35. Production of Virus-Specific Antiserum Corresponding to Sequences in the Lactate Dehydrogenase-Elevating Virus ORF6 Protein

Hiromi Takahashi-Omoe, Satoru Matsushita and Akihiro Kawano

Keywords: lactate dehydrogenase-elevating virus, ORF6 protein, virus-specific antiserum

The Arteriviridae are enveloped positive-stranded RNA viruses comprised of a variety of animal pathogens including lactate dehydrogenase-elevating virus (LDV), equine arteritis virus (EAV), simian hemorrhagic fever virus (SHFV), and porcine reproductive and respiratory syndrome virus (PRRSV). The ability to replicate in a variety of cell lines is characteristic of EAV, SHFV, and PRRSV but not of LDV, which has a strict host cell specificity. To date, no LDV receptors responsible for cell tropism have been identified. Thus, as a first step toward understanding the underlying mechanism of the restriction of LDV susceptibility, a system for the analysis of LDV envelope protein in mammalian cells was developed.

LDV possesses two major envelope proteins. The smaller is an 18- to 19-kDa nonglycosylated protein (M/VP-2; encoded by ORF6), and the other protein is a primary envelope glycoprotein (VP-3; encoded by ORF5). It has been shown that M/VP-2 and VP-3 of LDV are present in virions as heterodimers that are covalently linked by disulfide bond(s). Since breakage of the disulfide bond(s) in virions resulted in a loss of viral infectivity, the linkage between M/VP-2 and VP-3 seems to be required for host cell entry, perhaps by generating the virion receptor attachment site. Further analysis of the interaction between VP-3 -VP-2/M heterodimer envelope proteins of LDV and host cells will require antibodies directed specifically against each envelope protein. Although several specific antibodies against LDV VP-3 have been generated, despite much effort, no antibodies that react consistently with virion M/VP-2 have been generated. As a first step to examine the biological and functional properties of LDV M/VP-2, we prepared a newly generated specific antibody and analyzed the expression of ORF6 protein in mammalian cells.

A synthetic polypeptide corresponding to the C-terminal region of lactate dehydrogenase-elevating virus strain C (LDV-C) ORF6 which encodes virion structural protein M/VP-2 was chemically synthesized and coupled to keyhole limpet hemocyanin. The peptide was immunogenic in rabbits and induced antibody specific for viral protein. Western blotting and immunofluorescence analysis of virion M/VP-2 in infected macrophages or ORF6 protein in transfected COS-7 cells showed that the antibody was able to react with both authentic and expressed proteins. The immunoreactive antibody against both LDV M/VP-2 and expressed ORF6 protein described in this study provides the experimental basis for further studies of interaction between LDV envelope proteins and receptive host cells.