Helicase and capping enzyme active site mutations in brome mosaic virus protein 1a cause defects in template recruitment, negative-strand RNA synthesis, and viral RNA capping

Abstract

Brome mosaic virus (BMV) encodes two RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and 2a, which is related to polymerases. BMV 1a and 2a can direct virus-specific RNA replication in the yeast Saccharomyces cerevisiae, which reproduces the known features of BMV replication in plant cells. We constructed single amino acid point mutations at the predicted capping and helicase active sites of 1a and analyzed their effects on BMV RNA3 replication in yeast. The helicase mutants showed no function in any assays used: they were strongly defective in template recruitment for RNA replication, as measured by 1a-induced stabilization of RNA3, and they synthesized no detectable negative-strand or subgenomic RNA. Capping domain mutants divided into two groups. The first exhibited increased template recruitment but nevertheless allowed only low levels of negative-strand and subgenomic mRNA synthesis. The second was strongly defective in template recruitment, made very low levels of negative strands, and made no detectable subgenomes. To distinguish between RNA synthesis and capping defects, we deleted chromosomal gene XRN1, encoding the major exonuclease that degrades uncapped mRNAs. XRN1 deletion suppressed the second but not the first group of capping mutants, allowing synthesis and accumulation of large amounts of uncapped subgenomic mRNAs, thus providing direct evidence for the importance of the viral RNA capping function. The helicase and capping enzyme mutants showed no complementation. Instead, at high levels of expression, a helicase mutant dominantly interfered with the function of the wild-type protein. These results are discussed in relation to the interconnected functions required for different steps of positive-strand RNA virus replication.

FIG. 1

(A) Schematic of BMV 1a protein. The capping domain, the seven conserved helicase motifs, and the proline-rich linker sequence (PPP) separating the capping enzyme and helicase-like domains are indicated. The subregion of the capping enzyme domain showing similarity with methyltransferases (5) is shaded. The mutations studied in this work are marked by arrows at the top. (B) Enzymatic activity of BMV 1a mutant L52P. Extracts of yeast transformants expressing wt 1a or mutant L52P were assayed in the presence of S-adenosylmethionine and [α-³²P]GTP for covalent binding of methylated guanylate (3) (left). The reaction mixtures were analyzed by SDS-PAGE and autoradiography to visualize covalently radiolabeled proteins. Positions of molecular weight markers, in kilodaltons, are shown on the left. The arrow indicates the position of full-length 1a. The smaller labeled proteins are proteolytic fragments of 1a, commonly observed in these preparations (3). The same extracts were assayed for guanine-7-methyltransferase activity (3) (right). The bars show averages and standard deviations of an experiment performed in quadruplicate. (C) Schematic of the replication of BMV RNA3 derivatives in yeast. RNA3(+), originally derived from DNA template by RNA polymerase II-mediated transcription and ribozyme cleavage, gives rise to complementary negative strands, which can be used as templates for production of progeny positive strands or for transcription of subgenomic RNA4. The open reading frame (marked X) within RNA4 can be translated only after the steps previously described. In wt RNA3, the open reading frame encodes BMV coat protein, but it can be replaced by reporter genes, such as URA3 or GUS. Methylated cap structures at the 5′ end, tRNA-like structures at the 3′ end, and the intergenic RE are indicated. (D) RNA3 replication-dependent growth of yeast expressing wt BMV 1a or its derivatives. Yeast strain YMI04, which has an inactivating mutation in its chromosomal URA3 gene but contains BMV RNA3 derivatives B3URA3 and B3GUS integrated in the chromosome, was transformed with plasmids expressing BMV 2a and BMV 1a or its mutated derivatives or with a plasmid lacking the 1a open reading frame (−1a) as indicated. Individual transformants were streaked on a plate containing galactose as a carbon source (to induce RNA3 derivative expression), lacking histidine and leucine (to select for 1a and 2a plasmids), and lacking uracil. These yeast cells show sustained growth on this medium only if they are capable of BMV RNA replication, RNA4 transcription, and translation of the reporter gene URA3 from RNA4.

FIG. 2

RNA3 replication and negative-strand synthesis in yeast expressing BMV 1a or its mutated derivatives together with 2a. (A) Total RNA and protein were isolated from yeast expressing RNA3, 2a, and the 1a derivatives indicated at the top. Aliquots were analyzed by Western blotting for 1a expression (uppermost panel) and by Northern blotting using specific RNA probes to detect RNA3 positive strands or negative strands as indicated on the left. (B) Total RNA was isolated from yeast expressing an RNA3 derivative with substitution of GAL1 leader sequence for the wt 5′ noncoding sequence, together with 2a and the indicated 1a derivatives. Aliquots were analyzed by Northern blotting to detect RNA3 negative strands.

FIG. 3

RNA3 accumulation in yeast expressing BMV 1a or its mutated derivatives in the absence of 2a. Total RNA was isolated from yeast expressing RNA3 and the 1a derivatives indicated at the top. Aliquots were analyzed by Northern blotting to detect RNA3 positive strands.

FIG. 4

RNA3 replication in wt yeast and Δxrn1 yeast expressing BMV 1a or its mutated derivatives together with 2a. Total RNA was isolated, and aliquots were analyzed by Northern blotting to detect RNA3 positive or negative strands as indicated on the left.

FIG. 5

Primer extension analysis of the 5′ ends of BMV subgenomic RNA4 species. In vitro-transcribed RNA4, virion RNA, or total RNA from yeast (wt or Δxrn1 strain) containing the indicated 1a derivatives together with wt 2a was annealed with an oligonucleotide complementary to RNA4 bases 64 to 83. The primer was extended as described in Materials and Methods. Nucleotide position relative to the wt RNA4 cDNA sequence is shown on the left, and nucleotide position relative to the RNA4 derivative produced in yeast cells is shown on the right. The RNA4 produced in yeast in these experiments is four nucleotides longer than wt RNA4, due to an engineered insertion disrupting the coat protein open reading frame (see Materials and Methods).

FIG. 6

Effects of combining mutations within BMV 1a on RNA3 replication. Total RNA was isolated from yeast expressing RNA3 and the 1a derivatives indicated at the top. BMV 2a was either present (the two lower panels) or absent (uppermost panel), as indicated. Aliquots were analyzed by Northern blotting to detect RNA3 positive or negative strands, as indicated on the left.

FIG. 7

RNA3 replication in yeast expressing two BMV 1a derivatives together with 2a. Total RNA was isolated from yeast, and aliquots were analyzed by Northern blotting to detect RNA3 negative or positive strands as indicated on the left. (A) 1a derivatives were expressed from the ADH1 promoter. Above each lane, the derivative expressed from a HIS3 marker-containing plasmid is given first, followed by the derivative expressed from a URA3 marker-containing plasmid. “Empty” indicates that no 1a open reading frame was present on the plasmid. (B) The same 1a derivatives as in panel A were expressed from the GAL1 promoter.