The cloning and expression of the major core protein, VP3 from AHSV-3 and AHSV-9

Abstract

An important goal in AHSV biology, is understanding the way in which the virus structural proteins are assembled to produce the mature virion. Work done in several laboratories using the baculovirus expression system to express these structural proteins, alone or in combination, has gone a long way towards elucidating the problem. This strategy has demonstrated the in vivo assembly of core-like and virus-like particles which closely resemble the native virus. When the major core proteins VP3 and VP7 of BTV were co-expressed in insect cells using the baculovirus system, the formation of core-like particles resulted. It was therefore of interest to investigate whether this would also occur if the cognate VP3 and VP7 genes of AHSV were expressed using this system. However, in order to initiate this work, the VP3 gene of AHSV first needed to be cloned, and its expression in the baculovirus system tested. Full length cDNA copies of the VP3 genes of both AHSV-3 and AHSV-9 were cloned. Sequence analysis of their open reading frame termini revealed a similar high degree of conservation as is found amongst the VP3 genes of BTV. Bacterial expression and in vitro transcription and translation demonstrated that this gene could be expressed as a full-length protein. A baculovirus recombinant of this gene revealed the synthesis of a full-length gene product. However, upon production of high titre stocks of the virus, the levels of protein produced declined dramatically. This may have been attributable to an instability in the parental virus. Subsequent to this study, an alternative system was used to produce a VP3 baculovirus recombinant. This approach significantly improved the yield of recombinant VP3. When this recombinant was expressed together with a AHSV VP7 baculovirus recombinant, core like particles were produced which closely resembled those seen for BTV