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.

Keywords: nucleocapsid; sequestered genome; viral polymerase.

FIG 1

(A) Ribbon representation of three nucleocapsid protein subunits constructed from the structure reported under PDB accession number 3PTX (45). Shown in red is the N-terminal arm, which interacts with adjacent subunits. Shown in blue and magenta are the N lobe and the C lobe, respectively. Shown in yellow is the C-terminal loop, which also interacts with adjacent subunits. Furthermore, the backbone of the RNA is shown as a tan line sandwiched between the N and C lobes. (B) Cartoon representation of a tight or loose interaction of the genomic RNA in the nucleocapsid. This would regulate the accessibility of the sequestered RNA to vRdRp and cause possible disassociation or stalling of vRdRp. (C) Stick representation of nine nucleotides (adenosine) encapsidated in the nucleocapsid. Shown in red are the first 4 nucleotides that have bases stacked with each other. Shown in blue are nucleotides 5, 7, and 8 that also have bases stacked. Nucleotide 6 is shown in yellow and does not directly interact with other nucleotides.

FIG 2

(A) Vector for the minigenome based on the N gene of VSV. Under the control of the T7 promoter, the vector expresses the vRNA copy of the N gene, flanked by the leader and trailer sequences. (B) Highlighted sequence of the N gene showing the location of the high-frequency codons, in green, or the low-frequency codons, in red. Furthermore, highlighted in cyan is the placement of 3 stop codons to prevent translation of the mRNA transcripts. Also shown, in magenta, is the PCR tag utilized in qPCR to identify mRNA transcripts transcribed only from the minigenome. (C, top) Relative fold decreases in RNA levels from all the minigenomes quantitated by in situ vRdRp activity assays. N stands for the wt genome, H stands for the high-usage genome, and L stands for the low-usage genome. Notice the log₂ axis for ease of viewing the fold differences on similar scales. (Bottom) Fold increases in levels of shortened RNAs using primers that correspond to the beginning of each viral RNA moiety, as ratios to the full-length RNA moieties. Each experiment was carried out in triplicate, and the error bars represent the standard deviations between the experiments. (D) Construction of the chimeric minigenomes. Each minigenome was constructed based on the golden ratio, with the darker color representing the wt genome and gray representing the high- or low-usage genome. Each chimeric genome is shown in panel B as NH1 or NL1, etc. All sequences are presented in the supplemental material.

FIG 3

Calculated A+G% versus KIC. Each panel is a different window size ranging from 9 to 90 nucleotides as multiples of 9 due to the number of nucleotides in each N subunit. The wt genome is plotted in gray, the high-usage genome is plotted in red, and the low-usage genome is plotted in blue. The centers of mass are plotted in their respective color for each panel, and all three centers of mass are plotted on the wt plot. The KIC is a measurement of the probability of a repeating sequence within a set window. It is plotted against A+G% here for the purposes of investigating the purine/pyrimidine distribution throughout the wt, high-codon-usage, and low-codon-usage sequences. Altering these base-stacking patterns changes the distribution of the plot and corresponds to the decrease in vRdRp activity.

FIG 4

Thermal shift assays. A thermal shift assay was carried out on the randomly incorporated RNA in recombinantly expressed NLP, poly(rA), and poly(rU) in reconstituted NLPs. SYBR Safe, which monitors RNA thermo-release from the NLP, is shown in gray, while SYPRO orange, which monitors protein denaturation, is shown in black. Each trace is representative of data from a separate experiment. The top panels indicate the raw melting data. The middle panels are the first derivative graphs with the corresponding average T_m (from the SYPRO orange trace) and standard deviation calculated from the peak of each trace. The bottom panels show the second derivative of the SYBR Safe trace and the average T_free and standard deviation calculated from the peak of each trace, which is representative of the thermal release of RNA.