Replication of Chimeric Human Immunodeficiency Virus Type 1 (HIV-1) Containing HIV-2 Integrase (IN): Naturally Selected Mutations in IN Augment DNA Synthesis

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) integrase (IN) protein augments the initiation of reverse transcription. Chimeric HIV-1 containing HIV-2 IN (SG3IN2) is severely impaired in virus infectivity and DNA synthesis. To analyze the nature of this defect, we infected T cells with the chimeric SG3IN2 virus and by continuous passage in cell culture selected for virus with improved replication properties. Viruses from two different time points were chosen for further analysis, an early culture-adapted virus (CF-65) that exhibited an intermediate level of infectivity, and a later-passaged virus (CF-131) that was significantly more infectious. Sequence analysis of multiple clones derived from the CF-65 virus culture demonstrated a diversity of mutations in the reverse transcriptase (RT) and a common V204I IN mutation. Analysis of clones derived from the CF-131 virus indicated the selection of two additional IN mutations, Q96H and K127E, and a fixed V179I RT mutation. By cloning RT and/or IN sequences back into the original SG3IN2 chimeric virus, we demonstrated that mutations in both RT and IN contributed to the improvement in viral fitness. The effect of the HIV-2IN(IN2) mutations on virus DNA synthesis was analyzed by packaging IN2 mutants into HIV-1 as Vpr-IN2 fusion proteins. This analysis revealed that the Q96H, K127E and V204I mutations increased the infectivity of the chimeric virus by augmenting the initiation of viral cDNA synthesis in infected cells. The Q96H and K127E mutations are present in adjacent helical structures on the surface of the IN protein and together account for most of the increase observed in DNA synthesis. Our findings provide evidence that the IN protein augments the initiation of reverse transcription through specific interactions with other viral components comprising the initiation complex. Moreover, they implicate specific regions on the surface of IN that may help to elucidate mechanisms by which the HIV-1 IN protein augments the initiation of HIV-1 reverse transcription in vivo.

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Diminished RNA Primer Usage Associated with the L74V and M184V Mutations in the Reverse Transcriptase of Human Immunodeficiency Virus Type 1 Provides a Possible Mechanism for Diminished Viral Replication Capacity

Journal of Virology ◽

10.1128/jvi.77.16.8621-8632.2003 ◽

2003 ◽

Vol 77 (16) ◽

pp. 8621-8632 ◽

Cited By ~ 39

Author(s):

Karidia Diallo ◽

Bruno Marchand ◽

Xin Wei ◽

Luciano Cellai ◽

Matthias Götte ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcriptase ◽

Dna Synthesis ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Replication Capacity ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The emergence of drug resistance-conferring mutations can severely compromise the success of chemotherapy directed against human immunodeficiency virus type 1 (HIV-1). The M184V and/or L74V mutation in the reverse transcriptase (RT) gene are frequently found in viral isolates from patients treated with the nucleoside RT inhibitors lamivudine (3TC), abacavir (ABC), and didanosine (ddI). However, the effectiveness of combination therapy with regimens containing these compounds is often not abolished in the presence of these mutations; it has been conjectured that diminished fitness of HIV-1 variants containing L74V and M184V may contribute to sustained antiviral effects in such cases. We have determined that viruses containing both L74V and M184V are more impaired in replication capacity than viruses containing either mutation alone. To understand the biochemical mechanisms responsible for this diminished fitness, we generated a series of recombinant mutated enzymes containing either or both of the L74V and M184V substitutions. These enzymes were tested for their abilities to bypass important rate-limiting steps during the complex process of reverse transcription. We studied both the initiation of minus-strand DNA synthesis with the cognate replication primer human tRNA3 Lys and the initiation of plus-strand DNA synthesis, using a short RNA primer derived from the viral polypurine tract. We observed that the efficiencies of both reactions were diminished with enzymes containing either L74V or M184V and that these effects were significantly amplified with the double mutant. We also show that release from intrinsic pausing sites during reverse transcription appears to be a major obstacle that cannot be efficiently bypassed. Our data suggest that the efficiency of RNA-primed DNA synthesis represents an important consideration that can affect viral replication kinetics.

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Inhibition of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{3}^{\mathrm{Lys}}\) \end{document}-Primed Reverse Transcription by Human APOBEC3G during Human Immunodeficiency Virus Type 1 Replication

Journal of Virology ◽

10.1128/jvi.01038-06 ◽

2006 ◽

Vol 80 (23) ◽

pp. 11710-11722 ◽

Cited By ~ 147

Author(s):

Fei Guo ◽

Shan Cen ◽

Meijuan Niu ◽

Jenan Saadatmand ◽

Lawrence Kleiman

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Viral Infectivity ◽

Viral Dna ◽

Immunodeficiency Virus ◽

Dna Production ◽

Hiv 1

ABSTRACT Cells are categorized as being permissive or nonpermissive according to their ability to produce infectious human immunodeficiency virus type 1 (HIV-1) lacking the viral protein Vif. Nonpermissive cells express the human cytidine deaminase APOBEC3G (hA3G), and Vif has been shown to bind to APOBEC3G and facilitate its degradation. Vif-negative HIV-1 virions produced in nonpermissive cells incorporate hA3G and have a severely reduced ability to produce viral DNA in newly infected cells. While it has been proposed that the reduction in DNA production is due to hA3G-facilitated deamination of cytidine, followed by DNA degradation, we provide evidence here that a decrease in the synthesis of the DNA by reverse transcriptase may account for a significant part of this reduction. During the infection of cells with Vif-negative HIV-1 produced from 293T cells transiently expressing hA3G, much of the inhibition of early (≥50% reduction) and late (≥95% reduction) viral DNA production, and of viral infectivity (≥95% reduction), can occur independently of DNA deamination. The inhibition of the production of early minus-sense strong stop DNA is also correlated with a similar inability of tRNA3 Lys to prime reverse transcription. A similar reduction in tRNA3 Lys priming and viral infectivity is also seen in the naturally nonpermissive cell H9, albeit at significantly lower levels of hA3G expression.

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Effects of Varying Sequence Similarity on the Frequency of Repeat Deletion during Reverse Transcription of a Human Immunodeficiency Virus Type 1 Vector

Journal of Virology ◽

10.1128/jvi.76.15.7897-7902.2002 ◽

2002 ◽

Vol 76 (15) ◽

pp. 7897-7902 ◽

Cited By ~ 37

Author(s):

Wenfeng An ◽

Alice Telesnitsky

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Genetic Recombination ◽

Gene Segment ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Deletion Frequency

ABSTRACT Genetic recombination contributes to human immunodeficiency virus type 1 (HIV-1) diversity, with homologous recombination being more frequent than nonhomologous recombination. In this study, HIV-1-based vectors were used to assay the effects of various extents of sequence divergence on the frequency of the recombination-related property of repeat deletion. Sequence variation, similar in degree to that which differentiates natural HIV-1 isolates, was introduced by synonymous substitutions into a gene segment. Repeated copies of this segment were then introduced into assay vectors. With the use of a phenotypic screen, the deletion frequency of identical repeats was compared to the frequencies of repeats that differed in sequence by various extents. During HIV-1 reverse transcription, the deletion frequency observed with repeats that differed by 5% was 65% of that observed with identical repeats. The deletion frequency decreased to 26% for repeats that differed by 9%, and when repeats differed by 18%, the deletion frequency was about 5% of the identical repeat value. Deletion frequencies fell to less than 0.3% of identical repeat values when genetic distances of 27% or more were examined. These data argue that genetic variation is not as inhibitory to HIV-1 repeat deletion as it is to the corresponding cellular process and suggest that, for sequences that differ by about 25% or more, HIV-1 recombination directed by sequence homology may be no more frequent than that which is homology independent.

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Mutations in the 5′ End of the Human Immunodeficiency Virus Type 1 Polypurine Tract Affect RNase H Cleavage Specificity and Virus Titer

Journal of Virology ◽

10.1128/jvi.77.20.11150-11157.2003 ◽

2003 ◽

Vol 77 (20) ◽

pp. 11150-11157 ◽

Cited By ~ 18

Author(s):

Mary Jane McWilliams ◽

John G. Julias ◽

Stefan G. Sarafianos ◽

W. Gregory Alvord ◽

Edward Arnold ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Dna Synthesis ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Virus Titer ◽

Rnase H ◽

Cleavage Specificity ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The RNase H activity of retroviral reverse transcriptases (RTs) degrades viral genomic RNA after it has been copied into DNA, removes the tRNA used to initiate negative-strand DNA synthesis, and generates and removes the polypurine tract (PPT) primer used to initiate positive-strand DNA synthesis. The cleavages that remove the tRNA and that generate and remove the PPT primer must be specific to generate linear viral DNAs with ends that are appropriate for integration into the host cell genome. The crystal structure of human immunodeficiency virus type 1 (HIV-1) RT in a complex with an RNA/DNA duplex derived from the PPT revealed that the 5′ end of the PPT deviates from traditional Watson-Crick base pairing. This unusual structure may play a role in the proper recognition of the PPT by HIV-1 RT. We made substitution mutations in the 5′ end of the PPT and determined their effects on virus titer. The results indicated that single and double mutations in the 5′ end of the PPT had modest effects on virus replication in a single-cycle assay. More complex mutations had stronger effects on virus titer. Analysis of the two-long-terminal-repeat circle junctions derived from infecting cells with the mutant viruses indicated that the mutations affected RNase H activity, resulting in the retention of PPT sequences on viral DNA. The mutants tested preferentially retained specific segments of the PPT, suggesting an effect on cleavage specificity. These results suggest that structural features of the PPT are important for its recognition and cleavage in vivo.

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Dimerization and Template Switching in the 5′ Untranslated Region between Various Subtypes of Human Immunodeficiency Virus Type 1

Journal of Virology ◽

10.1128/jvi.77.5.3020-3030.2003 ◽

2003 ◽

Vol 77 (5) ◽

pp. 3020-3030 ◽

Cited By ~ 26

Author(s):

Ebbe Sloth Andersen ◽

Rienk E. Jeeninga ◽

Christian Kroun Damgaard ◽

Ben Berkhout ◽

Jørgen Kjems

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Untranslated Region ◽

Template Switching ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) particle contains two identical RNA strands, each corresponding to the entire genome. The 5′ untranslated region (UTR) of each RNA strand contains extensive secondary and tertiary structures that are instrumental in different steps of the viral replication cycle. We have characterized the 5′ UTRs of nine different HIV-1 isolates representing subtypes A through G and, by comparing their homodimerization and heterodimerization potentials, found that complementarity between the palindromic sequences in the dimerization initiation site (DIS) hairpins is necessary and sufficient for in vitro dimerization of two subtype RNAs. The 5′ UTR sequences were used to design donor and acceptor templates for a coupled in vitro dimerization-reverse transcription assay. We showed that template switching during reverse transcription is increased with a matching DIS palindrome and further stimulated proportional to the level of homology between the templates. The presence of the HIV-1 nucleocapsid protein NCp7 increased the template-switching efficiency for matching DIS palindromes twofold, whereas the recombination efficiency was increased sevenfold with a nonmatching palindrome. Since NCp7 did not effect the dimerization of nonmatching palindromes, we concluded that the protein most likely stimulates the strand transfer reaction. An analysis of the distribution of template-switching events revealed that it occurs throughout the 5′ UTR. Together, these results demonstrate that the template switching of HIV-1 reverse transcriptase occurs frequently in vitro and that this process is facilitated mainly by template proximity and the level of homology.

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Changes in Human Immunodeficiency Virus Type 1 Gag at Positions L449 and P453 Are Linked to I50V Protease Mutants In Vivo and Cause Reduction of Sensitivity to Amprenavir and Improved Viral Fitness In Vitro

Journal of Virology ◽

10.1128/jvi.76.15.7398-7406.2002 ◽

2002 ◽

Vol 76 (15) ◽

pp. 7398-7406 ◽

Cited By ~ 107

Author(s):

Michael F. Maguire ◽

Rosario Guinea ◽

Philip Griffin ◽

Sarah Macmanus ◽

Robert C. Elston ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Viral Fitness ◽

Wild Type Virus ◽

Wild Type ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Gag protease cleavage sites (CS) undergo sequence changes during the development of resistance to several protease inhibitors (PIs). We have analyzed the association of sequence variation at the p7/p1 and p1/p6 CS in conjunction with amprenavir (APV)-specific protease mutations following PI combination therapy with APV. Querying a central resistance data repository resulted in the detection of significant associations (P < 0.001) between the presence of APV protease signature mutations and Gag L449F (p1/p6 LP1′F) and P453L (p1/p6 PP5′L) CS changes. In population-based sequence analyses the I50V mutant was invariably linked to either L449F or P453L. Clonal analysis revealed that both CS mutations were never present in the same genome. Sequential plasma samples from one patient revealed a transition from I50V M46L P453L viruses at early time points to I50V M46I L449F viruses in later samples. Various combinations of the protease and Gag mutations were introduced into the HXB2 laboratory strain of HIV-1. In both single- and multiple-cycle assay systems and in the context of I50V, the L449F and P453L changes consistently increased the 50% inhibitory concentration of APV, while the CS changes alone had no measurable effect on inhibitor sensitivity. The decreased in vitro fitness of the I50V mutant was only partially improved by addition of either CS change (I50V M46I L449F mutant replicative capacity ≈ 16% of that of wild-type virus). Western blot analysis of Pr55 Gag precursor cleavage products from infected-cell cultures indicated accumulation of uncleaved Gag p1-p6 in all I50V viruses without coexisting CS changes. Purified I50V protease catalyzed cleavage of decapeptides incorporating the L449F or P453L change 10-fold and 22-fold more efficiently than cleavage of the wild-type substrate, respectively. HIV-1 protease CS changes are selected during PI therapy and can have effects on both viral fitness and phenotypic resistance to PIs.

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The tRNA Primer Activation Signal in the Human Immunodeficiency Virus Type 1 Genome Is Important for Initiation and Processive Elongation of Reverse Transcription

Journal of Virology ◽

10.1128/jvi.76.5.2329-2339.2002 ◽

2002 ◽

Vol 76 (5) ◽

pp. 2329-2339 ◽

Cited By ~ 57

Author(s):

Nancy Beerens ◽

Ben Berkhout

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Sequence Motif ◽

Immunodeficiency Virus ◽

Trna Primer ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) reverse transcription is primed by the cellular tRNA3 Lys molecule, which binds, with its 3"-terminal 18 nucleotides (nt), to a complementary sequence in the viral genome, the primer-binding site (PBS). Besides PBS-anti-PBS pairing, additional interactions between viral RNA sequences and the tRNA primer are thought to regulate the process of reverse transcription. We previously identified a novel 8-nt sequence motif in the U5 region of the HIV-1 RNA genome that is critical for tRNA3 Lys-mediated initiation of reverse transcription in vitro. This motif activates initiation from the natural tRNA3 Lys primer but is not involved in tRNA placement and was therefore termed primer activation signal (PAS). It was proposed that the PAS interacts with the anti-PAS motif in the TΨC arm of tRNA3 Lys. In this study, we analyzed several PAS-mutated viruses and performed reverse transcription assays with virion-extracted RNA-tRNA complexes. Mutation of the PAS reduced the efficiency of tRNA-primed reverse transcription. In contrast, mutations in the opposing leader sequence that trigger release of the PAS from base pairing stimulated reverse transcription. These results are similar to the reverse transcription effects observed in vitro. We also selected revertant viruses that partially overcome the reverse transcription defect of the PAS deletion mutant. Remarkably, all revertants acquired a single nucleotide substitution that does not restore the PAS sequence but that stimulates elongation of reverse transcription. These combined results indicate that the additional PAS-anti-PAS interaction is needed to assemble an initiation-competent and processive reverse transcription complex.

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Sequences in the 5′ and 3′ R Elements of Human Immunodeficiency Virus Type 1 Critical for Efficient Reverse Transcription

Journal of Virology ◽

10.1128/jvi.74.18.8324-8334.2000 ◽

2000 ◽

Vol 74 (18) ◽

pp. 8324-8334 ◽

Cited By ~ 17

Author(s):

Yuki Ohi ◽

Jared L. Clever

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Strand Transfer ◽

Minus Strand ◽

Immunodeficiency Virus ◽

Infected Cells ◽

Hiv 1

ABSTRACT The genome of human immunodeficiency virus type 1 (HIV-1) contains two direct repeats (R) of 97 nucleotides at each end. These elements are of critical importance during the first-strand transfer of reverse transcription, during which the minus-strand strong-stop DNA (−sssDNA) is transferred from the 5′ end to the 3′ end of the genomic RNA. This transfer is critical for the synthesis of the full-length minus-strand cDNA. These repeats also contain a variety of other functional domains involved in many aspects of the viral life cycle. In this study, we have introduced a series of mutations into the 5′, the 3′, or both R sequences designed to avoid these other functional domains. Using a single-round infectivity assay, we determined the ability of these mutants to undergo the various steps of reverse transcription utilizing a semiquantitative PCR analysis. We find that mutations within the first 10 nucleotides of either the 5′ or the 3′ R sequence resulted in virions that were markedly defective for reverse transcription in infected cells. These mutations potentially introduce mismatches between the full-length −sssDNA and 3′ acceptor R. Even mutations that would create relatively small mismatches, as little as 3 bp, resulted in inefficient reverse transcription. In contrast, virions containing identically mutated R elements were not defective for reverse transcription or infectivity. Using an endogenous reverse transcription assay with disrupted virus, we show that virions harboring the 5′ or the 3′ R mutations were not intrinsically defective for DNA synthesis. Similarly sized mismatches slightly further downstream in either the 5′, the 3′, or both R sequences were not detrimental to continued reverse transcription in infected cells. These data are consistent with the idea that certain mismatches within 10 nucleotides downstream of the U3-R junction in HIV-1 cause defects in the stability of the cDNA before or during the first-strand transfer of reverse transcription leading to the rapid disappearance of the −sssDNA in infected cells. These data also suggest that the great majority of first-strand transfers in HIV-1 occur after the copying of virtually the entire 5′ R.

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Association of Human Immunodeficiency Virus Type 1 Vif with RNA and Its Role in Reverse Transcription

Journal of Virology ◽

10.1128/jvi.74.19.8938-8945.2000 ◽

2000 ◽

Vol 74 (19) ◽

pp. 8938-8945 ◽

Cited By ~ 83

Author(s):

Markus Dettenhofer ◽

Shan Cen ◽

Bradley A. Carlson ◽

Lawrence Kleiman ◽

Xiao-Fang Yu

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcriptase ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Genomic Rna ◽

Immunodeficiency Virus ◽

Viral Genomic Rna ◽

Hiv 1

ABSTRACT The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication, although the functional target of Vif remains elusive. HIV-1 vif mutant virions derived from nonpermissive H9 cells displayed no significant differences in the amount, ratio, or integrity of their protein composition relative to an isogenic wild-type virion. The amounts of the virion-associated viral genomic RNA and tRNA3 Lyswere additionally present at normal levels in vif mutant virions. We demonstrate that Vif associates with RNA in vitro as well as with viral genomic RNA in virus-infected cells. A functionally conserved lentivirus Vif motif was found in the double-stranded RNA binding domain of Xenopus laevis, Xlrbpa. The natural intravirion reverse transcriptase products were markedly reduced invif mutant virions. Moreover, purified vifmutant genomic RNA-primer tRNA complexes displayed severe defects in the initiation of reverse transcription with recombinant reverse transcriptase. These data point to a novel role for Vif in the regulation of efficient reverse transcription through modulation of the virion nucleic acid components.

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Reverse Transcription of Human Immunodeficiency Virus Type 1 is Blocked by Retroviral Zinc Finger Inhibitors

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632029700800107 ◽

1997 ◽

Vol 8 (1) ◽

pp. 60-69 ◽

Cited By ~ 16

Author(s):

JA Turpin ◽

CA Schaeffer ◽

SJ Terpening ◽

L Graham ◽

M Bu ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Zinc Finger ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Zinc Fingers ◽

Immunodeficiency Virus ◽

Cross Linkage ◽

Hiv 1

The Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys zinc fingers of retroviral nucleocapsid (NC) proteins are prime antiviral targets due to conservation of the Cys and His chelating residues and the absolute requirement of these fingers in both early and late phases of retroviral replication. Certain 2,2′-dithiobisbenzamides (DIBAs) chemically modify the Cys residues of the fingers, thereby inhibiting in vitro replication of human immunodeficiency virus type 1 (HIV-1). We examined the consequences of DIBA interaction with cell-free virions and their subsequent ability to initiate new rounds of infection. The DIBAs entered intact virions and chemically modified the p7NC proteins, resulting in extensive disulphide cross-linkage among zinc fingers of adjacent p7NC molecules. Likewise, treatment of Pr55gag-laden pseudovirions, used as a model of virion particles, with DIBAs resulted in Pr55gag cross-linkage. In contrast, monomeric p7NC protein did not form cross-linkages after DIBA treatment, indicating that the retroviral zinc finger proteins must exist in close proximity for cross-linkage to occur. Cross-linkage of p7NC in virions correlated with loss of infectivity and decreased proviral DNA synthesis during acute infection, even though DIBAs did not inhibit virus attachment to host cells or reverse transcriptase enzymatic activity. Thus, DIBA-type molecules impair the ability of HIV-1 virions to initiate reverse transcription through their action on the retroviral zinc finger, thereby blocking further rounds of replication.

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