Identification of New Splice Sites Used for Generation of rev Transcripts in Human Immunodeficiency Virus Type 1 Subtype C Primary Isolates

Delgado, Elena; Carrera, Cristina; Nebreda, Paloma; Fernández-García, Aurora; Pinilla, Milagros; Garcia, Valentina E.; Pérez-Álvarez, Lucı́a; Thomson, Michael M.

doi:10.1371/journal.pone.0030574

Cited by 8 publications

(11 citation statements)

References 51 publications

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“…The splicing differences seen between the 4-kb and 1.8-kb sizes in NL4-3 are also seen in pZM247Fv2. A previous study found high variation in A4 splice site usage among subtypes and samples (16,38). In this single sample, we did not see any new splice site usage, although the pZM247Fv2 sequence has a point mutation that completely inactivates A4d.…”

Section: Figcontrasting

confidence: 44%

Characterizing HIV-1 Splicing by Using Next-Generation Sequencing

Emery

Zhou

Pollom

et al. 2017

J Virol

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Full-length human immunodeficiency virus type 1 (HIV-1) RNA serves as the genome or as an mRNA, or this RNA undergoes splicing using four donors and 10 acceptors to create over 50 physiologically relevant transcripts in two size classes (1.8 kb and 4 kb). We developed an assay using Primer ID-tagged deep sequencing to quantify HIV-1 splicing. Using the lab strain NL4-3, we found that A5 (/) is the most commonly used acceptor (about 50%) and A3 () the least used (about 3%). Two small exons are made when a splice to acceptor A1 or A2 is followed by activation of donor D2 or D3, and the high-level use of D2 and D3 dramatically reduces the amount of and transcripts. We observed distinct patterns of temperature sensitivity of splicing to acceptors A1 and A2. In addition, disruption of a conserved structure proximal to A1 caused a 10-fold reduction in all transcripts that utilized A1. Analysis of a panel of subtype B transmitted/founder viruses showed that splicing patterns are conserved, but with surprising variability of usage. A subtype C isolate was similar, while a simian immunodeficiency virus (SIV) isolate showed significant differences. We also observed transsplicing from a downstream donor on one transcript to an upstream acceptor on a different transcript, which we detected in 0.3% of 1.8-kb RNA reads. There were several examples of splicing suppression when the intron was retained in the 4-kb size class. These results demonstrate the utility of this assay and identify new examples of HIV-1 splicing regulation. During HIV-1 replication, over 50 conserved spliced RNA variants are generated. The splicing assay described here uses new developments in deep-sequencing technology combined with Primer ID-tagged cDNA primers to efficiently quantify HIV-1 splicing at a depth that allows even low-frequency splice variants to be monitored. We have used this assay to examine several features of HIV-1 splicing and to identify new examples of different mechanisms of regulation of these splicing patterns. This splicing assay can be used to explore in detail how HIV-1 splicing is regulated and, with moderate throughput, could be used to screen for structural elements, small molecules, and host factors that alter these relatively conserved splicing patterns.

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Section: Figcontrasting

confidence: 44%

Characterizing HIV-1 Splicing by Using Next-Generation Sequencing

Emery

Zhou

Pollom

et al. 2017

J Virol

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“…The great majority of studies on HIV-1 RNA splicing have been performed with isolates of subtype B, which is predominant in Western countries. 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 ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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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.

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“…Gag and Pol proteins are translated from the unspliced full-length viral mRNA, and the Env protein is translated from the spliced env -mRNA [1]. In contrast, it has been reported that human immunodeficiency virus (HIV) type 1, which is a complex retrovirus, could generate up to 40 different spliced RNAs using four 5’ss and nine 3’ss [2-4]. …”

Section: Introductionmentioning

confidence: 99%

The 0.3-kb fragment containing the R-U5-5’leader sequence of Friend murine leukemia virus influences the level of protein expression from spliced mRNA

Choo

Seki

Machinaga

et al. 2013

Virol J

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BackgroundA neuropathogenic variant of Friend murine leukemia virus (Fr-MLV) clone A8 induces spongiform neurodegeneration when infected into neonatal rats. Studies with chimeras constructed from the A8 virus and the non-neuropathogenic Fr-MLV clone 57 identified a 0.3-kb KpnI-AatII fragment containing a R-U5-5’leader sequence as an important determinant for inducing spongiosis, in addition to the env gene of A8 as the primary determinant. This 0.3-kb fragment contains a 17-nucleotide difference between the A8 and 57 sequences. We previously showed that the 0.3-kb fragment influences expression levels of Env protein in both cultured cells and rat brain, but the corresponding molecular mechanisms are not well understood.ResultsStudies with expression vectors constructed from the full-length proviral genome of Fr-MLV that incorporated the luciferase (luc) gene instead of the env gene found that the vector containing the A8-0.3-kb fragment yielded a larger amount of spliced luc-mRNA and showed higher expression of luciferase when compared to the vector containing the 57-0.3-kb fragment. The amount of total transcripts from the vectors, the poly (A) tail length of their mRNAs, and the nuclear-cytoplasm distribution of luc-mRNA in transfected cells were also evaluated. The 0.3-kb fragment did not influence transcription efficiency, mRNA polyadenylation or nuclear export of luc-mRNA. Mutational analyses were carried out to determine the importance of nucleotides that differ between the A8 and 57 sequences within the 0.3-kb fragment. In particular, seven nucleotides upstream of the 5’splice site (5’ss) were found to be important in regulating the level of protein expression from spliced messages. Interestingly, these nucleotides reside within the stem-loop structure that has been speculated to limit the recognition of 5’ss.ConclusionsThe 0.3-kb fragment containing the R-U5-5’leader sequence of Fr-MLV influences the level of protein expression from the spliced-mRNA by regulating the splicing efficiency rather than transcription, nuclear export of spliced-mRNA, or poly (A) addition to mRNA. Seven nucleotides in the 0.3-kb fragment, which reside within the stem-loop structure that has been speculated to limit recognition of the 5’ss, could pinpoint the function of this region.

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Identification of New Splice Sites Used for Generation of rev Transcripts in Human Immunodeficiency Virus Type 1 Subtype C Primary Isolates

Cited by 8 publications

References 51 publications

Characterizing HIV-1 Splicing by Using Next-Generation Sequencing

Characterizing HIV-1 Splicing by Using Next-Generation Sequencing

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

The 0.3-kb fragment containing the R-U5-5’leader sequence of Friend murine leukemia virus influences the level of protein expression from spliced mRNA

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