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

Abstract

Deletions in cDNA clones covering the 3' 201 nucleotides of brome mosaic virus RNA 3 were produced by S1 nuclease treatment of cloned DNA linearized at several different restriction sites. Transcription of these clones yielded RNAs containing structural alterations in the 3'-terminal tRNA-like structure that is involved in aminoacylation and replication. Replicase template activity, but not aminoacylation activity, was especially sensitive to deletions in arm C, which contains a tyrosyl anticodon. Deletions in arm B were detrimental to aminoacylation, but the proportion of replicase template activity lost depended on the site of the deletion. Removal of arm D had little effect on aminoacylation and, in some instances, resulted in a 2-fold stimulation of replicase template activity.The genomic RNAs of many RNA plant viruses possess at their 3' ends a highly conserved region responsible for several tRNA-specific activities (1, 2). 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. Although such observations suggest a significant role in infection processes, no function for the tRNA-like properties of any of the plant viral RNAs has as yet been defined. The failure to detect any involvement of charged viral RNA in translational functions (2, 5, 6) has focused attention on a possible role in replication (1, 7).The strongest evidence to date supporting an interaction between the tRNA-like properties and replication comes from our recent discovery that the 3'-terminal 134-nucleotide region of BMV RNA resonsible for aminoacylation (8) also contains the entire information needed for specific recognition and for initiation of (-) strand synthesis by BMV replicase in vitro (ref. 9; unpublished data). To explore the dual role of this region in aminoacylation and replication, we

Nucleotide Sequences of the Coat Protein Genes and Flanking Regions of Cucumber Mosaic Virus Strains C and WL RNA 3

“…Some of them lie in the 3' 200 base pairs and may, therefore, be conserved in order to maintain the secondary and tertiary structure of this region (Hall, 1979;Ahlquist et al, 1981 ;Haenni et HI., 1982;Rietveld et al, 1983). This region has been shown to affect such functions as amino acid charging of viral RNA, and viral replication (Kohl & Hall, 1974;Hall, 1979;Haenni et al, 1982;Bujarski et al, 1985;Miller et al, 1986;Weiner & Maizels, 1987;French & Ahlquist, 1987). On the other hand, conserved sequences outside this structure may also have some as yet undetermined functional importance.…”

tRNA-Like Structure Regulates Translation of Brome Mosaic Virus RNA

“…In the current three-dimensional model of the TLS BMV , hairpin D is positioned such that it may mimic the T⌿C hairpin of canonical tRNAs, and indeed the base of this hairpin structure makes strong contacts with the TyrRS enzyme. However, no functional role for this hairpin has been reported thus far (11,12), and it is absent from the related Tyr-accepting TLS BBMV of broad bean mottle virus (6).…”

“…Apparently, just as for the TLS TYMV , a 3Ј-terminal structure upstream of the final 3Ј acceptor arm is crucial for translation. This also implies that the complete TLS BMV may represent a compromise for efficient functioning in both translation and replication (6), whereas the folding of the shortened fragment may be more favorable for translation. For the distantly related Alfalfa mosaic virus (AMV), an Alfamovirus also belonging to the family Bromoviridae, a structural switch of the 3Ј UTR from a hairpin array into a sort of TLS regulates the functional transition from translation to replication (24,27).…”

Artificial Defective Interfering RNAs Derived from Brome Mosaic Virus

“…To determine whether the interference characteristics of M4 (Bujarski et al, 1985(Bujarski et al, , 1986, one of these mutations which exhibits approximately twice the replicase template activity of the wt sequence in vitro, but only about 50~o of that activity in vivo, were additive or synergistic to the pRNA effect, it was transferred to pRNA-2 M/S (see Methods) to yield M4 pRNA-2 M/S. The double mutant, M4 pRNA-2 M/S, was found to be deficient relative to the parental pRNA in vivo in both its replication and interference properties (Fig.…”

“…Deletions were confirmed by restriction enzyme analysis and sequencing. The 3' M4 deletion (Bujarski et al, 1985) was transferred to pT7B2 AMluI/StuI utilizing the HindlII site located 200 nucleotides (nt) from the 3" terminus of genomic RNA-2 and -3 (Ahlquist et al, 1984), and a 3' polylinker BamHI site, as described by Dreher et al (1989). A small deletion in the region of the translation initiation codon (nt 104) was made by linearizing pT7B2 at the BstBI site (nt 109) and Bal 31 nuclease digestion for 10 s. Recircularization produced construct pT7B2 A9@BstBI, from which RNA-2 A9 was transcribed.…”