scholarly journals Few Mutations in the 5′ Leader Region Mediate Fitness Recovery of Debilitated Human Immunodeficiency Type 1 Viruses

2005 ◽  
Vol 79 (9) ◽  
pp. 5421-5427 ◽  
Author(s):  
Eloísa Yuste ◽  
Antonio V. Bordería ◽  
Esteban Domingo ◽  
Cecilio López-Galíndez
Keyword(s):  
Reverse Transcription ◽  
Large Population ◽  
Primer Binding Site ◽  
Leader Region ◽  
Coding Regions ◽  
Immunodeficiency Virus ◽  
Large Populations ◽  
Large Virus ◽  
Hiv 1

ABSTRACT Repeated bottleneck passages of RNA viruses result in fitness losses due to the accumulation of deleterious mutations. In contrast, repeated transfers of large virus populations result in exponential fitness increases. Human immunodeficiency virus type 1 (HIV-1) manifested a drastic fitness loss after a limited number of plaque-to-plaque transfers in MT-4 cells. An analysis of the mutations associated with fitness loss in four debilitated clones revealed mutation frequencies in gag that were threefold higher than those in env. We now show an increase in the fitness of the debilitated HIV-1 clones by repeated passages of large populations. An analysis of the entire genomic nucleotide sequences of these populations showed that few mutations, from two to seven per clone, mediated fitness recovery. Eight of the 20 mutations affected coding regions, mainly by the introduction of nonsynonymous mutations (75%). However, most of the mutations accumulated during fitness recovery (12 of 20) were located in the 5′ untranslated leader region of the genome, and more specifically, in the primer binding site (PBS) loop. Two of the viruses incorporated the same mutation in the primer activation signal in the PBS loop, which is critical for the tRNA3 Lys-mediated initiation of reverse transcription. Moreover, 25% of the mutations observed were reversions. This fact, together with the presence of a large proportion of nonsynonymous replacements, may disclose the operation, during large population passages, of strong positive selection for optimal HIV-1 replication, which seems to be primarily affected by binding of the tRNA to the PBS and the initiation of reverse transcription.

scholarly journals Inability of Human Immunodeficiency Virus Type 1 Produced in Murine Cells To Selectively Incorporate Primer \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{3}^{\mathrm{Lys}}\) \end{document}

2008 ◽  
Vol 82 (24) ◽  
pp. 12049-12059 ◽  
Author(s):  
Min Wei ◽  
Yiliang Yang ◽  
Meijuan Niu ◽  
Laurie Desfosse ◽  
Robert Kennedy ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Leukemia Virus ◽  
Trna Synthetase ◽  
Primer Binding Site ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Attempts to use the mouse as a model system for studying AIDS are stymied by the multiple blocks to human immunodeficiency virus type 1 (HIV-1) replication that exist in mouse cells at the levels of viral entry, transcription, and Gag assembly and processing. In this report, we describe an additional block in the selective packaging of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} into HIV-1 produced in murine cells. HIV-1 and murine leukemia virus (MuLV) use \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} and tRNAPro, respectively, as primers for reverse transcription. Selective packaging of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} into HIV-1 produced in human cells is much stronger than that for tRNAPro incorporation into MuLV produced in murine cells, and different packaging mechanisms are used. Thus, both lysyl-tRNA synthetase and GagPol are required for \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} packaging into HIV-1, but neither prolyl-tRNA synthetase nor GagPol is required for tRNAPro packaging into MuLV. In this report, we show that when HIV-1 is produced in murine cells, the virus switches from an HIV-1-like incorporation of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} to an MuLV-like packaging of tRNAPro. The primer binding site in viral RNA remains complementary to \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} , resulting in a significant decrease in reverse transcription and infectivity. Reduction in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} incorporation occurs even though both murine lysyl-tRNA synthetase and HIV-1 GagPol are packaged into the HIV-1 produced in murine cells. Nevertheless, the murine cell is able to support the select incorporation of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} into another retrovirus that uses \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} as a primer, the mouse mammary tumor virus.

scholarly journals Probing the Importance of tRNA Anticodon: Human Immunodeficiency Virus Type 1 (HIV-1) RNA Genome Complementarity with an HIV-1 That Selects tRNAGlu for Replication

2003 ◽  
Vol 77 (16) ◽  
pp. 8756-8764 ◽  
Author(s):  
Lesley C. Dupuy ◽  
Nathan J. Kelly ◽  
Tricia E. Elgavish ◽  
Stephen C. Harvey ◽  
Casey D. Morrow
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Mononuclear Cells ◽  
Primer Binding Site ◽  
Wild Type Virus ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs at the primer binding site (PBS) that is complementary to the 3′-terminal nucleotides of tRNA3 Lys. Why all known strains of HIV-1 select tRNA3 Lys for replication is unknown. Previous studies on the effect of altering the PBS of HIV-1 on replication identified an HIV-1 with a PBS complementary to tRNAGlu. Since the virus was not initially designed to use tRNAGlu, the virus had selected tRNAGlu from the intracellular pool of tRNA for use in replication. Further characterization of HIV-1 that uses tRNAGlu may provide new insights into the preference for tRNA3 Lys. HIV-1 constructed with the PBS complementary to tRNAGlu was more stable than HIV-1 with the PBS complementary to tRNAMet or tRNAHis; however, all of these viruses eventually reverted back to using tRNA3 Lys following growth in SupT1 cells or peripheral blood mononuclear cells (PBMCs). New HIV-1 mutants with nucleotides in U5 complementary to the anticodon of tRNAGlu remained stable when grown in SupT1 cells or PBMCs, although the mutants grew more slowly than the wild-type virus. Sequence analysis of the U5 region and the PBS revealed additional mutations predicted to further promote tRNA-viral genome interaction. The results support the importance of the tRNA anticodon-genome interaction in the selection of the tRNA primer and highlight the fact that unique features of tRNA3 Lys are exploited by HIV-1 for selection as the reverse transcription primer.

scholarly journals Defective Replication in Human Immunodeficiency Virus Type 1 When Non-\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{3}^{\mathrm{Lys}}\) \end{document}Primers Are Used for Reverse Transcription

2005 ◽  
Vol 79 (14) ◽  
pp. 9081-9087 ◽  
Author(s):  
Min Wei ◽  
Shan Cen ◽  
Meijuan Niu ◽  
Fei Guo ◽  
Lawrence Kleiman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Primer Binding Site ◽  
Pcr Analysis ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} , the primer for reverse transcriptase in human immunodeficiency virus type 1 (HIV-1), anneals to the primer binding site (PBS) in HIV-1 RNA. It has been shown that altering the PBS and U5 regions upstream of the PBS in HIV-1 so as to be complementary to sequences in tRNAMet or tRNAHis will allow these tRNA species to be stably used as primers for reverse transcription. We have examined the replication of these mutant viruses in Sup-T1 cells. When Sup-T1 cells are infected by cocultivation with HIV-1-transfected 293T cells, viruses using tRNAHis or tRNAMet are produced at rates that are approximately 1/10 or 1/100, respectively, of rates for wild-type virions that use \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} . When Sup-T1 cells are directly infected with equal amounts of these different viruses isolated from the culture supernatant of transfected 293T cells, virions using tRNAMet are produced at 1/100 the rate of wild-type viruses, and production of virions using tRNAHis is not detected. Both wild-type and mutant virions selectively package tRNALys only, and examination of the ability of total viral RNA to prime reverse transcription in vitro indicates a >80% reduction in the annealing of tRNAHis or tRNAMet to the mutant viral RNAs. PCR analysis of which of the three primer tRNAs is used indicates that only \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} is detected as primer in wild-type virions and only tRNAHis is detected as primer in virions containing a PBS complementary to tRNAHis, while the mutant viruses containing a PBS complementary to tRNAMet use both tRNAMet and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{1,2}^{Lys}\) \end{document} as primer tRNAs.

scholarly journals Preferential Completion of Human Immunodeficiency Virus Type 1 Proviruses Initiated with tRNA3Lys rather than tRNA1,2Lys

1998 ◽  
Vol 72 (7) ◽  
pp. 5464-5471 ◽  
Author(s):  
Zhijun Zhang ◽  
Qin Yu ◽  
Sang-Moo Kang ◽  
James Buescher ◽  
Casey D. Morrow
Keyword(s):  
Human Immunodeficiency Virus ◽  
Single Cell ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Primer Binding Site ◽  
Proviral Dna ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT All retroviral genomes contain a nucleotide sequence designated as the primer binding site (PBS) which is complementary to the tRNA used for initiation of reverse transcription. For human immunodeficiency virus type 1 (HIV-1), all naturally occurring genomes have a PBS complementary to tRNA3 Lys. However, within HIV-1 virions, there are approximately equal amounts of tRNA1 Lys, tRNA2 Lys, and tRNA3 Lys. We have used an endogenous reverse transcription-PCR technique specific for the tRNA species within isolated HIV-1 virions to demonstrate that in addition to tRNA3 Lys, tRNA1 Lys and tRNA2 Lys could be used for initiation of HIV-1 reverse transcription. Using a single-round infection assay which employed an HIV-1 genome with a gpt gene encoding xanthine-guanine phosphoribosyl transferase in place of the env gene, we generated cell lines resistant to mycophenolic acid. Analysis of the U5-PBS from single-cell clones revealed PBS complementary to tRNA3 Lys, not tRNA1 Lys or tRNA2 Lys. A mutant HIV-1 genome was then created which would favor the completion of reverse transcription with tRNA1,2 Lys. Using this provirus in the complementation system, we again found only genomes with a PBS complementary to tRNA3 Lys from proviral DNA isolated fromgpt-resistant single-cell colonies. Finally, infection of cells with a mutant HIV genome with a PBS complementary to tRNA1,2 Lys resulted in gpt- resistant cell colonies which contained integrated provirions with a PBS complementary to tRNA1,2 Lys. The results of these studies suggest that the selection of tRNA3 Lys for initiation of HIV-1 reverse transcription occurs both at the initiation and at a postinitiation step in reverse transcription prior to integration of the proviral DNA.

scholarly journals Genetic Analysis of a Unique Human Immunodeficiency Virus Type 1 (HIV-1) with a Primer Binding Site Complementary to tRNAMet Supports a Role for U5-PBS Stem-Loop RNA Structures in Initiation of HIV-1 Reverse Transcription

1999 ◽  
Vol 73 (3) ◽  
pp. 1818-1827 ◽  
Author(s):  
Sang-Moo Kang ◽  
Casey D. Morrow
Keyword(s):  
Human Immunodeficiency Virus ◽  
In Vitro Culture ◽  
Reverse Transcription ◽  
Primer Binding Site ◽  
Rna Structures ◽  
Stem Loop ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) exclusively uses tRNA3 Lys to initiate reverse transcription. A novel HIV-1 mutant which stably utilizes tRNAMet rather than tRNA3 Lys as a primer was previously identified [HXB2(Met-AC] (S.-M. Kang, Z. Zhang, and C. D. Morrow, J. Virol. 71:207–217, 1997). Comparison of RNA secondary structures of the unique sequence (U5)-primer binding site (PBS) viral RNA genome alone or complexed with tRNAMet of HXB2(Met-AC) revealed structural motifs in common with the U5-PBS of the wild-type virus. In the current study, mutations were constructed to alter the U5-PBS structure and disrupt the U5-PBS-tRNAMet interaction of the virus derived from HXB2(Met-AC). All of the mutant viruses were infectious following transfection and coculture with SupT1 cells. Analysis of the initiation of reverse transcription revealed that some of the mutants were impaired compared to HXB2(Met-AC). The genetic stability of the PBS from each virus was determined following in vitro culture. Two mutant proviral constructs, one predicted to completely disrupt the stem-loop structure in U5 and the other predicted to destabilize contact regions of U5 with tRNAMet, reverted back to contain a PBS complementary to tRNA3 Lys. All other mutants maintained a PBS complementary to tRNAMetafter in vitro culture, although all contained multiple nucleotide substitutions within the U5-PBS from the starting proviral clones. Most interestingly, a viral mutant containing a 32-nucleotide deletion between nucleotides 142 and 173, encompassing regions in U5 which interact with tRNAMet, maintained a PBS complementary to tRNAMet following in vitro culture. All of the proviral clones recovered from this mutant, however, contained an additional 19-nucleotide insertion in U5. RNA modeling of the U5-PBS from this mutant demonstrated that the additional mutations present in U5 following culture restored RNA structures similar to those modeled from HXB2(Met-AC). These results provide strong genetic evidence that multiple sequence and structural elements in U5 in addition to the PBS are involved in the interaction with the tRNA used for initiation of reverse transcription.

scholarly journals Alternative tRNA Priming of Human Immunodeficiency Virus Type 1 Reverse Transcription Explains Sequence Variation in the Primer-Binding Site That Has Been Attributed to APOBEC3G Activity

2005 ◽  
Vol 79 (5) ◽  
pp. 3179-3181 ◽  
Author(s):  
Atze T. Das ◽  
Monique Vink ◽  
Ben Berkhout
Keyword(s):  
Human Immunodeficiency Virus ◽  
Binding Site ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Sequence Variation ◽  
Primer Binding Site ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT It is generally assumed that human immunodeficiency virus type 1 (HIV-1) uses exclusively the cellular \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} molecule as a primer for reverse transcription. We demonstrate that HIV-1 uses not only \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} but also an alternative tRNA primer. This tRNA was termed \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{5}^{Lys}\) \end{document} , and the near completion of the human genome project has allowed the identification of four \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{5}^{Lys}\) \end{document} encoding genes. Priming with \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{5}^{Lys}\) \end{document} results in a single nucleotide polymorphism in the viral primer-binding site that is present in multiple natural and laboratory HIV isolates. This sequence variation was recently attributed to APOBEC3G activity. However, our results show that alternative tRNA priming can cause this mutation in the absence of APOBEC3G.

scholarly journals Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity

2020 ◽  
Vol 22 (1) ◽  
pp. 58
Author(s):  
Thomas Gremminger ◽  
Zhenwei Song ◽  
Juan Ji ◽  
Avery Foster ◽  
Kexin Weng ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Potential Interaction ◽  
Viral Rna ◽  
Primer Binding Site ◽  
Rna Integrity ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Extended Interaction ◽  
Hiv 1

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3′-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

scholarly journals Kinetics of Human Immunodeficiency Virus Type 1 Decay following Entry into Resting CD4+ T Cells

2005 ◽  
Vol 79 (4) ◽  
pp. 2199-2210 ◽  
Author(s):  
Yan Zhou ◽  
Haili Zhang ◽  
Janet D. Siliciano ◽  
Robert F. Siliciano
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Human Immunodeficiency Virus Type ◽  
Reverse Transcription ◽  
Half Life ◽  
Immunodeficiency Virus ◽  
Kinetics Of ◽  
Hiv 1

ABSTRACT In untreated human immunodeficiency virus type 1 (HIV-1) infection, most viral genomes in resting CD4+ T cells are not integrated into host chromosomes. This unintegrated virus provides an inducible latent reservoir because cellular activation permits integration, virus gene expression, and virus production. It remains controversial whether HIV-1 is stable in this preintegration state. Here, we monitored the fate of HIV-1 in resting CD4+ cells by using a green fluorescent protein (GFP) reporter virus carrying an X4 envelope. After virus entry into resting CD4+ T cells, both rescuable virus gene expression, visualized with GFP, and rescuable virion production, assessed by p24 release, decayed with a half-life of 2 days. In these cells, reverse transcription goes to completion over 2 to 3 days, and 50% of the viruses that have entered undergo functional decay before reverse transcription is complete. We distinguished two distinct but closely related factors contributing to loss of rescuable virus. First, some host cells undergo virus-induced apoptosis upon viral entry, thereby reducing the amount of rescuable virus. Second, decay processes directly affecting the virus both before and after the completion of reverse transcription contribute to the loss of rescuable virus. The functional half-life of full-length, integration-competent reverse transcripts is only 1 day. We propose that rapid intracellular decay processes compete with early steps in viral replication in infected CD4+ T cells. Decay processes dominate in resting CD4+ T cells as a result of the slow kinetics of reverse transcription and blocks at subsequent steps. Therefore, the reservoir of unintegrated HIV-1 in recently infected resting CD4+ T cells is highly labile.

scholarly journals Determination of the Ex Vivo Rates of Human Immunodeficiency Virus Type 1 Reverse Transcription by Using Novel Strand-Specific Amplification Analysis

2007 ◽  
Vol 81 (9) ◽  
pp. 4798-4807 ◽  
Author(s):  
David C. Thomas ◽  
Yegor A. Voronin ◽  
Galina N. Nikolenko ◽  
Jianbo Chen ◽  
Wei-Shau Hu ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Reverse Transcription ◽  
Ex Vivo ◽  
Dna Transfer ◽  
Minus Strand ◽  
Immunodeficiency Virus ◽  
Specific Amplification ◽  
Kinetics Of ◽  
Hiv 1

ABSTRACT Replication of human immunodeficiency virus type 1 (HIV-1), like all organisms, involves synthesis of a minus-strand and a plus-strand of nucleic acid. Currently available PCR methods cannot distinguish between the two strands of nucleic acids. To carry out detailed analysis of HIV-1 reverse transcription from infected cells, we have developed a novel strand-specific amplification (SSA) assay using single-stranded padlock probes that are specifically hybridized to a target strand, ligated, and quantified for sensitive analysis of the kinetics of HIV-1 reverse transcription in cells. Using SSA, we have determined for the first time the ex vivo rates of HIV-1 minus-strand DNA synthesis in 293T and human primary CD4+ T cells (∼68 to 70 nucleotides/min). We also determined the rates of minus-strand DNA transfer (∼4 min), plus-strand DNA transfer (∼26 min), and initiation of plus-strand DNA synthesis (∼9 min) in 293T cells. Additionally, our results indicate that plus-strand DNA synthesis is initiated at multiple sites and that several reverse transcriptase inhibitors influence the kinetics of minus-strand DNA synthesis differently, providing insights into their mechanism of inhibition. The SSA technology provides a novel approach to analyzing DNA replication processes and should facilitate the development of new antiretroviral drugs that target specific steps in HIV-1 reverse transcription.

scholarly journals 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

2006 ◽  
Vol 80 (23) ◽  
pp. 11710-11722 ◽  
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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