Rous Sarcoma Virus (RSV) Integration In Vivo: a CA Dinucleotide Is Not Required in U3, and RSV Linear DNA Does Not Autointegrate

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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: 68%

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

Chang

Alvord

et al. 2008

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“…Host proteins within the preintegration complex of retroviruses serve to stimulate efficient intermolecular integration and suppress autointegration (44). The hallmarks of autointegration are the creation of 1-and 2-LTR circles with heterogeneity resulting from differences in the orientation of the intasome attack and the creation of junctions joining processed 5= and 3= LTRs to viral DNA (45). One can distinguish the 2-LTR circles that result from autointegration from "true" 2-LTR circles formed by end-to-end ligation by PCR amplification across the 2-LTR junction and gel analysis of the products.…”

Section: ) (B)mentioning

confidence: 99%

Enhanced Autointegration in Hyperstable Simian Immunodeficiency Virus Capsid Mutants Blocked after Reverse Transcription

Tipper

Sodroski

2013

cAfter entering a host cell, retroviruses such as simian immunodeficiency virus (SIV) uncoat, disassembling the viral capsid. Rates of uncoating that are too high and too low can be detrimental to the efficiency of infection. Rapid uncoating typically leads to blocks in reverse transcription, but the basis for replication defects associated with slow uncoating is less clear. Here we characterize the phenotypes of two SIVmac239 mutants with changes, A87E and A87D, in the helix 4/5 loop of the capsid protein. These mutant viruses exhibited normal capsid morphology but were significantly attenuated for infectivity. The infectivity of wild-type and mutant SIVmac239 was not decreased by aphidicolin-induced growth arrest of the target cells. In the cytosol of infected cells, the A87E and A87D capsids remained in particulate form longer than the wild-type SIVmac239 capsid, suggesting that the mutants uncoat more slowly than the wild-type capsid. Both mutants exhibited much higher levels of autointegrated DNA forms than wild-type SIVmac239. Thus, some changes in the helix 4/5 loop of the SIVmac239 capsid protein result in capsid hyperstability and an increase in autointegration.

“…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. Full-length integrated viral DNAs were recovered from cells infected with these mutant viruses as described previously (18). Recovered plasmids were sequenced, and the chicken genomic sequences were analyzed by BLAT searches (http://genome .ucsc.edu/cgi-bin/hgBlat).…”

mentioning

confidence: 99%

“…When the aberrant virus/host junctions were examined, there were, in many cases, microhomologies involving one to nine nucleotides between the virus and host sequence (see Table S1 in the supplemental material). We previously showed that, at this type of aberrant junction, there frequently are microhomologies between the host and virus DNA (17,18). Given that the fraction of 2-LTR circle junctions with large deletions in the U3 sequences (these circles would arise from linear viral DNAs with large deletions in U3) was much lower in both mutants than in the wild type, the fact that infections with both viruses with U3 duplications led to the generation of proviruses in which there was a moderate fraction (20%) with defects at the U3 junction suggests that the last six nucleotides in U3 are sufficient to direct integration with moderate efficiency in vivo but that a larger segment makes contributions to efficient/accurate integration.…”

mentioning

confidence: 99%

“…We previously showed that about 60% of the proviruses derived from infections with RSVP(U3TCTT), in which the CA of the U3 was mutated to TC, were integrated normally, suggesting that RSV IN can recognize this mutated end sequence, remove the two nucleotides beyond the mutated TC sequence at the terminus of U3, and insert the linear DNA which has both nucleotides in the canonical CA at the mutated U3 terminus (18). Here, we show that RSV IN can process and insert linear viral DNAs that have, in place of the canonical CA, a CG, a TA, and an AT dinucleotide.…”

mentioning

confidence: 99%

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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

Chang

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.