Sequence Analysis of In Vivo-Expressed HIV-1 Spliced RNAs Reveals the Usage of New and Unusual Splice Sites by Viruses of Different Subtypes

Abstract

HIV-1 RNAs are generated through a complex splicing mechanism, resulting in a great diversity of transcripts, which are classified in three major categories: unspliced, singly spliced (SS), and doubly spliced (DS). Knowledge on HIV-1 RNA splicing in vivo and by non-subtype B viruses is scarce. Here 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 HIV-1 spliced RNAs, derived from viruses of five subtypes (A, B, C, F, G), were identified. In four samples, three of non-B subtypes, five 3’ splice sites (3’ss) mapping to unreported positions in the HIV-1 genome were identified. Two, designated A4i and A4j, were used in 22% and 25% of rev RNAs in two viruses of subtypes B and A, respectively. Given their close proximity (one or two nucleotides) to A4c and A4d, respectively, they could be viewed as variants of these sites. Three 3’ss, designated A7g, A7h, and A7i, located 20, 32, and 18 nucleotides downstream of A7, respectively, were identified in a subtype C (A7g, A7h) and a subtype G (A7i) viruses, each in around 2% of nef RNAs. The new splice sites or variants of splice sites were associated with the usual sequence features of 3’ss. Usage of unusual 3’ss A4d, A4e, A5a, A7a, and A7b was also detected. A4f, previously identified in two subtype C viruses, was preferentially used by rev RNAs of a subtype C virus. These results highlight the great diversity of in vivo splice site usage by HIV-1 RNAs. The fact that four of five newly identified splice sites or variants of splice sites were detected in non-subtype B viruses allows anticipating an even greater diversity of HIV-1 splice site usage than currently known.

Unusual Fusion Proteins of HIV-1

“…In addition to the canonical splice sites, several studies have reported the presence of alternative or cryptic splice sites in different clades of HIV-1 group M (Purcell and Martin, 1993; Ocwieja et al, 2012; Vega et al, 2016). While some of these sites are conserved among diverse HIV-1 isolates and seem to be used regularly, others have only been identified in single clones of HIV-1 and/or become only active upon mutation of canonical splice sites.…”

“…They hypothesized that at least one of these two proteins may represent an alternative Tat-Rev fusion product that is expressed if tat1 is fused in frame to rev2 , without any additional env sequences. Notably, comprehensive analyses of mRNA species in HIV-1 infected cells identified several neighboring splice acceptor sites at the 5′ end of rev2/tat2 that introduce a frameshift and may result in the expression of various chimeric Rev1-Tat2 or Tat1-Rev2 proteins (Figure 2, Table 1) (Schwartz et al, 1990a; Purcell and Martin, 1993; Ocwieja et al, 2012; Vega et al, 2016). Although at least some of these splice sites can be found in diverse subtypes of (primary) HIV-1 group M isolates (Vega et al, 2016), the expression of chimeric Tat/Rev proteins and their possible role in viral replication has never been investigated.…”

“…For example, usage of acceptors A7g and h in conjunction with donor D4 results in a +2 frameshift that enables the expression of a Tat1-Env protein (Vega et al, 2016). Conversely, splicing at A7e induces a +1 frameshift and the resulting RNA species have the potential to express a Rev1-Env fusion protein (Ocwieja et al, 2012).…”

“…Conversely, splicing at A7e induces a +1 frameshift and the resulting RNA species have the potential to express a Rev1-Env fusion protein (Ocwieja et al, 2012). Due to preferential usage of acceptor A5, however, the majority of mRNA species using alternative splice sites of A7 may lack the rev1 and tat1 initiation codons and express Nef instead (Ocwieja et al, 2012; Vega et al, 2016). …”

“…For example, cryptic exon 6D, which is required for the generation of TNV/p28 tev and p18 6D rev , has only been described for HIV-1 HXB2 and a few closely related viruses (Feinberg et al, 1986; Wright et al, 1986; Benko et al, 1990; Salfeld et al, 1990; Schwartz et al, 1990a; Göttlinger et al, 1992; Neumann et al, 1994; Wentz et al, 1997). Follow-up studies including in vivo transcriptome analyses of patient-derived cells failed to detect 6D transcripts in other HIV-1 strains and subtypes suggesting that they might represent an artifact of laboratory-adapted viruses (Furtado et al, 1991; Smith et al, 1992; Purcell and Martin, 1993; Vega et al, 2016). To our knowledge, Rev1-Vpu is the only unusual fusion protein known to be expressed by intact primary isolates of HIV-1 (Langer et al, 2015).…”