Carvalho, C. P. | Search bioRxiv

bioRxiv2025

Rescue of tomato yellow leaf curl virus mutants with heterologous iterons through in planta evolution

The single-stranded, circular DNA genomes of geminiviruses contain iterated motifs of 5-6 nucleotides, known as iterons, upstream of the replication protein (Rep) coding region. Iterons were previously found to interact with cognate Rep in a sequence-specific manner, and the iteron-Rep interaction was needed for viral DNA replication. Nonetheless, iterons of closely related viruses often have different sequences, suggesting diversifying selection. To identify selection pressures driving iteron diversification, we constructed tomato yellow leaf curl virus (TYLCV, isolate SH2) mutants in which the iteron motifs were replaced with those of closely related tobacco curly shoot virus (TbCSV, isolate Y35). All mutants replicated in inoculated leaves of Nicotiana benthamiana, but many failed to spread systemically. However, the systemic movement defects were mostly rescued by de novo mutations. Intriguingly, these de novo mutations did not restore the iterons to SH2 sequences. Rather, they likely enabled viral escape from repression exerted by the heterogenous Y35 iterons absent of a matching Rep. These results are consistent with iterons acting as sites of competitive binding by host-encoded transcription factors (TFs) and the cognate Rep. The iteron-TF binding commences as soon as viral genomes enter cell nuclei, committing genome copies to Rep mRNA transcription and protein translation; but also blocking them from replication. Conversely, iteron-Rep binding is possible only after Rep is produced, and likely repels TFs from some genome copies, permitting replication initiation. Testing this model through future research should clarify the intricate evolutionary interplays between geminiviruses and their crop hosts, and inform novel management strategies. Author SummaryGeminiviruses are important crop pathogens worldwide for which effective control measures are lacking, due to incomplete understanding of their evolutionary dynamics in infected plants. The current study focuses on a class of short sequence repeats in geminiviral genomic DNA, known as iterons, sitting immediately upstream of the viral gene encoding replication protein (Rep). Iterons are interesting because even though their positions and repeat patterns are conserved across all geminiviruses, their sequence identities are highly diverse. Our investigations revealed that contrary to previous reports, the sequence identity of iterons is non-essential for tomato yellow leaf curl virus (TYLCV) to replicate. Rather, they are repressors of replication, and this repression is overcome by their binding with cognate Rep. Our findings led to a new model postulating that the genome section encompassing iterons likely evolved specific sequence motifs to entice host-encoded transcription factors (TFs), facilitating rapid Rep production. Conversely, Rep promotes viral replication by removing TFs from genome copies through competitive iteron binding. Future testing of this new model will likely unveil novel targets for more effective management of crop diseases caused by geminiviruses.

evolutionary biology↗

bioRxiv2023

Single-cell mutation rate of turnip crinkle virus (-)-strand replication intermediates

Viruses with single-stranded, positive-sense (+) RNA genomes incur high numbers of errors during replication, thereby creating diversified genome populations from which new, better adapted viral variants can emerge. However, a definitive error rate is known for a relatively few (+) RNA plant viruses, due to challenges to account for perturbations caused by natural selection and/or experimental set-ups. To address these challenges, we developed a new approach that exclusively profiled errors in the (-)-strand replication intermediates of turnip crinkle virus (TCV), in singly infected cells. A series of controls and safeguards were devised to ensure errors inherent to the experimental process were accounted for. This approach permitted the estimation of a TCV error rate of 8.47 X 10-5 substitution per nucleotide site per cell infection. Importantly, the characteristic error distribution pattern among the 50 copies of 2,363-base-pair cDNA fragments predicted that nearly all TCV (-) strands were products of one replication cycle per cell. Furthermore, some of the errors probably elevated error frequencies by lowering the fidelity of TCV RNA-dependent RNA polymerase, and/or permitting occasional re-replication of progeny genomes. In summary, by profiling errors in TCV (-)-strand intermediates incurred during replication in single cells, this study provided strong support for a stamping machine mode of replication employed by a (+) RNA virus. Author SummaryMost (+) RNA viruses introduce replication errors at relatively high frequencies. As a result, it is of vital importance for these viruses to purge lethal errors in a timely manner. TCV, a plant-infecting small (+) RNA virus, was proposed to encode a Bottleneck, Isolate, Amplify, Select (BIAS) mechanism that compel swift clearance of lethal errors by bottlenecking the number of replicating genome copies to one per cell. A crucial prediction of this BIAS model is that such bottlenecking also acts on progeny genome copies, preventing them from repeating replication in the cells of their own genesis. The current study tested this prediction by developing a carefully controlled, readily reproducible approach to profile errors and error distributions in (-)-stranded replication intermediates of TCV. We found that most of replication-generated (-) strands descended from the primary (+) strands through a single replication cycle. This finding adds fresh support to the BIAS model.

evolutionary biology↗