Intracellular production of African horsesickness virus core-like particles by expression of the two major core proteins, VP3 and VP7, in insect cells.

Abstract

To gain more insight into the structure of the African horsesickness virus (AHSV) core particle, we have cloned, partially characterized and expressed the two major core proteins, VP3 and VP7, of AHSV-9. VP7 was found to be highly conserved amongst different serotypes. The VP3 and VP7 genes were subsequently expressed in insect cells by means of recombinant baculoviruses. VP7 was synthesized to very high levels and aggregated into distinctive crystals. Co-expression of VP3 and VP7 resulted in the intracellular formation of core-like particles which structurally resembled empty AHSV cores.African horsesickness is a highly infectious non-contagious disease of equines and in horses the mortality rate exceeds 90 %. The aetiological agent, African horsesickness virus (AHSV), belongs to the genus Orbivirus in the family Reoviridae. Like bluetongue virus (BTV), the prototype orbivirus, the AHSV virion is composed of seven structural proteins organized into a double-layered capsid containing the 10 double-stranded (ds) RNA segments of the viral genome (Bremer, 1976 ;Roy et al., 1994). The icosahedral core is composed of two major proteins, VP3 and VP7, and three minor structural proteins (VP1, 4 and 6). It has been shown that VP7 trimers constitute the surface of the BTV core : hence the prominent knob-like protrusions which overlay a scaffold of VP3 (Prasad et al., 1992 ;Grimes et al., 1995). The outer capsid layer is composed of the two major proteins, VP2 and VP5, of which VP2 is the serotype-specific antigen (Burrage et al

Silencing of African horse sickness virus VP7 protein expression in cultured cells by RNA interference

“…A truncated AHSV-9 VP7, lacking a stop codon, was obtained by PCR-amplification using plasmid pBSS7PCR, containing a full-length cDNA copy of the VP7 gene [21], as template DNA together with oligonucleotides VP7-F (5′-CACAGATCTATGGACGCGATAGC-3′) and VP7-R (5′-CACAAGCTTGTGGTAGGCTGCTA-3′), which contain a BglII and HindIII site, respectively (underlined). The amplicon was cloned into pGEM …”

The use of soluble African horse sickness viral protein 7 as an antigen delivery and presentation system

“…Pure recombinant viral stocks were prepared from single plaques using established procedures. A recombinant plaque-purified baculovirus, capable of expressing wild-type (WT) AHSV-9 VP7 in Sf9 cells (Maree et al, 1998) was available at the start of the investigation. A baculovirus recombinant that expressed the BTV-10 VP7 gene was also available.…”

“…All amplicons and constructs were verified by chain termination sequencing (Applied Biosystems). Plasmid vector, pFastBac1-VP7, constructed to generate a recombinant baculovirus expressing the VP7 gene of AHSV-9 (Maree et al, 1998), was used as template in the cloning strategy to generate the three vector proteins. The strategy involved amplifying the VP7 vector genes as two fragments from the pFastBac1-VP7 DNA template using the primers outlined in Table 1.…”

“…The vector proteins, as well as an unmodified wild-type (WT) VP7 control (Maree et al, 1998), were expressed by baculovirus recombinants in Sf9 insect cells. SDS-PAGE analysis of the expressed proteins indicated in each case the presence of a unique 39 kDa protein that reacted positively with VP7-specific antibodies in an immunoblot (results not shown).…”

“…These particles have been utilized in a subunit vaccine strategy to protect against a virulent viral challenge (Wade-Evans et al, 1997). This suggested the use of chimeric VP7 particles for the display of foreign peptides instead of a chimeric CLP strategy which is not viable due to the low yield of CLPs when AHSV VP7 and VP3 proteins are coexpressed in insect cells (Maree et al, 1998). Although the structural elements and amino acids that play a role in BTV VP7 solubility, trimerization and CLP formation have been analyzed in great detail (Limn et al, 2000), little is known about the factors that underlie AHSV VP7 insolubility and particle formation.…”