Engineering of African horse sickness virus vaccine candidates by targeting the VP4 protein responsible for capping and methylation of viral mRNA

Abstract

African horse sickness virus (AHSV), a member of the Orbivirus genus in the family Reoviridae, is the causative agent of the most devastating and rapidly fatal disease of horses. Control of the disease is based on the use of a polyvalent live-attenuated vaccine, but this vaccine is associated with several risks and drawbacks that prevents its use in non-endemic countries. Over the last decade, reverse genetics technology has enabled manipulation of viral genomes for vaccine production. Studies regarding the viral RNA cap and cap methyltransferases suggested that mRNA cap formation may be an attractive target for vaccine development. Consequently, this study aimed to generate potential vaccine candidates by engineering methyltransferase-defective viruses through a reverse genetics approach. An RNA-based reverse genetics platform developed for AHSV-4 was augmented through the construction of recombinant mammalian expression plasmids in order to replace inefficient primary protein expression from synthetic T7 transcripts transfected into mammalian cells. Subsequently, BSR mammalian cells were transfected first with helper viral protein expression plasmids containing only the coding regions of different core proteins (VP1, VP3, iv VP4 and VP7) and a non-structural protein (NS2). This was followed by a second transfection of the cells with in vitro-synthesized uncapped T7 transcripts of all ten viral genome segments. This approach resulted in the recovery of viable AHSV-4 and the transfection-derived virus retained the properties of wild-type AHSV-4. The augmented RNA-based reverse genetics platform and an entirely plasmid DNA-based reverse genetics platform were then used in a proof-of-principle study to generate vaccine strains by inhibiting viral cap methyltransferase activities. To this end, the VP4 protein of AHSV-4 was targeted for mutagenesis. The VP4 protein, together with VP1, VP6 and VP3, is an essential component of the primary replication complex and is solely responsible for the capping and methylation of the viral mRNA. Deletions, insertions and targeted substitutions were introduced into the coding region of VP4 in order to abrogate different enzymatic activities involved in the capping and methylation of viral mRNA. Irrespective of the reverse genetics platform used and despite using a complimenting cell line that expresses the native VP4 protein in trans, mutant viruses could not be recovered and therefore indicate that the introduced mutations were lethal to virus replication. Failure to assemble functional replication or transcriptional complexes, coupled with the greater instability of uncapped viral mRNA and their enhanced sensitivity to the antiviral effects of the interferon-mediated innate immune response may account for the inability to recover replication-competent mutant viruses. The results of this study highlight the importance of proper mRNA capping for viral gene replication and overall fitness. Moreover, this study also provides information for the design of VP4 protein expression constructs and targets for mutagenesis that can be used as a springboard for biochemical studies of the AHSV VP4 protein. It can be envisaged that an improved understanding of the enzymatic mechanics that govern AHSV transcription, including capping and methylation of the viral mRNA, may provide ideas for the design of vaccine candidates in the future.