scholarly journals The Role of Nucleocapsid and U5 Stem/A-Rich Loop Sequences in tRNA3Lys Genomic Placement and Initiation of Reverse Transcription in Human Immunodeficiency Virus Type 1

1998 ◽  
Vol 72 (5) ◽  
pp. 3907-3915 ◽  
Author(s):  
Yue Huang ◽  
Ahmad Khorchid ◽  
Juliana Gabor ◽  
Jing Wang ◽  
Xuguang Li ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Wild Type ◽  
Extracellular Virus ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We have studied the effect of mutations in the human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) sequence on tRNA3 Lys genomic placement, i.e., the in vivo placement of primer tRNA3 Lys on the HIV-1 primer binding site (PBS). HIV-1 produced from COS cells transfected with wild-type or mutant proviral DNA was used in this study. We have found that mutations in the amino acid sequences flanking the first Cys-His box in the NC sequence produce the maximum inhibition of genomic placement. A similar finding was obtained when the NC-facilitated annealing of primer tRNA3 Lys to the HIV PBS in vitro was studied. However, since the genomic placement of tRNA3 Lys occurs independently of precursor protein processing, the NC mutations studied here have probably exerted their effect through one or both of the precursor proteins, Pr55 gag and/or Pr160 gag-pol . One mutation in the linker region between the two Cys-His boxes, P31L, prevented packaging of both Pr160 gag-pol and tRNA3 Lys and prevented the genomic placement of tRNA3 Lys. Both packaging and genomic placement were rescued by cotransfection with a plasmid coding for wild-type Pr160 gag-pol . For other linker mutations [R7R10K11 S, R32G, and S3(32-34)], packaging of Pr160 gag-pol and tRNA3 Lyswas not affected, but genomic placement was, and placement could not be rescued by cotransfection with plasmids coding for either Pr55 gag or Pr160 gag-pol . After placement, the initiation of reverse transcription within extracellular virions is characterized by a 2-base DNA extension of the placed tRNA3 Lys. This process requires precursor processing, and those NC mutations which showed the most inhibition of initiation were in either of the two NC Cys-His boxes. Destabilization of a U5 stem-A-rich loop immediately upstream of the PBS (through deletion of four consecutive A’s in the loop) did not affect the in vivo genomic placement of tRNA3 Lys but resulted in the presence in the extracellular virus of longer cDNA extensions of tRNA3 Lys, with a corresponding decrease in the presence of unextended and 2-base-extended tRNA3 Lys.

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 Human Immunodeficiency Virus Type 1 Nucleocapsid Zn2+ Fingers Are Required for Efficient Reverse Transcription, Initial Integration Processes, and Protection of Newly Synthesized Viral DNA

2003 ◽  
Vol 77 (2) ◽  
pp. 1469-1480 ◽  
Author(s):  
James S. Buckman ◽  
William J. Bosche ◽  
Robert J. Gorelick
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Full Length ◽  
Viral Dna ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) containing mutations in the nucleocapsid (NC) Zn2+ finger domains have greatly reduced infectivity, even though genome packaging is largely unaffected in certain cases. To examine replication defects, viral DNA (vDNA) was isolated from cells infected with viruses containing His-to-Cys changes in their Zn2+ fingers (NCH23C and NCH44C), an integrase mutant (IND116N), a double mutant (NCH23C/IND116N), or wild-type HIV-1. In vitro assays have established potential roles for NC in reverse transcription and integration. In vivo results for these processes were obtained by quantitative PCR, cloning of PCR products, and comparison of the quantity and composition of vDNA generated at discrete points during reverse transcription. Quantitative analysis of the reverse transcription intermediates for these species strongly suggests decreased stability of the DNA produced. Both Zn2+ finger mutants appear to be defective in DNA synthesis, with the minus- and plus-strand transfer processes being affected while interior portions of the vDNA remain more intact. Sequences obtained from PCR amplification and cloning of 2-LTR circle junction fragments revealed that the NC mutants had a phenotype similar to the IN mutant; removal of the terminal CA dinucleotides necessary for integration of the vDNA is disabled by the NC mutations. Thus, the loss of infectivity in these NC mutants in vivo appears to result from defective reverse transcription and integration processes stemming from decreased protection of the full-length vDNA. Finally, these results indicate that the chaperone activity of NC extends from the management of viral RNA through to the full-length vDNA.

scholarly journals Mutational Analysis of the C-Terminal Gag Cleavage Sites in Human Immunodeficiency Virus Type 1

2007 ◽  
Vol 81 (18) ◽  
pp. 10047-10054 ◽  
Author(s):  
Lori V. Coren ◽  
James A. Thomas ◽  
Elena Chertova ◽  
Raymond C. Sowder ◽  
Tracy D. Gagliardi ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Mutational Analysis ◽  
Cleavage Sites ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Gag is expressed as a polyprotein that is cleaved into six proteins by the viral protease in a maturation process that begins during assembly and budding. While processing of the N terminus of Gag is strictly required for virion maturation and infectivity, the necessity for the C-terminal cleavages of Gag is less well defined. To examine the importance of this process, we introduced a series of mutations into the C terminus of Gag that interrupted the cleavage sites that normally produce in the nucleocapsid (NC), spacer 2 (SP2), or p6Gag proteins. Protein analysis showed that all of the mutant constructs produced virions efficiently upon transfection of cells and appropriately processed Gag polyprotein at the nonmutated sites. Mutants that produced a p9NC/SP2 protein exhibited only minor effects on HIV-1 infectivity and replication. In contrast, mutants that produced only the p8SP2/p6 or p15NC/SP2/p6 protein had severe defects in infectivity and replication. To identify the key defective step, we quantified reverse transcription and integration products isolated from infected cells by PCR. All mutants tested produced levels of reverse transcription products either similar to or only somewhat lower than that of wild type. In contrast, mutants that failed to cleave the SP2-p6Gag site produced drastically less provirus than the wild type. Together, our results show that processing of the SP2-p6Gag and not the NC-SP2 cleavage site is important for efficient viral DNA integration during infection in vitro. In turn, this finding suggests an important role for the p9NC/SP2 species in some aspect of integration.

scholarly journals Human Immunodeficiency Virus Type 1 (HIV-1) Viral Protein R (Vpr) Interacts with Lys-tRNA Synthetase: Implications for Priming of HIV-1 Reverse Transcription

1998 ◽  
Vol 72 (4) ◽  
pp. 3037-3044 ◽  
Author(s):  
Lesley A. Stark ◽  
Ronald T. Hay
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Viral Protein ◽  
Trna Synthetase ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a 96-amino-acid 14-kDa protein (viral protein R [Vpr]), which is produced late in the viral life cycle and is incorporated into the virion. Although Vpr is not required for viral replication in transformed cell lines and primary T lymphocytes, it is essential for productive infection of macrophages and monocytes and appears to be important for pathogenesis in vivo. To establish the role of Vpr in HIV-1 replication and pathogenesis, we have isolated cellular proteins with which Vpr interacts. By using the yeast two-hybrid system, Lys-tRNA synthetase (LysRS) was identified as a Vpr-interacting protein. The interaction between Vpr and LysRS was characterized both in vitro and in vivo, and the domains of Vpr required for the interaction were defined. In the presence of Vpr, LysRS-mediated aminoacylation of tRNALys is inhibited. Since tRNALys is the primer for reverse transcription of the HIV-1 genome, this suggests that the interaction between Vpr and LysRS may influence the initiation of HIV-1 reverse transcription.

scholarly journals Restoration of Wild-Type Infectivity to Human Immunodeficiency Virus Type 1 Strains Lacking nef by Intravirion Reverse Transcription

2001 ◽  
Vol 75 (24) ◽  
pp. 12081-12087 ◽  
Author(s):  
Mahfuz Khan ◽  
Minerva Garcia-Barrio ◽  
Michael D. Powell
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Envelope Protein ◽  
Wild Type ◽  
Virus Envelope ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Nef protein exerts several effects, both on infected cells and as a virion protein, which work together to enhance viral replication. One of these activities is the ability to enhance infectivity and the formation of proviral DNA. The mechanism of this enhancement remains incompletely understood. We show that virions with nef deleted can be restored to wild-type infectivity by stimulating intravirion reverse transcription. Particle composition and measures of reverse transcriptase activity remain the same for Nef+ and Nef− virions both before and after natural endogenous reverse transcription (NERT) treatment. The effect of NERT treatment on virions pseudotyped with murine leukemia virus envelope protein was similar to that on particles pseudotyped with HIV-1 envelope protein. However, virions pseudotyped with vesicular stomatitis virus G envelope protein showed no influence of Nef on NERT enhancement of infectivity. These observations suggest that Nef may function at a level prior to reverse transcription. Since NERT treatment results in partial disassembly of the viral core, we speculate that Nef may function at the level of core particle disassembly.

scholarly journals Requirement for Integrase during Reverse Transcription of Human Immunodeficiency Virus Type 1 and the Effect of Cysteine Mutations of Integrase on Its Interactions with Reverse Transcriptase

2004 ◽  
Vol 78 (10) ◽  
pp. 5045-5055 ◽  
Author(s):  
Kai Zhu ◽  
Charles Dobard ◽  
Samson A. Chow
Keyword(s):  
Human Immunodeficiency Virus ◽  
Life Cycle ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Retroviral integrase catalyzes the essential step of integrating a double-stranded DNA copy of the viral genome into a host cell chromosome. Mutational studies have revealed that integrase is involved in additional steps of viral replication, but the mechanism for the pleiotropic effect is not well characterized. Since Cys residues generally play crucial roles in protein structure and function, we introduced Cys-to-Ser substitutions at positions 56, 65, and 130 of human immunodeficiency virus type 1 (HIV-1) integrase to determine their effects on integration activity and viral replication. None of the substitutions significantly affected the enzymatic activities in vitro. When introduced into the NL4-3 molecular clone of HIV-1, mutant viruses encoding Cys mutations at positions 56 and 65 of integrase replicated similarly to the wild-type virus in CD4+-T-cell lines, whereas the C130S-containing virus was noninfectious. The entry and postintegration steps of the viral life cycle for all mutant viruses were normal, and all had particle-associated reverse transcriptase (RT) activity. However, early reverse-transcribed DNA products were absent in the lysate of cells infected with the C130S mutant virus, indicating that the mutation abolished the ability of the virus to initiate endogenous reverse transcription. Coimmunoprecipitation using purified integrase and RT showed that the C-terminal domain of wild-type HIV-1 integrase interacted with RT. The interaction between integrase and RT was not affected in the presence of a reducing or alkylating agent, suggesting that the interaction did not involve a disulfide linkage. The C130S substitution within the core region may disrupt the protein recognition interface of the C-terminal domain and abolish its ability to interact with RT. Our results indicate that integrase plays an important role during the reverse-transcription step of the viral life cycle, possibly through physical interactions with RT.

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

2008 ◽  
Vol 82 (19) ◽  
pp. 9318-9328 ◽  
Author(s):  
James A. Thomas ◽  
William J. Bosche ◽  
Teresa L. Shatzer ◽  
Donald G. Johnson ◽  
Robert J. Gorelick
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Nucleocapsid Protein ◽  
Productive Infection ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT 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 Pr55Gag processing. Critically, our results also suggest that the presence of intravirion vDNA could serve as a diagnostic for identifying replication-defective HIV-1.

scholarly journals Functional Domains of Tat Required for Efficient Human Immunodeficiency Virus Type 1 Reverse Transcription

1999 ◽  
Vol 73 (3) ◽  
pp. 2499-2508 ◽  
Author(s):  
Catherine Ulich ◽  
Amanda Dunne ◽  
Emma Parry ◽  
C. William Hooker ◽  
Richard B. Gaynor ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Tat expression is required for efficient human immunodeficiency virus type 1 (HIV-1) reverse transcription. In the present study, we generated a series of 293 cell lines that contained a provirus with atat gene deletion (Δtat). Cell lines that contained Δtat and stably transfected vectors containing either wild-type tat or a number of tat mutants were obtained so that the abilities of these tat genes to stimulate HIV-1 gene expression and reverse transcription could be compared. tat genes with mutations in the amino terminus did not stimulate either viral gene expression or HIV-1 reverse transcription. In contrast, tat mutants in the activation, core, and basic domains of Tat did not stimulate HIV-1 gene expression but markedly stimulated HIV-1 reverse transcription. No differences in the levels of virion genomic RNA or tRNA3 Lys were seen in the HIV-1 Δtat viruses complemented with either mutant or wild-type tat. Finally, overexpression of the Tat-associated kinases CDK7 and CDK9, which are involved in Tat activation of HIV-1 transcription, was not able to complement the reverse transcription defects associated with the lack of a functionaltat gene. These results indicate that the mechanism by which tat modulates HIV-1 reverse transcription is distinct from its ability to activate HIV-1 gene expression.

scholarly journals Allogeneic leukocytes but not therapeutic blood elements induce reactivation and dissemination of latent human immunodeficiency virus type 1 infection: implications for transfusion support of infected patients

Blood ◽  
1992 ◽  
Vol 80 (8) ◽  
pp. 2128-2135 ◽  
Author(s):  
MP Busch ◽  
TH Lee ◽  
J Heitman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Blood Components ◽  
Route Of Transmission ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Donor Lymphocytes

Abstract Various immunologic stimuli and heterologous viral regulatory elements have been shown to increase susceptibility to, and replication of, human immunodeficiency virus type 1 (HIV-1) in lymphocytes and monocytes in vitro. Transfusion of allogeneic blood components from heterologous donors constitutes a profound immunologic stimulus to the recipient, in addition to being a potential route of transmission of lymphotropic viral infections. To investigate the hypothesis that transfusions, and particularly those containing leukocytes, activate HIV-1 replication in infected recipient cells, we cocultured peripheral blood mononuclear cells (PBMC) from three anti-HIV-1-positive individuals with allogeneic donor PBMC, as well as partially purified populations of donor lymphocytes, monocytes, granulocytes, platelets, and red blood cells (RBC) and allogeneic cell-free plasma. Allogeneic PBMC induced a dose-related activation of HIV-1 expression in in vivo infected cells, followed by dissemination of HIV-1 to previously uninfected patient cells. Activation of HIV-1 replication was observed with donor lymphocytes, monocytes, and granulocytes, whereas no effect was seen with leukocyte-depleted RBC, platelets, or plasma (ie, therapeutic blood constituents). Allogeneic donor PBMC were also shown to upregulate HIV-1 expression in a “latently” infected cell line, and to increase susceptibility of heterologous donor PBMC to acute HIV-1 infection. Studies should be performed to evaluate whether transfusions of leukocyte-containing blood components accelerate HIV-1 dissemination and disease progression in vivo. If so, HIV-1-infected patients should be transfused as infrequently as possible and leukocyte-depleted (filtered) blood components should be used to avoid this complication.

scholarly journals The Interaction of Vpr with Uracil DNA Glycosylase Modulates the Human Immunodeficiency Virus Type 1 In Vivo Mutation Rate

2000 ◽  
Vol 74 (15) ◽  
pp. 7039-7047 ◽  
Author(s):  
Louis M. Mansky ◽  
Sandra Preveral ◽  
Luc Selig ◽  
Richard Benarous ◽  
Serge Benichou
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mutation Rate ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Dna Glycosylase ◽  
Uracil Dna Glycosylase ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The Vpr protein of human immunodeficiency virus type 1 (HIV-1) influences the in vivo mutation rate of the virus. Since Vpr interacts with a cellular protein implicated in the DNA repair process, uracil DNA glycosylase (UNG), we have explored the contribution of this interaction to the mutation rate of HIV-1. Single-amino-acid variants of Vpr were characterized for their differential UNG-binding properties and used to trans complement vpr null mutant HIV-1. A striking correlation was established between the abilities of Vpr to interact with UNG and to influence the HIV-1 mutation rate. We demonstrate that Vpr incorporation into virus particles is required to influence the in vivo mutation rate and to mediate virion packaging of the nuclear form of UNG. The recruitment of UNG into virions indicates a mechanism for how Vpr can influence reverse transcription accuracy. Our data suggest that distinct mechanisms evolved in primate and nonprimate lentiviruses to reconcile uracil misincorporation into lentiviral DNA.

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