Broad-Spectrum In Vitro Activity and In Vivo Efficacy of the Antiviral Protein Griffithsin against Emerging Viruses of the Family Coronaviridae

Viruses of the family Coronaviridae have recently emerged through zoonotic transmission to become serious human pathogens. The pathogenic agent responsible for severe acute respiratory syndrome (SARS), the SARS coronavirus (SARS-CoV), is a member of this large family of positive-strand RNA viruses that cause a spectrum of disease in humans, other mammals, and birds. Since the publicized outbreaks of SARS in China and Canada in 2002-2003, significant efforts successfully identified the causative agent, host cell receptor(s), and many of the pathogenic mechanisms underlying SARS. With this greater understanding of SARS-CoV biology, many researchers have sought to identify agents for the treatment of SARS. Here we report the utility of the potent antiviral protein griffithsin (GRFT) in the prevention of SARS-CoV infection both in vitro and in vivo. We also show that GRFT specifically binds to the SARS-CoV spike glycoprotein and inhibits viral entry. In addition, we report the activity of GRFT against a variety of additional coronaviruses that infect humans, other mammals, and birds. Finally, we show that GRFT treatment has a positive effect on morbidity and mortality in a lethal infection model using a mouse-adapted SARS-CoV and also specifically inhibits deleterious aspects of the host immunological response to SARS infection in mammals.The Coronaviridae are a group of enveloped positive-strand RNA viruses of the group Nidovirales. This group of viruses was not, until recently, of major concern as a matter of public health, although they were long recognized as important agents of serious disease in domestic and companion animals. The recent evidence of zoonotic transfer of this family of viruses from bats to animals such as palm civet cats and then to humans during the 2002-2003 outbreak greatly increased scientific interest in the Coronaviridae (7, 14, 19). The best-known coronavirus (CoV) is the causative agent of severe acute respiratory syndrome (SARS), termed the SARS-related coronavirus (SARS-CoV) (7, 14, 19). The lethal SARS outbreaks in China and Canada in 2002-2003 first brought SARS-CoV to public attention. The subsequent identification of two new human coronaviruses associated with acute respiratory infections in humans further illuminated the continuing potential threat that coronaviruses present to public health (31, 36).Infection with SARS-CoV results from the binding of SARS-CoV spike glycoprotein (S) to angiotensin-converting enzyme 2 (ACE2) on the surface of susceptible cells in the lung followed by viral fusion with host cell membranes and transfer of virion contents into the cell (12, 25, 27). The infection stimulates significant cytokine responses in lung tissue that, together with pathologies associated with rapidly replicating virus, cause damage to the airway epithelium and alveolar membranes resulting in edema, respiratory distress, and (in ∼10% of cases) death (5). Due to the proven threat from SARS-CoV infections and the possibility of future zoonotic transmission of coronaviruses, efforts have been initiated to identify agents that could either reduce infection or suppress the deleterious cytokine response to SARS-CoV infection (8, 29).The molecular physiology of the SARS-CoV life cycle and the host response to infection have provided numerous potential targets for chemotherapeutic intervention. In addition to vaccine development strategies, various research groups have targeted the SARS-CoV-specific main protease or viral attachment, entry, and fusion for intervention. SARS-CoV protease inhibitors which inhibit the enzyme at concentrations from 0.5 to 7 μM have been reported (2). The SARS-CoV papain-like protease (PLP) has also been successfully developed as a target for small-molecule antivirals, some of which are active in the 100 nM range (22). Viral entry inhibitors include SARS-CoV S glycoprotein heptad repeat peptides identified as potential inhibitors of viral fusion (3). Another broad-spectrum antiviral approach involves targeting the high-mannose oligosaccharides that are commonly found on viral surface glycoproteins. For example, carbohydrate-binding lectins, including Urtica dioica agglutinin (UDA), have been reported to bind to the SARS-CoV S protein and inhibit viral fusion and entry (33).The antiviral protein griffithsin (GRFT) was originally isolated from the red alga Griffithsia sp. based upon its activity against the human immunodeficiency virus (HIV) (17). This unique 12.7-kDa protein was shown to bind specifically to oligosaccharides on the surface of the HIV envelope glycoprotein gp120. GRFT was shown to possess three largely identical carbohydrate-binding domains orientated as an equatorial triangle and affording multivalent binding and thereby increasing potency (37) (Fig. ​(Fig.1).1). Due to GRFT''s ability to bind to specific oligosaccharides on envelope glycoproteins and block viral entry, it was hypothesized that GRFT might show broad-spectrum antiviral activity against other viruses, including SARS-CoV (38). Here we report the testing of GRFT for antiviral activity against a spectrum of coronaviruses, including SARS-CoV. In addition we present data on the specific binding interactions between GRFT and the SARS-CoV S protein. Finally, we evaluate the in vivo efficacy of intranasal administration of GRFT against infection with SARS-CoV in a lethal mouse model of pulmonary infection and explore the effects that GRFT treatment has on the induction of host cytokine response to SARS-CoV infection.Open in a separate windowFIG. 1.The amino acid sequence and carbohydrate binding domains of griffithsin. Griffithsin monomers contain three distinct, nonlinear, and uniform binding sites for monosaccharides such as mannose and glucose. The binding sites (red, blue, and yellow) are shown both in the amino acid sequence of griffithsin (A) and binding to the disaccharide maltose in a three-dimensional representation derived from the X-ray crystal structure (B).