Avian Leukosis Virus Subgroup J(ALV-J)Hijacks Collagen Triple Helix Repeat Containing 1(CTHRC1)to Facilitate Its Replicaiton

Avain leukosis virus subgroup J is a typical tumorigenic retrovirus that mainly encodes three genes,including viral capsid protein(gag),polymerase(pol)and envelope protein(env).When ALV-J infects host cell,it first adsorbs the receptor of cell-specific and then enteres cytoplasm.ALV-J is able to mediem the transcripation of viral genome from viral RNA into proviral DNA carried by itself Reverse transcriptase,proviral DNA then insecte host genome that utilizes host' RNA polymerase to generate profeny virus RNA.These profeny virus RNA is then translated to product various precursor proteins of the virus and mature proteins assembled with virions.Finally,ALV-J is releasing to extracellular by budding.ALV-J life cycle involves in complex physiological processes from adsorption to shelling(budding),and like other viruses,ALV-J lacks complete enzyme system required for self-replication for the simple reason that it genomes are relatively simple.Therefore,the life cycle of ALV-J requires for hijacking host proteins,which are utilized by virus to help its competitive virus replication.But,so far,there have been few reports on how ALV-J hijacks host protein to facilicate its replication.In order to clarify the host protein that plays a key role in ALV-J replication,proteomis(Tandem mass tag,TMT)was performed in that investagete.Protein sample of DF-1 cells of ALV-J infected and normal were analysised by mass spectrometry.Collagen triple helix repeat containing 1(CTHRC1)was then screened,for it may involve in ALV-J replication.To further confirm the role that ALV-J activated the expression of CTHRC1,ALV-J infected cells model and ALV-J congenitally infected chicken embryo model were established in this study.The results showed that ALV-J can activate the expression of CTHRC1 in vitro by qPRC,western blot and ELISA.Meanwhile,Immunohistochemistry(IHC)results showed that ALV-J can also activate the expression of CTHRC1 in vivo.The above results indicate that ALV-J infection activates the expression of CTHRC1 both in vitro and in vivo.ALV-J infection can specifically activate the expression of CTHRC1.Is high expression of CTHRC1 beneficial for ALV-J replication? In order to confirm the relationship between CTHRC1 expression and ALV-J replication,the plasmid of CTHRC1 overexpression and CTHRC1 interference were constructed in that investagete.The results demonstrated that compared with mock cells,the expression of CTHRC1 has significantly decreaed in transfected shRNA cells,and then ALV-J load was also distinctly down-regulated.Meanwhile,the expression of CTHRC1 was significantly increased when cells transfected the plasmid of overexpression CTHRC1,ALV-J load was then up-regulated for the inceased of CTHRC1 expression,and both CTHRC1 level and ALV-J load are positive correlation.The above results indicate that there may a close relationship between CTHRC1 and ALV-J,but it has been no understanding that whether CTHRC1 and ALV-J are interaction.In order to verify the interaction between the two,Co-immunoprecipitation and laser confocal were performed,respectively.CTHRC1 was precipitated by the ALV-J SU monoclonal antibody 1D4 as a bait.The precipitated product was then analyzed by western blot and found that CTHRC1 interacte with ALV-J SU potein in that test.Meanwhile,laser confocal results found that the subcellular localization of ALV-J and CTHRC1 in ALV-J-infected cells is in the cytoplasm,and the fluorescence signals of the two can overlap to produce yellow fluorescence,laser confocal experiments is also auxiliart to confirm the interaction between the two;laser confocal experiments also found that CTHRC1 was mainly diffusely distributed in the nucleus in normal DF-1 cells;whereas CTHRC1 was distributed around the nucleus in the cytoplasm in ALV-J-infected cells.Therefore,the results indicate that the subcellular localization of CTHRC1,a shuttle protein,can be changed from nuclear to cytoplasm due to ALV-J infection.Taken together,this study found that ALV-J can significantly activate CTHRC1 expression,and activated CTHRC1 is directly interacted with ALV-J SU protein to promote its replication;CTHRC1 acts as a shuttle protein,ALV-J can drive the subcellular localization of CTHRC1 from the nucleus to the cytoplasm.This study provides a new theoretical basis for the basic research of ALV-J replication;it provides a new idea for the development of biological products for prevention and treatment of ALV-J;meanwhile,ALV-J has significant lentiviral properties and CTHRC1 can be used as a replication enhancer for ALV-J.