Abstract
Dendrolimus punctatus cypovirus (DpCPV) is an important pathogen of D. punctatus, but little is known about the mechanisms of DpCPV infection. Here, we investigated the effects of VP3, VP4 and VP5 structural proteins on the viral invasion. Both the C-terminal of VP3 (methyltransferase (MTase) domain) and VP4 (A-spike) bound to Spodoptera exigua midgut brush border membrane vesicles (BBMVs) in a dose-dependent manner, and the binding was inhibited by purified DpCPV virions. Importantly, anti-MTase and anti-VP4 antibodies inhibited viral binding to S. exigua BBMVs. Using far-Western blots, a 65 kDa protein in Bombyx mori BBMVs, identified as alkaline phosphatase protein (BmALP) by mass spectrometry, specifically interacted with DpCPV MTase. The interaction between MTase and BmALP was verified by co-immunoprecipitation in vitro. Pretreatment of B. mori BBMVs with an anti-ALP antibody or incubation of DpCPV virions with prokaryotically expressed BmALP reduced viral attachment. Additionally, BmALP inhibited DpCPV infection in S. exigua larvae. Our data provide evidence that the MTase domain and A-spike function as viral attachment proteins during the DpCPV infection process, and ALP is the ligand that interacts with DpCPV via the MTase domain. These results augment our understanding of the mechanisms used by cypoviruses to enter their hosts.
Highlights
Cypovirus (CPV), a segmented double-stranded ribonucleic acid virus within the genusCypovirus (Reoviridae family), possesses a single capsid layer and is commonly embedded in the characteristic crystalline inclusion body called a polyhedron, which is formed in the cell cytoplasm of insects [1]
We showed that MTase and VP4 functioned as the viral attachment proteins in the Dendrolimus punctatus cypovirus (DpCPV) infection process and alkaline phosphatase protein (ALP) served as the MTase ligand
To confirm whether DpCPV virions can bind to SeBBMVs, an ELISA was performed in which
Summary
Cypovirus (CPV), a segmented double-stranded ribonucleic acid (dsRNA) virus within the genus. Cypovirus (Reoviridae family), possesses a single capsid layer and is commonly embedded in the characteristic crystalline inclusion body called a polyhedron, which is formed in the cell cytoplasm of insects [1]. CPV infection occurs via the fecal-oral route and is mainly restricted to the larvae midgut. After the consumption of contaminated food by the larvae, the polyhedra dissolve in the highly alkaline environment of the midgut, releasing virions that adhere to and penetrate the plasma membrane of microvilli and settle in the cytoplasm of the columnar epithelial cells [2]. The recent study showed that CPV enter the cell by clathrin-mediated endocytosis employing integrin beta and receptor for activated protein kinase C (RACK1) [3]. The digestive and absorptive functions are severely affected and the larvae die one after another
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