A new mouse-adapted strain of SARS-CoV as a lethal model for evaluating antiviral agents in vitro and in vivo

Virology
Volume 395, Issue 2, 20 December 2009, Pages 210-222

Craig W. Day, Ralph Baric, Sui Xiong Cai, Matt Frieman, Yohichi Kumaki John D. Morrey, Donald F. Smee, Dale L. Barnard.

Abstract

Severe acute respiratory syndrome (SARS) is a highly lethal emerging disease caused by coronavirus SARS-CoV. New lethal animal models for SARS were needed to facilitate antiviral research. We adapted and characterized a new strain of SARS-CoV (strain v2163) that was highly lethal in 5- to 6-week-old BALB/c mice. It had nine mutations affecting 10 amino acid residues. Strain v2163 increased IL-1α, IL-6, MIP-1α, MCP-1, and RANTES in mice, and high IL-6 expression correlated with mortality. The infection largely mimicked human disease, but lung pathology lacked hyaline membrane formation. In vitro efficacy against v2163 was shown with known inhibitors of SARS-CoV replication. In v2163-infected mice, Ampligen™ was fully protective, stinging nettle lectin (UDA) was partially protective, ribavirin was disputable and possibly exacerbated disease, and EP128533 was inactive. Ribavirin, UDA, and Ampligen™ decreased IL-6 expression. Strain v2163 provided a valuable model for anti-SARS research.

Keywords

SARS-CoV, Lethal, IL-6, Ribavirin, Ampligen™, UDA, Protease inhibitor, Cytokine, Chemokine, Mouse

Antibody-mediated synergy and interference in the neutralization of SARS-CoV at an epitope cluster on the spike protein

Biochemical and Biophysical Research Communications
Volume 390, Issue 3, 18 December 2009, Pages 1056-1060

Lilin Zhong, Lia Haynes, Evi Budo Struble, Azaibi Tamin, Maria Luisa Virata-Theimer, Pei Zhang.

Abstract

Incomplete neutralization of virus, especially when it occurs in the presence of excess neutralizing antibody, represents a biological phenomenon that impacts greatly on antibody-mediated immune prophylaxis of viral infection and on successful vaccine design. To understand the mechanism by which a virus escapes from antibody-mediated neutralization, we have investigated the interactions of non-neutralizing and neutralizing antibodies at an epitope cluster on the spike protein of severe acute respiratory syndrome coronavirus (SARS-CoV). The epitope cluster was mapped at the C-terminus of the spike protein; it consists of structurally intertwined epitopes recognized by two neutralizing monoclonal antibodies (mAbs), 341C and 540C, and a non-neutralizing mAb, 240C. While mAb 341C binds to a mostly linear epitope composed of residues 507PAT509 and V349, mAb 240C binds to an epitope that partially overlaps the former by at least two residues (P507 and A508). The epitope corresponding to mAb 540C is a conformational one, involving residues L504 and N505. In neutralization assays, non-neutralizing 240C disrupted virus neutralization by mAb 341C and/or mAb 540C, whereas a combination of mAbs 341C and 540C blocked virus infectivity synergistically. These findings indicate that the epitope cluster on the spike protein may serve as an evolutionarily conserved platform at which a dynamic interplay between neutralizing and non-neutralizing antibodies occurs, thereby determining the outcome of SARS-CoV infection.

Keywords

SARS-CoV, Monoclonal antibody, Neutralization, Epitope

Identification of a New Region of SARS-CoV S Protein Critical for Viral Entry

Journal of Molecular Biology
Volume 394, Issue 4, 11 December 2009, Pages 600-605

Ying Guo, Jennifer Tisoncik, Susanna McReynolds, Michael Farzan, Bellur S. Prabhakar, Thomas Gallagher, Lijun Rong, Michael Caffrey.

Abstract

Infection by severe acute respiratory syndrome coronavirus (SARS-CoV) is initiated by specific interactions between the SARS-CoV spike (S) protein and its receptor ACE2. In this report, we screened a peptide library representing the SARS-CoV S protein sequence using a human immunodeficiency virus-based pseudotyping system to identify specific regions that affect viral entry. One of the 169 peptides screened, peptide 9626 (S residues 217–234), inhibited SARS-CoV S-mediated entry of the pseudotyped virions in 293T cells expressing a functional SARS-CoV receptor (human angiotensin-converting enzyme 2) in a dose-dependent manner (IC50 ∼ 11 μM). Alanine scanning mutagenesis was performed to assess the roles of individual residues within this region of S, which was previously uncharacterized. The effects included significant reductions in expression (K223A), viral incorporation (L218A, I230A, and N232A), and reduced viral entry (L224A, L226A, I228A, T231A, and F233A). Taken together, these results reveal a new region of the S protein that is crucial for SARS-CoV entry.

Keywords

viral entry, SARS-CoV, envelope, mutagenesis, spike

Dual effect of nitric oxide on SARS-CoV replication: Viral RNA production and palmitoylation of the S protein are affected

Virology
Volume 395, Issue 1, 5 December 2009, Pages 1-9

Sara Åkerström, Vithiagaran Gunalan, Choong Tat Keng, Yee-Joo Tan, Ali Mirazimi

Abstract

Nitric oxide is an important molecule playing a key role in a broad range of biological process such as neurotransmission, vasodilatation and immune responses. While the anti-microbiological properties of nitric oxide-derived reactive nitrogen intermediates (RNI) such as peroxynitrite, are known, the mechanism of these effects are as yet poorly studied. Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) belongs to the family Coronaviridae, was first identified during 2002-2003. Mortality in SARS patients ranges from between 6 to 55%. We have previously shown that nitric oxide inhibits the replication cycle of SARS-CoV in vitro by an unknown mechanism. In this study, we have further investigated the mechanism of the inhibition process of nitric oxide against SARS-CoV. We found that peroxynitrite, an intermediate product of nitric oxide in solution formed by the reaction of NO with superoxide, has no effect on the replication cycle of SARS-CoV, suggesting that the inhibition is either directly effected by NO or a derivative other than peroxynitrite. Most interestingly, we found that NO inhibits the replication of SARS-CoV by two distinct mechanisms. Firstly, NO or its derivatives cause a reduction in the palmitoylation of nascently expressed spike (S) protein which affects the fusion between the S protein and its cognate receptor, angiotensin converting enzyme 2. Secondly, NO or its derivatives cause a reduction in viral RNA production in the early steps of viral replication, and this could possibly be due to an effect on one or both of the cysteine proteases encoded in Orf1a of SARS-CoV.

Keywords

SARS-CoV, Palmitoylation, Nitric oxide, Spike protein

Investigation of the pharmacophore space of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) NTPase/helicase by dihydroxychromone derivatives

Bioorganic & Medicinal Chemistry Letters
Volume 19, Issue 16, 15 August 2009, Pages 4538-4541

Chaewoon Lee, Jin Moo Lee, Na- Ra Lee, Dong-Eun Kim, Yong-Joo Jeong, You hoon Chong.

Abstract

Aryl diketoacids have been identified as the first SARS-CoV NTPase/helicase inhibitors with a distinct pharmacophore featuring an arylmethyl group attached to a diketoacid. In order to search for the pharmacophore space around the diketoacid core, three classes of dihydroxychromone derivatives were prepared. Based on SAR study, an extended feature of the pharmacophore model of SARS-CoV NTPase/helicase was proposed which is constituted of a diketoacid core, a hydrophobic arylmethyl substituent, and a free catechol unit.

Keywords

SARS (Severe Acute Respiratory Syndrome), NTPase/helicase, Dihydroxychromone, Pharmacophore