Abstract
The 7.5-kb plus-stranded genomic RNA of rabbit hemorrhagic disease virus contains two open reading frames of 7 kb (ORF1) and 351 nucleotides (ORF2) that cover nearly 99% of the genome. The aim of the present study was to identify the proteins encoded in these open reading frames. To this end, a panel of region-specific antisera was generated by immunization of rabbits with bacterially expressed fusion proteins that encompass in total 95% of the ORF1 polyprotein and almost the complete ORF2 polypeptide. The antisera were used to analyze the in vitro translation products of purified virion RNA of rabbit hemorrhagic disease virus. Our studies show that the N-terminal half of the ORF1 polyprotein is proteolytically cleaved to yield three nonstructural proteins of 16, 23, and 37 kDa (p16, p23, and p37, respectively). In addition, a cleavage product of 41 kDa which is composed of VPg and a putative nonstructural protein of approximately 30 kDa was identified. Together with the results of previous studies which identified a trypsin-like cysteine protease (TCP) of 15 kDa, a putative RNA polymerase (pol) of 58 kDa, and the major capsid protein VP60, our data establish the following gene order in ORF1: NH2-p16-p23-p37 (helicase)-p30-VPg-TCP-pol-VP60-COOH. Immunoblot analyses showed that a minor structural protein of 10 kDa is encoded in ORF2. The data provide the first complete genetic map of a calicivirus. The map reveals a remarkable similarity between caliciviruses and picornaviruses with regard to the number and order of the genes that encode the nonstructural proteins.