Pseudodiploid Genome Organization Aids Full-Length Human Immunodeficiency Virus Type 1 DNA Synthesis

Abstract

Template switching between copackaged human immunodeficiency virus type 1 (HIV-1) genomic RNAs is genetically silent when identical RNAs are copackaged but yields recombinants when virions contain two distinct RNAs. Sequencing has revealed that errors at retroviral recombination junctions are infrequent, suggesting that template switching is not intrinsically mutagenic. Here, we tested the hypothesis that template switching may instead contribute to replication fidelity. This hypothesis predicts that reverse transcription of a single-copy gene will be more error prone than replication in the presence of a second copy. To test this, HIV-1-based vectors containing both lacZ and the puromycin resistance marker were expressed either alone or with an excess of an "empty" vector lacking lacZ and puro. This resulted in virions with either RNA homodimers or haploid genomes with only a single lacZ-puro RNA. In untreated cells, lacZ inactivation rates suggested that haploid vector reverse transcription was slightly more error prone than that of homodimerized pseudodiploid vectors. Haploid reverse transcription was at least threefold more error prone than pseudodiploid-templated synthesis when slowed by hydroxyurea treatment or stopped prematurely with zidovudine. Individual products of one-and two-copy genes revealed both nucleotide substitutions and deletions, with deletions more frequent than point mutations among haploid genome products. Similar spectra of defective products were observed at early reverse transcription time points and among products of haploid virions. These results indicate that faithful, full-length reverse transcription products were underrepresented in the absence of a reserve of genetic information and suggest that template switching contributes to HIV-1 genomic integrity.

The Remarkable Frequency of Human Immunodeficiency Virus Type 1 Genetic Recombination

“…When replication was halted before the average half-time of provirus completion, deletion products like those observed for one-gRNA virions were abundant. These findings suggest that HIV-1 replication errors occur less frequently when a second gRNA is present and that full-length proviruses are synthesized more efficiently in the presence of a second gRNA (166). Figure 7 presents a model for RT elongation that considers these observations in light of the model of minus-strand exchange.…”

“…Further support for the notion that recombination aids replication fidelity comes from a study where recombination was prevented by engineering HIV-1 virions to contain only single intact gRNAs (166). The frequency of successful provirus generation was compared to that for normal pseudodiploid genomes.…”

Structural Determinants and Mechanism of HIV-1 Genome Packaging

“…Both RNA molecules are utilized for strand transfer-mediated recombination during reverse transcription 18,19 , but only one DNA allele is generated, and retroviruses are therefore considered “pseudodiploid.” Recombination appears to enhance viral fitness in several ways. It enables strand transfer-mediated read through at sites of RNA damage 20,21 , which could serve as a mechanism for dealing with what appears to be a relatively fragile genome 22 , and as a defense against restriction nucleases 23 . In addition, although cells containing a single integrated provirus can only produce homozygous virions, cells from infected individuals have sometimes been observed to contain several integrated proviruses 24–26 , which probably explains the very high number of circulating recombinant forms of HIV-1 27 .…”

Cis–Acting RNA Elements of Human Immunodeficiency Virus

“…It was shown that SL1 deletion impairs plus-strand HIV-1 DNA transfer in RT (Paillart et al, 1996a;Shen et al, 2000). In addition, template-switching is restricted in a 2-kb region immediately downstream of SL1 mutations (Chin et al, 2008), which affects the efficiency of RT and the synthesis of full-length HIV-1 DNA (King et al, 2008). The second observation is intriguing because most HIV-1 RNA secondary structures that are thought to stall RT and thereby increase recombination are limited to a very short region (Derebail and DeStefano, 2004;Galetto et al, 2004;Moumen et al, 2001).…”