HIV-1 single-transcription start-site mutants display complementary replication functions that are restored by reversion

HIV-1 transcription initiates at two positions, generating RNAs with either ^cap1G or ^cap3G 5' ends. The replication fates of these RNAs differ, with viral particles encapsidating almost exclusively ^cap1G RNAs and ^cap3G RNAs retained in cells where they are enriched on polysomes and among spliced viral RNAs. Here, we studied replication properties of virus promoter mutants that produced only one RNA 5' isoform or the other: separately, in combination, and during spreading infection. Results showed that either single-start RNA could serve as both mRNA and genomic RNA when present as the only form in cells, although ^cap3G RNA was more efficiently translated and spliced, while ^cap1G RNA was packaged into nascent virions slightly better than RNAs from the parental virus. When co-expressed from separate vectors, ^cap1G RNA was preferentially packaged into virions. During spreading infection, the ^cap1G-only virus displayed only minor defects, but the ^cap3G-only virus showed severe replication delays in both the highly permissive MT-4 cell line and in primary human CD4+ T cells. Passage of ^cap3G-only virus yielded revertants that replicated as well as the twinned (^cap1G+ ^cap3G) transcription start-site parent. These revertants displayed restored packaging and splicing levels and had regained multiple transcription start-site use.IMPORTANCEHIV-1 generates two RNAs during its replication that differ by only two nucleotides in length. Despite this very minor difference, the RNAs perform different and complementary replication functions. When mutants that expressed only one RNA were forced to revert, they regained functions associated with the second RNA.