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

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.

Treatment with suboptimal doses of raltegravir leads to aberrant HIV-1 integrations

“…We recovered three aberrant proviruses that had deletions of the host chromosome at the site of integration, fewer than those with duplications. This is consistent with the results obtained in the experiments done with the RCAS vector system and suggests that the hostmediated insertion has a preference for one side of the original (IN-mediated) insertion (16). In the case of the proviruses in Fig.…”

“…Of the 12 aberrant integrations obtained at suboptimal concentrations of RAL, only 4 showed no homology at the virus-host junction. This is higher than the frequency at which microhomologies are seen in normal integrations, where the presence of matching hostviral sequences corresponds to what would be expected from chance (16).…”

“…The results obtained in the experiments done in PBMCs were similar to the results obtained in HOS cells. There were no aberrant integrations in the absence of RAL; however, in the presence of 5.1 nM RAL, ∼7% of the proviruses were aberrant ( stretches of homology (16). We proposed that these regions of microhomology were the result of the participation of a host DNA polymerase in the generation of the aberrant integrations (Discussion).…”

“…However, most of the resulting proviruses were abnormal (16). The data strongly suggested that the IN mediated the insertion of the "good" end of the viral DNA.…”

“…The data strongly suggested that the IN mediated the insertion of the "good" end of the viral DNA. However, if the mutation of the other end prevented IN from carrying out the ST reaction, the insertion of the blocked end could still occur with moderate efficiency (approximately 30% of WT), but this insertion was mediated by host enzymes and usually involved rearrangements of the host DNA, which included duplications, deletions, inversions, and the acquisition of sequences from other chromosomes (16).…”