Temperature-sensitive RNA polymerase mutants of a picornavirus.

Abstract

Temperature-sensitive (ts) RNA polymerase mutants of a picornavirus are reported, Two foot-and-mouth disease virus (FMDV) mutants designated ts 22 and to 115 have been characterized. As judged by isoelectric focusing, both have charge alterations in P56a, the FMDV RNA polymerase protein. Virus RNA synthesis in cells infected with the mutants is severely impaired at the nonpermissive temperature. RNA polymerase purified from baby hamster kidney cells infected with these mutants exhibits a marked to transcribing activity in vitro. Spontaneous revertants of both mutants have P56a polypeptides that are indistinguishable from the parental proteins on the basis of charge. The revertants regain the ability to synthesize virus RNA in vivo at the nonpermissive temperature. RNA polymerase purified from the revertants remains transcriptionally active at the nonpermissive temperature.The genome of foot-and-mouth disease virus (FMDV) is a single-stranded "positive"-sense RNA molecule of approximately 8 kilobases with a small protein termed VPg covalently attached to its 5' end, an internal poly(C) tract, and a poly(A) sequence at the 3' end (1) (Fig. 1) (14,15). Hence, a tentative alignment of the genetic and biochemical maps has been constructed (15). We describe how a modified EF technique was used to screen mutants with ts loci at the 3' end of the FMDV genetic map for an altered P56a polypeptide. Two candidate RNA polymerase mutants (ts 22 and.ts 115) were identified by these criteria. Cells infected with these mutants synthesize only trace amounts of virus RNA at the nonpermissive temperature. Furthermore, FMDV RNA polymerase purified from the cytoplasm of baby hamster kidney cells infected with the mutants showed a ts transcribing activity in vitro when compared with the enzyme extracted from cells infected with the parental virus. MATERIALS AND METHODSViruses. The parental FMDV strain from which all mutants were derived was 0 Pacheco (ts+). Mutants mapping at the 3' end of the FMDV genome (5-fluorouracil-induced ts 22 and spontaneous ts 115) have been described (12, 13). The isolation ofa revertant ofts 22 (designated ts 22R) also has been described (15 The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Poliovirus RNA-Dependent RNA Polymerase Synthesizes Full-Length Copies of Poliovirion RNA, Cellular mRNA, and Several Plant Virus RNAs In Vitro

“…Similar results have also been obtained with the footand-mouth disease virus RNA polymerase (18). In addition, several RNA-negative mutations have been shown to map in the foot-and-mouth disease virus RNA polymerase protein, p56a, which appears to be the foot-and-mouth disease virus equivalent to the poliovirus polymerase protein, p63 (19).…”

Multiple sites of recombination within the RNA genome of foot-and-mouth disease virus

“…Recombinants between the two FMDV subtypes, 0, and O,, were isolated by (McCahon et al, 1977). ' Based on covariation with a protein or oligonucleotide: ts16, Saunders et al, (1985); ts22, Lowe et al (1981); ls33, King and Newman (1980); ts302, 0303, King et al (1982 infecting cells with a mixture of ts mutants, and selecting t.sf progeny in an infectious centre assay (McCahon and Slade, 1981). A variety of ts mutants was used, with the aim of generating as many different types of recombinant as possible.…”

The genetics of foot and mouth disease virus

“…Two ts mutants were found to have electrophoretic mutations in their p56a which co-varied with their ts mutation, so demonstrating that their ts mutations were in p56a. The isolated p56a from these two mutants was shown to be temperature-sensitive in polymerase activity in vitro and so provided independent proof that p56a was the viral polymerase (21). A variety of such mutants are now available and could be used to analyse further the function of this protein.…”