Constraints of Viral RNA Synthesis on Codon Usage of Negative-Strand RNA Virus

Abstract

Negative-strand RNA viruses (NSVs) include some of the most pathogenic human viruses known. NSVs completely rely on the host cell for protein translation, but their codon usage bias is often different from that of the host. This discrepancy may have originated from the unique mechanism of NSV RNA synthesis in that the genomic RNA sequestered in the nucleocapsid serves as the template. The stability of the genomic RNA in the nucleocapsid appears to regulate its accessibility to the viral RNA polymerase, thus placing constraints on codon usage to balance viral RNA synthesis. By in situ analyses of vesicular stomatitis virus RNA synthesis, specific activities of viral RNA synthesis were correlated with the genomic RNA sequence. It was found that by simply altering the sequence and not the amino acid that it encoded, a significant reduction, up to an ∼750-fold reduction, in viral RNA transcripts occurred. Through subsequent sequence analysis and thermal shift assays, it was found that the purine/pyrimidine content modulates the overall stability of the polymerase complex, resulting in alteration of the activity of viral RNA synthesis. The codon usage is therefore constrained by the obligation of the NSV genome for viral RNA synthesis. IMPORTANCE Negative-strand RNA viruses (NSVs) include the most pathogenic viruses known. New methods to monitor their evolutionary trends are urgently needed for the development of antivirals and vaccines. The protein translation machinery of the host cell is currently recognized as a main genomic regulator of RNA virus evolution, which works especially well for positive-strand RNA viruses. However, this approach fails for NSVs because it does not consider the unique mechanism of their viral RNA synthesis. For NSVs, the viral RNA-dependent RNA polymerase (vRdRp) must gain access to the genome sequestered in the nucleocapsid. Our work suggests a paradigm shift that the interactions between the RNA genome and the nucleocapsid protein regulate the activity of vRdRp, which selects codon usage.

Nucleocapsid Structure of Negative Strand RNA Virus

“…To define which step in viral RNA synthesis is mostly affected by the genomic RNA sequence, the codon usage preference of VSV genome was mutated to that of the mammalian host [67]. The most profound impact is the reduction of viral transcription levels, consistent with previous observations.…”

“…Analyses by kappa index of coincidence (a number to indicate a degree of association not by random) revealed that clusters of purine or pyrimidine nucleotides are reciprocally related to the level of RNA transcription. By thermal shift assays of nucleocapsid stability, it was shown that a nucleocapsid-like particle (NLP) containing poly(rA) is more stable than that containing random RNA sequences [67]. This is consistent with the observation that a compound stabilizing the nucleocapsid also inhibits viral RNA synthesis by VSV polymerase [68].…”

From SARS and MERS CoVs to SARS‐CoV‐2: Moving toward more biased codon usage in viral structural and nonstructural genes

“…Codon usage bias is used in the analysis of genes composition and conclusion of the forces controlling evolution and functions 23,24 . It has been used in the analysis of viral structural 25,26 and nonstructural genes 25,27 .…”

Immune Pressure on Polymorphous Influenza B Populations Results in Diverse Hemagglutinin Escape Mutants and Lineage Switching

“…This indicates that the viruses emerged as much as a result of selection from the existing quasispecies pool as through mutation during replication. Recent analysis of negative-strand RNA virus genomes has found that their codon usage bias does not correlate well with the host codon bias because of constraints in viral RNA transcription [45]. It is possible that codon mutations, while limited by transcription and translation requirements, are also constrained by other features such as genomic RNA structures [46].…”