TEMPERATURE-SENSITIVE LESIONS IN THE CAPSID PROTEINS OF THE ROTAVIRUSMUTANTS TSF AND TSG THAT AFFECT VIRION ASSEMBLY

The SA11 rotavirus mutants tsF and tsG contain temperature sensitive ( ts) lesions in the capsid proteins VP2 and VP6, respectively, that int erfere with their ability to assemble. To understand the nature of the ir lesions, full-length cDNAs of tsF gene 2 and tsG gene 6 were prepar ed from viral mRNA by reverse transcription and polymerase chain react ion. Comparative sequence analysis indicated that the ts phenotype of tsF VP2 is due to an Ala --> Asp substitution at position 387. The mut ation falls outside of those regions of VP2 previously suggested to be of functional significance and therefore points to a previously unide ntified site in VP2 that is important for the assembly of viral cores. Comparative sequence analysis showed that tsG VP6 contains two mutant amino acids, i.e., Thr-10 and His-13, and therefore one or both of th ese mutations are responsible for the ts phenotype of the mutant VP6, In the case of other group A and group C VP6 sequences, these residues are Ser and Asp, respectively. Characterization of tsG-infected cells by indirect immunofluorescence staining showed that while viroplasmic inclusions are formed at the nonpermissive temperature, the mutant VP 6 accumulates in these structures only at the permissive temperature. While influencing intracellular accumulation, the Thr-10 --> Ser and H is-13 --> Asp mutations in tsG VP6 are probably not directly involved in the interaction of VP6 with VP2, as VP6 deletion mutants lacking re sidues 10 and 13 retain the ability to bind VP2 in vitro. Analysis of VP6 failed to confirm previous reports that the protein was myristylat ed and thus excludes the possibility that this cotranslational modific ation is temperature-dependent for tsG VP6. Together, these data sugge st that the amino terminus of VP6 plays an essential role in virus ass embly in vivo, perhaps by being necessary for the movement of the prot ein to viroplasmic inclusions, the site of core and single-shelled par ticle formation, (C) 1994 Academic Press, Inc.