Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry

Abstract

Severe acute respiratory syndrome-associated coronavirus (SARSCoV) is a rapidly emerging pathogen with potentially serious consequences for public health. Here we describe conditions that result not only in the efficient expression of the SARS-CoV spike (S) protein on the surface of cells, but in its incorporation into lentiviral particles that can be used to transduce cells in an S glycoproteindependent manner. We found that although some primate cell lines, including Vero E6, 293T and Huh-7 cells, could be efficiently transduced by SARS-CoV S glycoprotein pseudoviruses, other cells lines were either resistant or very poorly permissive to virus entry. Infection by pseudovirions could be inhibited by several lysosomotropic agents, suggesting a requirement for acidification of endosomes for efficient S-mediated viral entry. In addition, we were able to develop a cell-cell fusion assay that could be used to monitor S glycoprotein-dependent membrane fusion. Although proteolysis did not enhance the infectivity of cell-free pseudovirions, trypsin activation is required for cell-cell fusion. Additionally, there was no apparent pH requirement for S glycoprotein-mediated cell-cell fusion. Together, these studies describe important tools that can be used to study SARS-CoV S glycoprotein structure and function, including approaches that can be used to identify inhibitors of the entry of SARS-CoV into target cells.

Comparison between SARS CoV and MERS CoV Using Apriori Algorithm, Decision Tree, SVM

“…Spike glycoprotein(S protein) is one of common structural proteins of coronavirus, belonging to class I fusion protein [5]. It is arranged on the surface of the virus forming a unique "corona", or crown-like appearance.…”

Evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses

“…Pseudotyping with retroviral and lentiviral vectors has been used to analyze the mechanism and the specificity of entry, which faithfully models infection of different cell types by virus (6,7,16,17). S-dependent entry occurs through pHsensitive endocytosis (6,7,16) and can be inhibited by antisera that protect against pulmonary viral replication in mice (8,18). To analyze the sensitivity of alternative SARS-CoV S glycoproteins to antibody neutralization, S sequences from alternative human isolates and two palm civet strains were synthesized as codon-modified cDNAs and inserted into expression vectors as described in ref.…”

“…Recent studies indicate that human angiotensin-converting enzyme 2 (hACE-2) protein is its major cellular receptor (5). S mediates viral entry, and membrane fusion is pH-dependent (6,7). SARS-CoV S is a major target of vaccine and antiviral drug development.…”

Protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection

“…This idea is based on the following two findings: (i) SARS-CoV infection can be blocked by lysosomotropic agents, and (ii) S protein expressed on cells is activated for fusion by trypsin. These results were obtained by studies using pseudotype retroviruses harboring SARS-CoV S protein on the envelope and those using S protein expressed on cells by expression vectors (10).…”

“…However, the entry pathway of SARS-CoV appears to be distinct from that of the other coronaviruses. Simmons et al (10) hypothesized that SARS-CoV enters cells by an endosomal pathway, and S protein is activated for fusion by trypsin-like protease in an acidic environment. This idea is based on the following two findings: (i) SARS-CoV infection can be blocked by lysosomotropic agents, and (ii) S protein expressed on cells is activated for fusion by trypsin.…”