Viral replication is rapid and robust, but it is far from a chaotic process. Instead, successful production of infectious progeny requires that events occur in the correct place and at the correct ...time. Rotaviruses (segmented double-stranded RNA viruses of the Reoviridae family) seem to govern their replication through ordered disassembly and assembly of a triple-layered icosahedral capsid. In recent years, high-resolution structural data have provided unprecedented insight into these events. In this Review, we explore the current understanding of rotavirus replication and how it compares to replication of other Reoviridae family members.
Rotavirus outer-layer protein VP7 is a principal target of protective antibodies. Removal of free calcium ions (Ca²⁺) dissociates VP7 trimers into monomers, releasing VP7 from the virion, and ...initiates penetration-inducing conformational changes in the other outer-layer protein, VP4. We report the crystal structure at 3.4 angstrom resolution of VP7 bound with the Fab fragment of a neutralizing monoclonal antibody. The Fab binds across the outer surface of the intersubunit contact, which contains two Ca²⁺ sites. Mutations that escape neutralization by other antibodies suggest that the same region bears the epitopes of most neutralizing antibodies. The monovalent Fab is sufficient to neutralize infectivity. We propose that neutralizing antibodies against VP7 act by stabilizing the trimer, thereby inhibiting the uncoating trigger for VP4 rearrangement. A disulfide-linked trimer is a potential subunit immunogen.
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 ≥10⁴ 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.
► Structures of rotavirus have greatly informed studies of replication and entry. ► The Reoviridae may have a conserved capsid–polymerase interaction mechanism. ► The rotavirus polymerase orientation ...suggests the location of the capping enzyme. ► The conformation of the rotavirus spike is regulated by the outer capsid protein. ► New methods are needed to study the rotavirus spike precursor and capping enzyme.
Rotaviruses are members of the Reoviridae family of non-enveloped viruses and important etiologic agents of acute gastroenteritis in infants and young children. In recent years, high-resolution structures of triple-layered rotavirus virions and the constituent proteins have provided valuable insights into functions. Of note, structural studies have revealed the position of the viral RNA-dependent RNA polymerase, VP1, within the inner capsid, which in turn provides clues about the location of the viral capping machinery and the route of viral transcript egress. Mechanisms by which the viral spike protein, VP4, mediates receptor binding and membrane penetration have also been aided by high-resolution structural studies. Future work may serve to fill the remaining gaps in understanding of rotavirus particle structure and function.
Effective methods to engineer the segmented, double-stranded RNA genomes of Reoviridae viruses have only recently been developed. Mammalian orthoreoviruses (MRV) and bluetongue virus (BTV) can be ...recovered from entirely recombinant reagents, significantly improving the capacity to study the replication, pathogenesis, and transmission of these viruses. Conversely, rotaviruses (RVs), which are the major etiological agent of severe gastroenteritis in infants and children, have thus far only been modified using single-segment replacement methods. Reoviridae reverse genetics techniques universally rely on site-specific initiation of transcription by T7 RNA polymerase to generate the authentic 5′ end of recombinant RNA segments, but they vary in how the RNAs are introduced into cells: recombinant BTV is recovered by transfection of in vitro transcribed RNAs, whereas recombinant MRV and RV RNAs are transcribed intracellularly from transfected plasmid cDNAs. Additionally, several parameters have been identified in each system that are essential for recombinant virus recovery. Generating recombinant BTV requires the use of 5′ capped RNAs and is enhanced by multiple rounds of RNA transfection, suggesting that translation of viral proteins is likely the rate-limiting step. For RV, the efficiency of recovery is almost entirely dependent on the strength of the selection mechanism used to isolate the single-segment recombinant RV from the unmodified helper virus. The reverse genetics methods for BTV and RV are presented and compared to the previously described MRV methods. Analysis and comparison of each method suggest several key lines of research that might lead to a reverse genetics system for RV, analogous to those used for MRV and BTV.
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