Coronavirus nonstructural protein 1: Common and distinct functions in the regulation of host and viral gene expression

Abstract

The recent emergence of two highly pathogenic human coronaviruses (CoVs), severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, has ignited a strong interest in the identification of viral factors that determine the virulence and pathogenesis of CoVs. The nonstructural protein 1 (nsp1) of CoVs has attracted considerable attention in this regard as a potential virulence factor and a target for CoV vaccine development because of accumulating evidence that point to its role in the downregulation of host innate immune responses to CoV infection. Studies have revealed both functional conservation and mechanistic divergence among the nsp1 of different mammalian CoVs in perturbing host gene expression and antiviral responses. This review summarizes the current knowledge about the biological functions of CoV nsp1 that provides an insight into the novel strategies utilized by this viral protein to modulate host and viral gene expression during CoV infection.

Keywords: Coronavirus; Nsp1; RNA viruses; SARS; Translation inhibition; mRNA cleavage.

Fig. 1

Genome organization and proteolytic processing of ORF1a polyprotein of selected members in the α-CoV and β-CoV genera of Coronaviridae family. The open reading frames (ORFs) 1a and 1b constitute gene 1. One member of each genus is shown as a representative example. The sites in ORF1a polyprotein processed by the viral proteinases, PL^pro and 3CL^pro, are indicated. In the upper panel, the dotted lines indicate the putative processing at the nsp1–nsp2 and nsp3–nsp4 cleavage sites by TGEV PL1^pro. The predicted activity of TGEV PL1^pro at these cleavage sites remains to be determined. RFS: ribosomal frameshift; UTR: untranslated region. The figure is not drawn to scale.

Fig. 2

Analysis of the subcellular localization of TGEV nsp1 using confocal microscopy. HEK 293 cells, grown on 4-well Lab-Tek II chamber slides (Nalgene Nunc International), were transfected with a TGEV nsp1 expressing plasmid, pCAGGS-nsp1 (lower row) or an empty pCAGGS plasmid (mock; upper row). At 24 h post-transfection, the cells were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100. Subsequently, the cells were subjected to immunofluorescence analysis using a TGEV-nsp1 specific primary antibody, which was raised by immunizing rabbits with purified full-length C-terminally myc-tagged TGEV nsp1 protein, followed by Alexa Fluor 594-conjugated secondary antibody (Molecular Probes) and DAPI counterstaining (Cell Signaling Technology). Images were collected using a Zeiss LSM-510 META confocal laser-scanning microscope with a 100X oil immersion lens and processed with the LSM image browser (Zeiss) and ImageJ (NIH) software program. No signal was detected in the negative control (mock; upper row), confirming the specificity of the anti-TGEV nsp1 antibody.

Fig. 3

Two-pronged strategy of SARS-CoV nsp1 to inhibit host gene expression. SARS-CoV nsp1 gains access to host mRNAs through its association with the 40S ribosomal subunit. Consequently, nsp1 inhibits the translation of capped cellular mRNAs by primarily blocking the steps involved in the formation of elongation-competent 80S initiation complex. Nsp1 also recruits a cellular endonuclease to induce an endonucleolytic RNA cleavage in the 5′-UTR of host mRNAs that subsequently results in the accelerated degradation of the 5′-truncated intermediate by the cellular Xrn1-mediated 5′–3′ exonucleolytic mRNA decay pathway.

Fig. 4

Viral mRNAs are not spatially separated from nsp1 in SARS-CoV-infected cells. RNA fluorescent in situ hybridization (RNA-FISH) and confocal microscopy analyses of viral mRNAs and nsp1 in SARS-CoV-infected cells. Vero E6 cells, grown on 4-well Lab-Tek II chamber slides (Nalgene Nunc International), were either mock-infected (Mock) or infected with wt SARS-CoV (SCoV-WT) at a multiplicity of infection (MOI) of 5. At 12 h p.i., the cells were fixed in 4% paraformaldehyde for 16 h at 4 °C and permeabilized with 0.5% Triton X-100 for 5 min at room temperature. Subsequently, the cells were precipitated with 70% ethanol at 4 °C overnight and subjected to RNA-FISH and immunofluorescence analyses. A digoxigenin (DIG)-labeled antisense riboprobe, corresponding to the nucleotides (nt) 29,084–29,608 at the 3′-end of the SARS-CoV genome, was used to detect viral mRNAs, including mRNAs 1–9. Viral mRNAs (green) were visualized with a primary sheep anti-DIG antibody and Alexa488-conjugated anti-sheep secondary antibody (Invitrogen). Nsp1 (red) was visualized using an affinity-purified rabbit anti-nsp1 polyclonal antibody, which was raised by immunizing rabbits with purified full-length nsp1 protein, followed by Alexa594-conjugated secondary antibody (Invitrogen). Images were collected using a Zeiss LSM-510 META confocal microscope with a 63×, 1.40 numerical aperture oil immersion lens and processed with the LSM image browser and Metamorph software (Molecular Devices, Downingtown, PA). In the lower panel (SCoV-WT), the inset to the right of the merged image represents the indicated region (small square panel) in the merged image. The histogram displays the fluorescence signal intensities along the white arrow in the inset, which is a representative line for a single line scan showing the fluorescence signal intensity (y axis) in both channels (red and green) in an arbitrary scale versus distance (x axis) (in pixel) over that line.

Fig. 5

Viral growth kinetics and mRNA accumulation are similar in SARS-CoV and SARS-CoV-mt-infected cells. Vero E6 cells were either mock-infected (Mock) or infected with wt SARS-CoV (WT) or SARS-CoV-mt (mt), carrying the mutations K164A and H165A in nsp1, at an MOI of 5. (A) Culture supernatants were collected at the indicated times p.i., and virus titers were determined by TCID₅₀ analysis in Vero E6 cells. The results represent the average of three independent experiments. (B) Total RNAs were extracted at 10 h and 12 h p.i. The viral mRNAs were detected by Northern blot analysis using a riboprobe that binds to the 3′-end of SARS-CoV genome, as described in the legend for Fig. 1. Representative data from three independent experiments are shown. (C) At 10 h and 12 h p.i., total proteins were extracted, and Western blot analysis was performed to detect Nsp1 protein using a rabbit anti-nsp1 polyclonal antibody. Representative data from three independent experiments are shown.

Fig. 6

SARS-CoV nsp1 inhibited the translation of viral mRNAs in infected cells. Vero E6 cells were either mock-infected (Mock) or infected with wt SARS-CoV (WT) or SARS-CoV-mt (mt), carrying the mutations K164A and H165A in nsp1, at an MOI of 5. (A) Cells were radiolabeled for 15 min with 500 μCi of Tran[³⁵S] label (MP Biomedicals) at 10 h and 12 h p.i. Equivalent amounts of the intracellular proteins were analyzed on a 12% SDS-PAGE and visualized by autoradiography. Arrowhead indicates the position of N protein. Representative data from three independent experiments are shown. (B) Total proteins, extracted at 10 h and 12 h p.i. in (A), were subjected to Western blot analysis to detect the viral structural and accessory proteins, S, N, 3a, 6 and 7a. Anti-SARS-CoV S antibodies (IMG-541; Imgenex and AP6000a; Abgent) and anti-SARS-CoV N antibody (IMG-548; Imgenex) were used to detect S and N proteins, respectively. Rabbit anti-SCoV 3a, 6 and 7a antibodies were used to detect 3a, 6 and 7a proteins, respectively, as described previously (Huang et al., 2006a, Huang et al., 2006b, Huang et al., 2007). Anti-actin antibody (Santa Cruz Biotechnology) was used to detect β-actin and the detection of similar amounts of β-actin in both SARS-CoV and SARS-CoV-mt-infected cells confirmed the loading of similar amounts of cell extracts in each lane. Representative data from three independent experiments are shown.