tRNALys3: The Primer tRNA for Reverse Transcription in HIV‐1

Kleiman, Lawrence

doi:10.1080/15216540211469

Cited by 42 publications

(40 citation statements)

References 52 publications

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“…However, the human 7SL derivative scAlu RNA and the hY1 and hY3 RNAs are not present in appreciable amounts in HIV-1 (42,54,56). With the exception of the tRNAs that retroviruses use to prime reverse transcription (31,38), any functions of these packaged host RNAs in viral replication remain unknown.…”

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confidence: 99%

cis -Acting Determinants of 7SL RNA Packaging by HIV-1

Keene

Telesnitsky

2012

J Virol

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The host noncoding RNA 7SL is highly enriched in the virions of retroviruses. We examined the regions of 7SL that mediate packaging by HIV-1. Both the Alu domain and the S domain were sufficient to mediate specific packaging when expressed separately as truncations of 7SL. However, while the Alu domain competed with endogenous 7SL for packaging in proportion to Gag, the S domain was packaged additively, implying that the Alu and S domains are packaged via separate mechanisms and that the Alu domain is packaged by the same mechanism as endogenous 7SL. Further truncations of the Alu domain or mutation of the Alu domain helix 5c region significantly reduced packaging efficiency, implicating helix 5c as critical for packaging, reinforcing the finding that 7SL packaging is highly selective, and confirming that 7SL is not passively acquired. Surprisingly, when the Alu domain was mutated so that it no longer contained a binding site for the SRP protein heterodimer SRP9/14, it was no longer packaged in a competitive manner but instead was packaged additively with endogenous 7SL. These data support a model in which 7SL RNA is packaged via interactions between Gag and a 7SL RNA structure that exists transiently at a discrete stage of SRP biogenesis. Our data further indicate that a secondary "additive" pathway exists that can result in the packaging of certain 7SL derivatives in molar excess to endogenously packaged 7SL.

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cis -Acting Determinants of 7SL RNA Packaging by HIV-1

Keene

Telesnitsky

2012

J Virol

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“…Eighteen nucleotides at the 3Ј end of the cellular tRNA Lys,3 hybridize to a specific site on the RNA genome termed the primer binding site (PBS; see ref. 2 and references therein). RT uses tRNA Lys,3 to prime synthesis of a singlestranded DNA product termed minus-strand strong-stop DNA.…”

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confidence: 99%

Identification of specific HIV-1 reverse transcriptase contacts to the viral RNA:tRNA complex by mass spectrometry and a primary amine selective reagent

Kvaratskhelia

Miller

Budihas

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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We have devised a high-resolution protein footprinting methodology to dissect HIV-1 reverse transcriptase (RT) contacts to the viral RNA:tRNA complex. The experimental strategy included modification of surface-exposed lysines in RT and RT-viral RNA:tRNA complexes by the primary amine selective reagent NHS-biotin, SDS͞PAGE separation of p66 and p51 polypeptides, in gel proteolysis, and comparative mass spectrometric analysis of peptide fragments. The lysines modified in free RT but protected from biotinylation in the nucleoprotein complex were readily revealed by this approach. Results of a control experiment examining the RT-DNA:DNA complex were in excellent agreement with the crystal structure data on the identical complex. Probing the RT-viral RNA:tRNA complex revealed that a majority of protein contacts are located in the primer-template binding cleft in common with the RT-DNA:DNA and RT-RNA:DNA species. However, our footprinting data indicate that the p66 fingers subdomain makes additional contacts to the viral RNA:tRNA specific for this complex and not detected with DNA:DNA. The protein footprinting method described herein has a generic application for high-resolution solution structural studies of multiprotein-nucleic acid contacts.

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“…The majority of cellular RNAs known to be packaged by retroviruses are small, noncoding polymerase III (Pol III) products that serve structural or enzymatic functions in the cell (29). The only cellular RNA present in retroviral particles whose viral function is known is the tRNA used to prime reverse transcription (20,24).…”

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7SL RNA Is Retained in HIV-1 Minimal Virus-Like Particles as an S-Domain Fragment

Keene

King

Telesnitsky

2010

J Virol

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HIV-1 is known to package several small cellular RNAs in addition to its genome. Previous work consistently demonstrated that the host structural RNA 7SL is abundant in HIV-1 virions but has yielded conflicting results regarding whether 7SL is present in minimal, assembly-competent virus-like particles (VLPs). Here, we demonstrate that minimal HIV-1 VLPs retain 7SL RNA primarily as an endoribonucleolytic fragment, referred to as 7SL remnant (7SLrem). Nuclease mapping showed that 7SLrem is a 111-nucleotide internal portion of 7SL, with 5 and 3 ends corresponding to unpaired loops in the 7SL two-dimensional structure. Analysis of VLPs comprised of different subsets of Gag domains revealed that all NC-positive VLPs contained intact 7SL while the presence of 7SLrem correlated with the absence of the NC domain. Because 7SLrem, which maps to the 7SL S domain, was not detectable in infected cells, we propose a model whereby the species recruited to assembling VLPs is intact 7SL RNA, with 7SLrem produced by an endoribonuclease in the absence of NC. Since recruitment of 7SL RNA was a conserved feature of all tested minimal VLPs, our model further suggests that 7SL's recruitment is mediated, either directly or indirectly, through interactions with conserved features of all tested VLPs, such as the C-terminal domain of CA.

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tRNALys3: The Primer tRNA for Reverse Transcription in HIV‐1

Abstract: SummaryDuring the assembly of HIV-1, tRNA Lys isoacceptors are selectively packaged into the virion, and one of these, tRNA Lys3 , is annealed to the viral RNA genome where it acts to prime the reverse transcriptase (

Cited by 42 publications

References 52 publications

cis -Acting Determinants of 7SL RNA Packaging by HIV-1

cis -Acting Determinants of 7SL RNA Packaging by HIV-1

Identification of specific HIV-1 reverse transcriptase contacts to the viral RNA:tRNA complex by mass spectrometry and a primary amine selective reagent

7SL RNA Is Retained in HIV-1 Minimal Virus-Like Particles as an S-Domain Fragment

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