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

• Published in: Scientific reports Vol. 6; no. 1; p. 34573
• Main Authors: Baeyens, Ann, Naessens, Evelien, Van Nuffel, Anouk, Weening, Karin E., Reilly, Anne-Marie, Claeys, Eva, Trypsteen, Wim, Vandekerckhove, Linos, Eyckerman, Sven, Gevaert, Kris, Verhasselt, Bruno
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 10-10-2016; Nature Publishing Group
• Subjects: 631/326/596/1787; 631/326/596/2148; Alternative splicing; Alternative Splicing - physiology; Cell Line; Cloning; Deoxyribonucleic acid; DNA; Genome, Viral - physiology; Genomes; HIV-1 - genetics; HIV-1 - metabolism; Humanities and Social Sciences; Humans; Infectivity; mRNA; multidisciplinary; Nucleotide sequence; Proteins; Regulatory sequences; RNA Folding - physiology; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Viral - genetics; RNA, Viral - metabolism; Science; Science (multidisciplinary); vpr Gene Products, Human Immunodeficiency Virus - genetics; vpr Gene Products, Human Immunodeficiency Virus - metabolism; Vpr protein
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep34573

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.