scholarly journals The Interaction of APOBEC3G with Human Immunodeficiency Virus Type 1 Nucleocapsid Inhibits tRNA3Lys Annealing to Viral RNA

2007 ◽  
Vol 81 (20) ◽  
pp. 11322-11331 ◽  
Author(s):  
Fei Guo ◽  
Shan Cen ◽  
Meijuan Niu ◽  
Yiliang Yang ◽  
Robert J. Gorelick ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Transcription Initiation ◽  
Rna Binding ◽  
Viral Rna ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) containing human APOBEC3G (hA3G) has a reduced ability to produce viral DNA in newly infected cells. At least part of this hA3G-facilitated inhibition is due to a cytidine deamination-independent reduction in the ability to initiate reverse transcription. HIV-1 nucleocapsid (NCp7) is required both for the incorporation of hA3G into virions and for the annealing between viral RNA and tRNA3 Lys, the primer tRNA for reverse transcription. Herein we present evidence that the interaction of hA3G with nucleocapsid is required for the inhibition of reverse transcription initiation. A tRNA3 Lys priming complex was produced in vitro by the NCp7-facilitated annealing of tRNA3 Lys to synthetic viral RNA in the absence or presence of hA3G. The effect of hA3G on the annealing of tRNA3 Lys to viral RNA and the ability of tRNA3 Lys to initiate reverse transcription was measured. Our results show the following. (i) Electrophoretic band shift and primer binding site assays show that hA3G reduces the annealing of tRNA3 Lys 44 and 60%, respectively, but does not disrupt the annealed complex once formed. (ii) hA3G inhibits tRNA3 Lys priming 70 to 80%. (iii) Inhibition of tRNA3 Lys priming by hA3G requires an interaction between hA3G and NCp7 during annealing. Thus, annealing of tRNA3 Lys is insensitive to hA3G inhibition when facilitated by a zinc finger mutant of NCp7 unable to interact with hA3G. NCp7-independent annealing of DNA to viral RNA also is insensitive to hA3G inhibition. These results indicate that hA3G does not sterically block tRNA3 Lys annealing by binding to viral RNA. Annealing and priming are not affected by another RNA binding protein, QKI-6.

scholarly journals The Tat Protein of Human Immunodeficiency Virus Type 1 (HIV-1) Can Promote Placement of tRNA Primer onto Viral RNA and Suppress Later DNA Polymerization in HIV-1 Reverse Transcription

2002 ◽  
Vol 76 (8) ◽  
pp. 3637-3645 ◽  
Author(s):  
Masanori Kameoka ◽  
Max Morgan ◽  
Marc Binette ◽  
Rodney S. Russell ◽  
Liwei Rong ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Viral Rna ◽  
Dna Polymerization ◽  
Functional Homology ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type-1 Tat has been proposed to play a role in the regulation of reverse transcription. We previously demonstrated that wild-type Tat can augment viral infectivity by suppressing the reverse transcriptase (RT) reaction at late stages of the viral life cycle in order to prevent the premature synthesis of potentially deleterious viral DNA products. Here we have performed a detailed analysis of the cell-free reverse transcription reaction to elucidate the mechanism(s) whereby Tat can affect this process. Our results show that Tat can suppress nonspecific DNA elongation while moderately affecting the specific initiation stage of reverse transcription. In addition, Tat has an RNA-annealing activity and can promote the placement of tRNA onto viral RNA. This points to a functional homology between Tat and the viral nucleocapsid (NC) protein that is known to be directly involved in this process. Experiments using a series of mutant Tat proteins revealed that the cysteine-rich and core domains of Tat are responsible for suppression of DNA elongation, while each of the cysteine-rich, core, and basic domains, as well as a glutamine-rich region in the C-terminal domain, are important for the placement of tRNA onto the viral RNA genome. These results suggest that Tat can play at least two different roles in the RT reaction, i.e., suppression of DNA polymerization and placement of tRNA onto viral RNA. We believe that the first of these activities of Tat may contribute to the overall efficiency of reverse transcription of the viral genome during a new round of infection as well as to enhanced production of infectious viral particles. We hypothesize that the second activity, illustrating functional homology between Tat and NC, suggests a potential role for NC in the displacement of Tat during viral maturation.

scholarly journals Dissociation of Genome Dimerization from Packaging Functions and Virion Maturation of Human Immunodeficiency Virus Type 1

2002 ◽  
Vol 76 (3) ◽  
pp. 959-967 ◽  
Author(s):  
Jun-ichi Sakuragi ◽  
Aikichi Iwamoto ◽  
Tatsuo Shioda
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Rna ◽  
Rna Packaging ◽  
Virus Particles ◽  
Immunodeficiency Virus ◽  
Rna Interaction ◽  
Hiv 1

ABSTRACT The dimer initiation site/dimer linkage sequence (DIS/DLS) region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is thought to play important roles at various stages of the virus life cycle. Recently we showed that the DIS/DLS region affects RNA-RNA interaction in intact virus particles, by demonstrating that duplication of the region in viral RNA caused the production of virus particles containing partially monomeric RNAs. We have extended this finding and succeeded for the first time in creating mutant particles which contain only monomeric RNAs without modifying any viral proteins. In terms of RNA encapsidation ability, virion density, and protein processing, the mutant particles were comparable to wild-type particles. The level of production of viral DNA by the mutant virus construct in infected cells was also comparable to that of the constructs that produced exclusively dimeric RNA, indicating that monomeric viral RNA could be the template for strand transfer. These results indicated that the RNA dimerization of HIV-1 could be separated from viral RNA packaging and was not absolutely required for RNA packaging, virion maturation, and reverse transcription.

scholarly journals Cholesterol Depletion of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus with β-Cyclodextrin Inactivates and Permeabilizes the Virions: Evidence for Virion-Associated Lipid Rafts

2003 ◽  
Vol 77 (15) ◽  
pp. 8237-8248 ◽  
Author(s):  
David R. M. Graham ◽  
Elena Chertova ◽  
Joanne M. Hilburn ◽  
Larry O. Arthur ◽  
James E. K. Hildreth
Keyword(s):  
Human Immunodeficiency Virus ◽  
Lipid Rafts ◽  
Simian Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Rna ◽  
Host Proteins ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Recent evidence suggests that human immunodeficiency virus type 1 (HIV-1) particles assemble and bud selectively through areas in the plasma membrane of cells that are highly enriched with glycosylphosphatidylinositol-anchored proteins and cholesterol, called lipid rafts. Since cholesterol is required to maintain lipid raft structure and function, we proposed that virion-associated cholesterol removal with the compound 2-hydroxy-propyl-β-cyclodextrin (β-CD) might be disruptive to HIV-1 and simian immunodeficiency virus (SIV). We examined the effect of β-CD on the structure and infectivity of cell-free virions. We found that β-CD inactivated HIV-1 and SIV in a dose-dependent manner and permeabilized the viral membranes, resulting in the loss of mature Gag proteins (capsid, matrix, nucleocapsid, p1, and p6) without loss of the envelope glycoproteins. SIV also lost reverse transcriptase (RT), integrase (IN), and viral RNA. IN appeared to be only slightly diminished in HIV-1, and viral RNA, RT, matrix, and nucleocapsid proteins were retained in HIV-1 but to a much lesser degree. Host proteins located internally in the virus (actin, moesin, and ezrin) and membrane-associated host proteins (major histocompatibility complex classes I and II) remained associated with the treated virions. Electron microscopy revealed that under conditions that permeabilized the viruses, holes were present in the viral membranes and the viral core structure was perturbed. These data provide evidence that an intact viral membrane is required to maintain mature virion core integrity. Since the viruses were not fixed before β-CD treatment and intact virion particles were recovered, the data suggest that virions may possess a protein scaffold that can maintain overall structure despite disruptions in membrane integrity.

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.

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

2002 ◽  
Vol 76 (15) ◽  
pp. 7897-7902 ◽  
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.

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 Viral RNA Is Required for the Association of APOBEC3G with Human Immunodeficiency Virus Type 1 Nucleoprotein Complexes

2005 ◽  
Vol 79 (9) ◽  
pp. 5870-5874 ◽  
Author(s):  
Mohammad A. Khan ◽  
Sandra Kao ◽  
Eri Miyagi ◽  
Hiroaki Takeuchi ◽  
Ritu Goila-Gaur ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cytidine Deaminase ◽  
Viral Rna ◽  
Target Sequence ◽  
Immunodeficiency Virus ◽  
Nucleoprotein Complexes ◽  
Hiv 1

ABSTRACT APOBEC3G (APO3G) is a host cytidine deaminase that is incorporated into human immunodeficiency virus type 1 (HIV-1) particles. We report here that viral RNA promotes stable association of APO3G with HIV-1 nucleoprotein complexes (NPC). A target sequence located within the 5′-untranslated region of the HIV-1 RNA was identified to be necessary and sufficient for efficient APO3G packaging. Fine mapping revealed a sequence normally involved in viral genomic RNA dimerization and Gag binding to be important for APO3G packaging and association with viral NPC. Our data suggest that packaging of APO3G into HIV-1 NPC is enhanced by viral RNA.

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

2003 ◽  
Vol 77 (5) ◽  
pp. 3020-3030 ◽  
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.

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 Transcriptional Activation of the Integrated Chromatin-Associated Human Immunodeficiency Virus Type 1 Promoter

1998 ◽  
Vol 18 (5) ◽  
pp. 2535-2544 ◽  
Author(s):  
Aboubaker El Kharroubi ◽  
Graziella Piras ◽  
Ralf Zensen ◽  
Malcolm A. Martin
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Chromatin Structure ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The regulation of human immunodeficiency virus type 1 (HIV-1) gene expression involves a complex interplay between cellular transcription factors, chromatin-associated proviral DNA, and the virus-encoded transactivator protein, Tat. Here we show that Tat transactivates the integrated HIV-1 long terminal repeat (LTR), even in the absence of detectable basal promoter activity, and this transcriptional activation is accompanied by chromatin remodeling downstream of the transcription initiation site, as monitored by increased accessibility to restriction endonucleases. However, with an integrated promoter lacking both Sp1 and NF-κB sites, Tat was unable to either activate transcription or induce changes in chromatin structure even when it was tethered to the HIV-1 core promoter upstream of the TATA box. Tat responsiveness was observed only when Sp1 or NF-κB was bound to the promoter, implying that Tat functions subsequent to the formation of a specific transcription initiation complex. Unlike Tat, NF-κB failed to stimulate the integrated transcriptionally silent HIV-1 promoter. Histone acetylation renders the inactive HIV-1 LTR responsive to NF-κB, indicating that a suppressive chromatin structure must be remodeled prior to transcriptional activation by NF-κB. Taken together, these results suggest that Sp1 and NF-κB are required for the assembly of transcriptional complexes on the integrated viral promoter exhibiting a continuum of basal activities, all of which are fully responsive to Tat.

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