Rotaviruses (RVs) are nonenveloped viruses that cause gastroenteritis in infants and young children. Sialic acid is an initial receptor, especially for animal RVs, including rhesus RV. Sialic acid ...binds to the VP8* subunit, a part of the outer capsid protein VP4 of RV. Although interactions between virus and glycan receptors influence tissue and host tropism and viral pathogenicity, research has long been limited to biochemical and structural studies due to the unavailability of an RV reverse genetics system. Here, we examined the importance of sialic acid in RV infections using recombinant RVs harboring mutations in sialic acid-binding sites in VP4 via a simian RV strain SA11-based reverse genetics system. RV VP4 mutants that could not bind to sialic acid had replicated to decreased viral titer in MA104 cells. Wild-type virus infectivity was reduced, while that of VP4 mutants was not affected in sialic acid-deficient cells. Unexpectedly, in vivo experiments demonstrated that VP4 mutants suppressed mouse pups' weight gain and exacerbated diarrhea symptoms compared to wild-type viruses. Intestinal contents enhanced VP4 mutants' infectivity. Thus, possibly via interactions with other unknown receptors and/or intestinal contents, VP4 mutants are more likely than wild-type viruses to proliferate in the murine intestine, causing diarrhea and weight loss. These results suggest that RVs binding sialic acid notably affect viral infection in vitro and viral pathogenesis in vivo.
Various studies have been conducted on the binding of VP8* and glycans, and the direct interaction between purified VP8* and glycans has been investigated by crystalline structure analyses. Here, we used a reverse genetics system to generate rotaviruses (RVs) with various VP4 mutants. The generated mutant strains clarified the importance of glycan binding
and
. Moreover, even when VP4 mutants could not bind to sialic acid, they were able to bind to an unknown receptor. As RVs evolve, pathogenicity can also be modified by easily altering the glycans to which VP4 binds.
Rotavirus (RV), the most common cause of gastroenteritis in children, carries a high economic and health burden worldwide. RV encodes six structural proteins and six nonstructural proteins (NSPs) ...that play different roles in viral replication. NSP4, a multifunctional protein involved in various viral replication processes, has two conserved N-glycosylation sites; however, the role of glycans remains elusive. Here, we used recombinant viruses generated by a reverse genetics system to determine the role of NSP4 N-glycosylation during viral replication and pathogenesis. The growth rate of recombinant viruses that lost one glycosylation site was as high as that of the wild-type virus. However, a recombinant virus that lost both glycosylation sites (glycosylation-defective virus) showed attenuated replication in cultured cell lines. Specifically, replications of glycosylation-defective virus in MA104 and HT29 cells were 10- and 100,000-fold lower, respectively, than that of the wild-type, suggesting that N-glycosylation of NSP4 plays a critical role in RV replication. The glycosylation-defective virus showed NSP4 mislocalization, delay of cytosolic Ca
elevation, and less viroplasm formation in MA104 cells; however, these impairments were not observed in HT29 cells. Further analysis revealed that assembly of glycosylation-defective virus was severely impaired in HT29 cells but not in MA104 cells, suggesting that RV replication mechanism is highly cell type dependent.
mouse experiments also showed that the glycosylation-defective virus was less pathogenic than the wild-type virus. Taken together, the data suggest that N-glycosylation of NSP4 plays a vital role in viral replication and pathogenicity.
Rotavirus is the main cause of gastroenteritis in young children and infants worldwide, contributing to 128,500 deaths each year. Here, we used a reverse genetics approach to examine the role of NSP4 N-glycosylation. An N-glycosylation-defective virus showed attenuated and cell-type-dependent replication
. In addition, mice infected with the N-glycosylation-defective virus had less severe diarrhea than mice infected with the wild type. These results suggest that N-glycosylation affects viral replication and pathogenesis. Considering the reduced pathogenicity
and the high propagation rate in MA104 cells, this glycosylation-defective virus could be an ideal live attenuated vaccine candidate.
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 G4P8 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.
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
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.
Rotaviruses (RVs) are an important cause of acute gastroenteritis in young children. Recently, versatile plasmid-based reverse genetics systems were developed for several human RV genotypes; however, ...these systems have not been developed for all commonly circulating human RV genotypes. In this study, we established a reverse genetics system for G2P4 human RV strain HN126. Nucleotide sequence analysis, including that of the terminal ends of the viral double-stranded RNA genome, revealed that HN126 possessed a DS-1-like genotype constellation. Eleven plasmids, each encoding 11 gene segments of the RV genome, and expression plasmids encoding vaccinia virus RNA capping enzyme (D1R and D12L), Nelson Bay orthoreovirus FAST, and NSP2 and NSP5 of HN126, were transfected into BHK-T7 cells, and recombinant strain HN126 was generated. Using HN126 or simian RV strain SA11 as backbone viruses, reassortant RVs carrying the outer and intermediate capsid proteins (VP4, VP7 and VP6) of HN126 and/or SA11 (in various combinations) were generated. Viral replication analysis of the single, double and triple reassortant viruses suggested that homologous combination of the VP4 and VP7 proteins contributed to efficient virus infectivity and interaction between other viral or cellular proteins. Further studies of reassortant viruses between simian and other human RV strains will contribute to developing an appropriate model for human RV research, as well as suitable backbone viruses for generation of recombinant vaccine candidates.
Species A rotaviruses (RVs) are a leading cause of severe acute gastroenteritis in infants and children younger than 5 years. Currently available RV vaccines were adapted from wild-type RV strains by ...serial passage of cultured cells or by reassortment between human and animal RV strains. These traditional methods require large-scale screening and genotyping to obtain vaccine candidates. Reverse genetics is a tractable, rapid, and reproducible approach to generating recombinant RV vaccine candidates carrying any VP4 and VP7 genes that provide selected antigenicity. Here, we developed a vaccine platform by generating recombinant RVs carrying VP4 (P4 and P8), VP7 (G1, G2, G3, G8, and G9), and/or VP6 genes cloned from human RV clinical samples using the simian RV SA11 strain (G3P2) as a backbone. Neutralization assays using monoclonal antibodies and murine antisera revealed that recombinant VP4 and VP7 monoreassortant viruses exhibited altered antigenicity. However, replication of VP4 monoreassortant viruses was severely impaired. Generation of recombinant RVs harboring a chimeric VP4 protein for SA11 and human RV gene components revealed that the VP8* fragment was responsible for efficient infectivity of recombinant RVs. Although this system must be improved because the yield of vaccine viruses directly affects vaccine manufacturing costs, reverse genetics requires less time than traditional methods and enables rapid production of safe and effective vaccine candidates.
Although vaccines have reduced global RV-associated hospitalization and mortality over the past decade, the multisegmented genome of RVs allows reassortment of VP4 and VP7 genes from different RV species and strains. The evolutionary dynamics of novel RV genotypes and their constellations have led to great genomic and antigenic diversity. The reverse genetics system is a powerful tool for manipulating RV genes, thereby controlling viral antigenicity, growth capacity, and pathogenicity. Here, we generated recombinant simian RVs (strain SA11) carrying heterologous VP4 and VP7 genes cloned from clinical isolates and showed that VP4- or VP7-substituted chimeric viruses can be used for antigenic characterization of RV outer capsid proteins and as improved seed viruses for vaccine production.
Recombinant viruses expressing fluorescent or luminescent reporter proteins are used to quantitate and visualize viral replication and transmission. Here, we used a split NanoLuc luciferase (NLuc) ...system comprising large LgBiT and small HiBiT peptide fragments to generate stable reporter rotaviruses (RVs). Reporter RVs expressing NSP1-HiBiT fusion protein were generated by placing an 11 amino acid HiBiT peptide tag at the C-terminus of the intact simian RV NSP1 open reading frame or truncated human RV NSP1 open reading frame. Virus-infected cell lysates exhibited NLuc activity that paralleled virus replication. The antiviral activity of neutralizing antibodies and antiviral reagents against the recombinant HiBiT reporter viruses were monitored by measuring reductions in NLuc expression. These findings demonstrate that the HiBiT reporter RV systems are powerful tools for studying the viral life cycle and pathogenesis, and a robust platform for developing novel antiviral drugs.
•Recombinant rotaviruses harboring HiBiT tag were generated.•The function of viral NSP1 was not affected by addition of HiBiT tag.•Replication of HiBiT reporter rotaviruses was monitored by NanoLuc activity.•HiBiT reporter rotavirus system is a powerful tool for antiviral screening.
Rotavirus causes severe diarrhea in infants. Although live attenuated rotavirus vaccines are available, vaccine-derived infections have been reported, which warrants development of next-generation ...rotavirus vaccines. A single-round infectious virus is a promising vaccine platform; however, this platform has not been studied extensively in the context of rotavirus. Here, we aimed to develop a single-round infectious rotavirus by impairing the function of the viral intermediate capsid protein VP6. Recombinant rotaviruses harboring mutations in VP6 were rescued using a reverse genetics system. Mutations were targeted at VP6 residues involved in virion assembly. Although the VP6-mutated rotavirus expressed viral proteins, it did not produce progeny virions in wild-type cells; however, the virus did produce progeny virions in VP6-expressing cells. This indicates that the VP6-mutated rotavirus is a single-round infectious rotavirus. Insertion of a foreign gene, and replacement of the VP7 gene segment with that of human rotavirus clinical isolates, was successful. No infectious virions were detected in mice infected with the single-round infectious rotavirus. Immunizing mice with the single-round infectious rotavirus induced neutralizing antibody titers as high as those induced by wild-type rotavirus. Taken together, the data suggest that this single-round infectious rotavirus has potential as a safe and effective rotavirus vaccine. This system is also applicable for generation of safe and orally administrable viral vectors.IMPORTANCERotavirus, a leading cause of acute gastroenteritis in infants, causes an annual estimated 128,500 infant deaths worldwide. Although live attenuated rotavirus vaccines are available, they are replicable and may cause vaccine-derived infections. Thus, development of safe and effective rotavirus vaccine is important. In this study, we report the development of a single-round infectious rotavirus that can replicate only in cells expressing viral VP6 protein. We demonstrated that (1) the single-round infectious rotavirus did not replicate in wild-type cells or in mice; (2) insertion of foreign genes and replacement of the outer capsid gene were possible; and (3) it was as immunogenic as the wild-type virus. Thus, the mutated virus shows promise as a next-generation rotavirus vaccine. The system is also applicable to orally administrable viral vectors, facilitating development of vaccines against other enteric pathogens.
Mammalian reovirus (MRV) strain type 3 Dearing (T3D) is a naturally occurring oncolytic virus that has been developed as a potential cancer therapeutic. However, MRV treatment cannot be applied to ...cancer cells expressing low levels of junctional adhesion molecule A (JAM-A), which is the entry receptor of MRV. In this study, we developed a reverse genetics system for MRV strain T3D-L, which showed high oncolytic potency. To modify the cell tropism of MRV, an arginine-glycine-aspartic acid (RGD) peptide with an affinity to integrin was inserted at the C terminus or loop structures of the viral cell attachment protein σ1. The recombinant RGD σ1-modified viruses induced remarkable cell lysis in human cancer cell lines with marginal JAM-A expression and in JAM-A knockout cancer cell lines generated by a CRISPR/Cas9 system. Pretreatment of cells with anti-integrin antibody decreased cell death caused by the RGD σ1-modified virus, suggesting the infection to the cells was via a specific interaction with integrin αV. By using mouse models, we assessed virulence of the RGD σ1-modified viruses
This system will open new avenues for the use of genetically modified oncolytic MRV for use as a cancer therapy.
Oncolytic viruses kill tumors without affecting normal cells. A variety of oncolytic viruses are used as cancer therapeutics. Mammalian reovirus (MRV), which belongs to the genus
, family
, is one such natural oncolytic virus. The anticancer effects of MRV are being evaluated in clinical trials. Unlike other oncolytic viruses, MRV has not been genetically modified for use as a cancer therapeutic in clinical trials. Here, we used a reverse genetic approach to introduce an integrin-affinity peptide sequence into the MRV cell attachment protein σ1 to alter the natural tropism of the virus. The recombinant viruses were able to infect cancer cell lines expressing very low levels of the MRV entry receptor, junctional adhesion molecule A (JAM-A), and cause tumor cell death while maintaining its original tropism via JAM-A. This is a novel report of a genetically modified oncolytic MRV by introducing a peptide sequence into σ1.
The family
is a nonenveloped virus group with a double-stranded (ds) RNA genome comprising 9 to 12 segments. In the family
, the genera
,
,
,
, and
contain virus species having 12-segmented dsRNA ...genomes. Reverse genetics systems used to generate recombinant infectious viruses are powerful tools for investigating viral gene function and for developing vaccines and therapeutic interventions. Generally, this methodology has been utilized for
viruses such as
,
,
, and
, which have genomes with 10 or 11 segments, respectively. However, no reverse genetics system has been developed for
viruses with a genome harboring 12 segments. Herein, we describe development of an entire plasmid-based reverse genetics system for Tarumizu tick virus (TarTV) (genus
family
), which has a genome of 12 segments. Recombinant TarTVs were generated by transfection of 12 cloned complementary DNAs encoding the TarTV genome into baby hamster kidney cells expressing T7 RNA polymerase. Using this technology, we generated VP12 mutant viruses and demonstrated that VP12 is an
-glycosylated protein. We also generated a reporter virus expressing the HiBiT-tagged VP8 protein. This reverse genetics system will increase our understanding of not only the biology of the genus
but also the replication machinery of the family
.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes severe acute respiratory symptoms in humans. Controlling the coronavirus disease pandemic is a worldwide priority. The number of ...SARS-CoV-2 studies has dramatically increased, and the requirement for analytical tools is higher than ever. Here, we propose monolayered-intestinal epithelial cells (IECs) derived from human induced pluripotent stem cells (iPSCs) instead of three-dimensional cultured intestinal organoids as a suitable tool to study SARS-CoV-2 infection. Differentiated IEC monolayers express high levels of angiotensin-converting enzyme 2 and transmembrane protease serine 2 (TMPRSS2), host factors essential for SARS-CoV-2 infection. SARS-CoV-2 efficiently grows in IEC monolayers. Using this propagation system, we confirm that TMPRSS2 inhibition blocked SARS-CoV-2 infection in IECs. Hence, our iPSC-derived IEC monolayers are suitable for SARS-CoV-2 research under physiologically relevant conditions.
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