Phosphoramidate,Phosphorothioate, and Methylphosphonate Analogs of Oligodeoxynucleotide : Inhibitors of Replication of Human Immunodeficiency Virus

Abstract

Modified oligodeoxynucleotides complementary to RNA of human immunodeficiency virus (HIV-1) were tested for their ability to inhibit virally induced syncytium formation and expression of viral p24 protein. The modification of oligomers include replacement of phophodiester backbone with phosphorothioate, methylphosphonate and various phosphoramidates. Cells infected for four days, then treated with the antisense oligomers also showed inhibition of viral expression.     

Nitric oxide synthesis enhances human immunodeficiency virus replication in primary human macrophages

Macrophages are suspected to play a major role in human immunodeficiency virus (HIV) infection pathogenesis, not only by their contribution to virus dissemination and persistence in the host but also through the dysregulation of immune functions. The production of NO, a highly reactive free radical, is thought to act as an important component of the host immune response in several viral infections. The aim of this study was to evaluate the effects of HIV type 1 (HIV-1) Ba-L replication on inducible nitric oxide synthase (iNOS) mRNA expression in primary cultures of human monocyte-derived macrophages (MDM) and then examine the effects of NO production on the level of HIV-1 replication. Significant induction of the iNOS gene was observed in cultured MDM concomitantly with the peak of virus replication. However, this induction was not accompanied by a measurable production of NO, suggesting a weak synthesis of NO. Surprisingly, exposure to low concentrations of a NO-generating compound (sodium nitroprusside) and L-arginine, the natural substrate of iNOS, results in a significant increase in HIV replication. Accordingly, reduction of L-arginine bioavailability after addition of arginase to the medium significantly reduced HIV replication. The specific involvement of NO was further demonstrated by a dose-dependent inhibition of viral replication that was observed in infected macrophages exposed to N(G)-monomethyl L-arginine and N(G)-nitro-L-arginine methyl ester (L-NAME), two inhibitors of the iNOS. Moreover, an excess of L-arginine reversed the addition of L-NAME, confirming that an arginine-dependent mechanism is involved. Finally, inhibitory effects of hemoglobin which can trap free NO in culture supernatants and in biological fluids in vivo confirmed that endogenously produced NO could interfere with HIV replication in human macrophages.     

Fate of the human immunodeficiency virus type 1 provirus in infected cells: a role for vpr.

We investigated the fate of human immunodeficiency virus type 1 (HIV-1) viral DNA in infected peripheral blood lymphocytes and immortalized T-cell lines by using a replication-defective HIV-1. We observed that integrated HIV-1 DNA and viral gene expression decrease over time. A frameshift mutation in vpr resulted in maintenance of the HIV-1 provirus and stable persistence of viral expression. Transfection of vpr together with the neomycin resistance gene in the absence of other viral genes decreased the formation of geneticin-resistant colonies, indicating either a cytotoxic or a cytostatic effect upon cells. Therefore, maintenance of HIV-1 infection within an infected proliferating population is due to two competing processes, the rate of viral spread and the degree of cell growth inhibition and/or death induced by Vpr.     

Vpr and Vpu are important for efficient human immunodeficiency virus type 1 replication and CD4+ T-cell depletion in human lymphoid tissue ex vivo

The relevance of the accessory vpr, vpu, and nef genes for human immunodeficiency virus type 1 (HIV-1) replication in human lymphoid tissue (HLT), the major site of viral replication in vivo, is largely unknown. Here, we show that an individual deletion of nef, vpr, or vpu significantly decreases HIV-1 replication and prevents CD4+ T-cell depletion in ex vivo HLT. However, only combined defects in all three accessory genes entirely disrupt the replicative capacity of HIV-1. Our results demonstrate that nef, vpr, and vpu are all essential for efficient viral spread in HLT, suggesting an important role in AIDS pathogenesis.     

Induction of interleukin-10 by human immunodeficiency virus type 1 and its gp120 protein in human monocytes/macrophages.

In this study, we evaluated the effects of human immunodeficiency virus type 1 (HIV-1) and its gp120 protein on interleukin-10 (IL-10) expression in cultured human monocytes/macrophages. Infection of either 1-day monocytes or 7-day monocyte-derived macrophages with HIV-1 strain Ba-L resulted in clear-cut accumulation of IL-10 mRNA at 4 and 24 h. Likewise, treatment of these cells with recombinant gp120 induced IL-10 mRNA expression and caused a marked increase in IL-10 secretion. Monoclonal antibodies to gp120 strongly inhibited recombinant gp120-induced IL-10 secretion by monocytes/macrophages. Moreover, the addition of IL-10 to monocytes/macrophages resulted in a significant inhibition of HIV-1 replication 7 and 14 days after infection. On the whole, these results indicate that HIV-1 (possibly through its gp120 protein) up-regulates IL-10 expression in monocytes/macrophages. We suggest that in vivo production of IL-10 by HIV-primed monocytes/macrophages can play an important role in the early response to HIV-1 infection.     

Vpx and Vpr proteins of HIV-2 up-regulate the viral infectivity by a distinct mechanism in lymphocytic cells

Mutants of human immunodeficiency virus type 2 (HIV-2) carrying a frame-shift mutation in vpx, vpr, and in both genes were monitored for their growth potentials in a newly established lymphocytic cell line, HSC-F. Worthy of note, the replication of a vpx single mutant, but not vpr, was severely impaired in these cells, and that of a vpx-vpr double mutant was more damaged. Defective replication sites of the vpx single and vpx-vpr double mutants were demonstrated to be mapped, respectively, to the nuclear import of viral genome, and to both, this process and the virus assembly/release stage. While the mutational effect of vpr was small, the replication efficiency in one cycle of the vpx mutant relative to that of wild-type virus was estimated to be 10%. The growth phenotypes of the vpx, vpr, and vpx-vpr mutant viruses in HSC-F cells were essentially repeated in primary human lymphocytes. In primary human macrophages, whereas the vpx and vpx-vpr mutants did not grow at all, the vpr mutant grew equally as well as the wild-type virus. These results strongly suggested that Vpx is critical for up-regulation of HIV-2 replication in natural target cells by enhancing the genome nuclear import, and that Vpr promotes HIV-2 replication somewhat, at least in lymphocytic cells, at a very late replication phase.     

Dual role of the HIV-1 vpr protein in the modulation of the apoptotic response of T cells

We investigated the effect of vpr, physiologically expressed during the course of an acute HIV-1 infection, on the response of infected cells to apoptotic stimuli as well as on the HIV-induced apoptosis. At 48 h after infection, Jurkat cells exhibited a lower susceptibility to undergo apoptosis with respect to uninfected cells. This effect was not observed following infection with either a vpr-mutated virus or a wild-type strain in the presence of antisense oligodeoxynucleotides targeted at vpr mRNA. Single-cell analysis, aimed at simultaneously identifying apoptotic and infected cells, revealed that resistance to apoptosis correlated with productive infection. Notably, vpr-dependent protection from induced apoptosis was also observed in HIV-1-infected PBMC. In contrast, at later stages of infection, a marked increase in the number of cells spontaneously undergoing apoptosis was detected in infected cultures. This virus-induced apoptosis involved vpr expression and predominantly occurred in productively infected cells. These results indicate that HIV-1 vpr can exert opposite roles in the regulation of apoptosis, which may depend on the level of its intracellular expression at different stages of HIV-1 infection. The dual function of vpr represents a novel mechanism in the complex strategy evolved by HIV to influence the turnover of T lymphocytes leading to either viral persistence or virus release and spreading.     

Extracellular Vpr protein increases cellular permissiveness to human immunodeficiency virus replication and reactivates virus from latency.

The vpr gene product of human immunodeficiency virus (HIV) and simian immunodeficiency virus is a virion-associated regulatory protein that has been shown using vpr mutant viruses to increase virus replication, particularly in monocytes/macrophages. We have previously shown that vpr can directly inhibit cell proliferation and induce cell differentiation, events linked to the control of HIV replication, and also that the replication of a vpr mutant but not that of wild-type HIV type 1 (HIV-1) was compatible with cellular proliferation (D. N. Levy, L. S. Fernandes, W. V. Williams, and D. B. Weiner, Cell 72:541-550, 1993). Here we show that purified recombinant Vpr protein, in concentrations of < 100 pg/ml to 100 ng/ml, increases wild-type HIV-1 replication in newly infected transformed cell lines via a long-lasting increase in cellular permissiveness to HIV replication. The activity of extracellular Vpr protein could be completely inhibited by anti-Vpr antibodies. Extracellular Vpr also induced efficient HIV-1 replication in newly infected resting peripheral blood mononuclear cells. Extracellular Vpr transcomplemented a vpr mutant virus which was deficient in replication in promonocytic cells, restoring full replication competence. In addition, extracellular Vpr reactivated HIV-1 expression in five latently infected cell lines of T-cell, B-cell, and promonocytic origin which normally express very low levels of HIV RNA and protein, indicating an activation of translational or pretranslational events in the virus life cycle. Together, these results describe a novel pathway governing HIV replication and a potential target for the development of anti-HIV therapeutics.     

Synthesis of phosphorothioate analogues of oligodeoxyribonucleotides and their antiviral activity against human immunodeficiency virus (HIV)

Nuclease-resistant phosphorothioate analogues of oligodeoxynucleotides (oligos) were synthesized by sulfurization of either internucleoside phosphite linkages, in a repetitive manner during chain extension, or internucleoside hydrogen phosphonate linkages, in a single step following chain assembly. These analogues were tested as antiviral agents against human immunodeficiency virus (HIV). In a cytopathic effect inhibition assay using HIV-uninfected susceptible T cells (tetanus toxoid-specific normal T cells) co-cultured with irradiated chronically HIV-infected cells, phosphorothioate oligomers inhibited the cytopathic effect and replication of several isolates of HIV-1 and HIV-2. Thus phosphorothioate analogues of oligos could inhibit cell-to-cell transmission of the virus as well as the infection by cell-free virus particles and also could inhibit a variety of isolates of human retroviruses.     

Inhibition of clinical human immunodeficiency virus (HIV) type 1 isolates in primary CD4+ T lymphocytes by retroviral vectors expressing anti-HIV genes.

Gene therapy may be of benefit in human immunodeficiency virus type 1 (HIV-1)-infected individuals by virtue of its ability to inhibit virus replication and prevent viral gene expression. It is not known whether anti-HIV-1 gene therapy strategies based on antisense or transdominant HIV-1 mutant proteins can inhibit the replication and expression of clinical HIV-1 isolates in primary CD4+ T lymphocytes. We therefore transduced CD4+ T lymphocytes from uninfected individuals with retroviral vectors expressing either HIV-1-specific antisense-TAR or antisense-Tat/Rev RNA, transdominant HIV-1 Rev protein, and a combination of antisense-TAR and transdominant Rev. The engineered CD4+ T lymphocytes were then infected with four different clinical HIV-1 isolates. We found that replication of all HIV-1 isolates was inhibited by all the anti-HIV vectors tested. Greater inhibition of HIV-1 was observed with transdominant Rev than with antisense RNA. We hereby demonstrated effective protection by antisense RNA or transdominant mutant proteins against HIV-1 infection in primary CD4+ T lymphocytes using clinical HIV-1 isolates, and this represents an essential step toward clinical anti-HIV-1 gene therapy.     

Human immunodeficiency virus vpr product is a virion-associated regulatory protein.

The vpr product of the human immunodeficiency virus type 1 (HIV-1) acts in trans to accelerate virus replication and cytopathic effect in T cells. Here it is shown that the HIV-1 viral particle contains multiple copies of the vpr protein. The vpr product is the first regulatory protein of HIV-1 to be found in the virus particle. This observation raises the possibility that vpr acts to facilitate the early steps of infection before de novo viral protein synthesis occurs.     

Induction of beta interferon by human immunodeficiency virus type 1 and its gp120 protein in human monocytes-macrophages: role of beta interferon in restriction of virus replication.

In vitro cultivated human monocytes show a time-dependent differentiation into macrophages, characterized by an increased expression of macrophage-specific antigens. Monocytes-macrophages were infected with human immunodeficiency virus type 1 strain Ba-L (HIV-1Ba-L) at different stages of differentiation. When 7-day cultured macrophages were infected in the presence of antibodies to beta interferon (IFN-beta), a significant increase in HIV-1 p24 release was detected. This effect was not observed in 1-day monocytes. This finding suggests that IFN-beta secreted by the infected macrophages inhibits p24 release. Treatment of cultured macrophages with recombinant gp120 (rgp120) protein resulted in the induction of IFN-beta mRNA and in an antiviral state to vesicular stomatitis virus. This rgp120-induced antiviral state was largely neutralized by antibodies to IFN-beta, whereas anti-IFN-alpha antibodies were ineffective. In cultured macrophages, 0.1 IU of IFN-beta per ml was sufficient to induce a marked inhibition of vesicular stomatitis virus yield, whereas this dose was ineffective in 1-day monocytes. These results indicate that (i) HIV-1 (possibly in part through its gp120 protein) induces low levels of IFN-beta in macrophages and (ii) this IFN-beta is very effective in inducing an antiviral state in differentiated macrophages.     

Tat- and Rev-directed antisense RNA expression inhibits and abolishes replication of human immunodeficiency virus type 1: a temporal analysis.

Replication of human immunodeficiency virus type 1 (HIV-1) was inhibited by stable intracellular expression of antisense RNA in the human T-lymphoid cell line Jurkat. When the viral subregion encoding the HIV-1 activator proteins was targeted, the extent of antisense RNA-mediated inhibition was greater than 97% during the first 2 weeks postinfection. Later in the time course, productive HIV-1 infection broke through at high initial infective doses. However, at initial multiplicities of infection equal to or smaller than 0.1, HIV-1 production was not detectable during the 5 weeks of observation. The results underline the effectiveness of stable intracellular antisense RNA expression in inhibiting HIV-1 replication.     

In vivo inhibition of duck hepatitis B virus replication and gene expression by phosphorothioate modified antisense oligodeoxynucleotides.

Antisense oligodeoxynucleotide strategies have been employed in a variety of eukaryotic systems both to understand normal gene function and to block gene expression. Pharmacologically, 'code blockers' are ideal agents for antitumour and antimicrobial treatments because of their specific mode of action. Here we report the inhibition of duck hepatitis B virus (DHBV) by antisense oligodeoxynucleotides in primary duck hepatocyte cultures in vitro as well as in DHBV-infected Pekin ducks in vivo. The most effective antisense oligodeoxynucleotide was directed against the 5' region of the pre-S gene and resulted in a complete inhibition of viral replication and gene expression in vitro and in vivo. These results demonstrate the application of antisense oligodeoxynucleotides in vivo and exemplify their potential as human antiviral therapeutics.     

Alpha interferon inhibits early stages of the human immunodeficiency virus type 1 replication cycle.

In this study, we have analyzed the effect of human alpha interferon (IFN-alpha) on a single replication cycle of human immunodeficiency virus type 1 (HIV-1) infection in the lymphocytic cell line CEM-174, which is highly sensitive to the antiviral effects of IFN. Pretreatment of cells with 50 to 500 U of recombinant human IFN-alpha per ml resulted in a marked reduction in viral RNA and protein synthesis. The effect of IFN-alpha was dose dependent and was amplified in multiple infection cycles. IFN-induced inhibition of viral protein synthesis could be detected only when cells were treated with IFN-alpha prior to infection or when IFN-alpha was added up to 10 h postinfection, but not if IFN-alpha was added at the later stages of HIV-1 replication cycle or after the HIV-1 infection was already established. Analysis of the integrated HIV-1 provirus showed a marked decrease in the levels of proviral DNA in IFN-treated cells. Thus, in contrast to the previous studies on established HIV-1 infection in T cells, in which the IFN block appeared to be at the posttranslational level, during de novo infection, IFN-alpha interferes with an early step of HIV-1 replication cycle that occurs prior to the integration of the proviral DNA. These results indicate that the early IFN block of HIV-1 replication, which has been previously observed only in primary marcophages, can also be detected in the IFN-sensitive T cells, indicating that the early IFN block is not limited to macrophages.