CHARACTERIZATION OF VIRAL-VIRAL AND VIRAL HOST INTERACTIONS THAT CONTRIBUTE TO THE REPLICATION AND PATHOGENESIS OF THE SEVERE ACUTE RESPIRATORY SYNDROME VIRUS (SARS-COV)

Ph.DDOCTOR OF PHILOSOPH

SARS coronavirus 8b reduces viral replication by down-regulating E via an ubiquitin-independent proteasome pathway

10.1016/j.micinf.2010.10.017Microbes and Infection 132179-18

The human severe acute respiratory syndrome coronavirus (SARS-CoV) 8b protein is distinct from its counterpart in animal SARS-CoV and down-regulates the expression of the envelope protein in infected cells

The severe acute respiratory syndrome coronavirus (SARS-CoV), isolated from humans infected during the peak of epidemic, encodes two accessory proteins termed as 8a and 8b. Interestingly, the SARS-CoV isolated from animals contains an extra 29-nucleotide in this region such that these proteins are fused to become a single protein, 8ab. Here, we compared the cellular properties of the 8a, 8b and 8ab proteins by examining their cellular localizations and their abilities to interact with other SARS-CoV proteins. These results may suggest that the conformations of 8a and 8b are different from 8ab although nearly all the amino acids in 8a and 8b are found in 8ab. In addition, the expression of the structural protein, envelope (E), was down-regulated by 8b but not 8a or 8ab. Consequently, E was not detectable in SARS-CoV-infected cells that were expressing high levels of 8b. These findings suggest that 8b may modulate viral replication and/or pathogenesi

Generation of escape SARS-CoV mutant virus in the presence of mAb 1A9.

<p>Human SARS-CoV HKU39849 strain was cultured in Vero E6 cells in the absence or presence of mAb 1A9 (0.25 mg/ml). Supernatant from the wells containing cells that exhibited cytopathic effect (CPE) at the highest dilution of SARS-CoV was harvested as passage 1. The virus was then passaged 3 times in the presence of mAb 1A9 (0.25 mg/ml) and the virus supernatant harvested at each passage was titrated on Vero E6 cells.</p

Determination of cell surface expression of wild-type and mutant D1128A S proteins by FACS analysis and determination of wild-type and mutant D1128A S protein incorporation in S-pp by ELISA.

<p>(A) FACS analysis was performed with 293 FT cells were transfected with empty vector (full line) or with plasmids expressing full length wild-type S (dotted line) or full length mutant D1128A S (dash line). Cells were harvested 72 hours post-transfection and stained with mouse mAb 7G12, followed by FITC-conjugated goat anti-mouse antibody. Results shown are representative of 3 independent experiments. (B) Pseudoviral particles not expressing S (pNL43-R-E-Luc virus), S-pp expressing wild-type S (S-WT-pp) and mutant D1128A S (S-D1128A-pp) were coated on a 96-well plate at 16 ng/well, as previously quantitated using P24 ELISA, and detected using mAb 7G12 (top) and mAb P24 (bottom) at 4-fold serial dilutions. Optical density (OD) was measured at 450 nm. Bars represent SD of the experiment carried out in triplicates. MAb P24 was used as a control antibody to ensure equal amounts of S-pp were coated onto each well.</p

Neutralization of wild-type, mutant D1128A, N1056K and D1128A/N1056K S-pps by mAb 1A9 and mAb 1G10.

<p>S-pp containing wild-type (S-WT-pp) or mutant S (S-D1128A-pp, S-N1056K-pp and S-D1128A/N1056K-pp) were pre-incubated with different concentrations of (A) mAb 1A9 or (B) mAb 1G10 at 25, 50, 100 and 200 µg/ml for 1 hour before infecting CHO-ACE2 cells. An anti-S1, non-neutralizing mAb 7G12 was used as control antibody at 200 µg/ml. Cells were harvested 48 hours post-infection and luciferase activities were measured. Viral entry, as indicated by the luciferase activity measured in relative light units (RLU), was expressed as a percentage of the reading obtained in the absence of antibody, which was set at 100%. Data shown represents that obtained from 3 independent experiments. Bars represent SD of the experiment carried out in triplicates. *indicates statistically significant difference of <i>p</i><0. 01 when compared to S-WT-pp.</p

Neutralization of wild-type and 1A9 escape SARS-CoV virus by mAb 1A9 and mAb 1G10.

<p>Wild-type human SARS-CoV HKU39849 strain (A and C) and escape mutant SARS-CoV virus generated in the presence mAb 1A9 (B and D) were used at 1000 TCID₅₀ to infect Vero E6 cells in the presence of two mAbs, namely mAb 1A9 (A and B) and mAb 1G10 (C and D). MAb 1A9 was used at concentrations 0, 0.25, 0.5, 1.0 and 2.0 mg/ml while mAb 1G10 was used at concentrations 0, 0.125, 0.25, 0.5 and 1.0 mg/ml. Percentage cytopathic effects (% CPE) of the infected cells was observed. MAb 1G10, a SARS-CoV-neutralizing, anti-S2 mAb that binds to S2 at residues 1151–1192, was used as the control mAb.</p

Binding of mAb 1A9 to wild-type, mutant D1128A, mutant N1056K and mutant D1128A/N1056K S proteins by Western Blot and immunoprecipitation.

<p>293(mock) or with plasmids expressing full length wild-type S (S-WT) or full length mutant S (S-D1128A, S-N1056K and S-D1128A/N1056K). (A) Western Blot analysis was performed on the cell lysates using mAb 1A9 to determine if it can bind to the different S proteins. MAb 7G12, which binds to the S1 of the S protein, was used as a control antibody to detect protein expression. (B) Cell lysates containing S-WT, S-D1128A, S-N1056K or S-D1128A/N1056K were subjected to immunoprecipitation (IP) using mAb 1A9 or 7G12 and protein A beads. S proteins immunoprecipitated by mAbs 1A9 and 7G12 were detected using rabbit anti-SΔ1 antibody in Western blot analysis (WB). The rabbit anti-SΔ1 antibody binds to amino acids 48–358 of the S1 subunit.</p

Binding and neutralization of human, civet SARS-CoVs and bat SL-CoVs to mAb 1A9.

<p>(A) Schematic representation of chimeric S protein of civet SARS-CoV SZ3, bat SL-CoVs Rp3 and Rf1. The civet SARS-CoV SZ3 chimeric S contains four mutations (R344K, S360F, K479N and S485T) at its RBD that is different from the RBD of human SARS-CoV HKU39849. The bat SL-CoVs Rp3 and Rf1 chimeric S proteins had their entire RBD (amino acids 322–496) replaced by the RBD of human SARS-CoV HKU39849 (amino acids 318–518). (B) 293 FT cells were transfected with no plasmid (mock) or with plasmids expressing S of human SARS-CoV HKU39849, RBD-modified chimeric S of civet SARS-CoV SZ3, bat SL-CoV Rp3 and Rf1 respectively. Western Blot analysis was performed on the cell lysates using mAb 1A9 to determine if it can bind to different S proteins. MAb 7G12, which binds to the S1 of the S protein, was used as a control antibody to detect protein expression. (C) S-pps containing S of human HKU39849, civet SZ3, bat Rp3 or Rf1, were pre-incubated with different concentrations of mAb 1A9 at 100, 150 and 200 µg/ml for 1 hour before infecting CHO-ACE2 cells. Cells were harvested 48 hours post-infection and luciferase activities were measured. Viral entry, as indicated by the luciferase activity measured in relative light units (RLU), was expressed as a percentage of the reading obtained in the absence of antibody, which was set at 100%. Data shown represents that obtained from 3 independent experiments. Bars represent SD of the experiment carried out in triplicates.</p

Amino Acids 1055 to 1192 in the S2 Region of Severe Acute Respiratory Syndrome Coronavirus S Protein Induce Neutralizing Antibodies: Implications for the Development of Vaccines and Antiviral Agents

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-SΔ10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells