Reactivity of the18 Neutralizing HmAbs with SARS CoV 12-510-S1 proteins.

<p>Medisorp ELISA plates were coated with 100 ng/well of Urbani and RBD mutant 12-510S1-IgG proteins and 2.5 µg/ml of each HmAb was used as the primary antibody. Anti-human IgG2 HRP mouse monoclonal antibody was used as secondary antibody. OD was measured at 450 nm. Error bars represent SD of a representative experiment performed in triplicates. (A) Urbani versus Sin845 mutant. (B) Urbani versus GD01 mutant. (C) Urbani versus GZ0402 mutant. (D) Urbani versus GZ-C mutant.</p

Combinations of HmAbs more efficiently inhibit the entry of SARS-CoV RBD surrogate clinical isolates.

<p>Neutralizing HmAbs binding to different regions of S protein 4D4 (S1), 1F8 (HR1), 5E9 (HR2)) were tested for their ability to neutralize pseudoviruses in different combinations as well as individually at a concentration of 6.25 µg/ml each. The virus/Ab mixture was incubated for 1 hr at 37°C then added to 293/ACE2 stable cell line. Seventy two hours later, the virus entry was determined by luciferase expression. The percentage entry inhibitions by individual antibodies as well as combinations of antibodies were calculated. Error bars represent SD of representative experiment performed in triplicates. Statistical analysis was done using Student-t test, significant differences are indicated by asterisks,<i>* p<0.05.</i></p

HmAbs to HR1 and HR2 can efficiently neutralize surrogate clinical isolates.

a<p>Likely binding region of antibodies.</p>b<p>S glycoprotein ectodomain.</p>c<p>Anti-SARS-S protein polyclonal antibody.</p

Reactivity of Urbani SARS-CoV-S protein antibodies with Urbani S1 protein and mutant S1 proteins.

<p>(A) Different dilutions of a rabbit anti-Urbani SARS-CoV-S protein immune serum were tested in an ELISA against Urbani as well as mutant S1-IgG proteins. Anti-rabbit donkey polyclonal HRP antibody was used as the secondary antibody. (B) Competitive ELISA assay: Different protein concentrations of Urbani or GZ-C proteins were pre-incubated with 5A7 antibody then the protein/Ab mixtures were tested for binding to the other protein by ELISA. OD was measured at 450 nm.</p

Proposed recognition models of K48-Ub₂ and ISG15 by PLpro.

<p>PLpro is shown as a surface representation and colored grey. The ubiquitin binding subsites distal to the isopeptide bond are indicated in red for SUb1 and blue for SUb2. Ubiquitin molecules in the chain are indicated as circles in yellow and numbering follows conventional protease substrates numbering with ubiquitins distal to the isopeptide bond as Ub2, Ub1 and those proximal as Ub1′ etc. Ubiquitin lysines are labeled as K48 or K63. The greater than symbols (>>) designate the relative affinity of one complex over the other from data presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004113#ppat-1004113-g003" target="_blank">Figure 3</a>.</p

<i>In vitro</i> pseudovirus neutralization assay.

<p>Eighteen neutralizing HmAbs were tested against different mutant as well as Urbani pseudoviruses. Pseudoviruses equivalent to 10 ng of HIVp24 were incubated for 1 hr with 25 µg/ml of each of the HmAbs at 37°C. The virus/Ab mixtures were then added to 293/ACE2 stable cell line. Seventy two hours later, the virus entry was determined by luciferase expression. The percentage entry inhibitions obtained with Abs were calculated and normalized to HIV/Urbani-S inhibitions (A) HIV/GZ-C and HIV/Sin845 inhibitions (B) HIV/GZ0402 and HIV/GD01 inhibitions. Polyclonal rabbit immune serum (PolyAb) was used as a positive control. Error bars represent SD of a representative experiment performed in triplicates.</p

Deubiquitinating activity and NF-κB antagonism are reduced by mutation of SARS-CoV PLpro residue F70.

<p>(<b>A</b>) HEK293T cells were transfected with expression plasmids encoding FLAG-Ub and the indicated PLpro. At 18 hours post transfection, cells were lysed and immunoblotted for FLAG-Ub, calnexin, and PLpro-V5. (B) HEK293T cells were transfected with constructs expressing nsp2-3-GFP and SARS-CoV PLpro-V5. Lysates were immunoblotted with anti-GFP, anti-calnexin, and anti-V5. (C) HEK293 cells were transfected with IkBα-HA and the indicated PLpro. After 16 hours incubation, cells were stimulated with TNFα (20 ng/ml). Lysates were analyzed by 10% SDS-PAGE and immunoblotted for anti-HA, anti-calnexin, and anti-V5. (D) 293HEK cells were transfected with NFkB-reporter and Renilla luciferase control constructs and the indicated PLpro. After 12 hours, TNFα was added to a final concentration of 10 ng/mL and the cells were incubated for an additional 4 hours. Results are normalized to induction of NFkB reporter activity by TNFα. Panels below are western blots of the lysates using anti-V5 for detection of PLpro and anti-actin as a protein loading control. Experiments were performed in triplicate and repeated twice. * = p<.05 statistical difference from mock transfected cells by student t-test.</p

Structural Basis for the Ubiquitin-Linkage Specificity and deISGylating Activity of SARS-CoV Papain-Like Protease

<div><p>Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a papain-like protease (PLpro) with both deubiquitinating (DUB) and deISGylating activities that are proposed to counteract the post-translational modification of signaling molecules that activate the innate immune response. Here we examine the structural basis for PLpro's ubiquitin chain and interferon stimulated gene 15 (ISG15) specificity. We present the X-ray crystal structure of PLpro in complex with ubiquitin-aldehyde and model the interaction of PLpro with other ubiquitin-chain and ISG15 substrates. We show that PLpro greatly prefers K48- to K63-linked ubiquitin chains, and ISG15-based substrates to those that are mono-ubiquitinated. We propose that PLpro's higher affinity for K48-linked ubiquitin chains and ISG15 stems from a bivalent mechanism of binding, where two ubiquitin-like domains prefer to bind in the palm domain of PLpro with the most distal ubiquitin domain interacting with a “ridge” region of the thumb domain. Mutagenesis of residues within this ridge region revealed that these mutants retain viral protease activity and the ability to catalyze hydrolysis of mono-ubiquitin. However, a select number of these mutants have a significantly reduced ability to hydrolyze the substrate ISG15-AMC, or be inhibited by K48-linked diubuiquitin. For these latter residues, we found that PLpro antagonism of the nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) signaling pathway is abrogated. This identification of key and unique sites in PLpro required for recognition and processing of diubiquitin and ISG15 versus mono-ubiquitin and protease activity provides new insight into ubiquitin-chain and ISG15 recognition and highlights a role for PLpro DUB and deISGylase activity in antagonism of the innate immune response.</p></div

Updated models of (A) ISG15 (yellow) and (B) K48-Ub₂ (orange) and bound to PLpro (blue).

<p>The ubiquitin hydrophobic patch residues are shown in magenta. PLpro residues identified through site-directed mutagenesis as important for K48-Ub₂ and ISG15 binding are highlighted in a yellow circle. The two distal regions involved in binding Ub2 or ISG15 are labeled as distal-1 (closest to active site, location of single ubiquitin binding) and distal-2 (binding of second ubiquitin-like domain).</p

The crystal structure of the PLpro-Ubal complex reveals a dense hydrogen-bonding pattern between the active site of PLpro and the C-terminus of ubiquitin.

<p><b>A.</b> Stereoview of PLpro active-site interactions with ubiquitin-aldehyde. PLpro residues are shown in blue and labeled in black, and ubiquitin residues are shown and labeled in orange. Hydrogen bonds between PLpro and ubiquitin are shown as dashed lines. <b>B.</b> Electron density associated with the region surrounding the C-terminal residues of ubiquitin (orange density) and their interactions with the PLpro active site in the region of the mobile loop (blue density). The residues shown and the view depicted are similar to those in panels A and C. The electron density maps were calculated by omitting ubiquitin from the structure factor calculations. The Fo-Fc map for ubiquitin (orange) is contoured at 3σ and the 2Fo-Fc map for PLpro (blue density) is contoured at 1.5σ. The figure was generated using the program Pymol. <b>C.</b> Comparison of the PLpro active site loop in bound (blue with white surface) and unbound (yellow) conformations. The C-terminus of ubiquitin is shown in orange. The orientation of the structure is similar to that shown in panels A and B.</p