Adaptive evolution influences the infectious dose of MERS-CoV necessary to achieve severe respiratory disease

Virology
Volume 517, April 2018, Pages 98-107

Madeline G. Douglas, Jacob F. Kocher, Trevor Scobey, Ralph S. Baric, Adam S. Cockrell

Abstract

We recently established a mouse model (288–330+/+) that developed acute respiratory disease resembling human pathology following infection with a high dose (5 × 106 PFU) of mouse-adapted MERS-CoV (icMERSma1). Although this high dose conferred fatal respiratory disease in mice, achieving similar pathology at lower viral doses may more closely reflect naturally acquired infections. Through continued adaptive evolution of icMERSma1 we generated a novel mouse-adapted MERS-CoV (maM35c4) capable of achieving severe respiratory disease at doses between 103 and 105 PFU. Novel mutations were identified in the maM35c4 genome that may be responsible for eliciting etiologies of acute respiratory distress syndrome at 10–1000 fold lower viral doses. Importantly, comparative genetics of the two mouse-adapted MERS strains allowed us to identify specific mutations that remained fixed through an additional 20 cycles of adaptive evolution. Our data indicate that the extent of MERS-CoV adaptation determines the minimal infectious dose required to achieve severe respiratory disease.

Keywords

Coronavirus, MERS-CoV, Middle East respiratory syndrome, Respiratory disease, Acute respiratory distress syndrome, Spike protein

MERS-CoV spike protein: Targets for vaccines and therapeutics

Antiviral Research
Volume 133, September 2016, Pages 165-177

Qihui Wang, Gary Wong, Guangwen Lu, Jinghua Yan, George F. Gao

Abstract

The disease outbreak caused by Middle East respiratory syndrome coronavirus (MERS-CoV) is still ongoing in the Middle East. Over 1700 people have been infected since it was first reported in September 2012. Despite great efforts, licensed vaccines or therapeutics against MERS-CoV remain unavailable. The MERS-CoV spike (S) protein is an important viral antigen known to mediate host-receptor binding and virus entry, as well as induce robust humoral and cell-mediated responses in humans during infection. In this review, we highlight the importance of the S protein in the MERS-CoV life cycle, summarize recent advances in the development of vaccines and therapeutics based on the S protein, and discuss strategies that can be explored to develop new medical countermeasures against MERS-CoV.

Keywords

Coronavirus, MERS-CoV, Spike protein, Vaccines, Therapeutics, Animal models

A hexapeptide of the receptor-binding domain of SARS corona virus spike protein blocks viral entry into host cells via the human receptor ACE2

Antiviral Research
Volume 94, Issue 3, June 2012, Pages 288-296

Anna-WinonaStruck, Marco Axmanna, Susanne Pfefferle, ChristianDrosten, Bernd Meyera

Abstract

In vitro infection of Vero E6 cells by SARS coronavirus (SARS-CoV) is blocked by hexapeptide Tyr-Lys-Tyr-Arg-Tyr-Leu. The peptide also inhibits proliferation of coronavirus NL63. On human cells both viruses utilize angiotensin-converting enzyme 2 (ACE2) as entry receptor. Blocking the viral entry is specific as alpha virus Sindbis shows no reduction in infectivity. Peptide 438YKYRYL443 is part of the receptor-binding domain (RBD) of the spike protein of SARS-CoV. Peptide libraries were screened by surface plasmon resonance (SPR) to identify RBD binding epitopes. 438YKYRYL443 carries the dominant binding epitope and binds to ACE2 with KD = 46 μM. The binding mode was further characterized by saturation transfer difference (STD) NMR spectroscopy and molecular dynamic simulations. Based on this information the peptide can be used as lead structure to design potential entry inhibitors against SARS-CoV and related viruses.

Keywords

SARS coronavirus, Spike protein, ACE2, Entry inhibitor, Virus proliferation assay, SPR screening

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

Lipid rafts are involved in SARS-CoV entry into Vero E6 cells

Biochemical and Biophysical Research Communications
Volume 369, Issue 2, 2 May 2008, Pages 344-349

Yanning Lu, Ding Xiang Liu, James P. Tam.

Abstract

Lipid rafts often serve as an entry site for certain viruses. Here, we report that lipid rafts in Vero E6 cells are involved in the entry of severe acute respiratory syndrome coronavirus (SARS-CoV). Infectivity assay showed the integrity of lipid rafts was required for productive infection of pseudotyped SARS-CoV. Depletion of plasma membrane cholesterol with MβCD relocalized raft-resident marker caveolin-1 as well as SARS-CoV receptor ACE2 to a nonraft environment, but did not significantly change the surface expression of ACE2. MβCD-treatment inhibited infectivity of pseudotyped SARS-CoV by 90%. Biochemical fractionation and confocal imaging confirmed that ACE2 colocalized with raft-resident markers. Furthermore, an ectodomain of SARS-CoV S protein (S1188HA) could associate with lipid rafts after binding to its receptor, and colocalize with raft-resident marker ganglioside GM1. The binding of S1188HA was not affected by depleting plasma membrane cholesterol. Taken together, our results support that lipid rafts serve as an entry port for SARS-CoV.

Keywords

Lipid rafts, SARS-CoV, Vero E6, ACE2, Spike protein, Entry

Inhibition of SARS-CoV replication by siRNA

Antiviral Research
Volume 65, Issue 1, January 2005, Pages 45-48

Chang-Jer Wu, Hui-Wen Huang, Chiu-Yi Liu, Cheng-Fong Hong, Yi-LinChan

Abstract

Serious outbreaks of severe acute respiratory syndrome (SARS), caused by the newly discovered coronavirus SARS-CoV, occurred between late 2002 and early 2003 and there is an urgent need for effective antiviral agents. RNA interference in animals and post-transcriptional gene silencing plants is mediated by small double-stranded RNA molecules named small interfering RNA (siRNA). Recently, siRNA-induced RNA interference(RNAi) may provide a new approach to therapy for pathogenic viruses, e.g. HIV and HCV. In this study, the silencing potential of seven synthetic siRNAs against SARS-CoV leader, TRS, 3′-UTR and Spike coding sequence have been applied to explore the possibility for prevention of SARS-CoV infection. We demonstrate that siRNAs directed against Spike sequences and the 3′-UTR can inhibit the replication of SARS-CoV in Vero-E6 cells, and holds out promise for the development of an effective antiviral agent against SARS-CoV.

Keywords

SARS-CoV, siRNA, Spike protein, Antiviral agent

Inactivated SARS-CoV vaccine elicits high titers of spike protein-specific antibodies that block receptor binding and virus entry

Biochemical and Biophysical Research Communications
Volume 325, Issue 2, 10 December 2004, Pages 445-452

Yuxian He, Yusen Zhou, Pamela Siddiqui, Shibo Jiang

Abstract

The only severe acute respiratory syndrome (SARS) vaccine currently being tested in clinical trial consists of inactivated severe acute respiratory syndrome-associate coronavirus (SARS-CoV). However, limited information is available about host immune responses induced by the inactivated SARS vaccine. In this study, we demonstrated that SARS-CoV inactivated by β-propiolactone elicited high titers of antibodies in the immunized mice and rabbits that recognize the spike (S) protein, especially the receptor-binding domain (RBD) in the S1 region. The antisera from the immunized animals efficiently bound to the RBD and blocked binding of RBD to angiotensin-converting enzyme 2, the functional receptor on the susceptible cells for SARS-CoV. With a sensitive and quantitative single-cycle infection assay using pseudovirus bearing the SARS-CoV S protein, we demonstrated that mouse and rabbit antisera significantly inhibited S protein-mediated virus entry with mean 50% inhibitory titers of 1:7393 and 1:2060, respectively. These data suggest that the RBD of S protein is a major neutralization determinant in the inactivated SARS vaccine which can induce potent neutralizing antibodies to block SARS-CoV entry. However, caution should be taken in using the inactivated SARS-CoV as a vaccine since it may also cause harmful immune and/or inflammatory responses.

Keywords

SARS-CoV, Vaccine, Spike protein, Receptor-binding domain, Antibodies