Entirely plasmid-based reverse genetics system for rotaviruses

Significance

Rotaviruses (RVs) are a group of viruses that cause severe gastroenteritis in infants and young children. Until now, no strategy has been developed to generate infectious RVs entirely from cloned cDNAs. The absence of a reliable reverse genetics platform has been a major roadblock in the RV field, precluding numerous studies of RV replication and pathogenesis and hampering efforts to develop the next generation of RV vaccines. Here, we developed a plasmid-based reverse genetics system that is free from helper viruses and independent of any selection for RV. This technology will accelerate studies of RV pathobiology, allow rational design of RV vaccines, and yield RVs suitable for screening small molecules as potential antivirals.

Abstract

Rotaviruses (RVs) are highly important pathogens that cause severe diarrhea among infants and young children worldwide. The understanding of the molecular mechanisms underlying RV replication and pathogenesis has been hampered by the lack of an entirely plasmid-based reverse genetics system. In this study, we describe the recovery of recombinant RVs entirely from cloned cDNAs. The strategy requires coexpression of a small transmembrane protein that accelerates cell-to-cell fusion and vaccinia virus capping enzyme. We used this system to obtain insights into the process by which RV nonstructural protein NSP1 subverts host innate immune responses. By insertion into the NSP1 gene segment, we recovered recombinant viruses that encode split-green fluorescent protein–tagged NSP1 and NanoLuc luciferase. This technology will provide opportunities for studying RV biology and foster development of RV vaccines and therapeutics.

Group A rotaviruses (RVs), members of the family Reoviridae, are a highly prevalent cause of severe diarrhea in infants and young children worldwide and are responsible for ∼215,000 deaths annually, mostly in developing countries (1). RVs are nonenveloped icosahedral viruses containing a genome of 11 gene segments composed of double-stranded (ds) RNA.

Reverse genetics systems for manipulating viral genomes provide key critical insights into viral replication and pathogenesis and facilitate development of novel vaccines and viral vectors through direct gene modification and attenuation. Entirely plasmid- or RNA transcript-based reverse genetics systems have now been established for several genera of Reoviridae, including mammalian orthoreovirus (MRV), Nelson Bay orthoreovirus (NBV) (Orthoreovirus genus), and bluetongue virus, African horse sickness virus, and epizootic hemorrhagic disease virus (Orbivirus genus) (2–9). The development of the plasmid-based reverse genetics system for MRV (2) raised expectations that this technology could be readily applied to genus Rotavirus. Partial plasmid-based reverse genetics systems that are dependent on helper viruses have been developed for RV, and these strategies have been used to generate recombinant RVs containing a single recombinant gene segment derived from cloned cDNAs (10–13). The breakthrough for generating recombinant RVs was developed to manipulate the gene segment that encodes outer-capsid protein VP4 (13). In this system, a plasmid cDNA containing the VP4 gene segment was transfected into monkey kidney epithelial COS-7 cells expressing T7 RNA polymerase (T7pol) from attenuated vaccinia virus (VV) recombinant-strain rDIs-T7pol. The cells were then infected with human RV strain KU as a helper virus. Distinct recombinant VP4 monoreassortant viruses were isolated using neutralizing monoclonal antibodies specific for helper virus VP4. Subsequently, other helper-virus–dependent techniques were developed by modification of the first system. Troupin et al. reported a reverse genetics method for RVs based on preferential packaging of rearranged gene segments. In this system, a recombinant monoreassortant virus containing the nonstructural protein NSP3 gene segment was engineered by extensive serial selective passage at a high multiplicity of infection (MOI) (11). A method for generating a recombinant virus containing the NSP2 gene segment uses independent selection mechanisms: a temperature-sensitive (ts) mutant in which NSP2 is defective at nonpermissive temperature as a helper virus, and siRNA-mediated gene silencing against the NSP2 message-sense ssRNA of the ts mutant (12). However, despite extensive efforts in many laboratories, no entirely plasmid-based reverse genetics system that does not require a selection method against helper virus and is applicable to all gene segments of RV strains has been developed (14).

Here, we demonstrate that recombinant RV can be recovered following transfection of baby hamster kidney cells constitutively expressing T7pol (BHK-T7) with 11 RV cDNA plasmids and expression plasmids encoding NBV fusion-associated small transmembrane (FAST) protein and VV capping enzyme. We tested the plasmid-based reverse genetics system by generating a recombinant virus lacking the C-terminal region of NSP1 and used it to investigate the function of this protein as an antagonist of the innate immune response in infected cells. In addition, we established efficient gene transfer systems for use in live-cell imaging, trafficking, and antiviral screening.

Results

Development of a Reverse Genetics System for RV.

In the efforts to develop improved reverse genetics systems for Reoviridae viruses, we discovered two important modifications that significantly increase nonfusogenic MRV and RV replication and enhance recombinant virus recovery. Fusogenic orthoreovirus FAST proteins are the smallest known nonenveloped viral fusogenic proteins (15) and promote viral replication and pathogenesis in vivo (16). Based on these findings, we speculated that FAST proteins could accelerate replication of other Reoviridae viruses, including MRV and RV, which do not encode a FAST homolog. As expected, yields of MRV and RV were significantly increased (by ∼15-fold and ∼40-fold, respectively) in infected cells transfected with a FAST expression plasmid relative to mock-transfected cells (Fig. S1 A and B). To determine whether FAST expression increases the efficiency of the MRV rescue system, we cotransfected BHK-T7 cells with the rescue vector set of the reverse genetics system for MRV strain T1L (9) and a FAST expression plasmid. Coexpression of FAST protein (0.005 µg) resulted in an ∼600-fold increased viral yield compared with that in cells transfected with the MRV rescue vector set alone (Fig. S1C). However, no infectious virus was synthesized in the presence of the highest concentration of FAST protein (0.05 µg) (Fig. S1C). MRV and RV mRNAs are capped at their 5′ ends, but their 3′ ends are not polyadenylated. In a VV-based reverse genetics system for Reoviridae viruses (2, 9, 11–13), T7pol transcripts are efficiently capped in the cytoplasm by the VV capping enzyme, which consists of two subunits, D1R and D12L (17–19), thereby increasing translation efficiency. By contrast, primary transcripts synthesized from rescue plasmids in the cytoplasm of cells stably expressing T7pol are presumably not capped and thus poorly translated, suggesting that viral recovery might benefit from the VV capping enzyme. Coexpression of the VV capping enzyme allowed more efficient virus recovery (∼125-fold) relative to the original MRV rescue system (Fig. S1C). An additional ∼1,150-fold increase in yield was achieved by coexpression of FAST and the VV capping enzyme along with the MRV rescue plasmids (Fig. S1C). Thus, the reverse genetics systems for MRV using FAST and the VV capping enzyme greatly improved rescue efficiency.

Fig. S1.

Development of an improved reverse genetics system for MRV. Monolayers of Vero cells cultured in 24-well plates were transfected with 0.25–2 µg of FAST expression plasmid (black bar) or empty vector (white bar). Two hours after transfection, cells were infected with MRV strain T1L (A) or RV strain SA11 (B) at an MOI of 0.001 pfu per cell. Infected cells were lysed by freeze/thaw 16 h postinfection, and viral titers in cell lysates were determined by plaque assay. Data are expressed as the means ± SD, n = 3. *P < 0.05 (t test). (C) BHK-T7/P5 cells (2 × 10⁵ cells) were transfected with the indicated plasmids as follows: 0.4 µg of each T1L rescue plasmid (pT7-L1-M2T1L, pT7-L2-M3T1L, pT7-L3-S3T1L, and pT7-S1-S2-S4-M1T1L), 0.05–0.0005 µg of FAST expression plasmid, and 0.2 µg of each capping enzyme expression plasmid (pCAG-D1R and pCAG-D12L). Forty-eight hours after transfection, transfected cells were lysed by freeze/thaw, and viral titers in cell lysates were determined by plaque assay. Results are presented as mean viral titers for quadruplicate experiments. Asterisks indicate statistically significant differences with respect to the control sample (P < 0.05, t test). Error bars indicate the SD.

Based on these improved systems for MRV, we sought to develop a plasmid-based system for RV. To this end, cDNAs representing each of the 11 RV dsRNA gene segments from strain SA11 were introduced into plasmids at sites flanked by the T7 promoter sequence and the antigenomic hepatitis delta virus (HDV) ribozyme (Fig. 1A). Transcription using T7pol and self-cleavage by the HDV ribozyme generated RNA transcripts corresponding to viral positive-sense RNAs containing the authentic viral 5′ and 3′ ends, respectively. BHK-T7 cells were cotransfected with 11 plasmids, each corresponding to a single RV gene segment, along with expression plasmids encoding FAST and VV capping enzyme (Fig. 1A). After 3–5 d of incubation, transfected cells were subjected to three cycles of freezing and thawing and lysates were passaged in MA104 cells. A few days after the first passage, a significant cytopathic effect (CPE) was observed in MA104 cells, suggesting recovery of recombinant strain (rs) SA11 derived from cloned cDNAs. By contrast, we did not recover any recombinant virus following several passages from cells transfected with the 11 RV cDNA rescue vectors in the absence of FAST protein and VV capping enzyme. To exclude the possibility of contamination with parental virus, a unique MluI site was engineered (as a genetic marker) into the NSP4 gene segment of rsSA11 derived from cloned cDNAs (Fig. 1B). We also engineered BamHI, EcoRV, and EcoRI sites into the NSP1, NSP2, and NSP3 gene segments, respectively, of another recombinant SA11 (rsSA11-3) generated by reverse genetics (Fig. 1B). The amplified NSP gene fragments derived from rsSA11 and rsSA11-3 were cleaved by the corresponding restriction enzymes, whereas the RT-PCR products from the parental virus were resistant to digestion (Fig. 1C). Direct sequencing of the RT-PCR products demonstrated that the expected mutations were present as genetic markers in the targeted gene segments (Fig. S2A), confirming that rsSA11 and rsSA11-3 originated from cloned cDNAs. The electropherotypes of rsSA11 and rsSA11-3 were indistinguishable from that of parental SA11 (Fig. S2B). Replication kinetics and peak titers for these viruses were virtually identical (Fig. 1D), demonstrating similar replication characteristics of native and recombinant viruses.

Fig. 1.

Development of a plasmid-based reverse genetics 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 genes of rescued viruses from cloned cDNAs. One of two substitutions in each segment creates a unique restriction enzyme site (underlined). (C) Restriction enzyme digestion analysis 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 incubated 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. Results are expressed as the mean viral titer from triplicate experiments. Error bars denote the SD.

Fig. S2.

Sequence analysis of gene segments from rsSA11 and rsSA11-3. (A) NSP1–NSP4 gene segments of recombinant RVs were amplified by RT-PCR using specific primers and viral dsRNAs extracted from purified virions. Direct sequence analysis was performed on the amplified fragments. Nucleotide differences between native and recombinant viruses are indicated by asterisks. (B) Electropherotypes of native virus, rsSA11, and rsSA11-3. Viral dsRNAs extracted from purified virions were separated in 10% polyacrylamide gel and visualized using ethidium bromide staining. Numbers at Left indicate the order of the SA11 gene segments.

Reassortment is the process by which segmented RNA viruses exchange gene segments during coinfection of a single host cell with different strains. Using the newly developed reverse genetics system, we recovered a monoreassortant virus, rsSA11/KUVP6, containing the human strain KU VP6 gene segment in an otherwise simian strain SA11 genetic background (Fig. 2A). The electrophoretic pattern of rsSA11/KUVP6 revealed comigration of the VP6 RNA with that of strain KU (Fig. 2B), and the monoreassortant 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.

Fig. 2.

Generation of a monoreassortant virus between strains SA11 and KU. (A) Representative monoreassortant, rsSA11/KUVP6, which contains the strain KU VP6 gene segment on an otherwise SA11 genetic background. (B) Coelectrophoretic pattern of rsSA11/KUVP6. Viral genomic dsRNAs extracted from native SA11, KU, and rsSA11/KUVP6 virions were separated in 10% polyacrylamide gels and visualized by silver staining. Numbers indicate the order of the SA11 and KU gene segments. (C) Monolayers of MA104 cells were infected with native SA11, KU, and rsSA11/KUVP6 at an MOI of 0.01 pfu per cell for various intervals. Results are expressed as the mean viral titer from triplicate experiments. Error bars denote the SD. *P < 0.05 (t test).

The C-Terminal Region of NSP1 Is Essential for Targeting of Interferon 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 importance 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 spontaneous mutants encoding C-terminal truncations in duplicate sequences of NSP1 (20–22). To determine whether the C-terminal region of NSP1 is essential for inhibiting IFN signaling by antagonizing 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 analysis of viral dsRNA. The truncated NSP1 gene segment was smaller than the wild-type NSP1 gene segment (Fig. 3B). Expression of NSP1 in cells infected with rsSA11 or rsSA11-dC103 was confirmed by immunoblotting with NSP1-specific antiserum (Fig. S3A). A comparison of the truncated NSP1 mutant and wild-type viruses revealed that replication of rsSA11-dC103 was hampered in simian and human cell lines (Fig. 3C). We observed significant degradation of IRF3 in HT29 cells infected with 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 TANK-binding kinase 1 (TBK1), which is required for the nuclear translocation of IRF3 (23). Replication of rsSA11-dC103 was significantly greater in TBK1-deficient (TBK1^−/−) MEFs than in TBK1^+/− MEFs, even though the titers of rsSA11-dC103 were lower than those of rsSA11 in TBK^−/− cells (Fig. 3E). Taken together, these results indicate that the C-terminal region of NSP1 is required to antagonize the innate immune response by inducing the degradation of IRF3, a transcription factor required for IFN expression.

Fig. 3.

Analysis of the role of the C-terminally truncated NSP1 mutant virus in the innate immune response. (A) Construction of the C-terminally truncated NSP1 mutant. The NSP1-dC103 cDNA construct was generated by deleting 103 residues (nucleotides 1,192–1,490) from the C-terminal region of NSP1. (B) Electrophoretic pattern of rsSA11-dC103. Viral genomic dsRNAs extracted from recombinant RV virions were separated in 10% polyacrylamide 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 actin-specific antibody was used as a loading control. Molecular masses were determined by coelectrophoresis 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).

Fig. S3.

IFN-β promoter activation by the NSP1 mutant. (A) Expression of NSP1 protein from a recombinant virus. MA104 cells were infected with rsSA11 or rsSA11-dC103 at an MOI of 0.1 pfu per cell. As a control, cells were transfected with NSP1 expression plasmid (pCAG-NSP1). Viral protein expression in cells was assessed by immunoblotting using anti-NSP1 antibody after 48 h of incubation. An actin-specific antibody was used as a loading control. Molecular masses were determined by coelectrophoresis of prestained protein markers. (B) IFN-β promoter activation assay. The 293T cells were transfected with a firefly luciferase (Fluc) reporter plasmid under the control of the IFN-β promoter (pIFN-β–Fluc) and a pSV40-Rluc reporter plasmid that expresses Renilla luciferase (Rluc) under the control of the SV40 promoter (Promega). Twelve hours after transfection, cells were infected with rsSA11 or rsSA11-dC103 at an MOI of 10 pfu per cell. Cells were harvested 10 h postinfection, and luciferase activities were determined. Relative luminescence units (Fluc/Rluc) are shown. *P < 0.05, **P < 0.01 (Tukey’s multiple comparison test).

Development of RV Efficient Gene Transduction Systems.

To determine whether RVs capable of expressing an exogenous small fragment could be generated from cloned cDNAs, we exploited the split-green fluorescent protein (split-GFP) system for labeling NSP1. The split-GFP system is based on the autoassembly capacity of two GFP components, the GFP1–10 detector fragment and GFP11 tag fragment, to restore a fully fluorescent signal (24). We introduced the small GFP11 fragment (16 residues) at the C-terminal end of the NSP1 ORF (Fig. 4A). Electropherotype analysis of rescued rsSA11-GFP11 virus expressing NSP1 as a GFP11-tagged fusion protein revealed the predicted migration of the NSP1 gene segment relative to that of rsSA11 (Fig. 4B). rsSA11-GFP11 exhibited replication kinetics similar to those of rsSA11 in MA104 cells (Fig. S4). To determine whether the GFP tag is functional, BSR cells were transfected with the GFP1–10 expression plasmid and infected with rsSA11-GFP11 or rsSA11 viruses. A GFP signal was observed in rsSA11-GFP11–infected cells expressing GFP1–10, but not in mock-transfected cells or rsSA11-infected cells (Fig. 4C). This observation suggests that efficient complementation of NSP1-GFP11 with GFP1–10 occurred to generate a specific GFP signal. NSP1-GFP11 protein exhibited a diffuse distribution pattern and localized at the perinuclear region of virus-infected cells, whereas NSP5 was detected in viroplasms (Fig. 4C). These observations are consistent with those of previous studies (25–27) and indicate that the distribution of rsSA11-GFP11 NSP1 is the same as that of native NSP1.

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 protein 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 incubation, 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 detected using rabbit anti-NSP5 antiserum and CF 594-conjugated anti-rabbit IgG. (D) Luciferase imaging of plaques from cells infected with rsSA11-NLuc. Monolayers 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 determined 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 supplemented 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.

Fig. S4.

Replication of rsSA11-GFP11 in MA104 cells. MA104 cells were infected with rsSA11 or rsSA11-GFP11 viruses at an MOI of 0.01 pfu per cell and incubated for various intervals. After freezing/thawing, viral titer in cell lysates was determined by plaque assay. Results are presented as mean viral titers from triplicate experiments. Error bars indicate SD.

RV mutants harboring significant sequence variation in NSP1 replicate efficiently in cell lines (28, 29), and RNA interference targeting of gene 5 to inhibit NSP1 mRNA expression has confirmed the nonessential role of this protein in viral replication (30), suggesting that the NSP1 gene segment can tolerate large insertions without a significant effect on viral replication. To generate a recombinant RV expressing a reporter gene, we incorporated the gene encoding NanoLuc luciferase (NLuc) into the NSP1 gene segment of rsSA11 (Fig. 4A). The electrophoretic pattern of the NSP1-NLuc gene from rsSA11-NLuc revealed that it migrated slower than the corresponding NSP1 gene from rsSA11 (Fig. 4B). Moreover, the plaques formed by rsSA11-NLuc on CV-1 monolayer cells were smaller than those of rsSA11. However, cells infected with rsSA11-NLuc were positive for bioluminescent signals (Fig. 4D). rsSA11-NLuc replicated efficiently in MA104 cells (over 10⁶ pfu/mL at 48 h postinfection), although its replication kinetics were slightly impaired compared with rsSA11 (Fig. 4E). The activities of NLuc in cell lysates infected with rsSA11-NLuc could be detected as early as 12 h postinfection and increased over time (Fig. 4E). Finally, we demonstrated the utility of rsSA11-NLuc for antiviral screening using a known RV inhibitor, ribavirin (31). CV-1 cells infected with rsSA11 or rsSA11-NLuc were cultured with ribavirin at increasing concentrations; the cells did not exhibit significant cytotoxicity in the range of concentrations used in the experiment (Fig. S5A). Strong bioluminescence signals were observed in cells infected with rsSA11-NLuc at low concentrations (0–10 µM) of ribavirin and decreased in a dose-dependent manner (Fig. 4F). Similarly, viral titers in infected cell lysates also exhibited dose-dependent inhibition (Fig. S5B), supporting the validity of screening with rsSA11-NLuc. The genetic stability of rsSA11-NLuc to express NLuc was unchanged through five passages (Fig. S6 A and B). These results demonstrate that replication-competent RVs encoding a reporter gene can be recovered by plasmid rescue and used for antiviral screening.

Fig. S5.

Inhibitory effect of ribavirin on RV replication. (A) Cytotoxicity of ribavirin in CV-1 cells. Monolayers of CV-1 cells cultured in 96-well plates were incubated in DMEM supplemented with different amounts of ribavirin for 13 h. To determine the cytotoxicity of ribavirin, WST-1 reagent (Roche) was added to each well, and the samples were incubated at 37 °C for 1 h, followed by measurement of optical density (OD) at 440 nm. Relative OD values (%) vs. ribavirin-free cells are shown. Data are expressed as means ± SD, n = 2. (B) CV-1 cells were infected with rsSA11 or rsSA11-NLuc at an MOI of 0.001 and then incubated in DMEM supplemented with trypsin (0.5 µg/mL) and ribavirin (0–1,000 µM) for 14 h. After freezing/thawing, viral titer in cell lysates was determined by plaque assay. Data are expressed as means ± SD, n = 3. Asterisks indicate statistically significant differences with respect to the control sample (P < 0.05, t test).

Fig. S6.

Genetic stability of recombinant RVs. rsSA11-3 and rsSA11-NLuc were serially passaged (five times) in MA104 cells. (A) Viral dsRNAs extracted from passage 1 (P1) and 5 (P5) stocks of SA11-3 and rsSA11-NLuc were separated in 10% polyacrylamide gels and visualized using ethidium bromide staining. (B) Monolayers of MA104 cells were infected with P1 and P5 stocks of rsSA11-NLuc or rsSA11 at an MOI of 0.01 pfu per cell and incubated with trypsin (0.5 µg/mL). NLuc activity in cell lysates was quantified at 24 h postinfection. Data are expressed as the mean ± SD, n = 3. *P < 0.05 (Tukey’s multiple comparison test), n.s., not significant.

Discussion

We succeeded in developing an entirely plasmid-based RV reverse genetics system in which FAST protein and capping enzyme were coexpressed along with rescue plasmids in BHK-T7 cells (Fig. 1). The mechanism underlying the remarkable effects of FAST protein, which induce cell-to-cell fusion and syncytium formation in nonfusogenic RV and MRV replication in infected and transfected cells, remains unclear. However, previous studies (15, 16, 32) suggest that the cell fusion activity of FAST proteins accelerates cell-to-cell transmission of virus infection and ensures rapid release of progeny virions from apoptotic syncytia, thereby promoting systematic infection. FAST proteins also may increase cotransfection efficiency, resulting in increased virus production in reverse genetic systems via fusion of transfected cells with neighboring cells, leading to formation of syncytia carrying all 14 transfected plasmids (11 RV cDNA plasmids and expression plasmids encoding FAST and VV capping enzyme). The currently available reverse genetics systems for RVs are based on vaccinia-driven T7pol expression (11–13). Although it is possible to rescue Reoviridae viruses that have capped and nonpolyadenylated mRNA using VV expressing T7pol, VV infection has negative effects on RV replication and rescue efficiency in reverse genetics systems. Accordingly, the improved FAST- and VV capping enzyme-based reverse genetics system free of any helper virus for RV and MRV described herein is applicable to the recovery of any member of the Reoviridae family, particularly attenuated recombinant viruses that replicate poorly.

Two licensed RV vaccines, Rotarix (GlaxoSmithKline) and RotaTeq (Merck), are currently available. Rotarix is based on a single human strain, and RotaTeq is a combination of five bovine × human strain monoreassortants. In addition, a new RV vaccine, Rotavac (Bharat Biotech International), was licensed in India in 2014 (33). Although these vaccines are effective against RV-associated severe gastroenteritis, concerns about their efficacy, safety, and cost have inspired the development of new vaccines. We generated a recombinant RV containing silent mutations in three gene segments (NSP1, NSP2, and NSP3) and a monoreassortant virus harboring the human RV strain KU VP6 gene on the strain SA11 genetic background (Figs. 1 and 2). Thus, in contrast to earlier helper virus-based reverse genetics systems, the RV rescue system described here can be easily used for rapid generation of infectious RVs containing multiple mutations in several different gene segments simultaneously, as well as reassortants with any desired gene segment combination and features that could serve as vaccine candidates.

We confirmed that the C-terminal 103 residues of NSP1 are required to inhibit IFN signaling by inducing proteasome-dependent degradation of IRFs (Fig. 3). According to previous studies, NSP1 also inhibits NF-κB activation by inducing degradation of β-TrCP and down-regulating p53, which induces apoptosis and transactivates several genes involved in antiviral responses (34, 35). Additionally, NSP1 interacts with the p85 subunit of the phosphoinositide 3-kinase (PI3K)-mediated antiapoptotic PI3K/Akt pathway (36). Taken together, these observations indicate that NSP1 can interfere with multiple antiviral pathways, including IFN and apoptosis signaling, to promote efficient viral replication and infection. NSP1 mutants, including a C-terminal truncation incapable of blocking IFN signaling and apoptosis pathways, may be attractive candidates for the development of new attenuated RV vaccines.

We used the reverse genetics system to modify the NSP1 gene segment to engineer RVs expressing reporter genes. A recombinant RV harboring the split-GFP system was generated by inserting a small GFP11 tag into the C terminus of the NSP1 ORF (Fig. 4). Thus, the split-GFP–based recombinant NSP1 mutants will be useful tools for understanding NSP1 trafficking and interactions with host proteins, including IFN signaling components, in infected cells. Furthermore, a similar approach using the split-GFP system could be used to study other RV proteins in living cells. We also applied the reverse genetics system to generate a replication-competent recombinant RV expressing the NLuc gene fused to the N-terminal 27 residues of NSP1 (Fig. 4). The results confirm that NSP1 is not required for viral replication, a finding consistent with a previous study that used siRNA gene silencing (30), and that the NSP1 gene segment is suitable for insertion of a heterologous sequence. The attenuated replication kinetics of rsSA11-NLuc could be explained by the defect in the IFN suppressor activity of NSP1, or by the influence of reduced packaging efficiency. In addition, the replication and luciferase activity of rsSA11-NLuc were inhibited by ribavirin, a known anti-RV inhibitor, suggesting that the reporter virus would be a useful tool for high-throughput screening for antiviral therapeutics (Fig. 4). Furthermore, the replication-competent RV carrying the NLuc gene makes it possible to track RV infection in vivo and develop an oral RV vector.

In this study, we developed a plasmid-only–based reverse genetics system for RV. This technique opens new horizons for the study of RV replication and pathogenesis, as well as for the development of antiviral drugs and new vaccines that protect against this important gastrointestinal pathogen.

Materials and Methods

Cells and Viruses.

Monkey kidney epithelial MA104, CV-1, Vero, murine fibroblast L929, and human colon epithelial Caco-2 cells were cultured in DMEM (Nacalai Tesque) supplemented to contain 5% (vol/vol) fetal bovine serum (FBS) (Gibco), 100 units/mL penicillin, and 100 µg/mL streptomycin (Nacalai Tesque). BHK/T7-9 cells were grown in DMEM supplemented to contain 5% FBS, 10% (wt/vol) tryptose phosphate broth, 100 units/mL penicillin, and 100 µg/mL streptomycin (37). To establish another BHK-T7 cell line (BHK-T7/P5), BSR cells (3), a derivative of BHK cells, were selected by transfection with a eukaryotic expression plasmid encoding T7pol under the control of a strong CMV early enhancer/chicken β-actin (CAG) promoter (38), followed by incubation in the presence of 4 µg/mL puromycin (Sigma-Aldrich). Immortalized MEFs derived from TBK1^+/− IKKi^−/− (TBK1^+/−) and TBK1^−/− IKKi^−/− (TBK1^−/−) mice were prepared as previously described (39, 40) and grown in DMEM supplemented to contain 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin. Human cancer cell lines, HT29, PC3, HCC-2998, and OVCAR-4, were cultured in RPMI1640 (Nacalai Tesque) supplemented to contain 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin. Simian RV strain SA11 (SA11-L2) (G3P[2]) (41) and human RV strain KU (G1P[8]) (42) were propagated in MA104 cells cultured in DMEM supplemented with 0.5 µg/mL trypsin (Sigma-Aldrich). MRV strain T1L, a laboratory stock originally obtained from Bernard Fields, Harvard Medical School, Boston, was propagated in L929 cells. Infectious titers of RV and MRV were determined by plaque assay using CV-1 cells and L929 cells, respectively, as previously described (43, 44).

Recovery of Recombinant RVs from Cloned cDNAs.

Monolayers of BHK-T7 cells (8 × 10⁵) in six-well plates were cotransfected with plasmids using 2 μL of TransIT-LT1 transfection reagent per microgram of plasmid DNA, as follows: 0.8 µg of each strain SA11 rescue plasmid [pT7-VP1SA11, pT7-VP2SA11, pT7-VP3SA11, pT7-VP4SA11, pT7-VP6SA11 (or pT7-VP6KU), pT7-VP7SA11, pT7-NSP1SA11 (pT7-NSP1-BamHI, pT7-NSP1-dC103, or pT7-NSP1-GFP11), pT7-NSP2SA11 (or pT7-NSP2-EcoRV), pT7-NSP3SA11 (or pT7-NSP3-EcoRI), pT7-NSP4SA11 (or pT7-NSP4-MluI), and pT7-NSP5SA11], 0.015 µg of pCAG-FAST, and 0.8 µg of each capping enzyme expression plasmid. After 2 d of incubation in FBS-free medium, MA104 cells (10⁵ cells) were added to the transfected cells and cocultured for 3 d in FBS-free medium supplemented with trypsin (0.5 µg/mL). After incubation, transfected cells were lysed by freeze/thaw, trypsin was added to cell lysates at a final concentration of 10 µg/mL, and the samples were incubated at 37 °C for 30 min to activate infectious RVs. The lysates were then transferred to fresh MA104 cells. 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 incubated at 37 °C for 7 d. When CPE was observed following RV infection of monolayer cells, recombinant viruses were isolated from passaged cells by plaque purification using CV-1 cells.

SI Materials and Methods

Plasmid Construction.

To construct the RV rescue plasmids pT7-VP1SA11, pT7-VP2SA11, pT7-VP3SA11, pT7-VP4SA11, pT7-VP6SA11, pT7-VP7SA11, pT7-NSP1SA11, pT7-NSP2SA11, pT7-NSP3SA11, pT7-NSP4SA11, and pT7-NSP5SA11, each of which contains the full-length cDNA of the corresponding gene segment from strain SA11, full-length RV cDNAs were amplified by RT-PCR from viral dsRNAs extracted from purified virions. Amplified cDNA fragments were subcloned into pT7-S3T1L, which contains the full-length cDNA of the S3 gene segment of MRV strain T1L (9). Following complete replacement of plasmid sequences encoding T1L S3 gene, viral cDNAs were flanked by T7 promoter and HDV ribozyme sequences in pT7-S3T1L. Rescue plasmids containing signature substitutions to create unique restriction enzyme sites [BamHI, EcoRV, EcoRI, and MluI, respectively, in the NSP1 (positions 1,053 and 1,059), NSP2 (positions 409 and 418), NSP3 (positions 406 and 412), and NSP4 (positions 389 and 395) gene segments] were generated using a KOD-Plus-Mutagenesis kit (Toyobo). To generate rescue plasmid pT7-VP6KU encoding a full-length VP6 gene segment derived from strain KU, VP6 cDNA amplified by RT-PCR from viral dsRNAs extracted from purified virions was inserted into T7 expression vector pX8dT (45). The C-terminally truncated NSP1 mutant plasmid pT7-NSP1-dC103, which lacks nucleotide sequences 1,192–1,490, was generated by standard site-directed mutagenesis. To generate pT7-NSP1-GFP11, which encodes the NSP1 protein C-terminally fused to the small split GFP11 fragment (amino acids RDHMVLHEYVNAAGIT), the nucleotide sequence encoding the GFP11 fragment was inserted between nucleotides 1,518 and 1,519 of NSP1 of pT7-NSP1SA11. To create pT7-NSP1-NLuc, which encodes a full-length NLuc gene (accession no. KM359774), NLuc gene was amplified by PCR and inserted between nucleotides 111 and 112 in the NSP1 gene of pT7-NSP1 using the In-Fusion Cloning kit (Clontech). The NLuc gene was expressed as a fusion protein harboring amino acids 1–27 of NSP1 at the N terminus. Genes encoding FAST derived from NBV strain Miyazaki-Bali/2007 (accession no. AB908284), VV capping enzyme subunits D1R (NC006998) and D12L (NC006998), and superfolder GFP (sfGFP) (JQ341914) were synthesized by gene synthesis services (Eurofins Genomics). Artificially synthesized genes amplified by PCR were inserted into the EcoRI site of pCAGGS (38) using the In-Fusion HD Cloning kit to create pCAG-FAST, pCAG-D1R, pCAG-D12L, and pCAG-GFP1–10 (encoding amino acids 1–213 of sfGFP). The NSP1 gene from SA11 was amplified by RT-PCR and inserted into the EcoRI site of pCAGGS to yield the pCAG-NSP1 expression vector. All plasmids were confirmed by DNA sequencing. Primer sequences used for plasmid construction are available upon request.

Sequence Determination of Strain SA11.

Viral dsRNAs were extracted from purified SA11 virions using Sepazol RNA I Super (Nacalai Tesque). Viral genomic cDNAs including intact 5′ and 3′ termini were amplified from viral dsRNA by full-length amplification of cDNA as previously described (46). Briefly, a self-priming oligo DNA linker, C9 anchor primer, was attached to the 3′ ends of viral dsRNAs using T4 RNA ligase (Thermo Fisher Scientific), and adaptor-ligated RV dsRNAs were purified by 1% agarose gel electrophoresis. Purified viral cDNAs were synthesized using ThermoScript reverse transcriptase (Thermo Fisher Scientific). Full-length viral cDNAs were amplified with a single primer complementary to the C9 anchor primer using KOD-Plus-Neo polymerase (Toyobo). Amplified viral cDNAs were subcloned into pBluescript KS (+) and subjected to sequencing. Cycle sequencing reactions were performed using BigDye terminator (Applied Biosystems), and viral sequences were determined on an ABI 3130 genetic analyzer (Life Technologies). The complete genome sequences of strain SA11 have been deposited in the GenBank database and assigned accession nos. LC178564–LC178574.

Electrophoresis of Viral dsRNA Genomes.

Viral dsRNAs were extracted from virions and mixed with an equal volume of 2× sample buffer [125 mM Tris⋅HCl (pH 6.8), 10% (vol/vol) 2-mercaptoethanol, 4% (wt/vol) SDS, and 10% (wt/vol) sucrose]. The dsRNAs were separated on 10% (wt/vol) precast polyacrylamide gels (Atto) and visualized by silver or ethidium bromide staining.

Generation of RV-Specific Antisera.

To generate antiserum against SA11 NSP1, rabbits were immunized with mixed synthetic peptides (corresponding to 234-VIFNTYTKTPGRSIYRN-250 and 216-LPSSKLKQIYFSDFTKE-232 of SA11 NSP1) conjugated to keyhole limpet hemocyanin (Medical and Biological Laboratories). Antiserum against SA11 NSP5 was generated by immunizing rabbits with recombinant NSP5 protein. To construct the NSP5 expression vector, the NSP5 coding region was amplified by RT-PCR with specific primers containing BamHI and SalI restriction enzyme sites. The resultant PCR product was digested with both enzymes and ligated into the corresponding sites of pGEX6P-3 vector (GE Healthcare) to yield pGEX/NSP5 encoding NSP5-GST (GST) fusion protein. Escherichia coli XL-1 blue cells were transformed with pGEX/NSP5 and incubated with 1 mM isopropyl β-d-1-thiogalactopyranoside. NSP5-GST fusion protein was purified using Sepharose 4B column chromatography (GE Healthcare). Rabbits were immunized with purified GST-NSP5 by standard procedures (Medical and Biological Laboratories).

Immunoblotting.

MA104 cells were infected with wild-type rsSA11 or rsSA11-dC103 at an MOI of 0.1 pfu per cell. Twenty-four hours after infection, cells were lysed, and proteins were size fractionated by SDS/PAGE and electroblotted onto polyvinylidene difluoride membranes. Viral proteins were detected using 1:2,000 dilution of anti-NSP1 antiserum and 1:2,000 dilution of HRP-conjugated anti-rabbit IgG as secondary antibody (Sigma). To detect IRF3 and viral proteins, infected HT29 cells were harvested 6 h postinfection. Proteins were detected using Super Signal West Femto Maximum Sensitivity Substrate (Pierce) and LAS-3000 (Fujifilm) following incubation with anti-IRF3 antiserum (Santa Cruz Biotechnology) or antibody specific for NSP5 and the appropriate secondary antibodies.

IFN-β Promoter Reporter Assay.

The 293T cells were cotransfected with pIFN-β–Luc, which expresses firefly luciferase under the control of the IFN-β promoter (47), and pRL-SV40 (Promega), which expresses Renilla luciferase under the control of the SV40 promoter (as an internal control). Twelve hours after transfection, cells were infected with recombinant viruses at an MOI of 10 pfu per cell. After 10 h, luciferase activities were detected using the Dual-Luciferase Reporter Assay system (Promega) and Luminometer AB-2200 (Atto).

Indirect Immunofluorescence Assay.

BSR cells transfected with pCAG-GFP1–10 were infected with rsSA11 or rsSA11-GFP11 at an MOI of 0.1 pfu per cell. Sixteen hours after infection, cells were fixed with 10% (vol/vol) formaldehyde and permeabilized with 0.1% Triton X-100, and viral proteins were stained with anti-NSP5 antiserum, followed by CF 594 goat anti-rabbit IgG (Nacalai Tesque). Cells were stained with 4′,6-diamidino-2-phenylindole to label nuclei. Images were analyzed on a FluoView FV1000 laser scanning confocal microscope (Olympus).

Luciferase Assay.

MA104 cells were infected with rsSA11 or rsSA11-NLuc at an MOI of 0.01 pfu per cell and incubated with 0.5 µg/mL trypsin. Following 0–3 d of incubation, NLuc activity in cell lysates was quantified using the Nano-Glo Luciferase Assay system (Promega). Luciferase signals of plaques formed by rsSA11-NLuc on CV-1 monolayer cells were visualized using the Nano-Glo Luciferase Assay system and an in vivo imaging system (IVIS) (Xenogen). After observation, cells were fixed with 10% formaldehyde and stained with crystal violet.

Antiviral Assay.

Monolayers of CV-1 cells cultured in 96-well plates were infected with wild-type or recombinant viruses at an MOI of 0.001 pfu per cell. After adsorption for 1 h at 37 °C, cells were washed twice with PBS and the medium was replaced with DMEM supplemented with 0.5 µg/mL trypsin and 0–200 µM ribavirin (Sigma-Aldrich). Fourteen hours after infection, viral titers in cell lysates were determined by plaque assay. To visualize luminescence signals in infected cells, NanoLuc substrate was added to each well, and luciferase signals were detected by IVIS. To monitor cell cytotoxicity during ribavirin treatment, monolayers of CV-1 cells were incubated with DMEM supplemented with 0.5 µg/mL trypsin and 0–1,000 µM ribavirin (Sigma-Aldrich). Thirteen hours after infection, WST-1 reagent (Roche) was added to each well, and the samples were incubated at 37 °C for 1 h. Absorbance at 440 nm was measured using a PowerScan HT microplate reader (DS Pharma Biomedical).

Statistics.

Viral titers were analyzed by t test, and IFN-β reporter assays were analyzed by ANOVA and Tukey’s multiple comparison test using GraphPad Prism (version 5.01). P < 0.05 was considered statistically significant.