The potent antiviral lectin GRFT displayed low nanomolar activity against SARS-CoV (Urbani strain) with an EC50
of 0.61 μg/ml (48 nM). Previously, we had speculated that GRFT would be likely to have activity against SARS-CoV due to its carbohydrate specificity and the known carbohydrate components of the SARS-CoV envelope glycoprotein S (17
). Extending our previous work (38
), here we detail the activity of GRFT against several strains of SARS-CoV and show consistent advantageous selectivity indices for all of the tested strains (Table ). The carbohydrate-binding agent Urtica dioica
agglutinin (UDA) has been reported by others to display anti-SARS-CoV activity (33
), but the concentrations necessary for inhibition were significantly higher than those that we report here for GRFT. This difference in activity could be attributed to the multivalent interactions that the three independent carbohydrate-binding sites afford GRFT (Fig. ). Other agents reported to show activity against SARS-CoV, including SARS-CoV protease inhibitors (2
) and SARS-specific heptad repeat peptides (3
), also show such activity only at significantly higher molar concentrations than GRFT. Though human recombinant alpha interferon (multiferon) and other host-targeted agents have demonstrated anti-SARS activity at low concentrations (Table ), their potency does not significantly exceed that displayed by GRFT, which directly targets viral envelope glycoproteins.
In addition to GRFT's activity against SARS-CoV, GRFT demonstrated broad-spectrum activity against a variety of Coronaviridae
, including those recently identified as human pathogens (Table ). Both group 1 and group 2 coronaviruses were susceptible to GRFT with similarly low nanomolar sensitivities. GRFT was active against coronavirus strains that utilize protein-protein interactions for viral targeting (e.g., ACE2 as a cellular receptor, SARS-CoV, and HCoV-NL63) and those that utilize protein-carbohydrate interactions for viral attachment (i.e., α-2,3-linked sialic acid moieties, IBV-CoV, and HCoV-OC43). The broad range of Coronaviridae
species sensitive to GRFT is a significant attribute for this antiviral protein, as this group of viruses appears to be capable of continuing zoonotic evolution and transfer to human hosts (24
). GRFT was active against several coronaviruses at concentrations less than the lowest tested concentration, with HCoV-NL63, a strain recently identified as a human pathogen (31
), displaying the greatest sensitivity (EC50
of <0.0032 μg/ml [0.25 nM]).
The molecular target through which GRFT appears to mediate its anticoronavirus activity is the surface envelope glycoprotein spike (S). GRFT binds directly to S as shown by ELISA studies showing the concentration-dependent binding to recombinant S (Fig. ). This mechanism is consistent with our previous studies of the mechanism of GRFT inhibition of HIV that revealed that GRFT binds to HIV-1 gp120 and prevents viral entry (18
). As with gp120, GRFT appears to bind to S via
interaction with oligosaccharide moieties. Here we show that the binding of GRFT to S can indeed be inhibited by millimolar concentrations of mannose (Fig. ). GRFT is known to bind to select monosaccharides (mannose, glucose, and N
-acetylglucosamine) in a multivalent manner via
its three independent carbohydrate-binding domains (Fig. ) (37
). The unique equatorial triangular orientation of these three sites has been shown to be ideally situated so as to allow for engagement of multiple triantennary arms of specific high-mannose oligosaccharides such as oligomannose 9 (38
). The oligosaccharide component of SARS-CoV S has been previously reported to contain multiple high-mannose oligosaccharides to which GRFT might bind (11
). In isothermal titration calorimetry studies of the GRFT/S binding interactions, we determined that GRFT binds to S with a stoichiometry of 3:1, indicating that there are multiple binding sites for GRFT on S (Table ). In this same study GRFT was shown to bind to S with a dissociation constant of 24.9 nM (Table ). This affinity is weaker than that between GRFT and HIV gp120, an interaction that was shown to have a stoichiometry of ~10:1. The difference in the affinity for GRFT between the two envelope glycoproteins is likely responsible for the difference seen in the antiviral activity of GRFT against HIV (0.048 to 0.63 nM) (17
) and SARS-CoV (48 to 94 nM). Finally, we found that GRFT does not inhibit the interaction between the SARS-CoV S protein and the cellular receptor ACE2 (data not shown). Thus, the interaction between GRFT and S results in a complex that, though still able to bind to ACE2, may prevent the subsequent steps necessary for viral entry. The activity of GRFT against IBV suggests that this perturbation of viral entry by GRFT may be independent of the specific cellular receptor to which S binds. This mechanism would be similar to that seen with GRFT and HIV wherein GRFT binding to gp120 does not prevent the subsequent binding of gp120 to the cellular receptor CD4 and where GRFT activity is independent of the chemokine receptor tropism (CCR5 or CXCR4) of individual strains of HIV-1 (17
The potent in vitro
activity of GRFT against SARS-CoV was confirmed using a mouse model system recently shown to recapitulate several aspects of the physiology of human disease, including a robust pulmonary disease component (23
). This mouse model was previously used to examine the impact of prior infection with SARS-CoV on immune responses and survival following subsequent reexposure to the virus (23
). Previously our group has shown that intranasal administration of the antiviral lectin cyanovirin-N (CV-N) was efficacious in the prevention of mortality in mice infected with a lethal strain of influenza virus H1N1 (28
), and so, for the current study, GRFT was administered to mice via
an intranasal route.
These studies showed that GRFT protected mice from a lethal inoculum of mouse-adapted SARS-CoV. Remarkably, 100% of the animals treated with 10 mg/kg/day GRFT survived viral challenge, in contrast to only 30% of control animals (Fig. ). The improved survival was further mirrored by prevention of weight loss and an improvement in lung histopathology scores and a reduction in lung tissue virus titers (Fig. and Fig. ), which indicated that, though GRFT-treated animals were infected by SARS-CoV, drug treatment significantly modified the disease course and outcome.
While studies of the prevention or treatment of SARS-CoV lung disease have been hampered by the lack of animal models that faithfully recapitulate the features of human disease, several agents have shown some efficacy in modifying disease outcomes. These include alpha interferon (10
), small interfering RNA (siRNA) (15
), and passive immunization (16
). Furthermore, in a previous study we showed that, during a simultaneous experiment, the cyclic antimicrobial peptide rhesus theta-defensin did not afford the complete protection from the morbidity of SARS-CoV infections seen with GRFT (34
). Our studies with a robust model of SARS-CoV lung disease suggest that GRFT may modify disease outcome by more than one mechanism. First, by binding to the spike glycoprotein and interfering with productive infection, GRFT may reduce the overall virus burden during the first and subsequent rounds of infection. The reduced virus load in the lung at days 2 and 4 is consistent with this idea. In addition, enhanced peribronchial mononuclear cell infiltration and modification of cytokine responses suggest that GRFT also is immunomodulatory. Stimulation of leukocyte infiltration has been reported following high-dose topical application of GRFT to rabbit (but not human) cervical mucosa (18
GRFT is currently being developed for potential use as an anti-HIV microbicide, and a recent report demonstrates the feasibility of large-scale production and purification from Nicotiana benthamia
). This new production stream for GRFT greatly enables future development efforts based on GRFT's activity against enveloped viruses. Here we report that GRFT shows remarkable efficacy against lethal SARS-CoV infection and was potently active against a broad spectrum of human coronaviruses and other animal coronaviruses. Several questions remain, however, for the potential development of GRFT for use in the treatment of respiratory infections by coronaviruses. Though GRFT completely protected animals from SARS-CoV-induced death, the presence of perivascular infiltrates in GRFT-treated animals will need to be further characterized. Since GRFT-treated infected animals recover, this suggests that these cellular infiltrates may mediate protective immunity to SARS-CoV. Further, we used 10-mg/kg/day GRFT treatment in the in vivo
studies reported here, but it is possible that lower doses of GRFT treatment would be equally efficacious. In summary, the antiviral protein GRFT shows noteworthy activity against Coronaviridae
a novel mechanism of action. Its outstanding in vivo
efficacy in SARS-CoV-infected mice suggests that this antiviral agent merits further investigation for the prophylaxis or treatment of respiratory infection by susceptible viruses.