Genetic Studies on the Mechanism of Chemical and Physical Inactivation of Reovirus

Abstract

SUMMARYThe three serotypes of reovirus differ markedly in their response to a variety of chemical inactivating agents. We used intertypic recombinants containing various combinations of genes derived from the parental serotypes to study the basis of these differences. In addition to recombinants derived from types 1 and 3, and 2 and 3, we were able to isolate recombinants derived from types 1 and 2, suggesting that these two serotypes also undergo unrestricted reassortment. The intertypic recombinants behaved like one parent or the other in the presence of the inactivating agents and allowed us to determine the genes responsible for each difference. Recombinants derived from crosses between wild-type parental serotypes produced straightforward results, while recombinants derived from mutagenized, temperature-sensitive parents often did not. Sensitivity to 2.5 M-guanidine-HC1 and pH 11 was determined by the S1 gene, sensitivity to 55 °C and 1 ~o SDS was determined by the $4 gene, and sensitivity to 33~ ethanol and to 1 ~ phenol was determined by the M2 gene. Thus, relatively nonspecific chemical agents appear to have their predominant effect on specific proteins of the reovirus virion. INTRODUCTIONThe mammalian reoviruses are classified as enteric viruses by their site of isolation and by their chemical and physical properties. These properties include resistance to proteolytic enzymes, acid, detergents and heat (Bitton, 1980). The biochemical and structural properties that allow the reoviruses, and other enteric viruses, to resist inactivation by these agents are not known. The genetic organization of mammalian reoviruses presents a unique opportunity to gain insight into the nature of the virus components responsible for determining the responses to such treatments. The three serotypes of human reoviruses are able to reassort dsRNA gene segments in mixed infection to produce recombinants (reassortants). These recombinants carry various combinations of discrete genes derived from the two parental serotypes. When two serotypes of reovirus differ in a biological or structural property, we have shown that intertypic recombinants can segregate the difference and thereby allow us to identify the gene(s) which specifies the difference (Fields, 1981). In this report, we show that the three reovirus serotypes differ in their sensitivity to detergents, heat, high pH and several organic denaturants. Intertypic recombinants segregate these differences and have allowed us to determine the genes responsible.

Mammalian reovirus, a nonfusogenic nonenveloped virus, forms size-selective pores in a model membrane

“…ISVP3 ISVP* conversion is correlated with membraneperforation activities both in vivo and in vitro (19,20). Other hallmarks of this conversion include 1 rearrangement to a protease-sensitive conformer and elution of the adhesion protein 1 (18,19,21,22). In addition, a myristoylated N-terminal fragment of 1, 1N, is generated by autocleavage and released from particles (23)(24)(25)(26).…”

Characterization of the Nucleoside Triphosphate Phosphohydrolase and Helicase Activities of the Reovirus λ1 Protein

“…During reovirus infection, the viral genome remains in the inner capsid (core) of the virus, composed of two major (1 and 2) and two minor (3 and 2) proteins. Gene reassortment experiments have resulted in the assignment of transcriptase activity to 3 (2). Although functions of other core proteins have not been firmly established, it is suspected that additional enzymatic functions are needed to achieve transcription and replication of the viral genome.…”

“…2 Furthermore, analysis of gene reassortment has recently resulted in the assignment of NTPase activity present in reovirus cores to the L3 gene encoding 1 (19). In this study, we present a biochemical characterization of the enzymatic activities exhibited by the 1 protein encoded by the cloned L3 gene.…”

Requirements for the Formation of Membrane Pores by the Reovirus Myristoylated μ1N Peptide

“…The core remains intact, and mRNA synthesis and 5Ј capping are primarily accomplished by core proteins 3 and 2, respectively (18,34,44,52). Translocation of the reovirus core from an extracellular milieu to the cytoplasm probably occurs in an endosome or related structure, and 1 appears to be a principal agent of this process (1,3,10,11,19,27,29,31,33,36,37,38,42,56).…”