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HIV-1 Vpr N-terminal tagging affects alternative splicing of the viral genome

Ann Baeyens (UGent) , Evelien Naessens (UGent) , Anouk Van Nuffel (UGent) , Karin Weening (UGent) , Anne-Marie Reilly, Eva Claeys, Wim Trypsteen (UGent) , Linos Vandekerckhove (UGent) , Sven Eyckerman (UGent) , Kris Gevaert (UGent) , et al.
(2016) SCIENTIFIC REPORTS. 6.
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Abstract
To facilitate studies on Vpr function in replicating HIV-1, we aimed to tag the protein in an infectious virus. First we showed that N-, but not C-terminal HA/FLAG tagging of Vpr protein preserves Vpr cytopathicity. Cloning the tags into proviral DNA however ablated viral production and replication. By construction of additional viral variants we could show this defect was not protein-but RNA-dependent and sequence specific, and characterized by oversplicing of the genomic RNA. Simulation of genomic RNA folding suggested that introduction of the tag sequence induced an alternative folding structure in a region enriched in splice sites and splicing regulatory sequences. In silico predictions identified the HA/His(6)-Vpr tagging in HIV-1 to affect mRNA folding less than HA/FLAG-Vpr tagging. In vitro infectivity and mRNA splice pattern improved but did not reach wild-type values. Thus, sequence-specific insertions may interfere with mRNA splicing, possibly due to altered RNA folding. Our results point to the complexity of viral RNA genome sequence interactions. This should be taken into consideration when designing viral manipulation strategies, for both research as for biological interventions.
Keywords
SITE, REPLICATION, U1 SNRNA, IN-VITRO, REV FUNCTION, STRUCTURE PREDICTION, IMMUNODEFICIENCY-VIRUS, RNA SECONDARY STRUCTURE, PRE-MESSENGER-RNA, INFECTION

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MLA
Baeyens, Ann, et al. “HIV-1 Vpr N-Terminal Tagging Affects Alternative Splicing of the Viral Genome.” SCIENTIFIC REPORTS, vol. 6, 2016, doi:10.1038/srep34573.
APA
Baeyens, A., Naessens, E., Van Nuffel, A., Weening, K., Reilly, A.-M., Claeys, E., … Verhasselt, B. (2016). HIV-1 Vpr N-terminal tagging affects alternative splicing of the viral genome. SCIENTIFIC REPORTS, 6. https://doi.org/10.1038/srep34573
Chicago author-date
Baeyens, Ann, Evelien Naessens, Anouk Van Nuffel, Karin Weening, Anne-Marie Reilly, Eva Claeys, Wim Trypsteen, et al. 2016. “HIV-1 Vpr N-Terminal Tagging Affects Alternative Splicing of the Viral Genome.” SCIENTIFIC REPORTS 6. https://doi.org/10.1038/srep34573.
Chicago author-date (all authors)
Baeyens, Ann, Evelien Naessens, Anouk Van Nuffel, Karin Weening, Anne-Marie Reilly, Eva Claeys, Wim Trypsteen, Linos Vandekerckhove, Sven Eyckerman, Kris Gevaert, and Bruno Verhasselt. 2016. “HIV-1 Vpr N-Terminal Tagging Affects Alternative Splicing of the Viral Genome.” SCIENTIFIC REPORTS 6. doi:10.1038/srep34573.
Vancouver
1.
Baeyens A, Naessens E, Van Nuffel A, Weening K, Reilly A-M, Claeys E, et al. HIV-1 Vpr N-terminal tagging affects alternative splicing of the viral genome. SCIENTIFIC REPORTS. 2016;6.
IEEE
[1]
A. Baeyens et al., “HIV-1 Vpr N-terminal tagging affects alternative splicing of the viral genome,” SCIENTIFIC REPORTS, vol. 6, 2016.
@article{8159575,
  abstract     = {{To facilitate studies on Vpr function in replicating HIV-1, we aimed to tag the protein in an infectious virus. First we showed that N-, but not C-terminal HA/FLAG tagging of Vpr protein preserves Vpr cytopathicity. Cloning the tags into proviral DNA however ablated viral production and replication. By construction of additional viral variants we could show this defect was not protein-but RNA-dependent and sequence specific, and characterized by oversplicing of the genomic RNA. Simulation of genomic RNA folding suggested that introduction of the tag sequence induced an alternative folding structure in a region enriched in splice sites and splicing regulatory sequences. In silico predictions identified the HA/His(6)-Vpr tagging in HIV-1 to affect mRNA folding less than HA/FLAG-Vpr tagging. In vitro infectivity and mRNA splice pattern improved but did not reach wild-type values. Thus, sequence-specific insertions may interfere with mRNA splicing, possibly due to altered RNA folding. Our results point to the complexity of viral RNA genome sequence interactions. This should be taken into consideration when designing viral manipulation strategies, for both research as for biological interventions.}},
  articleno    = {{34573}},
  author       = {{Baeyens, Ann and Naessens, Evelien and Van Nuffel, Anouk and Weening, Karin and Reilly, Anne-Marie and Claeys, Eva and Trypsteen, Wim and Vandekerckhove, Linos and Eyckerman, Sven and Gevaert, Kris and Verhasselt, Bruno}},
  issn         = {{2045-2322}},
  journal      = {{SCIENTIFIC REPORTS}},
  keywords     = {{SITE,REPLICATION,U1 SNRNA,IN-VITRO,REV FUNCTION,STRUCTURE PREDICTION,IMMUNODEFICIENCY-VIRUS,RNA SECONDARY STRUCTURE,PRE-MESSENGER-RNA,INFECTION}},
  language     = {{eng}},
  pages        = {{11}},
  title        = {{HIV-1 Vpr N-terminal tagging affects alternative splicing of the viral genome}},
  url          = {{http://doi.org/10.1038/srep34573}},
  volume       = {{6}},
  year         = {{2016}},
}
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