Sequences in the 5 ' and 3 ' R elements of human immunodeficiency virus type 1 critical for efficient reverse transcription

The genome of human immunodeficiency virus type 1 (HIV-1) contains two dire ct repeats (R) of 97 nucleotides at each end. These elements are of critica l importance during the first-strand transfer of reverse transcription, dur ing which the minus-strand strong-stop DNA (-sssDNA) is transferred from th e 5' end to the 3' end of the genomic RNA. This transfer is critical for th e synthesis of the full-length minus-strand cDNA, These repeats also contai n a variety of other functional domains involved in many aspects of the vir al life cycle. In this study, we have introduced a series of mutations into the 5', the 3', or both R sequences designed to avoid these other function al domains. Using a single-round infectivity assay, we determined the abili ty of these mutants to undergo the various steps of reverse transcription u tilizing a semiquantitative PCR analysis, We find that mutations within the first 10 nucleotides of either the 5' or the 3' R sequence resulted in vir ions that were markedly defective for reverse transcription in infected tel ls. These mutations potentially introduce mismatches between the full-lengt h -sssDNA and 3' acceptor R, Even mutations that would create relatively sm all mismatches, as little as 3 bp, resulted in inefficient reverse transcri ption. In contrast, virions containing identically mutated R elements were not defective for reverse transcription or infectivity. Using an endogenous reverse transcription assay with disrupted virus, we show that virions har boring the 5' or the 3' R mutations were not intrinsically defective for DN A synthesis. Similarly sized mismatches slightly further downstream in eith er the 5', the 3', or both R sequences were not detrimental to continued re verse transcription in infected cells. These data are consistent with the i dea that certain mismatches within 10 nucleotides downstream of the U3-R ju nction in HIV-1 cause defects in the stability of the cDNA before or during the first-strand transfer of reverse transcription leading to the rapid di sappearance of the -sssDNA in infected cells. These data also suggest that the great majority of first-strand transfers in HIV-1 occur after the copyi ng of virtually the entire 5' R.