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.
Alphaviruses are positive-sense RNA arboviruses that can cause either a chronic arthritis or a 24 potentially lethal encephalitis. Like other RNA viruses, alphaviruses produce truncated, defective 25 genomes featuring large deletions during replication. Defective RNAs (D-RNAs) have primarily been 26 isolated from virions after high-multiplicity of infection passaging. Here, we aimed to characterize both 27 intracellular and packaged viral D-RNA populations during early passage infections under the 28 hypothesis that D-RNAs arise de novo intracellularly that may not be packaged and thus have remained 29 undetected. To this end, we generated NGS libraries using RNA derived from passage 1 (P1) stock 30 chikungunya virus (CHIKV) 181/clone 25, intracellular virus, and encapsidated P2 virus and analyzed 31 samples for D-RNA expression, followed by diversity and differential expression analyses. We found 32 that the diversity of D-RNA species is significantly higher for intracellular D-RNA populations than 33 encapsidated and specific populations of D-RNAs are differentially expressed between intracellular and 34 encapsidated compartments. Importantly, these trends were likewise observed in a murine model of 35 CHIKV 15561 infection, as well as in vitro studies using related Mayaro, Sindbis, and Aura viruses. 36Additionally, we identified a novel subtype of subgenomic D-RNA that are conserved across 37 arthritogenic alphaviruses. D-RNAs specific to intracellular populations were defined by recombination 38 events specifically in the subgenomic region, which was confirmed by direct RNA nanopore sequencing 39 of intracellular CHIKV RNAs. Together, these studies show that only a portion of D-RNAs generated 40 intracellularly are packaged and D-RNAs readily arise de novo in the absence of transmitted template. 41 3 IMPORTANCE 42 Our understanding of viral defective RNAs (D-RNAs), or truncated viral genomes, comes largely from 43 passaging studies in tissue culture under artificial conditions and/or packaged viral RNAs. Here, we 44 show that specific populations of alphavirus D-RNAs arise de novo and that they are not packaged into 45 virions, thus imposing a transmission bottleneck and impeding their prior detection. This raises 46 important questions about the roles of D-RNAs, both in nature and in tissue culture, during viral infection 47 and whether their influence is constrained by packaging requirements. Further, during the course of 48 these studies, we found a novel type of alphavirus D-RNA that is enriched intracellularly; dubbed 49 subgenomic D-RNAs (sgD-RNAs), they are defined by deletion boundaries between capsid/E3 and 50 E1/3'UTR regions and are common to chikungunya, Mayaro, Sindbis, and Aura viruses. These sgD-51 RNAs are enriched intracellularly and do not appear to be selectively packaged, and additionally may 52 exist as subgenome-derived transcripts. 53 Although D-RNAs have been considered an epi-phenomenon of cell-culturing practices, emerging deep 79 sequencing technologies have enabled researchers t...
The relationship between certain chromosomal aberration (CA) types and cell lethality is well established. On that basis we used multi-fluor in situ hybridization (mFISH) to tally the number of mitotic human lymphocytes exposed to graded doses of gamma rays that carried either lethal or nonlethal CA types. Despite the fact that a number of nonlethal complex exchanges were observed, the cells containing them were seldom deemed viable, due to coincident lethal chromosome damage. We considered two model variants for describing the dose responses. The first assumes independent linear-quadratic (LQ) dose response shapes for the yields of both lethal and nonlethal CAs. The second (simplified) variant assumes that the mean number of nonlethal CAs per cell is proportional to the mean number of lethal CAs per cell, meaning that the shapes and magnitudes of both aberration types differ only by a multiplicative proportionality constant. Using these models allowed us to assemble dose response curves for the frequency of aberration-bearing cells that would be expected to survive. This took the form of a joint probability distribution for cells containing ≥1 nonlethal CAs but having zero lethal CAs. The simplified second model variant turned out to be marginally better supported than the first, and the joint probability distribution based on this model yielded a crescent-shaped dose response reminiscent of those observed for mutagenesis and transformation for cells “at risk” (i.e. not corrected for survival). Among the implications of these findings is the suggestion that similarly shaped curves form the basis for deriving metrics associated with radiation risk models.
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