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.
Highlights
HIV-1 RNAs are transcribed from a single promoter at the 5’ long terminal repeat and their relative expression is regulated through the alternative usage of splice sites
HIV-1 doubly and singly spliced RNAs were amplified in separate reactions by reverse transcriptase polymerase chain reaction (RT-PCR), followed by nested PCR, as previously described [12], with these modifications: (1) the antisense primer used for nested PCR of SS RNAs was SSD1 (CTCTCATTGCCACTGTCT TCTGCTC, HXB2 positions 6207–6231); (2) a different multiplex identifier (MID) sequence was appended to the 5’ of nested PCR primers used for each reaction, allowing for multiplexed analysis; and (3) 35 cycles were used for the nested PCR
This study provides new data on in vivo HIV-1 RNA splice site usage in viruses of different subtypes (A, B, C, F, and G), for which previously available data were scarce
Summary
HIV-1 RNAs are transcribed from a single promoter at the 5’ long terminal repeat and their relative expression is regulated through the alternative usage of splice sites. Published reports on non-subtype B viruses are limited to the study of two group O [11,34] and three subtype C [12] isolates In these studies, RNA splice sites used by rev RNAs previously unreported in subtype B viruses were identified: A4e in a group O virus [11], A4f in two subtype C viruses, and A4g in one subtype C virus [12]. We analyze in vivo HIV-1 splice site usage within the DS and SS categories through pyrosequencing using a greater number of clinical samples from HIV-1-infected individuals than in previous in vivo studies [8,49]. We isolated CD4+CD25+ lymphocytes, representing the activated T-lymphocyte population, obtained from 19 HIV-1-infected individuals at different stages of the infection These cells were chosen because, among circulating cells, they represent the source of the great majority of HIV-1 virions [56,57,58]. The results allowed the detection of the in vivo usage of five new HIV-1 RNA splice sites, four of them in non-subtype B viruses, and several unusual ones
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