The A5 gene of alcelaphine herpesvirus 1 encodes a constitutively active G-protei n-coupled receptor that is non-essential for the induction of malignant catarrhal fever in rabbits

Many gammaherpesviruses encode G-protein-coupled receptors (GPCRs). Several in vivo studies have revealed that gammaherpesvirus GPCRs are important for viral replication and for virus-induced pathogenesis. The gammaherpesvirus alcelaphine herpesvirus 1 (AIHV-1) is carried asymptomatically by wildebeest, but causes malignant catarrhal fever (MCF) following crossspecies transmission to a variety of susceptible species. The A5 ORF of the AIHV-1 genome encodes a putative GPCR. In the present study, we investigated whether A5 encodes a functional GPCIR and addressed its role in viral replication and in the pathogenesis of MCF. In silico analysis supported the hypothesis that A5 could encode a functional GPCR as its expression product contained several hallmark features of GPCRs. Expression of A5 as tagged proteins in various cell lines revealed that A5 localizes in cell membranes, including the plasma membrane. Using [S-35]GTP gamma S and reporter gene assays, we found that A5 is able to constitutively couple to alpha(i)-type G-proteins in transfected cells, and that this interaction is able to inhibit forskolin-triggered cAMP response element-binding protein (CREB) activation. Finally, using an AIHV-1 BAC clone, we produced a strain deleted for A5 and a revertant strain. Interestingly, the strain deleted for A5 replicated comparably to the wild-type parental strain and induced MCF in rabbits that was indistinguishable from that of the parental strain. The present study is the first to investigate the role of an individual gene of AIHV-1 in MCF pathogenesis.