Study On Mechanism Of Internalization Of African Swine Fever Virus Virions

African swine fever(ASF)is an acute,highly contagious infectious disease affecting domestic pigs and wild boars,which is classified as a notifiable disease by the World Organization for Animal Health(WOAH).Since the ASF was reported in the 1920 s,it has been spread around the world for more than one hundred years,resulting in huge economic losses to the global pig industry.ASF is caused by the African swine fever virus(ASFV)and it is the only member of the genus ASFV.Due to its huge genome structure and complex escape host antiviral mechanisms,there are no safe,effective vaccines and drugs to prevent and control ASF disease.Exploring the invasion mechanism of ASFV is helpful to further understand the ASFV infection,pathogenesis,which will provide a clue to develop new vaccines and antiviral breeding.The virus invasion is mainly mediated by the interaction between viral capsid proteins and receptor(s)on the cell members.p72 protein is the most abundant capsid protein on the surface of ASFV particles.Previous studied showed that p72 can induce neutralizing antibodies to prevent ASFV infection and block attachment of ASFV virions to the surface of macrophages.It has also been reported that ASFV virions without outer capsular membrane still have infection ability.Therefore,we speculated that ASFV p72 may interact with viral entry-related host factors on the cell membrane to mediate viral entry.Previous results showed that labeled-ASFV virions were adhered to the surface of cell membrane,and anti-p72 antibody can block the process and inhibit ASFV infection.Therefore,p72 was selected as a “bait” to screen for ASFV entry-related host factors.Several p72 binding proteins were identified by immunoprecipitation-mass spectrum(IP-MS).Among them,CD1 d,a member of a family of glycoproteins expressed on the surface of multiple antigen-presenting cells,had the highest score,and the interaction between the two proteins and co-localization were confirmed.In order to verify whether CD1 d was involved in ASFV infection,we verified in PAMs via RNA interference CD1 d expression,CD1 d antibody blocking and CD1 d protein neutralization tests,and the results showed that the infection level of the virus was significantly inhibited.In addition,overexpression of CD1 d was performed in ASFV non-susceptible cell lines,and the results showed that overexpression of CD1 d significantly increased the ASFV infection.Moreover,the knockout and complementation of CD1 d was performed in MA104 cells and the results showed that the deletion of CD1 d significantly inhibited ASFV infection and the infection could be restored after the complementation.These results suggested that CD1 d played an important role in the infection of ASFV.In order to further verify which step of ASFV invasion was mainly affected by CD1 d,the adhesion and internalization tests were performed.The results showed that after CD1 d knockout or knockdown,the number of attached virions was not reduced,but the number of internalized virions was significantly reduced.Consistent results were also obtained by observing the attachment and internalization of Di D-labeled ASFV virions(Di D-ASFV).Further the study found that CD1 d participated in the internalization of ASFV via clathrin-mediated endocytosis(CME).Mechanically,I found that the adaptation protein EPS15 in the CME pathway interacted with the intracellular region of CD1 d.It is worth noting,CD1 d is necessary for the formation of p72-CD1d-EPS15 complex.These results suggest that CD1 d might act as a bridging protein to mediate the interaction between p72 and EPS15 and regulate the internalization process of ASFV.ASFV completes its entry process via various ways.Besides CD1d-mediated ASFV entry process via CME,there are other entry routes.It has been reported that many viruses can invade the host cells via viral apoptotic mimicry.This pathway is characterized by the presence of phosphatidylserine(PS)on the surface of virions,which is a marker of apoptosis and can be recognized by phagocytes to mediate virus invasion.To verify whether ASFV can invade the host cells via this pathway,we tested PS expression by flow cytometry and dot blot assay.The results showed that PS was indeed present on the surface of purified ASFV virions.At the same time,I found that anti-phosphatidylserine antibodies can inhibit viral infection in a dose-dependent manner,suggesting that ASFV entry into host cells may be mediated by viral apoptotic mimicry.Through RNA interference assay,I screened phosphatidylserine receptors(PSRs)that mediated ASFV invasion,and found that AXL,a member of the TAM Receptor Tyrosine Kinase(RTK)family,was the most obvious PSR that affected ASFV infection,so AXL was selected for further study.Through AXL knockout,AXL overexpression and AXL antibody blocking assay,the results showed that AXL played a crucial role in ASFV infection.Moreover,we tested which stage of ASFV invasion AXL may be affected by RNA interference and knockout cells,and the results showed that AXL could affect the attachment and internalization stage of the virus.Further studies showed that AXL affected the internalization of ASFV mainly through macropinocytosis.Mechanistically,we found that kinase activity of AXL is essential for the internalization of ASFV.In addition,during the internalization of ASFV,AXL phosphorylated to activate downstream endocytosis signals.In summary,I found that capsid protein p72 can bind to CD1 d on the cell membrane during the ASFV infection,and then EPS15 was recruited to form p72-CD1d-EPS15 complex to mediate the internalization of ASFV virions.Additionally,I found that ASFV can invade the host cells via viral apoptotic mimicry,and found that AXL participated in internalization of ASFV virions through macropinocytosis.Our findings reveal new molecular mechanisms involved in invasion of ASFV virions entry,which provides a new theoretical basis for the prevention and control of ASF.