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

Abstract

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...

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

“…1C). However, the percentage of consensus 2-LTR circle junctions was significantly higher than that in an infection with a wild-type virus, and there was a significant decrease in the percentage of large deletions in ends of linear viral DNA compared to that for the wild type (4). Although most of the proviruses that derived from infections with both mutants (RSV U3ϩ4 and RSV U3ϩ6) were integrated normally using the CA dinucleotide in the duplicated insert, about 20% of proviruses were integrated aberrantly.…”

“…The position of the RNase H cleavage was not affected by the mutations in the CA dinucleotide in the 5Ј-TACAT, generating either 5Ј-TACGT or 5Ј-TATAT on the U3 end of the linear viral DNA from DuckHepBFlipPPT2 or DuckHepBFlipPPT3, respectively ( Fig. 1C) (4). However, when the A residue one nucleotide downstream of the 5Ј-TACAT sequence was changed to a C (DuckHepBFlipPPT5), RSV RNase H no longer cleaved at the position (U3ϩ5) favored in the parental DuckHepBFlipPPT virus.…”

“…In infections with RSVP(HIV2), RSV IN removed three nucleotides beyond the CA and properly integrated the processed U3 end, although the titer of this virus titer was only 3.5% of the wild-type level (16). However, surprisingly, the titer of DuckHepBFlipPPTP virus was comparable to that of the wild-type virus (93%) (4). Most of the proviruses derived from infections with the DuckHepBFlipPPTP virus were integrated normally and flanked by a 5-or 6-bp duplication at the target site; in one provirus, there was a 7-bp duplication.…”

“…Although there was preferential cleavage at position U3ϩ4, RNase H cleaved the PPT sequences at various positions, including U3ϩ5, U3ϩ7, and U3ϩ9 (Fig. 1C) (4). We wanted to test how aberrant U3 ends are treated by RSV IN and to ask whether a CA was required for the efficient integration of the U3 end.…”

“…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).…”