First round of mutagenesis reveals nsp1-m5 mutant with partial loss of inhibition of host signaling.

<p>SARS-CoV mutants nsp1-m1 through nsp1-m8 were tested for (A) inhibition of host IFN- and virus-dependent signaling using the ISREx3-CAT reporter, followed by inhibition of gene expression using (B) luciferase and (C) β-galactosidase assays. CAT activity values correspond to percent chloramphenicol acetylation using cell extracts diluted 100-fold, luciferase activity is determined in straight extracts and is expressed in RLU and β-galactosidase activity corresponds to released ortho-nitrophenol absorption at 405 nm using extracts diluted 10-fold. Immunoblots of nsp1 mutants are quantitated in (D). Error bars are ± standard error; P-values are result of a t-Test. Mutant nsp1-m5 exhibited attenuated inhibition of host IFN- and virus-dependent signaling (A) while maintaining wildtype inhibition of β-galactosidase (C); P-values for nsp1-m5 are indicated in figure, significance for other mutants is listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062416#pone-0062416-t001" target="_blank">Table 1</a>.</p

Mapping of SARS-CoV nsp1 mutants onto its 3D-structure.

<p>The structure of SARS-CoV nsp1 was solved from a.a. 13 to 127 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062416#pone.0062416-Almeida1" target="_blank">[32]</a> and is displayed using the PyMOL software. Top row (A–C), the backbone structure is displayed in cartoon form, it consists of a mixed parallel/antiparallel six strand β-barrel, with the α-helix (cyan) at one barrel opening and the 3₁₀-helix (green) alongside the barrel. Middle row (D–F) displays protein electrostatic surface charge colored blue for positive regions and red for negative regions, with scale (G). Bottom row (H–J), atoms from amino acids that were mutated in this study are shown as space-filling spheres on top of the backbone structure in cartoon form; purple amino acids correspond to mutations that did not affect nsp1 function; blue amino acids correspond to mutations that partially attenuated both inhibition of signal transduction and inhibition of gene expression; green amino acids correspond to mutations that abolished both inhibition of signal transduction and inhibition of gene expression; yellow amino acids correspond to nsp1-m5/−m16, which maintained inhibition of gene expression but lost inhibition of IFN- and antiviral-signal transduction; orange amino acids correspond to mutations that maintained inhibition of gene expression but increased inhibition of signal transduction. Red mutations displayed increased inhibition of ISREx3CAT, β-galactosidase and luciferase reporters. The structures in each row are rotated along the vertical axis by 120°. A putative amphipathic α-helix in the C-terminus of nsp1 (a.a.169–177) is displayed in a wheel representation, a.a. are color coded: hydrophobic, grey; hydrophilic, purple; acid, red; and basic, blue (K). Primary sequence of SARS-CoV nsp1 (L): grayed amino acids correspond to regions disordered in the published NMR structure (a.a. 1–12, a.a. 128–180); amino acids were color-coded as in (H–J); the boxed sequence corresponds to the putative amphipathic α-helix displayed in (K).</p

Raw and normalized inhibitory activities for all nsp1 mutants.

<p>The inhibitory functions for all nsp1 mutants are shown in this table expressed as averaged percent compared to nsp1-wt (set to 100 percent). Percent inhibitions were also normalized to expression levels. No values are reported for mutants nsp1-m8, -m25, and -m26 as they had no significant inhibitory activities and were expressed at a level too low to be measured. After expression normalization, many nsp1 mutants exhibited increased inhibitory abilities compared to nsp1-wt; for example, nsp1-m19 exhibits nearly four-fold more inhibitory functions over nsp1-wt. Even after normalization, nsp1-m16 continues to show a loss of inhibition of host IFN- and virus-dependent signaling while maintaining strong inhibition of host gene expression. (*indicates significant as compared to empty vector; ^#indicates significant as compared to nsp1-wt; significant is defined as a t-Test P-value ≤ 0.05).</p

Graphical representation of experimental design.

<p>(A) SARS-CoV nsp1 was cloned into a plasmid driven by the CMV enhancer with a N-terminal triple FLAG tag. Surface residues that could be important for function were identified and mutated. (B) SARS-CoV nsp1-wt and mutants were cotransfected into 293T cells with reporter plasmids. Plasmid expressing eGFP was used to visually inspect for high transfection efficiency. (C) Standard assays were performed: luciferase and β-galactosidase assays were used as a proxy to measure level of nsp1 inhibition of gene expression; CAT gene under the control of three ISRE copies was used as a proxy for nsp1 inhibition of interferon- and virus-dependent signaling; immunoblots were run to directly measure nsp1 inhibition of STAT1 phosphorylation and IRF3 dimerization, and nsp1 levels.</p

Second round of mutagenesis reveals nsp1-m16 mutant with complete loss of inhibition of host signaling.

<p>SARS-CoV nsp1-m9 through nsp1-m26 mutants were tested for (A) inhibition of host IFN- and virus-dependent signaling using the ISREx3-CAT reporter, followed by inhibition of host gene expression using (B) luciferase and (C) β-galactosidase assays. CAT activity values correspond to percent chloramphenicol acetylation using cell extracts diluted 100-fold, luciferase activity is determined in straight extracts and is expressed in RLU and β-galactosidase activity corresponds to released ortho-nitrophenol absorption at 405 nm using extracts diluted 10-fold. Immunoblots of nsp1 mutants are quantitated in (D). Mutant nsp1-m16 had completely lost its ability to inhibit host IFN- and virus-dependent signaling (A) while retaining wildtype levels of inhibition of β-galactosidase expression (C). Mutant nsp1-m14 consistently exhibited stronger inhibitory effects than nsp1-wt in all assays (A,B,C). Error bars are ± standard error; P-values are result of a t-Test. P-values for nsp1-m14 and -m16 are indicated in figure, significance for other mutants is listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062416#pone-0062416-t001" target="_blank">Table 1</a>.</p

Vero E6-derived SNV stocks induce innate responses via a Jak/STAT pathway and contain IFNλ.

<p>(A) Hec 1b Jak/STAT knockout cells were treated with poly (I∶C) (50 µg/mL) or the indicated stock of virus from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011159#pone-0011159-g001" target="_blank">Figure 1</a> at an MOI = 1 for 3 h. Total cellular RNA was isolated and used for qRT-PCR for ISG56 and MxA. Real-time qRT-PCR results are reported as the mean ± SEM from triplicate biological experiments. (B) Medium supplemented with 50 µg/mL of rhIL29, and SNV derived from Vero E6 8 dpi and 20 dpi were applied to a 100-kDa filter and centrifuged for 10 min. The filtrate was then applied to a 3-kDa filter and centrifuged for 30 min. Equal volumes of the retentate were then used for SDS-PAGE. Western blotting was then performed using an anti-IL29 antibody.</p

SNV stocks induce innate responses in Huh7 independent of titer, replication, and intact virus particles.

<p>(A) Huh7 cells were infected with SNV at an MOI = 1 for 1 h. At the time points indicated, cellular mRNA was isolated and used to quantitate viral S-segment RNA (right y-axis) and ISG expression (left y-axis) using qRT-PCR. (B) Vero E6 cells grown in 75 cm2 tissue culture flasks were inoculated with SNV (10 ffu) for 1 h and supernatants were collected at the time points indicated on the x-axis. Virus was then titrated on fresh Vero E6 cells (left y-axis) or used to infect Huh7 cells at equal volumes. ISG56 and MxA expression was measured by qRT-PCR 3 hpi (right y-axis). (C) SNV from 20 dpi Vero E6 stocks was applied to a 100-kDa filter and centrifuged for 10 min. The retentate and filtrate was resuspended to the loading volume and applied to Huh7 cells for 3 h. MxA expression was then measured using by qRT-PCR of total cellular RNA and compared to untreated control (Con) cells. All real-time qRT-PCR results are reported as the mean ± SEM from triplicate biological experiments.</p

Endothelial cells specifically respond to SNV and are refractory to IFNλ.

<p>HUVEC were treated with SNV at an MOI = 1 (50 µL of stock virus), rhIL29 (50 ng/mL), rhIL28A (50 ng/mL), or left untreated (con) with or without pre-incubation with the indicated neutralizing antibody for 1 h. Cellular RNA was used to quantitate ISG expression 3 h post exposure. Real-time qRT-PCR results are reported as the mean ± SEM from triplicate biological experiments.</p