Research Article: Substitution at Aspartic Acid 1128 in the SARS Coronavirus Spike Glycoprotein Mediates Escape from a S2 Domain-Targeting Neutralizing Monoclonal Antibody

Date Published: July 14, 2014

Publisher: Public Library of Science

Author(s): Oi-Wing Ng, Choong-Tat Keng, Cynthia Sau-Wai Leung, J. S. Malik Peiris, Leo Lit Man Poon, Yee-Joo Tan, Stefan Pöhlmann.


The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is the etiological agent for the infectious disease, SARS, which first emerged 10 years ago. SARS-CoV is a zoonotic virus that has crossed the species barriers to infect humans. Bats, which harbour a diverse pool of SARS-like CoVs (SL-CoVs), are believed to be the natural reservoir. The SARS-CoV surface Spike (S) protein is a major antigenic determinant in eliciting neutralizing antibody production during SARS-CoV infection. In our previous work, we showed that a panel of murine monoclonal antibodies (mAbs) that target the S2 subunit of the S protein are capable of neutralizing SARS-CoV infection in vitro (Lip KM et al, J Virol. 2006 Jan; 80(2): 941–50). In this study, we report our findings on the characterization of one of these mAbs, known as 1A9, which binds to the S protein at a novel epitope within the S2 subunit at amino acids 1111–1130. MAb 1A9 is a broadly neutralizing mAb that prevents viral entry mediated by the S proteins of human and civet SARS-CoVs as well as bat SL-CoVs. By generating mutant SARS-CoV that escapes the neutralization by mAb 1A9, the residue D1128 in S was found to be crucial for its interaction with mAb 1A9. S protein containing the substitution of D1128 with alanine (D1128A) exhibited a significant decrease in binding capability to mAb 1A9 compared to wild-type S protein. By using a pseudotyped viral entry assay, it was shown that the D1128A substitution in the escape virus allows it to overcome the viral entry blockage by mAb 1A9. In addition, the D1128A mutation was found to exert no effects on the S protein cell surface expression and incorporation into virion particles, suggesting that the escape virus retains the same viral entry property as the wild-type virus.

Partial Text

The Severe Acute Respiratory Syndrome (SARS) first emerged as an infectious disease ten years ago, manifesting itself as a severe form of pneumonia. Its etiological agent was identified as a then novel coronavirus known as the SARS coronavirus (SARS-CoV) [1], [2]. Within a short span of time from December 2002 to July 2003, the newly emerged virus spread quickly to infect more than 8000 people across 25 countries with an overall fatality rate of approximately 10% [3]. SARS-CoV is a zoonotic virus that has crossed the species barrier to infect humans. Small animals such as palm civets (Paguma larvata) and raccoon dogs (Nyctereutes procynonoides) sold in live-animal wet markets in Guangdong Province of Southern China are believed to be the zoonotic source of the virus transmitted to humans [4]. In 2005, the complete sequences of SARS-like coronaviruses (SL-CoVs) of genetic homology of 87–92% to SARS-CoV were identified from horseshoe bats of the genus Rhinolophus in China [5], [6]. However, these SL-CoVs display significant differences in sequences at the receptor-binding domain (RBD) compared to SARS-CoV and are unable to use the SARS-CoV receptor, the human angiotensin-converting enzyme 2 (ACE2), for cellular entry [7], rendering them unlikely to be the immediate progenitor of SARS-CoV. More recently, a bat SL-CoV capable of using the human ACE2 receptor for cellular entry was characterized and isolated from Chinese horseshoe bats, providing strong evidence that bats are the natural reservoirs of SARS-CoV [8].

Although there has been no reported case of SARS-CoV infection in humans since 2004, the development of anti-SARS-CoV treatments and vaccines remains crucial as the threat of a re-emergence of SARS exists till today. Human and civet SARS-CoVs are believed to have originated from SL-CoVs residing in bats [42]. As coronaviruses are known to be capable of frequent cross-species transmission [12], the continual persistence of SL-CoVs in animal hosts and reservoirs poses a threat to humans should a cross-species transmission occurs. The development of broadly neutralizing mAbs that confer cross-protection not only against human SARS-CoV, but also zoonotic strains of SARS-CoV and SL-CoV, is therefore important. The putative S1 subunit of bats SL-CoVs has a low sequence homology of about 63% to that of SARS-CoV, especially in the RBD, indicating the usage of different host cell receptors and different tissue tropisms [43]. On the other hand, the high sequence homology in the S2 subunit of about 92–96% suggests that the fusion mechanism during viral infection is well-conserved [44]. Broadly neutralizing mAbs usually target conserved epitopes required for highly conserved process, such as the post-attachment fusion process [45]. A majority of SARS-CoV-neutralizing mAbs reported bind and target the S1 protein at the RBD region [46]. Nonetheless, neutralizing mAbs that bind to the S2 subunit have been reported. The epitopes of these mAbs are found to be located at the S2 subunit upstream of HR1 (residues 787–809, 791–805) [47], [48], within the loop region in between HR1 and HR2 (residues 1023–1189) [31], [40] and within the HR2 domain (residues 1151–1192) [31]. It has also been shown that anti-S2 mAbs that bind to the highly conserved HR1, HR2 and ectodomain of the SARS-CoV S protein were able to neutralize a wider range of clinical isolates, including human and zoonotic strains of SARS-CoVs [28], [30]. In this current study, mAb 1A9, an anti-S2 mAb that binds to the S2 subunit at the highly conserved loop region at residues 1111–1130, was demonstrated to be able to cross-neutralize pseudotyped S-pp viruses of the human SARS-CoV, civet SARS-CoV and bat SL-CoV strains. This is consistent with the sequence conservation of the mAb 1A9 binding epitope in S. In addition, sequence alignment (not shown) revealed that the mAb 1A9 binding site is also conserved in other bat CoVs such as the Bulgarian SARS-related CoV strain [49], indicating the potential cross-protective effect of mAb 1A9 against not only bat SL-CoV from China, but also from other parts of the world such as Europe.




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