Differential alphavirus defective rna diversity between intracellular and extracellular compartments is driven by subgenomic recombination events

R. M. Langsjoen, A. E. Muruato, S. R. Kunkel, E. Jaworski, A. Routh

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


Alphaviruses are positive-sense RNA arboviruses that can cause either a chronic arthritis or a potentially lethal encephalitis. Like other RNA viruses, alphavi-ruses produce truncated, defective viral RNAs featuring large deletions during repli-cation. 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 chikun-gunya virus (CHIKV) 181/clone 25, intracellular virus, and P2 virions and analyzed samples for D-RNA expression, followed by diversity and differential expression anal-yses. 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. Impor-tantly, these trends were likewise observed in a murine model of CHIKV AF15561 in-fection, 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. To-gether, 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 tem-plate. 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 al-phavirus D-RNAs arise de novo and that they are not packaged into virions, thus im-posing 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 re-quirements. Further, during the course of these studies, we found a novel type of al-phavirus 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, Sind-bis, 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.

Original languageEnglish (US)
Article numbere00731-20
Pages (from-to)1-20
Number of pages20
Issue number4
StatePublished - Jul 1 2020


  • Alphavirus
  • Chikungunya
  • Defective RNA
  • Viral recombination

ASJC Scopus subject areas

  • Microbiology
  • Virology


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