Placement of tRNA primer on the primer-binding site requires pol gene expression in avian but not murine retroviruses.

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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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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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

Virology ◽

10.1006/viro.1998.9542 ◽

1999 ◽

Vol 254 (1) ◽

pp. 160-168 ◽

Cited By ~ 6

Author(s):

Qin Yu ◽

Casey D. Morrow

Keyword(s):

Binding Site ◽

Reverse Transcription ◽

Major Determinant ◽

Primer Binding Site ◽

Trna Primer ◽

Hiv 1 ◽

Selection Of

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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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