scholarly journals Forced Selection of a Human Immunodeficiency Virus Type 1 Variant That Uses a Non-Self tRNA Primer for Reverse Transcription: Involvement of Viral RNA Sequences and the Reverse Transcriptase Enzyme

2004 ◽  
Vol 78 (19) ◽  
pp. 10706-10714 ◽  
Author(s):  
Truus E. M. Abbink ◽  
Nancy Beerens ◽  
Ben Berkhout
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Viral Rna ◽  
Replication Capacity ◽  
Immunodeficiency Virus ◽  
Trna Primer

ABSTRACT Human immunodeficiency virus type 1 uses the tRNA3 Lys molecule as a selective primer for reverse transcription. This primer specificity is imposed by sequence complementarity between the tRNA primer and two motifs in the viral RNA genome: the primer-binding site (PBS) and the primer activation signal (PAS). In addition, there may be specific interactions between the tRNA primer and viral proteins, such as the reverse transcriptase (RT) enzyme. We constructed viruses with mutations in the PAS and PBS that were designed to employ the nonself primer tRNAPro or tRNA1,2 Lys. These mutants exhibited a severe replication defect, indicating that additional adaptation of the mutant virus is required to accommodate the new tRNA primer. Multiple independent virus evolution experiments were performed to select for fast-replicating variants. Reversion to the wild-type PBS-lys3 sequence was the most frequent escape route. However, we identified one culture in which the virus gained replication capacity without reversion of the PBS. This revertant virus eventually optimized the PAS motif for interaction with the nonself primer. Interestingly, earlier evolution samples revealed a single amino acid change of an otherwise well-conserved residue in the RNase H domain of the RT enzyme, implicating this domain in selective primer usage. We demonstrate that both the PAS and RT mutations improve the replication capacity of the tRNA1,2 Lys-using virus.

scholarly journals 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

2003 ◽  
Vol 77 (16) ◽  
pp. 8621-8632 ◽  
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.

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

2002 ◽  
Vol 76 (5) ◽  
pp. 2329-2339 ◽  
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.

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

2000 ◽  
Vol 74 (19) ◽  
pp. 8938-8945 ◽  
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.

scholarly journals Roles of Pr55gag and NCp7 in tRNA3Lys Genomic Placement and the Initiation Step of Reverse Transcription in Human Immunodeficiency Virus Type 1

2000 ◽  
Vol 74 (22) ◽  
pp. 10796-10800 ◽  
Author(s):  
Shan Cen ◽  
Ahmad Khorchid ◽  
Juliana Gabor ◽  
Liwei Rong ◽  
Mark A. Wainberg ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Viral Rna ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Initiation Step

ABSTRACT To study in vivo tRNA3 Lys genomic placement and the initiation step of reverse transcription in human immunodeficiency virus type 1, total viral RNA isolated from either wild-type or protease-negative (PR−) virus was used as the source of primer tRNA3 Lys/genomic RNA templates in an in vitro reverse transcription assay. At low dCTP concentrations, both the rate and extent of the first nucleotide incorporated into tRNA3 Lys, dCTP, were lower with PR− than with wild-type total viral RNA. Transient in vitro exposure of either type of primer/template RNA to NCp7 increased PR− dCTP incorporation to wild-type levels but did not change the level of wild-type dCTP incorporation. Exposure of either primer/template to Pr55 gag had no effect on initiation. These results indicate that while Pr55 gag is sufficient for tRNA3 Lys placement onto the genome, exposure of this complex to mature NCp7 is required for optimum tRNA3 Lys placement and initiation of reverse transcription.

scholarly journals Human Immunodeficiency Virus Type 1 N-Terminal Capsid Mutants Containing Cores with Abnormally High Levels of Capsid Protein and Virtually No Reverse Transcriptase

2003 ◽  
Vol 77 (23) ◽  
pp. 12592-12602 ◽  
Author(s):  
Shixing Tang ◽  
Tsutomu Murakami ◽  
Naiqian Cheng ◽  
Alasdair C. Steven ◽  
Eric O. Freed ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Capsid Protein ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Cyclophilin A ◽  
Virus Infectivity ◽  
Immunodeficiency Virus

ABSTRACT We previously described the phenotype associated with three alanine substitution mutations in conserved residues (Trp23, Phe40, and Asp51) in the N-terminal domain of human immunodeficiency virus type 1 capsid protein (CA). All of the mutants produce noninfectious virions that lack conical cores and, despite having a functional reverse transcriptase (RT), are unable to initiate reverse transcription in vivo. Here, we have focused on elucidating the mechanism by which these CA mutations disrupt virus infectivity. We also report that cyclophilin A packaging is severely reduced in W23A and F40A virions, even though these residues are distant from the cyclophilin A binding loop. To correlate loss of infectivity with a possible defect in an early event preceding reverse transcription, we modeled disassembly by generating viral cores from particles treated with mild nonionic detergent; cores were isolated by sedimentation in sucrose density gradients. In general, fractions containing mutant cores exhibited a normal protein profile. However, there were two striking differences from the wild-type pattern: mutant core fractions displayed a marked deficiency in RT protein and enzymatic activity (<5% of total RT in gradient fractions) and a substantial increase in the retention of CA. The high level of core-associated CA suggests that mutant cores may be unable to undergo proper disassembly. Thus, taken together with the almost complete absence of RT in mutant cores, these findings can account for the failure of the three CA mutants to synthesize viral DNA following virus entry into cells.

scholarly journals HuR interacts with human immunodeficiency virus type 1 reverse transcriptase, and modulates reverse transcription in infected cells

Retrovirology ◽  
2008 ◽  
Vol 5 (1) ◽  
pp. 47 ◽  
Author(s):  
Julie Lemay ◽  
Priscilla Maidou-Peindara ◽  
Thomas Bader ◽  
Eric Ennifar ◽  
Jean-Christophe Rain ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Immunodeficiency Virus ◽  
Infected Cells

scholarly journals Impaired Rescue of Chain-Terminated DNA Synthesis Associated with the L74V Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase

2005 ◽  
Vol 49 (7) ◽  
pp. 2657-2664 ◽  
Author(s):  
Fernando A. Frankel ◽  
Bruno Marchand ◽  
Dan Turner ◽  
Matthias Götte ◽  
Mark A. Wainberg
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Replication Capacity ◽  
Viral Dna ◽  
Immunodeficiency Virus ◽  
Template Substrate ◽  
Hiv 1

ABSTRACT The L74V and M184V mutations in the reverse transcriptase (RT) gene of human immunodeficiency virus type 1 (HIV-1) are frequently associated with resistance to the nucleoside reverse transcriptase inhibitors abacavir, didanosine, and lamivudine. Yet viruses containing any of these mutations often display hypersusceptibility to zidovudine (ZDV). Two distinct mechanisms have been described to explain HIV-1 drug resistance. One of these involves diminished rates of incorporation of the nucleotide analogue by mutated RT, while the other mechanism involves increased rates of phosphorolytic excision of the drug-terminated primer. To understand the biochemical mechanisms responsible for the hypersensitization of L74V-containing viruses to ZDV, we studied the efficiency of excision of ZDV-monophosphate (ZDV-MP)-terminated primers by recombinant wild-type and mutated HIV-1 RTs in cell-free assays. We observed that the L74V mutation in RT caused reductions in ATP-dependent removal of ZDV-MP from newly synthesized viral DNA. In addition, we determined that the L74V and M184V mutations did not affect the ratio between the populations of RT-DNA/DNA complexes found at pre- and posttranslocational stages; however, they might have affected proper alignment between incorporated chain terminator and pyrophosphate donor, substrate orientation, affinity for ATP, and/or primer-template substrate. Finally, we confirmed previous findings that L74V-containing viruses display diminished replication capacity and that this is associated with reduced levels of synthesis of early reverse-transcribed viral DNA molecules.

Human Immunodeficiency Virus Type 1 Infection Requires Reverse Transcription of Nascent Viral RNA

1993 ◽  
Vol 12 (8) ◽  
pp. 685-693 ◽  
Author(s):  
MARY JANE POTASH ◽  
GONGRONG LI ◽  
MUHAMMAD SHAHABUDDIN ◽  
MICHAEL G. PELLEGRINO ◽  
DAVID J. VOLSKY
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Viral Rna ◽  
Immunodeficiency Virus

scholarly journals Amino Acid Substitutions at Position 190 of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Increase Susceptibility to Delavirdine and Impair Virus Replication

2003 ◽  
Vol 77 (2) ◽  
pp. 1512-1523 ◽  
Author(s):  
Wei Huang ◽  
Andrea Gamarnik ◽  
Kay Limoli ◽  
Christos J. Petropoulos ◽  
Jeannette M. Whitcomb
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Antiretroviral Drugs ◽  
Replication Capacity ◽  
Amino Acid Substitutions ◽  
Immunodeficiency Virus

ABSTRACT Suboptimal treatment of human immunodeficiency virus type 1 (HIV-1) infection with nonnucleoside reverse transcriptase inhibitors (NNRTI) often results in the rapid selection of drug-resistant virus. Several amino acid substitutions at position 190 of reverse transcriptase (RT) have been associated with reduced susceptibility to the NNRTI, especially nevirapine (NVP) and efavirenz (EFV). In the present study, the effects of various 190 substitutions observed in viruses obtained from NNRTI-experienced patients were characterized with patient-derived HIV isolates and confirmed with a panel of isogenic viruses. Compared to wild-type HIV, which has a glycine at position 190 (G190), viruses with 190 substitutions (A, C, Q, S, V, E, or T, collectively referred to as G190X substitutions) were markedly less susceptible to NVP and EFV. In contrast, delavirdine (DLV) susceptibility of these G190X viruses increased from 3 to 300-fold (hypersusceptible) or was only slightly decreased. The replication capacity of viruses with certain 190 substitutions (C, Q, V, T, and E) was severely impaired and was correlated with reduced virion-associated RT activity and incomplete protease (PR) processing of the viral p55 gag polyprotein. These defects were the result of inadequate p160 gagpol incorporation into virions. Compensatory mutations within RT and PR improved replication capacity, p55 gag processing, and RT activity, presumably through increased incorporation of p160 gagpol into virions. We observe an inverse relationship between the degree of NVP and EFV resistance and the impairment of viral replication in viruses with substitutions at 190 in RT. These observations may have important implications for the future design and development of antiretroviral drugs that restrict the outgrowth of resistant variants with high replication capacity.

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