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

Kleiman, Lawrence

doi:10.1080/15216540211469

Cited by 40 publications

(36 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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“…Even in the absence of viral genomic RNA, retroviral particles contain RNA, and it has been shown both in vitro (13) and in cell culture (40) that RNA, although not necessarily of viral origin, is required for virion assembly. Previous studies of the RNA content of retroviral particles have demonstrated packaging of short RNA species, initially designated 4S to 7S RNAs based on their sedimentation properties, in virions of avian myeloblastosis virus (55), equine infectious anemia virus (15), feline leukemia virus (11), Visna virus (35), Rous sarcoma virus (RSV) (9), MLV (20,33,34,43), and human immunodeficiency virus type 1 (27,28,31). Early work on the RNA content of RSV particles demonstrated that along with the dimer of viral RNA, 7S RNA (now known as 7SL or SRP RNA, the RNA component of the signal recognition particle [29]), 4S RNA, 5S rRNA, and trace amounts of 28S and 18S ribosomal RNAs were also present (9,21,36,46).…”

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Nonrandom Packaging of Host RNAs in Moloney Murine Leukemia Virus

Onafuwa-Nuga

King

Telesnitsky

2005

J Virol

107

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Moloney murine leukemia virus (MLV) particles contain both viral genomic RNA and an assortment of host cell RNAs. Packaging of virus-encoded RNA is selective, with virions virtually devoid of spliced env mRNA and highly enriched for unspliced genome. Except for primer tRNA, it is unclear whether packaged host RNAs are randomly sampled from the cell or specifically encapsidated. To address possible biases in host RNA sampling, the relative abundances of several host RNAs in MLV particles and in producer cells were compared. Using 7SL RNA as a standard, some cellular RNAs, such as those of the Ro RNP, were found to be enriched in MLV particles in that their ratios relative to 7SL differed little, if at all, from their ratios in cells. Some RNAs were underrepresented, with ratios relative to 7SL several orders of magnitude lower in virions than in cells, while others displayed intermediate values. At least some enriched RNAs were encapsidated by genome-defective nucleocapsid mutants. Virion RNAs were not a random sample of the cytosol as a whole, since some cytoplasmic RNAs like tRNA Met were vastly underrepresented, while U6 spliceosomal RNA, which functions in the nucleus, was enriched. Real-time PCR demonstrated that env mRNA, although several orders of magnitude less abundant than unspliced viral RNA, was slightly enriched relative to actin mRNA in virions. These data demonstrate that certain host RNAs are nearly as enriched in virions as genomic RNA and suggest that ⌿ ؊ mRNAs and some other host RNAs may be specifically excluded from assembly sites.Host cell RNA packaging has been observed in several viruses, including DNA viruses such as human cytomegalovirus (54) and herpes simplex virus type 1 (50), RNA viruses such as flock house virus (49), and several retroviruses (9,11,15,16,20). In the herpesviruses, cellular mRNAs are reportedly packaged in a random fashion, most likely representative of their intracellular abundance (50, 54). For retroviruses, the primer tRNA is specifically recruited, and mRNAs are thought to a represent a random sampling (37, 41), but it is unclear whether encapsidation is random among most remaining groups of cellular RNAs.From the mixture of viral and cellular transcripts in the infected cell, two copies of genomic RNA are selected for incorporation into each retroviral particle (7, 36). Virus-encoded RNAs within a Moloney murine leukemia virus (MLV)-infected cell include the capped and polyadenylated unspliced primary transcript as well as spliced env mRNA. Unspliced MLV RNAs contain a ϳ350-nucleotide (nt) region near their 5Ј ends termed the packaging signal, or ⌿ (38), which lies downstream of the splice donor site and upstream of the gag start codon. MLV RNAs that contain ⌿ are packaged at least 100-fold more efficiently than RNAs that lack ⌿ (38). Although other viral sequences and motifs have been implicated in facilitating the specific recruitment of RNAs for packaging (6,7,42,57), the selective encapsidation of MLV and other retroviral genomes is largely attributed to the...

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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 40 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

Nonrandom Packaging of Host RNAs in Moloney Murine Leukemia Virus

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