Characterizing HIV-1 splicing by using next-generation sequencing

Ann Emery, Shuntai Zhou, Elizabeth Pollom, Ronald Swanstrom

Research output: Research - peer-reviewArticle

  • 5 Citations

Abstract

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 (env/nef) is the most commonly used acceptor (about 50%) and A3 (tat) 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 vif and vpr 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 env 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.

LanguageEnglish (US)
Article numbere02515-16
JournalJournal of Virology
Volume91
Issue number6
DOIs
StatePublished - 2017

Fingerprint

HIV-1
Human immunodeficiency virus 1
varespladib methyl
RNA
assays
RNA Splicing
High-Throughput Nucleotide Sequencing
Simian Immunodeficiency Virus
Introns
Exons
Genome
Viruses
Messenger RNA
Temperature
RNA splicing
Simian immunodeficiency virus
exons
introns
viruses
genome

Keywords

  • HIV-1
  • Next-generation sequencing
  • Primer ID
  • RNA splicing
  • Simian immunodeficiency virus

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

Characterizing HIV-1 splicing by using next-generation sequencing. / Emery, Ann; Zhou, Shuntai; Pollom, Elizabeth; Swanstrom, Ronald.

In: Journal of Virology, Vol. 91, No. 6, e02515-16, 2017.

Research output: Research - peer-reviewArticle

Emery, Ann ; Zhou, Shuntai ; Pollom, Elizabeth ; Swanstrom, Ronald. / Characterizing HIV-1 splicing by using next-generation sequencing. In: Journal of Virology. 2017 ; Vol. 91, No. 6.
@article{59904f049496472c8b27b5ccaa9d977a,
title = "Characterizing HIV-1 splicing by using next-generation sequencing",
abstract = "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 (env/nef) is the most commonly used acceptor (about 50%) and A3 (tat) 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 vif and vpr 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 env 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.",
keywords = "HIV-1, Next-generation sequencing, Primer ID, RNA splicing, Simian immunodeficiency virus",
author = "Ann Emery and Shuntai Zhou and Elizabeth Pollom and Ronald Swanstrom",
year = "2017",
doi = "10.1128/JVI.02515-16",
volume = "91",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "6",

}

TY - JOUR

T1 - Characterizing HIV-1 splicing by using next-generation sequencing

AU - Emery,Ann

AU - Zhou,Shuntai

AU - Pollom,Elizabeth

AU - Swanstrom,Ronald

PY - 2017

Y1 - 2017

N2 - 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 (env/nef) is the most commonly used acceptor (about 50%) and A3 (tat) 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 vif and vpr 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 env 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.

AB - 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 (env/nef) is the most commonly used acceptor (about 50%) and A3 (tat) 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 vif and vpr 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 env 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.

KW - HIV-1

KW - Next-generation sequencing

KW - Primer ID

KW - RNA splicing

KW - Simian immunodeficiency virus

UR - http://www.scopus.com/inward/record.url?scp=85014093459&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85014093459&partnerID=8YFLogxK

U2 - 10.1128/JVI.02515-16

DO - 10.1128/JVI.02515-16

M3 - Article

VL - 91

JO - Journal of Virology

T2 - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 6

M1 - e02515-16

ER -