Repair of the tRNA-like CCA sequence in a multipartite positive-strand RNA virus

Abstract

The 3' portions of plus-strand brome mosaic virus (BMV) RNAs mimic cellular tRNAs. Nucleotide substitutions or deletions in the 3'CCA of the tRNA-like sequence (TLS) affect minus-strand initiation unless repaired. We observed that 2-nucleotide deletions involving the CCA 3' sequence in one or all BMV RNAs still allowed RNA accumulation in barley protoplasts at significant levels. Alterations of CCA to GGA in only BMV RNA3 also allowed RNA accumulation at wild-type levels. However, substitutions in all three BMV RNAs severely reduced RNA accumulation, demonstrating that substitutions have different repair requirements than do small deletions. Furthermore, wild-type BMV RNA1 was required for the repair and replication of RNAs with nucleotide substitutions. Results from sequencing of progeny viral RNA from mutant input RNAs demonstrated that RNA1 did not contribute its sequence to the mutant RNAs. Instead, the repaired ends were heterogeneous, with one-third having a restored CCA and others having sequences with the only commonality being the restoration of one cytidylate. The role of BMV RNA1 in increased repair was examined.

FIG. 1.

One or more BMV RNAs are required for the repair of nucleotide substitutions but not for small deletions. (A) Sequence of the 3′ end of all BMV positive-strand RNAs and the mutations that affect the CCA site. The horizontal bars denote the nucleotide deleted, with the names of the deletion written above the bar. The box identifies the CCA sequence, which was changed to GGA when nucleotide substitutions were studied. (B) Effects of mutations in one or more BMV RNAs. The gel images were all taken from one Northern blot experiment by using the Molecular Dynamics program Data Storm. The sense of the RNAs is denoted to the left, and the time at which the protoplasts were harvested is denoted in parentheses (12 h in this experiment). The identities of individual RNAs are listed to the left and right of the images. The barley protoplasts were transfected with a mixture of three RNAs, either wild type (WT) (denoted by a plus sign) or with a mutation. The two slices of gel near the bottom are the 18S rRNA stained with ethidium bromide. In general, all of the gel images in this work are arranged in the same format. (C) Effect of short deletions in one or more BMV RNA repairs and replications. In this experiment, all three of the BMV RNAs had the deletion from the 3′ end. The names of the mutants indicate the number of nucleotides deleted. Protoplasts were transfected and then harvested at 0, 6, and 12 h posttransfection, as labeled below the gel image. The identities of the RNA bands are indicated to the side of the gel images.

FIG. 2.

R1 is required to increase the repair of base substitutions in R2 and R3. (A) Gel image of a Northern blot containing different combinations of wild-type and mutant RNAs, as denoted above the gel image. The polarity of the RNAs is shown to the left of the image, and the identity of each RNA is indicated to the right. Similar results were observed for the plus-strand RNAs (data not shown). (B) A time course experiment to confirm that R1 is required for increased repair of R2 and R3 with base substitutions at their 3′ ends and that R3 is a preferred substrate for repair in comparison to R2. The times, in hours, at which the transfected protoplasts were harvested are stated at the bottom of the gel image.

FIG. 3.

Sequencing analysis of repaired RNAs in the presence of wild-type R1. (A) Gel images that demonstrate that R1 with a marked fourth position from the 3′ end (a change from A to U) is capable of replication as well as increased repair and that similarly marked R2GGA and R3GGA are substrates for the repair process. (B) Protocol used to sequence RT-PCR fragments directly or to clone and sequence individual sequences of the 3′ ends of R2GGA and/or R3GGA. RNAs that contained a CCA site that did not require repair were sequenced directly from PCR products, while the products of the repair were heterogeneous and required cloning into pGEM-TEasy. (C) Examples of the sequencing results from the RT-PCR product from R1^m (left-hand graph) and from RT-PCR of R2 (right-hand graph). These results demonstrate that there is predominantly only one sequence in each of the two reactions. (D) Compilation of the DNA sequences from three independent experiments where different RNA combinations were transfected into barley protoplasts. The input RNAs are listed to the left. The column headed cDNA (form) identifies the origin of the RNAs used to generate the cDNA and whether they were sequenced after PCR or after cloning into pGEM-TEasy. The sequences observed and the number of independent reactions in which a sequence was observed is in the last column. Wherever the −4 position was marked to distinguish an RNA from the others, the marked nucleotide is underlined. The retention of the mutated residues is denoted with a boldface G. The number of sequences that originated from a mutant RNA is in boldface. The asterisks identify sequences that were restored to CCA and could be due to the activity of the cellular tRNA nucleotidyltransferase. The frequency of this repair to CCA is summarized at the bottom of the table.

FIG. 4.

Increased repair depends on R1 concentration and replication competence. (A) Effects of increasing amounts of R1 on the accumulation of different BMV RNAs. The RNAs transfected consisted of the amount of R1 indicated in the horizontal axis and 0.5 μg each of R2GGA and R3GGA. The amount of each class of RNA synthesized is represented in the vertical axis. (B) Mutations that affect minus-strand R1 synthesis prevented increased repair. The RNA stem-loop shown is the SLC within the tRNA-like region of all BMV plus-strand RNAs. The circled A is the clamped adenine that is required for minus-strand RNA initiation. A change to a G that would eliminate minus-strand RNA synthesis is identified by the name SLC^m. To the right of the schematic is the gel image from a Northern blot demonstrating that SLC^m in R1 eliminated both BMV replication and the repair of nucleotide substitutions. Wild-type RNAs are identified by a plus sign, while nucleotide substitutions in the CCA site of each RNA are identified by GGA.

FIG. 5.

Effects of a chimeric RNA on BMV replication and increased repair. (A) A schematic of chimeric RNA R2/1/2 used in this experiment. (B) Demonstration that R2/1/2 is capable of directing RNA replication and transcription. The levels of the RNAs were examined over a 2-day period to ensure that there was not a defect in RNA levels associated with R2/1/2 that is more obvious over time. (C) Examination of the ability of R2/1/2 to increase repair of R2GGA and R3GGA. The gel image is from a Northern blot of RNA harvested at 12 h posttransfection. The mixtures of the RNAs used in each transfection are identified above the gel image. (D) R2/1/2 and a replication-incompetent R1 can lead to the repair of R2GGA. Whether R2/1/2 or a wild-type RNA is present (+) or absent (−) is indicated. The presence of mutant versions of RNA is denoted by names instead of plus signs.

FIG. 6.

Effects of mutations in the 1a coding sequence on increased repair. (A) Effect of replication-defective mutant PK6 on repair of R1GGA and R2GGA in the presence or absence of R2/1/2. A plus sign denotes the presence of RNA in the column. Specific names, such as PK6 and GGA, indicate that the RNA has a mutation. (B) Effects of replication-defective mutant PK17 on increased repair in the presence or absence of R2/1/2. The arrangement of the gel is the same as that for panel A. The identities of the RNAs are indicated to the sides of the gel images.