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

TACE antagonists blocking ACE2 shedding caused by the spike protein of SARS-CoV are candidate antiviral compounds

Antiviral Research
Volume 85, Issue 3, March 2010, Pages 551-555

Shiori Haga, Noriyo Nagata, Tadashi Okamura, Norio Yamamoto Tetsutaro Sata, Naoki Yamamoto, Takehiko Sasazuki Yukihito Ishizaka

Abstract

Because outbreaks of severe acute respiratory syndrome coronavirus (SARS-CoV) might reemerge, identifying antiviral compounds is of key importance. Previously, we showed that the cellular factor TNF-α converting enzyme (TACE), activated by the spike protein of SARS-CoV (SARS-S protein), was positively involved in viral entry, implying that TACE is a possible target for developing antiviral compounds. To demonstrate this possibility, we here tested the effects of TACE inhibitors on viral entry. In vitro and in vivo data revealed that the TACE inhibitor TAPI-2 attenuated entry of both pseudotyped virus expressing the SARS-S protein in a lentiviral vector backbone and infectious SARS-CoV. TAPI-2 blocked both the SARS-S protein-induced shedding of angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV, and TNF-α production in lung tissues. Since the downregulation of ACE2 by SARS-S protein was proposed as an etiological event in the severe clinical manifestations, our data suggest that TACE antagonists block SARS-CoV infection and also attenuate its severe clinical outcome.

Keywords

SARS-CoV, TACE, ACE2, Shedding

TACE antagonists blocking ACE2 shedding caused by the spike protein of SARS-CoV are candidate antiviral compounds

Antiviral Research
Volume 85, Issue 3, March 2010, Pages 551-555

Shiori Haga, Noriyo Nagata, Tadashi Okamura, Norio Yamamoto, Tetsutaro Sata, Naoki Yamamoto, Takehiko Sasazuki, Yukihito Ishizaka

Abstract

Because outbreaks of severe acute respiratory syndrome coronavirus (SARS-CoV) might reemerge, identifying antiviral compounds is of key importance. Previously, we showed that the cellular factor TNF-α converting enzyme (TACE), activated by the spike protein of SARS-CoV (SARS-S protein), was positively involved in viral entry, implying that TACE is a possible target for developing antiviral compounds. To demonstrate this possibility, we here tested the effects of TACE inhibitors on viral entry. In vitro and in vivo data revealed that the TACE inhibitor TAPI-2 attenuated entry of both pseudotyped virus expressing the SARS-S protein in a lentiviral vector backbone and infectious SARS-CoV. TAPI-2 blocked both the SARS-S protein-induced shedding of angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV, and TNF-α production in lung tissues. Since the downregulation of ACE2 by SARS-S protein was proposed as an etiological event in the severe clinical manifestations, our data suggest that TACE antagonists block SARS-CoV infection and also attenuate its severe clinical outcome.

Keywords

SARS-CoV, TACE, ACE2, Shedding

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