Binding and entry of a non-enveloped T=4 insect RNA virus is triggered by alkaline pH

Tetraviruses are small, non-enveloped, RNA viruses that exclusively infect lepidopteran insects. Their particles comprise 240 copies of a single capsid protein precursor (CP), which undergoes autoproteolytic cleavage during maturation. The molecular mechanisms of capsid assembly and maturation are well understood, but little is known about the viral infectious lifecycle due to a lack of tissue culture cell lines that are susceptible to tetravirus infection. We show here that binding and entry of the alphatetravirus, Helicoverpa armigera stunt virus (HaSV), is triggered by alkaline pH. At pH 9.0, wild-type HaSV virus particles undergo conformational changes that induce membrane-lytic activity and binding to Spodoptera frugiperda Sf9 cells. Binding is followed by entry and infection, with virus replication complexes detected by immunofluorescence microscopy within 2h post-infection and the CP after 12h. HaSV particles produced in S. frugiperda Sf9 cells are infectious. Helicoverpa armigera larval virus biofeed assays showed that pre-treatment with the V-ATPase inhibitor, Bafilomycin A1, resulted in a 50% decrease in larval mortality and stunting, while incubation of virus particles at pH 9.0 prior to infection restored infectivity. Together, these data show that HaSV, and likely other tetraviruses, requires the alkaline environment of the lepidopteran larval midgut for binding and entry into host cells.