Environment determines fidelity for an RNA virus replicase

Abstract

The rate of insertion and deletion mutations of the replicase of Cucumber mosaic virus (CMV) was determined in planta by using a parasitic satellite RNA (satRNA) as a reporter. We found that the CMV replicase had different fidelity in different environments, with important implications in viral disease evolution. Insertions were very rare events, irrespective of the region of the satRNA genome assayed and independent of the hosts tested. On the other hand, deletion events were more frequent but were restricted to a highly structured region of the reporter. Deletion mutation rates were different for the two hosts tested, although the mutation distribution was not influenced by the hosts. Moreover, hot spots with high mutation rates were identified on the satRNA genome.

FIG. 1.

The cDNA sequence of D4 satRNA plus-strand. Stop codons in the plus strand are shown in boldface, and stop codons in the minus strand are underlined. Primer sites for the nonstop A region are shown above the sequence line (AF, A region forward; AR, A region reverse). The forward primer contains two changes from the wild-type sequence to eliminate stop codons. The TGA in the reverse primer is in the same frame as the engineered stop codon and will be shifted out of frame in the event of an insertion or a deletion. Following the same strategy, primers BF (forward) and BR (reverse) were designed for the nonstop region B. The forward and reverse primers contain a PstI site and an EcoRI site (respectively) for directional cloning. The regions assayed include the sequences between (exclusive of) the primers.

FIG. 2.

D4 satRNA cDNA and Δ5′ deletion mutant. The putative plus-strand promoter was deleted to allow only half a round of replication. MCS, multiple cloning site.

FIG. 3.

Strand specificity. Lane a, positive control reaction using plus-strand-specific RT-PCR and the plus-strand transcript used in lane d; lane b, 1-kb ladder marker; lane c, minus-strand-specific RT-PCR on minus-strand transcript; lane d, minus-strand-specific RT-PCR on plus-strand transcript. The size of the targeted fragment is indicated with the black arrow.

FIG. 4.

Distribution of indels observed in planta and relationship to D4 satRNA secondary structure (revised from reference 18). The nonstop regions A and B are shown in red and blue, respectively. Deletions are indicated with blue circles or red circles (for hot spots). The nucleotide number is arbitrary when deletions occurred in runs of a single nucleotide. The box marks the nucleotides involved in the 4-base insertion event.

FIG. 5.

Estimates of deletion rates and 95% confidence interval for each experiment. Samples are labeled according to the following key: P, pepper plant; T, tobacco plant; C, control. Samples were from inoculated leaves (Inoc.) or systemic leaves (Sys.). A description of the statistical analysis is available on request.