Deletion of Internal Sequences Results in Tobacco Mosaic Virus Defective RNAs That Accumulate to High Levels without Interfering with Replication of the Helper Virus

AbstractDeletion of certain internal sequences of the tobacco mosaic tobamovirus (TMV) genome was required to create replication-defective RNAs (dRNA) that were replicatedin transby TMV. All dRNAs that accumulated to detectable levels were missing nucleotides 3420–4902, which appeared to constitute a core region that inhibited replicationin trans.Deletion of additional sequences resulted in dRNAs that varied tremendously in ability to be replicated from none to levels exceeding that of the helper viral RNA. Accumulation of dRNA negative- and positive-stranded RNAs of each dRNA paralleled those of the helper virus. Negative-stranded RNA accumulation of both helper and dRNA ceased at the same early time in the infection while synthesis of both positive-stranded RNAs continued, suggesting that both dRNAs and helper virus RNAs were synthesized from the same pool of replicase complexes. Positive- to negative-stranded RNA ratios for the dRNAs were similar to, or slightly greater than the wild-type helper virus. Full-length dRNAs were not supportedin transby a replication-competent helper virus. Even though some of the artificially constructed dRNAs accumulated to levels exceeding the level of the helper virus, none appreciably affected the replication of the helper virus, suggesting that the dRNAs are produced from “excess” replicase capacity