Complementarity between 3′ Terminal Nucleotides of tRNA and Primer Binding Site Is a Major Determinant for Selection of the tRNA Primer Used for Initiation of HIV-1 Reverse Transcription

ABSTRACT Initiation of retrovirus reverse transcription requires the selection of a tRNA primer from the intracellular milieu. To investigate the features of primer selection, a human immunodeficiency virus type 1 (HIV-1) and a murine leukemia virus (MuLV) were created that require yeast tRNAPhe to be supplied in trans for infectivity. Wild-type yeast tRNAPhe expressed in mammalian cells was transported to the cytoplasm and aminoacylated. In contrast, tRNAPhe without the D loop (tRNAPheD−) was retained within the nucleus and did not complement infectivity of either HIV-1 or MuLV; however, infectivity was restored when tRNAPheD− was directly transfected into the cytoplasm of cells. A tRNAPhe mutant (tRNAPheUUA) that did not have the capacity to be aminoacylated was transported to the cytoplasm and did complement infectivity of both HIV-1 and MuLV, albeit at a level less than that with wild-type tRNAPhe. Collectively, our results demonstrate that the tRNA primer captured by HIV-1 and MuLV occurs after nuclear export of tRNA and supports a model in which primer selection for retroviruses is coordinated with tRNA biogenesis at the intracellular site of protein synthesis.

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Mutations in the U5 Region Adjacent to the Primer Binding Site Affect tRNA Cleavage by Human Immunodeficiency Virus Type 1 Reverse Transcriptase In Vivo

Journal of Virology ◽

10.1128/jvi.02611-06 ◽

2007 ◽

Vol 82 (2) ◽

pp. 719-727 ◽

Cited By ~ 11

Author(s):

Jangsuk Oh ◽

Mary Jane McWilliams ◽

John G. Julias ◽

Stephen H. Hughes

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcriptase ◽

Binding Site ◽

Rnase H ◽

Primer Binding Site ◽

Cleavage Specificity ◽

Immunodeficiency Virus ◽

Trna Primer ◽

Hiv 1

ABSTRACT In retroviruses, the first nucleotide added to the tRNA primer defines the end of the U5 region in the right long terminal repeat, and the subsequent removal of this tRNA primer by RNase H exactly defines the U5 end of the linear double-stranded DNA. In most retroviruses, the entire tRNA is removed by RNase H cleavage at the RNA/DNA junction. However, the RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase cleaves the tRNA 1 nucleotide from the RNA/DNA junction at the U5/primer binding site (PBS) junction, which leaves an rA residue at the U5 terminus. We made sequence changes at the end of the U5 region adjacent to the PBS in HIV-1 to determine whether such changes affect the specificity of tRNA primer cleavage by RNase H. In some of the mutants, RNase H usually removed the entire tRNA, showing that the cleavage specificity was shifted by 1 nucleotide. This result suggests that the tRNA cleavage specificity of the HIV-1 RNase domain H depends on sequences in U5.

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How the HIV-1 Nucleocapsid Protein Binds and Destabilises the (−)Primer Binding Site During Reverse Transcription

Journal of Molecular Biology ◽

10.1016/j.jmb.2008.08.046 ◽

2008 ◽

Vol 383 (5) ◽

pp. 1112-1128 ◽

Cited By ~ 73

Author(s):

Sarah Bourbigot ◽

Nick Ramalanjaona ◽

Christian Boudier ◽

Gilmar F.J. Salgado ◽

Bernard P. Roques ◽

...

Keyword(s):

Binding Site ◽

Reverse Transcription ◽

Nucleocapsid Protein ◽

Primer Binding Site ◽

Hiv 1

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A novel mechanism of self-primed reverse transcription defines a new family of retroelements.

Molecular and Cellular Biology ◽

10.1128/mcb.15.6.3310 ◽

1995 ◽

Vol 15 (6) ◽

pp. 3310-3317 ◽

Cited By ~ 60

Author(s):

H L Levin

Keyword(s):

Binding Site ◽

Reverse Transcription ◽

Primer Binding Site ◽

New Family ◽

Highly Active ◽

Priming Process ◽

Trna Primer ◽

Compensatory Mutations ◽

Transcript Evidence

Retroviruses and long terminal repeat (LTR)-containing retrotransposons initiate reverse transcription by using a specific tRNA primer than anneals to the primer-binding site of the retroelement transcript. Sequences from a large number of retroviruses and LTR-containing retrotransposons had indicated that the role of tRNAs in priming reverse transcription is universal among these LTR-containing retroelements. Data presented here strongly support the surprising conclusion that Tf1, a highly active LTR-containing retrotransposon isolated from Schizosaccharomyces pombe, undergoes a novel self-priming process that requires hybridization between the primer-binding site and the first 11 bases of the Tf1 transcript. Single-base mutations in these regions block transposition and reverse transcription, while compensatory mutations that reestablish complementarily rescue both defects. In addition, the sequence of the minus-strand RNA primer of reverse transcription was consistent with its being derived from the 5' end of the Tf1 transcript. Evidence that this mechanism defines a new family of retroelements is presented.

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Mutations in the U5 Sequences Adjacent to the Primer Binding Site Do Not Affect tRNA Cleavage by Rous Sarcoma Virus RNase H but Do Cause Aberrant Integrations In Vivo

Journal of Virology ◽

10.1128/jvi.80.1.451-459.2006 ◽

2006 ◽

Vol 80 (1) ◽

pp. 451-459 ◽

Cited By ~ 18

Author(s):

Jangsuk Oh ◽

Kevin W. Chang ◽

Stephen H. Hughes

Keyword(s):

Binding Site ◽

Rous Sarcoma Virus ◽

Virus Titer ◽

Rnase H ◽

Primer Binding Site ◽

Rous Sarcoma ◽

Trna Primer ◽

Sarcoma Virus ◽

The Right ◽

Hiv 1

ABSTRACT In most retroviruses, the first nucleotide added to the tRNA primer becomes the right end of the U5 region in the right long terminal repeat (LTR); the removal of this tRNA primer by RNase H defines the right end of the linear double-stranded DNA. Most retroviruses have two nucleotides between the 5′ end of the primer binding site (PBS) and the CA dinucleotide that will become the end of the integrated provirus. However, human immunodeficiency virus type 1 (HIV-1) has only one nucleotide at this position, and HIV-2 has three nucleotides. We changed the two nucleotides (TT) between the PBS and the CA dinucleotide of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z to match the HIV-1 sequence (G) and the HIV-2 sequence (GGT), and we changed the CA dinucleotide to TC. In all three mutants, RNase H removes the entire tRNA primer. Sequence analysis of RSVP(HIV2) proviruses suggests that RSV integrase can remove three nucleotides from the U5 LTR terminus of the linear viral DNA during integration, although this mutation significantly reduced virus titer, suggesting that removing three nucleotides is inefficient. However, the results obtained with RSVP(HIV1) and RSVP(CATC) show that RSV integrase can process and integrate the normal U3 LTR terminus of a linear DNA independently of an aberrant U5 LTR terminus. The aberrant end can then be joined to the host DNA by unusual processes that do not involve the conserved CA dinucleotide. These unusual events generate either large duplications or, less frequently, deletions in the host genomic DNA instead of the normal 5- to 6-base duplications.

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Polyamide Nucleic Acid Targeted to the Primer Binding Site of the HIV-1 RNA Genome Blocksin VitroHIV-1 Reverse Transcription†

Biochemistry ◽

10.1021/bi972197m ◽

1998 ◽

Vol 37 (3) ◽

pp. 900-910 ◽

Cited By ~ 53

Author(s):

Reaching Lee ◽

Neerja Kaushik ◽

Mukund J. Modak ◽

Ravi Vinayak ◽

Virendra N. Pandey

Keyword(s):

Nucleic Acid ◽

Binding Site ◽

Reverse Transcription ◽

Primer Binding Site ◽

Rna Genome ◽

Hiv 1

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

Journal of Virology ◽

10.1128/jvi.79.5.3179-3181.2005 ◽

2005 ◽

Vol 79 (5) ◽

pp. 3179-3181 ◽

Cited By ~ 11

Author(s):

Atze T. Das ◽

Monique Vink ◽

Ben Berkhout

Keyword(s):

Human Immunodeficiency Virus ◽

Binding Site ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Sequence Variation ◽

Primer Binding Site ◽

Immunodeficiency Virus ◽

Hiv 1

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

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HIV-1 Employs Multiple Mechanisms To Resist Cas9/Single Guide RNA Targeting the Viral Primer Binding Site

Journal of Virology ◽

10.1128/jvi.01135-18 ◽

2018 ◽

Vol 92 (20) ◽

Cited By ~ 11

Author(s):

Zhen Wang ◽

Wenzhou Wang ◽

Ya Cheng Cui ◽

Qinghua Pan ◽

Weijun Zhu ◽

...

Keyword(s):

Binding Site ◽

Reverse Transcription ◽

Gene Editing ◽

Viral Escape ◽

Primer Binding Site ◽

Minus Strand ◽

Guide Rna ◽

Target Dna ◽

Almost All ◽

Hiv 1

ABSTRACTThe clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (Cas9) gene-editing technology has been used to inactivate viral DNA as a new strategy to eliminate chronic viral infections, including HIV-1. This utility of CRISPR-Cas9 is challenged by the high heterogeneity of HIV-1 sequences, which requires the design of the single guide RNA (sgRNA; utilized by the CRISPR-Cas9 system to recognize the target DNA) to match a specific HIV-1 strain in an HIV patient. One solution to this challenge is to target the viral primer binding site (PBS), which HIV-1 copies from cellular tRNA3Lysin each round of reverse transcription and is thus conserved in almost all HIV-1 strains. In this study, we demonstrate that PBS-targeting sgRNA directs Cas9 to cleave the PBS DNA, which evokes deletions or insertions (indels) and strongly diminishes the production of infectious HIV-1. While HIV-1 escapes from PBS-targeting Cas9/sgRNA, unique resistance mechanisms are observed that are dependent on whether the plus or the minus strand of the PBS DNA is bound by sgRNA. Characterization of these viral escape mechanisms will inform future engineering of Cas9 variants that can more potently and persistently inhibit HIV-1 infection.IMPORTANCEThe results of this study demonstrate that the gene-editing complex Cas9/sgRNA can be programmed to target and cleave HIV-1 PBS DNA, and thus, inhibit HIV-1 infection. Given that almost all HIV-1 strains have the same PBS, which is copied from the cellular tRNA3Lysduring reverse transcription, PBS-targeting sgRNA can be used to inactivate HIV-1 DNA of different strains. We also discovered that HIV-1 uses different mechanisms to resist Cas9/sgRNAs, depending on whether they target the plus or the minus strand of PBS DNA. These findings allow us to predict that a Cas9 variant that uses the CCA sequence as the protospacer adjacent motif (PAM) should more strongly and persistently suppress HIV-1 replication. Together, these data have identified the PBS as the target DNA of Cas9/sgRNA and have predicted how to improve Cas9/sgRNA to achieve more efficient and sustainable suppression of HIV-1 infection, therefore improving the capacity of Cas9/sgRNA in curing HIV-1 infection.

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

International Journal of Molecular Sciences ◽

10.3390/ijms22010058 ◽

2020 ◽

Vol 22 (1) ◽

pp. 58

Author(s):

Thomas Gremminger ◽

Zhenwei Song ◽

Juan Ji ◽

Avery Foster ◽

Kexin Weng ◽

...

Keyword(s):

Reverse Transcription ◽

Potential Interaction ◽

Viral Rna ◽

Primer Binding Site ◽

Rna Integrity ◽

Immunodeficiency Virus ◽

Infected Cells ◽

Extended Interaction ◽

Hiv 1

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

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