Revealing the interactions among cypovirus proteins would facilitate our understanding of the replication and assembly of this virus. In the present study, interactions among proteins encoded by the 10 segments of Dendrolimus punctatus cypovirus (DpCPV) were identified using yeast two-hybrid (Y2H) and far-Western blotting assays. In total, 24 pairs of interactions were detected. Twelve pairs of one-direction interactions, four pairs of binary interactions and four pairs of selfassociations were identified in the Y2H assays. Another four pairs of interactions were identified by far-Western blotting. The interactions between the methyltransferase domain of the turret protein (VP3) and VP4 as well as between polyhedrin and VP4 were further confirmed by farWestern blotting and pull-down assays, respectively. In addition, immunogold labelling showed that the A-spike of DpCPV is formed by VP4. In conclusion, we obtained a protein-protein interaction network of DpCPV and showed that its A-spike is formed by VP4 encoded by genomic segment 6.

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.