Mutagenesis of Coronavirus nsp14 Reveals Its Potential Role in Modulation of the Innate Immune Response

Abstract

Coronavirus (CoV) nonstructural protein 14 (nsp14) is a 60-kDa protein encoded by the replicase gene that is part of the replication-transcription complex. It is a bifunctional enzyme bearing 3'-to-5' exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) activities. ExoN hydrolyzes single-stranded RNAs and double-stranded RNAs (dsRNAs) and is part of a proofreading system responsible for the high fidelity of CoV replication. nsp14 N7-MTase activity is required for viral mRNA cap synthesis and prevents the recognition of viral mRNAs as "non-self" by the host cell. In this work, a set of point mutants affecting different motifs within the ExoN domain of nsp14 was generated, using transmissible gastroenteritis virus as a model of Alphacoronavirus Mutants lacking ExoN activity were nonviable despite being competent in both viral RNA and protein synthesis. A specific mutation within zinc finger 1 (ZF-C) led to production of a viable virus with growth and viral RNA synthesis kinetics similar to that of the parental virus. Mutant recombinant transmissible gastroenteritis virus (TGEV) ZF-C (rTGEV-ZF-C) caused decreased cytopathic effect and apoptosis compared with the wild-type virus and reduced levels of dsRNA accumulation at late times postinfection. Consequently, the mutant triggered a reduced antiviral response, which was confirmed by evaluating different stages of the dsRNA-induced antiviral pathway. The expression of beta interferon (IFN-β), tumor necrosis factor (TNF), and interferon-stimulated genes in cells infected with mutant rTGEV-ZF-C was reduced compared to the levels seen with the parental virus. Overall, our data revealed a potential role for CoV nsp14 in modulation of the innate immune response.

Importance: The innate immune response is the first line of antiviral defense that culminates in the synthesis of interferon and proinflammatory cytokines to control viral replication. CoVs have evolved several mechanisms to counteract the innate immune response at different levels, but the role of CoV-encoded ribonucleases in preventing activation of the dsRNA-induced antiviral response has not been described to date. The introduction of a mutation in zinc finger 1 of the ExoN domain of nsp14 led to production of a virus that induced a weak antiviral response, most likely due to the accumulation of lower levels of dsRNA in the late phases of infection. These observations allowed us to propose a novel role for CoV nsp14 ExoN activity in counteracting the antiviral response, which could serve as a novel target for the design of antiviral strategies.

FIG 1

RNA synthesis of TGEV nsp14 mutants. (A) Schematic representation of TGEV nsp14. Exonuclease (ExoN) and N7 methyltransferase (N7-MTase) domains are indicated. Motifs I, II, and III conferring the ExoN active site are shown (gray boxes). The three zinc fingers (ZF1, ZF2, and ZF3) are indicated (black boxes), as are the amino acids forming the S-adenosylmethionine (SAM) binding pocket in the N7-MTase domain (white box). Within the partial sequence alignments, positions of key amino acids in each motif (white letters in black boxes) are indicated. Those mutated in each rTGEV construct with respect to the wild-type (WT) sequence are shown as black letters in gray boxes. (B) Quantification of replication and transcription levels in each rTGEV mutant replicon. Nonreplicative (NR) and wild-type (WT) replicons were included as controls. Genomic RNA (gRNA) and subgenomic mRNA from gene 7 (mRNA-7) were analyzed by RT-qPCR using specific TaqMan assays after transfection of each replicon in BHK-N cells. Mean values from eight independent transfection experiments are plotted; error bars represent standard deviations. *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001.

FIG 2

Analysis of rTGEV ExoN mutants. (A) RT-PCR analysis of viral RNA from passage 0 and passage 1 of the rTGEV-ExoI and rTGEV-ExoIII mutants, compared with the rTGEV-WT. Genomic RNA (gRNA) and subgenomic mRNA of gene N (mRNA-N) were analyzed. Lanes A and B indicate duplicate infectious cDNAs, tested individually for each mutant. Weight-average molecular weight markers (M_w) are shown on the right. (B) Quantification of genomic RNA (gRNA) and mRNA of gene 7 (mRNA-7). Both negative and positive strands were measured by RT-qPCR using specific TaqMan assays 24 h posttransfection of infectious cDNAs into BHK-N cells. Means of results from four independent transfection experiments are plotted; error bars represent standard deviations. (C) Detection of viral proteins by Western blotting. Total protein was extracted 24 h after transfection of BHK-N cells with infectious cDNAs from ExoI, ExoIII, WT, nonreplicative rTGEV (NR), or mock-transfected (M) cells. Viral protein nsp3 and β-actin (as a loading control) were detected using specific antibodies. The right panel represents the quantification of the bands by densitometry, corrected by the amount of β-actin. Means of results from two independent transfection experiments are plotted; error bars represent standard deviations. *, P value < 0.05; ***, P value < 0.001.

FIG 3

Growth of nsp14 mutant rTGEV-ZF-C in tissue cultures. (A) Lysis plaques produced by mutant rTGEV-ZF-C and rTGEV-WT at 48 h postinfection in ST cells. The right panel represents the mean diameter of 10 individual lysis plaques; error bars indicate standard deviations. (B) Growth kinetics of rTGEV-WT and rTGEV-ZF-C viruses. ST cells were infected at low (0.05; left) and high (5; right) MOIs with mutant rTGEV-ZF-C (ZF-C) or rTGEV-WT (WT); supernatants were collected at different times postinfection, and infectious titers were determined by plaque titration on ST cells. Means of results from three independent experiments are plotted; error bars represent standard deviations. (C) RNA synthesis of rTGEV-WT and rTGEV-ZF-C viruses. ST cells were infected at an MOI of 1 with mutant rTGEV-ZF-C or rTGEV-WT; intracellular RNA was collected at different times postinfection, and genomic RNA (gRNA) was quantified by RT-qPCR using specific TaqMan assays. Means of results from three independent experiments are plotted; error bars represent standard deviations. *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001.

FIG 4

Induction of apoptosis by mutant rTGEV-ZF-C. (A) Detection of active caspase 3 (casp3) and TGEV N protein (N) by Western blotting. Total protein was extracted from ST cells 24 h after infection with mutant rTGEV-ZF-C, infection with rTGEV-WT, or mock infection (M). Lanes A and B indicate duplicate viral clones that were tested individually. Casp3, N, and β-actin (loading control) were detected using specific antibodies; procaspase 3 (procasp3) and the form of the TGEV N protein cleaved by caspases (N-cl) are also indicated. (B) Quantification of the bands by densitometry, corrected by amount of the β-actin. Relative levels of protein (r.u.) were based on comparison with the WT virus protein level, which was considered to be 100%. Means of results from three independent experiments are plotted; error bars represent standard deviations. *, P value < 0.05; **, P value < 0.01.

FIG 5

Innate immune response induced by mutant rTGEV-ZF-C. ST cells were infected with rTGEV-ZF-C or rTGEV-WT virus at an MOI of 1, and intracellular RNA was collected at different times postinfection. (A) Quantification of IFN-β and TNF mRNA was performed by RT-qPCR using specific TaqMan assays; relative mRNA levels were based on comparison with mock-infected cells. Means of results from three independent experiments are plotted; error bars represent standard deviations. (B) Induction of interferon-stimulated genes by rTGEV-ZF-C. Quantification of IRF-1, OAS, RIG-I, MDA5, and TGF-ß mRNAs was performed by RT-qPCR using specific TaqMan assays; relative mRNA levels were based on comparison with mock-infected cells. Numbers below asterisks indicate the fold change in induction by rTGEV-WT relative to that of mutant rTGEV-ZF-C. Means of results from four independent experiments are plotted; error bars represent standard deviations. *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001.

FIG 6

Accumulation of dsRNA in cells infected with mutant rTGEV-ZF-C. Confocal microscopy analysis was performed on synchronized ST cells infected with the rTGEV-ZF-C or rTGEV-WT virus at an MOI of 1. (A) Immunofluorescence images of ST cells subjected to mock infection (mock) or to infection with rTGEV-ZF-C (ZF-C) or rTGEV-WT at 8 and 16 h postinfection. nsp14 was detected using a specific polyclonal antisera and a secondary antibody staining green; dsRNA was detected using monoclonal antibody MAb-J2 and a secondary antibody staining red; DAPI (4′,6-diamidino-2-phenylindole) (blue) was used to stain the nuclear DNA. Colocalization is indicated by yellow pixels in the merge panels. (B) Quantification of the mean intensity of fluorescence and normalized standard deviation of fluorescence intensity (intensity variance) of nsp14 and dsRNA. Means of 30 individual cells are plotted; error bars represent standard deviations. ***, P value < 0.001.

FIG 7

Modulation of dsRNA-induced antiviral response by mutant rTGEV-ZF-C. (A) ST cells were mock infected (white bars) or infected with mutant rTGEV-ZF-C (gray bars) or rTGEV-WT (black bars) at an MOI of 1 and were subsequently transfected at 12 h postinfection with poly(I·C). At 4 h posttransfection, total intracellular RNA was collected, and quantification of viral genomic RNA (gRNA), IFN-β, and TNF mRNAs was performed by RT-qPCR using specific TaqMan assays. Relative mRNA levels were based on comparison with mock-infected, nontransfected cells. Means of results from three independent experiments are plotted; error bars represent standard deviations. (B) Cellular RNA integrity. Total RNA was extracted from mock-infected (M) or rTGEV-WT (WT)- and rTGEV-ZF-C (ZF)-infected ST cells that were left untreated (−PolyIC) or treated (+PolyIC) with poly(I·C). The RNA was then analyzed using a Bioanalyzer. The positions of 28S and 18S rRNAs are indicated (left panel). The data in the graph represent 28S rRNA integrity, as measured by the Bioanalyzer. Error bars indicate standard deviations of results from four independent experiments. f.u., fluorescence units. *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001.

FIG 8

Modulation of rTGEV-WT-induced antiviral response by rTGEV-ZF-C mutant. ST cells were mock infected or individually infected with mutant rTGEV-ZF-C or rTGEV-WT or coinfected with each virus at two different ratios (1:1 or 5:1 ZF-C/WT ratio; the MOIs of each virus used are indicated by numbers on the x axis). At 16 h postinfection, total intracellular RNA was collected, and quantification of viral genomic RNA (gRNA), IFN-β, and TNF mRNAs was performed by RT-qPCR using specific TaqMan assays. Relative mRNA levels were based on comparison with mock-infected cells. Means of results from three independent experiments are plotted; error bars represent standard deviations. *, P value < 0.05; **, P value < 0.01.

FIG 9

Working model for the role of nsp14 in the counteracting of antiviral responses during CoV infection. A schematic overview of the host cell dsRNA-induced antiviral pathway is shown. The proposed mechanism of action for CoV nsp14 is indicated.