Endogenous Reverse Transcription Research Articles

Internal RNA 2'-O-methylation on the HIV-1 genome impairs reverse transcription.

Viral RNA genomes are modified by epitranscriptomic marks, including 2'-O-methylation that is added by cellular or viral methyltransferases. 2'-O-Methylation modulates RNA structure, function and discrimination between self- and non-self-RNA by innate immune sensors such as RIG-I-like receptors. This is illustrated by human immunodeficiency virus type-1 (HIV-1) that decorates its RNA genome through hijacking the cellular FTSJ3 2'-O-methyltransferase, thereby limiting immune sensing and interferon production. However, the impact of such an RNA modification during viral genome replication is poorly understood. Here we show by performing endogenous reverse transcription on methylated or hypomethylated HIV-1 particles, that 2'-O-methylation negatively affects HIV-1 reverse transcriptase activity. Biochemical assays confirm that RNA 2'-O-methylation impedes reverse transcriptase activity, especially at low dNTP concentrations reflecting those in quiescent cells, by reducing nucleotide incorporation efficiency and impairing translocation. Mutagenesis highlights K70 as a critical amino acid for the reverse transcriptase to bypass 2'-O-methylation. Hence, the observed antiviral effect due to viral RNA 2'-O-methylation antagonizes the FTSJ3-mediated proviral effects, suggesting the fine-tuning of RNA methylation during viral replication.

The Host Cell Metabolite Inositol Hexakisphosphate Promotes Efficient Endogenous HIV-1 Reverse Transcription by Stabilizing the Viral Capsid.

A defining activity of retroviruses is reverse transcription, the process by which the viral genomic RNA is converted into the double-stranded DNA required for virus replication. Reverse transcriptase (RT), the viral enzyme responsible for this process, was identified in 1970 by assaying permeabilized retrovirus particles for DNA synthesis in vitro Such reactions are inefficient, with only a small fraction of viral genomes being converted to full-length double-stranded DNA molecules, possibly owing to disruption of the structure of the viral core. Here, we show that reverse transcription in purified HIV-1 cores is enhanced by the addition of the capsid-binding host cell metabolite inositol hexakisphosphate (IP6). IP6 potently enhanced full-length minus-strand synthesis, as did hexacarboxybenzene (HCB), which also stabilizes the HIV-1 capsid. Both IP6 and HCB stabilized the association of the viral CA and RT proteins with HIV-1 cores. In contrast to the wild type, cores isolated from mutant HIV-1 particles containing intrinsically hyperstable capsids exhibited relatively efficient reverse transcription in the absence of IP6, further indicating that the compound promotes reverse transcription by stabilizing the viral capsid. We also observed that the capsid-destabilizing antiviral compound PF74 inhibited endogenous reverse transcription with a potency that mirrors its ability to inhibit reverse transcription during infection. Our results show that the stabilization of the HIV-1 capsid permits efficient reverse transcription in HIV-1 cores, providing a sensitive experimental system for analyzing the functions of viral and host cell molecules and the role of capsid disassembly (uncoating) in the process.IMPORTANCE HIV-1 infection requires reverse transcription of the viral genome. While much is known about the biochemistry of reverse transcription from simplified biochemical reactions, reverse transcription during infection takes place within a viral core. However, endogenous reverse transcription reactions using permeabilized HIV-1 virions or purified viral cores have been inefficient. Using viral cores purified from infectious HIV-1 particles, we show that efficient reverse transcription is achieved in vitro by addition of the capsid-stabilizing metabolite inositol hexakisphosphate. The enhancement of reverse transcription was linked to the capsid-stabilizing effect of the compound, consistent with the known requirement for an intact or semi-intact viral capsid for HIV-1 infection. Our results establish a biologically relevant system for dissecting the function of the viral capsid and its disassembly during reverse transcription. The system should also prove useful for mechanistic studies of capsid-targeting antiviral drugs.

EEF1A demonstrates paralog specific effects on HIV-1 reverse transcription efficiency

The eukaryotic translation elongation factor 1A (eEF1A) has two cell-type specific paralogs, eEF1A1 and eEF1A2. Both paralogs undertake a canonical function in delivering aminoacyl-tRNA to the ribosome for translation, but differences in other functions are emerging. eEF1A1 has been reported to be important for the replication of many viruses, but no study has specifically linked the eEF1A2 paralog. We have previously demonstrated that eEF1A1 directly interacts with HIV-1 RT and supports efficient reverse transcription. Here, we showed that RT interacted more strongly with eEF1A1 than with eEF1A2 in immunoprecipitation assay. Biolayer interferometry using eEF1A paralogs showed different association and dissociation rates with RT. Over expressed eEF1A1, but not eEF1A2, was able to restore HIV-1 reverse transcription efficiency in HEK293T cells with endogenous eEF1A knocked-down and HIV-1 reverse transcription efficiency correlated with the level of eEF1A1 mRNA, but not to eEF1A2 mRNA in both HEK293T and primary human skeletal muscle cells.

Host SAMHD1 protein restricts endogenous reverse transcription of HIV-1 in nondividing macrophages

BackgroundSAM domain and HD domain containing protein 1 (SAMHD1) is a host anti-HIV-1 restriction factor known to suppress viral reverse transcription in nondividing myeloid cells by its dNTP triphosphorylase activity that depletes cellular dNTPs. However, HIV-2 and some SIV strains rapidly replicate in macrophages due to their accessory protein, viral protein X (Vpx), which proteosomally degrades SAMHD1 and elevates dNTP levels. Endogenous reverse transcription (ERT) of retroviruses is the extra-cellular reverse transcription step that partially synthesizes proviral DNAs within cell-free viral particles before the viruses infect new cells. ERT activity utilizes dNTPs co-packaged during budding from the virus-producing cells, and high ERT activity is known to enhance HIV-1 infectivity in nondividing cells. Here, since Vpx elevates cellular dNTP levels in macrophages, we hypothesize that HIV-2 should contain higher ERT activity than HIV-1 in macrophages, and that the Vpx-mediated dNTP elevation should enhance both ERT activity and infectivity of HIV-1 particles produced in macrophages.ResultsHere, we demonstrate that HIV-2 produced from human primary monocyte derived macrophages displays higher ERT activity than HIV-1 produced from macrophages. Also, HIV-1 particles produced from macrophages treated with virus like particles (VLPs) containing Vpx, Vpx (+), displayed large increases of ERT activity with the enhanced copy numbers of early, middle and late reverse transcription products within the viral particles, compared to the viruses produced from macrophages treated with Vpx (−) VLPs. Furthermore, upon the infection with an equal p24 amount to fresh macrophages, the viruses produced from the Vpx (+) VLP treated macrophages demonstrated higher infectivity than the viruses from the Vpx (−) VLP treated macrophages.ConclusionsThis finding identifies the viral ERT step as an additional step of HIV-1 replication cycle that SAMHD1 restricts in nondividing myeloid target cells.

Sequence and structural determinants of human APOBEC3H deaminase and anti-HIV-1 activities.

BackgroundHuman APOBEC3H (A3H) belongs to the A3 family of host restriction factors, which are cytidine deaminases that catalyze conversion of deoxycytidine to deoxyuridine in single-stranded DNA. A3 proteins contain either one (A3A, A3C, A3H) or two (A3B, A3D, A3F, A3G) Zn-binding domains. A3H has seven haplotypes (I-VII) that exhibit diverse biological phenotypes and geographical distribution in the human population. Its single Zn-coordinating deaminase domain belongs to a phylogenetic cluster (Z3) that is different from the Z1- and Z2-type domains in other human A3 proteins. A3H HapII, unlike A3A or A3C, has potent activity against HIV-1. Here, we sought to identify the determinants of A3H HapII deaminase and antiviral activities, using site-directed sequence- and structure-guided mutagenesis together with cell-based, biochemical, and HIV-1 infectivity assays.ResultsWe have constructed a homology model of A3H HapII, which is similar to the known structures of other A3 proteins. The model revealed a large cluster of basic residues (not present in A3A or A3C) that are likely to be involved in nucleic acid binding. Indeed, RNase A pretreatment of 293T cell lysates expressing A3H was shown to be required for detection of deaminase activity, indicating that interaction with cellular RNAs inhibits A3H catalytic function. Similar observations have been made with A3G. Analysis of A3H deaminase substrate specificity demonstrated that a 5′ T adjacent to the catalytic C is preferred. Changing the putative nucleic acid binding residues identified by the model resulted in reduction or abrogation of enzymatic activity, while substituting Z3-specific residues in A3H to the corresponding residues in other A3 proteins did not affect enzyme function. As shown for A3G and A3F, some A3H mutants were defective in catalysis, but retained antiviral activity against HIV-1vif (−) virions. Furthermore, endogenous reverse transcription assays demonstrated that the E56A catalytic mutant inhibits HIV-1 DNA synthesis, although not as efficiently as wild type.ConclusionsThe molecular and biological activities of A3H are more similar to those of the double-domain A3 proteins than to those of A3A or A3C. Importantly, A3H appears to use both deaminase-dependent and -independent mechanisms to target reverse transcription and restrict HIV-1 replication.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0130-8) contains supplementary material, which is available to authorized users.

HIV-1-associated PKA acts as a cofactor for genome reverse transcription

BackgroundHost cell proteins, including cellular kinases, are embarked into intact HIV-1 particles. We have previously shown that the Cα catalytic subunit of cAMP-dependent protein kinase is packaged within HIV-1 virions as an enzymatically active form able to phosphorylate a synthetic substrate in vitro (Cartier et al. J. Biol. Chem. 278:35211 (2003)). The present study was conceived to investigate the contribution of HIV-1-associated PKA to the retroviral life cycle.ResultsNL4.3 viruses were produced from cells cultured in the presence of PKA inhibitors H89 (H89-NL4.3) or Myr-PKI (PKI-NL4.3) and analyzed for viral replication. Despite being mature and normally assembled, and containing expected levels of genomic RNA and RT enzymatic activity, such viruses showed poor infectivity. Indeed, infection generated reduced amounts of strong-strop minus strand DNA, while incoming RNA levels in target cells were unaffected. Decreased cDNA synthesis was also evidenced in intact H89-NL4.3 and PKI-NL4.3 cell free particles using endogenous reverse transcription (ERT) experiments. Moreover, similar defects were reproduced when wild type NL4.3 particles preincubated with PKA inhibitors were subjected to ERT reactions.ConclusionsAltogether, our results indicate that HIV-1-associated PKA is required for early reverse transcription of the retroviral genome both in cell free intact viruses and in target cells. Accordingly, virus-associated PKA behaves as a cofactor of an intraviral process required for optimal reverse transcription and for early post-entry events.

Murine leukemia virus glycosylated Gag blocks apolipoprotein B editing complex 3 and cytosolic sensor access to the reverse transcription complex

Pathogenic retroviruses have evolved multiple means for evading host restriction factors such as apolipoprotein B editing complex (APOBEC3) proteins. Here, we show that murine leukemia virus (MLV) has a unique means of counteracting APOBEC3 and other cytosolic sensors of viral nucleic acid. Using virus isolated from infected WT and APOBEC3 KO mice, we demonstrate that the MLV glycosylated Gag protein (glyco-Gag) enhances viral core stability. Moreover, in vitro endogenous reverse transcription reactions of the glyco-Gag mutant virus were substantially inhibited compared with WT virus, but only in the presence of APOBEC3. Thus, glyco-Gag rendered the reverse transcription complex in the viral core resistant to APOBEC3. Glyco-Gag in the virion also rendered MLV resistant to other cytosolic sensors of viral reverse transcription products in newly infected cells. Strikingly, glyco-Gag mutant virus reverted to glyco-Gag-containing virus only in WT and not APOBEC3 KO mice, indicating that counteracting APOBEC3 is the major function of glyco-Gag. Thus, in contrast to the HIV viral infectivity factor protein, which prevents APOBEC3 packaging in the virion, the MLV glyco-Gag protein uses a unique mechanism to counteract the antiviral action of APOBEC3 in vivo--namely, protecting the reverse transcription complex in viral cores from APOBEC3. These data suggest that capsid integrity may play a critical role in virus resistance to intrinsic cellular antiviral resistance factors that act at the early stages of infection.

Eukaryotic elongation factor 1 complex subunits are critical HIV-1 reverse transcription cofactors

Cellular proteins have been implicated as important for HIV-1 reverse transcription, but whether any are reverse transcription complex (RTC) cofactors or affect reverse transcription indirectly is unclear. Here we used protein fractionation combined with an endogenous reverse transcription assay to identify cellular proteins that stimulated late steps of reverse transcription in vitro. We identified 25 cellular proteins in an active protein fraction, and here we show that the eEF1A and eEF1G subunits of eukaryotic elongation factor 1 (eEF1) are important components of the HIV-1 RTC. eEF1A and eEF1G were identified in fractionated human T-cell lysates as reverse transcription cofactors, as their removal ablated the ability of active protein fractions to stimulate late reverse transcription in vitro. We observed that the p51 subunit of reverse transcriptase and integrase, two subunits of the RTC, coimmunoprecipitated with eEF1A and eEF1G. Moreover eEF1A and eEF1G associated with purified RTCs and colocalized with reverse transcriptase following infection of cells. Reverse transcription in cells was sharply down-regulated when eEF1A or eEF1G levels were reduced by siRNA treatment as a result of reduced levels of RTCs in treated cells. The combined evidence indicates that these eEF1 subunits are critical RTC stability cofactors required for efficient completion of reverse transcription. The identification of eEF1 subunits as unique RTC components provides a basis for further investigations of reverse transcription and trafficking of the RTC to the nucleus.

Blocking premature reverse transcription fails to rescue the HIV-1 nucleocapsid-mutant replication defect

BackgroundThe nucleocapsid (NC) protein of HIV-1 is critical for viral replication. Mutational analyses have demonstrated its involvement in viral assembly, genome packaging, budding, maturation, reverse transcription, and integration. We previously reported that two conservative NC mutations, His23Cys and His44Cys, cause premature reverse transcription such that mutant virions contain approximately 1,000-fold more DNA than wild-type virus, and are replication defective. In addition, both mutants show a specific defect in integration after infection.ResultsIn the present study we investigated whether blocking premature reverse transcription would relieve the infectivity defects, which we successfully performed by transfecting proviral plasmids into cells cultured in the presence of high levels of reverse transcriptase inhibitors. After subsequent removal of the inhibitors, the resulting viruses showed no significant difference in single-round infective titer compared to viruses where premature reverse transcription did occur; there was no rescue of the infectivity defects in the NC mutants upon reverse transcriptase inhibitor treatment. Surprisingly, time-course endogenous reverse transcription assays demonstrated that the kinetics for both the NC mutants were essentially identical to wild-type when premature reverse transcription was blocked. In contrast, after infection of CD4+ HeLa cells, it was observed that while the prevention of premature reverse transcription in the NC mutants resulted in lower quantities of initial reverse transcripts, the kinetics of reverse transcription were not restored to that of untreated wild-type HIV-1.ConclusionsPremature reverse transcription is not the cause of the replication defect but is an independent side-effect of the NC mutations.

Subtype-associated differences in HIV-1 reverse transcription affect the viral replication

BackgroundThe impact of the products of the pol gene, specifically, reverse transcriptase (RT) on HIV-1 replication, evolution, and acquisition of drug resistance has been thoroughly characterized for subtype B. For subtype C, which accounts of almost 60% of HIV cases worldwide, much less is known. It has been reported that subtype C HIV-1 isolates have a lower replication capacity than B; however, the basis of these differences remains unclear.ResultsWe analyzed the impact of the pol gene products from HIV-1 B and C subtypes on the maturation of HIV virions, accumulation of reverse transcription products, integration of viral DNA, frequency of point mutations in provirus and overall viral replication. Recombinant HIV-1 viruses of B and C subtypes comprising the pol fragments encoding protease, integrase and either the whole RT or a chimeric RT from different isolates of the C and B subtypes, were used for infection of cells expressing CXCR4 or CCR5 co-receptors. The viruses carrying different fragments of pol from the isolates of B and C subtypes did not reveal differences in Gag and GagPol processing and viral RNA incorporation into the virions. However, the presence of the whole RT from subtype C, or the chimeric RT containing either the polymerase or the connection and RNase H domains from C isolates, caused significantly slower viral replication regardless of B or C viral backbone. Subtype C RT carrying viruses displayed lower levels of accumulation of strong-stop cDNA in permeabilized virions during endogenous reverse transcription, and decreased accumulation of both strong-stop and positive strand reverse transcription products in infected cells and in isolated reverse transcription complexes. This decreased accumulation correlated with lower levels of viral DNA integration in cells infected with viruses carrying the whole RT or RT domains from subtype C isolates. The single viral genome assay analysis did not reveal significant differences in the frequency of point mutations between the RT from B or C subtypes.ConclusionsThese data suggest that the whole RT as well as distinct polymerase and connection-RNase H domains from subtype C HIV-1 confer a lower level of accumulation of reverse transcripts in the virions and reverse transcription complexes as compared to subtype B, resulting in a lower overall level of virus replication.

Strand Transfer and Elongation of HIV-1 Reverse Transcription Is Facilitated by Cell Factors In Vitro

Recent work suggests a role for multiple host factors in facilitating HIV-1 reverse transcription. Previously, we identified a cellular activity which increases the efficiency of HIV-1 reverse transcription in vitro. Here, we describe aspects of the activity which shed light on its function. The cellular factor did not affect synthesis of strong-stop DNA but did improve downstream DNA synthesis. The stimulatory activity was isolated by gel filtration in a single fraction of the exclusion volume. Velocity-gradient purified HIV-1, which was free of detectable RNase activity, showed poor reverse transcription efficiency but was strongly stimulated by partially purified cell proteins. Hence, the cell factor(s) did not inactivate an RNase activity that might degrade the viral genomic RNA and block completion of reverse transcription. Instead, the cell factor(s) enhanced first strand transfer and synthesis of late reverse transcription suggesting it stabilized the reverse transcription complex. The factor did not affect lysis of HIV-1 by Triton X-100 in the endogenous reverse transcription (ERT) system, and ERT reactions with HIV-1 containing capsid mutations, which varied the biochemical stability of viral core structures and impeded reverse transcription in cells, showed no difference in the ability to be stimulated by the cell factor(s) suggesting a lack of involvement of the capsid in the in vitro assay. In addition, reverse transcription products were found to be resistant to exogenous DNase I activity when the active fraction was present in the ERT assay. These results indicate that the cell factor(s) may improve reverse transcription by facilitating DNA strand transfer and DNA synthesis. It also had a protective function for the reverse transcription products, but it is unclear if this is related to improved DNA synthesis.

RETRACTED ARTICLE: A U5 repressor of reverse transcription is required for optimal HIV-1 infectivity and replication

Here we provide strong evidence that a highly conserved stem loop structure in the U5 region of the HIV-1 RNA leader harbours a repressor of reverse transcription (RRT). We showed that two sequences in U5, at +143-145 and +151-153, are essential for RRT function. Mutation of either site strongly and unexpectedly increased endogenous reverse transcription, and cell infection assays showed that both mutations dramatically increased negative strand strong stop DNA synthesis. Early, late, 1-LTR and 2-LTR reverse transcription products were present proportionally, indicating that the downstream reverse transcription events were not affected. In vitro structural probing of the wild type and mutant RNA revealed an unexpected destabilization effect of the mutations on the whole U5 stem loop, which would explain the loss of regulation of reverse transcription. This functional effect was not observed in vitro, where, in the absence of viral proteins other than RT and cellular factors, all RNA performed similarly. These U5 mutations decreased virus replication in Jurkat and primary T-cells, which could be attributed to a marked defect in viral integration. Analysis of 1-LTR and 2-LTR circular DNA isolated from infected cells revealed that substantial deletions were present, indicating that the viral DNA was degraded by cellular nucleases. Together, our experiments suggest that regulated reverse transcription initiation is essential to allow synthesis of the viral DNA in a cellular environment that supports the assembly of a functional HIV-1 pre-integration complex, which also protects the proviral DNA from cellular degradation processes.

Mutations in Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Zinc Fingers Cause Premature Reverse Transcription

Human immunodeficiency virus type 1 (HIV-1) requires that its genome be reverse transcribed into double-stranded DNA for productive infection of cells. This process requires not only reverse transcriptase but also the nucleocapsid protein (NC), which functions as a nucleic acid chaperone. Reverse transcription generally begins once the core of the virion enters the cytoplasm of a newly infected cell. However, some groups have reported the presence of low levels of viral DNA (vDNA) within particles prior to infection, the significance and function of which is controversial. We report here that several HIV-1 NC mutants, which we previously identified as being replication defective, contain abnormally high levels of intravirion DNA. These findings were further reinforced by the inability of these NC mutants to perform endogenous reverse transcription (ERT), in contrast to the readily measurable ERT activity in wild-type HIV-1. When either of the NC mutations is combined with a mutation that inactivates the viral protease, we observed a significant reduction in the amount of intravirion DNA. Interestingly, we also observed high levels of intravirion DNA in the context of wild-type NC when we delayed budding by means of a PTAP((-)) (Pro-Thr-Ala-Pro) mutation. Premature reverse transcription is most probably occurring before these mutant virions bud from producer cells, but we fail to see any evidence that the NC mutations alter the timing of Pr55(Gag) processing. Critically, our results also suggest that the presence of intravirion vDNA could serve as a diagnostic for identifying replication-defective HIV-1.

Cell Factors Stimulate Human Immunodeficiency Virus Type 1 Reverse Transcription In Vitro

After fusion of the human immunodeficiency virus type 1 (HIV-1) envelope with the host cell membrane, the HIV-1 core enters the cell cytoplasm. Core components are then restructured to form the reverse transcription complex (RTC); the biochemical details of this process are currently unclear. To investigate early RTC formation, we characterized the endogenous reverse transcription activity of virions, which was less efficient than reverse transcription during cell infection and suggested a requirement for a cell factor. The addition of detergent to virions released reverse transcriptase and capsid, and reverse transcription products became susceptible to the action of exogenous nucleases, indicating virion disruption. Disruption was coincident with the loss of the endogenous reverse transcription activity of virions, particularly late reverse transcription products. Consistent with this observation, the use of a modified "spin thru" method, which uses brief detergent exposure, also disrupted virions. The addition of lysates made from mammalian cell lines (Jurkat, HEK293T, and NIH 3T3 cells) to virions delipidated by detergent stimulated late reverse transcription efficiency. A complex with reverse transcription activity that was slower sedimenting than virions on a velocity gradient was greatly stimulated to generate full-length reverse transcription products and was associated with only relatively small amounts of capsid. These experiments suggest that cell factors are required for efficient reverse transcription of HIV-1.

Fully-spliced HIV-1 RNAs are reverse transcribed with similar efficiencies as the genomic RNA in virions and cells, but more efficiently in AZT-treated cells

We have shown previously that HIV actively and selectively packages the spliced HIV RNAs into progeny virions. In the present study, by using a RT-QPCR and QPCR strategies, we show that spliced viral RNAs are present in infectious particles and consequently participate, along with the unspliced genomic RNA, to some of the early steps of infection such as the reverse transcription step. This work provides the first quantitative data on reverse transcription of the fully spliced viral RNAs, also called the early transcripts, in target cells but also inside virions. The latter results were obtained by measuring the natural endogenous reverse transcription activity directly on intact HIV-1 particles. Our study reveals that spliced HIV RNAs are reverse transcribed as efficiently as the genomic RNA, both in cells and virions. Interestingly, we also show that reverse transcription of spliced RNAs is 56-fold less sensitive to the inhibitor AZT than reverse transcription of the genomic RNA. Therefore, the selection mediated by inhibitors of reverse transcription used to treat patients could lead to increased representativeness of spliced forms of HIV, thus favoring recombination between the HIV DNA species and facilitating HIV recovery.

Analysis of the replication of HIV-1 forced to use tRNAMet(i) supports a link between primer selection, translation and encapsidation

BackgroundPrevious studies have suggested that the process of HIV-1 tRNA primer selection and encapsidation of genomic RNA might be coupled with viral translation. In order to further investigate this relationship, proviruses were constructed in which the primer-binding site (PBS) was altered to be complementary to elongator tRNAMet (tRNAMet(e)) (HXB2-Met(e)) or initiator tRNAMet (tRNAMet(i)) (HXB2-Met(i)). These tRNAMet not only differ with respect to the 3' terminal 18-nucleotides, but also with respect to interaction with host cell proteins during protein synthesis.ResultsConsistent with previous studies, HXB2-Met(e) were infectious and maintained this PBS following short-term in vitro culture in SupT1 cells. In contrast, transfection of HBX2-Met(i) produced reduced amounts of virus (as determined by p24) and did not establish a productive infection in SupT1 cells. The low infectivity of the virus with the PBS complementary to tRNAMet(i) was not due to differences in endogenous levels of cellular tRNAMet(i) compared to tRNAMet(e); tRNAMet(i) was also capable of being selected as the primer for reverse transcription as determined by the endogenous reverse transcription reaction. The PBS of HXB2-Met(i) contains an ATG, which could act as an upstream AUG and syphon scanning ribosomes thereby reducing initiation of translation at the authentic AUG of Gag. To investigate this possibility, a provirus with an A to G change was constructed (HXB2-Met(i)AG). Transfection of HXB2-Met(i)AG resulted in increased production of virus, similar to that for the wild type virus. In contrast to HXB2-Met(i), HXB2-Met(i)AG was able to establish a productive infection in SupT1 cells. Analysis of the PBS following replication revealed the virus favored the genome with the repaired PBS (A to G) even though tRNAMet(i) was continuously selected as the primer for reverse transcription.ConclusionThe results of these studies suggest that HIV-1 has access to both tRNAMet for selection as the replication primer and supports a co-ordination between primer selection, translation and encapsidation during virus replication.

Protein methylation is required to maintain optimal HIV-1 infectivity

Background:Protein methylation is recognized as a major protein modification pathway regulating diverse cellular events such as protein trafficking, transcription, and signal transduction. More recently, protein arginine methyltransferase activity has been shown to regulate HIV-1 transcription via Tat. In this study, adenosine periodate (AdOx) was used to globally inhibit protein methyltransferase activity so that the effect of protein methylation on HIV-1 infectivity could be assessed.Results:Two cell culture models were used: HIV-1-infected CEM T-cells and HEK293T cells transfected with a proviral DNA plasmid. In both models, AdOx treatment of cells increased the levels of virion in culture supernatant. However, these viruses had increased levels of unprocessed or partially processed Gag-Pol, significantly increased diameter, and displayed reduced infectivity in a MAGI X4 assay. AdOx reduced infectivity equally in both dividing and non-dividing cells. However, infectivity was further reduced if Vpr was deleted suggesting virion proteins, other than Vpr, were affected by protein methylation. Endogenous reverse transcription was not inhibited in AdOx-treated HIV-1, and infectivity could be restored by pseudotyping HIV with VSV-G envelope protein. These experiments suggest that AdOx affects an early event between receptor binding and uncoating, but not reverse transcription.Conclusion:Overall, we have shown for the first time that protein methylation contributes towards maximal virus infectivity. Furthermore, our results also indicate that protein methylation regulates HIV-1 infectivity in a complex manner most likely involving the methylation of multiple viral or cellular proteins and/or multiple steps of replication.

Development of a New Methodology for Screening of Human Immunodeficiency Virus Type 1 Microbicides Based on Real-Time PCR Quantification

Potential topical retrovirucides or vaginal microbicides against human immunodeficiency virus type 1 (HIV-1) include nonnucleoside reverse transcriptase inhibitors (NNRTIs). To be successful, such agents have to be highly active against cell-free virions. In the present study, we developed a new real-time PCR-based assay to measure the natural endogenous reverse transcription (NERT) activity directly on intact HIV-1 particles in the presence of reverse transcriptase (RT) inhibitors. We further evaluated the permeability to nevirapine (NVP) and efavirenz (EFV) and their retention within nascent viral particles. We also demonstrated the NVP and EFV inhibitory effects on NERT activity and the impact of resistance mutations measured directly by this new strategy. Furthermore, the results showed a clear correlation between NERT activity and classical infectivity assays. The 50% inhibitory concentrations (IC50s) of NVP and EFV were demonstrated to be up to 100-fold higher for cell-free than for cell-associated virions, suggesting that cell-free virions are less permeable to these drugs. Our results suggest that NVP and EFV penetrate both the envelope and the capsid of HIV-1 particles and readily inactivate cell-free virions. However, the characteristics of these NNRTIs, such as lower permeability and lower retention during washing procedures, in cell-free virions reduce their efficacies as microbicides. Here, we demonstrate the usefulness of the NERT real-time PCR as an assay for screening novel antiretroviral compounds with unique mechanisms of action.

Inhibition of endogenous reverse transcription of human and nonhuman primate lentiviruses: Potential for development of lentivirucides

In the current study, we extended our previous works on natural endogenous reverse transcription (NERT) and further examined its potential as a virucide molecular target in sexual transmission of primate lentiviruses. HIV-1 and SIV virions were pretreated with select nucleoside (NRTIs) and nonnucleoside RT inhibitors (NNRTIs), either alone or in combination with NERT-stimulating substances. The effects of these antiretrovirals on virion inactivation were analyzed in human T cell lines and primary cell cultures. Pretreatment of HIV-1 virions with physiologic NERT-stimulants and 3′-azido-3′-deoxythymidine 5′-triphosphate (AZT-TP) or nevirapine potently inactivated cell-free HIV-1 virions and resulted in strong inhibition of the viral infectivity. Pretreatment of chimeric SHIV-RT virions with NERT-stimulating cocktail and select antiretrovirals also resulted in virion inactivation and inhibition of viral infectivity in T cell lines. Our findings demonstrate the potential clinical utility of approaches based on inhibiting NERT in sexual transmission of HIV-1, through the development of effective anti-HIV-1 microbicides, such as NRTIs and NNRTIs.

Dawn of non-nucleoside inhibitor-based anti-HIV microbicides

The emergence of HIV/AIDS as a disease spread through sexual intercourse has prompted the search for safe and effective vaginal and rectal microbicides for curbing mucosal viral transmission via semen. Since endogenous reverse transcription is implicated in augmenting the sexual transmission of HIV-1 infection, potential microbicides should have the inherent ability to optimally inhibit both wild-type and drug-escape mutants. The non-nucleoside reverse transcriptase inhibitors (NNRTIs), which bind to an allosteric site on RT, are an important arsenal of drugs against HIV-1. The clinical success of NNRTI-based HIV/AIDS therapies has led to extensive structural and molecular modelling studies of enzyme complexes and chemical synthesis of second- and third-generation NNRTIs. Rationally designed NNRTIs deduced from changes in binding pocket size, shape and residue character that result from clinically observed NNRTI resistance-associated mutations exhibit high binding affinity for HIV-1 RT and robust anti-HIV activity against the wild-type and drug-escape mutants without cytotoxicity. Notably, membrane permeable tight binding NNRTIs have the ability to inactivate cell-free as well as cell-associated HIV-1 in semen without metabolic activation. Consequently, NNRTIs currently under development as experimental microbicides include thiourea-PETT (where PETT stands for phenethylthiazolylthiourea) derivatives (PHI-236, PHI-346 and PHI-443), urea-PETT derivatives (MIV-150), oxypyrimidines (S-DABOs), thiocarboxanilides (UC-781) and diarylpyrimidines (TMC-120). Mucoadhesive formulations of these NNRTIs have been studied for safety and efficacy in animal models and some have entered Phase I safety testing in humans. This review focuses on the structural, biological and preclinical studies relevant to the clinical development of these NNRTIs as molecular virucides intended to prevent the sexual transmission of HIV-1.