CA or CPSF6 polypeptides is sought to be determined, and thereafter monitoring said cells for changes caused by said modulation. Such an assay enables the identification of modulators, such as agonists, antagonists and allosteric modulators, of the interaction between HIV CA and CPSF6.

In addition to, or as well as, transfected HIV CA or CPSF6 polypeptides, the endogenous CPSF6 may be used to detect interaction with transfected HIV CA or CA derived from a viral infection.

Cell-based screening assays can be designed by constructing cell lines in which the expression of a reporter protein, i. e. an easily assayable protein, such as p galactosidase, chloramphenicol acetyltransferase (CAT) or luciferase, is dependent on the interaction between HIV CA and CPSF6 polypeptides. For example, a reporter gene encoding one of the above polypeptides may be placed under the control of an enhancer which is activated by a factor assembled ni a two-hybrid reaction between HIV CA and CPSF6 polypeptides.

Alternative assay formats include assays which directly assess HIV infectivity in a biological system. Such systems are known in the art, and further described below.

Compounds which modulate the interaction between HIV CA and CPSF6 polypeptides. As noted above, assays may be configured to detect binding between HIV CA and CPSF6 polypeptides, or the modulation of viral infectivity by disruption of the indirect interaction between CPSF6 and nuclear transport factors. Examples of compounds which are capable of modulating the interaction between HIV CA and CPSF6 polypeptides include compounds such as PF-3450074. Compounds

In a still further aspect, the invention relates to a compound or compounds identifiable by an assay method as defined in the previous aspect of the invention.

Accordingly, there is provided the use of a compound identifiable by an assay as described herein, for the modulation of the infectivity of HIV.

Compounds which influence the HIV CA CPSF6 interaction may be of almost any general description, including low molecular weight compounds, including organic compounds which may be linear, cyclic, polycyclic or a combination thereof, peptides, polypeptides including antibodies, or proteins. In general, as used herein, "peptides", "polypeptides" and "proteins" are considered equivalent.

Inhibitor compounds preferably comprise molecules that are hydrophobic competitors for residues on the N-terminal capsid domain that are themselves hydrophobic but partially exposed to solvent. These residues include L56, L69, 1134, I73, W133, A105, A77, T107, Y130 and M66. Inhibiting compounds form stacking interactions and desolvate these residues. They may be mimics of the structure of the natural CPSF6 ligand, which uses three discrete hydrophobic side-chains (residues F330, L324 and V323) to occupy three sub-sites on the capsid. Ligands are likely to mediate binding through a composite of weaker interactions, which when added together drive interaction. In addition to hydrophobic interactions, competitive inhibitors should form hydrogen bond and electrostatic interactions with residues K70, Q67, N74, N53 and S102. Ligands should compete for the CPSF6 interface but not necessarily occlude binding to other cellular co-factors such as Cyclophilin A. Competitors will reduce infectious titre of HIV and related lentiviruses by inhibiting post-entry, pre-integration steps in the viral life cycle. Specifically, compounds will prevent recruitment of cellular co-factors either directly or indirectly to the interface on the N-terminal capsid domain defined by CPSF6 and including those residues listed above. Interfering with this recruitment will lead to prevention of nuclear entry of the virus and integration into sites in the genome that are consistent with productive infection. Antibodies

Antibodies, as used herein, refers to complete antibodies or antibody fragments capable of binding to a selected target, and including Fv, ScFv, Fab' and F (ab') 2, monoclonal and polyclonal antibodies, engineered antibodies including chimeric, CDRgrafted and humanized antibodies, and artificially selected antibodies produced using phage display or alternative techniques. Small fragments, such Fv and ScFv, possess advantageous properties for diagnostic and therapeutic applications on account of their small size and consequent superior tissue distribution.

The antibodies according to the invention are especially indicated for diagnostic and therapeutic applications. Accordingly, they may be altered antibodies comprising an effector protein such as a toxin or a label. Especially preferred are labels which allow the imaging of the distribution of the antibody in vivo. Such labels may be radioactive labels or radioopaque labels, such as metal particles, which are readily visualizable within the body of a patient. Moreover, the may be fluorescent labels or other labels which are visualizable on tissue samples removed from patients.

Recombinant DNA technology may be used to improve the antibodies of the invention. Thus, chimeric antibodies may be constructed in order to decrease the immunogenicity thereof in diagnostic or therapeutic applications. Moreover, immunogenicity may be minimized by humanizing the antibodies by CDR grafting [see European Patent Application 0 239 400 (Winter)] and, optionally, framework modification [see international patent application WO 90/07861 (Protein Design Labs)].

Antibodies according to the invention may be obtained from animal serum, or, in the case of monoclonal antibodies or fragments thereof, produced in cell culture. Recombinant DNA technology may be used to produce the antibodies according to established procedure, in bacterial or preferably mammalian cell culture. The selected cell culture system preferably secretes the antibody product.

Therefore, the present invention includes a process for the production of an antibody according to the invention comprising culturing a host, e. g. E. coli or a mammalian cell, which has been transformed with a hybrid vector comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding said protein, and isolating said protein.

Multiplication of hybridoma cells or mammalian host cells in vitro is carried out in suitable culture media, which are the customary standard culture media, for example Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium, optionally replenished by a mammalian serum, e. g. fetal calf serum, or trace elements and growth sustaining supplements, e. g. feeder cells su suspension culture, e. g. in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e. g. in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges.

Large quantities of the desired antibodies can also be obtained by multiplying mammalian cells in vivo. For this purpose, hybridoma cells producing the desired antibodies are injected into histocompatible mammals to cause growth of antibodyproducing tumors. Optionally, the animals are primed with a hydrocarbon, especially mineral oils such as pristane (tetramethyl-pentadecane), prior to the injection. After one to three weeks, the antibodies are isolated from the body fluids of those mammals. For example, hybridoma cells obtained by fusion of suitable myeloma cells with antibody-producing spleen cells from Balb/c mice, or transfected cells derived from hybridoma cell line Sp2/0 that produce the desired antibodies are injected intraperitoneally into Balb/c mice optionally pre-treated with pristane, and, after one to two weeks, ascitic fluid is taken from the animals. The foregoing, and other, techniques are discussed in, for example, Kohler and Milstein, (1975) Nature 256 : 495-497 ; US 4, 376, 1 10 ; Harlow and Lane,

Antibodies: a Laboratory Manual, (1988) Cold Spring Harbor, incorporated herein by reference. Techniques for the preparation of recombinant antibody molecules is described in the above references and also in, for example, EP 0623679 ; EP 0368684 and EP 0436597, which are incorporated herein by reference.

Antibodies and antibody fragments according to the invention are useful in targeting HIV CA or CPSF6 polypeptides, and can inhibit binding between these molecules. Peptides

Peptides according to the present invention are usefully derived from HIV CA or CPSF6 or another polypeptide involved in the functional interaction between HIV CA and the nuclear transport factors involved in HIV infection.

Preferably, the peptides are derived from the domains in HIV CA or CPSF6 which are responsible for HIV CA CPSF6 interaction. For example, Thornberry et al., (1994) Biochemistry 33: 39343940 and Milligan et al., (1995) Neuron 15 : 385-393 describe the use of modified tetrapeptides to inhibit ICE protease. In an analogous fashion, peptides derived from HIV CA or CPSF6 or an interacting protein may be modified, for example with an aldehyde group, chloromethylketone, (acyloxy) methyl ketone or CH20C(0)-DCB group to inhibit the HIV CA/CPSF6 interaction.

In order to facilitate delivery of peptide compounds to cells, peptides may be modified in order to improve their ability to cross a cell membrane. For example, US 5, 149, 782 discloses the use of fusogenic peptides, ion-channel forming peptides, membrane peptides, long-chain fatty acids and other membrane blending agents to increase protein transport across the cell membrane. These and other methods are also described in WO 97/37016 and US 5, 108, 921 , incorporated herein by reference.

Many compounds according to the present invention may be lead compounds useful for drug development. Useful lead compounds are especially antibodies and peptides, and particularly intracellular antibodies expressed within the cell in a gene therapy context, which may be used as models for the development of peptide or low molecular weight therapeutics. In a preferred aspect of the invention, lead compounds and HIV CA or CPSF6 or other target peptide may be co-crystallized in order to facilitate the design of suitable low molecular weight compounds which mimic the interaction observed with the lead compound.

Crystallization involves the preparation of a crystallization buffer, for example by mixing a solution of the peptide or peptide complex with a "reservoir buffer", preferably in a 1 : 1 ratio, with a lower concentration of the precipitating agent necessary for crystal formation. For crystal formation, the concentration of the precipitating agent is increased, for example by addition of precipitating agent, for example by titration, or by allowing the concentration of precipitating agent to balance by diffusion between the crystallization buffer and a reservoir buffer. Under suitable conditions such diffusion of precipitating agent occurs along the gradient of precipitating agent, for example from the reservoir buffer having a higher

concentration of precipitating agent into the crystallization buffer having a lower concentration of precipitating agent. Diffusion may be achieved for example by vapor diffusion techniques allowing diffusion in the common gas phase. Known techniques are, for example, vapor diffusion methods, such as the "hanging drop" or the "sitting drop" method. In the vapor diffusion method a drop of crystallization buffer containing the protein is hanging above or sitting beside a much larger pool of reservoir buffer. Alternatively, the balancing of the precipitating agent can be achieved through a semipermeable membrane that separates the crystallization buffer from the reservoir buffer and prevents dilution of the protein into the reservoir buffer.

In the crystallization buffer the peptide or peptide/binding partner complex preferably has a concentration of up to 30 mg/ml, preferably from about 2 mg/ml to about 4 mg/ml.

Formation of crystals can be achieved under various conditions which are essentially determined by the following parameters : pH, presence of salts and additives, precipitating agent, protein concentration and temperature. The pH may range from about 4. 0 to 9. 0. The concentration and type of buffer is rather unimportant, and therefore variable, e. g. in dependence with the desired pH. Suitable buffer systems include phosphate, acetate, citrate, Tris, MES and HEPES buffers. Useful salts and additives include e. g. chlorides, sulphates and other salts known to those skilled in the art. The buffer contains a precipitating agent selected from the group consisting of a water miscible organic solvent, preferably polyethylene glycol having a molecular weight of between 100 and 20000, preferentially between 4000 and 10000, or a suitable salt, such as a sulphates, particularly ammonium sulphate, a chloride, a citrate or a tartarate. A crystal of a peptide or peptide/binding partner complex according to the invention may be chemically modified, e. g. by heavy atom derivatization. Briefly, such derivatization is achievable by soaking a crystal in a solution containing heavy metal atom salts, or a organometallic compounds, e. g. lead chloride, gold thiomalate, thimerosal or uranyl acetate, which is capable of diffusing through the crystal and binding to the surface of the protein. The location (s) of the bound heavy metal atom (s) can be determined by X-ray diffraction analysis of the soaked crystal, which information may be used e. g. to construct a three-dimensional model of the peptide. A three-dimensional model is obtainable, for example, from a heavy atom derivative of a crystal and/or from all or part of the structural data provided by the crystallization. Preferably building of such model involves homology modeling and/or molecular replacement.

Computational software may also be used to predict the secondary structure of the peptide or peptide complex. The peptide sequence may be incorporated into the crystal structure. Structural incoherences, e. g. structural fragments around insertions/deletions can be modeled by screening a structural library for peptides of the desired length and with a suitable conformation. For prediction of the side chain conformation, a side chain rotamer library may be employed.

The final homology model is used to solve the crystal structure of the peptide by molecular replacement using suitable computer software. The homology model is positioned according to the results of molecular replacement, and subjected to further refinement comprising molecular dynamics calculations and modeling of the inhibitor used for crystallization into the electron density. Other Compounds

In a preferred embodiment, the above assay is used to identify peptide but also non- peptide-based test compounds that can modulate HIV CA or CPSF6 activity, as evidenced by HIV infectivity, or target polypeptide interactions. The test compounds of the present invention can be obtained using any of the numerous approaches involving combinatorial library methods known in the art, including: biological libraries, spatially addressable parallel solid phase or solution phase libraries ;

synthetic library methods requiring deconvolution; the One-bead one-compound' library method ; and synthetic library methods using affinity chromatography selection. These approaches are applicable to peptide, non-peptide oligomer, or small molecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des. 12: 145).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in : DeWitt et al. (1993) Proc. Natl. Acad. Sci. U. S. A. 90 : 6909 ; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91 : 1 1422 ; Zuckermann et al. (1994). J. Med. Chem. 37 : 2678 ; Cho et al. (1993) Science 261 : 1303 ; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33 : 2059 ; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33 : 2061 ; and in Gallop et al. (1994) J. Med. Chem. 37 : 1233.

Libraries of compounds may be presented in solution (e. g., Houghten (1992) ? Biotechniques 13 : 412-421 ), or on beads (Lam (1991 ) Nature 354:82-84), chips (Fodor (1993) Nature 364 : 555-556), bacteria (Ladner USP 5, 223, 409), spores (Ladner USP '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89 : 1865- 1869) or on phage (Scott and Smith (1990) Science 249 : 386-390) ; (Devlin (1990) Science 249 : 404-406) ; (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87 : 6378-6382); (Felici (1991 ) J. Mol. Biol. 222:301 -310); Ladner (supra.).

If desired, any of the compound libraries described herein may be divided into preselected libraries comprising compounds having, e. g., a given chemical structure, or a given activity, e. g., kinase inhibitory activity. Pre-selecting a compound library may further involve performing any art recognized molecular modeling in order to identify particular compounds or groups or combinations of compounds as likely to have a given activity, reactive site, or other desired chemical functionality. In one

embodiment, modulators of HIV CA or CPSF6 are pre-selected using molecular modeling designed to identify compounds having, or likely to have, activity on HIV infectivity.

Suitable methods, as are known in the art, can be used to select particular moieties for interacting with a particular domain of HIV CA or CPSF6 or target component. For example, visual inspection, particularly utilizing three-dimensional models, can be employed. Preferably, a computer modeling program, or software, is used to select one or more moieties which can interact with a particular domain.

Suitable computer modeling programs include QUANTA (Molecular Simulations, Inc., Burlington, MA (1992) ), SYBYL (Tripos Associates, Inc. , St. Louis, MO (1992)), AMBER (Weiner et al. , J. Am. Chem. Soc. 106 : 765-784 (1984) ) and CHARMM (Brooks et al., J. Comp. Chem. 4 : 187-217 (1983) ). Other programs which can be used to select interacting moieties include GRID (Oxford University, U. K.; Goodford et al. , J. Mod. Chem. 28 : 849-857 (1985)) ; MCSS (Molecular Simulations, Inc. , Burlington, MA ; Miranker, A. and M. Karplus, Proteins : Structure, Function and Genetics 1 1 : 29-34 (1991 )) ; AUTODOCK (Scripps Research Institute, La Jolla, CA ; Goodsell et al., Proteins : Structure, Function and Genetics : 195-202 (1990)) ; and DOCK (University of California, San Francisco, CA ; Kuntz et al. , J. Mol. Biol. 161 : 269-288 (1982). After potential interacting moieties have been selected, they can be attached to a scaffold which can present them in a suitable manner for interaction with the selected domains. Suitable scaffolds and the spatial distribution of interacting moieties thereon can be determined visually, for example, using a physical or computer-generated threedimensional model, or by using a suitable computer program, such as CAVEAT (University of California, Berkeley, CA ; Bartlett et al., in "Molecular Recognition of in Chemical and Biological Problems", Special Pub., Royal Chemical Society 78 : 182- 196 (1989)) ; three-dimensional database systems, such as MACCS-3D (MDL Information Systems, San Leandro, CA (Martin, Y. C, J. Mod. Chem. 35 : 2145-2154 (1992)) ; and HOOK (Molecular Simulations, Inc. ). Other computer programs which can be used in the design and/or evaluation of potential HIV CA CPSF6 inhibitors include LUDI (Biosym Technologies, San Diego, CA ; Bohm, H. J., J. Comp. Aid. Molec. Design : 61 -78 (1992) ), LEGEND (Molecular Simulations, Inc. ; Nishibata et al., Tetrahedron 47 : 8985 8990 (1991 )), and LeapFrog (Tripos Associates, Inc.).

In addition, a variety of techniques for modeling protein-drug interactions are known in the art and can be used in the present method (Cohen et al. , J. Med. Chem. 33 : 883-894 (1994) ; Navia et al. Current Opinions in Structural Biology 2 : 202-210 (1992); Baldwin et al. , J. Mod. Chem. 32 : 2510-2513 (1989) ; Appelt et al. ; J. Mod. Chem. 34: 1925-1934 (1991 ) ; Ealick et al. , Proc. Nat. Acad. Sci. USA 88 : 1 1540- 1 1544 (1991 )).

Thus, a library of compounds, e. g., compounds that are protein based,

carbohydrate based, lipid based, nucleic acid based, natural organic based, synthetically derived organic based, or antibody based compounds can be assembled and subjected, if desired, to a further preselection step involving any of the aforementioned modeling techniques. Suitable candidate compounds determined to be HIV CA or CPSF6 modulators using these modeling techniques may then be selected from art recognized sources, e. g., commercial sources, or, alternatively, synthesized using art recognized techniques to contain the desired moiety predicted by the molecular modeling to have an activity, e. g., HIV inhibitory activity. These compounds may then be used to form e. g., a smaller or more targeted test library of compounds for screening using the assays described herein. In one embodiment, an assay is a cell-based or cell-free assay in which either a cell that expresses, e. g., a HIV CA or CPSF6 polypeptide or cell lysate/or purified protein comprising HIV CA or CPSF6 is contacted with a test compound and the ability of the test compound to alter HIV CA or CPSF6 activity, e. g., binding activity or HIV inhibition is measured.

Any of the cell-based assays can employ, for example, a cell of eukaryotic or prokaryotic origin. Determining the ability of the test compound to bind to a HIV CA or CPSF6 polypeptide can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the polypeptide can be determined by detecting the labeled compound in a complex.

For example, test compounds can be labeled with ¹²⁵l, ³⁵S,¹⁴C,³³P or ³H, either directly or indirectly, and the radioisotope detected by direct counting of

radioemmission or by scintillation counting. Alternatively, test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline

phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

It is also within the scope of this invention to determine the ability of a test compound to interact with a target polypeptide without the labeling of any of the interactants. For example, a microphysiometer can be used to detect the interaction of a test compound with HIV CA or CPSF6 without the labeling of either the test compound, HIV CA or CPSF6 (McConnell, H. M. et al. (1992) Science 257 : 1906-1912). In yet another embodiment, an assay of the present invention is a cellfree assay in which, e. g., HIV CA or CPSF6 are contacted with a test compound and the ability of the test compound to alter the interaction is determined.

Determining the ability of the candidate compound to bind to either polypeptide can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA) (Sjolander, S. and Urbaniczky, C. (1991 ) Anal. Chem. 63 : 2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5 : 699-705). As used herein, "BIA" is a technology for studying bispecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed in cell- free systems, such as may be performed using purified or semi-purified proteins, are often preferred as "primary" screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with upstream or downstream elements. Accordingly, in an exemplary screening assay of the present invention, the compound of interest is contacted with the HIV CA or CPSF6 polypeptide as set forth above. The efficacy of the test compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound. Moreover, a control assay can also be performed to provide a baseline for comparison. In another embodiment, various candidate compounds are tested and compared to a control compound with a known activity, e. g., an inhibitor having a known generic activity, or, alternatively, a specific activity, such that the specificity of the test compound may be determined.

In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize the target polypeptide to facilitate separation of complexed from uncomplexed forms or accommodate automation of the assay.

Binding of HIV CA to CPSF6 polypeptide in the presence or absence of a test compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/target polypeptide fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound and incubated under conditions conducive to phosphorylation or complex formation (e. g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, and the complex is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of target polypeptide binding or

phosphorylation activity can be determined using standard techniques. Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention.

In yet another aspect of the invention, HIV CA or CPSF6 polypeptides can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e. g., U. S. Patent No. 5, 283, 317 ; Zervos et al. (1993) Cell 72 : 223-232 ; Madura et al. (1993) J. Biol. Chem. 268 : 12046-12054 ; Bartel et al. (1993) Biotechniques 14 : 920-924 ; Iwabuchi et al. (1993) Oncogene 8 : 1693-1696 ; and Brent W094/10300), to identify other proteins or compounds, which bind to or interact with HIV CA or CPSF6.

This invention further pertains to novel agents identified by the above-described screening assays and to processes for producing such agents by use of these assays. Accordingly, in one embodiment, the present invention includes a compound or agent obtainable by a method comprising the steps of any one of the aforementioned screening assays (e. g., cell-based assays or cell-free assays). For example, in one embodiment, the invention includes a compound or agent obtainable by any of the methods described herein.

Accordingly, it is within the scope of this invention to further use an agent, e. g., a HIV CA or CPSF6 polypeptide or compound identified as described herein in an appropriate animal model. For example, an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.

Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. In addition, such an agent if deemed appropriate, may be administered to a human subject.

The present invention also pertains to uses of novel agents identified by the above- described screening assays for diagnoses, prognoses, and treatments of any of the disorders described herein. Accordingly, it is within the scope of the present invention to use such agents in the design, formulation, synthesis, manufacture, and/or production of a drug or pharmaceutical composition for use in diagnosis, prognosis, or treatment of any of the disorders described herein. 4. Pharmaceutical Compositions

In a preferred embodiment, there is provided a pharmaceutical composition comprising a compound or compounds identifiable by an assay method as defined in the previous aspect of the invention.

A pharmaceutical composition according to the invention is a composition of matter comprising a compound or compounds capable of modulating the infectivity of HIV as an active ingredient. Typically, the compound is in the form of any pharmaceutically acceptable salt, or, e.g., where appropriate, an analog, free base form, tautomer, enantiomer racemate, or combination thereof. The active ingredients of a

pharmaceutical composition comprising the active ingredient according to the invention are contemplated to exhibit excellent therapeutic activity, for example, in the treatment or prevention of HIV infection. For example, the invention

encompasses any compound that can alter the binding of HIV CA to CPSF6.

Dosage regima may be adjusted to provide the optimum therapeutic response.

For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

The active ingredient may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implanting (e. g. using slow release molecules). Depending on the route of administration, the active ingredient may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said ingredient.

In order to administer the active ingredient by other than parenteral administration, it will be coated by, or administered with, a material to prevent its inactivation. For example, the active ingredient may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes. Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon. Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as

polyoxyethylene oleyl ether and nhexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes.

The active ingredient may also be administered parenterally or intraperitoneally.

Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the

extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid

polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants.

The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active ingredient in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When the active ingredient is suitably protected as described above, it may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active ingredient may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of active ingredient in such therapeutically useful compositions in such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain the following : a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier.

Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active ingredient may be incorporated into sustained-release preparations and formulations.

As used herein "pharmaceutically acceptable carrier and/or diluent" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated ; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such as active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired.

The principal active ingredients are compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients. In a further aspect there is provided the active ingredient of the invention as hereinbefore defined for use in the treatment of disease either alone or in

combination with art recognized compounds known to be suitable for treating the particular indication. The invention is further described, for the purpose of illustration only, in the following examples.

Examples METHODS

Protein expression and purification

HIV-1 and HIV-2 CAN were expressed in BL21 (DE3) E. coli cells and purified as described (price et al). SIVmac and FIV CAN were expressed with an N-terminal His tag in BL21 (DE3) E. coli cells and purified by capture on Ni-NTA resin (Qiagen) followed by gel filtration. All HIV-1 CAN mutants were purified as per the wild type protein. Isothermal titration calorimetry (ITC)

Proteins were prepared by dialysis against a buffer containing 50 mM potassium phosphate (pH 7.4), 100 mM NaCI and 1 mM DTT. The chemically synthesized CPSF6313-327 peptide (Designer Bioscience) was dissolved in the same buffer. ITC experiments were conducted on a MicroCal ITC-200, with CPSF6313-327 (10 mM) in the syringe and CAN (600 μΜ) in the cell. Drug PF-3450074 was synthesized in- house and binding to CAN proteins carried out as described [26], except with protein (200μΜ) in the syringe and drug (30 μΜ) in the cell. Data were analyzed using Origin data analysis software (MicroCal).

Crystallization, data collection, structure determination and refinement

Crystals of HIV-1 CAN:CPSF6313-327 grew at 17 °C in sitting drops. Protein/peptide solution (0.37 mM HIV-1 CAN and 4 mM CPSF6313-327 in 20 mM HEPES pH 7, 50 mM NaCI, 1 mM DTT) was mixed with reservoir solution (20% w/v PEG 3350, 0.2 M potassium phosphate dibasic) in a 1 :1 mix, producing 0.55 mm x 0.15 mm x 0.05 mm crystals within one week. Crystals were flash-frozen in liquid nitrogen and data collected on an in-house Mar-345 detector to a resolution of 1 .8 A. Crystal data collection and refinement statistics are provided in Supplementary Table 1. The dataset was processed using the CCP4 program suite (collab comp etc). Data were indexed and scaled in MOSFLM and SCALA, respectively. The structure was determined by molecular replacement in PHASER using HIV-1 CAN (pdb: 2GON) as a model. Structural figures were prepared using PyMOL (MacPyMOL Molecular Graphics System, 2009, DeLano Scientific LLC). Cells and viruses

HeLa cells were transfected with EXN-based expression plasmids containing HA- tagged CPSF6 constructs and transduced cells were selected with 1 mg/ml G418 (Gibco). Transgenic expression was confirmed by western blot using a-HA monoclonal antibody 16B12 (Covance). To optimize the CPSF6- 358-mediated restriction phenotype, HeLa cells expressing CPSF6-358 were FACS sorted into single cells and selected with 1 mg/ml G418 to generate monoclonal cell lines and the best-restricting clone was chosen for use in the indicated infection experiments. HeLa cells stably depleted for TNP03 or NUP358 were made using short hairpin sequences expressed from MLV vector pSIREN RetroQ (Clontech) and depletion confirmed using mouse transports 3 antibody ab54353 (Abeam) and a Nup358 antibody kindly given by Frauke Melchior. VSV-G pseudotyped GFP-encoding lentiviral vectors based on HIV-1 NL4.3 were prepared in HEK 293T cells, as described [32].

Infection assays

Cells were seeded in 6-well plates at 1x105 cells/well and inoculated with GFP- reporter virus in the presence of 5 μg/ml polybrene. The virus dose was selected so as to infect -30% of unmodified cells and the percentage of GFP-positive cells enumerated 48 h later by flow cytometry. Unless otherwise indicated, experiments were performed in triplicate and one representative experiment is shown in each case. Titers are plotted as infectious units per ng of reverse transcriptase activity ± standard deviation.

Immunofluorescence

Cells were plated on glass coverslips, washed with PBS and fixed with 4% PFA in PBS before being permeabilized with 0.5% Triton in PBS for 10 min at room temperature, washed with PBS and then blocked with 5% BSA in PBS containing 0.1 % Tween (PBST) for 1 h at room temperature. Cells were incubated for 1 h with the first antibody (α-ΗΑ 16B12) at 1 :250 dilution, washed three times with PBST and then incubated for 1 h with the secondary antibody (Alexa-488 conjugated anti-mouse IgG (Invitrogen)) at 1 :400 dilution. Coverslips were mounted onto glass slides using Vectashield mounting medium with DAPI (Vector Labs) and imaged using a Zeiss 780 confocal microscope equipped with a 63x/1 .4 NA Plan-Apochromat oil- immersion objective. Images were taken under identical conditions to aid

comparison. Images were prepared using ImageJ (NIH).

Accession codes.

Protein Data Bank: Coordinates for HIV-1 CAN:CPSF6313-327 have been deposited.

Example 1 : CPSF6 binds diverse lentiviral CAs

To test our hypothesis that there is an unidentified interaction interface on HIV-1 CA, we investigated CA interaction with CPSF6 using a combination of biophysical, structural and cellular infection approaches. CPSF6 residues 313-327 have been shown to be necessary for restriction of HIV- 1 by CPSF6-358 (manuscript submitted). A peptide corresponding to this putative HIV-1 -interacting region

(CPSF6313-327) was therefore synthesized and binding to recombinant HIV-1 CA N- terminal domain (CAN) tested by isothermal titration calorimetry (ITC). We found that CPSF6 residues 313-327 were sufficient for direct binding to HIV-1 CAN (Figure 2A) with low affinity (362 μΜ). Next, we tested binding of CPSF6313-327 to HIV-1 CA mutant N74D, which escapes CPSF6-358 restriction. This single mutation all but abolished binding to CPSF6313-327 (Kd > 5 mM) (Figure 2B). This suggests that CPSF6 binding to HIV-1 CA is specific and that N74D allows escape from CPSF6- 358 restriction by preventing CA interaction with CPSF6-358. To determine whether CPSF6 binding is conserved across diverse lentiviruses, we measured the interaction between CPSF6313-327 and CANs from HIV-2, SIVmac and FIV. All three lentiviral CANs bound to CPSF6313-327, with an affinity of 219-350 μΜ (Figure 2C). Binding of CPSF6313-327 to HIV-2 and SIVmac agreed with published data showing that these viruses are restricted by CPSF6-358; however, binding of CPSF6313-327 to FIV CAN was unexpected, given that FIV, like HIV-1 N74D, is insensitive to CPSF6- 358 restriction [14]. Indeed, FIV CAN paradoxically bound to CPSF6 with a higher affinity than HIV-1 (242 μΜ compared to 362 μΜ). This suggests that the mechanism of CPSF6-358 restriction is not a structural one (acting to accelerate or prevent uncoating) but rather competitive inhibition of a host cofactor necessary for HIV-1 , HIV-2 and SIVmac. The conservation of CPSF6 binding by FIV suggests that whilst this interaction is not neccesary for FIV infection of HeLa cells, it cannot be assumed that CPSF6 is not a cofactor for FIV infection in vivo.

Example 2: Crystal structure of HIV-1 CAN in complex with CPSF6313-327

To understand how CPSF6313-327 binds directly to HIV-1 CAN, we solved the crystal structure of the complex between HIV-1 CAN and CPSF6313-327 at 1 .8 A resolution (Figure 3). In the complexed structure, CPSF6313-327 lies in a binding site comprised of a narrow channel formed on one side by helix 4 and on the other by helices 3 and 5 and the helix 5/6 turn (Figure 3A). Three discrete pockets in the centre of the channel are filled by CPSF6 residues V314, L315 and F321 (Figure 3B). The channel is closed at one end around residue Q63 and extends the length of helix 4, until the beginning of the CypA-binding loop at V86 where it opens into solvent. The interface, as defined by CPSF6, is bordered by CA residues 53, 56-57, 66-67, 70, 73-74, 105, 107, 109 and 130 (Figure 3C). CPSF6313-327 itself does not possess any secondary structure but forms a relatively compact loop due to intramolecular interactions centering on the Q319 side chain, which hydrogen bonds to the amide nitrogen of F316 and the carbonyl oxygens of V314 and Q323, pinning the two halves of the peptide together (Figure 3D). Additional constraining intramolecular interactions are also made between the peptide oxygen of F316 and the amide nitrogen of Q319, and between the peptide oxygen of P320 and the amide nitrogen of Q323. Formation of these interactions is facilitated by proline residues P317 and P320, which introduce kinks into the backbone, and by glycine residues G318 and G322, which confer backbone flexibility. The N and C termini of CPSF6313-327 project directly out of the binding channel (Figure 3E), suggesting that CPSF6313-327 is a protruding structure within the full-length CPSF6 protein. This supports a model in which CPSF6 residues 313- 327 can access the CA interface in the context of intact, full-length CPSF6.

Interactions in the HIV-1 CAN :CPSF6313-327 complex

CPSF6313-327 is highly hydrophobic, containing only two polar residues (Q319 and Q323). Therefore, it makes a number of hydrophobic interactions with CA, including via V314, L315 and F321 , which project into the channel at the centre of the binding interface (Figure 3B). In addition to hydrophobic burial of CPSF6 side chains,

CPSF6 is also held in place by a number of hydrogen bonds between side chains in HIV-1 CAN and the backbone amide and carbonyl groups of CPSF6313-327, some of which are water mediated (Figure 4). Significantly, the side chain of CA residue N74 makes a bifurcated hydrogen bond with the main chain of L315 in CPSF6

(Figure 4B), which explains why the N74D mutation resulted in loss of binding to CPSF6 and escape from restriction by CPSF6-358 (Figure 2B and [14]). Two water-mediated interactions are also made between the backbone amide of V314 in CPSF6 and the main chain carbonyl of N74, and between the backbone carbonyl of V314 and the side chain of T107 (Figure 4B). CPSF6 makes two further interactions with side chains from helix 4 in HIV-1 CAN: one between the backbone nitrogen of G318 in CPSF6 and the CA Q67 side chain; and the other between the peptide oxygen of Q319 in CPSF6 and the CA K70 side chain (Figure 4C). CA N57 is another key interaction residue for CPSF6 binding. Similar to CA N74, the side-chain of N57 mediates a bifurcated hydrogen bond with the backbone of F321 in CPSF6. This positions the benzyl side chain of F321 for hydrophobic burial beneath the aliphatic side chain of K70. The identification of CA N57 as an important

residue for CPSF6 binding is of interest, as mutation N57A T54A impairs HIV-1 infection of nondividing cells [8]. Finally, several water-mediated interactions are made between the backbone of G322 and the side chains of N53 and Y130 and the main chain carbonyls of A105 and S109 (Figure 4D). It is also worth noting the similarity between the location of the CPSF6-binding interface and exposed CA mutations that have been shown to affect infectivity

(Figure 1 ). Many of the side chains that are directly involved in CPSF6 binding (N57, Q67, K70 and N74) were found to reduce HIV-1 infectivity in a comprehensive alanine-scan of CAN[10] (Figure 4 and Figure 5B and C). Furthermore, alanine-scan mutants that map to CPSF6 are distinct in that their reduced infectivity is not fully explained by structural or assembly defects. Mutants Q63A Q67A and N74A have 5- 35 fold decreased infectivity but normal levels of particle production and no assembly defects[10]. Similarly, while mutants T54A N57A and K70A have fewer conical capsids, this was a minor defect (~4-fold with respect to wild type HIV-1 ) compared to their effect on infectivity (which was reduced by 20-80 fold) [10]. This lack of correspondance between magnitude of structural defect and loss of infection supports the conclusion that CPSF6 defines an interface in which residues have a role in mediating protein interaction.

Example 3: CPSF6 F321 is essential for interaction with HIV-1 CAN

The aromatic side chain of F321 forms extensive hydrophobic interactions with CA, suggesting it may be essential for CPSF6 binding. To confirm the importance of F321 , we tested the ability of CPSF6-358 F321 N to restrict HIV-1 infection. CPSF6- 358 was cloned by introducing a stop codon after residue N358 in the CPSF6 isoform NM_007007 from HeLa cell cDNA. Of the clones that we sequenced, NM_007007 was the predominantly expressed transcript. Previous characterisation was with the BC000714 isoform [14]. BC000714 contains exon 6 whereas NM_007007 lacks exon 6; however, despite this difference, CPSF6-358 from NM_007007 potently restricted wild type HIV-1 but not the escape mutant HIV-1 N74D, the same as observed for CPSF6-358 from BC000714 (data not shown). We found that CPSF6-358 F321 N was unable to restrict HIV-1 , demonstrating that F321 is a key residue required for interaction with CA (Figure 4E). This also provides correlative data that the mode of binding as observed in the crystal structure is the same as that used by CPSF6-358 during cellular restriction.

Example 4: The CPSF6-binding interface is accessible and highly conserved in HIV-1 virions

To address the accessibility of the CA:CPSF6 interface in the context of the hexameric CA, we superposed our HIV-1 CAN:CPSF6313-327 complex structure onto the recently solved structure of the HIV-1 CA hexamer (pdb: 3H47 [1 1]) (Figure 5). The monomers of the hexamer are arranged radially from a centre comprised of the packed N-terminal CA domains. The CPSF6-binding interface is found on the outside edge of the hexamer, where it is exposed to solvent and highly accessible for proteinprotein interaction (Figure 5A). The CPSF6-binding interface is also unaffected by the inter-hexamer interactions that occur exclusively between C- terminal CA domains and which build up the capsid lattice found in assembled virions. As can be seen, the CPSF6 interface is not involved in hexamer-hexamer interactions and is fully solvent accessible (Figure 5D). This suggests that CPSF6 binding does not require the dissociation of inter-subunit interactions found in the assembled capsid lattice. Together with the ITC and restriction data, which show that although FIV binds CPSF6313-327 it is not restricted by CPSF6-358 (Figure 2C and [14]), this suggests that binding of CPSF6-358 to the CPSF6 interface on capsid does not in and of itself restrict virus replication, for example by directly affecting capsid stability and uncoating, but rather competitively inhibits recruitment of endogenous CPSF6 and/or other cofactors necessary for productive nuclear import and integration. Next, we used our HIV-1 CAN:CPSF6313-327 complex structure to gain some measure of the importance of the CPSF6 interface in HIV-1 infection. Physiologically relevent protein-protein interfaces are more conserved than non- interacting surfaces [23]. Sequence mapping of -100 unique CAN sequences onto the complexed structure showed that the CPSF6-binding interface is very highly conserved within HIV-1 CA (Figure 5E) suggesting that it is a functionally important interface that may be required for efficient HIV-1 infection.

Mutation of the CPSF6-binding interface alters nuclear entry co-factor dependence

Mutation N74D is located at the centre of the CPSF6-binding interface and abolishes binding of CPSF6313-327 to CA. N74D also results in loss of dependence on TNP03, suggesting that the CPSF6- binding interface may be involved in HIV-1 nuclear entry. To test this, we investigated whether there is a correlation between mutation of CPSF6-binding interface residues, binding to CPSF6313-327, and viral dependence on nuclear entry co-factors TNP03 and RanBP2. Using our structure, we designed CA mutations with the aim of specifically knocking out CPSF6 binding. Five residues were selected for mutation (N57, Q67, K70, N74 and T107), on the basis that (1 ) they bind CPSF6 via their side chain and not their main chain and (2) are not obviously involved in maintaining CA structure. With the exception of N74D [14], all residues were mutated to alanine in accordance with previously published mutations [10]. We also made an additional M66F mutation in order to occlude the hydrophobic pocket filled by CPSF6 residue F321 , which we had found to be essential for CPSF6-358 restriction (Figure 4E). Modelling of this mutant on our structure suggested that the only F66 rotamer that would permit normal HIV-1 folding would be one that resulted in a steric clash with F321 . We tested the effect of these mutations on in vitro affinity to CPSF6313-327 (Figure 6A) and sensitivity of VSV-G pseudotyped HIV-1 infection to CPSF6-358 restriction and TNP03 and RanBP2 depletion (Figure 6B). All of the mutants showed reduced CPSF6313-327 affinity and CPSF6-358 restriction, confirming that the mutations had acted to impair CPSF6 binding. Strikingly, all of the mutations also resulted in either the loss (N57A and N74D) or reduction (Q67A, K70A and T107A) of dependence on TNP03 and

RanBP2, a phenotype previously only shown for N74D [14]. In broad agreement with previous observations [10], four out of the six mutations resulted in viruses with significantly impaired infectivity (N57A, M66F, Q67A and K70A), suggesting that they cause loss-of-function. Conversely, the wild type infectivity levels of mutants N74D and T107A suggest that they may alter co-factor usage, as proposed by Lee et al [14]. Importantly, the direct correlation between escape from CPSF6-358 restriction and the lack of sensitivity to TNP03 and RanBP2 depletion support a link between the CPSF6-binding interface in CA and the utilization of normal nuclear entry pathway components by HIV-1 . Example 5: Addition of an ectopic NLS to CPSF6-358 rescues CPSF6 nuclear localization and HIV-1 infection

Next, we investigated whether CPSF6 itself could be an HIV-1 co-factor. CPSF6 is known to shuttle in and out of the nucleus [18] and contains a C-terminal nuclear- targeting RS-domain [19, 20] of the type bound by TNP03 [21 , 22]. Therefore, binding of CPSF6 to capsid may facilitate viral nuclear entry. Indeed, deletion of the nuclear-targeting domain of CPSF6 results in a truncated cytosolic form (CPSF6- 358) that reduces viral titre [14, 20], suggesting that CPSF6-358 might act as a dominant negative, preventing the use of endogenous CPSF6 by HIV-1 . We hypothesised that retargeting truncated CPSF6 to the nucleus by attaching a different NLS motif might prevent this loss of titre. To test this, we determined the permissivity to HIV-1 infection of HeLa cells expressing full length CPSF6 (CPSF6-FL), a clonal line of HeLa cells expressing CPSF6-358 (CPSF6-358.4) and HeLa cells expressing CPSF6-358 with the SV40 NLS sequence 'PKKKRKVG' at the C-terminus (CPSF6- 358- NLS), and compared the subcellular localization of CPSF6 inside these cells. Whilst CPSF6-358.4 localized to both the cytosol and the nucleus, CPSF6-FL and CPSF6-358-NLS were entirely nuclear (Figure 8A). Furthermore, we observed that HIV-1 titre was reduced 50-fold in cells expressing CPSF6-358.4, whereas efficient infection was observed in cells expressing CPSF6-358-NLS (Figure 8B). The recovery of efficient infection upon restoration of CPSF6 nulcear transport is consistent with a model in which CPSF6 is a co-factor for HIV-1 nuclear import. Example 6: CPSF6-CA structure reveals antiviral drug mechanism

Several drugs have been identified that directly bind to HIV-1 CA [24-26]. In each case they are thought to inhibit viral replication by altering the stability of the capsid. Intriguingly, we observe that the most recently described drug, PF-3450074, binds within the CPSF6-binding interface [26]. Even more remarkably, one of the phenyl rings of the drug superposes almost exactly with the phenyl ring of CPSF6 residue F321 , a critical residue for CPSF6-CA interaction (Figure 7A). The reported mechanism of action of PF-3450074 is disruption of viral uncoating, however the evidence for this is inconsistent as both an increase in CA multimerization [26] and an increase in capsid destabilization [27] has been observed upon addition of PF- 3450074. Instead, we propose that PF-3450074 is a competitive inhibitor of a cellular cofactor, most likely CPSF6. To investigate the extent to which the drug occupies the same interface as CPSF6313-327, we measured the binding affinity of PF-3450074 to wild type HIV-1 CAN and each of the CAN mutants used in this study (Figure 7C). Whereas the drug bound wild type HIV-1 CAN with an affinity of 5 μΜ (similar to the previously reported affinity of 3 μΜ), mutations N57A and K70A abolished drug binding completely (Figure 7C). This is in agreement with the PF-3450074:HIV-1 CAN crystal structure, which shows that residues N57 and K70 form direct hydrogen bonds with the drug (Figure 7B). However, mutation of other key CPSF6 interface residues has little affect on drug binding; N74D and M66F reduced the affinity by 2- and 3-fold respectively, while Q67A had no effect despite Q67 forming a (weak) water-mediated hydrogen bond with the drug (Figure 7B and C). One mutation, T107A, even resulted in an increased affinity to the drug (Kd = 1 μΜ), possibly due to the removal of a slight steric repulsion between one of the aromatic moieties in PF- 3450074 and the T107 sidechain. These data show that PF-3450074 occupies only one pocket within a larger protein interface bound by CPSF6. Consequently, PF- 3450074 is unlikely to be as effective a drug as one that inhibits the entire CPSF6- binding interface. It may be possible to develop effective high-affinity drugs by addressing the CPSF6 interface as a drug target, either by compound or fragment screening or rational drug design. REFERENCES

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Sequence Listing

SEQ ID No 1 : CPSF6

MADGVDHIDIYADVGEEFNQEAEYGGHDQI DLYDDVI SPSANNG

DAPEDRDYMDTLPPTVGDDVGKGAAPNVVYTYTGKRIALYIGNLTWWTTDEDLTEAVH SLGVNDILEIKFFENRANGQSKGFALVGVGSEASSKKLMDLLPKRELHGQNPVVTPCN KQFLSQFEMQSRKTTQSGQMSGEGKAGPPGGSSRAAFPQGGRGRGRFPGAVPGGDRFP GPAGPGGPPPPFPAGQTPPRPPLGPPGPPGPPGPPPPGQVLPPPLAGPPNRGDRPPPP VLFPGQPFGQPPLGPLPPGPPPPVPGYGPPPGPPPPQQGPPPPPGPFPPRPPGPLGPP LTLAPPPHLPGPPPGAPPPAPHVNPAFFPPPTNSGMPTSDSRGPPPTDPYGRPPPYDR GDYGPPGREMDTARTPLSEAEFEEIMNRNRAI SSSAISRAVSDASAGDYGSAIETLVT AISLIKQSKVSADDRCKVLISSLQDCLHGIESKSYGSGSRRERSRERDHSRSREKSRR HKSRSRDRHDDYYRERSRERERHRDRDRDRDRERDREREYRHR

SEQ ID No 2: N- erminal CA

MPIVQNLQGQMVHQAISPRTLNAWVKWEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQM LKETINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILG LNKIVRMYS

Claims

1 . A method for identifying a compound capable of inhibiting infection by HIV, comprising contacting a HIV CA polypeptide with a CPSF6 polypeptide in the presence of the compound, and determining the influence of the compound on the binding of HIV CA to CPSF6, by measuring the interaction of CPSF6 with HIV CA at one or more of residues 53, 56-57, 66-67, 70, 73-74, 105, 107, 109 and 130 thereof.

2. A method according to claim 1 , wherein the CPSF6 polypeptide comprises residues 313-327 of CPSF6.

3. A method according to claim 2, wherein the CPSF6 polypeptide comprises at least residues V314, L315 and F321 .

4. A method according to any preceding claim, wherein the CA polypeptide comprises the structure of the HIV CA polypeptide residues 53-130.

5. A method according to any preceding claims, wherein the CPSF6 and HIV CA polypeptides are defined according to the sequences set for in SEQ ID Nos 1 and 2.

6. A method for identifying a compound or compounds capable, directly or indirectly, of modulating the interaction of HIV CA and CPSF6 and thereby the infectivity of HIV, comprising the steps of :

(a) incubating a CA polypeptide with the compound or compounds to be

assessed ; and

(b) identifying those compounds which bind to the CA polypeptide in the region defined by residues 53, 56-57, 66-67, 70, 73-74, 105, 107, 109 and 130.

7. A method according to claim 6, comprising:

incubating a compound or compounds to be tested with a CPSF6 polypeptide and a HIV CA polypeptide, under conditions in which, but for the presence of the compound or compounds to be tested, the interaction between CPSF6 and HIV CA induces a measurable chemical or biological effect;

determining the ability of the HIV CA polypeptide to interact, directly or indirectly, with the CPSF6 polypeptide to induce the measurable chemical or biological effect in the presence of the compound or compounds to be tested ; and selecting those compounds which modulate the interaction between CPSF6 and HIV CA.

8. A method according to claim 7, wherein the CPSF6 polypeptide includes an amino acid sequence having at least 75% identity with the amino acid sequence of residues 313-327 of wild-type CPSF6; and/or an HIV CA polypeptide that includes an amino acid sequence having at least 75% identity with residues 53-130 of the amino acid sequence of wild-type HIV CA.

9. A method for treating a condition associated with an HIV infection in a subject in need thereof by modulating the interaction of HIV CA and CPSF6, by administering a pharmaceutical composition capable of modulating interaction of HIV CA and CPSF6 in an amount sufficient to modulate the nuclear infection by HIV.

10. A compound which modulates the interaction of HIV CA and CPSF6 for use in modulating an HIV mediated condition.

1 1 . A method for developing an anti-HIV drug comprising the steps of (a) identifying one or more compounds which demonstrate anti-HIV infection activity; (b) screening said compounds and selecting one or more compounds which affect the interaction of HIV CA and CPSF6; (c) determining the structure of the compound and using structure-guided mutagenesis to prepare variants of the compound with improved activity.

12. A drug cocktail comprising two or more drugs for use in the treatment or prevention of an HIV infection, wherein at least one of said drugs is indicated for the disruption of the interaction between the HIV CA polypeptide and a nuclear entry cofactor.

13. The drug cocktail for use according to claim 12, wherein the nuclear entry factor is RanBP2 or TP03.

14. The drug cocktail for use according to claim 12 or claim 13, wherein the cocktail further comprises one or more anti-HIV drugs selected from the group consisting of efavirenz, emtricitabine, tenofovir, disoproxil fumarate, rilpivirine, lamivudine, zidovudine, emtricitabine, azidothymidine( AZT), nevirapine, amprenavir, tipranavir, indinavir, saquinavir mesylate, lopinavir, ritonavir, Fosamprenavir Calcium, darunavir, atazanavir sulfate, nelfinavir mesylate, raltegravir, maraviroc and enfuvirtide.

15. The drug cocktail for use according to any one of claims 9-14, wherein one or more drugs capable of disrupting of the interaction between the HIV CA polypeptide and a nuclear entry cofactor can be selected according to any one of claims 1 -10.

16. The drug cocktail for use according to claim 15, wherein the drug capable of disrupting of the interaction between the HIV CA polypeptide and a nuclear entry cofactor is PF-3450074.