Low-level alternative tRNA priming of reverse transcription of HIV-1 and SIV in vivo

ABSTRACT Several studies have indicated that the genetic diversity of human T-cell leukemia virus type 1 (HTLV-1), a virus associated with adult T-cell leukemia, is significantly lower than that of other retroviruses, including that of human immunodeficiency virus type 1 (HIV-1). To test whether HTLV-1 variation is lower than other retroviruses, a tractable vector system has been developed to measure reverse transcription accuracy in one round of HTLV-1 replication. This system consists of a HTLV-1 vector that contains a cassette with the neomycin phosphotransferase (neo) gene, a bacterial origin of DNA replication, and the lacZα peptide gene region (the mutational target). The vector was replicated bytrans-complementation with helper plasmids. The in vivo mutation rate for HTLV-1 was determined to be 7 × 10−6 mutations per target base pair per replication cycle. The majority of the mutations identified were base substitution mutations, namely, G-to-A and C-to-T transitions, frameshift mutations, and deletion mutations. Mutation of the methionine residue in the conserved YMDD motif of the HTLV-1 reverse transcriptase to either alanine or valine (i.e., M188A or M188V) led to a factor of two increase in the rate of mutation, indicating the role of this motif in enzyme accuracy. The HTLV-1 in vivo mutation rate is comparable to that of bovine leukemia virus (BLV), another member of the HTLV/BLV genus of retroviruses, and is about fourfold lower than that of HIV-1. These observations indicate that while the mutation rate of HTLV-1 is significantly lower than HIV-1, this lower rate alone would not explain the low diversity in HTLV-1 isolates, supporting the hypothesis that HTLV-1 replicates primarily as a provirus during cellular DNA replication rather than as a virus via reverse transcription.

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Targeting Human Immunodeficiency Virus (HIV) Type 2 Integrase Protein into HIV Type 1

Journal of Virology ◽

10.1128/jvi.73.10.8831-8836.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8831-8836 ◽

Cited By ~ 10

Author(s):

Hongmei Liu ◽

Xiaoyun Wu ◽

Hongling Xiao ◽

John C. Kappes

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcription ◽

Wild Type Virus ◽

Strand Transfer ◽

Wild Type ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Integrase (IN) is the only retroviral enzyme necessary for the integration of retroviral cDNA into the host cell’s chromosomes. The structure and function of IN is highly conserved. The human immunodeficiency virus type 2 (HIV-2) IN has been shown to efficiently support 3′ processing and strand transfer of HIV-1 DNA substrate in vitro. To determine whether HIV-2 IN protein (IN2) could substitute for HIV-1 IN function in vivo, we used HIV-1 Vpr to deliver the IN2 into IN mutant HIV-1 virions by expression intrans as a Vpr-IN fusion protein.Trans-complementation with IN2 markedly increased the infectivity of IN-minus HIV-1. Compared with the homologous trans-IN protein, infectivity was increased to a level of 16%. Since IN has been found to play a role in reverse transcription (Wu et al., J. Virol. 73:2126–2135, 1999), cells infected with IN2-complemented HIV-1 were analyzed for DNA products of reverse transcription. DNA levels of approximately 18% of that of wild type were detected. The homologous trans-IN protein restored the synthesis of viral cDNA to approximately 86% of that of wild-type virus. By complementing integration-defective HIV-1 IN mutant viruses, which were not impaired in cDNA synthesis, thetrans-IN2 protein was shown to support integration up to a level of 55% compared with that of the homologoustrans-IN protein. The delivery of heterologous IN protein into HIV-1 particles in trans offers a novel approach to understand IN protein function in vivo.

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Mutations altering acetylated residues in the CTD of HIV-1 integrase cause defects in proviral transcription at early times after integration of viral DNA

PLoS Pathogens ◽

10.1371/journal.ppat.1009147 ◽

2020 ◽

Vol 16 (12) ◽

pp. e1009147

Author(s):

Shelby Winans ◽

Stephen P. Goff

Keyword(s):

Life Cycle ◽

Histone Modifications ◽

Reverse Transcription ◽

Integration Site ◽

Viral Dna ◽

Large Defect ◽

Central Function ◽

Site Distribution ◽

Hiv 1

The central function of the retroviral integrase protein (IN) is to catalyze the integration of viral DNA into the host genome to form the provirus. The IN protein has also been reported to play a role in a number of other processes throughout the retroviral life cycle such as reverse transcription, nuclear import and particle morphogenesis. Studies have shown that HIV-1 IN is subject to multiple post-translational modifications (PTMs) including acetylation, phosphorylation and SUMOylation. However, the importance of these modifications during infection has been contentious. In this study we attempt to clarify the role of acetylation of HIV-1 IN during the retroviral life cycle. We show that conservative mutation of the known acetylated lysine residues has only a modest effect on reverse transcription and proviral integration efficiency in vivo. However, we observe a large defect in successful expression of proviral genes at early times after infection by an acetylation-deficient IN mutant that cannot be explained by delayed integration dynamics. We demonstrate that the difference between the expression of proviruses integrated by an acetylation mutant and WT IN is likely not due to altered integration site distribution but rather directly due to a lower rate of transcription. Further, the effect of the IN mutation on proviral gene expression is independent of the Tat protein or the LTR promoter. At early times after integration when the transcription defect is observed, the LTRs of proviruses integrated by the mutant IN have altered histone modifications as well as reduced IN protein occupancy. Over time as the transcription defect in the mutant virus diminishes, histone modifications on the WT and mutant proviral LTRs reach comparable levels. These results highlight an unexpected role for the IN protein in regulating proviral transcription at early times post-integration.

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HIV-1 Vpr- and Reverse Transcription-Induced Apoptosis in Resting Peripheral Blood CD4 T Cells and Protection by Common Gamma-Chain Cytokines

Journal of Virology ◽

10.1128/jvi.01770-15 ◽

2015 ◽

Vol 90 (2) ◽

pp. 904-916 ◽

Cited By ~ 13

Author(s):

Benjamin Trinité ◽

Chi N. Chan ◽

Caroline S. Lee ◽

David N. Levy

Keyword(s):

T Cells ◽

Cell Death ◽

T Cell ◽

Reverse Transcription ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Productive Infection ◽

Hiv 1

ABSTRACTHIV-1 infection leads to the progressive depletion of the CD4 T cell compartment by various known and unknown mechanisms.In vivo, HIV-1 infects both activated and resting CD4 T cells, butin vitro, in the absence of any stimuli, resting CD4 T cells from peripheral blood are resistant to infection. This resistance is generally attributed to an intracellular environment that does not efficiently support processes such as reverse transcription (RT), resulting in abortive infection. Here, we show thatin vitroHIV-1 infection of resting CD4 T cells induces substantial cell death, leading to abortive infection.In vivo, however, various microenvironmental stimuli in lymphoid and mucosal tissues provide support for HIV-1 replication. For example, common gamma-chain cytokines (CGCC), such as interleukin-7 (IL-7), render resting CD4 T cells permissible to HIV-1 infection without inducing T cell activation. Here, we find that CGCC primarily allow productive infection by preventing HIV-1 triggering of apoptosis, as evidenced by early release of cytochromecand caspase 3/7 activation. Cell death is triggered both by products of reverse transcription and by virion-borne Vpr protein, and CGCC block both mechanisms. When HIV-1 RT efficiency was enhanced by SIVmac239 Vpx protein, cell death was still observed, indicating that the speed of reverse transcription and the efficiency of its completion contributed little to HIV-1-induced cell death in this system. These results show that a major restriction on HIV-1 infection in resting CD4 T cells resides in the capacity of these cells to survive the early steps of HIV-1 infection.IMPORTANCEA major consequence of HIV-1 infection is the destruction of CD4 T cells. Here, we show that delivery of virion-associated Vpr protein and the process of reverse transcription are each sufficient to trigger apoptosis of resting CD4 T cells isolated from peripheral blood. While these 2 mechanisms have been previously described in various cell types, we show for the first time their concerted effect in inducing resting CD4 T cell depletion. Importantly, we found that cytokines such as IL-7 and IL-4, which are particularly active in sites of HIV-1 replication, protect resting CD4 T cells from these cytopathic effects and, primarily through this protection, rather than through enhancement of specific replicative steps, they promote productive infection. This study provides important new insights for the understanding of the early steps of HIV-1 infection and T cell depletion.

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

Journal of Virology ◽

10.1128/jvi.77.2.1469-1480.2003 ◽

2003 ◽

Vol 77 (2) ◽

pp. 1469-1480 ◽

Cited By ~ 105

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.

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Frequent Dual Initiation in Human Immunodeficiency Virus-Based Vectors Containing Two Primer-Binding Sites: a Quantitative In Vivo Assay for Function of Initiation Complexes

Journal of Virology ◽

10.1128/jvi.78.10.5402-5413.2004 ◽

2004 ◽

Vol 78 (10) ◽

pp. 5402-5413 ◽

Cited By ~ 10

Author(s):

Yegor A. Voronin ◽

Vinay K. Pathak

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcription ◽

Binding Sites ◽

Transcription Initiation ◽

Pcr Analysis ◽

Quantitative Real Time Pcr ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Dual Initiation

ABSTRACT We previously demonstrated that murine leukemia virus (MLV)-based vectors containing two primer-binding sites (PBSs) have the capacity to initiate reverse transcription more than once (Y. A. Voronin and V. K. Pathak, Virology 312:281-294, 2003). To determine whether human immunodeficiency virus (HIV)-based vectors also have the capacity to initiate reverse transcription twice, we constructed an HIV type 1 (HIV-1)-based vector containing the HIV-1 PBS, a green fluorescent protein reporter gene (GFP), and a second PBS derived from HIV-2 3′ of GFP. Simultaneous initiation of reverse transcription at both the 5′ HIV-1 PBS and 3′ HIV-2 PBS was predicted to result in deletion of GFP. As in the MLV-based vectors, GFP was deleted in approximately 25% of all proviruses, indicating frequent dual initiation in HIV-based vectors containing two PBSs. Quantitative real-time PCR analysis of early reverse transcription products indicated that HIV-1 reverse transcriptase efficiently used the HIV-2 PBS. To investigate tRNA primer-RNA template interactions in vivo, we introduced several mutations in the HIV-2 U5 region. The effects of these mutations on the efficiency of reverse transcription initiation were measured by quantitative real-time PCR analysis of early reverse transcription products, with initiation at the HIV-1 PBS used as an internal control. Disruption of the lower and upper parts of the U5-inverted repeat stem reduced the efficiency of initiation 20- and 6-fold, respectively. In addition, disruption of the proposed interactions between viral RNA and tRNALys3 thymidine-pseudouridine-cytidine and anticodon loops decreased the efficiency of initiation seven- and sixfold, respectively. These results demonstrate the relative influence of various RNA-RNA interactions on the efficiency of initiation in vivo. Furthermore, the two-PBS vector system provides a sensitive and quantitative in vivo assay for analysis of RNA-RNA and protein-RNA interactions that can influence the efficiency of reverse transcription initiation.

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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

Journal of Virology ◽

10.1128/jvi.72.5.3907-3915.1998 ◽

1998 ◽

Vol 72 (5) ◽

pp. 3907-3915 ◽

Cited By ~ 35

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.

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INI1/hSNF5-interaction defective HIV-1 IN mutants exhibit impaired particle morphology, reverse transcription and integration in vivo

Retrovirology ◽

10.1186/1742-4690-10-66 ◽

2013 ◽

Vol 10 (1) ◽

pp. 66 ◽

Cited By ~ 6

Author(s):

Sheeba Mathew ◽

Minh Nguyen ◽

Xuhong Wu ◽

Achintya Pal ◽

Vaibhav B Shah ◽

...

Keyword(s):

Reverse Transcription ◽

Particle Morphology ◽

Hiv 1

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Abasic Phosphorothioate Oligomers Inhibit HIV-1 Reverse Transcription and Block Virus Transmission across Polarized Ectocervical Organ Cultures

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02991-14 ◽

2014 ◽

Vol 58 (12) ◽

pp. 7056-7071

Author(s):

Joseph A. Fraietta ◽

Yvonne M. Mueller ◽

Karissa L. Lozenski ◽

Deena Ratner ◽

Alina C. Boesteanu ◽

...

Keyword(s):

Viral Entry ◽

Reverse Transcription ◽

Immune Cell ◽

Virus Transmission ◽

Molecular Size ◽

Mechanisms Of Action ◽

Resistant Isolate ◽

Tissue Samples ◽

Hiv 1

ABSTRACTIn the absence of universally available antiretroviral (ARV) drugs or a vaccine against HIV-1, microbicides may offer the most immediate hope for controlling the AIDS pandemic. The most advanced and clinically effective microbicides are based on ARV agents that interfere with the earliest stages of HIV-1 replication. Our objective was to identify and characterize novel ARV-like inhibitors, as well as demonstrate their efficacy at blocking HIV-1 transmission. Abasic phosphorothioate 2′ deoxyribose backbone (PDB) oligomers were evaluated in a variety of mechanistic assays and for their ability to inhibit HIV-1 infection and virus transmission through primary human cervical mucosa. Cellular and biochemical assays were used to elucidate the antiviral mechanisms of action of PDB oligomers against both lab-adapted and primary CCR5- and CXCR4-utilizing HIV-1 strains, including a multidrug-resistant isolate. A polarized cervical organ culture was used to test the ability of PDB compounds to block HIV-1 transmission to primary immune cell populations across ectocervical tissue. The antiviral activity and mechanisms of action of PDB-based compounds were dependent on oligomer size, with smaller molecules preventing reverse transcription and larger oligomers blocking viral entry. Importantly, irrespective of molecular size, PDBs potently inhibited virus infection and transmission within genital tissue samples. Furthermore, the PDB inhibitors exhibited excellent toxicity and stability profiles and were found to be safe for vaginal applicationin vivo. These results, coupled with the previously reported intrinsic anti-inflammatory properties of PDBs, support further investigations in the development of PDB-based topical microbicides for preventing the global spread of HIV-1.

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HIV-1 Accessory Protein Vpr Interacts with REAF/RPRD2 To Mitigate Its Antiviral Activity

Journal of Virology ◽

10.1128/jvi.01591-19 ◽

2019 ◽

Vol 94 (4) ◽

Cited By ~ 1

Author(s):

Joseph M. Gibbons ◽

Kelly M. Marno ◽

Rebecca Pike ◽

Wing-yiu Jason Lee ◽

Christopher E. Jones ◽

...

Keyword(s):

T Cells ◽

Viral Replication ◽

Nuclear Localization ◽

Reverse Transcription ◽

Early Phase ◽

Early Stage ◽

Target Cells ◽

Accessory Protein ◽

Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr enhances viral replication in both macrophages and, to a lesser extent, cycling T cells. Virion-packaged Vpr is released in target cells shortly after entry, suggesting it is required in the early phase of infection. Previously, we described REAF (RNA-associated early-stage antiviral factor; RPRD2), a constitutively expressed protein that potently restricts HIV replication at or during reverse transcription. Here, we show that a virus without an intact vpr gene is more highly restricted by REAF and, using delivery by virus-like particles (VLPs), that Vpr alone is sufficient for REAF degradation in primary macrophages. REAF is more highly expressed in macrophages than in cycling T cells, and we detected, by coimmunoprecipitation assay, an interaction between Vpr protein and endogenous REAF. Vpr acts quickly during the early phase of replication and induces the degradation of REAF within 30 min of viral entry. Using Vpr F34I and Q65R viral mutants, we show that nuclear localization and interaction with cullin 4A-DBB1 (DCAF1) E3 ubiquitin ligase are required for REAF degradation by Vpr. In response to infection, cells upregulate REAF levels. This response is curtailed in the presence of Vpr. These findings support the hypothesis that Vpr induces the degradation of a factor, REAF, that impedes HIV infection in macrophages. IMPORTANCE For at least 30 years, it has been known that HIV-1 Vpr, a protein carried in the virion, is important for efficient infection of primary macrophages. Vpr is also a determinant of the pathogenic effects of HIV-1 in vivo. A number of cellular proteins that interact with Vpr have been identified. So far, it has not been possible to associate these proteins with altered viral replication in macrophages or to explain why Vpr is carried in the virus particle. Here, we show that Vpr mitigates the antiviral effects of REAF, a protein highly expressed in primary macrophages and one that inhibits virus replication during reverse transcription. REAF is degraded by Vpr within 30 min of virus entry in a manner dependent on the nuclear localization of Vpr and its interaction with the cell’s protein degradation machinery.

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