N-Acetyl-tyrosine at the 3' End of Brome Mosaic Virus RNA has Little Effect on Infectivity

Abstract

SUMMARYDigestion of the N-acetylated-tyrosylated form of brome mosaic virus (BMV) RNA with proteinase K removed the T-blocking N-acetyl-tyrosine residue. Removal of the blocking group affected neither infectivity nor mRNA activity. The previously reported effect of acetylation of tyrosylated BMV RNA on infectivity appears to result from non-specific acetylation of the RNA.Brome mosaic virus (BMV) RNA is one of several plant viral RNAs capable of accepting an amino acid at its 3' terminus in a tRNA-like manner (for review, see Hall, 1979). In the case of bromoviruses, the reaction is specific for tyrosine (Kohl & Hall, 1974). Aminoacylation does not affect viral RNA translation (Chen& Hall, 1973), nor has it been shown to function in RNA replication or virion assembly. However, the demonstrations that BMV RNA is tyrosylated in barley protoplasts (Loesch-Fries & Hall, 1982) and that turnip yellow mosaic virus RNA is valylated in Xenopus laevis oocytes (Joshi et al., 1978) suggest that aminoacylation occurs during viral infection.Previously (Kohl & Hall, 1977), we sought to establish a function for aminoacylation by measuring the biological activity ofBMV RNA whose tyrosylation capacity had been destroyed by chemical modification. In that study, the 3' terminus of the RNA was blocked by acetylating tyrosylated RNA (tyr RNA). Since the hydrolysis rate of N-acetylated tyrosine (N-acetyl-tyr) from RNA is much slower than that of tyrosine, BMV RNA modified in this way was incapable of supporting de novo tyrosylation. The messenger function of the viral RNA appeared to be unaffected by the modification, but a significant loss of infectivity was observed. We now describe a set of experiments that involve the removal in vitro of N-acetyl-tyr from the 3' terminus of the viral genome, and the subsequent assay of the biological activity of the repaired BMV RNA.BMV RNA was tyrosylated in vitro (Kohl & Hall, 1977), using a wheat germ synthetase preparation enriched in tyrosyl-tRNA synthetase (Faulhammer & Cramer, 1977) that routinely yielded viral RNA which was 70 to 100% charged. The conditions for synthesis of the acetylating agent (the N-hydroxysuccinimide ester of acetic acid) and for acetylation of BMV tyr RNA were as described previously (Kohl & Hall, 1977). The extent of acetylation was estimated by comparing the rates of hydrolysis of N-acetyl[3H]tyr and [3H]tyr from BMV RNA incubated in 0.1 M-Tris-HCI pH 8.5 for 30 min at 37 °C. By this criterion, the acetylation reaction, was 65 to 80% complete.Although significant amounts of N-acetyl-tyr could be removed from BMV RNA by alkaline hydrolysis, this approach was unsatisfactory, because the conditions (0.25M-Tris-HC1 pH 9-0, 30 °C for 30 min) degraded the RNA (P. A. Kiberstis, unpublished observations). To test whether aminoacylated BMV RNA would serve as a substrate for proteinase K, which can specifically hydrolyse N-blocked amino acids from tRNAs (Vidales et al., 1979), we used the i Present address:

cDNA Cloning of Plant RNA Viruses and Viroids

“…In infected plants an additional RNA is abundantly present, RNA 4, which is a subgenomic mRNA of RNA3 and codes for the capsid protein. The viral RNAs all have a cap structure at their 5'-end and possess a tRNA-like structure at their 3'-end, including a CCA-terminus which can be aminoacetylated with tyrosine (Kiberstis and Hall, 1983;Loesch-Fries and Hall, 1982) (see Fig. 3).…”

“…Note that the central three nucleotides in the nonameric recognition sequence of the restriction enzyme Tth 1111 are not important for cleavage, allowing site-directed mutagenesis at these three nucleotides without affecting restriction enzyme digestion role in initiation of ( -) strand synthesis and amino acylation (Ahlquist et aI., 1984 b). To investigate sequences involved in ( -) strand synthesis and aminoacylation, mutant viral RNAs were synthesised in vitro and offered as templates to either extracts of BMV-infected barley leaves (Miller and Hall, 1983) or partially purified preparation of aminoacetyl-tRNA synthetase from wheat germ (Kiberstis and Hall, 1983).…”

Deletions in the 3'-terminal tRNA-like structure of brome mosaic virus RNA differentially affect aminoacylation and replication in vitro.

“…The tRNA-like structure formed by this region represents an integral part of the three genomic and single subgenomic RNAs of brome mosaic virus (BMV) and is capable of being stoichiometrically aminoacylated in vitro with tyrosine (3). Tyrosylation has also been observed in vivo during infection of barley protoplasts with BMV (4), indicating that the viral RNAs are able to compete with tRNAs for the cognate synthetases.…”

“…The production of substantial amounts of such modified RNAs was made possible by the development of a system for transcribing correctly terminated, biologically active 3'-terminal fragments of BMV RNA in vitro from cDNA (10). Transcripts may be readily assayed in vitro for template activity with the template-specific, template-dependent BMV replicase (11,12) and for aminoacylation with wheat germ tyrosyl-tRNA synthetase (3,13).…”