Differential Alphavirus Defective RNA Diversity between Intracellular and Extracellular Compartments Is Driven by Subgenomic Recombination Events

ABSTRACT Alphaviruses are positive-sense RNA arboviruses that can cause either a chronic arthritis or a potentially lethal encephalitis. Like other RNA viruses, alphaviruses produce truncated, defective viral RNAs featuring large deletions during replication. These defective RNAs (D-RNAs) have primarily been isolated from virions after high-multiplicity-of-infection passaging. Here, we aimed to characterize both intracellular and packaged viral D-RNA populations during early-passage infections under the hypothesis that D-RNAs arise de novo intracellularly that may not be packaged and thus have remained undetected. To this end, we generated next-generation sequencing libraries using RNA derived from passage 1 (P1) stock chikungunya virus (CHIKV) 181/clone 25, intracellular virus, and P2 virions and analyzed samples for D-RNA expression, followed by diversity and differential expression analyses. We found that the diversity of D-RNA species is significantly higher for intracellular D-RNA populations than P2 virions and that specific populations of D-RNAs are differentially expressed between intracellular and extracellular compartments. Importantly, these trends were likewise observed in a murine model of CHIKV AF15561 infection, as well as in vitro studies using related Mayaro, Sindbis, and Aura viruses. Additionally, we identified a novel subtype of subgenomic D-RNA that is conserved across arthritogenic alphaviruses. D-RNAs specific to intracellular populations were defined by recombination events specifically in the subgenomic region, which were confirmed by direct RNA nanopore sequencing of intracellular CHIKV RNAs. Together, these studies show that only a portion of D-RNAs generated intracellularly are packaged and D-RNAs readily arise de novo in the absence of transmitted template. IMPORTANCE Our understanding of viral defective RNAs (D-RNAs), or truncated viral genomes, comes largely from passaging studies in tissue culture under artificial conditions and/or packaged viral RNAs. Here, we show that specific populations of alphavirus D-RNAs arise de novo and that they are not packaged into virions, thus imposing a transmission bottleneck and impeding their prior detection. This raises important questions about the roles of D-RNAs, both in nature and in tissue culture, during viral infection and whether their influence is constrained by packaging requirements. Further, during the course of these studies, we found a novel type of alphavirus D-RNA that is enriched intracellularly; dubbed subgenomic D-RNAs (sgD-RNAs), they are defined by deletion boundaries between the capsid-E3 region and the E1-3′ untranslated region (UTR) and are common to chikungunya, Mayaro, Sindbis, and Aura viruses. These sgD-RNAs are enriched intracellularly and do not appear to be selectively packaged, and additionally, they may exist as subgenome-derived transcripts.

Differential alphavirus defective RNA diversity between intracellular and encapsidated compartments is driven by subgenomic recombination events

ABSTRACTAlphaviruses are positive-sense RNA arboviruses that can cause either a chronic arthritis or a potentially lethal encephalitis. Like other RNA viruses, alphaviruses produce truncated, defective genomes featuring large deletions during replication. Defective RNAs (D-RNAs) have primarily been isolated from virions after high-multiplicity of infection passaging. Here, we aimed to characterize both intracellular and packaged viral D-RNA populations during early passage infections under the hypothesis that D-RNAs arise de novo intracellularly that may not be packaged and thus have remained undetected. To this end, we generated NGS libraries using RNA derived from passage 1 (P1) stock chikungunya virus (CHIKV) 181/clone 25, intracellular virus, and encapsidated P2 virus and analyzed samples for D-RNA expression, followed by diversity and differential expression analyses. We found that the diversity of D-RNA species is significantly higher for intracellular D-RNA populations than encapsidated and specific populations of D-RNAs are differentially expressed between intracellular and encapsidated compartments. Importantly, these trends were likewise observed in a murine model of CHIKV 15561 infection, as well as in vitro studies using related Mayaro, Sindbis, and Aura viruses. Additionally, we identified a novel subtype of subgenomic D-RNA that are conserved across arthritogenic alphaviruses. D-RNAs specific to intracellular populations were defined by recombination events specifically in the subgenomic region, which was confirmed by direct RNA nanopore sequencing of intracellular CHIKV RNAs. Together, these studies show that only a portion of D-RNAs generated intracellularly are packaged and D-RNAs readily arise de novo in the absence of transmitted template.IMPORTANCEOur understanding of viral defective RNAs (D-RNAs), or truncated viral genomes, comes largely from passaging studies in tissue culture under artificial conditions and/or packaged viral RNAs. Here, we show that specific populations of alphavirus D-RNAs arise de novo and that they are not packaged into virions, thus imposing a transmission bottleneck and impeding their prior detection. This raises important questions about the roles of D-RNAs, both in nature and in tissue culture, during viral infection and whether their influence is constrained by packaging requirements. Further, during the course of these studies, we found a novel type of alphavirus D-RNA that is enriched intracellularly; dubbed subgenomic D-RNAs (sgD-RNAs), they are defined by deletion boundaries between capsid/E3 and E1/3’UTR regions and are common to chikungunya, Mayaro, Sindbis, and Aura viruses. These sgD-RNAs are enriched intracellularly and do not appear to be selectively packaged, and additionally may exist as subgenome-derived transcripts.

Impact of Cultivated Hosts on the Recombination ofCucumber Mosaic Virus

ABSTRACTCucumber mosaic virus(CMV) is one of the most successful viruses known, infecting over 1,200 species of plants. Like other single-stranded RNA viruses, CMV is known to have a high potential for population diversity due to error-prone replication and short generation times. Recombination is also a mechanism that allows viruses to adapt to new hosts. Host genes have been identified that impact the recombination of RNA viruses by using single-cell yeast systems. To determine the impact that the natural plant host has on virus recombination, we used a high-recombination-frequency strain of CMV, LS-CMV, which belongs to subgroup II, in three different cultivated hosts:Capsicum annuumcv. Marengo (pepper),Nicotiana tabacumcv. Xanthi nc (tobacco), andCucurbita pepocv. Black Beauty (zucchini). The recombination frequency was calculated by using an RNA 3 reporter carrying restriction enzyme sites created by introducing silent mutations. Our results show that the recombination frequency of LS-CMV is correlated with the infected host. The recombination events in pepper were 1.8-fold higher than those in tobacco and 5-fold higher than those in zucchini. Furthermore, we observed the generation of defective RNAs in inoculated pepper plants, but not in tobacco or zucchini. These results indicate that the host is involved in both intra- and intermolecular recombination events and that hosts like pepper could foster more rapid evolution of the virus. In addition, we report for the first time the production of defective RNAs in a CMV subgroup II isolate.IMPORTANCERecombination is an important mechanism used by viruses for their diversification and to adapt to diverse hosts. Understanding the host role in the mechanisms of evolution is important for virus disease management and controlling the emergence of new strains. This study shows the impact that cultivated hosts are playing in the evolution of CMV. Furthermore, our results and previous studies show how some specific hosts could be an ideal environment for the emergence of new viral strains.

5′ and 3′ modifications controlling RNA degradation: from safeguards to executioners

RNA degradation is a key process in the regulation of gene expression. In all organisms, RNA degradation participates in controlling coding and non-coding RNA levels in response to developmental and environmental cues. RNA degradation is also crucial for the elimination of defective RNAs. Those defective RNAs are mostly produced by ‘mistakes’ made by the RNA processing machinery during the maturation of functional transcripts from their precursors. The constant control of RNA quality prevents potential deleterious effects caused by the accumulation of aberrant non-coding transcripts or by the translation of defective messenger RNAs (mRNAs). Prokaryotic and eukaryotic organisms are also under the constant threat of attacks from pathogens, mostly viruses, and one common line of defence involves the ribonucleolytic digestion of the invader's RNA. Finally, mutations in components involved in RNA degradation are associated with numerous diseases in humans, and this together with the multiplicity of its roles illustrates the biological importance of RNA degradation. RNA degradation is mostly viewed as a default pathway: any functional RNA (including a successful pathogenic RNA) must be protected from the scavenging RNA degradation machinery. Yet, this protection must be temporary, and it will be overcome at one point because the ultimate fate of any cellular RNA is to be eliminated. This special issue focuses on modifications deposited at the 5′ or the 3′ extremities of RNA, and how these modifications control RNA stability or degradation. This article is part of the theme issue ‘5′ and 3′ modifications controlling RNA degradation’.