J. Mol. Biol. (1989) 206, 425-438 Replication in WJO of Mutant Brome Mosaic Virus RNAs Defective in Aminoacylation T. W. Dreher’f, A. L. N. Rao and T. C. Hallj: Department of Biology, Texas A&M University College Station, TX 77843-3258, U.S.A. (Received 17 October 1988) In order to understand the relationship between replication and aminoacylation of the genomic RNAs of brome mosaic virus, the replication of four mutants, whose RNAs were expected (on the basis of their properties in vitro) to be inefficiently tyrosylated in vivo, was studied in barley protoplasts and plants. Test inocula consisted of capped transcripts of wild-type RNAs 1 and 2, and of RNA 3 variants with defined mutations in the 3’ tRNA- like region. Mutant 5’PsK, which is defective in minus-strand promoter activity and a POOI substrate in vitro for both tyrosylation and 3’ adenylation, replicated in protoplasts to 20 y0 of wild-type even though only about 6% of the progeny molecules had correct 3’ termini that would permit tyrosylation. Mutant $GG: which is defective in vitro for 3’ adenylation and minus-strand promoter activities but accepts tyrosine at near-normal rates, replicated to 40% of wild-type in protoplasts although only 15% of the progeny molecules had correct 3’ termini. Two other mutants (A5 and 5’AGA), with 20-fold lower rates of tyrosylation in vitro than wild-type RNA, replicated to 60 to 70% of wild-type levels in protoplasts and gave similar yields to wild-type in systemic infections of plants. All mutant sequences were preserved in progeny RNAs, indicating that no recombination between homologous 3’ ends occurred. The 40% reduction of replication in protoplasts seen for mutant A$, whose only known functional lesion is depressed tyrosylation in vitro, may indicate that an indirect role for aminoacylation exists. However, the results obtained argue against an obligatory role for tyrosylation in RNA replication in vivo. 1. Introduction Several groups of RNA viruses infecting plants possess a tRNA-like domain at the 3’ end of their genomic RNAs (Hall, 1979; Haenni et al., 1982). In brome mosaic virus (BMVS), each of the four virion RNAs (RNA 1, 3.2 kb; RNA 2, 2.9 kb; RNA 3, 2.1 kb; RNA 4, 0.9 kb) contains this domain (Ahlquist et al., 1981a), which is responsible for the specific tyrosylation of the 3’.CCA,, terminus, catalyzed by host tyrosyl-tRNA synthetase (Kohl & Hall, 1974). In addition, viral RNAs lacking part of the 3’-terminal-CCA,, are substrates in vitro for the host enzyme (CTP,ATP) : tRNA nucleotidyl transferase, which is able to regenerate the normal -CGA,, from truncated 3’ termini of tRNA-like molecules (Deutscher, 1982). During replication in t Present address: Department of Agricultural Chemistry. Oregon State University, Corvallis, OR 97331-6502, U.S.A. $ Author for correspondence. 4 Abbreviation used: BMV, brome mosaic virus; kb, lo3 bases or base-pairs. TYMV, turnip yellow mosaic virus; TLC, thin-layer chromatography. barley cells, both of these host-supplied tRNA- associated enzymes are thought to be active on viral RNA. The tyrosylation of BMV RNAs during infection in barley protoplasts (Loesch-Fries & Hall, 1982) and valylation of turnip yellow mosaic virus (TYMV) RNAs in Chinese cabbage cells (Joshi et al., 1982) have been observed directly. In the latter case, where virion RNAs lack the 3’-terminal A residue, the activity of host nucleotidyl transferase is implicated in adenylating a free pool of TYMV RNAs prior to valylation (Yot et al., 1970). The roles of the tRNA-like activities of the viral RNA remain incompletely understood, but are thought to be intimately associated with the replication of the RNA (Dreher & Hall, 1988a). This is suggested by the fact that the same domain that functions as the core of the tRNA-like structure (Fig. 1) also acts as the promoter used by the BMV RNA-dependent RNA polymerase (replicase) during minus-strand initiation and synthesis (Miller et al., 1986). The interactions of BMV RNA with the two tRNA-associated enzyme activities and with the minus-strand promoter activity have been studied extensively in vitro, and a number of mutants OOZZ-2836/89/070425-14 $03.00/O 425 0 1989 Academic Press Limited