CA2014990A1 - Method of inhibiting induction of latent or chronic viral infection - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Disclosed are methods of inhibiting or controlling the induction of a viral infection from a latent or chronic infection to an active replicating infection. Oligomers complementary to a viral Enhancer Site inhibit such induction.

Description

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deposiled with ~ Uni~ed St.Jtes P~st31 Sen/lco "Express M~il Posl ONiceJo~4('dle~ir.ee" eervice under 37 CFII l.lo on ih~ ilcd .;bove and Is addressed ~o the Cl~:nr.lkslor.cl ol P~ler,ls and Trad~-METHOD OF INHIBITING INDUCTION O~arhs, Wasl~ loll, DC 20231.
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LATENT OR CHRONIC VIRAL INFECTIONS ~yped or Prin~ed Name ol Person BACKGROUND OF T~E INVENTION natur~ \

The present invention is directed to processes for the inhibition of induction of viruses in latently or chronically infected cells into the actively replicating form.
Retroviruses were so named because in their life cycle the normal flow of ge~etic information (DNA to mRNA to protein) is reversed. In cells the genetic material is DNA; when genes are expressed, the DNA is first transcribed into messenger RNA (mRNA) which then serves as a template for the production of proteins.
However, the genes of a retrovirus are encoded in RNA; the genes must be converted into DNA before they can be expressed. Only then are the viral genes transcribed into mRNA and translated lnto prote1ns in the usual sequence.
A retrovirus infects a cell by binding to the outside of the cell and in~ecting its core. The core contains the RNA encoding the viral genome as well as structural proteins ~g~ gene), viral enzymes and regulatory proteins ~ y, nef). One enzyme is responsible for converting the viral genetic information into DNA. This DNA polymerase (~ol gene) first makes a single-stranded DNA copy of the viral RNA. An associated enzyme, ribonuclease, destroys the original RNA, and the polymerase makes a second DNA strand, using the fir~t one as a template. ~The polymerase and ribonuclease together are often called ~'reverse transcriptase"). Thus, the viral genome, now in the form of double-stranded DNA ~the same form in which the cell carries its own genes), migrates to the cell nucleus. A third viral enzyme, called an integrase, may then splice the viral genome -- its full complement of genetic information -- into the host cell's DNA.

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Once so incorporated, the viral DNA (the "provirus") will be duplicated together with the cell's own genes every time the cell divides. Thus established, the infection is a permanent part of the cell. During this latent or chronic infection phase, new virus particles are not generally produced.
The production of new virus particles (virions) begins when nucleotide sequences in the long terminal repeat (LTR) region o the viral DNA direct enzymes belonging to the host cell to copy the DNA of the integrated virus into RNA. Some of the RN~ will provide the genetic information for a new generation of virus.
Certain other RNA strands serve as mRNAs that guide cellular machinery in producing the structural proteins and enzymes of the new vlrus. ~he conversion of the virus from the latent proviral stage to the active replicating stage is called induction or rescue.
Other viruses, besides retroviruses, such as Herpes viruses and the Hepatitls B virus have similar cycles with latent or chronic infection phases and active replicating phases.
With many viruses, especially retroviruses, after initially infecting a cell the viral genome is integrated into the DNA in the cell's nucleus, in what is called the provirus form. The provirus may remain integrated in the cell's DNA during a long period of chronic or latent infection. The latent or chronic infection period ends when the provirus ~virus) i8 induced and enter~ an active replicating pha~e. Mammalian viruses which exhibit such a life cycle often having long periods of latency (or chronic infection) as well as active replication phase~
include retroviruses such as the AIDS virus ~human immunodeficiency virus or HIV), the various Herpes viruses and Hepatitis B. Since an individual (or animal) may be virtually asymptomatic during this chronic or latent infection phase and .: :

2~390 suffer few of the ravages of the active replicating phase, once an individual was infected, it would be advantageous to maintain the virus in provirus form and inhibit its conversion (induction and rescue) into the active replicating phase.
The AIDS virus, HIV, is an example of a virus that would be advantageous to maintain in the provirus form. After initial infection with HIV, the virus often replicates abundantly, and free virus may appear in the brain and spinal cord and in the bloodstream~ Fevers, rashes, flu-like ~ymptoms and ~ometimes neurological complaints can accompany this first wave of HIV
replication. Then, within a few weeks, the amount of virus in the circulation and the cerebrospinal fluid drops precipitously and the initial svmptoms disappear. However, the virus is still present (primarily in the provirus form). It can be found not only in the T4 lymphocytes, the subset of immune system cells originally thought to be its only target, but also in other classes of immune system cells, in cells of the nervous system and the intestine and probably in some bone marrow cells. From about two to ten years after the start of this asymptomatic period, replication of the virus flareG again and the infection enters an active replicating phase, generally the final stage.
Vnderlying this variable course of infection are complex interactions between HIV and its host cells. The virus behaves differently depending on the kind of host cell and the cell's own level of activity. In T cells, HIV can lie dormant indefinitely, inextricable from the cell but hidden from the victim' 8 immune system. When the cells are stimulated (induced), however, it can destroy them in a burst of replication. In other cells, such as the immune system cells called macrophages and their precursors, called monocytes, the virus may be latent or grow continuously, but slowly, sparing the cell but probably altering its function.

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2~t 49~0 In HIV, a regulatory gene known as tat, for trans-activator, is one gene responsible for the burst of viral replication seen, for example, in T4 cells that have been stimulated by an encounter with an antigen. The tat gene is unusual in both its structure and its effects. It is made up of two widely separated sequences of nucleotides: after it is transcribed into mRNA th~
intervening genetic material must be spliced out before the transcript can be made into protein. The tat gene has 6ites termed splice/acceptor ("S/A") and splice/donor ("S/D") which allow the 6plicing out of non-transcribed material. The effect of the resulting small protein (from tat) is dramatic) it can increase the expression level of the viral genes to 1,000 times the baseline expression level.
Nonionic oligonucleoside alkyl- and aryl-phosphonate analogs complementary to a selected foreign nucleic acid sequence can ~electively inhibit the expression or function or expression of that particular nucleic acid without disturbing the function or expres6ion of other nucleic acids pxesent in the cell, by binding to or interfering with that nucleic acid. (See, e.g. U.S. Patent No. 4,469,863 and 4,511,713). The use of complementary nuclease-resistant nonionic oligonucleoside methylphosphonates which are taken up by mammalian cells to inhibit viral protein synthesis in certain contexts, includi~g Herpes simplex virus~ disclosed in U.S. Patent No. 4,757,055.
The use of anti-sense oligonucleotides or phosphorothioate analogs complementary to a part of viral mRNA to interrupt the transcription and translation of viral DNA into protein has been proposed. The anti~sense constructs can bind to viral RNA and were thought to obstruct the cells ribosomes from moving along the RNA and thereby halting the translation of mRNA into protein, a process called "translation arrest" or "ribosomal-hybridization arrest." (See, Yarchoan et al., "AIDS Therapies," Scientific American, pp. llo-lls (October 1988)).
The inhibition of infec~ion of cells by HTLV-III by administration of oligonucleotide complementary to highly conserved regions of the HTLV-III genome necessary for HTLV-III
replication and/or expression is disclosed in U.s. Patent No.
4,806,463. The oligonucleotides were found to affect viral replication and/or gene expression as assayed by reverse transcriptase activity (replication) and production of viral proteins pl5 and p24 (gene expression).
The ability of some antisense oligodeoxynucleotides containing intern~cleoside methylphosphonate linkages to inhibit HIV-induced syncytium formation and expression has been studied (See Sarin, et al., PNAS 85:7448-7451 (1988).) SUMMARY OF THE INVENTIQN
The present invention is directed to methods of inhibiting or controlling of the induction of a viral infection ~rom a latent or chronic infection to an active replicating infection.
'rhe present invention is also directed to novel methylphosphonate nucleoside oligomers which are useful for inhibitlng the induction of such viruses.
Accordingly, the present invention is directed to a method of treating virally infected cells to inhibit induction of a latent or chronic viral infection into an active replicating infection which comprises treating said cells or their growth environment with an Oligomer which ls complimentary to an Enhancer Site as defined hereinbelow. Suitable nucleoside oligomers ~nclude oligonucleotides, nonionic oligonucleoside alkyl- and aryl-phosphonate analogs, phosphorothioate analogs, neutral phosphate ester analogs of oligonucleotides, `- 20~990 phosphoroamidate analogs or other oligonucleotide analogs and modified oligonucleotides.
The present invention is also directed to the treatment of isolated cells, or individuals or animals whose cells or fluids have such latent or chronic viral infections or contain viruses capable of entering a latent or chronic infection state.
In particular, the method of the presen~ invention is especially suited to treating infections which typically have periods of latent or chronic infec~ion followed by especially virulent active replicating phases. Such viruses include retroviruses such as HIV, HTLV-I, HTLV-II and the like; the Herpes viruses; Hepatitis B virus; and the like. Although Oligomers having various internucleoside phosphorous linkages may be used according to the present invention, due to their increased resistance to enzymatic metabolism, it is particularly preferred to use oligonucleoside alkyl-and aryl-phosphonate analogs in to the methods of the present invention.
The present invention also provides certain novel oligonucleoside methyl phosphonate analogs ~"MP-Oligomers") which sre partlcularly active in inhibiting induction of virus replication in latent and/or chronic virus infections.
The present invention also provides Oligomers capable of hybridizing to an Enhancer Site of viral DNA of a virus which maintains or ~ B capable o~ maintaining a chronic or latent infection or to a viral RNA sequence which corresponds to said viral DNA.
The present invention also provides hybridization probee for a viru6 which maintains or is capable of maintaining a chronic or latent infection which comprises an Oligomer of at least 8 nucleosides wherein said Oligomer is 6ubstantially complementary to an Enhancer Site of said virus.

186f255 Also provided herein are diagnostic methods; including methods for detecting the presence of in a test sample of a virus which maintains or is capable of maintaining a chronic or latent infection.

Definitions As used herein, the following terms have the following meanings unless expressly stated to the contrary.
The term "latent infection" refers to a viral infection wherein the provirus i6 integrated into the genetic nuclear structure (DNA) of the host cell and wherein there is no unintegrated viral DNA, no viral RNA and no viral proteins.
The term "chronic infection" refers to a viral infection having the provirus or virus material in the nucleus or cytoplasm of the host cell and which, until induced, has little or no detectable viral RNA or protein.
The term "Enhancer Site" refers to all sequences of U3 of a retrovirus on the viral DNA or to the correspondlng sequences of the viral RNA which a~fect viral replication and to similar sites in Herpes or Hepatitis B viruses which have equivalent activity.
The term "Oligomer" refers to oligonucleotides, nonionic oligonucleos~de alkyl- and aryl-phosphonate analogs, phosphoro-thionate ana~ogs o~ oligonucleotides, phosphoamidate analogs of oligonucleotides, neutral phosphate ester oligonucleotide analogs, and other oligonucleotide analogs and modified oligonucleotides.
The term "methylphosphonate Oligomer" (or "MP-oligomer") refers to nucleotide oligomers (or oligonucleotide analogs) having internucleoside phosphorus group linkages wherein at least one methylphosphonate internucleoside linkage replaces a phosphodiester internucleo~ide linkage.

` 201~990 The term "nucleoside" includes a nucleosidyl unit and is used interchangeably therewith.
In the various oligomer sequences listed herein "p" in, e.g., as in ApA represents a phosphodiester linkage, and "~" in, e.g., as in C~G represents a methylphosphonate linkage.

BRIEF DESCRIPTION F THE DRAWINGS
FIG. 1 depicts a qeneralized genetic map of a retrovirus whose induction may be inhibited according to the present invention.
FIG. 2 depicts a genetic map of HIV.
FIG. 3 shows the position in the HIV genome of the sequences complimentary to some of the oligomers tested.

DETAILED DESC~ Q~LO~ NVENTION
The present invention i8 directed to methods of inhibiting the induction of a viral infection using Oligomers which are complimentary to and which bind to an Enhancer Site on the viral DNA or a corxesponding sequence of the viral RNA.
Sin¢e these Oligomers are complimentary to the (~j or "sense~ ~trand of the viral nucleic acid, they are called "anti-eense Oligomer6". These Oligomers are constructed to be complimentary to a specific region of the viral nucleic acid.
Accordingly, such anti-sen~e oligomers when complimentary to the firet ~plice acceptor region ~S/A-l) of the ~3~ gene may be termed "anti~ S/A-l" or anti-S/A-1, when complimentary to the secord splice acceptor region ~S/A-2)of the tat gene may be termed anti-tat-S/A-2 or anti-S/A-2, when complimentary to the target region of tat (TAR) may be termed "anti-TAR" or when complimentary to the initiator region of env may be termed "anti-env", and so forth.

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201~990 186~255 Preferred are Oligomers having at least 8 nucleosides, which is usually a sufficient number to allow for specific binding to a desired nucleic acid sequence. More preferred are Oligomers having from about 8 to about 40 nucleotides: especially preferred are Oligomers having from about lo to about 25 nucleosides. Due to a combination of ease of synthesis, with specificity for a selected sequence, coupled with minimization of intra-Oligomer, internucleoside interactions such as folding and coiling, it is believed that Oligomers having from about 12 to about 15 nucleosides comprise a particularly preferred group.

Preferred OligQ~Lers These Oligomers may comprise either oligoribonucleosides or oligodeoxyribonucleosidess however, oligodeoxyribonucleosides are preferred.
Although nucleotide oligomers ~i.e., having the phospho-diester internucleoside lin~ages present in natural nucleotide ollgomers, as well as other oligonucleotide analogs) may be used according to the present invention, preSerred Oligomers comprise oligonucleoside alkyl and aryl-phosphonate analogs, phosphorothioate oligonucleoside analogs, phosphoroamidate analogs and neutral phosphate ester oligonucleotide analogs.
However, especially preferred are oligonucleoside alkyl- and aryl-analogs which contain phosphonate linkages replacing the phosphodiester linkages which conne¢t two nucleosides. The preparation of such oligonucleoside alkyl and aryl-phosphonate analogs and their use to inhibit expression o~ preselected nucleic acid sequences is disclosed in U.S. Patent Nos.
4,469,863t 4,511,713; 4,757,055; 4,507,433: and 4,591,614, the disclosures of which are incorporated herein by reference. A

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- 20i4990 particularly preferred class of those phosphonate analogs are methylphosphonate Oligomers.
Preferred synthetic methods for methylphosphate Oligomers (~MP-Oligomers~) are described in Lee, B.L. et al, Biochemistry 27:3197-3203 ~1988) and Miller, P.S., et al., Biochemistry 2S:5092-5097 (1986), the disclosures of which are incorporated herein by reference.
Preferred are oligonucleoside alkyl- and aryl-phosphonate analogs wherein at least one of the phosphodiester internucleo-side linkage6 is replaced by a 3' - 5' linked internucleoside methylphosphonyl (MP) group (or "methylphosphonate"). The methylphosphonate linkage is isosteric with respect to the pho6phate groups of oligonucleotides. ~hus, these methylphos-phonate oligomers ("MP-oligomers") should present minimal steric re~trictions to interaction with complimentary polynucIeotides or ~ingle-~tranded regions of nucleic acid molecules. These MP-oligomer6 should be more resistant to hydrolysis by various nuclease and esterase activities, since the methylphosphonyl group is not found in naturally occurring nucleic acid molecules.
It has been found that certain MP-oligomers are more re6istant to nuclease hydroly~is, are taken up in intact form by mammalian cells in culture and can exert specific inhibitory effects on cellular DNA and protein synthesis (See, e.g., U.S. Patent No, 4,469,863).
If desired labeling groups 6uch as psoralen, chemiluminescent groups, cross-linking agents, intercalating agents such as acridine, or groups capable of cleaving the targeted portion of the viral nucleic acid such as molecular 6cissors li~e o-phenanthroline-copper or EDTA-iron may be incorporated in the NP-Oligomers.

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Preferred are MP-oligomers having at least about 8 nucleo-sides which is usually sufficient to allow for specific binding to the desired nucleic acid sequence. More preferxed are MP-oligomers having from about 8 to about 40 nucleosides, especially preferred are those having from about 10 to about 25 nucleosides.
DUQ to a combination of ease of preparation, with specificity for a selected sequence and minimization of intra-Oligomer, inter-nucleoside interactions such as folding and coiling, particularly preferred are MP-oligomers of from about 12 to 15 nucleosides.
Especially preferred are MP-oligomers where the 5' -internucleoside linkage is a phosphodiester linkage and the remainder of the internucleoside linkages are methylphosphonyl llnkages. Having a phosphodiester linkage on the 5' - end of the MP-oligomer permits ~inase labelling and electrophoresis of the oligomer and also improves its solubility.
Target regions of the viral nucleic acid were sequenced and MP-oligomers complimentary to the sense strands of those regions were prepared by the methods disclosed in the above noted patents.
In particular, in retroviruses we have found that MP-oligomers complimentary to an Enhancer Site of U3 of the viral DNA or the corresponding sequence in viral RNA are especially preferred. One preferred group of MP-oligomers may include a sequence complimentary to all or a portion of a tract immediately 5' to U3 having the sequence:
AAAAGAAAAGGGGGGACT

In one embodiment of the present invention, we have found that MP-oligomers which are complimentary to the portion of an Enhancer Site of U3 region which binds the NF-XB protein in the viral DNA or the corresponding sequence in the viral RNA to be especially effective in inhibiting the induction of HIV in chronically infected cells, even when the cellæ are treated with a strong inducer such as the protein kinase c activator phorbol myristate acetate (PMA). such MP-oligomers were effective in inhibiting viral induction without significantly inhibiting cell proliferation or other normal cellular functions. This is in contrast to MP-oligomers complimentary to some other regions of the HIV genome wherein activity in decreasing infection by HIV
appeared to correspond to activity in inhibiting cell proliforation and other normal cell functions.
Accordingly, in one embodiment, the present invention is directed to novel MP-oligomers which are complimentary to an Enhancer Site in HIV and which are capable of inhibiting induction of chronically infected cells without significantly inhibiting cell proliferation. In particular, examples of such preferred MP-oligomers of the present invention include:
dTpApA~A~G~T~C~C~C~C~A~G
wherein p - phosphodlester linkage ~ - methylphosphonate linkage Inhibitlon of H~V
In one embodiment, the present invention is directed to a method o~ inhibiting the induction of HIV virus in cells which have a chronic or latent HIV infection.
People have been looking at different ways of modifying nucleotides complimentary to different regions of the AIDS virus HIV.
Previously two strategies have been tried, either to make the nucleotide oligomers or modified oligomers complimentary to structural genes of the virus or complimentary to regulatory genes~ Oligomers complimentary to viral regulatory genes were , . . .' ' .' ' , ,. 1, ..

directed to two possible targets: (a) the initiation codon AUG
of a particular gene and the sequence that followed, or (b) the sequences around the site known as the splice/acceptor site, abbreviated S/A, which would give the added advantage of possibly being able to block expression of two different message~ simul-taneously. It has been shown that sequences complimentary to the particular sites can bind and inhibit expression of the virus.
However, it has been found that oligomers which bind to those sites and inhibit the virus inhibit normal cellular functions, including cell division. For example, when a T-cell line or macrophage cell line that proliferates autonomously (i.e. without added growth factors) is used, if viral replication is blocked by 50%, it has been found that the growth of those cells is blocked by 50%. In normal T-cells or other normal blood components, these anti-S/A oligomers, even though they are supposed to be ~pecific for viral nucleic acid sequences, seem to effect important cell funations. We believe that these oligomers, particularly ones complimentary to regulatory regions of the viral genome, also inhibit consensus S/A sequences in the normal human genome that are similar to virai S/A sequences. That effect would explain why such oligomers inhibit important house~eeping functions of the cell, while inhibiting viral replication.
According to the method of the present invention, we are able to inhibit induction and, thus, replication of the virus using oligomers capable of binding both to the viral ~NA and DNA.
The genetic materlal of HIV and other retroviruses is RNA. Once the virus lnfects a cell, it directs synthesis of a double-stranded DNA version of its genome which is incorporated into the cell's genome as the provirus. We decided to target the region of the viral genome ~nown as the U3 region, which is the farthest 2014~90 downstream part of the viral RNA, since oligomers complimentary to the DNA Enhancer Site represented once in ~NA would directly bind to the viral message and block reverse transcription, and if the viral DNA ~ynthesis had begun, the oligomers would bind to the DNA copy (when one of these viruses infects the cell, DNA
synthesis commences within the first two to three hours after infection). In virus-directed DNA synthesis in retroviruses, the U3 region is duplicated and copies form on either end of the viral gene sequence. The region including U3 is called the long terminal repeat or LTR, and includes an important enhancer site for the virus. Certain cellular or viral proteins bind to the enhancer sites in viral DNA, and cause the amount of viral replication to increase tremendously. Thus, Oligomers complimentary to these enhancer sites which can block binding of those proteins to the enhancer sites would inhibit the conversion o~ a chronic or latent virus infection into an active replicative state. Also by blocking this earlier step in the viral replication ~cheme, we would also block reverse transcription as well. Since the LTR region is long, up to about 400 bp of information, a DNase I protection reaction was used to determine what parts of the U3 region o~ the viral DNA were open and not protected by proteins (See, Wu, F., et al, J. Virol. 62: 218-225 (1988)) and thus were available to be bound by oligomer~. By comparing those open regions with regions we believed were related to viral replicat~on ln other cells, we selQated certain regions and made Oligomers complimentary to those regions. We chose region~ including the enhancer site that had the capacity to bind Oligomer~ in the both DNA form and the RNA form.
Previous work on oligomer~ complimentary to viral nucleic acid only targeted messenger RNA sequences in an attempt to block transcription. Normally, the viral DNA sequences are not .

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18~/255 available for binding by oligomers, since the DNA is unavailable due to supercoiling, being double stranded, or being bound by protein. However, we found a sequence that would be available for oligomer binding in the RNA form as many of the other sequences are, but would be available for binding in the DNA form as well.
According to the method of present invention, these Oligomers are complimentary to and capable of binding to the binding region for enhancer proteins rather than just bind to the RNA to block transcription, Thus, these oligomers complimentary to the enhancing region are capable of inhibiting viral replication by two means. First they can block transcription by targeting the sequence that is represented in the ~NA, and, ~econd they can inhibit induction and, thus, replication by binding to the vlral DNA in the enhancer site of U3 in order to block the binding of a regulatory protein to that region and thus inhibiting induction. We have found that when one of these Oligomers, in concentrations which are non-cytotoxic and which do not significantly depress cell replication, i5 put in a culture of cells with the addition of a viral activator, the Oligomer blocked the induction, of HIV-associated tat activity by greater than 90% and of viral replication by greater than 50% at a 50~m concentration (a number of different viral activators may be used but due to its strong and general effects, preferred is the protein kinase C-activator known as phorbol ester).
It was generally thought that the most important controlling region for ~IV was the tat gene, and that by inhibiting expres-sion of that gene, viral replication could be inhibited.
However, as noted above, the anti-S/A oligomers that have been found to inhibit expression of that gene were also found to inhibit normal cellular processes. In an attempt to overcome the 201~990 problem of inhibition of cellular functions, we also attempted to inh~bit the target for that gene ("TAR") rather ~han the gene itself using one oligomer complimentary to TAR. This~oligomer complimentary to TAR did not inhibit viral replication.
In an additional aspect, the present invention is directed to the use of these Oligomers in determining the presence or absence of a virus which maintains or is capable of maintaining a chronic or latent infection state or the presence of such a chronic or latent viral infection in samples including isolated cells, tissue samples or bodily fluids. For example, these Oligomers may be used in determining the presence or absence of HIV or infection by HIV. ~See, e.g. US Patent No. 4,806,463).
Thus, in one aspect, the preæent invention is directed to hybridization assay probes comprising these Oligomers and to detection assays using these Oligomers. These probes may also be used in diagnostic kits.
These Oligomers may be labelled by any of several well known methods. Useful labels include radioisotopes as well as non-radioacti~e reporting groups. Isotopic labels include ~H, 3GS, 92p, ~2sI, Cobalt and I~C. Most methods o$ isotopic labelling involve the u~e of enzymes and include the known methods of nick translation, end labelling, second strand synthesis, and reverse transcription. When using radio-labelled probes, hybridization can be detected by autoradiography, scintillation counting, or gamma counting. The detection method selected will depend upon the hybridization conditions and the particular radioisotope used for labelling.
Non-isotopic materials can also be used for labelling, and may be introduced by the incorporation of modified nucleosides or nucleoside analogs through the use of enzymes or by chemical modification of the Oligomer, for example, by the use o$ non-.. ` .

186/255nucleotide linker groups. Non-isotopic labels include fluorescent molecules, chemiluminescent molecules, enzymes, cofactors, enzyme substrates, haptens or other ligands. One preferred la~elling method is acridinium esters.
Such labelled Oligonucleotides are particularly suited as hybridization assay probes and for use in hybridization assays.
To assist in understanding the present invention, the following examples are included which descri~e the results of a 6eries of experiments. The following examples relating to this invention should not, of course, be construed in specifically limiting the invention and 6uch variations of the invention, now known or later developed, which would be within the purview of one skilled in the art are considered to fall within the scope of the present invention as hereinafter claimed.

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ACTIVITY OF CERTAIN ~P-OLIGOMERS IN
INHIBITING HIV REP~ICATION
Oligonucleotide methylphosphonate analog~ complimentary to the initiator codon regions of the gag-pol polypeptide and of tat were tested for their effect as 6pecific inhibitor6 of HIV repli-cation. An Oligomer ~peci~ic to B-globulin mRNA and a d-AT
polymer were used as negative controls. Varying concentrations of the Methylphosphonate ~"MP") oligomer~ were incubated with phytohemagglutin (IlPHA'') stimulated human peripheral blood mono-nuclear cells ~P~MC) for two hours of 37-C and then exposed to 100 ID-50 of Stock HIV from HTLV-IIIB-infected H9 cells.
Eighteen hour6 later, fresh medium and additional oligomer were added. Cells were harvested after seven days and evaluated for HIV-specific proteins in an indirect immunofluorescence assay employing IgG isolated from the serum of an HIV-infected indi-vidual. In parallel, reverse transcriptase (RT) activity w~s measured in the culture supernatants by standard techniques (See, Laurence, J., et al., Science ~ 1501-1504 (1987)).
Results are reported in Table I. As may be seen from Table I, oligomers specific to the initiator codon region of qaa-pol and tat were ineffective at inhibiting ~IV replication, although they were non-cytotox~c to activated PBMC at concentrations up ~o 200 ~M.

~FFECT OF ANTI-~A~ MP-OLIGOMERS
ON HIy-~$~0CIAT~p TRANS-ACTIVATION
Methylphosphonate oligomers complimentary to the tat initiation and splice/acceptor regions were tested for their ability to inhibit HIV-associated transactivation as measured by the ability of tat to enhance expression of the chloramphenicol acetyl transfer gene ~CAT) upon transfection of a human T4+
lymphoblastoid T cell line with either a CAT plasmid alone or together with a tat plasmid (See: Laurence, J., et ~l., ~- Çlin-Invest- 80:1631-1639 ~December 1987)). The CAT plasmid pC15CAT
and the control tat plasmid pCV-l, which lacked the splice/-acceptor sites, were p~ovided by Dr. Wong-Staal of the NCI. The experimental tat plasmid containing the two ~ associated exons, pBR322/pIIIextatlll was provided by Dr. Haseltine of Harvard Medical School.
CAT activity was assessed by TLC analysis of acetylated forms of ~I~C]-chloramphenical by standard techniques. (See, Laurence, J., ~ al., J. Clin. ~nvest. 80:1631-1639 (1987)).
Results are shown in Table II. The MP-oligomer directed against the tat initiation site had no effect, while the MP-2~1~9~0 186/255oligomer directed against the splice/acceptor site showed inhibition of transactivation at 50 ~M.

EFFECT OF MP-OLIGOMERS ON HIV-REPLICATION I~ VITRO
Anti-tat MP-oligomers were tested for their ability to block HIV infection of targe~ cells ~in order to see if that activity paralleled activity against trans-activation).
Cells and oligomer were incubated in the presence of HIV as de~cribed in Example 1.
Effects of oligomer on normal cell function, T-lymphocyte viability and PHA-mediated proliferation were measured (See, Laurence, et al., J. .Cli~. Invçst. 80:1631-1639 (1987)~. No adverse e~fect on viability, measured by the ability to exclude trypan blue wa~ noted. However, three different anti-~~plice/acceptor oligomers ~S/A-1, S/A-lA and S/A-2, see below) depres~ed PHA-mediated blastogenesis by about 40 to 50% at concentratlons >25 ~M in 3 ~eparate experiments.
Re~ult~ o~ ollgomerc in inhibiting HIV ln~ection are reported in Table III;
Oliaomer identification MP oliaomer Sequence anti-tat S/A-l dCpA~C~C~C~A~A~T~T~C~TpG
anti-tat S/A-lA dApApApApT~G~G~A~TDA~A~A
anti-~ S/A-2 dTpG~G~G~ApG~G~T
where p ~ pho~phodie~ter linkage where D - methylphosphonate linkage .

201~99~

EFFEC~ OF MP-OLIGOMERS 0~ PMA-INDUCTION OF HIV
MP-oligomers were assayed for their effect on phorbol-ester (13-phorbol-lZ-myri~tate acetate or "PMA") mediated induction of HIV.
In certain cells (including T4+ T-cells), NF- B, a factor that regulates transcription and binds to the HIV enhancer, also mediates phorbol-ester and mitogen or antigen activation.
MP-ol~gomers complimentary to the tat-linked target element TAR and to the enhancing region in U3 which binds NF- B were tested for their ability to inhibit PMA-induction of HIV
replication-related effects.
A stock solution of lOO~g/ml PMA (Sigma) was prepared in absolute ethanol diluted in RPMI-1640 and used in final concentrations of 5 to 500 ng/ml. U1.1 cells were obtained from T.M. Folks of the NIH; they were subcloned from Ul, a clone of the human monocytic cell line U937 which had been infected with the lymphadenopathy-as60ciated virus (LAV) strain of HIV. H9, a human CD3+, CD4+ lymphobla6toid cell line permis6ive for the replication of HIV annd partially resistant to its cytotoxic effect~ wa~ obtained from R.C. Gallo of the NIH. Stock Samples of these cells were cultured in RPMI-1640 (Flow Laboratories, McLean, VA1; plus 10% fetal bovine aerum (FBS), at a concentration of 5 x 106 cell6/ml.
Varying concentrations of Oligomer (2 to 100 ~M) wer0 incubated with U1.1 cells (a chronically infected macrophage cell line) a~ 37-C for one hour prior to exposure of the cells to PMA
(5 ng/ml). Cultures were evaluated for HIV activity after 48 hours.
NIV antigens were quantitated in supernatants by an ELISA-based assay for viral p24 core protein. Human immunoglobulin 201~99û

directed against p24 epitopes (Abbott Labs, Chicago, IL) contained in polystryene beads was added to supernatants and maintained overnight at room temperature. Plates were washed with citrate phosphate buffer; and rabbit anti-HIV IgG, followed by addition of horseradish peroxidase-labeled goat anti-rabbit antibody (Abbott). Color was developed with O-phenylenediamine as a substrate, followed by IN H2SO~ to stop the reaction.
Absorbance was read at 492 nm; data were expressed as pg/10 cells. The sensitivity of this assay was < 60 pg/ml.
Result6 are tabulated in Table IV. As may be seen, an ll-mer MP-oligomer complimentary to the enhancing sequence in U3 (which binds NF- B) was able to block induction of HIV by almost 60% at 50 ~M. That concentration inhibited replication of Ul.l cells a6 measured by ~H-Thymidine incorporation by le8s than 20 percent.
DNA synthetic response was assayed according to the following procedure. Cells were collected, wa6hed three times with P~S, and viability asses6ed by trypan blue dye exclusion.
x 10~ viable cells were resuspended in 0.2 ml of medicine in polystryene flat-bottom microwell plates. Selected cultures were treated with inducing agent. All groups were assayed in tripli-cate. Cells were incubated for 48 hours; eighteen hours before culture termination, they were pulsed with 0.1 mCi of tJH-methyl~-thymidine (1.9 Ci/~M sp. act., New England Nuclear). The contents of each well were harvested and incorporation of radioactivity was measued by liquld scintillation counting.
The cells were al50 assayed for PMA-induced enhancement o~
HIV-LTR driven CAT activity. The ability of the tat transcrip-tion unit of HIV to enhance the expression of the chloramphenical acetyl transferase ~CAT) gene when CAT is linked to the LTR of HIV was measured as described in Laurence, J. et al., J. 51~a-0 ~

186J255Invest. 80:16~1-1639 (1987) with the followin~ modifications. 2 X lo~ Ul.l cells per condition were washed with 6erum-free RPMI-1640 and resuæpended in lml of smM Tris (pH7.3) containing 250~g/ml DEAE-dextran (Sigma) and 2 or 4 ~g of total plasmid DNA.
Two plasmids (See Arya, S.K., et al., Science 229:69 (1985):
plasmids were obtained from the sources noted in Example 2) were used either singly ~HIV-LTR-CAT alone) or together (co-transfection of CAT and tat containing vectors). The ~ plasmid pCV-l contains a 1.8 kb fragment of HIV-l cDNA encompassing the tat gene. The CAT plasmid pC15CAT contains SV-40 regulatory sequences, and the LTR and a portion of L~ (3'-orf) of HIV-l.
After transfection, cells were washed with serum-free RPMI-1640 and incubated in 0.5 ml of culture medium at 37-C. Certain cultures al~o contained PMA (50 ng/ml). Cells were harvested, washed with PBS, resuspended in 100 ~1 of 0.25 M Tris (pH 7.08), and cellular extracts were prepareed by three cycles of freezing (in ethanol and dry ice) and thawing at 37-C. CAT activity was determined by incubating 50 ~1 aliquots of cell extract6 with tl~C]-chloramphenicol (New England Nuclear) and 2.5M acetyl coenzyme A (P-L Biochemicals, Inc., Piscataway, NJ) at 37C for 2 hours and extracting with ethyl acetate. The acetylated forms of chloramphenicol were separated from the unacetylated form by ascending thin layer chromatography using a chromatogram 6heet (Eastman Kodak, Rochester, NY) in a chamber containing chloroform and methanol (19:1, v/v). The chromatogram was then autoradio-graphed. Areas of radioactivity were marked, cut from the sheet, and counted in scintillation fluid.
Results are tabulated in Table V.

. 186/255 Mp-oligomer Nucleotide sequence position Identification dCpApC~C~C~A~A~T~T~C~T~G 5366-5355 tat splice-acceptor site=anti-tat S/A-l dApA~A~A~T~G~G~A~T~A~A~A 5356-5343 tat splice-acceptor site=anti-ta~ S/A-lA

dTpA~A~A~G~T~C~C~CC~A~G -83 to -94 enhancer (NF-~B
repeated element of U3 of HIV~ anti-enhancer dG~T~C~T~G~G~G~T~C~T A~A TAR = an~i-TAR

p=phosphodiester linkage ~=methylphosphonate linkage EXAMP~ 5 EFFECT OF OLIGOM~ ON SPONTANEOUS CEL~
GROWTH AND DNA SYNTHET~C RESPONSE
Spontaneous growth and DNA synthetic response, as measured by 3H-Thymidine incorporation, of an immortal T4+cell line (SK7) and normal, mitogen-exposed peripheral blood mononuclear cells (PBMC) in the presence of a MP-oligomer complimentary to the S/A-1 region of HIV and a MP-oligomer complimentary to the initiator codon of ~nY was measured, as described in Example 4.
Results are tabulated in Tables VI-A (SK7) and VI-B (PMBC).
As may be seen, the anti-S/A-l oligomer significantly inhibited cellular proliferation in both cell lines. The anti-env oligomer inhibited cell proliferation to a lesser effect, however, we have found that anti-~nv MP-oligomers have poor anti-viral activity.
Identification MP-Oligome~Sequençç
anti-S/A-l dCpC~C~A~A~T~TpC~T~G
anti-~n~ dCpA~G~G~C~A~ApG~ADA~T~C

20149~0 1~6/255 TABLE I
E~ 9~ rQIIgoM~s ~OMPLIMENTARY TO
HIV-SP~CIFIC INITIATION CO~ONS ON VIRAL INFECTION
A s s a y s for H I V
Replication Percent Inhibition MP-Oligomer MP- Oligomer Immuno- RT
sequence _ oligomer Concentration 1uorescence Activity d-CpA~T~T~C~T_G~T B-qlobin 20~M 0 0 d-CpADA~T~T~CpTpG~T B-glo~in 200~M 0 0 d-Ap(T~A~)n none 200~M 0 0 d-TpT_T~C~T~T~G~C~T~C tat 200~M ND o d-TpC~T~C~T~C~T~-C~C_T~T_C ~ol 200~M 22.7 0 TAB~E II
EFFECT OF ANTI-TAT MP-OLIGOMERS ON
HIV-ASSOCIATED TRANS~CTIVATION
Percent Inhibition of CAT
MP-Oligomer MP-oligomer Oligomer Control Exper.
~eauence 5PCÇi5iÇllY Conc. Plas~mid Plasmid d-TpTpTPC~TE2 TPG~c~T~c tat-initiation 50~M 0 0 d-CpC~C_A~A~T~
T~C_T~G tat-S/A-1 50~M 0 >50 TABL~
EF~E T OF ANTI-TAT MP-Oh~_OMERS ON
HIV-ASSOCIATED ~$~C~TI9~ Q
HIV Reverse Transcriptase ACtiv~ity MP-Oligomer MP-oligomer Ollgomer Mean cpm % Inhi-seguence spec ~ Conc. + SV* biti~
d-TpT~T~C~TDT~ tat-initiation 50~M 69,575 1.0 G~C~T~C +11,595 d-CpC~C~A_A~T~ tat-S/A-l 50~M 8,674 87.7 T C_T~G +2,245 None - - 70,293 +16,101 * Mean of two experiments .

2~14990 TABLE IV
E~FECT OF MP-OLIGOM~ Q~ I~DUCTION
OF HIV FROM CHRONICA~ INFEC~ED CE~LS*
HIV p24 antigen~
10 cells ~IV
inducer Concen-MP-Oligomer MP-oligomer Conc. PMA, tration % Inhi-sequence 6pecificity luM~ 5ng/ml fD~Jml~ ion _ _ - - <30 _ - - + 1,915 +308 anti-tat-S/A-1 tat S/A 50 - O
anti-enhancer enhancer 50 - O
anti-tat-S/A-1 tat S/A 50 + 1,250 34.7 anti-enhancer enbancer 50 + 785 59.0 * 1 x 10~ U1.1 ¢ell~ were plated in flat-bottom microwell~
~n 0.2ml ~PMI 1640 + 10% FBS. Selected cultures w~re exposed to PMA (5ng/ml phorbol myr~tate acetate) for 30 minutes prior to add$tion o~ MP-ollgomer. Cell-free ~upernatant~ were harvested at 48 hour~ for determination o~ p24 antigen determination (Abbott Lab Kit).

EFFECT QF ~p-QLIGOMERS O~ ~F ~EGULATION
OF ~IV-L~R ÇA~RI~ ACTIVITy BY TAT*
Plasmid OAT
PMA ~10 Oligomer Oligomer tran~fected acti-Ex~'t. nY~ml) sDecificity conc~ L 8IY~5~LL~B tat ~i~Y
A - - - + _ 1.9 _ _ - + + 9.8 _ anti-~-S/A-l 50 + ~ 10.3 - anti-~n~-S/A-l 100 ~ + 4.7 - anti-TAR 100 + + 9.4 B + - - + + 14.5 + anti-TAR 100 + + 14.7 + anti-enhancer 100 + ~ 0.3 '' " ' ' ' . ^

TABLE VI
~:FFECT OF OI,IGOMERS ON CELLUI~R PROI IFERATION
MFASURED F~Y THE INÇORPORATION OF ~-~YM~

TABLE VI--A -- EFFECT ON SK7 T--CEL~ HlrBRIDOMA I,INE

CAT~q~AT
Transec~ion CAT
Oligomer - - S/A-l S~A-l env Amount - - lO~M 50~M 50~M
CPM* 780 770 670 410 ~ 660 % Inhibition - - 14 50 16 TABLE VI-B - EFFECT ON PMBC
~ITLV--III -- ~ + +
oligomer (50~M) - - S/A-l çnY
CPM* 8600 9530 5240 8500 % Inhibition - - 45 11 -* Average of duplicate samples.

Claims (78)

1. A method of preventing induction of a latent or chronic virus infection into an actively replicating form in cells having a chronic or latent infection of said virus which comprises treating said cells or their growth environment with an oligomer which is complimentary to and which can bind to or interact with an Enhancer site of the viral DNA or a corresponding sequence in the viral RNA.

2. A method according to claim 1 wherein said virus is a retrovirus, a Herpes virus or a Hepatitis B virus.

3. A method according to claim 2 wherein said Oligomer comprises a nonionic oligonucleoside alkyl- or aryl-phosphonate analog, a phosphorothioate oligonucleotide analog, a phosphoramidate oligonucleotide analog, or a neutral phosphate ester oligonucleotide analog.

4. A method according to claim 3 wherein said virus comprises a retrovirus.

5. A method according to claim 4 wherein said Oligomer comprises an oligonucleoside alkyl- or aryl-phosphonate analog.

6. The method according to claim 5 wherein said Oligomer comprises a methylphosphonate Oligomer.

7. The method according to claim 6 wherein said virus is HIV.

8. The method according to claim 7 wherein said Oligomer comprises at least 10 nucleosides.

9. The method according to claim 8 wherein said Oligomer comprises from about 10 to about 25 nucleosides.

10. The method according to claim 9 wherein the Oligomer has a phosphodiester internucleoside linkage at its 5'-end and wherein the remainder of the internucleoside linkages are methylphosphonate linkages.

11. The method according to claim 10 wherein said Oligomer is complementary to the Enhancer Site of U3 which binds to NF- B.

12. The method according to claim 11 wherein said Oligomer has from about 12 to about 15 nucleosides.

13. The method according to claim 12 wherein said Oligomer has the sequence:
dTpApApApGpTpCpCpCpCpApG
wherein p is a phosphodiester internucleoside linkage and p is a methylphosphonate linkage.

14. A methylphosphonate Oligomer which is complementary to an Enhancer Site of viral DNA of a virus which maintains or is capable of maintaining a chronic or latent infection or to a viral RNA sequence which corresponds to said viral DNA.

15. An Oligomer according to claim 14 wherein said virus comprises a retrovirus, or Herpes virus or a Hepatitis B virus.

16. An Oligomer according to claim 15 which comprises an oligodeoxyribonucleoside.

17. An Oligomer according to claim 16 which comprises at least about 8 nucleosides.

18. An Oligomer according to claim 17 which comprises from about 10 to about 25 nucleosides.

19. An Oligomer according to claim 18 wherein said virus is a retrovirus.

20. An Oligomer according to claim 19 wherein said virus is HIV.

21. An Oligomer according to claim 20 which is complementary to at least a portion of a tract immediately 5' to U3.

22. An Oligomer according to claim 21 wherein said tract has the sequence:

23. An Oligomer according to claim 18 which comprises from about 12 to about 15 nucleosides.

24. An Oligomer according to claim 23 which has the following sequence:
dTpApApApGpTpCpCpCpCpApG
wherein p is a phosphodiester internucleoside linkage and p is a methylphosphonate internucleoside linkage.

25. A methylphosphonate Oligomer which is complimentary to the enhancer site which is capable of binding NF-?B in U3 of the LTR of a retrovirus.

26. An oligomer according to claim 25 wherein said virus is HIV.

27. An oligomer according to claim 26 having a 5'-phosphodiester internucleoside linkage at its 5'-end and wherein the remainder of internucleoside linkages are methylphosphonate linkages.

28. An oligomer according to claim 27 which comprises a deoxynucleoside oligomer.

29. An oligomer according to claim 28 having at least about 8 nucleosides.

30. A method of treating an organism or isolated cells thereof having a chronic or latent viral infection or a fluid from that organism or cells having a virus capable of entering a chronic or latent infection in order to maintain said infection in a chronic or latent phase and to inhibit its induction to a active replicating phase which comprises the administration to said organism of a therapeutically effective amount of an Oligomer which is complimentary to the base sequence of an Enhancer Site of said virus, effective to inhibit induction of said virus.

31. A method according to claim 30 where said virus comprises a virus which maintains a chronic or latent infection.

32. A method according to claim 31 wherein said virus comprises a retrovirus, a Herpes virus or a Hepatitis B virus.

33. A method according to claim 32 wherein said Oligomer comprises a nonionic oligonucleoside alkyl- or aryl-phosphonate analog, a phosphorothioate oligonucleotide analog a phosphoramidate oligonucleotide analog, or a neutral phosphate ester oligonucleotide analog.

34. A method according to claim 33 wherein said virus comprises a retrovirus.

35. A method according to claim 34 wherein said Oligomer comprises an oligonucleoside alkyl- or aryl-phosphonate analog.

36. The method according to claim 35 wherein said Oligomer comprises a methylphosphonate Oligomer.

37. The method according to claim 36 wherein said virus is HIV.

38. The method according to claim 37 wherein said Oligomer comprises at least 8 nucleosides.

39. The method according to claim 38 wherein said Oligomer comprises from about 10 to about 25 nucleosides.

40. The method according to claim 39 wherein the Oligomer has a phosphodiester internucleoside linkage at its 5'-end and wherein the remainder of the internucleoside linkages are methylphosphonate linkages.

41. The method according to claim 40 wherein said Oligomer is complementary to the Enhancer Site of U3 which binds to NF-?8.

42. The method according to claim 41 wherein said Oligomer has from about 12 to about 15 nucleosides.

43. The method according to claim 42 wherein said Oligomer has the sequence:

dTpApApApGpTpCpCpCpCpApG
wherein p is a phosphodiester internucleoside linkage and p is a methylphosphonate linkage.

44. An Oligomer capable of hybridizing to an Enhancer Site of viral DNA of a virus which maintains or is capable of maintaining a chronic or latent infection or to a viral RNA
sequence which corresponds to said viral DNA.

45. An Oligomer according to claim 44 wherein said virus comprises a retrovirus, or Herpes virus or a Hepatitis B virus.

46. An Oligomer according to claim 45 which comprises an oligodeoxyribonucleoside.

47. An Oligomer according to claim 46 which comprises at least about 8 nucleosides.

48. An Oligomer according to claim 47 which comprises from about 10 to about 25 nucleosides.

49. An Oligomer according to claim 48 wherein said virus is a retrovirus.

50. An Oligomer according to claim 49 wherein said virus is HIV.

51. An Oligomer according to claim 50 which is complementary to at least a portion of a tract immediately 5' to U3.

52. An Oligomer according to claim 51 wherein said tract has the sequences

53. An Oligomer according to claim 48 which comprises from about 12 to about 15 nucleosides.

54. An Oligomer according to claim 53 which has the following sequence:
dTAAAGTCCCCAG

55. An Oligomer according to claim 53 which is complementary to the Enhancer Site which is capable of binding NF- B in U3.

56. A hybridization probe for a Virus which maintains or is capable of maintaining a chronic or latent infection which comprises an Oligomer of at least 8 nucleosides wherein said Oligomer is substantially complementary to an Enhancer site of said virus.

57. A probe according to claim 56 wherein said virus comprises a retrovirus, or Herpes virus or a Hepatitis B virus.

58. A probe according to claim 57 which comprises an oligodeoxyribonucleoside.

59. A probe according to claim 58 which comprises at least about 8 nucleosides.

60. A probe according to claim 59 which comprises from about 10 to about 25 nucleosides.

61. A probe according to claim 60 wherein said virus is a retrovirus.

62. A probe according to claim 61 wherein said virus is HIV.

63. A probe according to claim 62 which is complementary to at least a portion of a tract immediately 5' to U3.

64. A probe according to claim 63 wherein said tract has the sequence:

65. A probe according to claim 60 which comprises from about 12 to about 15 nucleosides.

66. A probe according to claim 65 which is complementary to the Enhancer Site which is capable of binding NF-?B.

67. A method for detecting the presence in a test sample of a virus which maintains or is capable of maintaining a chronic or latent infection which comprises:
(a) bringing together test sample nucleic acid and an Oligomer sufficiently complementary to hybridize with an Enhancer Site of viral DNA or a viral RNA sequence which corresponds to said viral DNA;
(b) incubating said Oligomer and test sample under specified hybridization conditions such that said Oligomer hybridizes only to viral nucleic acid and does not detectably hybridize with cell nucleic acid; and (c) assaying for hybridization of Oligomer to test sample.

68. A method according to claim 67 wherein said virus is a retrovirus, a Herpes virus or a Hepatitis B virus.

69. A method according to claim 68 wherein said virus comprises a retrovirus.

70. A method according to claim 69 wherein said Oligomer comprises an oligonucleoside alkyl- or aryl-phosphonate analog.

71. The method according to claim 72 wherein said Oligomer comprises a methylphosphonate Oligomer.

72. The method according to claim 71 wherein said virus is HIV.

73. The method according to claim 72 wherein said Oligomer comprises at least about 8 nucleosides.

74. The method according to claim 73 wherein said Oligomer comprises from about 10 to about 25 nucleosides.

75. The method according to claim 74 wherein the Oligomer has a phosphodiester internucleoside linkage at its 5'-end and wherein the remainder of the internucleoside linkages are methylphosphonate linkages.

76. A detectably labelled Oligomer capable of hybridizing to an Enhancer Site of viral DNA of a virus which maintains or is capable of maintaining a chronic or latent infection or to a viral RNA sequence which corresponds to said viral DNA.

77. A detectably labelled methylphosphonate Oligomer which is complementary to an Enhancer Site of viral DNA of a virus which maintains or is capable of maintaining a chronic or latent infection or to a viral RNA sequence which corresponds to said viral DNA.

78. A diagnostic kit for detecting the presence or absence of a virus which maintains or is capable of maintaining a latent or chronic infection which comprises an Oligomer according to claim 14, 44, 76 or 77.