Inhibition Of Reverse Transcription Research Articles

ID: 26: PML-dependent inhibition of HIV-1 reverse-transcription by Daxx

PML, also known as TRIM19, belongs to the family of tripartite motif (TRIM) proteins. Like some other TRIM proteins, PML exerts a broad antiviral activity. PML is mainly expressed in the nucleus, where it forms dynamic structures known as PML nuclear bodies that recruit many proteins, such as Sp100 and Daxx. It was reported that HIV-1 infection induces the translocation of PML from the nucleus to the cytoplasm (Turelli et al., 2001). This paper also suggested that PML could interfere with HIV-1 infection but this was later contradicted (Berthoux et al., 2003). We sought to re-evaluate the implication of PML during HIV-1 infection. We show that infection by HIV-1 but also other retroviruses triggers the formation of PML cytoplasmic bodies, as early as 30 min post-infection. PML re-localization was blocked by reverse-transcription inhibitors and was not observed following infection with unrelated viruses, suggesting it is specifically triggered by retroviral reverse-transcription. Furthermore, we show that PML interferes with an early step of retroviral infection since PML knockdown dramatically increases RT efficiency. However, the effect of PML on retroviral infection was found to be indirect and to operate through the stabilization of one of its well-characterized partners, Daxx. In the presence of PML, Daxx inhibits an early step of reverse-transcription thereby interfering with HIV-1 infection. Like PML, Daxx is an interferon-inducible protein that provides intrinsic immunity against various viruses. Our study unravels for the first time its capacity to inhibit an early step of HIV-1 replication.

A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication

The process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting the in vitro data. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis. Reverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting the in vitro data. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.

The triple threat of HIV-1 protease inhibitors.

Newly released human immunodeficiency virus type 1 (HIV-1) particles obligatorily undergo a maturation process to become infectious. The HIV-1 protease (PR) initiates this step, catalyzing the cleavage of the Gag and Gag-Pro-Pol structural polyproteins. Proper organization of the mature virus core requires that cleavage of these polyprotein substrates proceeds in a highly regulated, specific series of events. The vital role the HIV-1 PR plays in the viral life cycle has made it an extremely attractive target for inhibition and has accordingly fostered the development of a number of highly potent substrate-analog inhibitors. Though the PR inhibitors (PIs) inhibit only the HIV-1 PR, their effects manifest at multiple different stages in the life cycle due to the critical importance of the PR in preparing the virus for these subsequent events. Effectively, PIs masquerade as entry inhibitors, reverse transcription inhibitors, and potentially even inhibitors of post-reverse transcription steps. In this chapter, we review the triple threat of PIs: the intermolecular cooperativity in the form of a cooperative dose-response for inhibition in which the apparent potency increases with increasing inhibition; the pleiotropic effects of HIV-1 PR inhibition on entry, reverse transcription, and post-reverse transcription steps; and their potency as transition state analogs that have the potential for further improvement that could lead to an inability of the virus to evolve resistance in the context of single drug therapy.

C3H strain of mouse mammary tumour virus, like GR strain, infects human mammary epithelial cells, albeit less efficiently than murine mammary epithelial cells.

Mouse mammary tumour virus (MMTV) is a member of the genus Betaretrovirus, infects rodent cells and uses mouse tranferrin receptor 1 for cell entry. Several MMTV strains have been shown to productively infect, in addition to murine cells, various heterologous cell lines including those of human origin, albeit less efficiently than murine cells. Here, we analysed whether MMTV from C3H mice [MMTV(C3H)], reported previously to be incapable of infecting human cells, could productively infect human cells. Using a recently described high-titre MMTV-based vector carrying MMTV(C3H) envelope protein (Env), we successfully transduced cells of human origin. Furthermore, WT MMTV(C3H) was able to infect human cells, albeit less efficiently than mouse cells. The established infection was, however, sufficient to enable virus spread to every cell in culture. The infectivity of WT MMTV(C3H) and MMTV-based vectors carrying MMTV(C3H)Env was blocked by heat inactivation, an inhibitor of reverse transcription (3'-azido-3'-deoxythymidine) and pre-incubation with neutralizing anti-MMTV antibodies that did not neutralize vectors pseudotyped with amphotropic murine leukemia virus Env, providing evidence for an authentic, receptor-mediated and reverse transcriptase-dependent infection process. Persistently infected human Hs578T cells produced infectious virions capable of infecting naïve human breast cells in culture, the infectivity of which could also be blocked by neutralizing anti-MMTV antibodies, demonstrating that virus particles released by the persistently infected Hs578T cells were related antigenically to the virus produced from murine cells. Taken together, our results show that MMTV(C3H), like MMTV(GR) and MMTV(RIII), is able not only to infect but also to replicate in cultured human breast cells.

Identification of capsid mutations that alter the rate of HIV-1 uncoating in infected cells.

After viral fusion with the cell membrane, the conical capsid of HIV-1 disassembles by a process called uncoating. We recently utilized the cyclosporine (CsA) washout assay, in which TRIM-CypA-mediated restriction of viral replication is used to detect the state of the viral capsid, to study the kinetics of uncoating in HIV-1-infected cells. Here we have extended this analysis to examine the effects of p24 capsid protein (p24(CA)) mutations and cellular environment on the kinetics of uncoating in infected cells. We found that p24(CA) mutations can significantly increase (A92E), delay (E45A and N74D), or have no effect (G94D) on the rate of uncoating and that these alterations are not due to changes in reverse transcription. Inhibition of reverse transcription delayed uncoating kinetics to an extent similar to that of the wild-type virus with all the p24(CA) mutant viruses tested. In addition, we observed differences in uncoating in two cell lines, which suggests that the cellular environment can differentially impact the disassembly of wild-type and mutant capsids. Collectively, these experiments suggest that viral and cellular factors are important for the process of uncoating. Finally, these data support the model whereby early steps in reverse transcription facilitate HIV-1 uncoating. The HIV-1 capsid is a cone-shaped structure, composed of the HIV-1-encoded protein p24(CA), which contains the viral RNA and other proteins needed for infection. After the virus enters a target cell, this capsid must disassemble by a process called uncoating. Uncoating is required for HIV-1 infection to progress, but the details of how this process occurs is not known. In this study, we used an in vivo assay to examine the uncoating process in HIV-1-infected cells. We determined that p24(CA) mutations could increase or decrease the rate of uncoating and that this rate varied in different cell lines. We also found that reverse transcription of the viral RNA altered the process of uncoating before the p24(CA) mutations. Collectively, these experiments provide a better understanding of how viral and cellular factors are involved with a poorly understood step in HIV-1 infection.

Selective binding and reverse transcription inhibition of single-strand poly(A) RNA by metal TMPyP complexes.

Ni-, Cu-, and Zn-TMPyP are capable of binding to single-strand poly(A) RNA with high preference and affinity and inhibiting the reverse transcription of RNA by both M-MuLV and HIV-1 reverse transcriptase. With 10 nM azidothymidine, the IC50 value of M-TMPyP could be lowered to 10(-1) μM order.

The allosteric HIV-1 integrase inhibitor BI-D affects virion maturation but does not influence packaging of a functional RNA genome.

The viral integrase (IN) is an essential protein for HIV-1 replication. IN inserts the viral dsDNA into the host chromosome, thereby aided by the cellular co-factor LEDGF/p75. Recently a new class of integrase inhibitors was described: allosteric IN inhibitors (ALLINIs). Although designed to interfere with the IN-LEDGF/p75 interaction to block HIV DNA integration during the early phase of HIV-1 replication, the major impact was surprisingly found on the process of virus maturation during the late phase, causing a reverse transcription defect upon infection of target cells. Virus particles produced in the presence of an ALLINI are misformed with the ribonucleoprotein located outside the virus core. Virus assembly and maturation are highly orchestrated and regulated processes in which several viral proteins and RNA molecules closely interact. It is therefore of interest to study whether ALLINIs have unpredicted pleiotropic effects on these RNA-related processes. We confirm that the ALLINI BI-D inhibits virus replication and that the produced virus is non-infectious. Furthermore, we show that the wild-type level of HIV-1 genomic RNA is packaged in virions and these genomes are in a dimeric state. The tRNAlys3 primer for reverse transcription was properly placed on this genomic RNA and could be extended ex vivo. In addition, the packaged reverse transcriptase enzyme was fully active when extracted from virions. As the RNA and enzyme components for reverse transcription are properly present in virions produced in the presence of BI-D, the inhibition of reverse transcription is likely to reflect the mislocalization of the components in the aberrant virus particle.

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.

Human immature Langerhans cells restrict CXCR4-using HIV-1 transmission.

BackgroundSexual transmission is the main route of HIV-1 infection and the CCR5-using (R5) HIV-1 is predominantly transmitted, even though CXCR4-using (X4) HIV-1 is often abundant in chronic HIV-1 patients. The mechanisms underlying this tropism selection are unclear. Mucosal Langerhans cells (LCs) are the first immune cells to encounter HIV-1 and here we investigated the role of LCs in selection of R5 HIV-1 using an ex vivo epidermal and vaginal transmission models.ResultsImmature LCs were productively infected by X4 as well as R5 HIV-1. However, only R5 but not X4 viruses were selectively transmitted by immature LCs to T cells. Transmission of HIV-1 was depended on de novo production of HIV-1 in LCs, since it could be inhibited by CCR5 fusion inhibitors as well as reverse transcription inhibitors. Notably, the activation state of LCs affected the restriction in X4 HIV-1 transmission; immune activation by TNF facilitated transmission of X4 as well as R5 HIV-1.ConclusionsThese data suggest that LCs play a crucial role in R5 selection and that immature LCs effectively restrict X4 at the level of transmission.

Understanding the Etiology and Management of HIV-Associated Peripheral Neuropathy

HIV may cause several forms of peripheral neuropathy, the most common of which is distal symmetric polyneuropathy (DSP) characterized by pain and sensory deficits in a stocking-glove distribution. The pathophysiology of DSP remains largely unknown but is thought to be related both to the neurotoxicity of HIV-through indirect immunomodulatory mechanisms-and to the neurotoxic effects of anti-retroviral therapies, most notably the dideoxynucleoside reverse transcription inhibitors or so-called d-drugs. Determining whether symptoms arise from the virus or the treatment poses a challenge to the clinician who must decide if a patient's HAART regimen should be altered. Treatment of symptoms related to HIV-DSP is a difficult task and there is no evidence that the traditional agents used in chronic neuropathic pain are efficacious in the HIV-DSP population. Indeed few pharmacologic agents have proven efficacy in HIV-DSP - these include cannabis and the capsaicin 8 % dermal patch. As such, alternative, non-pharmacologic therapies are being investigated. More research is needed to further elucidate the complex pathophysiology of HIV-DSP which may yield additional therapies for these patients.

Longer duration of combination antiretroviral therapy reduces the risk of Hodgkin lymphoma: A cohort study of HIV-infected male veterans

BackgroundHodgkin lymphoma (HL) incidence has increased since combined antiretroviral therapy (cART) introduction. It is unclear how different cART classes (e.g., protease inhibitors (PI), non-nucleoside reverse transcription inhibitors (NNRTI)) influence HL. This study aimed to determine the effects of cART duration on HL incidence among HIV-infected veterans. MethodsWe performed a retrospective cohort study utilizing the Veterans Affairs HIV Clinical Case Registry (1985–2010). HL cases were identified using ICD-9 codes (201.4-9). cART, PI, and NNRTI duration was the aggregate number of treatment days delivered. Incidence rates (IR) and rate ratios (IRR) were calculated from Poisson regression models to examine the effects of cART duration on HL. Results31,576 cART users contributed 288,736 person-years (PY) and 211 HL cases (IR=7.3/10,000 person-years). HL incidence decreased from 25.1/10,000 PY (95%CI=18.9–33.4) within the first year of cART to 0.6/10,000 PY (95%CI=0.3–1.6) after ≥10 years. In multivariable models, each additional year of cART was associated with decreased HL incidence (IRR=0.80; 95%CI=0.75–0.86); similar effects were observed in models assessing HL incidence by PI and NNRTI. ConclusionOur findings indicate long-term cART of any class is associated with decreased HL risk. High HL incidence directly following cART initiation supports a potential immune reconstitution mechanism in HIV-related HL. Further research is needed to evaluate the interaction between early cART, immune reconstitution, and HL.

A cell-intrinsic inhibitor of HIV-1 reverse transcription in CD4(+) T cells from elite controllers.

HIV-1 reverse transcription represents the predominant target for pharmacological inhibition of viral replication, but cell-intrinsic mechanisms that can block HIV-1 reverse transcription in a clinically significant way are poorly defined. We find that effective HIV-1 reverse transcription depends on the phosphorylation of viral reverse transcriptase by host cyclin-dependent kinase (CDK) 2 at a highly conserved Threonine residue. CDK2-dependent phosphorylation increased the efficacy and stability of viral reverse transcriptase and enhanced viral fitness. Interestingly, p21, a cell-intrinsic CDK inhibitor that is upregulated in CD4(+) T cells from "elite controllers," potently inhibited CDK2-dependent phosphorylation of HIV-1 reverse transcriptase and significantly reduced the efficacy of viral reverse transcription. These data suggest that p21 can indirectly block HIV-1 reverse transcription by inhibiting host cofactors supporting HIV-1 replication and identify sites of viral vulnerability that are effectively targeted in persons with natural control of HIV-1 replication.

Tenofovir Effect on the Kidneys of HIV-Infected Patients

Tenofovir disoproxil fumarate (TDF), the first nucleotidic inhibitor of HIV reverse transcription, became available in 2001. It has been extensively used worldwide and is now the most prescribed antiretroviral (ARV) drug. Its high antiviral activity and favorable metabolic profile are responsible for its success. Furthermore, TDF has been associated with other ARVs to form new combined antiretroviral treatments in only one tablet once-a-day, which increases treatment adherence. Fears of potential nephrotoxicity that tenofovir would have in common with two other drugs from the same family (adefovir, used to treat hepatitis B, and cidofovir, used to treat cytomegalovirus infections) were alleviated by the early clinical trials. Yet, in 2001, the first case of TDF-induced acute nephrotoxicity was published. Numerous cases have been published since then, and it is now established that TDF presents a tubular toxicity risk. Some facilitating factors have been identified, such as co-prescription of didanosine or boosted protease inhibitor, preexisting CKD, low body weight, and associated diabetes mellitus. Conversely, whether TDF is nephrotoxic in the long term is a highly debated question. Some studies suggest a decreased GFR when TDF is prescribed for a long period, while others indicate that TDF is safe for the kidneys even after many years of use. Here we review the differences in patient characteristics, study designs, and measured outcomes that can possibly explain these conflicting findings. We conclude with rational recommendation for appropriate TDF prescription.

HIV-1 infection induces a type I IFN response in primary CD4+ T cells

Background: Production of type 1 interferon (IFN) in response to viral infection requires the detection of viral nucleic acids or proteins by at least one cellular pattern recognition receptor. HIV-1 is capable of inducing an elaborate IFN response in plasmacytoid dendritic cells (pDCs) through Toll-like receptor mediated recognition of the entering viral RNA genome. However in T cells and macrophages, the main HIV target cells, IFN induction by HIV-1 is considered to be weak or undetectable. Here, we re-evaluate the occurrence of an innate immune response upon HIV-1 infection of primary CD4+ T cells (PBLs). Methods and results: Type 1 IFN induction was evaluated in activated purified PBLs during productive infection with HIV-1. In cells from several donors we observed a clear increase in IFNβ and IFNα mRNA levels, as well as induction of several interferon stimulated genes (ISGs) upon HIV-1 infection. The levels of induction progressed concurrently with the levels of HIV infection in the culture. Secreted type 1 IFN protein biological activity was furthermore detected in supernatants of HIV-1 infected cultures. To rule out residual contaminating pDCs as the source of IFN production in these cultures, we performed additional depletion of these cells from CD4+ T cell populations prior to infection and found that type 1 IFN induction was maintained in the absence of pDCs. Furthermore, we evaluated the biological relevance of the detected levels of type 1 IFN by addition of neutralizing antibodies to IFNβ and IFNα during infection. In presence of the both antibodies, we observed an increase in HIV-1 infection, indicating that the levels of HIV-induced IFN in the T cell cultures are sufficient to have an antiviral effect. To gain more insight in the mechanism of IFN induction by HIV-1, we used inhibitors of reverse transcription, integration and of the HIV-1 protease during single-cycle infection of VSV-pseudotyped HIV-1. We found that integration of the HIV provirus is required for full IFN induction in PBLs, indicating that newly expressed HIV RNA or newly produced HIV proteins are important for evoking an innate immune response in T cells. Conclusion: These data show that activated PBLs are capable of producing relevant levels of type 1 IFN in response to HIV-1 infection and suggest recognition of newly expressed HIV RNA or proteins as a main trigger of the innate response in these cells. Characterization of the responsible innate immune pathway and pattern recognition receptors will be subject of further investigation.

Evidence for biphasic uncoating during HIV-1 infection from a novel imaging assay

BackgroundUncoating of the HIV-1 core plays a critical role during early post-fusion stages of infection but is poorly understood. Microscopy-based assays are unable to easily distinguish between intact and partially uncoated viral cores.ResultsIn this study, we used 5-ethynyl uridine (EU) to label viral-associated RNA during HIV production. At early time points after infection with EU-labeled virions, the viral-associated RNA was stained with an EU-specific dye and was detected by confocal microscopy together with viral proteins. We observed that detection of the viral-associated RNA was specific for EU-labeled virions, was detected only after viral fusion with target cells, and occurred after an initial opening of the core. In vitro staining of cores showed that the opening of the core allowed the small molecule dye, but not RNase A or antibodies, inside. Also, staining of the viral-associated RNA, which is co-localized with nucleocapsid, decays over time after viral infection. The decay rate of RNA staining is dependent on capsid (CA) stability, which was altered by CA mutations or a small molecule inducer of HIV-1 uncoating. While the staining of EU-labeled RNA was not affected by inhibition of reverse transcription, the kinetics of core opening of different CA mutants correlated with initiation of reverse transcription. Analysis of the E45A CA mutant suggests that initial core opening is independent of complete capsid disassembly.ConclusionsTaken together, our results establish a novel RNA accessibility-based assay that detects an early event in HIV-1 uncoating and can be used to further define this process.

Constant drug dose in human immuno‐deficiency virus‐infected patients to induce long‐term non‐progressor status: bifurcation and controllability approach

The authors propose a therapy consisting of a constant dosage of reverse transcription inhibitor and protease inhibitor to achieve long-term non-progressor (LTNP) status in human immuno-deficiency virus (HIV) patients. Based on the authors analyses of cytotoxic T lymphocyte precursor (CTLp) concentration at several equilibrium points and the bifurcation of these equilibrium points, they find that administration of drugs with an efficacy lower than a certain level induces a higher CTLp concentration. As a result, drug doses of moderate efficacy result in more patients with LTNP status than doses with high efficacy. In analyses of controllability, they show that a treatment of moderate efficacy is more efficient than one of very high efficacy in terms of controlling the immune system. Using simulations, they demonstrate that their proposed method results in LTNP status in HIV patients.

A combination of provirus stimulants with blockers of regulators of complement activation represents a novel approach for purging HIV-1 latently infected cells (P6342)

Abstract HIV-1 latently infected cells are recognized as the last barrier towards viral eradication and cure. To purge these latently infected cells, we developed an “activation-killing” approach by combining provirus stimulants (SAHA, prostratin or both) with a blocker of human CD59, a key member of regulators of complement activation (RCA), to facilitate anti-HIV-1 antibodies to trigger antibody-dependent complement-mediated lysis (ADCML) of latently-infected cells. We found that both SAHA and prostratin activated proviruses in ACH-2 cells, a well-characterized cellular model of HIV-1 latency, resulting in expression of HIV-1 gp120 on the cell surface. To prevent new rounds of infections by virus released from the stimulated cells, we treated ACH-2 cells with provirus stimulants plus antiretroviral drugs. We found that reverse transcription inhibitors had no effect on virion production, whereas protease inhibitors (PIs) significantly reduced the production of mature virions, but not gp120 expression on the cell surface. ACH-2 cells post-activation of provirus became sensitive to ADCML in the presence of a human CD59 blocker plus anti-HIV-1 gp120 antibodies or sera from HIV-1-infected patients. The lysis resulted from specific cell killing by anti-HIV-1 antibodies because the lysis did not occur using irrelevant antibodies or inactivated complement. Thus, a combination of provirus stimulants with RCA blockers represents a novel approach for purging HIV-1 latently infected cells.

APOBEC3 Inhibition of Mouse Mammary Tumor Virus Infection: the Role of Cytidine Deamination versus Inhibition of Reverse Transcription

The apolipoprotein B editing complex 3 (APOBEC3) family of proteins is a group of intrinsic antiviral factors active against a number of retroviral pathogens, including HIV in humans and mouse mammary tumor virus (MMTV) in mice. APOBEC3 restricts its viral targets through cytidine deamination of viral DNA during reverse transcription or via deaminase-independent means. Here, we used virions from the mammary tissue of MMTV-infected inbred wild-type mice with different allelic APOBEC3 variants (APOBEC3(BALB) and APOBEC3(BL/6)) and knockout mice to determine whether cytidine deamination was important for APOBEC3's anti-MMTV activity. First, using anti-murine APOBEC3 antiserum, we showed that both APOBEC3 allelic variants are packaged into the cores of milk-borne virions produced in vivo. Next, using an in vitro deamination assay, we determined that virion-packaged APOBEC3 retains its deamination activity and that allelic differences in APOBEC3 affect the sequence specificity. In spite of this in vitro activity, cytidine deamination by virion-packaged APOBEC3 of MMTV early reverse transcription DNA occurred only at low levels. Instead, the major means by which in vivo virion-packaged APOBEC3 restricted virus was through inhibition of early reverse transcription in both cell-free virions and in vitro infection assays. Moreover, the different wild-type alleles varied in their ability to inhibit this step. Our data suggest that while APOBEC3-mediated cytidine deamination of MMTV may occur, it is not the major means by which APOBEC3 restricts MMTV infection in vivo. This may reflect the long-term coexistence of MMTV and APOBEC3 in mice.

RNA-mediated interference and reverse transcription control the persistence of RNA viruses in the insect model Drosophila

How persistent viral infections are established and maintained is widely debated and remains poorly understood. We found here that the persistence of RNA viruses in Drosophila melanogaster was achieved through the combined action of cellular reverse-transcriptase activity and the RNA-mediated interference (RNAi) pathway. Fragments of diverse RNA viruses were reverse-transcribed early during infection, which resulted in DNA forms embedded in retrotransposon sequences. Those virus-retrotransposon DNA chimeras produced transcripts processed by the RNAi machinery, which in turn inhibited viral replication. Conversely, inhibition of reverse transcription hindered the appearance of chimeric DNA and prevented persistence. Our results identify a cooperative function for retrotransposons and antiviral RNAi in the control of lethal acute infection for the establishment of viral persistence.

Identification of low molecular weight nuclear complexes containing integrase during the early stages of HIV-1 infection

BackgroundHIV-1 replication requires integration of its reverse transcribed viral cDNA into a host cell chromosome. The DNA cutting and joining reactions associated to this key step are catalyzed by the viral protein integrase (IN). In infected cells, IN binds the viral cDNA, together with viral and cellular proteins, to form large nucleoprotein complexes. However, the dynamics of IN complexes formation is still poorly understood.ResultsHere, we characterized IN complexes during the early stages of T-lymphocyte infection. We found that following viral entry into the host cell, IN was rapidly targeted to proteasome-mediated degradation. Interactions between IN and cellular cofactors LEDGF/p75 and TNPO3 were detected as early as 6 h post-infection. Size exclusion chromatography of infected cell extracts revealed distinct IN complexes in vivo. While at 2 h post-infection the majority of IN eluted within a high molecular weight complex competent for integration (IN complex I), IN was also detected in a low molecular weight complex devoid of full-length viral cDNA (IN complex II, ~440 KDa). At 6 h post-infection the relative proportion of IN complex II increased. Inhibition of reverse transcription or integration did not alter the elution profile of IN complex II in infected cells. However, in cells depleted for LEDGF/p75 IN complex II shifted to a lower molecular weight complex (IN complex III, ~150 KDa) containing multimers of IN. Notably, cell fractionation experiments indicated that both IN complex II and III were exclusively nuclear. Finally, IN complex II was not detected in cells infected with a virus harboring a mutated IN defective for LEDGF/p75 interaction and tetramerization.ConclusionsOur findings indicate that, shortly after viral entry, a significant portion of DNA–free IN that is distinct from active pre-integration complexes accumulates in the nucleus.