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

1997 ◽  
Vol 8 (1) ◽  
pp. 60-69 ◽  
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.

scholarly journals Inhibition of multiple phases of human immunodeficiency virus type 1 replication by a dithiane compound that attacks the conserved zinc fingers of retroviral nucleocapsid proteins.

1997 ◽  
Vol 41 (2) ◽  
pp. 419-426 ◽  
Author(s):  
W G Rice ◽  
D C Baker ◽  
C A Schaeffer ◽  
L Graham ◽  
M Bu ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Zinc Finger ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Zinc Fingers ◽  
Immunodeficiency Virus ◽  
Zinc Finger Domains ◽  
Hiv 1

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid p7 protein contains two retrovirus-type zinc finger domains that are required for multiple phases of viral replication. Chelating residues (three Cys residues and one His residue) of the domains are absolutely conserved among all strains of HIV-1 and other retroviruses, and mutations in these residues in noninfectious virions. These properties establish the zinc finger domains as logical targets for antiviral chemotherapy. Selected dithiobis benzamide (R-SS-R) compounds were previously found to inhibit HIV-1 replication by mediating an electrophilic attack on the zinc fingers. Unfortunately, reaction of these disulfide-based benzamides with reducing agents yields two monomeric structures (two R-SH structures) that can dissociated and no longer react with the zinc fingers, suggesting that in vivo reduction would inactivate the compounds. Through an extensive drug discovery program of the National Cancer Institute, a nondissociable tethered dithiane compound (1,2-dithiane-4,5-diol, 1,1-dioxide, cis; NSC 624151) has been identified. This compound specifically attacks the retroviral zinc fingers, but not other antiviral targets. The lead compound demonstrated broad antiretroviral activity, ranging from field isolates and drug-resistant strains of HIV-1 to HIV-2 and simian immunodeficiency virus. The compound directly inactivated HIV-1 virions and blocked production of infectious virus from cells harboring integrated proviral DNA. NSC 624151 provides a scaffold from which medicinal chemists can develop novel compounds for the therapeutic treatment of HIV infection.

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

2000 ◽  
Vol 74 (18) ◽  
pp. 8324-8334 ◽  
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.

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 The Human Factors YY1 and LSF Repress the Human Immunodeficiency Virus Type 1 Long Terminal Repeat via Recruitment of Histone Deacetylase 1

2000 ◽  
Vol 74 (15) ◽  
pp. 6790-6799 ◽  
Author(s):  
Jason J. Coull ◽  
Fabio Romerio ◽  
Jian-Min Sun ◽  
Janet L. Volker ◽  
Katherine M. Galvin ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Long Terminal Repeat ◽  
Histone Deacetylase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Zinc Fingers ◽  
Terminal Repeat ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Enigmatic mechanisms restore the resting state in activated lymphocytes following human immunodeficiency virus type 1 (HIV-1) infection, rarely allowing persistent nonproductive infection. We detail a mechanism whereby cellular factors could establish virological latency. The transcription factors YY1 and LSF cooperate in repression of transcription from the HIV-1 long terminal repeat (LTR). LSF recruits YY1 to the LTR via the zinc fingers of YY1. The first two zinc fingers were observed to be sufficient for this interaction in vitro. A mutant of LSF incapable of binding DNA blocked repression. Like other transcriptional repressors, YY1 can function via recruitment of histone deacetylase (HDAC). We find that HDAC1 copurifies with the LTR-binding YY1-LSF repressor complex, the domain of YY1 that interacts with HDAC1 is required to repress the HIV-1 promoter, expression of HDAC1 augments repression of the LTR by YY1, and the deacetylase inhibitor trichostatin A blocks repression mediated by YY1. This novel link between HDAC recruitment and inhibition of HIV-1 expression by YY1 and LSF, in the natural context of a viral promoter integrated into chromosomal DNA, is the first demonstration of a molecular mechanism of repression of HIV-1. YY1 and LSF may establish transcriptional and virological latency of HIV, a state that has recently been recognized in vivo and has significant implications for the long-term treatment of AIDS.

scholarly journals Characterization of Restrictions to Human Immunodeficiency Virus Type 1 Infection of Monocytes

2004 ◽  
Vol 78 (10) ◽  
pp. 5523-5527 ◽  
Author(s):  
Karine Triques ◽  
Mario Stevenson
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Immunodeficiency Virus ◽  
Exogenous Addition ◽  
Thymidine Phosphorylase Activity ◽  
Hiv 1

ABSTRACT Tissue macrophages are an important cellular reservoir for replication of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus. In vitro, the ability of macrophages to support viral replication is differentiation dependent in that precursor monocytes are refractory to infection. There is, however, no consensus as to the exact point at which infection is restricted in monocytes. We have revisited this issue and have compared the efficiencies of early HIV-1 replication events in monocytes and in differentiated macrophages. Although virus entry in monocytes was comparable to that in differentiated macrophages, synthesis of full-length viral cDNAs was very inefficient. Relative to differentiated macrophages, monocytes contained low levels of dTTP due to low thymidine phosphorylase activity. Exogenous addition of d-thymidine increased dTTP levels to that in differentiated macrophages but did not correct the reverse transcription defect. These results point to a restriction in monocytes that is independent of reverse transcription precursors and suggest that differentiation-dependent cellular cofactors of reverse transcription are rate limiting in monocytes.

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.

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