title: Expression strategy of a phlebovirus: biogenesis of proteins from the Rift Valley fever virus M segment.
abstract: The middle (M) RNA segment of Rift Valley fever virus (RVFV) encodes four proteins: the major viral glycoproteins G2 and G1, a 14-kilodalton (kDa) protein, and a 78-kDa protein. These proteins are derived from a single large open reading frame (ORF) present in the virus-complementary M-segment mRNA. We used recombinant vaccinia viruses in which sequences representing the M-segment ORF were engineered as a surrogate system to study phlebovirus protein expression. To investigate the translational initiation codon requirements for synthesis of these proteins, we constructed a series of vaccinia virus recombinants containing specific sequence changes which eliminated select ATG codons found in the region of the ORF preceding the mature glycoprotein-coding sequences (the preglycoprotein region). Examination of phleboviral proteins synthesized in cells infected with these vaccinia virus recombinants clearly showed that the first ATG of the ORF was required for the production of the 78-kDa protein, while synthesis of the 14-kDa protein was absolutely dependent on the second in-phase ATG codon. Efficient biosynthesis of glycoprotein G2 was shown to depend on one or more ATG codons within the preglycoprotein region, but not the first one of the ORF. Synthesis of about one-half of the total glycoprotein G1 was affected by the amino acid changes that eliminated ATG codons, while production of the remainder appeared to be independent of all ATG codons in the preglycoprotein region. These data indicated that the means for glycoprotein G1 biosynthesis was distinct from those of the other three M-segment gene products. The results presented herein suggest that a surprisingly complex expression strategy is employed by the RVFV M segment. Although the full nature of the mechanisms involved in the biogenesis of the four RVFV M-segment proteins remains unclear, it does involve the use of at least two (ATG codons 1 and 2), and likely more, distinct translation start sites within the same ORF to produce its complete complement of gene products.
authors.affiliation: Molecular Genetics, Inc., Minnetonka, Minnesota 55343.
authors.forename: J A
authors.initials: JA
authors.lastname: Suzich
authors.forename: L T
authors.initials: LT
authors.lastname: Kakach
authors.forename: M S
authors.initials: MS
authors.lastname: Collett
mesh_terms: D001483:Base Sequence; D002043:Bunyaviridae / Q000235:genetics*; D003001:Cloning, Molecular; D003062:Codon; D005822:Genetic Vectors; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000235:genetics*; D015531:Radioimmunoprecipitation Assay; D012296:Rift Valley fever virus / Q000235:genetics*; D014616:Vaccinia virus / Q000235:genetics; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D003062:Codon; D006023:Glycoproteins; D012367:RNA, Viral; D014764:Viral Proteins