Functional analysis of the stem loop S3 and S4 structures in the coronavirus 3'UTR

Abstract

We designed a series of mutations to separately destabilize two helical stems (designated S3 and S4) predicted by a covariation-based model of the coronavirus 3'UTR (Zust et al., 2008). Mouse hepatitis virus genomes containing three or four nucleotide mutations that destabilize either S3 or S4 were viable, whereas genomes carrying these mutations in both S3 and S4 were not viable. A genome carrying these mutations in S3 and S4 plus compensatory mutations restoring base-pairing yielded a virus with wild type phenotype. Larger mutations which completely disrupt S3 or S4 generated various phenotypes. Mutations opening up S3 were lethal. Disruptions of S4 generated both viable and lethal mutants. Genomes carrying the original mutations in S3 or S4 plus compensatory mutations restoring base pairing were viable and had robust growth phenotypes. These results support the Zust model for the coronavirus 3'UTR and suggest that the S3 stem is required for virus viability.

Fig. 1

Proposed RNA secondary structures and mutations in the MHV 3′ UTR. (A) 3′ UTR model proposed by Zust et al. (2008), including the region downstream of S2 comprised of the HVR and the two helical stem structures (S3 and S4). (B) Mutations made to locally disrupt S3 and S4. The C and D mutations were made in this study and the A and B were previously made by Johnson et al. (2005). The wild type sequence is shown and the nucleotides that are altered by mutation italicized. Arrows indicate the sequence of each mutant. (C) Mutated nucleotides are indicated by italicized lower case letters.

Fig. 2

Growth phenotypes for viable mutant viruses and RNA phenotypes for lethal mutant. (A) Plaque size (mm) of viable viruses. (B) Growth curve of viable viruses. (C) RT-PCR assays for negative sense RNA corresponding to genomic RNA, (D) subgenomic RNA3, and (E) RNA6 for wild type MHV-A59 and lethal mutant CD. Lanes labeled Negative represent RNA extracted from cells that were mock electroporated with buffer rather than with in vitro transcribed RNA. The positions of the expected PCR products for each RNA species are indicated by arrows.

Fig. 3

Mutations causing complete disruption of S3 or S4 helices or the L3 loop. Mutated nucleotides are indicated by italicized lower case letters.

Fig. 4

Growth and RNA phenotypes of S3, S4, and L3 mutations. (A) Plaque size (mm) of S3 and S4 viable viruses. (B) Plaque size (mm) of L3 viable viruses. (C) Growth curve of S3, S4 and L3 viruses. (D) RT-PCR assay for negative sense RNA corresponding to genomic RNA for wild type MHV-A59 and lethal mutants G and H. (E) RT-PCR assays for negative sense RNA corresponding to subgenomic RNA6 for wild type MHV-A59 and lethal mutants G and H. Lanes labeled Negative represent RNA extracted from cells that were mock electroporated with buffer rather than with in vitro transcribed RNA. The positions of the expected PCR products are indicated by arrows.