| The flavivirus West Nile virus (WNV) encodes a single polyprotein that is processed into three structural and seven nonstructural proteins. Various WNV bicistronic replicons that direct cap-dependent translation of an N-terminal viral capsid or capsid/Renilla luciferase fusion protein as well as IRES-dependent translation of the nonstructural proteins were constructed. An original replicon consisting of the WNV 5' NCR, the 5' 198 nts of the capsid coding sequence, which included the 5' cyclization sequence (Cyc), and an EMCV IRES followed by the WNV nonstructural genes and 3' NCR was generated. Real time qRT-PCR analysis of intracellular levels of this replicon RNA showed a 4 fold increase by 96 hr after transfection of BHK cells. Increasing the distance between the 5' Cyc and IRES by insertion of a 5' IRES flanking sequence alone or together with a Renilla luciferase reporter did not increase RNA replication. Addition of only a reporter decreased RNA replication. The insertion of an extended capsid coding sequence also did not enhance RNA replication, but did enhance both cap- and IRES-dependent translation of replicon RNA, as indicated by immunofluorescence and Western blot analysis. These results suggest the presence of a translation enhancer in the 3' portion of the capsid coding region.; Simian hemorrhagic fever virus (SHFV) is a member of the family Arteriviridae, order Nidovirales. SHFV is unique among Nidoviruses in having three instead of two papain-like cysteine protease (PCP) motifs designated alpha, beta, and gamma, within the N-terminal region of its ORF1a. Mutations of putative PCP cleavage sites showed that the most efficient cleavage was by PCPbeta at its downstream cleavage site. A large deletion located between the two catalytic residues of PCPalpha was hypothesized to render this protease inactive. However, processing was observed at the cleavage site following PCPalpha. Mutational analyses confirmed that PCPalpha is an inactive protease, and that the cleavage sites downstream of PCPalpha are cleaved by PCPgamma. When the catalytic residues of PCPgamma were mutated, PCPbeta was also able to back cleave at these sites. This "back" cleavage is a previously unreported activity for an arterivirus PCP.; INDEX WORDS: West Nile virus, Replicon, IRES, Capsid, RNA Replication, Translation, Simian hemorrhagic fever virus, Papain-like cysteine protease. |
