Identification Of Host Proteins Regulating Oncogenic Gammaherpesvirus Lytic Replication: PTK7 Enhances Viral Egress

Background:Epstein-Barr virus(EBV)is associated with post-transplant lymphoproliferative disorder(PTLD),which is a life-threatening complication following organ transplantation and leads to poor prognosis of the recipient and the graft.EBV,a human gammaherpesvirus,is the first oncogenic virus in human to be discovered and etiologically linked to a wide range of human tumors,including B cell malignancies,T-cell /natural killer-cell lymphomas,nasopharyngeal carcinoma and gastric carcinoma.Individuals with immunodeficiency resulting from receiving immunosuppression agents after organ transplantation,have an increased risk of lymphoid malignancies.Similarly,Kaposi’s sarcoma-associated herpesvirus(KSHV),another human gammaherpes virus,is also a tumor virus.The mechanisms involoved in tumorigenesis of gammaherpesvirus are complex.Investigations into the interactions and regulatory mechanisms between virus and host are of great significance for a better understanding of malignancies and therapy development.Unfortunately,in vivo studie of KSHV and EBV is hampered because of the restricted host range.Moreover,the study of lytic replication of the human gammaherpesviruses is also limited by the lack of a cell culture system that can support robust lytic replication.murine gammaherpesvirus 68(MHV-68),which is genetically and biologically related closely to EBV and KSHV,can undergo efficient lytic replication in a variety of common cell lines including those of human origins and is able to infect laboratory mice to establish an animal model.Therefore,it is a tractable system to study gammaherpesvirus lytic replication in vitro and in vivo.Our study aims to systematically identify regulatory cellular factors of MHV-68 lytic replication to provide new opportunities for the development of host factor-directed anti-viral and anti-tumor therapies.Methods:We screened a c DNA library of more than 1000 host factors(kinases and transcription factors)for the systematic identification of cellular factors that could enhance or inhibit MHV-68 lytic replication.STRING and Metascape were employed to perform protein-protein interaction(PPI)analysis,Gene Ontology(GO)and pathway enrichment analysis.We also conducted a small-scale pair-wise co-expression screening of 23 cellular factors to investigate their combined effects.Moreover,we further studied the role of protein tyrosine kinase 7(PTK7),a lytic replication promoting kinase,in different steps of lytic replication.Result:We identified 105 MHV-68 host factors(54 kinases and 51 transcription factors)and 164 restriction factors(66 kinases and 98 transcription factors).Many signaling pathways were involved in virus lytic replication.167 regulatory factors and 417 edges were displayed in PPI network.Some co-expression pairings,such as MAP3K5 and Fos,MAP2K3 and Fos,MAX and Fos,showed synergistic effects and amplified the enhancement potently,while the overexpression of CREB1 and Myc abolished the function of most positive regulators.Overexpression of host factor PTK7 did not affect MHV-68 gene transcription,genome replication and protein expression.However,it enhanced the egress of MHV-68 and herpes simplex virus type 1(HSV-1).Conclusions:Our high-throughput approach toward virus-host interaction analysis helps reveal cellular factors that coordinately regulate different steps of MHV-68 lytic replication including lytic gene expression,viral DNA replication,virus assembly,and egress.These cellular factors are research targets of futher study at molecular and cellular levels.Particularly,the finding of PTK7’s conserved function in promoting herpesvirus release from host cells provides a basis for exploring virus egress pathways to identify novel targets for anti-viral drug development.In conclusion,our work gives a new insight into the regulation of virus lytic replication and pathogenesis of gammaherpesviruses.Our findings provide new opportunities for the development of host factor-directed anti-viral and anti-tumor therapies.Background:Gammaherpesviruses depend on cellular machineries and multiple pathways in the host for their efficient infection,replication and propagation.Several studies on KSHV,EBV,or MHV-68 have elucidated that cellular proteins significantly impact gammaherpesvirus lytic replication through physical and functional interactions with viral proteins.Despite the progress made,most of the identified cellular factors affect gammaherpesvirus lytic replication by regulating the initial or early steps.Less is known about cellular factors’ functions during the later steps of gammaherpesvirus lytic replication such as viral DNA replication,late gene expression,virus assembly,and egress.Therefore,we conducted a high-throughout screening for a comprehensive identification of regulatory cellular factors functioning in various steps.Methods:We screened a c DNA library of approximately 350 kinases and 700 transcription factors for the systematic identification of cellular factors that could enhance or inhibit MHV-68 lytic replication.Luciferase activities of recombinant MHV-68 reporter virus were capable of indicating the relative virus titer after its productive infection in HEK293 T.The PPI network was constructed by STRING.Both Gene Ontology(GO)and pathway enrichment analysis was performed using Metascape.we conducted a pair-wise co-expression screening of 23 cellular factors functioning in herpesvirus infection to investigate their combined effects.Results:We identified 105 MHV-68 host factors(54 kinases and 51 transcription factor)and 164 restriction factors(66 kinases and 98 transcription factor).For host factors,MAPK signaling pathway,Erb B signaling pathway,Fox O signaling pathway and Gn RH signaling pathway were the main enriched pathways.Fox O signaling pathway,MAPK signaling pathway,PI3K-Akt signaling pathway,and Ras signaling pathway were significantly enriched with regard to the restriction factors.167 regulatory factors and 417 edges were displayed in PPI network.Moreover,8 clusters were identified in the PPI network and these genes were significantly enrich in gene transcription,protein localization,and activity.Some co-expression pairings,such as MAP3K5 and Fos,MAP2K3 and Fos,MAX and Fos,showed synergistic effects and amplified the enhancement potently,while the overexpression of CREB1 and Myc abolished the function of most positive regulators.Conclusions:Virus interacts with many intracellular pathways,in which both kinase and transcription factors are crucial modulators,to process signals and coordinate cell functions.We systematically identified 269 cellular factors that can regulate MHV-68 lytic replication through a screening of kinases and transcription factors.Signaling pathway enrichment analysis and protein-protein interaction analysis indicated the potential mechanisms and modules involved in viral lytic replication.Our work provides novel understanding and research targets for virus-host interactions.Background:PTK7,a member of receptor tyrosine kinases superfamily,was identified as a host factor in the high-throughout screening.It is involved in multiple cellular signaling pathways and associated with many cell activities including cell growth,differentiation,propagation,migration and apoptosis.It also have important impacts on the embryonic development and tumorigenesis in vertebrates.Here,we try to elucidate its function in MHV-68 lytic replication.Methods:HEK293T cells were transfected with different amount of plasmids,and infected with MHV-68-M3 FL virus.Viral supernatants were collected and were titered by luciferase assay.HEK293 T cells were transfected with PTK7 plasmid or the control plasmids then infected with WT MHV-68.The m RNA and expression levels of lytic genes,and viral DNA copy number were then measured.To establish a multi-step growth curve of MHV-68,virus amounts were measured at different time points.At 48 hours post-infection,virus amounts in infected cells,supernatants and also the total viral particles were titered by luciferase assay.Moreover,an RNA interference approach was used to knockdown the expression of PTK7 and the virus lytic production was measured.We also investidated the distribution of HSV-1 with PTK7 overexpression.Results:PTK7 promoted viral lytic replication in a dose-dependent manner.PTK7 overexpression did not affect lytic genes transcription and expression,as well as viral DNA replication.However,PTK7 enhanced viral egress.Knocking down PTK7 increased cell-associated virus.Ectopic expression of PTK7 also promoted HSV-1 egress.Conclusions:Our study illustrated the critical role of PTK7 in promoting the egress of the herpesvirus.It not only proves the effectiveness and significance of the high-throughput screening,which can identify a considerable number of cellular proteins at different steps of viral lytic replication at one time,but also provides a new target for the treatment of herpesvirus and related tumors.