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Entirely -based reverse system SEE COMMENTARY for

Yuta Kanaia, Satoshi Komotob, Takahiro Kawagishia,c, Ryotaro Noudaa,c, Naoko Nagasawaa, Misa Onishia, Yoshiharu Matsuurac, Koki Taniguchib, and Takeshi Kobayashia,1

aLaboratory of , International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871 Japan; bDepartment of 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 that cause severe extensive serial selective passage at a high multiplicity of among infants and young children worldwide. The under- (MOI) (11). A method for generating a recombinant containing standing of the molecular mechanisms underlying RV replication and the NSP2 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 system. In this study, we describe the recovery nonpermissive temperature as a , 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 that accelerates - 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 innate immune responses. By insertion into the gene segments of RV strains has been developed (14). NSP1 gene segment, we recovered recombinant that encode Here, we demonstrate that recombinant RV can be recovered – split-green fluorescent protein tagged NSP1 and NanoLuc luciferase. following of baby hamster kidney cells constitutively This technology will provide opportunities for studying RV biology expressing T7pol (BHK-T7) with 11 RV cDNA and and foster development of RV and therapeutics. expression plasmids encoding NBV fusion-associated small transmembrane (FAST) protein and VV capping enzyme. We | reverse genetics | | reporter virus tested the plasmid-based reverse genetics system by generating a recombinant virus lacking the C-terminal region of NSP1 and 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 of 11 gene segments composed of double-stranded (ds) RNA. Results Reverse genetics systems for manipulating viral Development of a Reverse Genetics System for RV. In the efforts to provide key critical insights into viral replication and - 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 (MRV), (NBV) (Orthoreovirus ), and bluetongue virus, virus, and epizootic hemorrhagic disease virus Rotaviruses (RVs) are a group of viruses that cause severe gas- ( 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 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 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) (Fig. 1A). 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 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 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 MluI site was engineered (as a genetic marker) into but their 3′ ends are not polyadenylated. In a VV-based reverse the NSP4 gene segment of rsSA11 derived from cloned cDNAs genetics system for Reoviridae viruses (2, 9, 11–13), T7pol tran- (Fig. 1B). We also engineered BamHI, EcoRV, and EcoRI sites scripts are efficiently capped in the cytoplasm by the VV capping into the NSP1, NSP2, and NSP3 gene segments, respectively, of enzyme, which consists of two subunits, D1R and D12L (17–19), another recombinant SA11 (rsSA11-3) generated by reverse ge- thereby increasing efficiency. By contrast, primary netics (Fig. 1B). The amplified NSP gene fragments derived from transcripts synthesized from rescue plasmids in the cytoplasm of rsSA11 and rsSA11-3 were cleaved by the corresponding restriction cells stably expressing T7pol are presumably not capped and thus enzymes, whereas the RT-PCR products from the parental virus poorly translated, suggesting that viral recovery might benefit were resistant to digestion (Fig. 1C). Direct sequencing of the RT- from the VV capping enzyme. Coexpression of the VV capping PCR products demonstrated that the expected mutations were enzyme allowed more efficient virus recovery (∼125-fold) rela- present as genetic markers in the targeted gene segments (Fig. tive to the original MRV rescue system (Fig. S1C). An additional S2A), confirming that rsSA11 and rsSA11-3 originated from cloned ∼1,150-fold increase in yield was achieved by coexpression of cDNAs. The electropherotypes of rsSA11 and rsSA11-3 were in- FAST and the VV capping enzyme along with the MRV rescue distinguishable from that of parental SA11 (Fig. S2B). Replication plasmids (Fig. S1C). Thus, the reverse genetics systems for MRV kinetics and peak titers for these viruses were virtually identical using FAST and the VV capping enzyme greatly improved (Fig. 1D), demonstrating similar replication characteristics of na- rescue efficiency. tive and recombinant viruses. Based on these improved systems for MRV, we sought to is the process by which segmented RNA viruses develop a plasmid-based system for RV. To this end, cDNAs exchange gene segments during coinfection of a single host cell representing each of the 11 RV dsRNA gene segments from with different strains. Using the newly developed reverse genetics

Fig. 1. Development of a plasmid-based reverse ge- netics system for RV. (A) Strategy for a plasmid-based reverse genetics system to recover infectious RVs from cloned cDNAs. RV cDNAs representing each of the 11 full-length SA11 gene segments are flanked by the T7 promoter (T7P) and the antigenomic hepatitis delta virus ribozyme (Rib). BHK-T7 cell lines were transfected with the 11 SA11 cDNAs and polymerase II promoter (Pol II)-driven expression plasmids encoding FAST and VV capping enzyme (D1R and D12L). (B) Nucleotide mutations in the NSP1–NSP4 of rescued viruses from cloned cDNAs. One of two substitutions in each segment creates a unique site (underlined). (C) Restriction enzyme digestion anal- ysis of NSP1–NSP4 gene segments of recombinant viruses. PCR amplicons from viral cDNA were purified and digested with the indicated restriction enzymes. (D) Replication kinetics of native SA11, rsSA11, and rsSA11-3. Monolayers of MA104 cells were infected with RVs at an MOI of 0.01 pfu per cell and in- cubated in the presence of trypsin (0.5 μg/mL) for various times. After freezing/thawing, the viral titer in cell lysates was determined by plaque assay. Re- sults are expressed as the mean viral titer from triplicate experiments. Error bars denote the SD.

2350 | www.pnas.org/cgi/doi/10.1073/pnas.1618424114 Kanai et al. Downloaded by guest on September 26, 2021 system, we recovered a monoreassortant virus, rsSA11/KUVP6, containing the human strain KU VP6 gene segment in an other- SEE COMMENTARY wise simian strain SA11 genetic background (Fig. 2A). The elec- trophoretic pattern of rsSA11/KUVP6 revealed comigration of the VP6 RNA with that of strain KU (Fig. 2B), and the mono- reassortant virus exhibited replication kinetics similar to those of native SA11 (Fig. 2C). The recovery of monoreassortant viruses demonstrated the utility of the reverse genetics system for rescue of reassortant viruses with unique genetic combinations that had not been reported previously.

The C-Terminal Region of NSP1 Is Essential for Targeting of Regulatory Factor 3 and Evasion of Innate Immune Surveillance. RV NSP1 antagonizes interferon (IFN) signaling by promoting degradation of IFN regulatory factor 3 (IRF3) (20). The im- portance of the C-terminal region of NSP1, including the pLxIS motif, for interaction with IRF3, followed by degradation, was demonstrated using recombinant NSP1 proteins and spontane- ous mutants encoding C-terminal truncations in duplicate se- quences of NSP1 (20–22). To determine whether the C-terminal region of NSP1 is essential for inhibiting IFN signaling by an- tagonizing the function of IRF3, we engineered isogenic rsSA11- dC103, which differs from wild-type rsSA11 only by harboring a C-terminal 103-residue truncation of NSP1 (Fig. 3A). Genotypes of recombinant viruses were confirmed by electrophoretic anal- ysis of viral dsRNA. The truncated NSP1 gene segment was smaller than the wild-type NSP1 gene segment (Fig. 3B). Ex- pression of NSP1 in cells infected with rsSA11 or rsSA11-dC103 was confirmed by immunoblotting with NSP1-specific antiserum Fig. 3. Analysis of the role of the C-terminally truncated NSP1 mutant virus (Fig. S3A). A comparison of the truncated NSP1 mutant and in the innate immune response. (A) Construction of the C-terminally trun- wild-type viruses revealed that replication of rsSA11-dC103 was cated NSP1 mutant. The NSP1-dC103 cDNA construct was generated by de- hampered in simian and human cell lines (Fig. 3C). We observed leting 103 residues (nucleotides 1,192–1,490) from the C-terminal region of significant degradation of IRF3 in HT29 cells infected with NSP1. (B) Electrophoretic pattern of rsSA11-dC103. Viral genomic dsRNAs extracted from recombinant RV virions were separated in 10% poly- acrylamide gels. Numbers indicate the order of the SA11 gene segments. (C) Replication of rsSA11 and rsSA11-dC103 in simian and human cell lines. Monolayers of cells were infected with rsSA11 or rsSA11-dC103 at an MOI of 0.001 pfu per cell. Cells were harvested 48 h postinfection, and the titer of infectious virus in the cell lysate was determined by plaque-forming assay in CV-1 cells. Data are expressed as the means ± SD n = 4, *P < 0.05 and **P < 0.01 (t test). (D) Impaired degradation of IRF3 in cells infected by rsSA11- dC103. HT29 cells were infected with rsSA11 or rsSA11-dC103 at an MOI of 10 pfu per cell. Six hours after infection, cells were harvested and primary antibodies raised against IRF3 and NSP5 were used to detect the intrinsic IRF3 and SA11 NSP5 proteins, respectively. An -specific antibody was MICROBIOLOGY used as a loading control. Molecular masses were determined by coelec- trophoresis of prestained protein markers. (E) Replication of rsSA11 and + − − − rsSA11-dC103 in IFN signaling-deficient cells. MEFs from TBK1 / or TBK1 / mice were infected with rsSA11 or rsSA11-dC103 at an MOI of 0.001 pfu per cell and incubated with 0.25 μg/mL trypsin. Cells were harvested, and the titer of infectious virus was determined by plaque assay. Fold increases in the pfu in TBK1−/− MEFs versus TBK1+/− MEFs are shown. Data are expressed as the mean ± SD, n = 3. *P < 0.05 (t test).

rsSA11 (Fig. 3D). By contrast, rsSA11-dC103 did not induce IRF3 degradation (Fig. 3D). Luciferase assays revealed that the IFN-β promoter element was activated in cells infected with rsSA11-dC103, but not in cells infected with rsSA11 (Fig. S3B). In addition, we assessed the replication of rsSA11 and rsSA11- dC103 in mouse embryonic fibroblasts (MEFs) deficient in Fig. 2. Generation of a monoreassortant virus between strains SA11 and TANK-binding kinase 1 (TBK1), which is required for the nu- KU. (A) Representative monoreassortant, rsSA11/KUVP6, which contains the clear translocation of IRF3 (23). Replication of rsSA11-dC103 − − strain KU VP6 gene segment on an otherwise SA11 genetic background. (B) was significantly greater in TBK1-deficient (TBK1 / ) MEFs + − Coelectrophoretic pattern of rsSA11/KUVP6. Viral genomic dsRNAs extracted than in TBK1 / MEFs, even though the titers of rsSA11-dC103 − − from native SA11, KU, and rsSA11/KUVP6 virions were separated in 10% were lower than those of rsSA11 in TBK / cells (Fig. 3E). polyacrylamide gels and visualized by silver staining. Numbers indicate the order of the SA11 and KU gene segments. (C) Monolayers of MA104 cells were Taken together, these results indicate that the C-terminal region infected with native SA11, KU, and rsSA11/KUVP6 at an MOI of 0.01 pfu per of NSP1 is required to antagonize the innate immune response cell for various intervals. Results are expressed as the mean viral titer from by inducing the degradation of IRF3, a transcription factor re- triplicate experiments. Error bars denote the SD. *P < 0.05 (t test). quired for IFN expression.

Kanai et al. PNAS | February 28, 2017 | vol. 114 | no. 9 | 2351 Downloaded by guest on September 26, 2021 Development of RV Efficient Gene Systems. To de- suggests that efficient complementation of NSP1-GFP11 with termine whether RVs capable of expressing an exogenous small GFP1–10 occurred to generate a specific GFP signal. NSP1-GFP11 fragment could be generated from cloned cDNAs, we exploited protein exhibited a diffuse distribution pattern and localized at the the split-green fluorescent protein (split-GFP) system for label- perinuclear region of virus-infected cells, whereas NSP5 was de- ing NSP1. The split-GFP system is based on the autoassembly tected in (Fig. 4C). These observations are consistent capacity of two GFP components, the GFP1–10 detector frag- with those of previous studies (25–27) and indicate that the distri- ment and GFP11 tag fragment, to restore a fully fluorescent bution of rsSA11-GFP11 NSP1 is the same as that of native NSP1. signal (24). We introduced the small GFP11 fragment (16 resi- RV mutants harboring significant sequence variation in NSP1 dues) at the C-terminal end of the NSP1 ORF (Fig. 4A). Elec- replicate efficiently in cell lines (28, 29), and RNA interference tropherotype analysis of rescued rsSA11-GFP11 virus expressing targeting of gene 5 to inhibit NSP1 mRNA expression has con- NSP1 as a GFP11-tagged fusion protein revealed the predicted firmed the nonessential role of this protein in viral replication migration of the NSP1 gene segment relative to that of rsSA11 (30), suggesting that the NSP1 gene segment can tolerate large (Fig. 4B). rsSA11-GFP11 exhibited replication kinetics similar to insertions without a significant effect on viral replication. To those of rsSA11 in MA104 cells (Fig. S4). To determine whether generate a recombinant RV expressing a , we in- the GFP tag is functional, BSR cells were transfected with the corporated the gene encoding NanoLuc luciferase (NLuc) into GFP1–10 expression plasmid and infected with rsSA11-GFP11 the NSP1 gene segment of rsSA11 (Fig. 4A). The electrophoretic or rsSA11 viruses. A GFP signal was observed in rsSA11- pattern of the NSP1-NLuc gene from rsSA11-NLuc revealed that GFP11–infected cells expressing GFP1–10, but not in mock- it migrated slower than the corresponding NSP1 gene from transfected cells or rsSA11-infected cells (Fig. 4C). This observation rsSA11 (Fig. 4B). Moreover, the plaques formed by rsSA11-NLuc

Fig. 4. Generation of RVs expressing reporter genes. (A) Construction of NSP1 genes containing the split- GFP tag or NLuc gene. The GFP11 small fragment was inserted into the C-terminal region of the NSP1 ORF. The NLuc ORF is flanked by SA11 NSP1 gene sequences (nucleotides 1–111 and 112–1,610). (B) Electropherotypes of rsSA11-GFP11 and rsSA11-NLuc. Viral dsRNAs were separated in 8% polyacrylamide gels. The numbers on the Left indicate the order of the SA11 gene segments. (C) Subcellular localization of NSP1-GFP11 fusion pro- tein in infected cells. BSR cells were transfected with the GFP1–10 fragment expression plasmid. Two hours after transfection, cells were infected with rsSA11 or rsSA11- GFP11 at an MOI of 0.5 pfu per cell. After 14 h of in- cubation, infected cells were fixed and self-assembled GFP (NSP1-GFP11 and GFP1–10) was detected as green fluorescent signal in cells transfected with or without the GFP1–10 expression plasmid. RV antigen was de- tected using rabbit anti-NSP5 antiserum and CF 594- conjugated anti-rabbit IgG. (D) Luciferase imaging of plaques from cells infected with rsSA11-NLuc. Mono- layers of MA104 cells were infected with rsSA11 or rsSA11-NLuc viruses. Five days after infection, plaques formed by RVs were visualized as luminescent signals using the In Vivo Imaging System (IVIS). (E) Replication kinetics of rsSA11-NLuc virus. MA104 cells were infected with rsSA11 or rsSA11-NLuc viruses at an MOI of 0.01 pfu per cell. Infectious virus titers (Upper) were de- termined by plaque assay, and NLuc activity (Lower) was quantified by luminometry. Data are expressed as the mean ± SD, n = 3. *P < 0.05 (t test). (F) Effect of ribavirin on rsSA11-NLuc virus infection. CV-1 cells infected with rsSA11 or rsSA11-NLuc at an MOI of 0.001 pfu per cell were incubated in DMEM supple- mented with trypsin (0.5 μg/mL) and ribavirin (0–200 μM). Fourteen hours after infection, NLuc substrate was added to each well and luminescent signals were visualized by IVIS.

2352 | www.pnas.org/cgi/doi/10.1073/pnas.1618424114 Kanai et al. Downloaded by guest on September 26, 2021 on CV-1 monolayer cells were smaller than those of rsSA11. as reassortants with any desired gene segment combination and However, cells infected with rsSA11-NLuc were positive features that could serve as vaccine candidates. SEE COMMENTARY for bioluminescent signals (Fig. 4D). rsSA11-NLuc replicated We confirmed that the C-terminal 103 residues of NSP1 are efficiently in MA104 cells (over 106 pfu/mL at 48 h post- required to inhibit IFN signaling by inducing proteasome- infection), although its replication kinetics were slightly impaired dependent degradation of IRFs (Fig. 3). According to previous compared with rsSA11 (Fig. 4E). The activities of NLuc in cell studies, NSP1 also inhibits NF-κB activation by inducing degra- lysates infected with rsSA11-NLuc could be detected as early as dation of β-TrCP and down-regulating p53, which induces apo- 12 h postinfection and increased over time (Fig. 4E). Finally, we ptosis and transactivates several genes involved in antiviral demonstrated the utility of rsSA11-NLuc for antiviral screening responses (34, 35). Additionally, NSP1 interacts with the p85 using a known RV inhibitor, ribavirin (31). CV-1 cells infected subunit of the phosphoinositide 3-kinase (PI3K)-mediated anti- with rsSA11 or rsSA11-NLuc were cultured with ribavirin at apoptotic PI3K/Akt pathway (36). Taken together, these obser- increasing concentrations; the cells did not exhibit significant vations indicate that NSP1 can interfere with multiple antiviral cytotoxicity in the range of concentrations used in the experiment pathways, including IFN and apoptosis signaling, to promote (Fig. S5A). Strong bioluminescence signals were observed in cells efficient viral replication and infection. NSP1 mutants, including infected with rsSA11-NLuc at low concentrations (0–10 μM) a C-terminal truncation incapable of blocking IFN signaling and ofribavirinanddecreasedinadose-dependent manner (Fig. 4F). apoptosis pathways, may be attractive candidates for the devel- Similarly, viral titers in infected cell lysates also exhibited dose- opment of new attenuated RV vaccines. dependent inhibition (Fig. S5B), supporting the validity of screening We used the reverse genetics system to modify the NSP1 with rsSA11-NLuc. The genetic stability of rsSA11-NLuc to express gene segment to engineer RVs expressing reporter genes. A NLuc was unchanged through five passages (Fig. S6 A and B). recombinant RV harboring the split-GFP system was generated These results demonstrate that replication-competent RVs encod- by inserting a small GFP11 tag into the C terminus of the NSP1 ing a reporter gene can be recovered by plasmid rescue and used for ORF (Fig. 4). Thus, the split-GFP–based recombinant NSP1 antiviral screening. mutants will be useful tools for understanding NSP1 trafficking and interactions with host proteins, including IFN signaling Discussion components, in infected cells. Furthermore, a similar approach We succeeded in developing an entirely plasmid-based RV re- using the split-GFP system could be used to study other RV verse genetics system in which FAST protein and capping en- proteins in living cells. We also applied the reverse genetics zyme were coexpressed along with rescue plasmids in BHK-T7 system to generate a replication-competent recombinant RV cells (Fig. 1). The mechanism underlying the remarkable effects expressing the NLuc gene fused to the N-terminal 27 residues of of FAST protein, which induce cell-to-cell fusion and NSP1 (Fig. 4). The results confirm that NSP1 is not required for formation in nonfusogenic RV and MRV replication in infected viral replication, a finding consistent with a previous study that and transfected cells, remains unclear. However, previous studies used siRNA gene silencing (30), and that the NSP1 gene segment (15, 16, 32) suggest that the cell fusion activity of FAST proteins is suitable for insertion of a heterologous sequence. The attenu- accelerates cell-to-cell transmission of virus infection and en- ated replication kinetics of rsSA11-NLuc could be explained by sures rapid release of progeny virions from apoptotic syncytia, the defect in the IFN suppressor activity of NSP1, or by the in- thereby promoting systematic infection. FAST proteins also may fluence of reduced packaging efficiency. In addition, the replica- increase cotransfection efficiency, resulting in increased virus tion and luciferase activity of rsSA11-NLuc were inhibited by production in reverse genetic systems via fusion of transfected ribavirin, a known anti-RV inhibitor, suggesting that the reporter cells with neighboring cells, leading to formation of syncytia virus would be a useful tool for high-throughput screening for carrying all 14 transfected plasmids (11 RV cDNA plasmids antiviral therapeutics (Fig. 4). Furthermore, the replication-com- and expression plasmids encoding FAST and VV capping en- petent RV carrying the NLuc gene makes it possible to track RV zyme). The currently available reverse genetics systems for RVs infection in vivo and develop an oral RV vector. are based on vaccinia-driven T7pol expression (11–13). Al- In this study, we developed a plasmid-only–based reverse ge- though it is possible to rescue Reoviridae viruses that have netics system for RV. This technique opens new horizons for the MICROBIOLOGY capped and nonpolyadenylated mRNA using VV expressing study of RV replication and pathogenesis, as well as for the T7pol, VV infection has negative effects on RV replication and development of antiviral drugs and new vaccines that protect rescue efficiency in reverse genetics systems. Accordingly, the against this important gastrointestinal pathogen. improved FAST- and VV capping enzyme-based reverse ge- netics system free of any helper virus for RV and MRV de- Materials and Methods scribed herein is applicable to the recovery of any member of Cells and Viruses. Monkey kidney epithelial MA104, CV-1, Vero, murine fi- the Reoviridae family, particularly attenuated recombinant broblast L929, and human colon epithelial Caco-2 cells were cultured in viruses that replicate poorly. DMEM (Nacalai Tesque) supplemented to contain 5% (vol/vol) fetal bovine Two licensed RV vaccines, Rotarix (GlaxoSmithKline) and serum (FBS) (Gibco), 100 units/mL penicillin, and 100 μg/mL streptomycin RotaTeq (Merck), are currently available. Rotarix is based on a (Nacalai Tesque). BHK/T7-9 cells were grown in DMEM supplemented to × contain 5% FBS, 10% (wt/vol) tryptose phosphate broth, 100 units/mL pen- single human strain, and RotaTeq is a combination of five bovine icillin, and 100 μg/mL streptomycin (37). To establish another BHK-T7 cell line human strain monoreassortants. In addition, a new RV vac- (BHK-T7/P5), BSR cells (3), a derivative of BHK cells, were selected by trans- cine, Rotavac (Bharat Biotech International), was licensed in fection with a eukaryotic expression plasmid encoding T7pol under the India in 2014 (33). Although these vaccines are effective against control of a strong CMV early enhancer/chicken β-actin (CAG) promoter (38), RV-associated severe gastroenteritis, concerns about their effi- followed by incubation in the presence of 4 μg/mL puromycin (Sigma- cacy, safety, and cost have inspired the development of new Aldrich). Immortalized MEFs derived from TBK1+/− IKKi−/− (TBK1+/−)and − − − − − − vaccines. We generated a recombinant RV containing silent TBK1 / IKKi / (TBK1 / ) mice were prepared as previously described (39, 40) mutations in three gene segments (NSP1, NSP2, and NSP3) and and grown in DMEM supplemented to contain 10% FBS, 100 units/mL μ a monoreassortant virus harboring the human RV strain KU penicillin, and 100 g/mL streptomycin. Human lines, HT29, PC3, HCC-2998, and OVCAR-4, were cultured in RPMI1640 (Nacalai Tesque) sup- VP6 gene on the strain SA11 genetic background (Figs. 1 and 2). plemented to contain 10% FBS, 100 units/mL penicillin, and 100 μg/mL Thus, in contrast to earlier helper virus-based reverse genetics streptomycin. Simian RV strain SA11 (SA11-L2) (G3P[2]) (41) and human RV systems, the RV rescue system described here can be easily used strain KU (G1P[8]) (42) were propagated in MA104 cells cultured in DMEM for rapid generation of infectious RVs containing multiple mu- supplemented with 0.5 μg/mL trypsin (Sigma-Aldrich). MRV strain T1L, a tations in several different gene segments simultaneously, as well laboratory stock originally obtained from Bernard Fields, Harvard Medical

Kanai et al. PNAS | February 28, 2017 | vol. 114 | no. 9 | 2353 Downloaded by guest on September 26, 2021 School, Boston, was propagated in L929 cells. Infectious titers of RV and MRV concentration of 10 μg/mL, and the samples were incubated at 37 °C for 30 min were determined by plaque assay using CV-1 cells and L929 cells, re- to activate infectious RVs. The lysates were then transferred to fresh MA104 cells. spectively, as previously described (43, 44). After adsorption at 37 °C for 1 h, the lysate-adsorbed MA104 cells were washed and cultured in FBS-free DMEM supplemented with 0.5 μg/mL trypsin and in- Recovery of Recombinant RVs from Cloned cDNAs. Monolayers of BHK-T7 cells cubated at 37 °C for 7 d. When CPE was observed following RV infection of 5 (8 × 10 ) in six-well plates were cotransfected with plasmids using 2 μLofTransIT- monolayer cells, recombinant viruses were isolated from passaged cells by pla- μ LT1 transfection reagent per microgram of plasmid DNA, as follows: 0.8 gof que purification using CV-1 cells. each strain SA11 rescue plasmid [pT7-VP1SA11, pT7-VP2SA11, pT7-VP3SA11, pT7- VP4SA11, pT7-VP6SA11 (or pT7-VP6KU), pT7-VP7SA11, pT7-NSP1SA11 (pT7- ACKNOWLEDGMENTS. We thank Terence S. Dermody for reviewing the NSP1-BamHI, pT7-NSP1-dC103, or pT7-NSP1-GFP11), pT7-NSP2SA11 (or pT7-NSP2- manuscript, Kaede Yukawa for secretarial work, Toru Okamoto for technical EcoRV), pT7-NSP3SA11 (or pT7-NSP3-EcoRI), pT7-NSP4SA11 (or pT7-NSP4-MluI), advice, Saori Fukuda for technical assistance, Polly Roy for providing BSR and pT7-NSP5SA11], 0.015 μg of pCAG-FAST, and 0.8 μg of each capping enzyme cells, and Naoto Ito for providing BHK/T7-9 cells. This work was supported 5 expression plasmid. After 2 d of incubation in FBS-free medium, MA104 cells (10 in part by grants-in-aid for the Research Program on Emerging and Re- cells) were added to the transfected cells and cocultured for 3 d in FBS-free Emerging Infectious Diseases from the Japan Agency for Medical Research medium supplemented with trypsin (0.5 μg/mL). After incubation, transfected and Development and Japanese Society for the Promotion of Science cells were lysed by freeze/thaw, trypsin was added to cell lysates at a final (JSPS) KAKENHI Grants JP16K19138, JP15J04209, and JP26292149.

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