Biology subjects

Perdoncini Carvalho, C.

Publications and source records attributed to Perdoncini Carvalho, C..

2 recordsLinked to original sources

bioRxiv2025

Naturally occurring mutations in replication proteins of a small RNA virus that alter the number, sizes, and relative abundances of subgenomic RNAs

Many positive-strand (+) RNA viruses produce subgenomic RNAs (sgRNAs) in infected cells. sgRNAs are synthesized by virus-encoded replication proteins (RPs), but whether RPs regulate the number and sizes of sgRNAs remains largely unknown. We report multiple naturally occurring mutations within the RPs of turnip crinkle virus (TCV) that alter the number, sizes, and relative abundances of TCV sgRNAs. TCV is a (+) RNA virus that normally produces two sgRNAs: the 1,724-nucleotide (nt) sgRNA1 expressing movement proteins, and the 1,449-nt sgRNA2 expressing capsid protein. A single amino acid change, A113V, within a region shared by TCV RPs p28 and p88, diminished sgRNA1 levels and delayed viral systemic spread. Interestingly, three second-site RP mutations emerged in infected plants that, alone or in combination with A113V, resulted in over-production of sgRNA1 or accumulation of two alternative sgRNAs of 1,876 and 1,601 nt, and rescued A113V defects. The alternative sgRNAs originated from nearly identical recombination events, their size difference reflecting varying 5 extensions. Structural modeling of the TCV replication complex showed a conical ring architecture containing p28, p88, and a partially double-stranded RNA. While A113 may interact with TCV genomic RNA or host factors to promote (-) sgRNA1 synthesis, the second-site mutations likely influenced binding and entry of RNA template into the p88 active site. They may stall RNA synthesis at specific hotspots and stimulate template switching, thereby generating alternative (-) sgRNAs. Our findings reveal previously unrecognized constraints on viral RPs that ensure production of sgRNAs with precise sizes and abundances. Author summaryMany (+)-strand viruses, such as SARS-CoV-1 and -2, Chikungunya virus, and tomato mosaic virus, synthesize subgenomic RNAs (sgRNAs) during cellular infections. Targeting sgRNA production could prove to be an effective antiviral strategy, as sgRNAs are needed to express diverse proteins critical for viral survival and transmission. Although sgRNA production requires virus-encoded replication proteins (RPs), whether RPs also dictate the number, sizes, and relative abundances of viral sgRNAs remain to be thoroughly investigated. We identified and characterized four naturally occurring mutations in RPs of the plant-infecting turnip crinkle virus that specifically perturbed one of the sgRNAs. Our findings uncover novel constraints on viral RPs that safeguard sgRNA integrity, and avail them as potential targets for controlling pathogenic viruses.

plant biology↗

bioRxiv2022

A small DNA virus initiates replication with no more than three genome copies per cell

Cellular organisms purge lethal mutations as they occur (in haploids), or as soon as they become homozygous (in sexually reproducing diploids), thus making the mutation-carrying genomes the sole victims of lethality. How lethal mutations in viruses are purged remains an unresolved question because numerous viral genomes could potentially replicate in the same cell, sharing their encoded proteins, hence shielding lethal mutations from selection. Previous investigations by us and others suggest that viruses with plus strand (+) RNA genomes may compel such selection by bottlenecking the replicating genome copies in each cell to low single digits. However, it is unclear if similar bottlenecks also occur in cells invaded by DNA viruses. Here we investigated whether tomato yellow leaf curl virus (TYLCV), a small virus with a single-stranded DNA genome, underwent population bottlenecking in cells of its host plants. We engineered the TYLCV genome to produce two replicons that express green fluorescent protein and mCherry, respectively, in a replication-dependent manner. We found that less than 65% of cells penetrated by both replicons replicated both, whereas at least 35% of cells replicated either of them alone, illustrating an intracellular population bottleneck size of no more than three. Furthermore, sequential inoculations unveiled strong mutual exclusions of these two replicons in most cells. Collectively our data demonstrated for the first time that DNA viruses like TYLCV are subject to stringent intracellular population bottlenecks, suggesting that such population bottlenecks may be a virus-encoded, evolutionarily conserved trait that assures timely elimination of lethal mutations. Significance statementAn important unresolved issue in virus life cycles is how natural selection acts on individual virus copies in the same cells. Unlike cellular organisms in which genome copies with lethal or advantageous mutations usually share their hosts with no more than one homologous genome copy, viruses could potentially reproduce with numerous sister genomes per cell, permitting sharing of protein products, thereby greatly diminishing phenotypic impacts of otherwise eventful mutations. Previous investigations suggest that (+) RNA viruses solve this problem by bottlenecking the number of replicating genome copies to low single digits. The current study reveals strikingly similar intracellular population bottlenecks for a DNA virus. Further mechanistic interrogations could avail the virus-encoded bottleneck-enforcing apparatus as targets for antiviral therapy and prevention.

microbiology↗