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Dual selection mechanisms drive efficient single-gene reverse genetics for rotavirus.

ABSTRACT: Current methods for engineering the segmented double-stranded RNA genome of rotavirus (RV) are limited by inefficient recovery of the recombinant virus. In an effort to expand the utility of RV reverse genetics, we developed a method to recover recombinant viruses in which independent selection strategies are used to engineer single-gene replacements. We coupled a mutant SA11 RV encoding a temperature-sensitive (ts) defect in the NSP2 protein with RNAi-mediated degradation of NSP2 mRNAs to isolate a virus containing a single recombinant gene that evades both selection mechanisms. Recovery is rapid and simple; after two rounds of selective passage the recombinant virus reaches titers of ?10(4) pfu/mL. We used this reverse genetics method to generate a panel of viruses with chimeric NSP2 genes. For one of the chimeric viruses, the introduced NSP2 sequence was obtained from a pathogenic, noncultivated human RV isolate, demonstrating that this reverse genetics system can be used to study the molecular biology of circulating RVs. Combining characterized RV ts mutants and validated siRNA targets should permit the extension of this "two-hit" reverse genetics methodology to other RV genes. Furthermore, application of a dual selection strategy to previously reported reverse genetics methods for RV may enhance the efficiency of recombinant virus recovery.

SUBMITTER: Trask SD

PROVIDER: S-EPMC2972929 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Dual selection mechanisms drive efficient single-gene reverse genetics for rotavirus.

Trask Shane D SD Taraporewala Zenobia F ZF Boehme Karl W KW Dermody Terence S TS Patton John T JT

Proceedings of the National Academy of Sciences of the United States of America 20101011 43

Current methods for engineering the segmented double-stranded RNA genome of rotavirus (RV) are limited by inefficient recovery of the recombinant virus. In an effort to expand the utility of RV reverse genetics, we developed a method to recover recombinant viruses in which independent selection strategies are used to engineer single-gene replacements. We coupled a mutant SA11 RV encoding a temperature-sensitive (ts) defect in the NSP2 protein with RNAi-mediated degradation of NSP2 mRNAs to isola ...[more]

PMID: 20937889

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Project description: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.

Project description:The notoriously low efficiency of Paramyxoviridae reverse genetics systems has posed a limiting barrier to the study of viruses in this family. Previous approaches to reverse genetics have utilized a wide variety of techniques to overcome the technical hurdles. Although robustness (i.e., the number of attempts that result in successful rescue) has been improved in some systems with the use of stable cell lines, the efficiency of rescue (i.e., the proportion of transfected cells that yield at least one successful rescue event) has remained low. We have substantially increased rescue efficiency for representative viruses from all five major Paramyxoviridae genera (from ~1 in 106-107 to ~1 in 102-103 transfected cells) by the addition of a self-cleaving hammerhead ribozyme (Hh-Rbz) sequence immediately preceding the start of the recombinant viral antigenome and the use of a codon-optimized T7 polymerase (T7opt) gene to drive paramyxovirus rescue. Here, we report a strategy for robust, reliable, and high-efficiency rescue of paramyxovirus reverse genetics systems, featuring several major improvements: (i) a vaccinia virus-free method, (ii) freedom to use any transfectable cell type for viral rescue, (iii) a single-step transfection protocol, and (iv) use of the optimal T7 promoter sequence for high transcription levels from the antigenomic plasmid without incorporation of nontemplated G residues. The robustness of our T7opt-HhRbz system also allows for greater latitude in the ratios of transfected accessory plasmids used that result in successful rescue. Thus, our system may facilitate the rescue and interrogation of the increasing number of emerging paramyxoviruses. IMPORTANCE The ability to manipulate the genome of paramyxoviruses and evaluate the effects of these changes at the phenotypic level is a powerful tool for the investigation of specific aspects of the viral life cycle and viral pathogenesis. However, reverse genetics systems for paramyxoviruses are notoriously inefficient, when successful. The ability to efficiently and robustly rescue paramyxovirus reverse genetics systems can be used to answer basic questions about the biology of paramyxoviruses, as well as to facilitate the considerable translational efforts being devoted to developing live attenuated paramyxovirus vaccine vectors.

Project description: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 (P[4] and P[8]), VP7 (G1, G2, G3, G8, and G9), and/or VP6 genes cloned from human RV clinical samples using the simian RV SA11 strain (G3P[2]) 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.IMPORTANCE 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.

Project description:Rotavirus is a segmented double-stranded RNA (dsRNA) virus that causes severe gastroenteritis in young children. We have established an efficient simplified rotavirus reverse genetics (RG) system that uses 11 T7 plasmids, each expressing a unique simian SA11 (+)RNA, and a cytomegalovirus support plasmid for the African swine fever virus NP868R capping enzyme. With the NP868R-based system, we generated recombinant rotavirus (rSA11/NSP3-FL-UnaG) with a genetically modified 1.5-kb segment 7 dsRNA encoding full-length nonstructural protein 3 (NSP3) fused to UnaG, a 139-amino-acid green fluorescent protein (FP). Analysis of rSA11/NSP3-FL-UnaG showed that the virus replicated efficiently and was genetically stable over 10 rounds of serial passaging. The NSP3-UnaG fusion product was well expressed in rSA11/NSP3-FL-UnaG-infected cells, reaching levels similar to NSP3 levels in wild-type recombinant SA11-infected cells. Moreover, the NSP3-UnaG protein, like functional wild-type NSP3, formed dimers in vivo Notably, the NSP3-UnaG protein was readily detected in infected cells via live-cell imaging, with intensity levels ∼3-fold greater than those of the NSP1-UnaG fusion product of rSA11/NSP1-FL-UnaG. Our results indicate that FP-expressing recombinant rotaviruses can be made through manipulation of the segment 7 dsRNA without deletion or interruption of any of the 12 open reading frames (ORFs) of the virus. Because NSP3 is expressed at higher levels than NSP1 in infected cells, rotaviruses expressing NSP3-based FPs may be more sensitive tools for studying rotavirus biology than rotaviruses expressing NSP1-based FPs. This is the first report of a recombinant rotavirus containing a genetically engineered segment 7 dsRNA.IMPORTANCE Previous studies generated recombinant rotaviruses that express FPs by inserting reporter genes into the NSP1 ORF of genome segment 5. Unfortunately, NSP1 is expressed at low levels in infected cells, making viruses expressing FP-fused NSP1 less than ideal probes of rotavirus biology. Moreover, FPs were inserted into segment 5 in such a way as to compromise NSP1, an interferon antagonist affecting viral growth and pathogenesis. We have identified an alternative approach for generating rotaviruses expressing FPs, one relying on fusing the reporter gene to the NSP3 ORF of genome segment 7. This was accomplished without interrupting any of the viral ORFs, yielding recombinant viruses that likely express the complete set of functional viral proteins. Given that NSP3 is made at moderate levels in infected cells, rotaviruses encoding NSP3-based FPs should be more sensitive probes of viral infection than rotaviruses encoding NSP1-based FPs.

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Dual selection mechanisms drive efficient single-gene reverse genetics for rotavirus.

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Dual selection mechanisms drive efficient single-gene reverse genetics for rotavirus.

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