Tomato Bushy Stunt Virus Recombination Guided by Introduced MicroRNA Target Sequences

Previously we described Tomato bushy stunt virus (TBSV) vectors, which retained their capsid protein gene and were engineered with magnesium chelatase (ChlH) and phytoene desaturase (PDS) gene sequences from Nicotiana benthamiana. Upon plant infection, these vectors eventually lost the inserted sequences, presumably as a result of recombination. Here, we modified the same vectors to also contain the plant miR171 or miR159 target sequences immediately 3′ of the silencing inserts. We inoculated N. benthamiana plants and sequenced recombinant RNAs recovered from noninoculated upper leaves. We found that while some of the recombinant RNAs retained the microRNA (miRNA) target sites, most retained only the 3′ 10 and 13 nucleotides of the two original plant miRNA target sequences, indicating in planta miRNA-guided RNA-induced silencing complex cleavage of the recombinant TBSV RNAs. In addition, recovered RNAs also contained various fragments of the original sequence (ChlH and PDS) upstream of the miRNA cleavage site, suggesting that the 3′ portion of the miRNA-cleaved TBSV RNAs served as a template for negative-strand RNA synthesis by the TBSV RNA-dependent RNA polymerase (RdRp), followed by template switching by the RdRp and continued RNA synthesis resulting in loss of nonessential nucleotides.Several plant viruses have been developed as tools for various biotechnology applications, including expression platforms for protein production in plants (1, 2, 6) and as gene silencing systems as part of reverse genetics approaches toward understanding host plant gene function (4, 5). For both of these applications, nonviral sequences conferring the desired function are cloned into the virus genome in order to be expressed during replication in plants. One advantage of using viruses engineered with nonviral sequences is flexibility in manipulating these “extra” sequences, which are not essential for viral replication or movement (1). However, recombinant viruses also tend to lose these sequences, causing instability at the insertion site and resulting in loss of function of the recombinant viral vector. The relatively high error rates of viral replicases (7, 14, 24) and the propensity for recombination events (9) contribute to the instability often seen with some viral vector systems.Recombination events in RNA viruses typically result in joining of two noncontiguous RNA segments (16). These could be sequences from two separate RNA molecules or distant regions of the same molecule. Retention by viruses of favorable sequences is selection driven and eliminates sequences that are unnecessary or negatively affect fitness (11, 31), hence making recombination critical to virus evolution (13, 29). Although phylogenetic analyses predict that recombination events have affected evolution for essentially all groups of RNA viruses (3), some viruses appear to be more prone to recombination than others. For example, plant-infecting supergroup II viruses of the family Tombusviridae appear to undergo frequent recombination, as is supported by the many well-characterized defective-interfering (DI) RNAs of Tomato bushy stunt virus (TBSV) (10, 30). The TBSV DI RNAs, derived entirely from the parental viral RNA, are not replication competent alone and depend on the parent virus to replicate them in trans. Recent developments of in vitro systems (19, 21) have further enhanced dissection of recombination mechanisms giving rise to TBSV DI RNAs.Of the proposed mechanisms for viral recombination (12, 20), the copy choice or template-switching mechanism is the most widely reported (8, 16, 18). This occurs when the viral replicase and its attached nascent polynucleotide chain switches viral RNA templates (or jumps locations on the same template) when making cRNA. Some properties for preferred donor/acceptor sites (sequences in the RNA molecule at which viral replicase switches from one template to another) are known for various RNA viruses (3, 20, 27), suggesting that recombination is not entirely random.The previously described TBSV vectors were efficient silencers of host genes but only while the inserted sequences were retained (23). Thus, optimizing viral vectors requires a better understanding of factors responsible for recombination and consequent loss of insert sequences. In order to address possible recombination mechanisms, we used previously characterized sequence-specific microRNA (miRNA)-guided cleavage determinants as parts of our TBSV vectors. We introduced the miRNA target site sequences for miR171 or miR159 3′ to the silencing inserts of our TBSV vectors (23). After plant inoculation, we analyzed recombinant virus sequences, determining specific recombination patterns, and demonstrated miRNA-mediated recombination events in vivo for the recombinant TBSV vectors. We also showed miRNA-guided RNA-induced silencing complex (RISC)-mediated cleavage for recombinant TBSV RNA and evidence supporting the TBSV RNA-dependent RNA polymerase (RdRp) switching templates during cRNA synthesis.