Inhibit HIV-1 Reverse Transcription Research Articles

No antagonism or cross-resistance and a high barrier to the emergence of resistance in vitro for the combination of islatravir and lenacapavir.

Islatravir (ISL) is a deoxyadenosine analog that inhibits HIV-1 reverse transcription by multiple mechanisms. Lenacapavir (LEN) is a novel capsid inhibitor that inhibits HIV-1 at multiple stages throughout the viral life cycle. ISL and LEN are being investigated as once-weekly combination oral therapy for the treatment of HIV-1. Here, we characterized ISL and LEN in vitro to assess combinatorial antiviral activity, cytotoxicity, and the potential for interactions between the two compounds. Bliss analysis revealed ISL with LEN demonstrated additive inhibition of HIV-1 replication, with no evidence of antagonism across the range of concentrations tested. ISL exhibited potent antiviral activity against variants encoding known LEN resistance-associated mutations (RAMs) with or without the presence of M184V, an ISL RAM in reverse transcriptase (RT) . Static resistance selection experiments were conducted with ISL and LEN alone and in combination, initiating with either wild-type virus or virus containing the M184I RAM in RT to further assess their barrier to the emergence of resistance. The combination of ISL with LEN more effectively suppressed viral breakthrough at lower multiples of the compounds' IC50 (half-maximal inhibitory concentration) values and fewer mutations emerged with the combination compared to either compound on its own. The known pathways for development of resistance with ISL and LEN were not altered, and no novel single mutations emerged that substantially reduced susceptibility to either compound. The lack of antagonism and cross-resistance between ISL and LEN support the ongoing evaluation of the combination for treatment of HIV-1.

The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive-strand RNA viruses.

Genome-wide screens are powerful approaches to unravel regulators of viral infections. Here, a CRISPR screen identifies the RNA helicase DDX42 as an intrinsic antiviral inhibitor of HIV-1. Depletion of endogenous DDX42 increases HIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42 overexpression inhibits HIV-1 infection, whereas expression of a dominant-negative mutant increases infection. Importantly, DDX42 also restricts LINE-1 retrotransposition and infection with other retroviruses and positive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 does not impact the replication of several negative-strand RNA viruses, arguing against an unspecific effect on target cells, which is confirmed by RNA-seq analysis. Proximity ligation assays show DDX42 in the vicinity of viral elements, and cross-linking RNA immunoprecipitation confirms a specific interaction of DDX42 with RNAs from sensitive viruses. Moreover, recombinant DDX42 inhibits HIV-1 reverse transcription in vitro. Together, our data strongly suggest a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Our results identify DDX42 as an intrinsic viral inhibitor, opening new perspectives to target the life cycle of numerous RNA viruses.

SUMOylation of SAMHD1 at Lysine 595 is required for HIV-1 restriction in non-cycling cells

SAMHD1 is a cellular triphosphohydrolase (dNTPase) proposed to inhibit HIV-1 reverse transcription in non-cycling immune cells by limiting the supply of the dNTP substrates. Yet, phosphorylation of T592 downregulates SAMHD1 antiviral activity, but not its dNTPase function, implying that additional mechanisms contribute to viral restriction. Here, we show that SAMHD1 is SUMOylated on residue K595, a modification that relies on the presence of a proximal SUMO-interacting motif (SIM). Loss of K595 SUMOylation suppresses the restriction activity of SAMHD1, even in the context of the constitutively active phospho-ablative T592A mutant but has no impact on dNTP depletion. Conversely, the artificial fusion of SUMO2 to a non-SUMOylatable inactive SAMHD1 variant restores its antiviral function, a phenotype that is reversed by the phosphomimetic T592E mutation. Collectively, our observations clearly establish that lack of T592 phosphorylation cannot fully account for the restriction activity of SAMHD1. We find that SUMOylation of K595 is required to stimulate a dNTPase-independent antiviral activity in non-cycling immune cells, an effect that is antagonized by cyclin/CDK-dependent phosphorylation of T592 in cycling cells.

Daxx Inhibits HIV-1 Reverse Transcription and Uncoating in a SUMO-Dependent Manner.

Death domain-associated protein 6 (Daxx) is a multifunctional, ubiquitously expressed and highly conserved chaperone protein involved in numerous cellular processes, including apoptosis, transcriptional repression, and carcinogenesis. In 2015, we identified Daxx as an antiretroviral factor that interfered with HIV-1 replication by inhibiting the reverse transcription step. In the present study, we sought to unravel the molecular mechanism of Daxx-mediated restriction and, in particular, to identify the protein(s) that Daxx targets in order to achieve its antiviral activity. First, we show that the SUMO-interacting motif (SIM) located at the C-terminus of the protein is strictly required for Daxx to inhibit HIV-1 reverse transcription. By performing a quantitative proteomic screen combined with classical biochemical analyses, we found that Daxx associated with incoming HIV-1 cores through a SIM-dependent interaction with cyclophilin A (CypA) and capsid (CA). Daxx was found to reside within a multiprotein complex associated with viral capsids, also containing TNPO3, TRIM5α, and TRIM34. Given the well-known influence of these cellular factors on the stability of HIV-1 cores, we investigated the effect of Daxx on the cytoplasmic fate of incoming cores and found that Daxx prevented HIV-1 uncoating in a SIM-dependent manner. Altogether, our findings suggest that, by recruiting TNPO3, TRIM5α, and TRIM34 and possibly other proteins onto incoming HIV-1 cores through a SIM-dependent interaction with CA-bound CypA, Daxx increases their stability, thus preventing uncoating and reverse transcription. Our study uncovers a previously unknown function of Daxx in the early steps of HIV-1 infection and further illustrates how reverse transcription and uncoating are two tightly interdependent processes.

Proteomic profiling of HIV-1 infection of human CD4+ T cells identifies PSGL-1 as an HIV restriction factor.

Human immunodeficiency virus (HIV) actively modulates the protein stability of host cells to optimize viral replication. To systematically examine this modulation in HIV infection, we used isobaric tag-based mass spectrometry to quantify changes in the abundance of over 14,000 proteins during HIV-1 infection of human primary CD4+ T cells. We identified P-selectin glycoprotein ligand 1 (PSGL-1) as an HIV-1 restriction factor downregulated by HIV-1 Vpu, which binds to PSGL-1 and induces its ubiquitination and degradation through the ubiquitin ligase SCFβ-TrCP2. PSGL-1 is induced by interferon-γ in activated CD4+ T cells to inhibit HIV-1 reverse transcription and potently block viral infectivity by incorporating in progeny virions. This infectivity block is antagonized by Vpu via PSGL-1 degradation. We further show that PSGL-1 knockdown can significantly abolish the anti-HIV activity of interferon-γ in primary CD4+ T cells. Our study identifies an HIV restriction factor and a key mediator of interferon-γ's anti-HIV activity.

Cyclophilin A potentiates TRIM5α inhibition of HIV-1 nuclear import without promoting TRIM5α binding to the viral capsid.

The host immunophilin cyclophilin A (CypA) binds to the capsid protein (CA) of HIV-1 and regulates its infectivity. Depending on the target cell type, CypA can either promote or inhibit HIV-1 infection. The ability of CypA to promote HIV-1 infection has been extensively studied and linked to several steps in early replication including uncoating, reverse transcription and nuclear import. By contrast, the mechanism by which CypA inhibits infection is less well understood. We investigated the mechanism by which CypA potentiates restriction of HIV-1 by the tripartite motif-containing protein 5 (TRIM5α). Depletion of TRIM5α in the African green monkey cell line Vero, resulted in a loss of inhibition of infection by CypA, demonstrating that inhibition by CypA is mediated by TRIM5α. Complementary genetic and biochemical assays failed to demonstrate an ability of CypA to promote binding of TRIM5α to the viral capsid. TRIM5α inhibits HIV-1 reverse transcription in a proteasome-dependent manner; however, we observed that inhibition of proteasome activity did not reduce the ability of CypA to inhibit infection, suggesting that CypA acts at a step after reverse transcription. Accordingly, we observed a CypA-dependent reduction in the accumulation of nuclear HIV-1 DNA, indicating that CypA specifically promotes TRIM5α inhibition of HIV-1 nuclear import. We also observed that the ability of CypA to inhibit HIV-1 infection is abolished by amino acid substitutions within the conserved CPSF6-binding surface in CA. Our results indicate that CypA inhibits HIV-1 infection in Vero cells not by promoting TRIM5α binding to the capsid but by blocking nuclear import of the HIV-1 preintegration complex.

A mutant tat protein inhibits HIV-1 reverse transcription by targeting the reverse transcription complex.

Previously, we reported that a mutant of Tat referred to as Nullbasic inhibits HIV-1 reverse transcription although the mechanism of action is unknown. Here we show that Nullbasic is a reverse transcriptase (RT) binding protein that targets the reverse transcription complex rather than directly inhibiting RT activity. An interaction between Nullbasic and RT was observed by using coimmunoprecipitation and pulldown assays, and a direct interaction was measured by using a biolayer interferometry assay. Mixtures of recombinant 6×His-RT and Nullbasic-FLAG-V5-6×His at molar ratios of up to 1:20,000 did not inhibit RT activity in standard homopolymer primer template assays. An analysis of virus made by cells that coexpressed Nullbasic showed that Nullbasic copurified with virus particles, indicating that it was a virion protein. In addition, analysis of reverse transcription complexes (RTCs) isolated from cells infected with wild type or Nullbasic-treated HIV-1 showed that Nullbasic reduced the levels of viral DNA in RTC fractions. In addition, a shift in the distribution of viral DNA and CAp24 to less-dense non-RTC fractions was observed, indicating that RTC activity from Nullbasic-treated virus was impaired. Further analysis showed that viral cores isolated from Nullbasic-treated HIV undergo increased disassembly in vitro compared to untreated HIV-1. To our knowledge, this is the first description of an antiviral protein that inhibits reverse transcription by targeting the RTC and affecting core stability. HIV-1 infection is treated by using combinations of antiretroviral drugs that target independent steps of virus replication. A newly described antiviral protein called Nullbasic can also inhibit a combination of different steps in virus replication (transcription, reverse transcription, and Rev-mediated viral mRNA transport), although the precise mechanism of action is unknown. This study shows that Nullbasic can inhibit reverse transcription by binding to the viral enzyme called reverse transcriptase, which results in accelerated uncoating of the viral core and instability of the viral apparatus called the reverse transcription complex (RTC). This unique antiviral activity may inform development of other RTC inhibitors, as well as providing a unique investigative tool for dissecting the RTC cellular composition.

Palbociclib, a selective inhibitor of cyclin-dependent kinase4/6, blocks HIV-1 reverse transcription through the control of sterile α motif and HD domain-containing protein-1 (SAMHD1) activity.

Sterile α motif and HD domain-containing protein-1 (SAMHD1) inhibits HIV-1 reverse transcription by decreasing the pool of intracellular deoxynucleotides. SAMHD1 is controlled by cyclin-dependent kinase (CDK)-mediated phosphorylation. However, the exact mechanism of SAMHD1 regulation in primary cells is unclear. We explore the effect of palbociclib, a CDK6 inhibitor, in HIV-1 replication. Human primary monocytes were differentiated into macrophages with monocyte-colony stimulating factor and CD4 T lymphocytes stimulated with phytohaemagglutinin (PHA)/interleukin-2. Cells were treated with palbociclib and then infected with a Green fluorescent protein-expressing HIV-1 or R5 HIV-1 BaL. Viral DNA was measured by quantitative PCR and infection assessed by flow cytometry. Deoxynucleotide triphosphate (dNTP) content was determined using a polymerase-based method. Pan-CDK inhibitors AT7519, roscovitine and purvalanol A reduced SAMHD1 phosphorylation. HIV-1 replication was blocked by AT7519 (66.4 ± 3.8%; n = 4), roscovitine (47.3 ± 3.9%; n = 4) and purvalanol A (55.7 ± 15.7%; n = 4) at subtoxic concentrations. Palbociclib, a potent and selective CDK6 inhibitor, blocked SAMHD1 phosphorylation, intracellular dNTP levels, HIV-1 reverse transcription and HIV-1 replication in primary macrophages and CD4 T lymphocytes. Notably, treatment of macrophages with palbociclib led to reduced CDK2 activation, measured as the phosphorylation of the T-loop at the Thr160. The antiviral effect was lost when SAMHD1 was degraded by Vpx, providing further evidence for a role of SAMHD1 in mediating the antiretroviral effect. Our results indicate that SAMHD1-mediated HIV-1 restriction is controlled by CDK as previously suggested but point to a preferential role for CDK2 and CDK6 as mediators of SAMHD1 activation. Our study provides a new signaling pathway susceptible for the development of new therapeutic approaches against HIV-1 infection.

Abasic phosphorothioate oligomers inhibit HIV-1 reverse transcription and block virus transmission across polarized ectocervical organ cultures.

In the absence of universally available antiretroviral (ARV) drugs or a vaccine against HIV-1, microbicides may offer the most immediate hope for controlling the AIDS pandemic. The most advanced and clinically effective microbicides are based on ARV agents that interfere with the earliest stages of HIV-1 replication. Our objective was to identify and characterize novel ARV-like inhibitors, as well as demonstrate their efficacy at blocking HIV-1 transmission. Abasic phosphorothioate 2' deoxyribose backbone (PDB) oligomers were evaluated in a variety of mechanistic assays and for their ability to inhibit HIV-1 infection and virus transmission through primary human cervical mucosa. Cellular and biochemical assays were used to elucidate the antiviral mechanisms of action of PDB oligomers against both lab-adapted and primary CCR5- and CXCR4-utilizing HIV-1 strains, including a multidrug-resistant isolate. A polarized cervical organ culture was used to test the ability of PDB compounds to block HIV-1 transmission to primary immune cell populations across ectocervical tissue. The antiviral activity and mechanisms of action of PDB-based compounds were dependent on oligomer size, with smaller molecules preventing reverse transcription and larger oligomers blocking viral entry. Importantly, irrespective of molecular size, PDBs potently inhibited virus infection and transmission within genital tissue samples. Furthermore, the PDB inhibitors exhibited excellent toxicity and stability profiles and were found to be safe for vaginal application in vivo. These results, coupled with the previously reported intrinsic anti-inflammatory properties of PDBs, support further investigations in the development of PDB-based topical microbicides for preventing the global spread of HIV-1.

Phase I/II study of the pharmacokinetics, safety and antiretroviral activity of tenofovir alafenamide, a new prodrug of the HIV reverse transcriptase inhibitor tenofovir, in HIV-infected adults

Tenofovir alafenamide (formerly GS-7340) is a new oral prodrug of tenofovir, a nucleotide analogue that inhibits HIV-1 reverse transcription. Unlike the currently marketed tenofovir prodrug, tenofovir disoproxil fumarate, tenofovir alafenamide is stable in plasma and then rapidly converted into tenofovir once inside cells. The pharmacokinetics, safety and antiviral activity of 40 or 120 mg of tenofovir alafenamide compared with 300 mg of tenofovir disoproxil fumarate when administered as monotherapy once daily for 14 days in HIV-1-infected, treatment-naive subjects was studied. Administration of 40 mg of tenofovir alafenamide for 14 days resulted in lower tenofovir Cmax (13 versus 207 ng/mL) and lower systemic exposures (AUC0-t, 383 versus 1810 ng · h/mL) compared with subjects who received tenofovir disoproxil fumarate. There were higher intracellular tenofovir concentrations within peripheral blood mononuclear cells with both 40 mg of tenofovir alafenamide (8.2 μM) and 120 mg of tenofovir alafenamide (16.9 μM) compared with 300 mg of tenofovir disoproxil fumarate (0.9 μM). The most commonly observed adverse events were headache, nausea and flatulence, which occurred similarly across the three groups. After 14 days, the mean changes in HIV-1 RNA were -0.94 log₁₀copies/mL for the tenofovir disoproxil fumarate group, -1.57 log₁₀ copies/mL for the 40 mg of tenofovir alafenamide group and -1.71 log₁₀ copies/mL for the 120 mg of tenofovir alafenamide group. The mean first-phase HIV-1 RNA decay slopes were -0.36, -0.63 and -0.64 for the tenofovir disoproxil fumarate group, the 40 mg of tenofovir alafenamide group and the 120 mg of tenofovir alafenamide group, respectively. No resistance mutations to either tenofovir alafenamide or tenofovir disoproxil fumarate were detected. Tenofovir alafenamide, a new once-daily oral prodrug of tenofovir, showed more potent anti-HIV-1 activity and higher intracellular tenofovir levels compared with tenofovir disoproxil fumarate, while maintaining lower plasma tenofovir exposure at 40 mg with good tolerability over 14 days of monotherapy.

The cellular antiviral protein APOBEC3G interacts with HIV-1 reverse transcriptase and inhibits its function during viral replication.

The cytidine deaminase APOBEC3G (A3G) exerts a multifaceted antiviral effect against HIV-1 infection. First, A3G was shown to be able to terminate HIV infection by deaminating the cytosine residues to uracil in the minus strand of the viral DNA during reverse transcription. Also, a number of studies have indicated that A3G inhibits HIV-1 reverse transcription by a non-editing-mediated mechanism. However, the mechanism by which A3G directly disrupts HIV-1 reverse transcription is not fully understood. In the present study, by using a cell-based coimmunoprecipitation (Co-IP) assay, we detected the direct interaction between A3G and HIV-1 reverse transcriptase (RT) in produced viruses and in the cotransfected cells. The data also suggested that their interaction did not require viral genomic RNA bridging or other viral proteins. Additionally, a deletion analysis showed that the RT-binding region in A3G was located between amino acids 65 and 132. Overexpression of the RT-binding polypeptide A3G(65-132) was able to disrupt the interaction between wild-type A3G and RT, which consequently attenuated the anti-HIV effect of A3G on reverse transcription. Overall, this paper provides evidence for the physical and functional interaction between A3G and HIV-1 RT and demonstrates that this interaction plays an important role in the action of A3G against HIV-1 reverse transcription.

Ribonucleoside Triphosphates as Substrate of Human Immunodeficiency Virus Type 1 Reverse Transcriptase in Human Macrophages

We biochemically simulated HIV-1 DNA polymerization in physiological nucleotide pools found in two HIV-1 target cell types: terminally differentiated/non-dividing macrophages and activated/dividing CD4+ T cells. Quantitative tandem mass spectrometry shows that macrophages harbor 22–320-fold lower dNTP concentrations and a greater disparity between ribonucleoside triphosphate (rNTP) and dNTP concentrations than dividing target cells. A biochemical simulation of HIV-1 reverse transcription revealed that rNTPs are efficiently incorporated into DNA in the macrophage but not in the T cell environment. This implies that HIV-1 incorporates rNTPs during viral replication in macrophages and also predicts that rNTP chain terminators lacking a 3′-OH should inhibit HIV-1 reverse transcription in macrophages. Indeed, 3′-deoxyadenosine inhibits HIV-1 proviral DNA synthesis in human macrophages more efficiently than in CD4+ T cells. This study reveals that the biochemical landscape of HIV-1 replication in macrophages is unique and that ribonucleoside chain terminators may be a new class of anti-HIV-1 agents specifically targeting viral macrophage infection.

Mechanisms of inhibition of HIV replication by non-nucleoside reverse transcriptase inhibitors

The non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are a therapeutic class of compounds that are routinely used, in combination with other antiretroviral drugs, to treat HIV-1 infection. NNRTIs primarily block HIV-1 replication by preventing RT from completing reverse transcription of the viral single-stranded RNA genome into DNA. However, some NNRTIs, such as efavirenz, have been shown to inhibit the late stages of HIV-1 replication by interfering with HIV-1 Gag-Pol polyprotein processing, while others, such as the pyrimidinediones, have been shown to inhibit both HIV-1 RT-mediated reverse transcription and HIV-1/HIV-2 viral entry. Accordingly, in this review we describe the multiple mechanisms by which NNRTIs inhibit HIV-1 reverse transcription (and in some cases HIV-2 reverse transcription) and other key steps involved in HIV-1/HIV-2 replication.

Deaminase-independent inhibition of HIV-1 reverse transcription by APOBEC3G

APOBEC3G (A3G), a host protein that inhibits HIV-1 reverse transcription and replication in the absence of Vif, displays cytidine deaminase and single-stranded (ss) nucleic acid binding activities. HIV-1 nucleocapsid protein (NC) also binds nucleic acids and has a unique property, nucleic acid chaperone activity, which is crucial for efficient reverse transcription. Here we report the interplay between A3G, NC and reverse transcriptase (RT) and the effect of highly purified A3G on individual reactions that occur during reverse transcription. We find that A3G did not affect the kinetics of NC-mediated annealing reactions, nor did it inhibit RNase H cleavage. In sharp contrast, A3G significantly inhibited all RT-catalyzed DNA elongation reactions with or without NC. In the case of (−) strong-stop DNA synthesis, the inhibition was independent of A3G's catalytic activity. Fluorescence anisotropy and single molecule DNA stretching analyses indicated that NC has a higher nucleic acid binding affinity than A3G, but more importantly, displays faster association/disassociation kinetics. RT binds to ssDNA with a much lower affinity than either NC or A3G. These data support a novel mechanism for deaminase-independent inhibition of reverse transcription that is determined by critical differences in the nucleic acid binding properties of A3G, NC and RT.

Conformational Changes in HIV-1 Reverse Transcriptase Induced by Nonnucleoside Reverse Transcriptase Inhibitor Binding

Nonnucleoside reverse transcriptase inhibitors (NNRTI) are a group of small hydrophobic compounds with diverse structures that specifically inhibit HIV-1 reverse transcriptase (RT). NNRTIs interact with HIV-1 RT by binding to a single site on the p66 subunit of the p66/p51 heterodimeric enzyme, termed the NNRTI-binding pocket (NNRTI-BP). This binding interaction results in both short-range and long-range distortions of RT structure. In this article, we review the structural, computational and experimental evidence of the NNRTI-induced conformational changes in HIV-1 RT and relate them to the mechanism by which these compounds inhibit HIV-1 reverse transcription.

Nucleotides, Part LIX, Synthesis, Characterization, and Biological Activities of New Potential Antiviral Agents: (2′ - 5′)Adenylate Trimer Analogs Containing 3′-Deoxy-3′-(hexadecanoylamino)adenosine at the 2′-Terminus

Based upon 3′-amino-3′-deoxyadenosine (15), its protected 3′-hexadecanoylamino derivative 22 was chosen as starting material for the synthesis of a series of new modified 2′ – 5′-adenylate trimers 33 – 36 as potential antiviral agents. All (2′ – 5′)A trimer analogs 33 – 36 inhibit HIV-1 replication as measured by the inhibition of syncytia formation and inhibition of HIV-1 reverse transcriptase activity. Compound 34 inhibits HIV-1 reverse transcription by 100% and subsequently inhibits expression of HIV-1 p24. However, compound 35 acts differently, since it does not inhibit HIV-1 reverse transcription, HIV-1 integrase, or HIV-1 p24 expression. Therefore, 35 appears to exert its inhibitory effect at a later stage of HIV-1 replication, i.e., the budding process.

Inhibition of HIV type 1 production by hygromycin B.

HIV infection alters the cellular uptake of ions and other small molecules. This study was designed to determine whether hygromycin B, a low molecular weight (MW 527) aminoglycoside protein synthesis inhibitor that is normally impermeable to mammalian cells at micromolar concentrations, can selectively inhibit HIV expression and cytopathology. CD4+ T lymphoblastoid cells (H9) and peripheral blood mononuclear cells (PBMCs) were infected with HIV-1, then incubated in medium containing various concentrations of hygromycin B. HIV-1-induced formation of multinucleated giant cells and single cell killing were dramatically reduced in the presence of micromolar concentrations of hygromycin B. Hygromycin B also inhibited HIV-1 production in a dose-dependent manner during acute infection. G418, a larger and more hydrophobic aminoglycoside (MW 692), did not display the same selective inhibition of HIV-1 production as hygromycin B. Relative to mock-infected cells, protein synthesis in acutely infected H9 cells was selectively inhibited by hygromycin B. Hygromycin B also reduced HIV production in PBMCs and in H9 cells persistently infected with HIV. PCR analysis demonstrated that hygromycin B did not inhibit HIV-1 reverse transcription. These results demonstrate that HIV-1 infection renders cells more sensitive to hygromycin B than uninfected cells, and provides support for the hypothesis that HIV-1 induces an alteration of plasma membrane permeability. The HIV-modified cell membrane may be a potential target for antiviral intervention and chemotherapy.