Porcine Deltacoronavirus Modulates Calcium Influx To Favor Viral Replication

Ionic calcium(Ca²⁺)is a versatile intracellular second messenger,which regulates various important physiological functions of cells,such as cell proliferation and differentiation,energy metabolism,autophagy,apoptosis and so on.In the resting state,intracellular free calcium concentration is about 100 n M,but the concentration of calcium pools such as mitochondria and endoplasmic reticulum can reach several hundred?M.Calcium is maintained in the intracellular and extracellular milieu with the existence of a concentration gradient that depends on a series of channels,transporters and pumps.Once the homeostasis of calcium modulated in cell is breaked down,and it may cause disease.It has been proved that many viruses can promote their own infection by regulating the calcium channel or calcium level of cells.The clinical manifestations of vomiting and diarrhea caused by rotavirus infection in animals are closely related to the influx of calcium.PDCoV is a new type of porcine intestinal coronavirus that discovered in recent years,which mainly causes clinical symptoms including vomiting,diarrhea,dehydration and even the death of piglets.PDCoV has caused severe economic losses to the pig industry.However,whether PDCoV infection promotes its own replication and pathogenicity by regulating calcium channels and calcium levels has not been reported.Therefore,our research were carried out on the role of Ca²⁺during PDCoV infection.The specific research content is as follows:2.1.PDCoV infection increases the cytosolic Ca²⁺concentrationIPI-2I and LLC-PK1 cells were preloaded with Fluo-3AM,and cells were infected with PDCoV at a multiplicity of infection?MOI?of 3.The fluorescence was measured at5-min intervals.It was found that the cytosolic Ca²⁺concentration was increased at 80min or 120 min after PDCoV infection.In order to explore intracellular Ca²⁺levels during entire infection cycle of PDCoV,the fluorescence was measured from 2 h postinfection to12 hpi in IPI-2I cells.The results showed that cytosolic Ca²⁺levels progressively increased after PDCoV infection,and the high concentration of intracellular calcium may be important for viral infection.2.2 Chelating extracellular Ca²⁺by EGTA inhibits PDCoV infectionIPI-2I cells were pretreated with EGTA?0.5,1.0,2.0 m M?for 1 h,and then mock infected or infected with PDCoV?MOI=0.5?.The unabsorbed viruses were removed and then cells were inoculated with medium with or without EGTA for an additional 6 h or 12h.The cells were collected to detect the genomic m RNA of PDCoV and the expression of the viral nucleocapsid?N?protein.The results showed that EGTA treatment decreased the PDCoV yield significantly at both 6 hpi and 12 hpi.We further tested whether replenishment of extracellular Ca²⁺can recover virus yield.IPI-2I cell were treated with EGTA?2 m M?and then Ca Cl₂?1.0 or 2.0 m M?was added,and this was followed by infection with PDCoV?MOI=0.5?.The cells were collected at 6 and 12 hpi to determine viral RNA,protein expression and viral titers.The inhibitory effects were restored after the addition of Ca Cl₂.To further confirm the role of extracellular Ca²⁺in viral replication,we also compared the growth curve of PDCoV in normal medium and calcium-free medium.The results showed that the virus titers of PDCoV in calcium-free medium were significantly lower than those in normal medium.Taken together,extracellular Ca²⁺plays an important role in PDCoV infection.2.3 Intracellular Ca²⁺chelator and channel blockers inhibit PDCoV productionWe selected several commonly used calcium channel inhibitors:Diltiazem hydrochloride?DTZ?,the L-type calcium channel blocker;Bepridil hydrochloride,the long-acting and non-selective calcium channel blocker;2-APB,the IP3R antagonist.The intracellular Ca²⁺chelator BAPTA-AM was also included.All tested drugs inhibited the expression of PDCoV m RNA and N protein significantly,as well as cause a reduction in viral titers at 6 hpi and 12 hpi.Among the four drugs,the inhibitory effects of DTZ were the most pronounced.2.4 Diltiazem hydrochloride inhibits the replication step of PDCoV infectionOwing to the inhibitory effects of Diltiazem hydrochloride were the most pronounced.Thus,we chosed it for subsequent experiments.To further explore the possible mechanism by which DTZ inhibits PDCoV infection,the study on the adsorption,invasion,replication and release of PDCoV with drugs were evaluated.We found that DTZ inhibits the viral infection by reducing the synthesis of negative-strand RNA of the virus,and had no obvious effect on other steps.2.5 Knockdown of CACNA1S inhibits PDCoV replicationWe all known that the L-type Ca²⁺voltage-gated channel subunit alpha1?CACNA1?,which includes four splice isoforms:CACNA1S,CACNA1C,CACNA1D and CACNA1F.We analyzed the expression abundance of different isoforms in IPI-2I cells and found that the m RNA expression of CACNA1S was significantly higher than others.We packaged the recombinant lentivirus,which expressing CACNA1S-specific sh RNA in HEK-293T cells.IPI-2I and LLC-PK1 cells were transduced with recombinant lentivirussh CACNA1S for 72 h and infected with PDCoV?MOI=0.5?for 12 h.The resulted demonstrated that knockdown of CACNA1S inhibited PDCoV infection,which confirmed that the L-type calcium channels play an important role in PDCoV infection.