Hantavirus Infection Is Inhibited by Griffithsin in Cell Culture

Abstract

Andes virus (ANDV) and Sin Nombre virus (SNV), highly pathogenic hantaviruses, cause hantavirus pulmonary syndrome in the Americas. Currently no therapeutics are approved for use against these infections. Griffithsin (GRFT) is a high-mannose oligosaccharide-binding lectin currently being evaluated in phase I clinical trials as a topical microbicide for the prevention of human immunodeficiency virus (HIV-1) infection (ClinicalTrials.gov Identifiers: NCT04032717, NCT02875119) and has shown broad-spectrum in vivo activity against other viruses, including severe acute respiratory syndrome coronavirus, hepatitis C virus, Japanese encephalitis virus, and Nipah virus. In this study, we evaluated the in vitro antiviral activity of GRFT and its synthetic trimeric tandemer 3mGRFT against ANDV and SNV. Our results demonstrate that GRFT is a potent inhibitor of ANDV infection. GRFT inhibited entry of pseudo-particles typed with ANDV envelope glycoprotein into host cells, suggesting that it inhibits viral envelope protein function during entry. 3mGRFT is more potent than GRFT against ANDV and SNV infection. Our results warrant the testing of GRFT and 3mGRFT against ANDV infection in animal models.

Keywords: antiviral; griffithsin; haemorrhagic fever; hantaviridae; hantavirus.

Figure 1

Griffithsin inhibits Andes virus replication. Griffithsin (GRFT) inhibited Andes virus (ANDV) replication in a concentration-dependent manner. (A) Vero-E6 cells were treated for 1 h with varying concentrations of GRFT before infection with ANDV at a multiplicity of infection (MOI) of 0.1. At 72 h post infection, the cells were fixed, permeabilized, and stained with an antibody against Puumala virus nucleoprotein that is cross-reactive with ANDV nucleoprotein. Green, ANDV nucleoprotein; blue, cell nuclei; red, cell cytoplasm. (B,C) Dose-response curve showing the quantitation of ANDV-infected cells after GRFT (B) or 3mGRFT (C) treatment (% normalized to the vehicle-only control). (D,E) Inhibition (neutralization) of ANDV by GRFT. Increasing concentrations of GRFT were pre-incubated with 2,000 TCID₅₀ of ANDV at 37°C for 1 h, and virus-GRFT (D) or virus-3mGRFT (E) mixtures were added to Vero-E6 cells. After 2 h of incubation at 37°C, inoculum was removed, and cells were replenished with fresh medium. After 3 days, cells were fixed, imaged, and analyzed as in (B). (F,G) GRFT inhibited titers of infectious ANDV. 2000 TCID₅₀ of ANDV were pre-incubated with varying concentration of GRFT (F) or 3mGRFT (G) as in E. At 72 h, supernatants were harvested, and viral yield was determined by TCID₅₀ assays on Vero-E6 cells. Graphs represent the mean ± SD and are representative of three independent experiments, performed in quadruplicate.

Figure 2

GRFT and 3mGRFT minimally affect post viral entry steps. Vero-E6 cells were infected with ANDV at MOI of 0.2 for 2 h at 37°C. Cells were then treated with varying concentrations of GRFT (A) or 3mGRFT (B). At 72 h post infection, cells were fixed, stained, and analyzed as in Figure 1B. Dose-response curve shows quantitation of ANDV-infected cells treated with GRFT (A) or 3mGRFT (B) (% normalized to vehicle-only control). Graphs represent the mean ± SD and are representative of three independent experiments, performed in quadruplicate.

Figure 3

GRFT and 3mGRFT affect viral entry. HIV particles bearing glycoproteins of ANDV or vesicular stomatitis virus (VSV) were prepared and used as previously described. Viral glycoprotein-dependent entry assays were performed using HT1080 cells pre-treated with serial dilutions of GRFT and inoculated with either ANDV pseudo-particles (pp) or control VSVpp in the presence of GRFT (A) or 3mGRFT (B) at increasing concentrations. Intracellular firefly luciferase signal was measured 72 h post transduction and normalized to signal in untreated, untransduced cells. Graphs represent the mean ± SD and are representative of three independent experiments, performed in quadruplicate. (C) Vero-E6 cells were treated for 1 h with either 1 or 5 μg/mL of GRFT or 3mGRFT before infection with ANDV at MOI of either 1or 3. At 24 h post infection, the cells were fixed, permeabilized, and stained with an antibody against Puumala virus nucleoprotein as described in Figure 1. Column chart showing the quantitation of ANDV-infected cells treated with GRFT or 3mGRFT (% normalized to vehicle-only controls). Graphs represent the mean ± SD and are representative of 3 independent experiments, performed in quadruplicate. P^* < 0.001, P^*** < 0.0001.

Figure 4

Antiviral effects of GRFT and 3mGRFT on Sin Nombre virus replication. Vero-E6 cells were treated for 1 h with either 1 or 5 μg/mL of GRFT or 3mGRFT before infection with Sin Nombre virus (SNV) at MOI of 0.2. At 72 h post infection, the cells were fixed, permeabilized, and stained with an antibody against Puumala virus nucleoprotein that cross-reacts with SNV nucleoprotein. Dose-response curve showing the quantitation of SNV-infected cells treated with GRFT or 3mGRFT (% normalized to vehicle-only controls). Graphs represent the mean ± SD and are representative of three independent experiments, performed in quadruplicate. P^* < 0.05, P^*** < 0.001.