Identification of sequence elements required for replication and assembly of reovirus M1 gene and expression of the M1 gene in mammalian cells.

Title: Identification of sequence elements required for replication and assembly of reovirus M1 gene and expression of the M1 gene in mammalian cells.
Authors: Zou, Shimian.
Date: 1995
Abstract: Reoviruses are double-stranded (ds) RNA viruses containing a genome of 10 dsRNA segments. A unique feature of reovirus replication is that each progeny virus contains one, and only one, of each of the 10 segments of dsRNA. However, the genetic elements that control the sorting and assembly of genome segments were not known. The M1 gene encodes the $\mu$2 protein which is a minor component of the virus core. Genetic studies have associated the M1 gene with cytopathic effect in cultured cells and pathogenesis in infected animals. The genetic basis for this function was not clear either. Because of the segmented nature of reovirus genome, coinfection of cells with two different serotypes can produce progeny viruses that contain genome segments from both serotypes (reassortants). Type 1 and type 3 reassortants that possess the type 3 L2 segment and type 1 M3 segment generate M1 segment deletion (smaller M1 segments) on serial passage (Brown et al., 1983). By analyzing reovirus M1 deletion mutants, it was found that both termini of the M1 gene were conserved. The results indicate that these two terminal regions contain all the genetic signals sufficient for the replication and assembly of the M1 gene and that 344 nucleotides could be the minimum length required for packaging into virions. Fifty-one nucleotide substitutions and 10 amino acid substitutions were identified between type 1 and type 3 M1 segments. These substitutions must be responsible for the phenotypic differences in cytopathic effect and pathogenesis that map to the M1 gene by genetic analyses. The M1 gene was further characterized by expression in mammalian cells. One $\mu$2-expressing L929 cell line was tested for its ability to support the growth of a reovirus temperature-sensitive (ts) mutant with a defect in its M1 gene. The restricted replication of the ts mutant at nonpermissive temperature was complemented by the $\mu$2-expressing cell line, demonstrating the feasibility of cultivating reoviruses with defective M1 genes. Mutagenesis of the M1 gene-containing DNA constructs and expression studies also clarified the translation initiation site of the M1 gene. It was found that translation of the M1 gene initiates from the first AUG codon starting at nucleotide 14 and that if there is initiation from AUG$\sb{161}$ as in 5$\sp\prime$-terminus-truncated constructs, a protein about 5 kDa smaller than $\mu$2 protein is produced. With the information about the M1 gene obtained from this study, preliminary reverse genetics work was initiated in attempts to further characterize the genetic signals within the conserved M1 termini and to develop reovirus as an expression vector. (Abstract shortened by UMI.)
CollectionTh├Ęses, 1910 - 2010 // Theses, 1910 - 2010
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