The Roles Of Calcium Ion And Its Related Proteins In The Entry Process Of Newcastle Disease Virus Infection In HeLa Cells

Newcastle disease?ND?has higher mortality and is a highly pathogenic viral disease of avian species caused by Newcastle disease virus?NDV?,which cause great economic losses to poultry in world wide.NDV is classified as a member of genus Avulavirus in the family Paramyxoviridae,which is an enveloped virus with non-segmented,negative sense,single stranded RNA.Geting further study of the mechanism of NDV entry is not only good for us to understand its pathogenesis,but also gives more ideas and theoretical support to development of specific antiviral drugs.As one of the most important second messengers in cell,calcium ion(Ca²⁺)play key roles in many cell biological activities.For better unstanding the roles of Ca²⁺and related proteins in the entry process of NDV infection in HeLa cells,we use chemical inhibitors and agonists,small interfering RNA,overexpression of dominant negative mutants to detect the dynamic changes of virus genome,NP proterin and p rotein kinase by realtime PCR and western blot while NDV infection in HeLa cells.The results showed that:During the process of NDV infection in HeLa cells,increaseing extracellular Ca²⁺concentration can promote the virus into the cells in a certain range while removing the extracellular Ca²⁺reduces virus entry.When we treated cells with Ca²⁺chelator,inhibitor or agonists of Ca²⁺channel and proteins of Ca²⁺downstream pathway,the number of genome entry and NP changed.All these results suggested that Ca²⁺participates in the entry process of NDV infection in HeLa cells.C-type of transient receptor potential Ca²⁺channel?TRPC?is one of the important ion channels in cell plasma membrane.The results of real-time PC R showed that TRPC4 is the most abundant channel in TRPC family.By transfecting small interfering RNA to knockdown TRPC4,we found it decreased NDV entry while increasing Ca²⁺concentration restored part of NDV entry.By overexpresseing wild type TRPC4 and domain-negative mutants,we found that the wild type TRPC4 increased NDV entry while the domain-negative mutants don't.These results suggested that TRPC4 as an important ion channel of Hella cells to mediate extracellular calcium ions flow into the cells,which promotes NDV entry.AnnexinA2?AnxA2?is one of the calcium regulated proteins in Ca²⁺signal pathway and it plays an important role at the early stages of the endocytic pathway.We found that knockdown AnxA2 decreased NDV entry while overexpression of AnxA2 increased NDV entry.But the promotion was greater than inhibiton.These results suggested that the entry of NDV don't completely depend on AnxA2,there might be other entry pathways.Lipid raft is microdomain in plasma membrane rich in cholesterol and phosphosphingolipid,which could serve as the platform of cell signal transduction and assemble the receptor complex.Lipid raft could rearrange receptors such as EGFR,ion channels and its downstream proteins to regulate incellular signal pathway.EGFR,TRPC4 and AnxA2locate in lipid raft to perform their function,which showes TRPC4 interact with AnxA2 and it was confirmed by co-immunoprecipitation.The cell signal transduction is associated with the virus entry.We detected the dynamic changes of EGFR activation during N DV entry either under the treatment of inhibitor or activator of Ca²⁺channels and calcium regulated proteins.The results showed that the activation of EGFR is necessary for NDV entry which might be Ca²⁺-depended.Cofilin?CFL?regulates the depolymerization of actin to facilate endocytosis.The activity of CFL is bidirectional.This result showed that actin involves in the entry process of N DV infect HeLa cells.Conclusion:As an important ion channel of Hella cells,TRPC4 promotes extracellular calcium ions flow into cells.Ca²⁺involves in the regulation of the activity of EGFR during the entry process of NDV infection in HeLa cells.