The Reovirus Mutant tsA279 Has Temperature-Sensitive Lesions in the M2 and L2 Genes: The M2 Gene Is Associated with Decreased Viral Protein Production and Blockade in Transmembrane Transport

Abstract

Temperature-sensitive mutants provide an ideal means for dissecting viral assembly pathways. The morphological variants produced by end biological characteristics of tsA279, a previously uncharacterized mutant from the Fields' panel of temperature-sensitive mutants of reovirus, were determined under restrictive growth conditions. The mutant showed a distinctive pattern of increased temperature sensitivity as the temperature was raised from 39° to 40°. Wild-type reovirus type 1 Lang and the mutant were crossed to generate reassortants. Efficiency of plating analyses of the reassortants showed that tsA279 has temperature-sensitive lesions in two genes, a mildly temperature-sensitive one in L2, which encodes core spike protein λ2, and a stronger, dominant lesion in M2, which encodes major outer capsid protein μ1. Electron microscopic examination of thin-sectioned tsA279 -infected cells showed three ways in which the mutant phenotypes were expressed. The mutant appeared to be blocked in transmembrane transport of virions, a phenotype that mapped to the M2 gene; the mutant produced significantly reduced amounts of identifiable particles; and those particles that were produced appeared to be morphological variants. Immunofluorescent microscopy and Immunoprecipitations of tsA279- and various T1L × tsA279 reassortant-infected cells suggested that the reduction in observed progeny was caused by a decreased production of viral proteins at the nonpermissive temperature. This phenotype also mapped to the mutant M2 gene. © 1995 Academic Press. All rights reserved.