Abstract
During the replication cycle of double-stranded (ds) RNA viruses, the viral RNA-dependent RNA polymerase (RdRP) replicates and transcribes the viral genome from within the viral capsid. How the RdRP molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions with answers that most likely vary across the different dsRNA virus families. In this study, we have determined a 2.4 Å resolution structure of an RdRP from the human picobirnavirus (hPBV). In addition to the conserved polymerase fold, the hPBV RdRP possesses a highly flexible 24 amino acid loop structure located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays and site-directed mutagenesis showed that: (1) the hPBV RdRP is fully active using both ssRNA and dsRNA templates; (2) the insertion loop likely functions as an assembly platform for the priming nucleotide to allow de novo initiation; (3) RNA transcription by the hPBV RdRP proceeds in a semi-conservative manner; and (4) the preference of virus-specific RNA during transcription is dictated by the lower melting temperature associated with the terminal sequences. Co-expression of the hPBV RdRP and the capsid protein (CP) indicated that, under the conditions used, the RdRP could not be incorporated into the recombinant capsids in the absence of the viral genome. Additionally, the hPBV RdRP exhibited higher affinity towards the conserved 5’-terminal sequence of the viral RNA, suggesting that the RdRP molecules may be encapsidated through their specific binding to the viral RNAs during assembly.
Highlights
Double-stranded RNA viruses are a diverse group of viruses that vary widely in host range, genome segment number, and in the number of capsid layers, with many of them considered important pathogens of either agriculture or human health
Given our terminal nucleotidyl transferase (TNTase) activity data and the fact that the insertion loop is located near the C-terminus of the human picobirnavirus (hPBV) RNA-dependent RNA polymerase (RdRP), we propose that the removal of the insertion loop structure leaves the dsRNA exit channel of the protein permanently open (Fig 2B), allowing the 3’-end of the RNA molecules to reach the active site for nucleotidyl addition in an orientation that is compatible for nucleotide addition
Isotope incorporation from (γ-32P) GTP into the dsRNA product was only detected for reactions containing the WT RdRP, further indicating that this protein utilizes the de novo initiation mechanism (Fig 4C). These results show that the insertion loop structure of the hPBV RdRP can effectively block template back-priming and facilitates initiation via a primer-independent mechanism, possibly by providing a docking site for the 3’-end of the RNA template and a binding site for the priming nucleotide
Summary
Double-stranded (ds) RNA viruses are a diverse group of viruses that vary widely in host range (humans, animals, plants, fungi, and bacteria), genome segment number (one to twelve), and in the number of capsid layers, with many of them considered important pathogens of either agriculture or human health. During the viral replication cycle, dsRNA viruses have been shown to encapsidate up to twelve RNA-dependent RNA polymerase (RdRP) molecules in each virus particle [2,3,4,5] To date, several different mechanisms of incorporating the RdRP molecules into the capsid have been identified Those that possess multi-layered capsids, such as the bacteriophage φ6, rotavirus, and reovirus, as well as the single-layered capsids of cypoviruses have been shown to attach their polymerase molecules to the inner surface of the capsid through direct protein-protein interactions [6,7,8,9,10], suggesting that non-covalent protein-protein interaction plays an important role in RdRP incorporation. It has been proposed that the dependence of polymerase activity on the presence of capsid proteins may help to ensure that dsRNA products are preferentially produced only within a capsid enclosure [17]
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