Sequence analysis of in vivo-expressed HIV-1 spliced RNAs reveals the usage of new and unusual splice sites by viruses of different subtypes

We analyze HIV-1 RNA splice site usage in CD4+CD25+ lymphocytes from HIV-1-infected individuals through pyrosequencing. HIV-1 DS and SS RNAs were amplified by RT-PCR in 19 and 12 samples, respectively. 13,108 sequences from 68 HIV-1 RNA classes, derived from viruses of five subtypes (A, B, C, F, and G), were identified. In four samples, three of non-B subtypes, five new 3’ splice sites (3’ss) were identified. Two, designated A4i and A4j, located 16 and 4 nucleotides, respectively, upstream of A4a, were used in 22% and 25% rev RNAs in two viruses of subtypes B and A, respectively. Three, designated A7g, A7h, and A7i, located 20, 32, and 18 nucleotides, respectively, downstream of A7, were identified in a subtype C (A7g and A7h) and a subtype G (A7i) viruses, each in around 2% nef RNAs. The new splice sites were associated with the usual sequence features of 3’ss. Usage of unusual splice sites A4d, A4e, A5a, A7a, and A7b was also detected. A4f, previously identified in two subtype C viruses, was used predominantly by rev RNAs of a subtype C virus. These results highlight the great diversity of in vivo splice site usage by HIV-1 RNAs.