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Entirely Plasmid-Based Reverse Genetics System for Rotaviruses

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Entirely Plasmid-Based Reverse Genetics System for Rotaviruses Entirely plasmid-based reverse genetics system SEE COMMENTARY for rotaviruses Yuta Kanaia, Satoshi Komotob, Takahiro Kawagishia,c, Ryotaro Noudaa,c, Naoko Nagasawaa, Misa Onishia, Yoshiharu Matsuurac, Koki Taniguchib, and Takeshi Kobayashia,1 aLaboratory of Viral Replication, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871 Japan; bDepartment of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi, 470-1192 Japan; and cDepartment of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871 Japan Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved December 28, 2016 (received for review November 7, 2016) Rotaviruses (RVs) are highly important pathogens that cause severe extensive serial selective passage at a high multiplicity of infection diarrhea among infants and young children worldwide. The under- (MOI) (11). A method for generating a recombinant virus containing standing of the molecular mechanisms underlying RV replication and the NSP2 gene segment uses independent selection mechanisms: a pathogenesis has been hampered by the lack of an entirely plasmid- temperature-sensitive (ts) mutant in which NSP2 is defective at based reverse genetics system. In this study, we describe the recovery nonpermissive temperature as a helper virus, and siRNA-mediated of recombinant RVs entirely from cloned cDNAs. The strategy requires gene silencing against the NSP2 message-sense ssRNA of the ts coexpression of a small transmembrane protein that accelerates cell- mutant (12). However, despite extensive efforts in many laboratories, to-cell fusion and vaccinia virus capping enzyme. We used this system no entirely plasmid-based reverse genetics system that does not re- to obtain insights into the process by which RV nonstructural protein quire a selection method against helper virus and is applicable to all NSP1 subverts host innate immune responses. By insertion into the gene segments of RV strains has been developed (14). NSP1 gene segment, we recovered recombinant viruses that encode Here, we demonstrate that recombinant RV can be recovered – split-green fluorescent protein tagged NSP1 and NanoLuc luciferase. following transfection of baby hamster kidney cells constitutively This technology will provide opportunities for studying RV biology expressing T7pol (BHK-T7) with 11 RV cDNA plasmids and and foster development of RV vaccines and therapeutics. expression plasmids encoding NBV fusion-associated small transmembrane (FAST) protein and VV capping enzyme. We rotavirus | reverse genetics | vaccine | reporter virus tested the plasmid-based reverse genetics system by generating a recombinant virus lacking the C-terminal region of NSP1 and Reoviridae roup A rotaviruses (RVs), members of the family , used it to investigate the function of this protein as an antag- Gare a highly prevalent cause of severe diarrhea in infants and ∼ onist of the innate immune response in infected cells. In ad- young children worldwide and are responsible for 215,000 deaths dition, we established efficient gene transfer systems for use in annually, mostly in developing countries (1). RVs are nonenveloped live-cell imaging, trafficking, and antiviral screening. icosahedral viruses containing a genome of 11 gene segments composed of double-stranded (ds) RNA. Results Reverse genetics systems for manipulating viral genomes Development of a Reverse Genetics System for RV. In the efforts to provide key critical insights into viral replication and pathogen- develop improved reverse genetics systems for Reoviridae viruses, esis and facilitate development of novel vaccines and viral vec- we discovered two important modifications that significantly tors through direct gene modification and attenuation. Entirely increase nonfusogenic MRV and RV replication and enhance plasmid- or RNA transcript-based reverse genetics systems have now been established for several genera of Reoviridae, including Significance mammalian orthoreovirus (MRV), Nelson Bay orthoreovirus MICROBIOLOGY (NBV) (Orthoreovirus genus), and bluetongue virus, African horse sickness virus, and epizootic hemorrhagic disease virus Rotaviruses (RVs) are a group of viruses that cause severe gas- (Orbivirus genus) (2–9). The development of the plasmid-based troenteritis in infants and young children. Until now, no strategy reverse genetics system for MRV (2) raised expectations that this has been developed to generate infectious RVs entirely from technology could be readily applied to genus Rotavirus. Partial cloned cDNAs. The absence of a reliable reverse genetics plat- plasmid-based reverse genetics systems that are dependent on form has been a major roadblock in the RV field, precluding helper viruses have been developed for RV, and these strategies numerous studies of RV replication and pathogenesis and ham- have been used to generate recombinant RVs containing a single pering efforts to develop the next generation of RV vaccines. recombinant gene segment derived from cloned cDNAs (10–13). Here, we developed a plasmid-based reverse genetics system The breakthrough for generating recombinant RVs was de- that is free from helper viruses and independent of any selection veloped to manipulate the gene segment that encodes outer- for RV. This technology will accelerate studies of RV pathobiol- ogy, allow rational design of RV vaccines, and yield RVs suitable capsid protein VP4 (13). In this system, a plasmid cDNA con- for screening small molecules as potential antivirals. taining the VP4 gene segment was transfected into monkey kidney epithelial COS-7 cells expressing T7 RNA polymerase Author contributions: Y.K., Y.M., K.T., and T. Kobayashi designed research; Y.K., S.K., (T7pol) from attenuated vaccinia virus (VV) recombinant-strain T. Kawagishi, R.N., N.N., and M.O. performed research;Y.K.andT.Kobayashianalyzed rDIs-T7pol. The cells were then infected with human RV strain data; and Y.K., Y.M., K.T., and T. Kobayashi wrote the paper. KU as a helper virus. Distinct recombinant VP4 monoreassortant The authors declare no conflict of interest. viruses were isolated using neutralizing monoclonal antibodies This article is a PNAS Direct Submission. – specific for helper virus VP4. Subsequently, other helper-virus Data deposition: The complete genome sequences of strain SA11 reported in this paper dependent techniques were developed by modification of the first have been deposited in the GenBank database (accession nos. LC178564–LC178574). system. Troupin et al. reported a reverse genetics method for RVs See Commentary on page 2106. based on preferential packaging of rearranged gene segments. In 1To whom correspondence should be addressed. Email: [email protected]. this system, a recombinant monoreassortant virus containing the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. nonstructural protein NSP3 gene segment was engineered by 1073/pnas.1618424114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1618424114 PNAS | February 28, 2017 | vol. 114 | no. 9 | 2349–2354 Downloaded by guest on September 26, 2021 recombinant virus recovery. Fusogenic orthoreovirus FAST strain SA11 were introduced into plasmids at sites flanked by the proteins are the smallest known nonenveloped viral fusogenic T7 promoter sequence and the antigenomic hepatitis delta virus proteins (15) and promote viral replication and pathogenesis (HDV) ribozyme (Fig. 1A). Transcription using T7pol and self- in vivo (16). Based on these findings, we speculated that FAST cleavage by the HDV ribozyme generated RNA transcripts proteins could accelerate replication of other Reoviridae viruses, corresponding to viral positive-sense RNAs containing the au- including MRV and RV, which do not encode a FAST homolog. thentic viral 5′ and 3′ ends, respectively. BHK-T7 cells were As expected, yields of MRV and RV were significantly increased cotransfected with 11 plasmids, each corresponding to a single (by ∼15-fold and ∼40-fold, respectively) in infected cells trans- RV gene segment, along with expression plasmids encoding fected with a FAST expression plasmid relative to mock-trans- FAST and VV capping enzyme (Fig. 1A). After 3–5dofin- fected cells (Fig. S1 A and B). To determine whether FAST cubation, transfected cells were subjected to three cycles of expression increases the efficiency of the MRV rescue system, we freezing and thawing and lysates were passaged in MA104 cells. cotransfected BHK-T7 cells with the rescue vector set of the A few days after the first passage, a significant cytopathic effect reverse genetics system for MRV strain T1L (9) and a FAST (CPE) was observed in MA104 cells, suggesting recovery of expression plasmid. Coexpression of FAST protein (0.005 μg) recombinant strain (rs) SA11 derived from cloned cDNAs. By resulted in an ∼600-fold increased viral yield compared with that contrast, we did not recover any recombinant virus following in cells transfected with the MRV rescue vector set alone (Fig. several passages from cells transfected with the 11 RV cDNA S1C). However, no infectious virus was synthesized in the pres- rescue vectors in the absence of FAST protein and VV capping ence of the highest concentration of FAST protein (0.05 μg) enzyme. To exclude the possibility of contamination with parental (Fig. S1C). MRV and RV mRNAs are capped at their 5′ ends, virus, a unique
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