Rearranged Genomic RNA Segments Offer a New Approach to the Reverse Genetics of Rotaviruses

Abstract

Group A rotaviruses (RV), members of the Reoviridae family, are a major cause of infantile acute gastroenteritis. The RV genome consists of 11 double-stranded RNA segments. In some cases, an RNA segment is replaced by a rearranged RNA segment, which is derived from its standard counterpart by partial sequence duplication. We report here a reverse genetics system for RV based on the preferential packaging of rearranged RNA segments. Using this system, wild-type or in vitro-engineered forms of rearranged segment 7 from a human rotavirus (encoding the NSP3 protein), derived from cloned cDNAs and transcribed in the cytoplasm of COS-7 cells with the help of T7 RNA polymerase, replaced the wild-type segment 7 of a bovine helper virus (strain RF). Recombinant RF viruses (i.e., engineered monoreassortant RF viruses) containing an exogenous rearranged RNA were recovered by propagating the viral progeny in MA-104 cells, with no need for additional selective pressure. Our findings offer the possibility to extend RV reverse genetics to segments encoding nonstructural or structural proteins for which no potent selective tools, such as neutralizing antibodies, are available. In addition, the system described here is the first to enable the introduction of a mutated gene expressing a modified nonstructural protein into an infectious RV. This reverse genetics system offers new perspectives for investigating RV protein functions and developing recombinant live RV vaccines containing specific changes targeted for attenuation.

Complete Genome Characterization of Recent and Ancient Belgian Pig Group A Rotaviruses and Assessment of Their Evolutionary Relationship with Human Rotaviruses

“…The exact function(s) of these duplications remains elusive, but gene segments possessing duplications are thought to be incorporated preferentially into progeny viruses. This principle has therefore been applied to improve rotavirus reverse genetics systems (44).…”

The Rhesus Rotavirus Gene Encoding VP4 Is a Major Determinant in the Pathogenesis of Biliary Atresia in Newborn Mice

“…A rotaviral particle consists of 3 concentric protein layers surrounding a genome of 11 double-stranded RNA (dsRNA) segments encoding 6 structural viral proteins (VP1 to VP4, VP6, and VP7) and 6 nonstructural proteins (NSP1 to NSP6). Reverse genetics is difficult to perform in rotavirus; however, rotavirus can undergo genetic reassortment after mixed infection in vivo or in cell culture (11,18,19,28,30,37,38). When a host cell is coinfected with two strains of rotavirus, progeny viruses, termed reassortants, that contain different combinations of the parental genes are generated.…”

Generation of Genetically Stable Recombinant Rotaviruses Containing Novel Genome Rearrangements and Heterologous Sequences by Reverse Genetics

“…These viruses grew well and were genetically stable. Unlike the duplicated sequences engineered by Troupin et al (38) into the 3= UTR of gene 7 (NSP3), the duplicated gene 8 sequences engineered in our study were not based on previous descriptions of naturally occurring rearrangements. For the naturally occurring variant Wag7/8re, the gene 8 sequence duplication initiates 12 nt downstream of the NSP2 stop codon, while in the engineered gene 8 sequences of recombinant viruses, the duplication initiates immediately downstream of the stop codon (28).…”

“…The molecular basis for this phenomenon is not entirely clear but may result from duplication of packaging signals in the rearranged segment, thereby increasing its probability of encapsidation. Indeed, Troupin et al (38) were able to develop an RV reverse genetics system based on a previously described, spontaneously occurring rearrangement in the gene 7 RNA of the bovine RV strain RF (29). By extensive serial passage of lysates from cells infected with wild-type RV RF and transfected with a T7 expression plasmid producing the rearranged gene 7 RNA, the authors recovered a recombinant RV containing the rearranged RNA (38).…”