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  1. Wang, Aiming (Ed.)

    Viruses are constantly subject to natural selection to enrich beneficial mutations and weed out deleterious ones. However, it remains unresolved as to how the phenotypic gains or losses brought about by these mutations cause the viral genomes carrying the very mutations to become more or less numerous. 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. Nevertheless, it is unclear if similarly stringent reproductive 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 a TYLCV genome to produce two replicons that express green fluorescent protein and mCherry, respectively, in a replication-dependent manner. We found that among the cells entered by both replicons, less than 65% replicated both, whereas at least 35% replicated either of them alone. Further probability computation concluded that replication in an average cell was unlikely to have been initiated with more than three replicon genome copies. Furthermore, sequential inoculations unveiled strong mutual exclusions of these two replicons at the intracellular level. In conclusion, the intracellular population of the small DNA virus TYLCV is actively bottlenecked, and such bottlenecking may be a virus-encoded, evolutionarily conserved trait that assures timely selection of new mutations emerging through error-prone replication.

     
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  2. Geminiviruses possess single-stranded, circular DNA genomes and control the transcription of their late genes, including BV1 of many bipartite begomoviruses, through transcriptional activation by the early expressing AC2 protein. DNA binding by AC2 is not sequence-specific; hence, the specificity of AC2 activation is thought to be conferred by plant transcription factors (TFs) recruited by AC2 in infected cells. However, the exact TFs AC2 recruits are not known for most viruses. Here, we report a systematic examination of the BV1 promoter (PBV1) of the mungbean yellow mosaic virus (MYMV) for conserved promoter motifs. We found that MYMV PBV1 contains three abscisic acid (ABA)-responsive elements (ABREs) within its first 70 nucleotides. Deleting these ABREs, or mutating them all via site-directed mutagenesis, abolished the capacity of PBV1 to respond to AC2-mediated transcriptional activation. Furthermore, ABRE and other related ABA-responsive elements were prevalent in more than a dozen Old World begomoviruses we inspected. Together, these findings suggest that ABA-responsive TFs may be recruited by AC2 to BV1 promoters of these viruses to confer specificity to AC2 activation. These observations are expected to guide the search for the actual TF(s), furthering our understanding of the mechanisms of AC2 action. 
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  3. RNA secondary structures play diverse roles in positive-sense (+) RNA virus infections, but those located with the replication protein coding sequence can be difficult to investigate. Structures that regulate the translation of replication proteins pose particular challenges, as their potential involvement in post-translational steps cannot be easily discerned independent of their roles in regulating translation. In the current study, we attempted to overcome these difficulties by providing viral replication proteins in trans. Specifically, we modified the plant-infecting turnip crinkle virus (TCV) into variants that are unable to translate one (p88) or both (p28 and p88) replication proteins, and complemented their replication with the corresponding replication protein(s) produced from separate, non-replicating constructs. This approach permitted us to re-examine the p28/p88 coding region for potential RNA elements needed for TCV replication. We found that, while more than a third of the p88 coding sequence could be deleted without substantially affecting viral RNA levels, two relatively small regions, known as RSE and IRE, were essential for robust accumulation of TCV genomic RNA, but not subgenomic RNAs. In particular, the RSE element, found previously to be required for regulating the translational read-through of p28 stop codon to produce p88, contained sub-elements needed for efficient replication of the TCV genome. Application of this new approach in other viruses could reveal novel RNA secondary structures vital for viral multiplication. 
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  4. Simon, Anne E. (Ed.)
    ABSTRACT Long noncoding RNAs (lncRNAs) of virus origin accumulate in cells infected by many positive-strand (+) RNA viruses to bolster viral infectivity. Their biogenesis mostly utilizes exoribonucleases of host cells that degrade viral genomic or subgenomic RNAs in the 5′-to-3′ direction until being stalled by well-defined RNA structures. Here, we report a viral lncRNA that is produced by a novel replication-dependent mechanism. This lncRNA corresponds to the last 283 nucleotides of the turnip crinkle virus (TCV) genome and hence is designated tiny TCV subgenomic RNA (ttsgR). ttsgR accumulated to high levels in TCV-infected Nicotiana benthamiana cells when the TCV-encoded RNA-dependent RNA polymerase (RdRp), also known as p88, was overexpressed. Both (+) and (−) strand forms of ttsgR were produced in a manner dependent on the RdRp functionality. Strikingly, templates as short as ttsgR itself were sufficient to program ttsgR amplification, as long as the TCV-encoded replication proteins p28 and p88 were provided in trans . Consistent with its replicational origin, ttsgR accumulation required a 5′ terminal carmovirus consensus sequence (CCS), a sequence motif shared by genomic and subgenomic RNAs of many viruses phylogenetically related to TCV. More importantly, introducing a new CCS motif elsewhere in the TCV genome was alone sufficient to cause the emergence of another lncRNA. Finally, abolishing ttsgR by mutating its 5′ CCS gave rise to a TCV mutant that failed to compete with wild-type TCV in Arabidopsis . Collectively, our results unveil a replication-dependent mechanism for the biogenesis of viral lncRNAs, thus suggesting that multiple mechanisms, individually or in combination, may be responsible for viral lncRNA production. IMPORTANCE Many positive-strand (+) RNA viruses produce long noncoding RNAs (lncRNAs) during the process of cellular infections and mobilize these lncRNAs to counteract antiviral defenses, as well as coordinate the translation of viral proteins. Most viral lncRNAs arise from 5′-to-3′ degradation of longer viral RNAs being stalled at stable secondary structures. Here, we report a viral lncRNA that is produced by the replication machinery of turnip crinkle virus (TCV). This lncRNA, designated ttsgR, shares the terminal characteristics with TCV genomic and subgenomic RNAs and overaccumulates in the presence of moderately overexpressed TCV RNA-dependent RNA polymerase (RdRp). Furthermore, templates that are of similar sizes as ttsgR are readily replicated by TCV replication proteins (p28 and RdRp) provided from nonviral sources. In summary, this study establishes an approach for uncovering low abundance viral lncRNAs, and characterizes a replicating TCV lncRNA. Similar investigations on human-pathogenic (+) RNA viruses could yield novel therapeutic targets. 
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  5. Abstract Many positive-sense RNA viruses, especially those infecting plants, are known to experience stringent, stochastic population bottlenecks inside the cells they invade, but exactly how and why these populations become bottlenecked are unclear. A model proposed ten years ago advocates that such bottlenecks are evolutionarily favored because they cause the isolation of individual viral variants in separate cells. Such isolation in turn allows the viral variants to manifest the phenotypic differences they encode. Recently published observations lend mechanistic support to this model and prompt us to refine the model with novel molecular details. The refined model, designated Bottleneck, Isolate, Amplify, Select (BIAS), postulates that these viruses impose population bottlenecks on themselves by encoding bottleneck-enforcing proteins (BNEPs) that function in a concentration-dependent manner. In cells simultaneously invaded by numerous virions of the same virus, BNEPs reach the bottleneck-ready concentration sufficiently early to arrest nearly all internalized viral genomes. As a result, very few (as few as one) viral genomes stochastically escape to initiate reproduction. Repetition of this process in successively infected cells isolates viral genomes with different mutations in separate cells. This isolation prevents mutant viruses encoding defective viral proteins from hitchhiking on sister genome-encoded products, leading to the swift purging of such mutants. Importantly, genome isolation also ensures viral genomes harboring beneficial mutations accrue the cognate benefit exclusively to themselves, leading to the fixation of such beneficial mutations. Further interrogation of the BIAS hypothesis promises to deepen our understanding of virus evolution and inspire new solutions to virus disease mitigation. 
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