WO2001090159A2 - Compositions and methods for delivery of a molecule into a cell - Google Patents

Abstract

Provided is a composition comprising a Vpr polypeptide conjugated to a therapeutic molecule. Preferably, the Vpr comprises synthetic Vpr. The therapeutic molecule can comprise any molecule capable of being conjugated to Vpr or a fragment thereof, including a polypeptide, a polynucleotide, and/or a toxin. The invention additionally provides a method for delivering a molecule into a cell. The method comprises contacting the cell with a conjugate comprising a Vpr polypeptide conjugated to the molecule. The invention further provides a method for modulating the expression of a transgene in a cell, a method for killing a target cell population in a subject, a method for increasing the sensitivity of cells to radiation therapy, and a method for inhibiting cell proliferation.

Description

COMPOSITIONS AND METHODS FOR DELIVERY OF A MOLECULE

INTO A CELL

This application claims priority of United States patent applications 60/206,610, filed May 23, 2000, 60/267,827, filed February 9, 2001, and 09/839,329, filed April 20, 2001. The entire contents of these applications is incorporated herein by reference.

Throughout this application various publications are referenced. In some instances, the references are indicated by numerals in parentheses, which numerals refer to a list of citations for the corresponding references that appears at the end of the specification. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention pertains. This application is related to German patent applications 199 08 752.0 and 199 08 766.0, filed on February 19, 1999 and February 17, 2000, respectively, as well as to the corresponding PCT international patent application filed on February 17, 2000. The disclosures of each of these related patent applications is incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The invention relates to compositions and methods for delivery of a molecule into a cell. More particularly, the invention relates to a Vpr polypeptide or fragment thereof conjugated to a molecule, which conjugate is capable of entering the plasma membrane of a cell. The compositions include pharmaceutical and vaccine compositions, and can be used in a variety of methods, including methods to modulate the sensitivity of cells to radiation therapy and to modulate cell proliferation and apoptosis.

BACKGROUND OF THE INVENTION

Human immunodeficiency virus type 1 (HIV-1) is a lentivirus that encodes the canonical retroviral Gag, Pol, and Env proteins, as well as six regulatory or auxiliary proteins including Tat, Rev, Vpu, Vif, Nef, and Vpr. Although not essential for viral replication in tissue culture, the latter four proteins are highly conserved and likely exert important but less well understood functions in vivo that contribute to viral pathogenesis. Vpr, a ~14-kDa, 96— amino acid protein is conserved among the primate lentiviruses HIV-1, HIV-2, and the simian immunodeficiency virus (SIV), supporting the notion that it serves an important function in the viral life cycle in ivo.

Human immunodeficiency virus (HIV) Vpr contributes to nuclear import of the viral prerntegration complex and induces G2 cell-cycle arrest in infected proliferating T lymphocytes. Vpr is highly cytotoxic in pro- and eukaryotic cells and forms homo- and heterologous protein complexes, characteristics that limit the production of recombinant Vpr. Although partial sequences of Vpr have been synthesized for biological (25, 26) and structural studies (32, 33, 34), chemical synthesis of full-length soluble forms of Vpr has proven difficult. Others (27) have reported that Vpr is difficult to synthesize due to its tendency for incomplete coupling, matπx interaction and peptide aggregation. For example, a Vpr peptide derived from the HIV-IBRU sequence has been synthesized, but irreversible aggregation precluded purification of the product (38, 39, 40). The biological activity of such forms of Vpr could not be analyzed in solution. There remains a need for a protocol for high-yield production of synthetic Vpr (sVpr), in which the resultant protein remains both soluble and stable in aqueous solution.

In addition, there remains a need for vehicles capable of delivering molecules into cells Given that Vpr contains at least two nuclear localization signals (3, 4, 5, 6, 7) and is capable of delivering molecules to the cell nucleus, a vehicle having the nuclear delivery capability of Vpr and also capable of efficiently entering the plasma membrane, preferably without requiring denaturation, is desirable.

SUMMARY OF THE INVENTION

The invention meets the above needs and others by providing a composition comprising a Vpr polypeptide conjugated to a therapeutic molecule. In preferred embodiments, the Vpr comprises synthetic Vpr. The synthetic Vpr is preferably stable in aqueous solution, such as the synthetic Vpr produced by the method described herein The therapeutic molecule can comprise any molecule capable of being conjugated to a Vpr polypeptide, including a second polypeptide, a polynucleotide, and/or a toxin. The toxin can be selected or modified so as to be toxic only to a target cell population. In one embodiment, the toxin or other molecule is modified by conjugation to a regulatory molecule, wherein contact with a target cell affects the regulatory molecule in such a manner as to result in activation of the toxin. For example, the regulatory molecule may be recognized by a protease expressed in the target cell population. Upon contact with a target cell, the protease cleaves the Vpr-toxin con_jugate, resulting in activation of the toxin.

The invention additionally provides a method for delivering a molecule into a cell. The method comprises contacting the cell with a conjugate comprising a Vpr polypeptide conjugated to the molecule. The invention further provides a method for modulating the expression of a transgene in a cell. The method comprises contacting the cell with a Vpr polypeptide conjugated to a regulatory molecule. The Vpr:regulatory molecule conjugate, upon contact with the cell, enters the cell and the regulatory molecule modulates the expression of the transgene. In addition, the invention provides a method for killing a target cell population in a subject. The method comprises administering to the subject a Vpr ρolypeptide:toxιn con_jugate to the subject. In one embodiment, the toxin is an anti-proliferative agent and the target cell population is cancer cells. The target cell population may be a type of cell that is more susceptible than non-target cells to transduction by Vpr, or it may be a type of cell that is more susceptible than non-target cells to the toxin. In some embodiments, the toxin is further conjugated to a regulatory molecule. Upon contact with a target cell, the regulatory molecule is affected in such a manner as to result in activation of the toxin. In some embodiments, the target cell has been modified to contain a transgene or transgene product whose expression is regulated by the regulatory molecule. In this manner, the invention provides a method for modulating the expression of a transgene in the context of a gene therapy method that results in the killing of cells targeted by a gene therapy protocol.

The invention further provides compositions comprising Vpr, preferably sVpr. Such compositions include pharmaceutical and vaccine compositions, and can be used in a variety of methods. In one embodiment, the invention provides a method for increasing an immune response to an antigen in a subject exposed to the antigen comprising administering Vpr to the subject. The Vpr can be a Vpr polypeptide, and can be administered alone, together with the antigen, or as a Vpr:antigen conjugate. In another embodiment, the invention provides a method for increasing an immune response to HIV in a subject comprising administering a Vpr polypeptide to the subject.

In yet another embodiment, the invention provides a method for increasing the sensitivity of cells to radiation therapy. The invention additionally provides a method for inhibiting cell proliferation. The methods comprise contacting the cells with Vpr. Vpr acts synergistically with radiation to cause G2 arrest in cells, thereby providing a radiosensitizer for use in radiation treatment, such as for treatment of malignancies and other disorders associated with dysregulated cell growth. Vpr can also act alone to inhibit cell proliferation, thereby providing a treatment for hyperproliferative cell disorders, such as malignancies, psoriasis and other disorders associated with dysregulated cell growth. In addition, Vpr can induce apoptosis, and thus can be used in a method of killing cells.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1A-G. Synthesis, purification, and MS analysis of sVpr and the C-terminal fragment Vpr^47"96. (1 A) Primary sequence of sVpr derived from the isolate HIV-1NL4-3 IS shown below the model of secondary structures identified in Vpr fragments (32, 35) Positively or negatively charged residues at the termini and helical structures, and a leucine-rich (LR) zipper-like motif that is presumably involved in the ohgomerization of Vpr are indicated. Chromatograms of crude (IB) or purified (1C) sVpr obtained by reverse-phase acetonitrile gradient HPLC are shown, with UV detection at 214 nm. Positive-ion ESI mass spectra of purified sVpr: experimental mass spectrum showing the distribution of multiply charged ions (ID). Deconvoluted mass spectrum showing the intense envelope of the molecular ion at 11377.9 Da (IE). Experimental ESI mass spectra (IF) and chromatogram (1G) of purified Vpr⁴⁷"⁹⁶.

Figures 2A-C. Characterization of sVpr by SDS-PAGE, Western blot, and imrniinoprecipitation. (2A) A serial dilution of 250 to 60 ng of sVpr, or 20 to 5 μl of lysates of H1V-1 virions per lane were separated by SDS-PAGE on 16% gels, transferred to PVDF membrane, and stained with R-96 antibodies. Antibody binding was visualized by ECL reaction. Positions of molecular weight standard marker proteins are indicated to the left, positions of monomers and dimers of sVpr are indicated to the right. (2B) Silver-stained 18% SDS-PAGE after separation of 250 and 100 ng of sVpr in the presence or absence of 250 mM DTT. (2C) sVpr (0.1 to 10 ng) was mixed with human serum and immunoprecipitated with R-96 antibodies. Immunoprecipitates were separated by SDS-PAGE on 14% gels, electro transfered, and analyzed by Western blot with R-96 antibodies and ¹²⁵I-protein G for detection. On the right panel, sVpr (0.01—10 ng per lane) was direcdy separated in the gel before Western blot analysis. Autoradiograms of a 2-day-exposure are shown in both panels.

Figures 3A-D. Far-ultraviolet CD spectra of sVpr. Spectra were recorded in pure water at different TFE concentrations (3A) and at different pH values in Pi buffer alone (3B) or with 20% (3C) or 50% TFE (3D).

Figures 4A-D. Far-ultraviolet CD spectra of Vpr^47-96 recorded in pure water at different TFE concentrations (4A) and at different pH values in P; buffer alone (4B) or with 20% (4C) or 50% TFE (4D).

Figures 5A-C. ID ¹H NMR spectrum of sVpr. (5A) Η NMR spectrum in 50% TFE. (5B) The low-field region of the same spectrum. (5C) The corresponding low-field region of the ¹H NMR spectrum of sVpr in water alone. Figures 6A-B. 2D Η TOCSY spectrum of sVpr. NH region of the 2D TOCSY spectrum of sVpr in 50% TFE (6A) and in water alone (6B) displayed to show the sharp signals. Signal assignments are those described in the text.

Figures 7A-D. Cellular uptake and lntracellular localization of sVpr-488. Fluorescentiy labeled peptide sVpr-488 at a concentration of 0.1 μg/rnl was added to human macrophages (7A and 7B) or HeLa cells (7C and 7D). After 48 hours, cells were fixed and examined by phase-contrast (7 A) and epifluorescence (7B) microscopy or by scanning confocal microscopy using phase contrast (7C, top) or epifluorescence (7C, bottom, and 7D).

Figure 8. Receptor-independent uptake of sVpr. L2 cells were incubated with ¹²⁵I- labeled sVpr or Vpu^,2~81 (control) for 60 or 120 min, and the distribution of radioactivity in the cell layer and the medium was determined in triplicate. In parallel, cells were incubated for 120 mm with ¹²⁵I-lableled sVpr and a 100-fold molar excess of unlabeled sVpr.

Figures 9A-B. sVpr transduction and induction of G2 cell-cycle arrest. (9 A) HeLa cells were incubated in medium containing sVpr-Cy3 or similarly labeled p6^S-Cγ at the indicted concentrations, fixed and stained with propidium iodide, and analyzed by flow cytometry. There was a dose-dependent uptake of sVpr-Cy3 while no p6s^a8-Cy3 was found staining the cells. (9B) Cell-cycle analysis of HeLa cells incubated with 2 μg/ml of sVpr-Cy3 shows significantly more Cy3-ρosιtive cells than Cy3-negative cells in the G2/M phase.

Figures 10A-E. Cell transduction and nuclear import of sVpr. (10A-B) Macrophages visualized using standard microscopy. (10C-E) HeLa cells visualized using confocal imaging.

Figure 11. Urea-denatured Vpr-β-galactosidase no longer transduces. Bar graph shows percent of cells FITC+ for M9-FITC controls and Vpr-β-gal FITC with and without urea treatment. Figures 12A-F. sVpr transduces from the extracellular compartment into cells. Graphs plot counts for cells exposed to mock (A), Cy3 alone (B), p68^as-Cy3 (25 μg/ml) (C), sVpr-Cy3 (2μg/ml) (D), sVpr-Cy3 (5μg/ml) (E), and sVpr-Cy3 (10 μg/ml) (F).

Figures 13A-B. sVpr induces G2 cell cycle arrest. Cells transduced with sVpr-Cy3 (B) show a greater proportion of cells in G2/M compared to Cy3 negative cells (A).

Figures 14A-D. sVpr tranduces Jurkat T cells in a dose-dependent fashion. SSC height is plotted as a function of FLl-H: Alexa-488 for 30 μg/ml ρ6 (A), 7.5 μg/ml sVpr (B), 15 μg/ml sVpr (C), and 30 μg/ml sVpr (D).

Figures 15A-E. sVpr tranduces Jurkat T cells and induces a dose-dependent G2 cell cycle arrest. sVpr subpopulations are identified based on FLl-H: Alexa-488 (15A). sVpr dose-dependent G2 arrest is shown by comparison of number of cells in Gl /GO versus G2/M as indicated by propidium iodide for non-transduced (15B), transduced subpopulation A (15C), transduced subpopulation B (15D), and transduced subpopulation C (15E).

Figures 16A-H. sVpr tranduces peripheral blood mononuclear cells freshly isolated from healthy donors. Shown is SSC height as a function of FL4-H: Cy3 for PBMC isolated from donors A-D following exposure to p6-Cy3 at 30 μg/ml (16A-D) or sVpr-Cy3 at 30 μg/ml (16E-H).

Figure 17. sVpr is incorporated into newly produced HIV-1 virions. Shown is a western blot using anti-Vpr and anti-p24 antibodies, comparing HIV-1 virions exposed to differing concentrations of sVpr.

Figure 18. Schematic diagram showing potential model for protein transduction based on charge characteristics; adapted from Derossi et al., JBC 1996; 271:18188.

Figures 19A-B. sVpr cellular uptake is not inhibited in competition experiments. Percent cell uptake is plotted for 1251-labeled sVpr and Vpu^{32 81} at 60 min and 120 min and compared to uptake with the addition of 100X unlabeled sVpr. Figure 20. Schematic diagram showing proposed secondary structure of HIV-1 Vpr

Figure 21. The carboxy terminal import signal of Vpr contains a bipartite, arginine-rich motif, as shown in this schematic.

Figure 22. Schematic diagram of HTV-1 showing position of Vpr relative to other elements of HIV- 1.

Figures 23A-D. Addition of sVpr to the extracellular medium enhances H1V-1 ΔVpr replication in macrophages. RT activity as a function of days post infection is plotted for wild type (A) and ΔVpr (B-D) with and without addition of sVpr¹"⁹⁶.

Figure 24. sVpr uptake by Jurkat T cells is rapid. Percent Cy3-positive cells is plotted for mock, p6 controls and sVpr at 5, 15 and 30 min. Uptake is observed in more than 75% of cells at 5 min.

Figure 25. sVpr induces apoptosis in Jurkat T cells. Percent apoptotic cells is plotted as a function of days after control, Vpr and p6 treatment. By 3 days, approximately 90% of cells treated with sVpr are apoptotic.

Figure 26. sVpr transduces multiple cell types. Percent Cy3-positive cells is plotted for CD3-, CD4+, CD8+, and CD14+ cells treated with p6 (control) or sVpr.

Figure 27. sVpr enters cells at 4°C. Geometric mean fluorescence is plotted for cells treated with transferrin or sVpr in the presence or absence of trypsin at 37°C or 4°C.

Figure 28. Fragments of sVpr do not transduce. Geometric mean fluorescence is plotted for cells exposed to sVpr 1-47 or sVpr 52-96 at 37°C or 4°C in the presence or absence of trypsin.

Figure 29. Schematic demonstration that the arginine residues in the C-terminal domain of NL4-3 Vpr align on a common face as predicted by an alpha helical wheel. DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that synthetic Vpr (sVpr) enters cells when added to the culture media, and that this cell entry occurs in a receptor- independent manner. sVpr enters cells efficiendy, in nanomolar quantities, and without requiring denaturation. Because Vpr is immunogenic and also because it transduces cells, Vpr can be used as a vaccine (e.g., as an HIV vaccine) and/or as a vaccine adjuvant. For such uses, Vpr can be administered alone or conjugated to a second molecule, such as a protein, DNA or RNA. The invention further relates to the discovery that Vpr inhibits cell proliferation, induces apoptosis, and also acts synergistically with radiation to cause G2 arrest of cells, thereby providing a method for inhibiting cell proliferation, for inducing apoptosis or cell killing, and for enhancing sensitivity to radiation therapy.

Definitions

All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this appHcation, the following words or phrases have the meanings specified.

As used herein, "Vpr polypeptide" means a protein or polypeptide having the cell transducing activity of Vpr. Vpr polypeptides and functional fragments thereof are identified in WO99/09412 and in German patent applications 199 08 752.0 and 199 08 766.0, filed on February 19, 1999 and February 17, 2000, respectively, as well as the corresponding PCT international patent application filed on February 17, 2000.

As used herein, "subject" or "host" refers to the recipient of the therapy to be practiced according to the invention. The subject can be any vertebrate, but will preferably be a mammal. If a mammal, the subject will preferably be a human, but may also be a domestic livestock, laboratory subject or pet animal. For example, subjects can be bovine, ovine, porcine, equine, canine or feline. As used herein, "native", in the context of nucleotide or amino acid sequence, refers to wild type or unaltered sequence.

As used herein, "analogous codon" means a codon that encodes the same amino acid, but may comprise a different triplet of bases.

As used herein, "pharmaceutically acceptable carrier" includes any material which, when combined with an active ingredient of a composition, allows the ingredient to retain biological activity and without causing disruptive reactions with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/ water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral adrriinistration are phosphate buffered saline or normal (0.9%) saline.

Compositions comprising such carriers are formulated by well known conventional methods (see, for example, Remington's Pharmaceutical Sciences, Chapter 43, 14th Ed., Mack Publishing Co., Easton PA 18042, USA).

As used herein, "a" or "an" means one or more, unless clearly indicated otherwise.

Structure and Preparation of sVpr

The Vpr polypeptide for use in the invention is preferably synthetic Vpr (sVpr), and most preferably has the features of sVpr prepared in accordance with the methods disclosed in the Examples portion of the specification. Other methods of sVpr preparation may be used, but care should be taken to avoid protein aggregation. Preferably, the sVpr is soluble and stable in aqueous solution. Unlike other transducing proteins, sVpr does not require urea denaturation. Moreover, the transducing activity of sVpr is not enhanced by urea denaturation. Also unlike other transducing proteins, Vpr does not contain an arginine-rich transducing domain that functions in the absence of other portions of the protein. Thus, Vpr or sVpr in its native state is preferred. Those skilled in the art appreciate, however, that minor modifications, including substitutions and deletions, can be made to the Vpr without interfering with the biological activity of Vpr.

Adrriinistration and Dosage

In a preferred embodiment of the method, the Vpr or Vpr conjugate is administered via a systemic, enteral or topical route. Examples of systemic routes include, but are not limited to, intradermal, intramuscular, subcutaneous and intravenous administration. Examples of topical routes include, but are not limited to, intranasal, intravaginal, intrarectal, intratracheal, transdermal and ophthalmic adrriinistration. Examples of enteral routes include, but are not limited to, oral and gastric adrriinistration.

The Vpr or Vpr conjugate can be administered as a composition for treatment. Because treatment includes prophylaxis and therapy, the compositions of the invention include both pharmaceutical and vaccine compositions. Prophylaxis or therapy can be accomplished by a single direct adniinistration at a single time point or multiple time points. Administration can also be delivered to a single or to multiple sites.

The subject can be any vertebrate, but will preferably be a mammal. Mammals include human, bovine, equine, canine, feline, porcine, and ovine animals. If a mammal, the subject will preferably be a human, but may also be a domestic livestock, laboratory subject or pet animal.

The dose of Vpr or Vpr conjugate to be administered to a subject, in the context of the present invention, should be sufficient to effect a beneficial therapeutic response in the subject over time, or to alleviate symptoms. Thus, the conjugate or composition is administered to a patient in an amount sufficient to alleviate, reduce, cure or at least partially arrest symptoms and/or compUcations from the disease. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Although the dosage used will vary depending on the clinical goals to be achieved, a suitable dosage range can be derived by reference to other references relating to Vpr and similar molecules, such as WO99/09412 and U.S. Patent No. 5,804,604. The mvention provides compositions, both prophylactic and therapeutic, that optionally mclude a pharmaceutically acceptable carrier. The conjugates of the mvention can be prepared m a variety of formulations, including conventional pharmaceutically acceptable carriers, and, for example, microbeads, microspheres, capsules designed for oral dehvery, etc. The conjugate can optionally be administered in conjunction with a drug useful m the treatment of the subject's condition. Such additional agents can be administered separately or mcluded m the con_jugate composition.

In view of the teaching provided by this disclosure, those of ordinary skill m the clinical arts will be familiar with, or can readily ascertain, suitable parameters for administration of compositions according to the mvention, including combination of conjugate administration with conventional treatments.

Methods

The mvention provides a method for delivering a molecule mto a cell. The method comprises contacting the cell with a conjugate comprising a Vpr polypeptide conjugated to the molecule. Because sVpr rapidly and strongly transduces freshly isolated primary human cells at nanomolar concentrations, the methods of the mvention provide an attractive and effective means for dehvery of a molecule mto a cell.

The mvention further provides a method for modulating the expression of a transgene m a cell. The method comprises contacting the cell with a Vpr polypeptide conjugated to a regulatory molecule. The Vprregulatory molecule conjugate, upon contact with the cell, enters the cell and the regulatory molecule modulates the expression of the transgene.

In addition, the mvention provides a method for killing a target cell population m a subject. The method comprises administering to the subject a Vpr polypeptide:toxιn conjugate to the subject. In one embodiment, the toxin is an anti-prohferative agent and the target cell population is cancer cells. The target cell population may be a type of cell that is more susceptible than non-target cells to transduction by Vpr, or it may be a type of cell that is more susceptible than non-target cells to the toxm. In some embodiments, the toxm is further conjugated to a regulatory molecule. Upon contact with a target cell, the regulatory molecule is affected m such a manner as to result in activation of the toxm In some embodiments, the target cell has been modified to contam a transgene or transgene product whose expression is regulated by the regulatory molecule. In this manner, the mvention provides a method for modulating the expression of a transgene m the context of a gene therapy method that results in the killing of cells targeted by a gene therapy protocol. In an alternative embodiment, the method of killing a target cell population comprises adrriinistration of a Vpr polypeptide, without necessarily conjugating the Vpr polypeptide to a toxm or other molecule. Because sVpr can mduce apoptosis, those cells which are more susceptible than non-target cells to transduction by Vpr are selectively killed.

In one embodiment, the invention provides a method for mcreasmg an immune response to an antigen in a subject exposed to the antigen comprising administering Vpr to the subject. The Vpr can be a Vpr polypeptide, and can be administered alone, together with the antigen, or as a Vpr:antigen conjugate. In another embodiment, the mvention provides a method for mcreasmg an immune response to HIV m a subject comprising administering a Vpr polypeptide to the subject As discussed m the examples below, CD4+, CD8+ and CD3- lymphocytes as well as CD 14+ monocytes are equivalendy transduced by sVpr. Thus, the dehvery of Vpr and/or a Vpr:antigen conjugate to these cells can be particularly useful m the induction or enhancement of an immune response to HIV or other antigen.

In yet another embodiment, the mvention provides a method for mcreasmg the sensitivity of cells to radiation therapy. The mvention additionally provides a method for inhibiting cell proliferation. The methods comprise contacting the cells with Vpr. Vpr acts synergistically with radiation to cause G2 arrest m cells, thereby providing a radiosensitizer for use m radiation treatment, such as for treatment of malignancies and other disorders associated with dysregulated cell growth. Vpr can also act alone to inhibit cell proliferation, thereby providing a treatment for hyperproliferative cell disorders, such as malignancies, psoriasis and other disorders associated with dysregulated cell growth. In addition, Vpr can induce apoptosis, and thus can be used m a method of killing cells.

EXAMPLES

The following examples are presented to illustrate the present invention and to assist one of ordmary skill m makmg and us g the same. The examples are not mtended m any way to otherwise limit the scope of the mvention.

Example 1 : Production and Biological Activity of sVpr

This example describes a protocol for high-yield production of synthetic Vpr (sVpr), and demonstrates that the resultant protem remains both soluble and stable m aqueous solution even at the relatively high protem concentrations required for structural studies. Circular dichroism demonstrates that sVpr is unstructured at neutral pH. In contrast, under acidic conditions (pH <5.0) or after the addition of trifluorethanol (TFE), sVpr adopts α-hekcal structures. Dynamic light scattering reveals that sVpr forms dimers m aqueous TFE, and high-order aggregates m pure water. Analysis of a C-termtnal fragment, Vpr^47"96, mdicates that this subdomam participates m α-hehx formation and self-association. Η NMR signals allows the assignment of N- and C-termmal residues, however, the central section of the molecule is obscured by self-association. These findings suggest that the in vivo folding of sVpr may require structure stabilizing interacting factors such as previously described interacting cellular and viral protems or nucleic acids. Surprisingly, m biological studies it was found that sVpr is effectively taken up from the extracellular medium by cells and also effectively imported mto the nucleus of these transduced cells Extracellular addition of sVpr also mduces G2 cell cycle arrest m HeLa cells. Together, these findings raise the possibility that circulating forms of Vpr observed m HIV-mfected patients may exert biological effects on a broad range of host target cells.

Vpr has important biological properties that may facihtate viral rephcation, mcludmg the presence of at least two nuclear localization signals (3, 4, 5, 6, 7). Unlike most animal retroviruses, the primate lentiviruses are able to replicate efficiendy m non-dividmg cells. Although not essential for viral rephcation m T cells, Vpr significandy augments viral rephcation in terminally differentiated monocytes/macrophages in vitro, a function that probably relates to its karyophihc properties (3). Vpr is thought to participate m the import of the viral preintegration complex (PIC), facilitating its passage across the nuclear pore. This import may similarly mvolve the function of other karyophihc viral protems mcludmg the pl7^,ws Matrix and Integrase proteins (reviewed m 8, 9).

Vpr also induces G2 cell-cycle arrest in infected proliferating human T cells (reviewed m 8, 10, 11). Such G2 arrest may serve to mduce an lntracellular miheu that is more favorable for LTR directed transcription (12). In fact, sufficient quantities of Vpr are present within the viral particle to mduce G2 arrest prior to the de novo synthesis of proviral derived protems (13, 14). Other biological activities ascribed to Vpr mclude ion- channel formation (15), transcriptional activation of various heterologous promoters (16, 17, 18, 19), co-activation of the glucocorticoid receptor (20), regulation of cell differentiation (10) and induction of apoptosis (21, 22). The importance of these latter functions for maintenance of the HIV rephcative hfe cycle and the induction of disease m the mfected host remams uncertam

The participation of Vpr m HIV-1 rephcation m macrophages suggests that selective mterruption of Vpr function with small molecule mhibitors might yield a new class of antiviral agents. However, the design of effective Vpr antagonists requires more detailed knowledge of its molecular structure, function, and mode of action. The availability of essentially unlimited quantities of pure and biologically active Vpr could certamly accelerate progress m understanding its biological functions and propel the development of effective antagonists. Recombinant Vpr has been produced in insect cells infected with recombinant baculo virus. At concentrations as low as 100 pg/ml, the extracellular addition of recombinant Vpr activates HIV-1 rephcation in both leukemic cell lines and primary peripheral blood mononuclear cells (PBMC) (23, 24). Vpr has also been expressed as a glutathione S-transferase fusion protein in Escherichia coli (15). However, after cleavage from the fusion protein or when isolated at high concentrations, these Vpr preparations often undergo spontaneous aggregation. In addition, production of Vpr by recombinant genetics is limited by cytotoxic effects of the protein in both pro- and eukaryotic cells (25, 26).

This example describes the production of synthetic Vpr (sVpr), its purification to homogeneity, and the characterization of this synthetic protein by N-terminal sequencing, mass spectrometry (MS), and gel electrophoresis. In addition, the behavior of sVpr in aqueous solution under various conditions as analyzed by dynamic light scattering (DLS), circular dichroism (CD), and Η nuclear magnetic resonance (NMR) spectroscopy is presented. Finally, the example demonstrates that sVpr is effectively taken up from the extracellular medium, is imported into the nucleus of such transduced cells, and produces G2 cell cycle arrest.

Materials and Methods

Peptide synthesis and purification

Synthesis was performed on a Perkin-Elmer MilkGen 9050 automated peptide synthesizer at a 0.09 mM scale on a TentaGel R PHB Ser(tBu) Fmoc resin (capacity 0.19 mmol g ¹) using the Fmoc/tBu strategy. The following side-chain protecting groups were used: 2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl (Arg), t-butoxycarbonyl (Trp, Lys), t-butyl ether (Thr, Ser, Tyr), t-butyl ester (Asp, Glu) and trityl (Asn, Cys, Gin and His). Couplings were performed with

N- [1 H-benzotriazol(l -yl) (dimetlιylamino)methylene] -N-memylme anaminium hexafluorophosphate-N-oxide (HBTU), except that l-(l-pyrroHdinyl-lH-l,2,3-triazolo- [4,5-b-]-pyridin-l -ylmethylene)-ρyrrohdinium hexafluorophosphate-N-oxide (HAPyU) was used for the last 30 amino acids. Coupling was performed with lmmol Fmoc ammo acids using HBTU m N-methylpyrrolidone as coupling agent with a cycle time of 45 mm for smgle coupling and 75 mm for double coupling applied for the last 56 amino acids. In order to mcrease the efficiency of the final part of the synthesis HAPyU (48) was used mstead of HBTU as coupling reagent. In order to avoid aspartimide formation deprotection of the N-terminal Fmoc group was performed with piperidine/DMF/ formic acid durmg the entire course of the synthesis. Deprotection of the Fmoc group was performed durmg the complete synthesis with 20% pipeπdine m DMF, containing 0.1 M HCOOH to avoid aspartimide side reactions. The crude protem was purified by reverse phase HPLC on a VYDAC C18 column (40 x 300 mm,1520 μ, 300 A) with a linear gradient of 100% A to 100% B m 53 mm. (A, 1000ml of water, 2 ml of TFA; B, 500 ml of acetonitrile, 100 ml water, 1 ml of TFA) at a flow rate of 100 ml mm"¹ with spectrophotometπc monitoring at λ = 220 nm. The fractions were analyzed by HPLC (Shimadzu L10) on a VYDAC C 18 column (4.6 x 250, 5 μ, 300 A) with a linear gradient of 10 to 100% B over 45 mm. The peptide Vpr⁴⁷"⁹⁶ was synthesized under the same condition, the synthesis of peptide Vpu³²"⁸¹ has been described (37).

Fluorescent labeling of sVpr

sVpr was labeled with Alexa 488 labeling kit (A-10235, Molecular Probes). The manufacturer's procedure was modified as follows: 2 mg of sVpr was dissolved m 1 ml of dimethyl formamide (DMF), 1 vial of dye from the kit was added, and the pH was adjusted to 8.5—9.0 with di-isopropyl ethylamme. After a 2-hr incubation at room temperature, the reaction was diluted with water, and the pH was adjusted to 2.0. Labeled sVpr was purified on the resm supphed with the kit. For flow cytometry, sVpr was labeled with the fluorescent dye bιs-l,r-(4-sulfobutyl)mdodιcarbocyanme-5- carboxyhc acid (sodium salt) that was coupled m the last step of the peptide synthesis onto the N-terminal NH₂ of sVpr by incubating the resm with the peptide m DMF with HBTU and di-isopropyl ethylamme overmght. After completion, the resm was washed with DMF and methylene chloride, dried, and treated with 90% trifluoracetic acid (TFA) and 5% triisopropylsilane m water. TFA was then removed under vacuum, and sVpr was precipitated with diethyl ether purified by a standard HPLC procedure. This procedure selectively labels the N-terminal residue, leaving other side chains of the peptide functional, and allows a relatively easy purification of the peptide while still attached to the resin. Similar to Cy3, this novel fluorescent dye absorbs at 550 nm and emits at 585 nm. Its detailed synthesis will be described elsewhere.

Peptide sequencing and mass spectrometry

For sVpr, 30 sequencing steps were completed on an Applied Biosystems 473A pulsed- liquid-phase sequencer according to a standard protocol. Positive-ion ESI mass spectra were recorded on a triple quad Finnigan TSQ 700 mass spectrometer equipped with an electrospray source. Protein samples were dissolved in 70% aqueous methanol and infused at a flow rate of 1 μL min"¹ into the electrospray chamber with an ES needle voltage of 5.5 kV. The experimental spectrum showing multiply charged molecular ions with 8 to 13 positive charges was deconvoluted with standard software. MALDI/TOF mass spectra were recorded on a Bruker reflex MALDI/TOF mass spectrometer using an N₂ laser (337 nm).

Circular dichroism (CD) spectroscopy

CD spectra were recorded at room temperature in 0.5 mm cuvettes on a Jasco J-600 CD spectropolarimeter in a range 180 to 260 nm, and the resulting curves were smoothed with a high-frequency filter. Secondary structure content was quantified with the program VARSELEC.

'H NMR spectroscopy

Samples of the protein were dissolved in distilled water containing 10% D₂0 or containing 50% aqueousTFE-D₂ by volume to give a final volume of 0.6 ml. Spectra were recorded at 300°K on a Bruker AVANCE DMX 600 NMR spectrometer. The Η spectra were referenced to sodium 4,4-dimethyl-4-silapentane-l-sulphonate or internally to the residual methylene signal of TFE at 3.95 ppm. 2D phase-sensitive spectra of Η COSY (correlation spectroscopy), TOCSY (total correlation spectroscopy) with mixing times 110 ms, and NOESY (nuclear Overhauser and exchange spectroscopy) with mixing times 250 ms, were recorded without spinning and processed with standard Bruker software.

Light-scattering measurements

DLS was performed on a DynaPro-801 Molecular Sizing Instrument. Protein solutions (250 μl) prepared either in water or in 50% aqueous TFE at a concentration of ~3 mg ml ¹ (sVpr) or 4 mg ml"¹ (Vpr^{47 96}) were injected through a 0.1 -μm Whatman membrane filter. Samples were illuminated by a semiconductor laser (780 nm, 25 mW) generated by a miniature solid-state Gai-_yALAs-diode. The photons scattered at a 90° angle by the particles in the sample were collected by an avalanche photodiode, and the time- dependent fluctuation in intensity of the scattered light was analyzed. The translational diffusion coefficient DT was calculated with the manufacturer's software (Dynamics, version 2.1). DT was then used to calculate the degree of sample polydispersity and the hydrodynamic radius of gyration RH of the particles using the Stokes-Einstein equation: (RH— (kbT)*(6πηDτ)^_1 where kb=Boltzman's constant, T=absolute temperature in Kelvin, and η=solvent viscosity). M_r was calculated on a standard curve (M_r vs. RH) supplied by the manufacturer. Ten continuous measurements were made for each sample.

Antibodies. SDS-PAGE. Western blot, and immunoprecipitation

A rabbit polyclonal antiserum, R-96, was generated by immunization with sVpr. Immunoprecipitation of sVpr was carried out in Triton wash buffer (50 mM Tris/HCl, pH 7.4, 60 mM NaCl, 0.5% Triton XI 00), precleared with non-immune human and rabbit sera, followed by incubation with R-96 antibodies pre-loaded onto GammaBind- Plus-Sepharose beads. The immunoprecipitates were washed twice with Triton wash buffer, once with SDS-DOC buffer (50 mM Tris/HCl, pH 7.4, 300 mM NaCl, 0.1% SDS, 0.1% deo ycholate), boiled for 10 min at 95°C in sample buffer (2% SDS, 1% mercaptoethanol, 1% glycerol, 65 mM Tris/HCl, pH 6.8), and subjected to electrophoresis on 16% PROSIEVE SDS-PAGE gels. Virus stocks were generated in HeLa cells transfected with ρNL4-3 (49) and subsequendy used to infect MT 4 cells. Virions were pelleted from cell culture supernatant (30,000xg, 1.5 hrs, 4°C) and purified on a sucrose cushion. For immunoblotting, samples were transferred to Immobilon polyvenylidene difluoride (PVDF) membranes (Immobilon). Membranes were incubated with R-96 and binding of the antibodies was identified with ¹²⁵I-labeled protein G.

Cellular uptake of sVpr

sVpr and Vpu³²"⁸¹ were iodinated with the chloramine-T method. Briefly, ~20 μg of peptides were reacted with 5.5 xlO⁷ Bq (1.5 mCi) Na¹²⁵I. Free iodine was removed by gel filtration through a Dowex ion-exchange column saturated with bovine serum albumin (BSA). For studies on cellular uptake, rat yolk choriocarcinoma L2-RYC cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% (v/v) fetal bovine serum (FBS) to 75% confluence on 24-well plates. Cells were washed once with phosphate-buffered saline (PBS) and incubated in serum-free DMEM supplemented with 0.1% BSA and ¹²⁵I-labeled sVpr or, as a negative control, ¹²⁵I-labeled Vpu³²"⁸¹. Radioactive peptides were added to the medium at a specific activity of 18 kBq ml ¹. In parallel, cells were also treated with a 100-fold excess of unlabeled sVpr or Vpu³²"⁸¹. Cells were incubated for specified times at 37°C, and the distribution of intra- and extracellular radioactivity was determined as described (50). Briefly, medium was removed, and the cell layer was washed with PBS and lysed with 1% Triton X- 100 in PBS. Radioactivity was determined in triplicate in the medium and cell layer. To correct for nonspecific binding of peptides to the cell surface, the cell layer radioactivity determined at time point 0 min (time when radiolabeled peptides were added to the medium for less than 30 sec) was subtracted as background from the radioactivity detected in the cell layer.

For cellular localization studies, either a suspension of HeLa cells (2x10⁶ ml"¹) or a confluent monolayer of human macrophages cultivated in chamber shdes was incubated with fluorescent sVpr-488. After 48 hours, the cells were washed with PBS, fixed with 1 % paraformaldehyde for 10 min, and mounted. The specimens were examined by epifluorescence or scanning confocal microscopy (model MRC-600; Bio-Rad Labs). Macrophages were isolated from random HIV-1-seronegative healthy blood donors. First, PBMC were isolated using Ficoll-Paque (Amersham) and grown in slide chambers, containing DMEM, 10% FCS, and 10% human serum AB (Gemini Bio-Products). After 1 week, cells were washed, and the adherent monolayer of monocyte-derived macrophages was used for import studies with sVpr. The number of cells transduced by sVpr-Cy3 was estimated by flow cytometry.

Cell cycle analysis

HeLa cells were incubated with sVpr-Cy3 for 48 hrs, trypsinized, and fixed for 30 min in 2% formaldehyde followed by incubation with 1 mg ml ¹ RNaseA and 10 μg ml ¹ propidium iodide in PBS for 30 min. Cellular DNA content in the fixed cells was then assessed with a FACScan flow cytometer and analyzed with the ModFit LT program (Beckton Dickinson).

Results

Synthesis and purification of sVpr

Solid-phase peptide synthesis (SPPS) of full-length Vpr was performed with a sequence derived from the isolate HIV-1NL4-3 (FigurelA). HPLC profiles of the crude and purified protein products are shown in Figures IB and 1C, respectively. In contrast to a recenύy described SPPS procedure for the synthesis of a Vpr protein derived from a different HΓV-1 isolate (27), the procedure was optimized as described herein with respect to the use of coupling agents, protection groups, cleavage reagents, and duration of coupling reactions. The present protocol gave reproducibly high yields (usually 15%) of purified sVpr without encountering any of the previously reported (27) synthesis problems such as incomplete coupling and deprotection, inter- and intrachain reaction with the resin matrix, hydrogen bond mediated peptide aggregation, or side chain reactions. Various fragments of sVpr were also synthesized using the same SPPS protocol. The HPLC purification of the peptide Vpr^{47 96}, comprising the C-terminal domain of sVpr from positions Tyr-47 to Ser-96 is demonstrated in Figure 1 G. Analysis of sVpr by protein sequencing. MS. and Western blotting

The identity of purified sVpr was confirmed by sequencing of the N-terminal 30 amino acids. Positive-ion electrospray ionization (ESI) MS was used for molecular weight determination. The experimental data showed a well-defined multiply-charged spectrum (Figure ID) that was deconvoluted to give an intense envelope for the molecular ion cluster at a molecular weight of 11377.9 Da (Figure IE), corresponding exacdy with the molecular weight of 11377.9 Da calculated for sVpr. In addition, the correct molecular weight of sVpr was also established by matrix-assisted laser desorption/ionization time- of- flight (MALDI/TOF) MS which showed an intense molecular ion cluster at 11377.2 Da (not shown). In summary, the MS and sequence analyses indicated that sVpr was homogenous and showed no detectable evidence of by-products. Similar results were also obtained for the C-terminal fragment Vpr⁴⁷"⁹⁶. The peptide was purified to homogeneity (Figure 1G) and the correct molecular weight of 5829.7 Da was established by ESI MS (Figure IF).

Molecular properties of sVpr were further characterized by SDS-PAGE (Figure 2).

Dilutions of sVpr were separated and detected by Western blotting using Vpr specific antibodies. For comparison with viral Vpr, lysates of purified HIV-1 particles were analyzed in parallel (Figure 2A). In agreement with sequencing and MS data, sVpr migrated as a single band with an apparent molecular mass of ~14 kDa that was almost indistinguishable from the migration of viral Vpr. Peptide fragments that could result from proteolysis or incomplete synthesis were not detected by Western blot (Figure 2A) or direct silver staining of sVpr in SDS-PAGE (Figure 2B).

In addition to monomeric Vpr, a small percentage of sVpr was detected in an M_r range consistent with di ers and, trimers. Such candidate oligomers were only detected at concentrations of ≥ 250 μg sVpr per lane (Figure 2A). The fact that sVpr forms multimers (as shown below by DLS) is consistent with the previous demonstration of Vpr oligomers by chemical cross linking (28). These multiple forms were not observed with preparations of viral Vpr (Figure 2A). Two possibilities may explain these results. First, according to the detection of monomeric sVpr by Western blot, the highest amount of viral Vpr analyzed corresponded to ~125 ng of sVpr per lane; at this concentration mummers of sVpr were not detected. Second, physical interactions between the C-termmal p6^m domam of the Pr55 Gag polyprotem (29, 30) direct the incorporation of Vpr mto budding virus particles (31). Hence, the presence of at least one of the known Vpr binding partners, p6^m, m the virus preparation may prevent homo-ohgomerization of viral Vpr that was otherwise evident for isolated sVpr.

Vpr contams a smgle cystem at residue 76 that may potentially participate m intermolecular disulfide bond formation. Although disulfide-bπdged dimers have not been reported for viral or recombmant Vpr, SDS-PAGE analysis of sVpr m the absence of reducmg agents mdicated that ~10% of the molecules exist as disulfide-hnked dimers, the formation of these dimers was prevented by the addition of dithiothreitol (DTT) (Figure 2B).

sVpr was also used as an immunogen m rabbits to generate polyclonal anti-Vpr antibodies. The liter of the anti-Vpr antibody, R-96, was significandy mcreased when standard couphng of sVpr to keyhole hmpet hemocyanin was omitted. The resultant R96 antiserum reacts with Vpr protems from several different HIV-1 isolates and bmds to both virus-derived Vpr and sVpr with comparable efficiency. Furthermore, usmg R96 for immunoprecipitation sVpr was detected at concentrations as low as 10 ng ml diluted m human serum (Figure 2C). Together, this demonstrates the usefulness of R96 for detection of viral Vpr m serum samples of HiV-infected mdividuals (23).

DLS analysis of Vpr

The SDS-PAGE results (Figure 2A,B) and the previously published cross-hnkmg data (28) suggest that isolated Vpr tends to form ohgomenc structures. To study the multimerization of sVpr m its dynamic state m solution, as opposed to the artificial fixation of particular folding states by -chemical cross-hnkmg, DLS was used to examine sVpr under various solution conditions. In pure water at a concentration of 3.8 mg ml without pH ad_justment (pH ~ 3 0), deconvolution of the primary DLS data indicate the existence of at least two main components with RH values of ca 4.8 nm and 26.2 nm (with relative abundance of 14 % and 86 %) corresponding to complexes with molecular weights of 128 kDa and 8075 kDa, respectively. Thus, in aqueous solution, sVpr existed as high-order aggregates (>500-mers) with a lower percentage of smaller oligomers. Such high-order complexes of sVpr cannot be resolved by SDS-PAGE and may not be stabihzed by cross-linking. Although the majority of sVpr exists in high M_r aggregates, no precipitation of the peptide was observed, even at concentrations as high as 4 mg ml"

1

Next tested was whether multimers of sVpr could be reduced by an organic solvent such as TFE, which favors intramolecular interactions and suppresses hydrophobic intermolecular interactions that were implied to drive Vpr clustering (32). DLS data acquired in 50% TFE showed that sVpr exists as a single species with a particle size that deviates less than 15% from the average R_H of 2.3 nm. This value corresponds well to a molecular mass of 26 kDa and indicates that TFE induces the formation of stable sVpr dimers. Thus, the addition of TFE promotes a substantial loss of high-order aggregates and the formation of dimers. This could result from changes in secondary structure that reduce the tendency for aggregation or from suppression of hydrophobic interactions by TFE.

Recendy, it was suggested that a leucine-rich (LR) domain located within the third, C- terminal α-helix of Vpr provides the molecular constraints for homo-oligomerization of Vpr (32). Therefore investigated was whether the C-terminal fragment, Vpr⁴⁷"⁹⁶, also tends to self-associate. DLS analysis of Vpr^{47 96} in pure water showed a single component (98.5% abundance) with an R_H value of ~3 nm corresponding to a hexameric particle of ~43 kDa. Upon addition of 50% TFE, one major species (94.2% abundance) of monomer (RH — 1.25, 6.25 kDa) and small amounts of dimers and trimers were detected. These data indicate, that like full length sVpr the C-terminal fragment Vpr⁴⁷"⁹⁶ exhibits an inherent tendency for ohgomerization that depends on the hydrophobicity of the solvent. Characterization of sVpr by CD spectroscopy

To further analyze the effect of TFE on secondary structures m sVpr, the peptides were investigated by CD spectroscopy under various solution conditions. Initially, sVpr was analyzed m water alone, without buffer, at a pH of ~3 8. The corresponding CD curve demonstrated negative elhpticity at 208 and 222 nm, and a strong positive band at ~ 192 nm (Figure 3A). These findings suggested the presence of significant content of α- hehcal structure, accounting for ~ 18% according to deconvolution of the CD spectrum. Addition of up to 20% TFE resulted m an initial stabilization of these hehcal structures (up to ~31% hehcal content) while further addition of TFE up to 60% mduced smaller changes with the maximum hehcal content at approximately 50% TFE. In contrast to previous studies of Vpr protem fragments (32, 33, 34, 35), the findings described herem suggest that sVpr possesses structure even m pure water and that its hehcal structure is stabihzed but not mduced by TFE.

Compared to the calculated basic lsoelectric pomt, it was surprising that solutions of sVpr adopt a hehcal configuration at acidic pH smce this is mconsistent with the physiological pH present in the cytosol or nucleus of the cell where Vpr is predominandy expressed. Hence, sVpr was analyzed at a constant concentration in phosphate buffer (Pi) while varying the pH from 3.9 to 7.2 (Figure 3B). Remarkably, while mcreasmg pH up to 5.0 had almost no effect, at neutral pH 7.2 the protem adopted a completely random conformation. This transition occurred at a critical pH of approximately 5.0, some loss m the shape of CD curves started at pH 6.0 (data not shown) while deprivation of structure was complete at neutral pH. In agreement with the DLS measurements, no precipitation of sVpr was evident under any of the solution conditions investigated

Subsequentiy tested was whether the destabihzmg effect of neutral pH on sVpr structure could be reversed by addition of TFE, an agent that appears to have a subtle effect on the peptide structure at acidic pH. Addition of 20% TFE, which had near-maximal effect at pH 3.8 (Figure 3A), did not stabilize sVpr at neutral pH (Figure 3C). However, TFE concentrations as high as 50% clearly provided an environment where the hehcal structure of sVpr was present (Figure 3D), even at the critical neutral pH where sVpr exhibited no structure without TFE (Figure 3B). In 50% TFE, the change from pH 4 to 7.2 had only a small effect upon the CD curves, implying that the secondary structure remained mtact and was only shghdy destabilized on transition to the higher pH (Figure 3D).

Recent structural studies on a 51 -residue N-terminal fragment of Vpr revealed no consequences of pH variation on secondary structure (35). These findings implied that the structural motif contributing to the pH-dependent foldmg of sVpr (Figure 3) may be located within the C-termmal domam of Vpr. To test this hypothesis, the fragment Vpr^{47 %} (Figure 1F,G) was subjected to an identical CD analysis (Figure 4) Like sVpr, Vpr^{47 96} adopted an acidic pH of 4 1 m pure water and tended to have a hehcal conformation, although not as pronounced as sVpr (Figure 4A). Addition of TFE mcreased the hehcal content, but in contrast to sVpr, there was a linear response with TFE concentration that reached a maximum at 98% TFE. As with sVpr, a pH- dependent folding switch was observed for Vpr⁴⁷"⁹⁶ at pH 5.0 (Figure 4B). The effect of TFE was slightly different to the situation of sVpr as the unfolding of Vpr^{47 96} could be reversed to some extent by the addition of 20% TFE (Figure 5C). Again, m 50% TFE solution, the destabihzmg effect of neutral pH was almost absent (Figure 5D). Thus, the folding of the C-terminal fragment responded to changes in solvent conditions m a fashion similar to that of full-length sVpr.

In summary, solution conditions can profoundly affect the structure of full-length Vpr: The peptide is completely unstructured at neutral pH, while lowermg the pH to a critical threshold of pH 5.0 or adding a membrane mimetic, such as TFE, stabilizes secondary structure that is mamly α-hehcal m character. This phenomenon can be attributed, at least partially, to structures located m the C-termmus, most hkely in the LR-domain of Vpr.

^jH NMR spectroscopic characterization of sVpr The structure of sVpr was further analyzed by Η NMR spectroscopy under various solution conditions. ID and 2D Η NMR spectra were recorded in water alone without any salt or buffer at pH 3.1 and in TFE/H2O (1:1). Stable solutions of sVpr devoid of any sign of protein precipitation were obtained at concentrations considerably higher than those employed in the CD measurements. The ID spectra of sVpr (Figure 5) showed relatively broad lines for both solutions, although the majority of those in water alone were the broadest. This is readily seen in the low-field region of the spectrum where line widths of 10-12 Hz were measured for the Trp-NΗ signals at 9.4—9.9 ppm in the 50% TFE solution (Figure 5B), but these lines were not visible in the spectrum obtained in pure water (Figure 5C).

SDS-PAGE analysis (Figure 2B) indicated that a small fraction of sVpr forms disulfide- hnked dimers. However, the addition of an equimolar amount of DTT gave no visible alteration in the NMR spectrum, suggesting that the majority of the molecules were not present as disulfide-linked dimers. However, it must be remembered that the NMR data were obtained using a protein solution at pH ~3, while the SDS-PAGE was performed at pH 6.8. Consequendy the signal broadening (Figure 5), indicative of protein-protein interaction, most likely arises from non-covalent associations for which a leucine-zipper motif in the C-terminus has been implicated (32).

The ID and 2D NMR spectra (Figures 5 and 6) show a further phenomenon: in both pure aqueous as well as 50% TFE solutions the protein has some regions that show particularly broad lines while at least several parts of the molecule appear to be relatively flexible resulting in sharper lines. Thus, inspection of 2D TOCSY spectra at different contour levels (Figure 6) indicates that there are several residues with resolvable spin systems. NOESY spectra of sVpr in 50% TFE indicate that these signals belong to the first 7 N-terminal (Glu-2 to Gln-8) and 5 C-terminal (Gly-92 to Ser-96) residues of the protein (Figure 6A). Similarly, in water alone and at a lower concentration of sVpr, the C-terminal residues Gly-82 to Thr-84 and Gly-92 to Arg-95 were unambiguously identified (Figure 6B).

Extracellular sVpr transduces cells and locahzes to the nucleus Like the HIV-1 Tat protein, extracellular Vpr has been shown to posses biological activity. Recombinant Vpr or Vpr isolated from the serum of patients displaying high HIV-1 loads enhances viral rephcation in both infected cell lines and primary human PBMCs (23, 24). Further, recombinant Vpr added to cell culture medium appears to exert glucocorticoid like effects (22). However, it has not been formally determined whether virion-free Vpr actually enters cells or instead engages cell-surface receptors and initiates various signaling cascades. To address these questions and to test the biological activity of sVpr, cellular uptake and subcellular localization of sVpr were studied. The peptide was labeled with the fluorophor Alexa-488 (sVpr-488) to monitor its potential uptake and subcellular localization in both macrophages and HeLa cells. These studies revealed that sVpr-488 effectively entered cells following its addition in the extracellular medium (termed as protein transduction) and further accumulated in the nucleus of these transduced cells (Figure 7). This intracellular staining pattern was not observed with a 10-fold higher concentration of a labeled control peptide (p6ⁱ"ⁱ-488) or the unconjugated fluorescent dye itself. Confocal microscopy revealed that in HeLa cells the transduced peptide sVpr-488 appears to be occasionally concentrated in cytosohc spots, while the majority of the peptide was clearly localized in the nucleus (Figure 7C,D). These data, together with the preliminary observation that sVpr activates H1V-1 rephcation and is specifically incorporated into budding HIV-1 virions, provide evidence that sVpr possesses biological activities similar to those of viral Vpr.

Receptor-independent uptake of sVpr

Next, the specificity of sVpr uptake was analyzed. Because Vpr is cationic, the possibihty was considered that its uptake is mediated by megalin, a cell-surface receptor that is expressed in a variety of tissues and binds to positively charged molecules (36). In the carcinoma cell line L2-RYC, which expresses large amounts of megalin, significant and time-dependent uptake of sVpr was observed (Figure 8). The level of intracellular sVpr reached a maximum of 8—10% within 2 hours followed by a constant plateau for up to 16 hours. This plateau may reflect a steady state between uptake and secretion of radioactivity. In contrast, a 50-amino acid control peptide, Vpu³²"⁸¹, synthesized under the same conditions as full length sVpr and containing similar secondary structural elements (37) was not effectively internalized (Figure 8). Similar results were also obtained in HeLa cells.

Furthermore, the uptake of ¹²⁵I-lableled sVpr was not inhibited by a 100-fold excess of unlabeled sVpr (Figure 8), suggesting that this process either does not involve a saturable receptor system or alternatively is mediated through a very high capacity receptor system.

sVpr is transduced efficiendy and induces G2 cell-cycle arrest

Transfection of various proliferating human cells with expression vectors encoding HIV- 1 Vpr produces G2 cell-cycle in a majority of the transfected cells (10). Given the nucleophilic properties of sVpr, it was investigated whether cells transduced with extracellularly added sVpr undergo a similar cell-cycle arrest. HeLa cells were incubated with sVpr labeled with a Cy-3-like fluorophor that allows effective sorting of the transduced cells. When sVpr-Cy3 was added at concentrations of 2, 5, and 10 μg ml^-1, flow cytometric studies revealed dose-dependent uptake of sVpr-Cy3 from the medium (71%, 92%, and 97% of the cells, respectively) (Figure 9A). When cells were incubated with sVpr-Cy3 at 2 μg ml^-1 and sorted based on fluorescence, 30% of the positive cells were arrested in the G2/M phase of the cell cycle. In contrast, only 13% of cells of the non-transduced cell population were present in G2/M (Figure 9B). These data suggest that sVpr is biologically active and is able to induce G2 cell cycle arrest in susceptible ceUs.

Discussion

Synthesis of full-length Vpr

Biological and structural studies of Vpr have been hampered by the limited availability of purified protein due to the inherent propensity of Vpr to participate in homo- and heteromolecular interactions leading to aggregation. Although partial sequences of Vpr have been synthesized for biological (25, 26) and structural studies (32, 33, 34), chemical synthesis of full-length soluble forms of Vpr has proven difficult. For example, a Vpr peptide derived from the HIV-IBRU sequence has been synthesized, but irreversible aggregation precluded purification of the product (38, 39, 40). The biological activity of such forms of Vpr could not be analyzed in solution. Far-Western blotting demonstrated the binding of SDS-denatured Vpr to the viral nucleocapsid p7^NC (39), a finding not recapitulated with virus-derived Vpr (30). The optimized SPPS protocol described herein permits the synthesis of soluble Vpr in sufficient quantities to allow structural and biological analyses. This approach also circumvents the cytotoxic effects and attendant low yields obtained when Vpr is expressed in either prokaryotic or eukaryotic cells (21, 22, 25). While others (27) have claimed that Vpr is difficult to synthesize due to its tendency for incomplete couphng, matrix interaction and peptide aggregation, these difficulties were not encountered by this use of optimized Fmoc chemistry with no additional side chain protection.

Effect of solution conditions on structure and ohgomerization of sVpr

Several attempts have been made to identify and characterize the structural and functional domains of Vpr. This protein appears to contain at least four discrete domains: a negatively charged N-terminus, a central domain comprised of three hehces (N-terminal α-1 and α-2 and C-terminal α-3), and a positively charged C-terminus (Figure 1A). The best characterized region, α-3 (residues 53—78), overlaps with a leucine-rich domain that contains a short leucine zipper-like motif involved in self- association (32, 41). The assignment of functional domains largely derives from mutational analyses and is complicated by variable results. Nuclear localization and cell cycle arrest have been assigned to different domains in Vpr although mutations throughout the length of Vpr can alter various properties of this protein (4, 25, 34, 41, 42). Structural analyses of Vpr fragments have rehed on CD and NMR spectroscopy. In all cases, the membrane mimetic organic solvent TFE or micelle solutions were employed to obtain suitable solution conditions that afford structure-stabilizing effects. Full-length sVpr based on HIV-IBRU has been studied in 30% TFE, although structural details have not been reported (27). The initial NMR experiments on sVpr in water alone and in 50% TFE identified line broadening for Η signals from the central section of the molecule and 2D data allowed sequential assignments of only a limited number of C- or N-terminal residues. To gain further insights into the folding characteristics of sVpr, DLS and CD studies of sVpr were conducted in various solutions. In pure water and in the absence of other binding partners, sVpr formed large complexes that preserved a significant amount of α-helical structure at low pH. Above pH 5.0, the structure became random. Remarkably, this pH-induced switch was rninimized by the addition of TFE. Thus, TFE has three pronounced effects on sVpr: it prevents large complex formation, stabilizes secondary structure at low pH, and protects this secondary structure against disruption at physiologic pH. These characteristics are consistent with the tendency of Vpr to interact with other molecules, including cellular and viral proteins (reviewed in 8) or even HIV-1- derived DNA (32, 43). These interactions may, like TFE or pH, stabilize structure and folding of Vpr.

Cellular uptake and nuclear translocation of sVpr

It was important to demonstrate that sVpr exhibited biological activities similar to that characteristic of virus-derived Vpr. Indeed, sVpr displayed nucleophilic properties similar to virus-derived Vpr or Vpr expressed in transfected cells. Furthermore, sVpr also induced G2 cell cycle arrest in human cells. Perhaps most surprisingly, sVpr mediated these effects even when added to the extracellular medium of cell cultures. Biological functions have been attributed to extracellular Vpr, but it was unknown whether Vpr actually can enter cells independent of the viral context. These findings are the first evidence that isolated molecules of Vpr alone can effectively transduce cells and exert a biological effect.

The HΓV protein Tat has also been shown to contain an effective protein transduction domain (44, 45). Recent in vivo studies have demonstrated that Tat can promote cellular uptake of a variety of proteins fused to Tat in an array of different cells including the brain (46, 47). This finding suggests that Vpr and Tat share protein transduction properties. It is likely that the unusual dipole character of Vpr, in combination with the C-terminal basic residues, regulates binding of Vpr to charges on membrane phospholipids and that cell uptake may be mediated by amphipathic helices present in Vpr. The transduction domain of Tat has recently been localized to the sequence YGKKRRQRRR, which strongly resembles the motif RQRRAR centered in the basic C- terminus of Vpr (44). Interestingly, this arginine-rich domain is sufficient for nuclear translocation of a heterologous cytoplasmic protein through a novel low energy, RanGTP-independent pathway of nuclear import (4). It has been proposed that there are at least two import signals contained within Vpr, one consisting of the leucine-rich hehces and one present in the basic C-terminal region (4).

Transduction of cells by sVpr provides a novel mode of delivering proteins into the cytosol and the nucleus. This adds a new dimension to the possible role of cell transduction of designer proteins as therapeutic agents. Furthermore, this dehvery system is quite efficient at nanomolar concentrations of sVpr and does not require a denaturation step, a procedure that sharply increases the efficiency of Tat mediated transduction (47). Of note, sVpr retains its karyophihc properties and is able to induce G2 cell cycle arrest in transduced cells. These findings add support to the notion that in vivo extracellular Vpr is biologically active and may even target host cells that can not be infected by HIV.

sVpr is highly immunogenic. sVpr was used to generate high-titer and broadly reactive polyclonal and monoclonal antibodies reacting with Vpr. Furthermore, sVpr activates

HΓV-1 rephcation in primary cells and is effectively incorporated into viral particles.

This, together with the finding that sVpr is taken up from the extracellular medium, locahzes to the nucleus, and induces G2 cell cycle arrest, generates confidence that the peptide prepared displays biological activity. The availability of significant amounts of biologically active sVpr should enable further studies aimed at clarifying the precise function of this viral protein, its mechanism of action, and its contributions to HIV pathogenesis. Example 2: Further Elucidation of the Biological Activity of sVpr m HIV-1 Infected and Uninfected Cells

Protem transduction is the ability to cross a biological membrane in a receptor-, energy- independent process. Hehcal wheel alignment of the C-terminal portion of Vpr revealed an arginine-πch face, similar to the transduction domains of other characterized transducing protems. sVpr cellular uptake defined by trypsin lnsensitivity occurs effectively at 4°C, a property consistent with protein transduction previously described with the Drosophila antennapedia, HSV VP22, and HIV Tat protems.

This example shows that sVpr is optimized for rapid protem transduction and is biologically active in HIV-1 mfected and uninfected cells Briefly, the results show that (1) separating the putative transducmg domam from the remainder of the sVpr molecule does not result in a functional Vpr capable of transduction; (2) urea denaturation does not enhance the transduction properties of sVpr; (3) sVpr rapidly and strongly transduces freshly isolated primary human cells at nanomolar concentrations; (4) CD4+, CD8+ and CD3- lymphocytes and CD 14+ monocytes are equivalently transduced by sVpr; and (5) sVpr mduces apoptosis m cultured T cells.

Full length Vpr was chemically synthesized and fluorescently labeled, purified by reverse phase HPLC, and added to the extracellular media of various cell cultures. Flow cytometry and epifluorescence microscopy were employed to monitor cellular uptake of sVpr. The distribution of cells within different phases of the cell cycle was studied by To- Pro-3 dye staining of DNA followed by flow cytometric analysis. The results are summarized m Figs. 10-29.

Surprisingly, studies mapping the transduction domain of sVpr revealed that, m contrast to the previously identified transducmg protems, sVpr does not display an arginine rich transducmg domain that functions in the absence of other portions of the protem. The ability of sVpr fragments, specifically residues 1-47 and 52-96 of sVpr, to transduce was studied and revealed that these fragments lack the transduction capability of full-length sVpr (Fig. 28). It appears that full-length sVpr, or at least more than only the putative transducing domain, is necessary for efficient protem transduction. In addition, sVpr differs from other transducing proteins in that urea denaturation does not enhance its transduction properties (Fig. 11). Taken together, these results suggest that Vpr may be optimized for its transducing properties in its native state, further underscoring its potential importance in HIV biology.

Moreover, sVpr rapidly and strongly transduces freshly isolated primary human cells at nanomolar concentrations (Fig. 24). Analysis of freshly isolated mixed cell populations reveals that CD4+, CD8+, and CD3- lymphocytes as well as CD14+ monocytes are equivalendy transduced by sVpr (Fig. 16A-H; Fig. 26). Transduced human Jurkat T cells accumulate in the G2 phase of the cell-cycle (Fig. 15A-E). Additionally, full-length sVpr was found to induce apoptosis in cultured T-cells (Fig. 25). Further, transduced sVpr concentrates in the nuclei of monocyte-derived macrophages and significandy increases the rephcation of HIV viruses lacking Vpr in these cells (Figs. 10A-B and 23A-D).

These studies demonstrate that HIV-1 Vpr is optimized for protein transduction, including apparent receptor independence and rapid uptake under conditions of limited energy. Such protein transducing properties may allow HIV to extend its effects to host cells not direcdy infected with the virus. Fig. 22 is a schematic representation of the HΓV-1 genome, showing the relative position of Vpr. Vpr is a small protein of 96 amino acids, 14 kD, that likely multimerizes. Vpr is contained in the HIV-l particle, binds p6^Gas, and is incorporated mto virion at high stoichiometry through interaction with p6^Ga8. Its structure has been surmised by CD and NMR, and found to include two leucine-rich helices and a C-terminal arginine-rich domain. It has a highly conserved ORF in vivo, and contains at least two NLS signals: 1-71 (β-Gal;BSA) and 73-96 (β-Gal). Vpr contains at least two nuclear localization signals that likely contribute to nuclear targeting of the viral preintegration complex.

REFERENCES

1. Lang, S. M., Weeger, M., Stahl-Hennig, O, Coulibaly, C, Hunsmann, G., Muller, J., Muller-Hermelink, H., Fuchs, D., Wachter, H., Daniel, M. M., Desrosiers, R. C, and Fleckenstein, B. (1993) /. Virol., 67, 902-912 2. Gibbs, J. S., Lackner, A. A., Lang, S. M., Simon, M. A., Sehgal, P. K., Daniel, M. D. and Desrosiers,R.C. (1995) J. Virol., 69, 2378-2383

3. Heinzinger, N. K., Bukrinsky, M. I., Haggerty, S. A., Ragland, A. M., Kewakamani, V., Lee, M.-A., Gendelman, H. E., Ratner, L., Stevenson, M. and Emerman, M. (1994) Proc. Natl. Acad. Sci. USA, 91, 73 1-7315

4. Jenkins, Y, McEntee, M., Weis, K. and Greene, W. C. (1998) /. Cell. Biol., 143, 875-885

5. Vodicka, M. A., Koepp, D. M., Silver, P. A. and Emerman, M. (1998) Genes Dev., 12, 175-185

6. Popov, S., Rexach, M., Zybarth, G., Reiling, N., Lee, M. A., Ratner, L., Lane,

C. M., Moore, M. S., Blobel, G. and Bukrinsky, M. (1998) EMBO J., 17, 909-917

7. Popov, S., Rexach, M., Ratner, L, Blobel, G., and Bukrinsky, M. (1998) /. Biol. Chem. 273, 13347-13352

8. Bukrinsky, M. and Adzhubei, A. (1999) Rev. Med. Virol, 9, 39-49

9. Emerman, M. (1996) Curr. Biol, 6, 1096-1103

10. Levy, D. N., Fernandes, L. S., Wilhams, W. V. and Weiner, D. B. (1993) Cell, 12, 541-550

11. Rogel, M. E., Wu, L. I., and Emerman, M. (1995) / Virol, 69, 882-8

12. Goh, W. C, Rogel, M. E., Kinsey, C. M., Michael, S. F., Fultz, P. N., Nowak, M. A., Hahn, B. H. and Emerman, M (1998) Nat. Med, 4, 65-71

13. Poon, B., Grovit-Ferbas, K., Stewart, S. A. and Chen, I. S. Y. (1998) Science, 281, 266-269 14. Hrimech, M., Yao, X. J., Bachand, F., Rougeau, N., Cohen, E. A. (1999) /. Virol. 73, 4101-4109

15. Piller, S. C, Ewart, G. D., Premkumar, A., Cox, G. B. and Gage, P. W. (1996) Proc. Natl Acad. Sci. USA, 93, 111-115

16. Cohen, E. A., Terwilkger, E. F., Jalinoos, Y., Proulx, J., Sodroski, J. G., and

Haseltine, W. A. (1990) JAIDS 3,11-18

17. Stark, L. A. and Hay, R. T. (1998) /. Virol. 72, 3037-3044

18. Felzien, L. K, Woffendin, C, Hottiger, M. O., Subbramanian, R. A., Cohen, E. A., Nabel, G. J. (1998) Proc. Natl. Acad. Sci. USA, 95, 5281-5286

19. Wang, L., Mukherjee, S., Jia, F., Narayan, O. and Zhao, L.-J. (1995) /. Biol.

Chem., 270, 25564-25569

20. Kino, T., Gragerov, A., Kopp, J. B., Stauber, R. H., Pavlakis, G. N., Chrousos, G. P. (1999) /. Exp. Med, 1, 51-61

21. Stewart, S.A., Poon, B., Jowett, J. B. and Chen, I. S. (1997) /. Virol, 71, 5579- 5592

22. Ayyavoo, V. Mahboubi, A., Mahahngam, S., Ramahngam, R., Kudchodkar, S., Wilhams, W. V., Green, D. R. and Weiner, D. B. (1997) Nat. Med., 3, 1117-1123

23. Levy, D. N., Refaeh, Y., MacGregor, R. R. and Weiner, D. B. (1994) Proc. Natl Acad. Sci. USA, 91, 10873-10877

24. Levy, D. N., Refaeh, Y, and Weiner, D. B. (1995) /. Virol, 69, 1243-1252

25. Macreadie, I. G., Castelh, L. A., Hewish, D. R., Kirkpatrick, A., Ward, A. C. and Azad, A. A. (1995) Proc. Natl. Acad. Sci. USA, 91, 27770-27774 26. Macreadie, I. G., Arunagiri, C. K, Hewish, D. R., White, J. F., and Azad, A. A. (1996) Mol Microbiol 19, 1185-1192

27. CorniUe, F.,Wecker, K, Loffet, A., Genet, R. and Roques, B (1999) . Peptide Res., 54, 427-435

28. Zhao, L. J., Wang, L., Mukherjee, S. and Narayan, O. (1994) /. Biol Chem.,

269, 32131-32137

29. Sehg, L, Pages, J. C, Tanchou, V., Preveral, S., Berlioz-Torrent, C, Liu, L. X., Erdtmann, L, Darhx, J., Benarous, R. and Bemchou, S. (1999) /. Virol. 73, 592-600

30. Bachand, F., Yao, X. J., Hrimech, M., Rougeau, N. and Cohen, E. A. (1999) /. Biol. Chem., 274, 9083-9091

31. Kondo, E., Mammano, F., Cohen, E. A. and Gόttlinger, H. G. (1995) /. Virol, 9, 2759-2764

32. Schuler, W., Wecker, K, de Rocquigny, H., Baudat, Y., Sire, J. and Roques, B. P. (1999) /. Mol. Biol, 285, 2105-2117

33. Luo, Z., Butcher, D. J., Murali, R., Srinivasan, A. and Huang, Z. (1998)

Biochem. Biophys. Res. Commun., 244, 732—736

34. Yao, S., Azad, A. A., Macreadie, I. G. and Norton, R. S. (1998) Protein Peptide Lett., 5, 127-134

35. Wecker, K. and Roques, B. P. (1999) Eur. J. Biochem., 266, 359-369

36. Lundgren, S., Carling, T., Hjalm, G., Juhlin, C, Rastad, J., Pihlgren, U., Rask,

L., Akerstrom, G. and Hellman, P. (1997) /. Histochem. Cytochem., 45, 383-392

37. Wray, V., Federau, T., Henklein, P., Klabunde, S., Kunert, O., Schomburg, D. and Schubert, U. (1995) Int. J. Pept. Protein Res., 45, 35-43 38. Gras-Masse, H., Ameisen, J. C, Boutillon, C, Gesquiere, J. C, Vian, S., Neyrinck, J. L., Drobecq, H., Capron, A. and Tartar, A. (1990) Int. J. Pept. Protein. Res., 36, 219-226

39. De Rocquigny, H., Petitjean, P., Tanchou, V., Decimo, D., Drouot, L, Delaunay, T., Darlix, J.-L. and Roques, B. P. (1997) /. Biol. Chem., 272, 30753-30759

40. Roques, B. P., Morellet, N., de Rocquigny, H., Demene, H., Schύler, W., Julhan, N. (1997) Biochimie, 79, 673-680

41. Wang, L., Mukherjee, S., Narayan, O., Zhao, L.-J. (1996) Gene, 178, 7-13

42. Nie, Z., Bergeron, D., Subbramanian, R. A., Yao, X. J., Checroune, F., Rougeau, N., Cohen, E. A. (1998) /. Virol, 12, 4104-4115

43. Zhang, S., Pointer, D., Singer, G., Feng, Y., Park, K. and Zhao, L. J. (1998) Gene, 212, 157-166

44. Green, M., Loewenstem, P. M. (1988) Cell, 55, 1179-1188

45. Frankel, A. D. and Pabo, C. O. (1988) Cell, 55, 1189-1193

46. Schwarze, S. R., Ho, A., Vocero-Akbani, A. and Dowdy, S. F. (1999) Science,

285, 1569-1572

47. Nagahara, H., Vocero-Akbani, A. M., Snyder, E. L., Ho, A., Latham, D. G., Lissy, N. A., Becker-Hapak, M., Ezhevsky, S. A. and Dowdy, S. F. (1998) Nat. Med, 12, 1449-1452

48. Carpino, L. A. (1993) Am. Chem. Soc, 115, 4397-4398

49. Adachi, A., Gendeman, H. E., Kόnig, S., Folks, T., Willey, R., Rabson, A. and Martin, M. A. (1986) /. Virol, 59, 284-291 50. Moestrup, S. K, Birn, H., Fischer, P. B., Petersen, C. M., Verroust, P. J., Sim, R. B., Christensen, E. I. and Nexo, E. (1996) Proc. Natl. Acad. Sά. USA, 93, 8612- 8617

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention.

Claims

What is claimed is:

I . A composition comprising a Vpr polypeptide conjugated to a therapeutic molecule.

2. The composition of claim 1 , wherein the Vpr comprises synthetic Vpr.

3. The composition of claim 2, wherein the synthetic Vpr is stable in aqueous solution.

4. The composition of claim 1 , wherein the therapeutic molecule comprises a polypeptide.

5. The composition of claim 1 , wherein the therapeutic molecule comprises a polynucleotide.

6. The composition of claim 5, wherein the polynucleotide comprises DNA or RNA.

7. The composition of claim 1, wherein the therapeutic molecule comprises a toxin.

8. A method for dehvering a molecule into a cell comprising contacting the cell with a conjugate comprising a Vpr polypeptide conjugated to the molecule.

9. The method of claim 8, wherein the molecule is dehvered to the nucleus of the cell.

10. The method of claim 8, wherein the Vpr comprises synthetic Vpr.

I I . The method of claim 10, wherein the synthetic Vpr is stable in aqueous solution.

12. The method of claim 8, wherein the molecule comprises a polypeptide.

13. The method of claim 8, wherein the molecule comprises a polynucleotide.

14. The method of claim 13, wherein the polynucleotide comprises DNA or RNA.

15. The method of claim 8, wherein the molecule comprises a toxin.

16. The method of claim 8, wherein the cell is a cancer cell.

17. The method of claim 8, wherein the cell is infected with a pathogen.

18. The method of claim 17, wherein the pathogen is a virus, a bacterium or a parasite.

19. The method of claim 18, wherein the virus is a lentivirus or a retrovirus.

20. The method of claim 19, wherein the lentivirus is HIV.

21. The method of claim 8, wherein the cell is geneticaUy modified to express a transgene.

22. A method of killing a target cell in a subject comprising administering to the subject a Vpr polypeptide conjugated to a toxin.

23. The method of claim 22, wherein the toxin is further conjugated to a regulatory molecule whereby contact with the target cell exerts an effect on the regulatory molecule that results in activation of the toxin.

24. The method of claim 22, wherein the target cell is a cancer cell.

25. The method of claim 22, wherein the target cell is infected with a pathogen.

26. A method of inhibiting cell prohferation comprising contacting a cell with a Vpr polypeptide. The method of claim 26, further comprising contacting the cell with radioactivity.

A method of treating a disorder associated with dysregulated cell growth in a subject comprising administering to the subject a composition comprising Vpr.

A method of increasing sensitivity to radiation therapy in a subject undergoing radiation therapy comprising administering to the subject a composition comprising Vpr.

The method of claim 29, wherein the composition is administered within one week of the radiation therapy.