Rift Valley fever virus M segment: cell-free transcription and translation of virus-complementary RNA

A cell-free system has been used to study gene expression of the M segment RNA of the Phlebovirus Rift Valley fever virus (RVFV). RVFV sequence-containing plasmids were used to synthesize M segment mRNA-like transcripts. These transcripts were then translated in vitro in the absence or presence of microsomal membranes. Cell-free translation of a transcript which closely resembled authentic M segment mRNA (RNA-7) yielded a primary translation product of 133 kilodaltons (kDa), the size expected of a polypeptide encompassing the entire open reading frame (ORF) of the M segment. When translations were conducted in the presence of microsomal membranes, this primary protein was cotranslationally processed to yield the two viral glycoproteins, G1 and G2, as well as proteins of 78, 21, and 14 kDa. With one exception, these in vitro processed polypeptides comigrated with M segment-encoded proteins found in RVFV-infected cell lysates. A polypeptide corresponding to the in vitro 21-kDa protein was not detected in vivo. To investigate translational initiation and processing of the protein products of the M segment, additional transcripts were generated in which varying portions of the amino-terminal "preglycoprotein" region of the M segment ORF were deleted. Translation results indicated that the 78- and 21-kDa proteins were initiated from the first methionine codon of the ORF, and the 14-kDa polypeptide began from the second in-phase ATG. These products and a major portion of the preglycoprotein region sequence were not required for the proper synthesis and processing of the viral glycoproteins in vitro. In light of these results, possible expression strategies used by this Phlebovirus M segment RNA are discussed.