Generation of Recombinant Rotavirus Expressing NSP3-UnaG Fusion Protein by a Simplified Reverse Genetics System

Abstract

Rotavirus is a segmented double-stranded (ds)RNA virus that causes severe gastroenteritis in young children. We have established an efficient simplified rotavirus reverse genetics (RG) system that uses eleven T7 plasmids, each expressing a unique simian SA11 (+)RNA, and a CMV support plasmid for the African swine fever virus NP868R capping enzyme. With the NP868R-based system, we generated recombinant rotavirus (rSA11/NSP3-FL-UnaG) with a genetically modified 1.5-kB segment 7 dsRNA that encodes full-length NSP3 fused to UnaG, a 139-aa green fluorescent protein (FP). Analysis of rSA11/NSP3-FL-UnaG showed that the virus replicated efficiently and was genetically stable over 10 rounds of serial passage. The NSP3-UnaG fusion product was well expressed in rSA11/NSP3-FL-UnaG-infected cells, reaching levels similar to NSP3 in wildtype rSA11-infected cells. Moreover, the NSP3-UnaG protein, like functional wildtype NSP3, formed dimers in vivo. Notably, NSP3-UnaG protein was readily detected in infected cells via live cell imaging, with intensity levels ~3-fold greater than that of the NSP1-UnaG fusion product of rSA11/NSP1-FL-UnaG. Our results indicate that FP-expressing recombinant rotaviruses can be made through manipulation of the segment 7 dsRNA without deleting or interrupting any of the twelve open reading frames of the virus. Because NSP3 is expressed at levels higher than NSP1 in infected cells, rotaviruses expressing NSP3-based FPs may be a more sensitive tool for studying rotavirus biology than rotaviruses expressing NSP1-based FPs. This is the first report of a recombinant rotavirus containing a genetically engineered segment 7 dsRNA.\n\nImportancePrevious studies have generated recombinant rotaviruses that express fluorescent proteins (FPs) by inserting reporter genes into the NSP1 open reading frame (ORF) of genome segment 5. Unfortunately, NSP1 is expressed at low levels in infected cells, making viruses expressing FP-fused NSP1 less than ideal probes of rotavirus biology. Moreover, FPs were inserted into segment 5 in such a way as to compromise NSP1, an interferon antagonist affecting viral growth and pathogenesis. We have identified an alternative approach for generating rotaviruses expressing FPs, one relying on fusing the reporter gene to the NSP3 ORF of genome segment 7. This was accomplished without interrupting any of the viral ORFs, yielding recombinant viruses likely expressing the complete set of functional viral proteins. Given that NSP3 is made at moderate levels in infected cells, rotavirus encoding NSP3-based FPs should be more sensitive probes of viral infection than rotaviruses encoding NSP1-based FPs.