title: Mutational Analysis of the Rift Valley Fever Virus Glycoprotein Precursor Proteins for Gn Protein Expression.
abstract: The Rift Valley fever virus (RVFV) M-segment encodes the 78 kD, NSm, Gn, and Gc proteins. The 1st AUG generates the 78 kD-Gc precursor, the 2nd AUG generates the NSm-Gn-Gc precursor, and the 3rd AUG makes the NSm'-Gn-Gc precursor. To understand biological changes due to abolishment of the precursors, we quantitatively measured Gn secretion using a reporter assay, in which a Gaussia luciferase (gLuc) protein is fused to the RVFV M-segment pre-Gn region. Using the reporter assay, the relative expression of Gn/gLuc fusion proteins was analyzed among various AUG mutants. The reporter assay showed efficient secretion of Gn/gLuc protein from the precursor made from the 2nd AUG, while the removal of the untranslated region upstream of the 2nd AUG (AUG2-M) increased the secretion of the Gn/gLuc protein. Subsequently, recombinant MP-12 strains encoding mutations in the pre-Gn region were rescued, and virological phenotypes were characterized. Recombinant MP-12 encoding the AUG2-M mutation replicated slightly less efficiently than the control, indicating that viral replication is further influenced by the biological processes occurring after Gn expression, rather than the Gn abundance. This study showed that, not only the abolishment of AUG, but also the truncation of viral UTR, affects the expression of Gn protein by the RVFV M-segment.
authors.affiliation: Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555, USA. inphoeni@UTMB.EDU.
authors.forename: Inaia
authors.initials: I
authors.lastname: Phoenix
authors.affiliation: Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555, USA. nalokuga@UTMB.EDU.
authors.forename: Nandadeva
authors.initials: N
authors.lastname: Lokugamage
authors.affiliation: Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555, USA. shnishiy@UTMB.EDU.
authors.forename: Shoko
authors.initials: S
authors.lastname: Nishiyama
authors.affiliation: Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555, USA. teikegam@utmb.edu.|The Sealy Center for Vaccine Development, The University of Texas Medical Branch, Galveston, TX 77555, USA. teikegam@utmb.edu.|The Center for Biodefense and Emerging Infectious Diseases, The University of Texas Medical Branch, Galveston, TX 77555, USA. teikegam@utmb.edu.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Ikegami
mesh_terms: D000818:Animals; D002460:Cell Line; D018387:Codon, Initiator; D004252:DNA Mutational Analysis; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D012296:Rift Valley fever virus / Q000235:genetics* / Q000502:physiology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D018387:Codon, Initiator; D006023:Glycoproteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D008156:Luciferases
keywords: 78 kD; Gn; M-segment; NSm; Rift Valley fever virus; expression strategy; precursor; reporter assay; reverse genetics