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 1G or 3G 5' ends. The replication fates of these RNAs di\er, with viral particles encapsidating almost exclusively 1G RNAs and 3G 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 3G RNA was more efficiently translated and spliced while 1G RNA was packaged into nascent virions slightly better than RNAs from the parental virus. When co-expressed from separate vectors, 1G RNA was preferentially packaged into virions. During spreading infection 1G-only virus displayed only minor defects but 3G-only virus showed severe replication delays in both the highly permissive MT-4 cell line and in primary human CD4+ T cells. Passage of 3G-only virus yielded revertants that replicated as well as the twinned (1G+ 3G) transcription start site parent. These revertants displayed restored packaging and splicing levels and had regained multiple transcription start site use.