Alternate Polypurine Tracts (PPTs) Affect the Rous Sarcoma Virus RNase H Cleavage Specificity and Reveal a Preferential Cleavage following a GA Dinucleotide Sequence at the PPT-U3 Junction

Alvord

et al. 2008

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We previously reported that a mutant Rous sarcoma virus (RSV) with an alternate polypurine tract (PPT), DuckHepBFlipPPT, had unexpectedly high titers and that the PPT was miscleaved primarily at one position following a GA dinucleotide by the RNase H of reverse transcriptase (RT). This miscleavage resulted in a portion of the 3 end of the PPT (5-ATGTA) being added to the end of U3 of the linear viral DNA. To better understand the RNase H cleavage by RSV RT, we made a number of mutations within the DuckHepBFlipPPT and in the sequences adjacent to the PPT. Deleting the entire ATGTA sequence from the DuckHepBFlipPPT increased the relative titer to wild-type levels, while point mutations within the ATGTA sequence reduced the relative titer but had minimal effects on the cleavage specificity. However, mutating a sequence 5 of ATGTA affected the relative titer of the virus and caused the RNase H of RSV RT to lose the ability to cleave the PPT specifically. In addition, although mutations in the conserved stretch of thymidine residues upstream of the PPT did not affect the relative titer or cleavage specificity, the mutation of some of the nucleotides immediately upstream of the PPT did affect the titer and cleavage specificity. Taken together, our studies show that the structure of the PPT in the context of the cognate RT, rather than a specific sequence, is important for the proper cleavage by RSV RT.During retroviral infection, the virally encoded enzyme reverse transcriptase (RT) converts the single-stranded RNA genome of the virus into a linear double-stranded DNA that can be integrated into the host genome (31, 32). RT has two enzymatic activities: a DNA polymerase that can copy either an RNA or DNA template and an RNase H that cleaves RNA if, and only if, it is part of an RNA-DNA hybrid. Both activities are required for the synthesis of the linear viral DNA. Like many other DNA polymerases, RT cannot initiate DNA synthesis de novo. A host tRNA, hybridized to the viral RNA genome near the 5Ј end of the genome, is used to prime the synthesis of minus-strand DNA. As the polymerase activity of RT synthesizes minus-strand DNA, it generates an RNA-DNA hybrid that is a substrate for RNase H. After RT has copied the short segment of the retroviral RNA genome between the tRNA primer and the 5Ј end of the viral genome and RNase H has degraded the RNA strand, minus-strand DNA synthesis is translocated to the 3Ј end of the viral genome via the repeat or R sequence found at both the 5Ј and 3Ј ends of the viral RNA genome. The synthesis of minus-strand DNA is then able to proceed toward the 5Ј end of the RNA genome. The viral RNA genome contains a sequence called the polypurine tract (PPT), which is relatively resistant to degradation by RNase H. Because the PPT is relatively stable, it is used to prime plusstrand DNA synthesis. In general, the cleavages made by RNase H are thought to be relatively nonspecific; however, there are several steps during reverse transcription in which the cleavages are known to be specific. One s...

Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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The Effects of Alternate Polypurine Tracts (PPTs) and Mutations of Sequences Adjacent to the PPT on Viral Replication and Cleavage Specificity of the Rous Sarcoma Virus Reverse Transcriptase

Alvord

et al. 2008

Self Cite

“…Several studies have shown that the PPT sequence is important for the proper generation and removal of the PPT primer by RNase H (7,8,14,15). We previously reported that alternate PPTs affected the specific cleavages that remove the PPT in a Rous sarcoma virus (RSV)-derived vector, RSVP(A)Z (3,4). One of the alternate PPTs, the duck hepatitis B virus PPT in the reverse orientation (DuckHepBFlipPPT), was preferentially cleaved by the RNase H of RSV reverse transcriptase in a way that added 5Ј-TACAT to the end of U3 (because it is the minus strand on the U3 end that is joined to host DNA by integrase [IN], the minus-strand sequence is shown).…”

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Integration of Rous Sarcoma Virus DNA: a CA Dinucleotide Is Not Required for Integration of the U3 End of Viral DNA

Hughes

2008

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The two ends of RSV linear DNA are independently inserted into host DNA by integrase in vivo. We previously showed that the range of U3 sequences that are acceptable substrates for integrase appeared to be greater than the range of acceptable U5 sequences in vivo. We have done additional experiments to determine which U3 sequences are good integrase substrates. On the U3 end, there does not appear to be a stringent requirement for the canonical CA, integrase can efficiently remove three nucleotides, and six nucleotides are sufficient to allow integration with reasonable, albeit reduced, efficiency.

Alternate Polypurine Tracts Affect Rous Sarcoma Virus Integration In Vivo

Alvord

et al. 2006

Self Cite

When the endogenous polypurine tract (PPT) of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z was replaced with alternate retroviral PPTs, the fraction of unintegrated viral DNA with the normal consensus ends significantly decreased and the retention of part of the PPT significantly increased. If the terminus of the U3 long terminal repeat (LTR) is aberrant, RSV integrase can correctly process and integrate the normal U5 LTR into the host genome. However, the canonical CA is not involved in joining the aberrant U3 LTR to the host DNA, generating either large duplications or deletions of the host sequences instead of the normal 5-or 6-bp duplication.