ABSTRACT
Group A rotavirus (RV) is a major cause of acute gastroenteritis in infants and young children worldwide. Recently, we established an entirely plasmid-based reverse genetics system for simian RV strain SA11. Although that system was robust enough to generate reassortant RVs, including human RV gene segments, and enabled better understanding of the biological differences between animal and human RV strains, a complete reverse genetics system for human RV strains is desirable. Here, we established a plasmid-based reverse genetics system for G4P[8] human RV strain Odelia. This technology was used to generate a panel of monoreassortant viruses between human and simian RV strains for all of the 11 gene segments demonstrating full compatibility between human and simian RV strains. Furthermore, we generated recombinant viruses lacking the C-terminal region of the viral nonstructural protein NSP1 and used it to define the biological function of the interaction between NSP1 and its target protein β-transducin repeat-containing protein (β-TrCP) during viral replication. While the NSP1 truncation mutant lacking the C-terminal 13 amino acids displayed lower β-TrCP degradation activity, it replicated as efficiently as the wild-type virus. In contrast, the truncation mutant lacking the C-terminal 166 amino acids of NSP1 replicated poorly, suggesting that the C-terminal region of NSP1 plays critical roles in viral replication. The system reported here will allow generation of engineered recombinant virus harboring desired mutations, increase our understanding of the molecular biology of human RV, and facilitate development of novel therapeutics and vaccines.
IMPORTANCE Reverse genetics, an approach used to generate viruses from cloned cDNA, has increased our understanding of virus biology. Worldwide research led to the development of an entirely plasmid-based reverse genetics system for the simian RV laboratory strain. Although the technique allows generation of gene-modified recombinant RVs, biological differences between animal and human RVs mean that reverse genetics systems for human RV strains are still needed. Here, we describe a reverse genetics system for the high-yield human RV strain Odelia, which replicates efficiently and is suitable for in vitro molecular studies. Monoreassortant viruses between simian and human RV strains and NSP1 mutant viruses generated by the rescue system enabled study of the biological functions of viral gene segments. This human RV reverse genetics system will facilitate study of RV biology and development of vaccines and vectors.
INTRODUCTION
Group A rotaviruses (RVs) are one of the etiological agents that cause severe diarrhea in mammals and avian host species (1). In humans, RVs are a leading cause of acute gastroenteritis in young children under the age of 5 years. RV infection claims the lives of approximately 215,000 infants and young children annually, particularly in developing countries (2).
RV forms a nonenveloped triple-layered virion particle that contains an 11-segmented double-stranded RNA (dsRNA) genome (1). Two viral surface proteins define the RV genotypes; the outer layer protein VP7 defines the G type and the spike protein VP4 defines the P type. Currently, six serotypes (G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8]) account for more than 90% of globally circulating strains (3, 54, 55). Two live-attenuated vaccines (monovalent Rotarix [RV1] and pentavalent RotaTeq [RV5]) are effective in high-income countries (4–8); however, their efficacy is lower in low-income countries, mainly those in sub-Saharan Africa and Southeast Asia (2, 9–13). Although malnutrition, differences in commensal gut microbiota, and enteric viral infections are thought to underlie the lower vaccine efficacy in developing countries, the exact reasons remain unclear (13). To develop better treatments and vaccines, further studies must identify the mechanisms underlying replication and pathogenesis of human RV.
Reverse genetics systems are powerful tools for manipulating viral genomes to better understand the mechanisms underlying viral replication and pathogenesis and to facilitate development of artificially attenuated vaccines and viral vectors. Recently, we reported an entirely plasmid-based reverse genetics system for simian rotavirus strain SA11, based on transfection of cells with cloned cDNAs encoding each of all 11 SA11 gene segments along with expression plasmids encoding fusion-associated small transmembrane (FAST) and vaccinia virus capping enzymes (14, 15), thereby enabling manipulation of all 11 RV gene segments to expand our understanding of RV biology.
However, the biological diversity among human and animal RVs means that this system is limited in terms of understanding the characteristics of human RVs. For example, VP8* of animal RVs, including that of strain SA11 (P[2]), uses sialic acid for cell entry, whereas the majority of human RVs (P[4], P[6], and P[8]) use human histo-blood group antigens (16–19, 56, 57). In addition, the genotype constellation of strain SA11 suggests that it is not closely related to major RV strains circulating in humans (e.g., Wa-like and DS-1-like genogroups) (20). Homologous RVs are more pathogenic and replicate more efficiently than heterologous RVs in homologous animals (21–28). Although some RV strains isolated from humans harbor gene segments derived from animal RVs, most human RVs harbor gene segments similar to those of prototype strains Wa and DS-1 (3, 29–31). These differences between human and animal RVs suggest that developing an entirely human RV reverse genetics system is preferable if we are to better understand human RV biology.
NSP1 proteins encoded by RV gene segment 5 are highly divergent between RV strains. The common function of NSP1 is to inhibit interferon (IFN) signaling by degrading host proteins that induce IFN expression. For example, NSP1 encoded by simian RVs degrades interferon regulating factor 3 (IRF3), IRF5, IRF7, and/or IRF9, whereas NSP1 encoded by porcine RV strain OSU and human RVs target β-TrCP as well as IRF proteins (32–36, 45, 58). Previous studies demonstrate that NSP1 from human and porcine RVs harbor an IκB-like degron (ILD) motif within the C-terminal region, which recruits β-TrCP and degrades it in a ubiquitin-dependent manner (37–39). The NSP1 ILD motif is phosphorylated by casein kinase II, and phosphorylation is required for interaction between NSP1 and β-TrCP (40). Although study of recombinant NSP1 proteins has enabled us to elucidate the molecular mechanisms underlying interaction between RV NSP1 and the IFN signaling machinery, the contribution of NSP1 protein to viral replication remains unclear due to the lack of a reverse genetics system.
Human RV strain Odelia was isolated from an infant with gastroenteritis (41). Sequence analysis revealed that strain Odelia belongs to the G4P[8] serotype (41). Here, we describe a plasmid-based reverse genetics system for human RV strain Odelia. Recombinant strain Odelia demonstrated similar growth kinetics to those of the parental strain in cultured cells. Using a reverse genetics system for strains SA11 and Odelia, we recovered a panel of monoreassortant viruses harboring one gene segment from strain Odelia within the strain SA11 genetic backbone. Furthermore, to show the utility of this human RV reverse genetics system, we generated and analyzed recombinant viruses lacking the C-terminal region of NSP1, including the ILD motif that is conserved among human and porcine NSP1 proteins. The reverse genetics system described here will provide insight into the molecular mechanisms underlying replication and pathogenesis of human RVs.
RESULTS
Establishment of a reverse genetics system for human RV strain Odelia.Simian RV laboratory strain SA11 has long been used as an experimental model for in vitro and in vivo studies (1, 42, 43); however, biological differences between SA11 and human RV strains mean that strain SA11 is not a suitable model for examining replication and pathogenesis of human RV (16–20). Thus, a reverse genetics system for human RV is needed. To develop a reverse genetics system for human RV, we generated cloned cDNA encoding each of the 11 gene segments derived from strain Odelia (G4P[8]). Whole-genome sequences of all 11 gene segments of strain Odelia were subjected to RV genotyping using the RotaC v2.0 automated genotyping tool (http://rotac.regatools.be/) (30, 44). The results showed that strain Odelia exhibited a Wa-like genotype constellation (i.e., G4-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1). Next, cDNAs encoding each of the 11 Odelia dsRNA gene segments were introduced into plasmids at sites flanked by the T7 promoter and hepatitis delta virus ribozyme sequences. To generate recombinant strain Odelia (rsOdelia) from cloned cDNAs, BHK-T7 cells were transfected with the 11 Odelia cDNAs and polymerase II promoter-driven expression plasmids encoding Nelson Bay reovirus FAST protein, vaccinia virus capping enzyme (D1R and D12L), and strain SA11 NSP2 and NSP5 proteins. Following incubation, transfected cell lysates were passaged in MA104 cells and cultured for 3 days. After incubation, the cells were lysed by freezing/thawing and transferred to fresh MA104 cells. We observed significant cytopathic effects in MA104 cells, suggesting that recombinant RVs were generated from the cloned cDNAs. To confirm whether rsOdelia has the characteristics of the parental Odelia strain, we first examined the replication kinetics of Odelia and rsOdelia in MA104 cells. We found that rsOdelia replicated as well as the parental Odelia strain (Fig. 1A). Polyacrylamide gel electrophoresis of dsRNA genomes extracted from rsOdelia virions revealed migration patterns identical to those of the parental Odelia strain (Fig. 1B). To exclude the possibility that the rsOdelia preparation was contaminated by the parental Odelia strain, we confirmed the presence of a unique XbaI site, a marker genetic mutation introduced into the NSP2 gene segment of rsOdelia. We extracted dsRNA genomes from Odelia and rsOdelia virions and amplified the NSP2 gene segment of these viruses. Sequence analysis demonstrated that the NSP2 gene segment from rsOdelia possessed the introduced mutation at nucleotide position 584, whereas the NSP2 gene amplified from the wild-type virus did not (Fig. 1C). Furthermore, the PCR amplicon from rsOdelia was digested by XbaI, whereas that from the wild-type virus was not (Fig. 1D). These data suggest that rsOdelia was rescued from cloned cDNAs, and that the replication characteristics of rsOdelia reflect those of the parental Odelia strain.
Establishment of a reverse genetics system for human RV strain Odelia. (A) Growth kinetics of Odelia and recombinant strain Odelia (rsOdelia) in MA104 cells. Cells were infected with Odelia and rsOdelia at a multiplicity of infection (MOI) of 0.01 PFU/cell. Cells were frozen at −80°C at 12, 24, 48, and 72 h postinfection. Virus titers were determined in a plaque assay using MA104 cells. Data are expressed as the mean ± standard deviation (SD) of triplicate samples. (B) Electropherotype of Odelia and rsOdelia. Viral double-stranded RNA (dsRNA) genomes were extracted from purified virions, separated by polyacrylamide electrophoresis, and visualized by ethidium bromide staining. (C) Nucleotide sequence of the NSP2 gene segment of Odelia and rsOdelia. The NSP2 gene segment of rsOdelia contains a unique XbaI site (underlined). Viral dsRNA genomes were purified from virions, and the NSP2 gene segment was reverse transcribed and amplified by PCR. The sequence electrogram shows that rsOdelia contains a T-to-C mutation at nucleotide position 584. (D) Electrophoretic analysis of the NSP2 gene segment of Odelia and rsOdelia. The amplified NSP2 products were treated with XbaI. DNA marker sizes are shown on the right.
Generation of monoreassortant viruses between human RV Odelia and simian RV SA11 strains.Reassortment is fundamental to the evolution and diversity of segmented RNA viruses; this is because it allows direct exchange of segmented genes during coinfection events. Thus, classical reassortment techniques can segregate differences and allow identification of genes that confer these differences; they are therefore effective and rapid methods of generating vaccine candidates. However, it is difficult to generate reassortant viruses with the desired combination of segmented genes using the classical method, which involves coinfection by different strains. To demonstrate the utility of the human RV reverse genetics system, we generated a panel of SA11 × Odelia monoreassortant viruses on the strain SA11 genetic background to better understand the characteristics of strains SA11 and Odelia (rsSA11/Odelia-VP1, -VP2, -VP3, -VP4, -VP6, -VP7, -NSP1, -NSP2, -NSP3, -NSP4, and -NSP5/NSP6). The growth kinetics of rsOdelia in MA104 cells were compared with those of simian recombinant strain SA11 (rsSA11). The results showed that rsOdelia reached a peak titer of ∼1 × 107 PFU/ml in MA104 cells, although the replication efficiency of rsOdelia was slightly lower than that of rsSA11 (Fig. 2A). The gene segments from each Odelia monoreassortant virus were amplified and confirmed by DNA sequencing; the electropherotypes of the dsRNA genomes extracted from each monoreassortant virion showed the same migration pattern as that of rsSA11, except for one gene segment derived from rsOdelia (Fig. 2B). The monoreassortant viruses harboring Odelia-VP3, VP6, NSP1, NSP2, NSP3, NSP4, and NSP5/NSP6 (rsSA11/Odelia-VP3, -VP6, -NSP1, -NSP2, -NSP3, -NSP4, and -NSP5/NSP6) in the strain SA11 genetic background replicated as well as rsSA11 (Fig. 2C). However, the titer of other monoreassortant viruses (rsSA11/Odelia-VP1, -VP2, -VP4, and -VP7) was lower than that of rsSA11 (Fig. 2C and D). Among these monoreassortant viruses, the titer of rsSA11/Odelia-VP4 was reduced by approximately 100-fold in comparison with that of rsSA11 (Fig. 2D). These results suggest that genetic combination between the VP1, VP2, VP4, and VP7 gene segments of strains SA11 and Odelia affects replication of these monoreassortant viruses in MA104 cells. To investigate whether impaired replication of SA11 × Odelia monoreassortant viruses in the strain SA11 genetic background is a common characteristic of genetic combination between strains SA11 and Odelia, we rescued rsOdelia/SA11-VP4 (which contains the SA11 VP4 gene segment in the strain Odelia background). The electropherotypes of dsRNA genomes extracted from rsOdelia/SA11-VP4 showed the same migration pattern as those extracted from rsOdelia; the exception was gene segment 4 (Fig. 2B). rsOdelia/SA11-VP4 and rsOdelia replicated efficiently in MA104 cells (Fig. 2E), suggesting that replication of rsSA11/Odelia-VP4 was impaired by combination of viral proteins from strains SA11 and Odelia. Collectively, these results demonstrate that the reverse genetics system for human RV strain Odelia can be combined with that of simian strain SA11.
Generation of monoreassortant viruses between strains SA11 and Odelia. (A) Growth kinetics of recombinant strain SA11 (rsSA11) and rsOdelia in MA104 cells. Cells were infected with viruses at an MOI of 0.01 PFU/cell. Cells were frozen at −80°C at 12, 24, 48, 72, and 96 h postinfection. Virus titer was determined in a plaque assay using MA104 cells. Data are expressed as the mean ± SD of triplicate samples. (B) Electropherotype of monoreassortant viruses between strains SA11 and Odelia. Viral dsRNA genomes were extracted from purified virions, separated by polyacrylamide electrophoresis, and visualized by ethidium bromide staining. The gene segments derived from strain Odelia on a genetic background of strain SA11 are denoted by yellow asterisks. The SA11 VP4 gene segment from rsOdelia/SA11-VP4 is denoted by an orange asterisk. (C, D, and E) Replication of monoreassortant viruses in MA104 cells. Cells were infected with virus at an MOI of (C) 0.01 PFU/cell or (D and E) 0.01 FFU/cell. Cells were frozen at −80°C at 72 h postinfection. Virus titer was determined in a plaque assay (C) or focus assay (D and E) using MA104 cells. Foci were detected using antiserum against NSP4. Data are expressed as the mean ± SD of triplicate samples. Significant differences in replication between rsSA11 and monoreassortant viruses (C and D) and between rsOdelia and rsOdelia/SA11-VP4 (E) were determined using one-way analysis of variance (ANOVA). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Generation of a monoreassortant virus harboring a reporter gene introduced into the SA11 NSP1 gene segment in the genetic background of strain Odelia.Recently, we developed a stable rotavirus reporter expression system using SA11 strain rescue system (14, 15). To improve the utility of the rescue systems for strains SA11 and Odelia, we generated a monoreassortant virus harboring the reporter-NSP1 chimeric gene from strain SA11 in the genetic background of strain Odelia (rsOdelia/SA11-NSP1-ZsG). Migration of gene segment 5 from rsOdelia/SA11-NSP1-ZsG was slower than that of gene segment 5 from rsOdelia (Fig. 3A). As shown previously for the SA11-based NSP1 ZsG virus, we observed ZsGreen signal in cells infected with rsOdelia/SA11-NSP1-ZsG (Fig. 3B). Although the titer of rsOdelia/SA11-NSP1-ZsG was slightly lower than that of rsOdelia, growth kinetics showed that rsOdelia/SA11-NSP1-ZsG replicated in cultured cells (Fig. 3C). These results demonstrate that monoreassortant virus expressing a reporter gene can be rescued by combining the rescue systems for strains SA11 and Odelia.
Generation of a monoreassortant virus harboring a reporter gene in the genetic background of strain Odelia. (A) Electropherotype of rsOdelia/SA11-NSP1-ZsG. Viral dsRNA genomes were extracted from purified virions, separated by polyacrylamide electrophoresis, and visualized by ethidium bromide staining. (B) ZsGreen expression by rsOdelia/SA11-NSP1-ZsG in MA104 cells. Cells were infected with virus at an MOI of 1 FFU/cell and fixed at 18 h postinfection. Viral protein was detected by indirect immunofluorescence analysis using an antibody specific for the NSP4 protein. (C) Growth kinetics of rsOdelia and rsOdelia/SA11-NSP1-ZsG in MA104 cells. Cells were infected with viruses at an MOI of 0.01 FFU/cell and frozen at −80°C at 12, 24, 48, and 72 h postinfection. Virus titer was determined in a focus assay using MA104 cells. Data are expressed as the mean ± SD of triplicate samples.
Generation of rsOdelia harboring a mutation in the NSP1 gene.Previous studies based on recombinant NSP1 proteins demonstrate that human RV NSP1 interacts with β-TrCP via the ILD motif in the NSP1 C-terminal region, resulting in proteasome-dependent degradation (34, 35). To examine the role of human RV NSP1, we constructed a protein expression vector harboring RV Odelia NSP1 (pCXN2-HA-Odelia-NSP1-dC13) lacking the C-terminal 13 amino acids (i.e., the ILD motif) (Fig. 4A). While cotransfection of pCXN2-HA-Odelia-NSP1-dC13 did not induce degradation of β-TrCP, wild-type NSP1 did reduce expression of β-TrCP (Fig. 4B). To further examine the biological role of NSP1 protein during viral replication, we generated a recombinant Odelia strain lacking the C-terminal 13 amino acids of NSP1 (rsOdelia-NSP1-dC13) (Fig. 4A). We then infected HEK293T cells transiently expressing β-TrCP with rsOdelia and rsOdelia-NSP1-dC13, and then examined expression of β-TrCP. As observed in plasmid-transfected cells, expression of β-TrCP in cells infected with rsOdelia decreased, whereas that in cells infected with rsOdelia-NSP1-dC13 did not (Fig. 4C). Furthermore, we assessed degradation of endogenous β-TrCP by infecting MA104 and HT-29 cells with either rsOdelia or rsOdelia-NSP1-dC13. While we observed slight degradation of β-TrCP by rsOdelia infection, rsOdelia-NSP1-dC13 did not cause degradation of β-TrCP in MA104 and HT-29 cells (Fig. 4D and E). To investigate the effects of NSP1-mediated β-TrCP degradation on viral replication, we compared the replication kinetics of rsOdelia and rsOdelia-NSP1-dC13 in MA104 cells and HT-29 cells. A recombinant RV strain SA11 lacking a functional NSP1 domain (rsSA11-dC103) was used as a control. Replication of rsOdelia-NSP1-dC13 was not hampered (Fig. 4F and G). In contrast, as shown in a previous study (14), rsSA11-dC103 displayed impaired replication in HT-29 cells (Fig. 4G). To better understand the importance of the C-terminal ILD motif for viral replication, we generated monoreassortant virus harboring genes encoding wild-type Odelia NSP1 (rsSA11/Ode-NSP1) and truncated NSP1 (rsSA11/Ode-NSP1-dC13) in genetic background of strain SA11. There was no difference in the replication capacity of both viruses, although both replicated better than rsOdelia and rsOdelia-NSP1-dC13 in MA104 and HT-29 cells (Fig. 4F and G). These data imply that degradation of β-TrCP by the NSP1 protein of human RV does not have a marked effect on viral replication in these cells.
Generation of recombinant viruses lacking the IκB-like degron motif. (A) Schematic presentation of the plasmid encoding the NSP1 gene used for protein expression and recovery of recombinant virus. The C-terminal IκB-like degron motif, DSGΦS, is underlined. (B) Expression of β-TrCP protein in HEK293T cells transfected with plasmids. HEK293T cells were transfected with protein expression vector pCXN2-FLAG-β-TrCP, along with pCXN2-HA-Odelia-NSP1 or pCXN2-HA-Odelia-NSP1-dC13. Expression of β-TrCP, NSP1, and β-actin was detected by anti-FLAG, anti-hemagglutinin (HA), and anti-β-actin antibodies, respectively. Protein markers are shown on the right. The relative band intensity of FLAG-β-TrCP is shown under the band. (C) Expression of β-TrCP protein in HEK293T cells transfected with plasmids. HEK293T cells were transfected with protein expression vector pCXN2-FLAG-β-TrCP and then infected with rsOdelia and rsOdelia-NSP1-dC13. Expression of β-TrCP, VP6, and β-actin was detected using anti-FLAG, anti-VP6, and anti-β-actin antibodies, respectively. Protein markers are shown on the right. Relative band intensity of FLAG-β-TrCP was shown under the band. (D and E) Expression of endogenous β-TrCP in (D) MA104 and (E) HT-29 cells. Cells were infected with rsOdelia and rsOdelia-NSP1-dC13 at an MOI of 10 FFU/cell. Expression of β-TrCP, VP6, and β-actin was detected using anti-β-TrCP, anti-VP6, and anti-β-actin antibodies, respectively. Protein markers are shown on the right. Relative band intensity of β-TrCP is shown under the band. (F and G) Growth kinetics of wild-type and NSP1 mutant viruses. (F) MA104 cells or (G) HT-29 cells were infected with rsOdelia and rsOdelia-NSP1-dC13 (left panel), rsSA11/Ode-NSP1 and rsSA11/Ode-NSP1-dC13 (middle panel), or rsSA11 and rsSA11-dC103 (right panel), at an MOI of 1 FFU/cell. Cells were frozen at 12, 24, 48, and 72 h postinfection. Virus titer was determined in a focus assay using MA104 cells. Data are expressed as the mean ± SD of triplicate samples. Statistical differences in viral replication between rsOdelia and rsOdelia-NSP1-dC13, between rsSA11 and rsSA11-dC103, and between rsSA11/Ode-NSP1 and rsSA11/Ode-NSP1-dC13 were calculated using a t test. ns, not significant; ***, P < 0.001.
To further analyze the importance of NSP1 during viral replication, we generated an NSP1 C-terminal truncation mutant harboring a 166-amino acid deletion (rsOdelia-NSP1-dC166) (Fig. 5A). Previous studies suggest that the 166 amino acids deleted from the C terminus of NSP1 form part of the IRF binding domain in addition to the ILD motif (35, 45). The electropherotypes of the dsRNA genome extracted from rsOdelia-NSP1-dC166 showed that the migration pattern of gene segment 5 was different from that in rsOdelia (Fig. 5B). Next, to obtain clearer images, we amplified segments 5, 8, and 9 using reverse transcription-PCR (RT-PCR) with specific primers and then compared their size. We found that gene segment 5 of rsOdelia-NSP1-dC166 was about 1 kbp. Therefore, it would likely run along with segments 7 to 9, which are of similar size (Fig. 5C). In agreement with the previous result, β-TrCP was degraded by wild-type Odelia NSP1 in a cotransfection experiment, but not by Odelia NSP1 that lacked 166 amino acids (pCXN2-HA-Odelia-NSP1-dC166) (Fig. 5D). Transfection of β-TrCP expression plasmid, followed by infection with rsOdelia and rsOdelia-NSP1-dC166, showed similar results. Whereas rsOdelia degraded β-TrCP, rsOdelia-NSP1-dC166 did not (Fig. 5E). Additionally, we examined replication of rsOdelia and rsOdelia-NSP1-dC166 in MA104 and HT-29 cells. Replication of rsOdelia-NSP1-dC166 in MA104 and HT-29 cells was ∼10-fold and ∼3,000-fold lower, respectively, than that of the wild-type virus (Fig. 5F and G). These data suggest that the C-terminal region of human RV NSP1, including the IRF binding domain, plays an important role in viral replication in these cell lines.
Generation of a recombinant virus lacking the C-terminal 166 residues of NSP1. (A) Schematic representation of the plasmid encoding the NSP1 gene used for recovery of recombinant virus. (B) Electropherotype of rsOdelia-NSP1-dC166. Viral dsRNA genomes were extracted from purified virions, separated by polyacrylamide electrophoresis, and visualized by ethidium bromide staining. The yellow asterisk denotes the predicted migration position of gene segment 5 of rsOdelia-NSP1-dC166. (C) Reverse transcription-PCR (RT-PCR) analysis and amplification of gene segments 5, 8, and 9 of rsOdelia and rsOdelia-NSP1-dC166. DNA marker sizes are shown on the right. (D) Expression of β-TrCP protein in HEK293T cells transfected with plasmids. HEK293T cells were transfected with protein expression vectors pCXN2-FLAG-β-TrCP plus pCXN2-HA-Odelia-NSP1 or pCXN2-HA-Odelia-NSP1-dC166. Expression of β-TrCP, NSP1, and β-actin was detected by anti-FLAG, anti-HA, and anti-β-actin antibodies, respectively. Protein markers are shown on the right. The relative band intensity of FLAG-β-TrCP is shown under the band. (E) Expression of β-TrCP protein in HEK293T cells transfected with plasmids then infected with viruses. HEK293T cells were transfected with protein expression vector pCXN2-FLAG-β-TrCP and then infected with rsOdelia and rsOdelia-NSP1-dC166. Expression of β-TrCP, VP6, and β-actin was detected using anti-FLAG, anti-VP6, and anti-β-actin antibodies, respectively. Protein markers are shown on the right. The relative band intensity of FLAG-β-TrCP is shown under the band. (F and G) Replication of the NSP1 truncation mutant in (F) MA104 and (G) HT-29 cells. Cells were infected with rsOdelia and rsOdelia-NSP1-dC166 at an MOI of 0.1 FFU/cell and then frozen at 72 h postinfection. Virus titer was determined in a focus assay using MA104 cells. Data are expressed as the mean ± SD of triplicate samples. Statistical differences in replication between rsOdelia and rsOdelia-NSP1-dC166 were determined using a t test. **, P < 0.01; ***, P < 0.001.
DISCUSSION
Here, we established a reverse genetics system for the G4P[8] human RV strain Odelia. Strain Odelia grows quite efficiently in cultured cells; indeed, it reached a titer of more than 1 × 107 PFU/ml in MA104 cells. Since such an intrinsic replication advantage may contribute to efficiency of virus recovery from the cell lines used for RV reverse genetics systems, the strain Odelia rescue system provides a unique opportunity to undertake basic research into human RVs and to develop novel vaccines. Genome reassortment occurs frequently during coinfection of a single cell with different RV strains. Introduction of gene segments from different strains sometimes affects the phenotype of a strain, including its ability to adapt to different host species. Generation of monoreassortant viruses derived from different virus strains is a robust technique that allows analysis of the strain-specific functions of individual viral genes harbored by gene segmented viruses. Although genome reassortment of Reoviridae viruses is (theoretically) thought to result in random gene combinations, there is a preference in terms of combined gene segments; indeed, coinfection experiments show that some gene segments from different strains are less likely to be incorporated into virus particles. Here, we demonstrated complete genetic compatibility between simian and human RV strains by successfully generating all SA11 × Odelia SA11 monoreassortant viruses on the genetic background of strain SA11. Among these monoreassortant viruses, replication of rsSA11/Odelia-VP1, -VP2, -VP4, and -VP7, which possess the structural proteins of the strain Odelia, in MA104 cells was lower than that of rsSA11 and the Odelia wild type. In contrast, rsOdelia/SA11-VP4 (which is a reciprocal monoreassortant of rsSA11/Odelia-VP4) and rsOdelia replicated efficiently in MA104 cells. We speculate that the difference in the replication capacity of these monoreassortant viruses (rsSA11/Odelia-VP1, -VP2, -VP4, -VP7, and rsOdelia/SA11-VP4) is due to differences in the strength of protein-protein interactions within virion structures, since RV virion particles are generated through highly organized interactions between structural proteins (46).
In addition, we generated a rsOdelia/SA11-NSP1-ZsG reporter virus. Although the virus possesses the NSP1 gene derived from strain SA11, other genes were derived from strain Odelia. Therefore, the reporter virus generates complete virion particles formed by human RV structural and nonstructural proteins (except the NSP1 protein). Thus, the reporter virus will be a useful tool for screening antiviral compounds and for monitoring replication, infectivity, and antigenicity of recombinant human RVs. Development of a strain Odelia rescue system will allow systematic characterization of gene segment functions and coevolution of gene segments derived from human and simian RV strains.
For the first time, we have used an Odelia strain reverse genetics system to generate a recombinant human RV strain harboring a mutant NSP1 gene and used it to define the biological role of NSP1. NSP1 of strain Odelia harbors an ILD motif within the C-terminal region, which is predicted to induce degradation of β-TrCP (a regulator of NF-κB activation). We observed degradation of endogenous β-TrCP in cells infected with wild-type rsOdelia but not in cells infected with rsOdelia-NSP1-dC13. However, contrary to our expectation, the replication kinetics of rsOdelia-NSP1-dC13 and rsSA11/Ode-NSP1-dC13 in MA104 and HT-29 cells were similar to those of wild-type viruses. This suggests that induction of IFN via β-TrCP is not biologically important with respect to suppression of human RV infection in these cell lines.
In contrast, replication of rsOdelia-NSP1-dC166 was impaired markedly in HT-29 cells, suggesting that deletion of the C-terminal region of human RV NSP1 affects viral replication. Previous studies demonstrate that human RV NSP1 targets not only β-TrCP but also IRF5 and IRF7, which are key transcription factors in the IFN pathway (35); however, the functional domains required for degradation of IRF5 and IRF7 by human RV NSP1 remain unclear. The impaired replication of rsOdelia-NSP1-dC166 shown here suggests that the C-terminal region of Odelia NSP1 may be involved in degradation of transcription factors, including IRF5 and IRF7, that induce transcription of IFN and other cytokines. To increase our understanding of the biological functions of the human RV NSP1 protein, comprehensive study of the interactions between NSP1, β-TrCP, and IRF is required. In addition to NSP1, a recent study demonstrates that the RV structural protein VP3 induces degradation of mitochondrial antiviral-signaling protein (MAVS) to inhibit type III IFN signaling (47). Efficient replication of rsOdelia-NSP1-dC13 in HT-29 cells may be affected by VP3-mediated degradation of MAVS. Degradation of IRFs and MAVS by the virus should be addressed in future studies.
Recent studies used human intestinal enteroid (HIE) cultures as a mini-gut model to study human RVs (38, 47–52). HIEs contain multiple epithelial cell types and possess complexity and cell diversity comparable with that of the intestinal epithelium. Although replication of human RVs in transformed cell lines and animal models is limited, it is supported by HIEs. This suggests that HIEs closely mimic natural infection by human RV. Very recently, a reverse genetics system for G1P[8] human RV was developed (53). Reverse genetics systems now are available for simian RV SA11 and for two human RV strains (G1P[8] and G4P[8]). Development of the strain Odelia-based rescue system described here will expand the utility of reverse genetics systems for study of RV biology. Furthermore, combination of these technologies (e.g., human RV reverse genetics systems and HIE culture systems) will provide great opportunities to study the machinery of human rotavirus replication. It will also expand our understanding of the biology of RVs, as well as facilitate development of therapeutics and vaccines.
MATERIALS AND METHODS
Cells and viruses.Monkey kidney epithelial MA104 and human kidney HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) supplemented with 5% fetal bovine serum (FBS; Gibco), 100 units/ml penicillin, and 100 μg/ml streptomycin (Nacalai Tesque). The human colon cancer cell line HT-29 was cultured in RPMI medium (Nacalai Tesque) supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. BHK cells stably expressing T7 RNA polymerase (BHK-T7) (14) were cultured in DMEM supplemented with 5% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 4 μg/ml puromycin (Sigma-Aldrich). Recombinant strain SA11 (rsSA11) was generated by reverse genetics, as previously described (14). Simian RV strain SA11 and human RV strain Odelia (G4P[8]) were propagated in MA104 cells cultured in DMEM supplemented with 0.5 μg/ml trypsin (Sigma-Aldrich).
Plaque assay.Virus titers were determined in a plaque assay using MA104 cells. A monolayer of MA104 cells in 12-well plates was infected with virus. After 1 h, the inoculums were removed and cells were overlaid with DMEM supplemented with 0.8% SeaKem agarose (Lonza) and 0.5 μg/ml trypsin. After 4 days, the cells were overlaid with DMEM containing 0.8% SeaKem agarose and 0.0132% neutral red solution (Sigma-Aldrich). The virus titer was expressed in terms of PFU per ml.
Focus assay.A monolayer of MA104 cells seeded into 96-well plates (1.5 × 104 cells/well) was infected with recombinant virus for 18 h. The cells were then fixed for 15 min with 4% paraformaldehyde and permeabilized for 15 min with phosphate-buffered saline (PBS) containing 0.5% Triton X-100. Next, cells were incubated for 1 h at room temperature with antiserum specific for RV-NSP4 (1:3,000) diluted in PBS containing 2% FBS. After washing three times with PBS, cells were incubated for 1 h at room temperature with CF 488A goat anti-rabbit IgG (1:2,000; Biotium) diluted in PBS containing 2% FBS. The number of focus-forming units was counted, and the virus titer was expressed as focus-forming units per ml (FFU/ml).
Sequence determination.To obtain the genome sequence of strain Odelia, viral dsRNA genomes were extracted from purified virions using Sepasol-RNA I Super G (Nacalai Tesque) according to the manufacturer’s instructions. The self-anchoring primer C9 was ligated to the 3′ terminus of each gene segment using T4 RNA ligase (Thermo Fisher Scientific). The cDNA of each gene segment was obtained by reverse transcription of dsDNA using RevetraAce (Toyobo). Each gene segment was amplified using KOD-plus-Neo (Toyobo) and a C9 annealing primer, and then cloned into the pBluescript-KS(+) vector. The viral genome sequence was determined by DNA sequencing.
Plasmid construction.To construct the plasmid used for reverse genetics of strain Odelia, cDNA encoding each of 11 gene segments was inserted between the T7 promoter sequence and the hepatitis delta virus ribozyme sequence. The resulting plasmids were named pT7-VP1Odelia, pT7-VP2Odelia, pT7-VP3Odelia, pT7-VP4Odelia, pT7-VP6Odelia, pT7-VP7Odelia, pT7-NSP1Odelia, pT7-NSP2Odelia, pT7-NSP3Odelia, pT7-NSP4Odelia, and pT7-NSP5Odelia. To construct plasmids pT7-NSP1-dC13Odelia and pT7-NSP1-dC166Odelia, the Odelia NSP1 C-terminal peptide coding sequence (KTAEYDSGISDVE) at nucleotide positions 1451 to 1489 and the Odelia NSP1 C-terminal region at nucleotide positions 992 to 1489 were deleted from pT7-NSP1Odelia via PCR mutagenesis. To construct the pCXN2-HA vector, a nucleotide sequence encoding a hemagglutinin (HA) tag and a GGGS linker sequence (YPYDVPDYAGGGS) was cloned into the EcoRI site of the pCXN2 vector. To construct the protein expression vector pCXN2-HA-Odelia-NSP1, the Odelia NSP1 open reading frame (ORF) was cloned into the pCXN2-HA vector. To construct pCXN2-HA-Odelia-NSP1-dC13 and pCXN2-HA-Odelia-NSP1-dC166, the Odelia NSP1 C-terminal peptide coding sequences (KTAEYDSGISDVE) at nucleotide positions 1451 to 1489 and 992 to1489, respectively, were deleted from pCXN2-HA-Odelia-NSP1 by PCR mutagenesis. To construct the pCXN2-FLAG vector, a nucleotide sequence encoding a FLAG tag and a GGGS linker sequence (DYKDDDDKGGGS) was cloned into the EcoRI site of the pCXN2 vector. To construct protein expression vector pCXN2-FLAG-β-TrCP, the ORF of β-TrCP (GenBank accession number NM_001256856) was cloned into the pCXN2-FLAG vector. Plasmid sequences were confirmed by DNA sequencing.
Reverse genetics system.Recombinant viruses were recovered from cells following plasmid transfection, as previously described (14, 15). In short, a monolayer of BHK-T7 cells in a 12-well plate (2 × 105 cells/well) was transfected with all 16 plasmids using 2 μl TransIT-LT1 (Mirus) per microgram of plasmid; 11 plasmids encoding each gene segment of strain Odelia (0.25 μg of each) along with expression plasmids pCAG-NSP2SA11, pCAG-NSP5SA11, pCAG-D1R, pCAG-D12L (0.25 μg of each), and pCAG-FAST (0.005 μg) were used. After 48 h, MA104 cells (5 × 104 cells/well) were added to transfected BHK-T7 cells and incubated for 3 days in the presence of 0.5 μg/ml trypsin (Sigma-Aldrich). After incubation, the cells were lysed by freezing/thawing and transferred to fresh MA104 cells. Recombinant viruses were purified in a plaque assay using MA104 cells. To generate monoreassortant viruses (rsSA11/Odelia-VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, NSP5, and NSP1-dC13), one plasmid encoding each gene segment cDNA of strain SA11 was replaced with cDNA encoding a gene segment of strain Odelia. To generate a monoreassortant virus (rsOdelia/SA11-VP4), the plasmid encoding Odelia VP4 cDNA was replaced with the plasmid encoding SA11 VP4 cDNA. To generate a monoreassortant reporter virus (rsOdelia/SA11-NSP1-ZsG), the plasmid encoding Odelia NSP1 cDNA was replaced with the reporter rescue plasmid pT7-ZsG-Δ722, which was generated previously (15). To generate truncation mutants (rsOdelia-NSP1-dC13 and rsOdelia-NSP1-dC166), pT7-NSP1Odelia was replaced with pT7-NSP1-dC13Odelia or pT7-NSP1-dC166Odelia, respectively.
Growth kinetics.A monolayer of MA104 cells in a 24-well plate (7.5 × 104 cells/well) was infected with virus at a multiplicity of infection (MOI) of 0.01 PFU/cell. After 1 h, cells were washed twice with PBS and cultured with DMEM supplemented with 0.5 μg/ml trypsin. The cells were frozen at −80°C at 12, 24, 48, and 72 h postinfection, except where stated otherwise. After freezing/thawing twice, the virus titer in cells was determined in a plaque assay or a focus assay. To compare the growth kinetics of wild-type and NSP1 mutant viruses, monolayers of MA104 cells in a 24-well plate (7.5 × 104 cells/well) or HT-29 cells in a 24-well plate (1.5 × 105 cells/well) were infected with virus at an MOI of 1 or 0.1 FFU/cell. After 1 h, cells were washed twice with PBS and cultured with DMEM supplemented with 0.5 μg/ml trypsin. Cells were frozen at −80°C at 12, 24, 48, and/or 72 h postinfection. After freezing/thawing twice, the virus titer was determined in a focus assay.
Electropherotype of the viral dsRNA genomes.Viral dsRNA genomes were extracted from purified virions using Sepasol-RNA I Super G (Nacalai Tesque), according to the manufacturer’s instructions. Then, the viral dsRNA genomes were mixed with 2× loading buffer (125 mM Tris-HCl [pH 6.8], 10% sucrose, and 0.004% bromophenol blue) and separated in a 10% polyacrylamide gel (Bio-Rad). The gel was then stained with 5 μg/ml ethidium bromide solution. Images were acquired under a UV transilluminator.
Indirect immunofluorescence analysis.MA104 cells were infected for 18 h with rsOdelia or rsOdelia/SA11-NSP1-ZsG at an MOI of 1 FFU/cell in the presence of 0.5 μg/ml trypsin. Next, the cells were fixed for 15 min with PBS containing 4% paraformaldehyde, permeabilized for 15 min with PBS containing 0.5% Triton X-100, and blocked for 1 h with PBS containing 2% FBS. The cells were incubated for 1 h at room temperature with a primary antibody (diluted 1:2,000 in PBS containing 2% FBS) specific for NSP4. The cells were washed with PBS and then incubated for 1 h at room temperature with CF 594 goat anti-rabbit IgG (diluted 1:2,000 in PBS containing 2% FBS; Biotium). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired under an Axio Observer 7 fluorescence microscope (Zeiss).
β-TrCP degradation.A monolayer of HEK293T cells seeded onto a 12-well plate (2 × 105 cells/well) was used for plasmid transfection. Briefly, pCXN2-FLAG-β-TrCP, pCXN2-HA-Odelia-NSP1, pCXN2-HA-Odelia-NSP1-dC13, or pCXN2-HA-Odelia-NSP1-dC166 (1 μg of each) was mixed with Opti-MEM medium (100 μl) and 1 mg/ml polyethyleneimine (molecular weight [MW], 40,000, 5 μl) (catalog no. 24765; Polysciences, Inc.) and incubated for 15 min. The DNA complex was added to HEK293T cells, and at 4 h posttransfection the medium was replaced with DMEM containing 2% FBS. To assess degradation of β-TrCP by recombinant viruses, HEK293T cells were transfected with pCXN2-FLAG-β-TrCP (1 μg). Four hours posttransfection, medium was replaced with DMEM and recombinant viruses with an MOI of 10 FFU/cell were inoculated for 1 h. Cells were collected at 24 h posttransfection for immunoblot analysis. To assess degradation of β-TrCP by recombinant viruses, MA104 cells (1.5 × 105 cells/well) or HT-29 cells (3 × 105 cells/well) were infected with viruses at an MOI of 10 FFU/cell. Cells were collected at 12 h postinfection for immunoblot analysis.
Immunoblot analysis.HEK293T cell pellets were lysed with 1× Laemmli sample buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 6% 2-mercaptoethanol, 10% glycerol, and 0.01% bromophenol blue) and boiled at 95°C for 5 min. Proteins were separated by SDS-PAGE in 10% acrylamide gels (TGX FastCast acrylamide solutions; Bio-Rad) according to the manufacturer’s instructions. Proteins were transferred to an Immobilon-P polyvinylidene difluoride (PVDF) membrane (Merck) and blocked with PBS containing 0.05% Tween 20 and 3% skim milk. The membranes were then incubated with the following antibodies: FLAG-tagged β-TrCP (monoclonal anti-FLAG antibody, clone M2, diluted 1:5,000; Sigma-Aldrich); endogenous β-TrCP (monoclonal anti-β-TrCP antibody, clone D13F10, diluted 1:1,000; CST); HA-tagged NSP1 (monoclonal anti-HA antibody, clone HA-7, diluted 1:5,000; Sigma-Aldrich); cellular actin (monoclonal anti-β-actin, A2228, diluted 1:5,000; Sigma-Aldrich;); and RV VP6 (polyclonal anti-RV VP6, diluted 1:1,000). The proteins were incubated for 1 h at room temperature with primary antibodies diluted in PBS containing 0.05% Tween 20 and 3% skim milk. The proteins were washed with PBS containing 0.05% Tween 20 and then incubated with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. The immune complex was visualized using Chemi-Lumi One Ultra (Nacalai Tesque) according to the manufacturer’s instructions, and images were acquired by ImageQuant LAS 4000 (Fujifilm) or Amersham Imager 600 (GE Healthcare Life Sciences) instruments.
Image analysis.Expression of β-TrCP protein was quantified using ImageJ software. Band intensity relative to that of the mock sample was calculated.
Statistical analysis.Data analysis was performed using Prism 5 (GraphPad Software, Inc.). Data are expressed as the mean plus or minus standard deviation of triplicate samples. P values of <0.05 were considered statistically significant.
Data availability.The genome sequence of strain Odelia has been deposited in the DNA Data Bank of Japan (DDBJ) database. The segments and GenBank accession numbers are as follows: segment 1 (LC485131), segment 2 (LC485132), segment 3 (LC485133), segment 4 (LC485134), segment 5 (LC485137), segment 6 (LC485135), segment 7 (LC485139), segment 8 (LC485138), segment 9 (LC485136), segment 10 (LC485140), and segment 11 (LC485141).
ACKNOWLEDGMENTS
We thank Y. Saioka, and N. Negishi for technical assistance, K. Yukawa and M. Yoshida for secretarial work, and T. Shinozaki for providing human RV strain Odelia.
This work was supported in part by AMED grants JP18im0210610, JP18fk0108018, and JP18fk0108001, by KAKENHI grants JP19H04835, JP18H02663, JP18K07145, JP18K19444, JP18K15167, and JP16H05360, and by the Public Foundation of the Vaccination Research Center (grant 2018-38).
FOOTNOTES
- Received 26 June 2019.
- Accepted 14 October 2019.
- Accepted manuscript posted online 23 October 2019.
- Copyright © 2020 American Society for Microbiology.
