Rift Valley fever virus M segment: phlebovirus expression strategy and protein glycosylation

The M segment RNA of Rift Valley fever virus (RVFV) encodes four gene products: the two viral envelop glycoproteins G2 and G1, a glycosylated 78-kDa protein, and a nonglycosylated 14-kDa protein. These proteins are generated from a single open reading frame (ORF) by a strategy involving independent translational initiations at both the first and second in-phase ATG codons and co-translational processing of primary polyprotein products. The ORF encodes six sites for N-linked glycosylation: one present in the "preglycoprotein region" preceding the coding sequences of the mature envelop glycoproteins, and within the coding sequences of both the 78- and 14-kDa proteins; one site in the glycoprotein G2 coding region, also present in the 78-kDa protein; and four sites within glycoprotein G1. From analyses of RVFV proteins produced in cells infected with recombinant vaccinia viruses expressing various M segment regions, we show glycoprotein G2 was glycosylated at its single site and glycoprotein G1 at at least three sites. Both sites for N-linked glycosylation in the 78-kDa protein were occupied with glycan. This latter result indicated the preglycoprotein region glycosylation site was utilized in the 78-kDa protein, but this same site within the 14-kDa protein was not. Further analysis showed utilization of this glycosylation site, as well as proteolytic processing at the amino terminus of the mature glycoprotein G2, appeared to be determined by initiation codon usage. The two-site translational initiation expression strategy of this phlebovirus M segment and its role in the control of post-translational protein modification and processing are discussed.