MTase Domain of Dendrolimus punctatus cypovirus VP3 Mediates Virion Attachment and Interacts with Host ALP Protein

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.

Heat shock protein 70, glutamate dehydrogenase, and angiotensin-converting enzyme of Bombyx mori mediate the cell attachment of Cypovirus 1

“…In a previous study, the BBMVs of B. mori were used to screen the host proteins that bound to VAPs, and only ALP was identified to interact with the MTase domain of DpCPV [24]. To identify more host proteins that mediate DpCPV-1 attachment, we used B. mori HMPs to study viral attachment.…”

“…The brush border membrane vesicles (BBMVs) of B. mori were prepared according to the method provided previously [24]. The proteins of BBMVs were extracted based on differential magnesium precipitation.…”

“…To obtain DpCPV-1 virions from polyhedra, suspensions of the purified polyhedra were digested with 0.2 mol·l −1 Na 2 CO 3 –NaHCO 3 (pH 10.8) solution to obtain clear lysate. Then, 1 mol·l −1 Tris-HCl (pH 6.8) was added to adjust the pH to 7.5, and the crude extract of DpCPV-1 virions was then purified via linear 20–60 % (w/v) sucrose gradient centrifugation [24]. The concentration of the purified DpCPV-1 virions was measured using a UV-31000PC spectrophotometer (Mapada, Shanghai, China) at 280 nm.…”

“…The VP3 segment of DpCPV-1 was split into GTase (amino acids 1-366) and MTase (amino acids 406-1058) domains according to previous research [9,24]. DpCPV-1 segments encoding GTase, MTase, and VP4 were amplified from a cDNA library constructed previously [10] using corresponding PCR primer pairs (Sangon, Shanghai, China) (Table S1, available in the online version of this article).…”

“…One study found that integrin beta and receptor for activated protein kinase C play important roles in the cell entry of Bombyx mori cypovirus [23]. Previous research identified the MTase domain of B-spike and A-spike of DpCPV-1 as viral attachment proteins, and alkaline phosphatase (ALP) of B. mori is identified as the ligand for MTase during DpCPV-1 infection [24].…”