Inhibit Human Immunodeficiency Virus Type 1 Replication Research Articles

Selective inhibition of HIV-1 replication by the CDK9 inhibitor FIT-039

FIT-039 has recently been identified as a novel cyclin-dependent kinase 9 inhibitor with potent antiviral activity against a broad spectrum of DNA viruses, such as herpes simplex virus type 1 (HSV-1) and human cytomegaloviruses. In this study, FIT-039 was examined for its inhibitory effect on human immunodeficiency virus type 1 (HIV-1) replication in chronically infected cells. Its 50% effective concentration was 1.4–2.1μM, irrespective of the cells used for antiviral assays, while its 50% cytotoxic concentration was >20μM, indicating that FIT-039 is a selective inhibitor of HIV-1 replication. FIT-039 also inhibited HIV-1 RNA expression in a dose-dependent fashion. Since previous studies demonstrated that FIT-039 exhibited antiviral efficacy without noticeable adverse effects in HSV-1-infected mice, the compound should be further investigated for its clinical potential against HIV-1 infection.

Design and Synthesis of Novel N-Arylsulfonyl-3-(2-yl-ethanone)-6-methylindole Derivatives as Inhibitors of HIV-1 Replication.

Seven novel N-arylsulfonyl-3-(2-yl-ethanone)-6-methylindole derivatives 4a–f and 6 were readily synthesized and have been identified as inhibitors of human immunodeficiency virus type-1 (HIV-1) replication. Initial biological studies indicated that among these derivatives, N-(p-ethyl)phenylsulfonyl-3-[2-morpholinoethanone]-6-methylindole (4f) and N-(p-ethyl)phenylsulfonyl-3-[2-(5-phenyl-1,3,4-oxadiazole-2-yl-thio)ethanone]-6-methylindole (6) showed the most promising activity against HIV-1 replication. The effective concentration (EC50) and therapeutic index (TI) values of 4f and 6 were 9.42/4.62 μM, and >49.77/66.95, respectively. The cytotoxicity of these compounds has also been assessed. No significant cytotoxicities were found for any of these compounds.

Development of benzimidazole derivatives to inhibit HIV-1 replication through protecting APOBEC3G protein

Human APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G, A3G) is a potent restriction factor against human immunodeficiency virus type 1 (HIV-1) by inducing hypermutation of G to A in viral genome after its incorporation into virions. HIV-1 Vif (Virion Infectivity Factor) counteracts A3G by inducing ubiquitination and proteasomal degradation of A3G protein. Vif-A3G axis therefore is a promising therapeutic target of HIV-1. Here we report the screening, synthesis and SAR studies of benzimidazole derivatives as potent inhibitors against HIV-1 replication via protecting A3G protein. Based on the steep SAR of the benzimidazole scaffold, we identified compound 14 and 26 which provided the best potency, with IC50 values of 3.45 nM and 58.03 nM respectively in the anti-HIV-1 replication assay in H9 cells. Compound 14 and 26 also afforded protective effects on A3G protein level. Both compounds have been proved to be safe in acute toxicological studies. Taken together, we suggest that these two benzimidazole derivatives can be further developed as a new category of anti-HIV-1 leads.

Inhibition of the DNA polymerase and RNase H activities of HIV-1 reverse transcriptase and HIV-1 replication by Brasenia schreberi (Junsai) and Petasites japonicus (Fuki) components

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) possesses two distinct enzymatic activities: those of RNA- and DNA-dependent DNA polymerases and RNase H. In the current HIV-1 therapy, all HIV-1 RT inhibitors inhibit the activity of DNA polymerase, but not that of RNase H. We previously reported that ethanol and water extracts of Brasenia schreberi (Junsai) inhibited the DNA polymerase activity of HIV-1 RT [Hisayoshi et al. (2014) J Biol Macromol 14:59-65]. In this study, we screened 43 edible plants and found that ethanol and water extracts of Brasenia schreberi and water extract of Petasites japonicus strongly inhibit not only the activity of DNA polymerase to incorporate dTTP into poly(rA)-p(dT)15 but also the activity of RNase H to hydrolyze the RNA strand of an RNA/DNA hybrid. In addition, these three extracts inhibit HIV-1 replication in human cells, with EC50 values of 1-2µg/ml. These results suggest that Brasenia schreberi and Petasites japonicus contain substances that block HIV-1 replication by inhibiting the DNA polymerase activity and/or RNase H activity of HIV-1 RT.

Search for Low Molecular Weight Compounds that Inhibit Human Immunodeficiency Virus Type 1 Replication

Highly active antiretroviral (ARV) therapy has successfully reduced viral transmission, morbidity, and mortality associated with human immunodeficiency virus type 1 (HIV-1) disease; however, the emergence of drug-resistant viruses is a major obstacle associated with ARV therapy. Therefore, the development of a new class of ARV drugs is urgently required. Cyclophilin A (CypA) is a host factor required for HIV-1 replication, and plays a role in viral replication by interacting with the HIV-1 capsid protein (CA). As such, it represents a potential target for novel ARV drugs. We here searched for low molecular weight compounds that inhibited HIV-1 replication by interfering with binding between CypA and HIV-1 CA. A total of 106 compounds were screened in an in silico docking study as candidates that were predicted to interact with the HIV-1 CA binding pocket of CypA. Biological tests were then conducted to evaluate the anti-HIV-1 activities as well as cytotoxicities of these test compounds, and 4 compounds that efficiently inhibited viral replication without exhibiting strong cytotoxicity were subsequently selected. The molecular mechanisms underlying the inhibition of HIV-1 replication by the 4 selected compounds have not been elucidated in the present study; however, we consider that these compounds will become the lead compounds for developing novel ARV drugs once more detailed studies are performed on the molecular mechanisms responsible for their anti-HIV-1 activities.

Prothymosin α variants isolated from CD8+ T cells and cervicovaginal fluid suppress HIV-1 replication through type I interferon induction.

Soluble factors from CD8(+) T cells and cervicovaginal mucosa of women are recognized as important in controlling human immunodeficiency virus type 1 (HIV-1) infection and transmission. Previously, we have shown the strong anti-HIV-1 activity of prothymosin α (ProTα) derived from CD8(+) T cells. ProTα is a small acidic protein with wide cell distribution, to which several functions have been ascribed, depending on its intracellular or extracellular localization. To date, activities of ProTα have been attributed to a single protein known as isoform 2. Here we report the isolation and identification of 2 new ProTα variants from CD8(+) T cells and cervicovaginal lavage with potent anti-HIV-1 activity. The first is a splice variant of the ProTα gene, known as isoform CRA_b, and the second is the product of a ProTα gene, thus far classified as a pseudogene 7. Native or recombinant ProTα variants potently restrict HIV-1 replication in macrophages through the induction of type I interferon. The baseline expression of interferon-responsive genes in primary human cervical tissues positively correlate with high levels of intracellular ProTα, and the knockdown of ProTα variants by small interfering RNA leads to downregulation of interferon target genes. Overall, these findings suggest that ProTα variants are innate immune mediators involved in immune surveillance.

Triptolide inhibits human immunodeficiency virus type 1 replication by promoting proteasomal degradation of Tat protein.

BackgroundPlants remain an important source of new drugs, new leads and new chemical entities. Triptolide is a diterpenoid epoxide isolated from Tripterygium wilfordii Hook F that possesses a broad range of bioactivities, including anti-inflammatory, immunosuppressive and anti-tumor properties. The antiviral activity of triptolide against human immunodeficiency virus type 1 (HIV-1) has not been reported.ResultsIn this study, nanomolar concentrations of triptolide were shown to potently inhibit HIV-1 replication in vitro. To identify the step(s) of the HIV-1 replication cycle affected by triptolide, time-of-addition studies, PCR analysis and direct transfection of viral genomic DNA were performed. The results of these experiments indicated that triptolide acts at the stage of viral gene transcription. In addition, a luciferase-based reporter assay that allows quantitative analysis of long terminal repeat (LTR)-driven transcription showed that Tat-induced LTR activation was impaired in the presence of triptolide. Moreover, Western blot analysis of exogenous gene expression (driven by the human elongation factor 1 α subunit promoter) in transiently transfected cells revealed that triptolide specifically reduces the steady-state level of Tat protein, without suppressing global gene expression. Further studies showed that triptolide accelerates Tat protein degradation, which can be rescued by administration of the proteasome inhibitor MG132. Mutation analysis revealed that N-terminal domains of Tat protein and nuclear localization are required for triptolide to reduce steady-state level of Tat.ConclusionThis study suggests for the first time that triptolide exerts its anti-HIV-1 activity by specifically prompting the degradation of the virally encoded Tat protein, which is a novel mechanism of action for an anti-HIV-1 compound. This compound may serve as a starting point for developing a novel HIV-1 therapeutic approach or as a basic research tool for interrogating events during viral replication.

Heat-stable molecule derived from Streptococcus cristatus induces APOBEC3 expression and inhibits HIV-1 replication.

Although most human immunodeficiency virus type 1 (HIV-1) cases worldwide are transmitted through mucosal surfaces, transmission through the oral mucosal surface is a rare event. More than 700 bacterial species have been detected in the oral cavity. Despite great efforts to discover oral inhibitors of HIV, little information is available concerning the anti-HIV activity of oral bacterial components. Here we show that a molecule from an oral commensal bacterium, Streptococcus cristatus CC5A can induce expression of APOBEC3G (A3G) and APOBEC3F (A3F) and inhibit HIV-1 replication in THP-1 cells. We show by qRT-PCR that expression levels of A3G and A3F increase in a dose-dependent manner in the presence of a CC5A extract, as does A3G protein levels by Western blot assay. In addition, when the human monocytic cell line THP-1 was treated with CC5A extract, the replication of HIV-1 IIIB was significantly suppressed compared with IIIB replication in untreated THP-1 cells. Knock down of A3G expression in THP-1 cells compromised the ability of CC5A to inhibit HIV-1 IIIB infectivity. Furthermore, SupT1 cells infected with virus produced from CC5A extract-treated THP-1 cells replicated virus with a higher G to A hypermutation rate (a known consequence of A3G activity) than virus used from untreated THP-1 cells. This suggests that S. cristatus CC5A contains a molecule that induces A3G/F expression and thereby inhibits HIV replication. These findings might lead to the discovery of a novel anti-HIV/AIDS therapeutic.

Plasmacytoid dendritic cells suppress HIV-1 replication but contribute to HIV-1 induced immunopathogenesis in humanized mice.

The role of plasmacytoid dendritic cells (pDC) in human immunodeficiency virus type 1 (HIV-1) infection and pathogenesis remains unclear. HIV-1 infection in the humanized mouse model leads to persistent HIV-1 infection and immunopathogenesis, including type I interferons (IFN-I) induction, immune-activation and depletion of human leukocytes, including CD4 T cells. We developed a monoclonal antibody that specifically depletes human pDC in all lymphoid organs in humanized mice. When pDC were depleted prior to HIV-1 infection, the induction of IFN-I and interferon-stimulated genes (ISGs) were abolished during acute HIV-1 infection with either a highly pathogenic CCR5/CXCR4-dual tropic HIV-1 or a standard CCR5-tropic HIV-1 isolate. Consistent with the anti-viral role of IFN-I, HIV-1 replication was significantly up-regulated in pDC-depleted mice. Interestingly, the cell death induced by the highly pathogenic HIV-1 isolate was severely reduced in pDC-depleted mice. During chronic HIV-1 infection, depletion of pDC also severely reduced the induction of IFN-I and ISGs, associated with elevated HIV-1 replication. Surprisingly, HIV-1 induced depletion of human immune cells including T cells in lymphoid organs, but not the blood, was reduced in spite of the increased viral replication. The increased cell number in lymphoid organs was associated with a reduced level of HIV-induced cell death in human leukocytes including CD4 T cells. We conclude that pDC play opposing roles in suppressing HIV-1 replication and in promoting HIV-1 induced immunopathogenesis. These findings suggest that pDC-depletion and IFN-I blockade will provide novel strategies for treating those HIV-1 immune non-responsive patients with persistent immune activation despite effective anti-retrovirus treatment.

Bromotyrosine derivatives from marine sponges inhibit the HIV-1 replication in vitro

Background: Human immunodeficiency virus type 1 (HIV-1) infection and Acquired immunodeficiency syndrome are mayor global public health issues. HIV-1 infection is now manageable as a chronic disease thanks to the development of antiretroviral therapy; however, the existence of HIV drug resistance and collateral effects have increased the search for therapeutic alternatives. Compounds of marine resources have been studied for their antiviral potential. Objectives: To evaluate the antiviral activity of isolated bromotyrosine-derivative compounds from the Colombian marine sponges, Verongula rigida and Aiolochoria crassa against HIV-1 infection in vitro. Methods: Cytotoxicity of 11 bromotyrosine-derivative compounds was determined by the MTT assay. Inhibition of HIV-1 replication was performed using the U373-MAGI cell line, which was infected with recombinant green fluorescent protein (GFP)-expressing viruses pseudotyped, in the presence or absence of the compounds. The percentage of infected cells was evaluated by flow cytometry. In addition, the inhibition of reverse transcription and nuclear import was determined by quantification of early and late reverse transcription products and 2-LTR circles, respectively, using quantitative PCR. Results: Aeroplysinin-1, purealidin B and 3-bromo-5-hydroxy-Omethyltyrosine inhibited the HIV-1 replication in a dose-dependent manner, with a median maximum percentage of inhibition of 74% (20 μM), 57% (80 μM) and 47% (80 μM), respectively. Importantly, none of these concentrations were cytotoxic. Aeroplysinin-1, 19-deoxyfistularin 3, purealidin B, fistularin 3 and 3-bromo-5-hydroxy-O-methyltyrosine inhibited the nuclear import efficiently; while 3,5-dibromo-N,N,N,O-tetramethyltyraminium, aeroplysinin-1, purealidin B, fistularin 3 and 3-bromo-5-hydroxy-Omethyltyrosine inhibited X4 HIV-1 cell entry with a median maximum percentage of inhibition ranging between 2 to 30%. Conclusions: Aeroplysinin-1, 19-deoxyfistularin 3, purealidin B, fistularin 3 and 3-bromo-5-hydroxy-O-methyltyrosine inhibited HIV replication at different steps. This study opens the possibility of chemically synthesizing these compounds and evaluating them as alternative therapies against HIV-1.

Allo-antigen stimulated CD8+ T-cells suppress NF-κB and Ets-1 DNA binding activity, and inhibit phosphorylated NF-κB p65 nuclear localization in CD4+ T-cells.

CD8+ T-cells of asymptomatic HIV-1 carriers (AC) suppress human immunodeficiency virus type 1 (HIV-1) replication in a class I major histocompatibility complex (MHC-I)-restricted and -unrestricted manner. In order to investigate the mechanism of MHC-I-unrestricted CD8+ T-cell-mediated HIV-1 suppression, we previously established allo-antigen stimulated CD8+T-cells from HIV-1-uninfected donors. These allo-antigen stimulated CD8+ T-cells suppressed HIV-1 replication in acutely infected autologous CD4+ T-cells when directly co-cultured. To elucidate the mechanism of HIV-1 replication suppression, we analyzed DNA-binding activity and phosphorylation of transcriptional factors associated with HIV-1 replication by electrophoresis mobility shift assay and Western blotting. When CD4+ T-cells were cultured with allo-antigen stimulated CD8+ T-cells, the reduction of NF-κB and Ets-1 DNA-binding activity was observed. Nuclear localization of NF-κB p65 and Ets-1 was suppressed in CD4+ T-cells. Although NF-κB p65 and Ets-1 are known to be regulated by protein kinase A (PKA), no difference was observed in the expression and phosphorylation of the PKA catalytic subunit in CD4+ T-cells cultured with PHA-treated CD8+ T-cells or allo-antigen stimulated CD8+ T-cells. Cyclic AMP is also known to enter through gap junctions, but the suppression of HIV-1 replication mediated by allo-antigen stimulated CD8+ T-cells was not affected by the gap junction inhibitor. The nuclear transport of phosphorylated NF-κB p65 (Ser276) was inhibited only in CD4+ T-cells cultured with allo-antigen stimulated CD8+ T-cells. Our results indicate that allo-antigen stimulated CD8+ T-cells suppress the transcriptional activity of NF-κB p65 or Ets-1 in an antigen-nonspecific manner, and inhibit the nuclear transport of phosphorylated NF-κB p65 (Ser276).

Ruxolitinib and tofacitinib are potent and selective inhibitors of HIV-1 replication and virus reactivation in vitro.

The JAK-STAT pathway is activated in both macrophages and lymphocytes upon human immunodeficiency virus type 1 (HIV-1) infection and thus represents an attractive cellular target to achieve HIV suppression and reduced inflammation, which may impact virus sanctuaries. Ruxolitinib and tofacitinib are JAK1/2 inhibitors that are FDA approved for rheumatoid arthritis and myelofibrosis, respectively, but their therapeutic application for treatment of HIV infection was unexplored. Both drugs demonstrated submicromolar inhibition of infection with HIV-1, HIV-2, and a simian-human immunodeficiency virus, RT-SHIV, across primary human or rhesus macaque lymphocytes and macrophages, with no apparent significant cytotoxicity at 2 to 3 logs above the median effective antiviral concentration. Combination of tofacitinib and ruxolitinib increased the efficacy by 53- to 161-fold versus that observed for monotherapy, respectively, and each drug applied alone to primary human lymphocytes displayed similar efficacy against HIV-1 containing various polymerase substitutions. Both drugs inhibited virus replication in lymphocytes stimulated with phytohemagglutinin (PHA) plus interleukin-2 (IL-2), but not PHA alone, and inhibited reactivation of latent HIV-1 at low-micromolar concentrations across the J-Lat T cell latency model and in primary human central memory lymphocytes. Thus, targeted inhibition of JAK provided a selective, potent, and novel mechanism to inhibit HIV-1 replication in lymphocytes and macrophages, replication of drug-resistant HIV-1, and reactivation of latent HIV-1 and has the potential to reset the immunologic milieu in HIV-infected individuals.

Triptolide inhibits human immunodeficiency virus type 1 replication by promoting proteasomal degradation of Tat protein

Plants remain an important source of new drugs, new leads and new chemical entities. Triptolide is a diterpenoid epoxide isolated from Tripterygium wilfordii Hook F that possesses a broad range of bioactivities, including anti-inflammatory, immunosuppressive and anti-tumor properties. The antiviral activity of triptolide against human immunodeficiency virus type 1 (HIV-1) has not been reported. In this study, nanomolar concentrations of triptolide were shown to potently inhibit HIV-1 replication in vitro. To identify the step(s) of the HIV-1 replication cycle affected by triptolide, time-of-addition studies, PCR analysis and direct transfection of viral genomic DNA were performed. The results of these experiments indicated that triptolide acts at the stage of viral gene transcription. In addition, a luciferase-based reporter assay that allows quantitative analysis of long terminal repeat (LTR)-driven transcription showed that Tat-induced LTR activation was impaired in the presence of triptolide. Moreover, Western blot analysis of exogenous gene expression (driven by the human elongation factor 1 α subunit promoter) in transiently transfected cells revealed that triptolide specifically reduces the steady-state level of Tat protein, without suppressing global gene expression. Further studies showed that triptolide accelerates Tat protein degradation, which can be rescued by administration of the proteasome inhibitor MG132. Mutation analysis revealed that N-terminal domains of Tat protein and nuclear localization are required for triptolide to reduce steady-state level of Tat. This study suggests for the first time that triptolide exerts its anti-HIV-1 activity by specifically prompting the degradation of the virally encoded Tat protein, which is a novel mechanism of action for an anti-HIV-1 compound. This compound may serve as a starting point for developing a novel HIV-1 therapeutic approach or as a basic research tool for interrogating events during viral replication.

Design and synthesis of N1-aryl-benzimidazoles 2-substituted as novel HIV-1 non-nucleoside reverse transcriptase inhibitors

A series of novel N1-aryl-2-arylthioacetamido-benzimidazoles were synthesized and evaluated as inhibitors of human immunodeficiency virus type-1 (HIV-1). Some of them proved to be effective in inhibiting HIV-1 replication at submicromolar and nanomolar concentration acting as HIV-1 non-nucleoside RT inhibitors (NNRTIs), with low cytotoxicity. The preliminary structure–activity relationship (SAR) of these new derivatives was discussed and rationalized by docking studies.

Toward an effective AIDS vaccine development

Toward an effective AIDS vaccine development

New insights into inhibition of human immunodeficiency virus type 1 replication through mutant tRNALys3.

BackgroundHost cellular tRNALys3 is exclusively utilized by human immunodeficiency virus type 1 (HIV-1) as a primer for the replication step of reverse transcription (RTion). Consequently, the priming step of HIV-1 RT constitutes a potential target for anti-HIV-1 intervention. Previous studies indicated that a mutant tRNALys3 with 7-nucleotide substitutions in the 3′ terminus resulted in aberrant HIV-1 RTion from the trans-activation response region (TAR) and inhibition of HIV-1 replication. However, the mutant tRNALys3 also directed HIV-1 RTion from the normal primer-binding site (PBS) with potentially weakened anti-HIV-1 activity. To achieve improved targeting of HIV-1 RTion at sites not including the PBS, a series of mutant tRNALys3 with extended lengths of mutations containing up to 18 bases complementary to their targeting sites were constructed and characterized.ResultsA positive correlation between the length of mutation in the 3′ PBS-binding region of tRNALys3 and the specificity of HIV-1 RTion initiation from the targeting site was demonstrated, as indicated by the potency of HIV-1 inhibition and results of priming assays. Moreover, two mutant tRNALys3s that targeted the IN-encoding region and Env gene, respectively, both showed a high anti-HIV-1 activity, suggesting that not only the TAR, but also distant sites downstream of the PBS could be effectively targeted by mutant tRNALys3. To increase the expression of mutant tRNALys3, multiple-copy expression cassettes were introduced into target cells with increased anti-HIV-1 potency.ConclusionsThese results highlight the importance of the length of complementarity between the 3′ terminus of the mutant tRNALys3 and its target site, and the feasibility of targeting multiple sites within the HIV-1 genome through mutant tRNALys3. Intervention of the HIV-1 genome conversion through mutant tRNALys3 may constitute an effective approach for development of novel therapeutics against HIV-1 replication and HIV-1-associated diseases.

Cycloviolacin O2 (CyO2) suppresses productive infection and augments the antiviral efficacy of nelfinavir in HIV‐1 infected monocytic cells

Human immunodeficiency virus type-1 (HIV-1), the etiologic agent of acquired immune deficiency syndrome (AIDS), is a global pandemic causing millions of deaths annually. Highly active antiretroviral therapy (HAART) greatly enhances lifespan but eventually causes debilitating side effects, in part, due to their chronic administration required to suppress HIV-1 replication. If treatment is discontinued, viral suppression is lost and dormant replication-competent monocytic cell reservoirs become reactivated, leading to viral recrudescence and progression to AIDS. Therefore, novel strategies to circumvent obstacles to HIV-1 therapy are critically needed. We evaluated the potentially therapeutic effects of cycloviolacin O2 (CyO2) on cell viability (MTT assay), membrane disruption (SYTOX Green uptake), p24 production [enzyme-linked immunosorbent assays (ELISA)], and proviral integration (PCR amplification) in U1 cells; a monocytic cell model of HIV-1 latency and reactivation. We demonstrate, for the first time, that CyO2 (0.5-5.0 μM) kills productively infected cells. Sub-toxic concentrations (<0.5 μM) of CyO2 disrupted plasma membranes in both latently-infected and productively-infected U1 cells and enhanced the antiviral efficacy of nelfinavir, a HIV-1 protease inhibitor (HPI). Interestingly, CyO2 also decreased virus production by activated U1 cells; however, this effect was not due to suppression of integrated provirus in U1 cells. This suggested that, in addition to the known pore-forming ability of cyclotides, a novel mode of antiviral activity may exist for CyO2. Our data indicate that CyO2 may be a promising candidate for the targeting HIV-1 reservoirs in monocytes, and their inclusion in adjuvant therapy approaches may augment the efficacy of HPIs and ultimately facilitate virus elimination.

Induction of heat-shock protein 70 by prostaglandin A1 inhibits HIV-1 Vif-mediated degradation of APOBEC3G

Previous studies have demonstrated that cyclopentenone prostaglandins (cyPGs) inhibit human immunodeficiency virus type 1 (HIV-1) replication in various cell types. This antiviral activity has been associated with the induction of heat-shock protein 70 (HSP70) in infected cells. We investigated a new role of prostaglandin A1 (PGA1) in the replication of HIV-1 in non-permissive cells. Because overexpression of HSP70 blocks the viral infectivity factor (Vif)-mediated degradation of APOBEC3G (A3G) via the ubiquitin–proteasome pathway, we examined the effects of PGA1 on A3G and HIV-1 replication. The induction of HSP70 synthesis by PGA1 blocked Vif-mediated A3G degradation and enhanced the incorporation of A3G into both wild-type and Vif-deficient viruses. Furthermore, we determined the viral titer of HIV-1 particles produced from PGA1-treated 293T cells. The induction of HSP70 synthesis by PGA1 significantly reduced the viral titer in the presence of A3G. Additionally, the p24 Gag antigen levels were dramatically reduced in non-permissive cells treated once or repeatedly with PGA1. Thus, we showed that PGA1 inhibits HIV-1 replication, at least in part, by blocking Vif-mediated A3G degradation.

Sustained Inhibition of HIV-1 Replication by Conditional Expression of theE. coli-Derived Endoribonuclease MazF in CD4+T cells

Gene therapy using a Tat-dependent expression system of MazF, an ACA nucleotide sequence-specific endoribonuclease derived from Escherichia coli, in a retroviral vector appears to be an alternative approach to the treatment of human immunodeficiency virus type 1 (HIV-1) infection. MazF can cleave HIV-1 RNA, since it has more than 240 ACA sequences. Significant inhibition of viral replication, irrespective of HIV-1 strains, was observed in CD4(+) T cells that had been transduced with the MazF-expressing retroviral vector (MazF-T cells). The growth and viability of MazF-T cells were not affected by HIV-1 infection. Interestingly, the infectivity of HIV-1 produced from MazF-T cells was found to be lower than that from control CD4(+) T cells. A long-term culture experiment with HIV-1-infected cells revealed that viral replication was always lower in MazF-T cells than in CD4(+) T cells transduced with or without a control vector for more than 200 days. MazF was expressed and mainly localized in the cytoplasm of the infected cells. Unlike in CD4(+) T cells, the expression level of Tat gradually decreased rather than increased in MazF-T cells after HIV-1 infection. As a consequence, the expression level of MazF appeared to be well regulated and sustained during HIV-1 infection in MazF-T cells. Furthermore, the levels of cellular mRNA were not affected by HIV-1 infection. Thus, the Tat-dependent MazF expression system has great potential for inhibition of HIV-1 replication in vivo without apparent toxicity and may be able to avoid the emergence of resistant strains.

GB virus type C E2 protein inhibits human immunodeficiency virus type 1 assembly through interference with HIV-1 gag plasma membrane targeting.

GB virus type C (GBV-C) is a single-stranded positive-sense RNA virus classified in the Flaviviridae family. Persistent coinfection with GBV-C is associated with lower human immunodeficiency virus type 1 (HIV-1) load, higher CD4(+) T-cell count, and prolonged survival in HIV-1 coinfected patients. The GBV-C envelope glycoprotein E2 has been reported to interfere with HIV-1 entry. In this study, we showed that the expression of GBV-C E2 inhibited HIV-1 Gag assembly and release. Expression of glycosylated GBV-C E2 inhibited HIV-1 Gag precursor processing, resulting in lower production of CAp24 and MAp17, while the overall expression level of the Gag precursor Pr55 remained unchanged. Membrane floatation gradient and indirect immunofluorescence confocal microscopy analysis showed that glycosylated E2 disrupted HIV-1 Gag trafficking to the plasma membrane, resulting in Gag accumulation in subcellular compartments. This interference in HIV-1 Gag trafficking led to diminished HIV-1 particle production, which is a critical step for HIV-1 to infect new host cells. These findings shed light on a novel mechanism used by GBV-C E2 to inhibit HIV-1 replication and may provide insight into new approaches for suppressing HIV-1 replication.