The role of Bunyamwera virus nonstructural protein NSs in shutoff of host cell protein synthesis

• Main Author: Hart, Timothy J
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2004
• Subjects: Molecular biology; Virology
• ISBN: 9780438165168; 0438165160

Bunyamwera virus (BUNV) is the type virus of the family Bunyaviridae and of the genus Orthobunyavirus. Members of the Bunyaviridae contain a single stranded RNA genome, of negative polarity, in three segments. The nonstructural protein, NSs, that is expressed from the smallest segment, S, has been implicated in inhibiting host protein synthesis in infected cells. NSs has also been implicated in inhibiting the interferon mediated antiviral response, in inhibiting viral replication as measured by a minireplicon system and as having both pro- and anti-apoptotic effects. These properties were largely determined using a recombinant BUNV which does not express the NSs protein, BUNdelNSs. This project aimed to investigate the particular characteristics of NSs and the role of BUNV NSs in the shutoff of host protein synthesis. Attempts were made to produce a monoclonal antibody against BUNV NSs. NSs was expressed in E. coli and in an in vitro expression system. In neither case was NSs expressed to sufficient levels to enable an immune response in inoculated mice feasible. A previous attempt had been made to make polyclonal antibodies in rabbits, using synthetic peptides representing partial NSs sequences. No reactivity against BUNV NSs had been found. Re-screening of these sera, using bacterially expressed NSs, revealed that one was able to detect NSs in Western blotting and by immunoprecipitation, but not by immunofluorescence. This antiserum, designated anti-NSs, was also able to detect NSs proteins expressed by orthobunyaviruses other than BUNV. Use of anti-NSs revealed that, in mammalian cells, BUNV NSs was expressed early in infection, but that its concentration decreased rapidly at late times post-infection. Subsequent experiments revealed that NSs was sensitive to proteasome-mediated degradation, implying that such degradation may play a role in reducing NSs levels at late times of infection. Western blotting of cells fractionated into nuclear and cytoplasmic compartments indicated that NSs was a predominantly cytoplasmic protein, although some NSs localised to the nucleus at times of peak expression. A previously constructed recombinant virus, BUNdelNSs was observed to express a 9.23 kDa peptide that reacted in Western blotting with the anti-NSs antibody. It is likely that this small protein represented a peptide synthesised through alternative initiation of translation from the third AUG of the NSs ORF. A new recombinant virus, which did not express the 9.23 kDa peptide, BUNdelNSs2, was produced using reverse genetics technology. As with BUNdelNSs, BUNdelNSs2 did not induce shutoff to the same extent as wt BUNV. Other properties noted for BUNdelNSs relative to wt BUNV, such as a reduced titre and smaller plaque size, were also noted for BUNdelNSs2. Comparison of the levels of four mRNAs of 'housekeeping' genes in wt BUNV and BUNdelNSs2 infected cells, by quantitative PCR, revealed that mRNA instability did not play a role in the inhibition of protein synthesis by BUNV NSs. A luciferase reporter assay was developed to quantify the shutoff caused by NSs. Inhibition of luciferase expression was shown to be an accurate proxy for the more general inhibition of protein synthesis caused by wt BUNV. Using the luciferase assay, the NSs protein of BUNV, as well as those from other orthobunyaviruses, was shown to be capable of inhibiting protein synthesis when expressed in the absence of other viral proteins and was shown to do so in a dose-dependent manner. Co-expression of various combinations of viral proteins showed that NSs was the principal viral protein causing shutoff Mutation of specific residues in NSs resulted in a decrease in the inhibition of luciferase expression, further illustrating the importance of NSs to the process of shutoff Wt BUNV did not inhibit host protein synthesis in mosquito cells. Furthermore, the NSs protein expressed alone was not able to inhibit luciferase synthesis in mosquito cells. Finally, NSs was found to be able to inhibit expression of luciferase from transfected luciferase mRNA. This was the case whether NSs was expressed by wt BUNV or from plasmids in vitro, implying that NSs is able to specifically inhibit translation. The importance of these observations in terms of the mechanism(s) by which BUNV NSs causes shutoff is discussed.