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. 2001 Apr;75(7):3207-19.
doi: 10.1128/JVI.75.7.3207-3219.2001.

Brome mosaic virus Protein 1a recruits viral RNA2 to RNA replication through a 5' proximal RNA2 signal

J Chen  1 , A Noueiry, P Ahlquist
Affiliations

Brome mosaic virus Protein 1a recruits viral RNA2 to RNA replication through a 5' proximal RNA2 signal

J Chen et al. J Virol. 2001 Apr.

Abstract

Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication factors. Membrane-associated 1a protein contains a helicase-like domain and RNA capping functions. 2a, which is targeted to membranes by 1a, contains a central polymerase-like domain. In the absence of 2a and RNA replication, 1a acts through an intergenic replication signal in BMV genomic RNA3 to stabilize RNA3 and induce RNA3 to associate with cellular membrane. Multiple results imply that 1a-induced RNA3 stabilization reflects interactions involved in recruiting RNA3 templates into replication. To determine if 1a had similar effects on another BMV RNA replication template, we constructed a plasmid expressing BMV genomic RNA2 in vivo. In vivo-expressed RNA2 templates were replicated upon expression of 1a and 2a. In the absence of 2a, 1a stabilized RNA2 and induced RNA2 to associate with membrane. Deletion analysis demonstrated that 1a-induced membrane association of RNA2 was mediated by sequences in the 5'-proximal third of RNA2. The RNA2 5' untranslated region was sufficient to confer 1a-induced membrane association on a nonviral RNA. However, sequences in the N-terminal region of the 2a open reading frame enhanced 1a responsiveness of RNA2 and a chimeric RNA. A 5'-terminal RNA2 stem-loop important for RNA2 replication was essential for 1a-induced membrane association of RNA2 and, like the 1a-responsive RNA3 intergenic region, contained a required box B motif corresponding to the TPsiC stem-loop of host tRNAs. The level of 1a-induced membrane association of various RNA2 mutants correlated well with their abilities to serve as replication templates. These results support and expand the conclusion that 1a-induced BMV RNA stabilization and membrane association reflect early, 1a-mediated steps in viral RNA replication.

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Figures

FIG. 1
FIG. 1
Pathway for initiating BMV RNA2 replication from DNA plasmid pB2. The bracket at the top indicates the RNA2 cDNA copy within pB2, with the 2a ORF boxed. The yeast GAL1 promoter fused to 5′ end of RNA2 cDNA allows galactose-inducible, glucose-repressible transcription of RNA2, and the hepatitis delta virus ribozyme cleaves the transcripts at the natural 3′ end of RNA2 as indicated. RNA2 transcripts serve as templates for translation of 2a protein and for synthesis of the negative-strand RNA2 replication intermediate.
FIG. 2
FIG. 2
RNA2 replication in yeast. (A) Negative-strand RNA2 accumulation was assayed by a two-cycle RNase protection assay (38) using equal amounts of total RNA extracted from yeast expressing no BMV components (lane 1) or yeast expressing wt RNA2 (B2) or an RNA2 mutant with a deletion of the conserved GDD polymerase motif (B2ΔGDD), in the presence (+1a) or absence (−1a) of 1a as indicated. After initial hybridization and RNase treatment to remove excess positive-strand RNA2, the remaining double-stranded RNA was denatured, hybridized with a 32P-labeled RNA probe corresponding to nt 1441 to 1685 of positive-strand RNA2, and treated with RNases A and T1. The reaction products were electrophoresed and autoradiographed. Parallel strand-specific Northern blot analysis produced similar negative-strand RNA2 accumulation results, but with higher background signals apparently due to cross-hybridization. Neg. ctrl., negative control. (B) Positive-strand RNA2 accumulation in yeast expressing RNA2 alone, with 1a, with RNA3, or with both, as indicated. Total RNA was isolated from yeast and analyzed by Northern blotting with a probe specific for positive-strand RNA2. (C) Primer extension analysis of 5′ ends of RNA2 species in yeast expressing wt RNA2 with or without 1a. A 5′ 32P-labeled primer complementary to nt 47 to 73 of RNA2 was annealed with BMV virion RNA (vRNA) or total RNA from the indicated yeast. The primer was extended with reverse transcriptase, and the resulting cDNA products were analyzed in a 6% polyacrylamide sequencing gel. A sequencing ladder prepared by extending the same labeled primer on pB2 plasmid DNA was coelectrophoresed, and sequence corresponding to the sense of the RNA product is shown at right. As previously demonstrated, the major primer extension bands from BMV positive-strand RNA replication products migrate one nucleotide above the end of the viral sequence due to cap-dependent incorporation of an additional nucleotide (2, 6).
FIG. 3
FIG. 3
1a-induced stabilization and membrane association of wt RNA2 and RNA2 frameshift mutants. (A) On the left are diagrams of expression cassettes for wt RNA2, B2fs1, and B2fs2. In B2fs1, a 2-nt insertion after nt 110 results in translation of 3 wt 2a codons followed by 18 out-of-frame codons. In B2fs2, a 4-nt insertion after nt 882 results in translation of 261 wt 2a codons followed by 3 out-of-frame codons. The stabilities of these RNAs in the absence (−1a) and presence (+1a) of 1a were analyzed by transferring galactose-induced yeast to glucose to repress GALI-promoted RNA2 transcription. Equal amounts of total RNA prepared from yeast harvested at the indicated times following glucose repression were analyzed by Northern blotting to detect positive-strand RNA2 (middle). The results of three or more independent stability analyses of each RNA were averaged and plotted on a logarithmic scale (right). Standard error bars are included on all points but in some cases are obscured by the symbols used to plot the average values. (B) Effects of 1a on distribution of wt RNA2, B2fs1, and B2fs2 in cell fractionation. Yeast cells expressing these RNAs with or without 1a were spheroplasted and lysed osmotically to yield a total RNA fraction (Tot.). A portion of the lysate was then centrifuged at 10,000 × g to yield pellet (Pell.) and supernatant (Sup.) fractions. RNA was isolated from each fraction by phenol-chloroform extraction, and equal percentages of each fraction were analyzed by Northern blotting to detect positive-strand RNA2. For each RNA, the accumulation in each fraction was normalized to that in the total fraction for that RNA in the absence of 1a. Averages and standard errors from three or more independent experiments were plotted.
FIG. 4
FIG. 4
RNA2 nt 1 to 882 contain sequences required for 1a-induced membrane association. (A) Expression cassettes for wt RNA2 and RNA2 deletion derivatives are diagrammed at the left. Membrane association of these RNAs with or without 1a was assessed by cell fractionation as described in the legend to Fig. 3, and representative Northern blots are shown at the right. Sup., supernatant. (B) As a quantitative measure of 1a responsiveness, the ratio of RNA accumulation in the membrane-associated pellet fraction in the presence and absence of 1a was calculated for each RNA. Averages and standard errors from three or more independent experiments are shown.
FIG. 5
FIG. 5
Deletion analysis of 5′ proximal RNA2 sequences required for 1a responsiveness. (A) Expression cassettes for wt RNA2 and the indicated RNA2 deletion derivatives are diagrammed at the left. Membrane association abilities of these RNAs in the absence (−) or presence (+) of 1a were assessed by cell fractionation as described in the legend to Fig. 3, and representative Northern blots are shown at the right. Sup., supernatant. (B) As a measure of 1a responsiveness, the ratio of RNA accumulation in the membrane-associated pellet fraction in the presence and absence of 1a was calculated for each RNA. Averages and standard errors from three or more independent experiments are shown.
FIG. 6
FIG. 6
RNA2 5′ stem-loop is required for 1a-induced membrane association. (A) Alignment of BMV, cowpea chlorotic mottle virus (CCMV), and cucumber mosaic virus (CMV) RNA1 and RNA2 5′-terminal sequences, showing conservation of the box B motif and the potential for base pairing of the surrounding stem regions. (B) Predicted structure of the 5′-terminal stem-loop of BMV wt RNA2 and deletion or base substitution mutants within the stem-loop. Base substitutions are indicated by boxes, and deletions are indicated with dashes. Membrane association abilities of these RNA2 mutants in the absence (−) or presence (+) of 1a were assessed by cell fractionation as described in the legend to Fig. 3, and representative Northern blots are shown below each derivative. Sup., supernatant. (C) As a measure of 1a responsiveness, the ratio of RNA accumulation in the membrane-associated pellet fraction in the presence and absence of 1a was calculated for each RNA. Averages and standard errors from three or more independent experiments are shown.
FIG. 7
FIG. 7
RNA2 5′ UTR and flanking 2a ORF sequences confer 1a responsiveness on a nonviral RNA. (A) Expression cassettes for chimeric RNAs containing the indicated RNA2 sequences, β-globin ORF, and the ADH1 3′ UTR and polyadenylation site are diagrammed at the left. Base substitutions within the box B motif are indicated with boxes. Membrane association abilities of these RNAs in the absence (−) or presence (+) of 1a were assessed by cell fractionation as described in the legend to Fig. 3, and representative Northern blots are shown at the right. Sup., supernatant. (B) As a measure of 1a responsiveness, the ratio of RNA accumulation in the membrane-associated pellet fraction in the presence and absence of 1a was calculated for each RNA. Averages and standard errors from three or more independent experiments are shown.
FIG. 8
FIG. 8
RNA2 sequences required for 1a-induced membrane association are also required in cis for RNA2 replication. (A) RNA2 derivatives unable to produce functional 2a are efficiently replicated by 2a provided in trans from pB2YT1, which expresses 2a from a nonreplicatable 2a mRNA with 5′ and 3′ UTR sequences replaced by nonviral sequences. Wt RNA2, B2fs2 (Fig. 3A), and B2ΔGDD (Fig. 2A) were expressed in yeast also expressing 1a or both 1a and 2a in trans. Total RNA was isolated from these yeast cells, and negative-strand RNA2 accumulation was assessed by RNase protection as described in the legend to Fig. 2A. Averages and standard errors from three or more independent experiments are shown. (B) Comparison of 2a protein accumulation in yeast expressing wt RNA2 from pB2 or expressing chimeric 2a mRNA from 2a expression plasmid pB2YT1. Equal amounts of total protein extract from each cell type were analyzed by Western blotting with anti-2a antibodies. (C) Negative-strand RNA2 accumulation in yeast expressing 1a, 2a, and the indicated RNA2 derivatives, determined by RNase protection as described in the legend to Fig. 2A. Averages and standard errors from three or more independent experiments are shown. (D) Positive-strand RNA2 accumulation in the same yeast as panel C, determined by Northern blotting with a single-stranded, 32P-labeled RNA probe complementary to the conserved 3′ 200 bases of positive-strand BMV RNAs, which hybridizes to wt RNA2 but not the 2a mRNA from pB2YT1. The arrowhead indicates the front edge of the yeast 25S rRNA band. The high concentration of RNA in this band tends to sweep background signals ahead of the rRNA, creating the observed discontinuity in the background.
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References

    1. Ahlquist P. Bromovirus RNA replication and transcription. Curr Opin Genet Dev. 1992;2:71–76. - PubMed
    1. Ahlquist P, Janda M. cDNA cloning and in vitro transcription of the complete brome mosaic virus genome. Mol Cell Biol. 1984;4:2876–2882. - PMC - PubMed
    1. Ahola T, Ahlquist P. Putative RNA capping activities encoded by brome mosaic virus: methylation and covalent binding of guanylate by replicase protein 1a. J Virol. 1999;73:10061–10069. - PMC - PubMed
    1. Ahola T, den Boon J, Ahlquist P. 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. J Virol. 2000;74:8803–8811. - PMC - PubMed
    1. Allison R, Thompson C, Ahlquist P. Regeneration of a functional RNA virus genome by recombination between deletion mutants and requirement for cowpea chlorotic mottle virus 3a and coat genes for systemic infection. Proc Natl Acad Sci USA. 1990;87:1820–1824. - PMC - PubMed

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