Systematic Identification And Functional Evaluation Of Interacting Proteins Of Human Cytomegalovirus

Human cytomegalovirus(HCMV)is the leading viral cause of congenital abnormalities and mental retardation in newborns,and also causes severe life-threatening complications in HIV-infected patients.To better understand the molecular mechanism of HCMV viral infection and replication,three efforts have been made to identify and functional evaluate the interacting proteins of HCMV,which are:(1)Systematic yeast two-hybrid library screens for eight HCMV DNA replication proteins(UL44,UL54,UL57,UL70,UL84,UL102,UL105,UL114)were performed,partial of results were further confirmed by subsequent co-immunoprecipitation.This gave rise to 154 distinct protein-protein interactions involving 8 HCMV and 130 human proteins including cellular proteins UBC9,SNAPAP,PIAS3,FHL2,DNAJB6,CTBP2,POLD2,ATRX,PFAAP5 and ZMYND19.Together with literature-curated data,these results provide a comprehensive insight into the complexity of the host-viral protein interaction network involving in viral DNA replication,and a detailed pathway for these viral proteins from gene to function is proposed.(2)Using yeast mating protocol to systematically screen potential direct interactions between 174 HCMV viral proteins and 16 HIV viral proteins,which yielded 12 distinct protein-protein interactions involving 7 HCMV and 7 HIV proteins including PR-RL1,Nef-RLl,p17-RL1,p24-RL1,Tat-RL1,p6-RL1,p6-UL119,p6-UL125,IN-RL,IN-UL24,IN-UL45 and IN-UL77.These results provide novel insights into development of different strategies and approaches for control of HCMV-HIV coinfection and pathogenesis.(3)Confirmed the interaction of ppUL44 with the SUMO-conjugating enzyme UBC9 in vitro by His pull-down assay and in vivo by co-localization and co-immunoprecipitation assays,found that two regions between aa 11-16 and aa 260-269 was critical for the interaction of ppUL44 with UBC9.Further demonstrated that ppUL44 is a novel target for sumoylation and could be covalently modified by sumo-1,-2 and-3.Lys410 located at the ψ/KxE consensus motif is the major sumoylation site of ppUL44.These findings suggest that the interaction of UBC9 and sumoylation plays a important role in the regulation of ppUL44 functions and HCMV viral replication.The research here facilitated the elucidations of viral protein functions in HCMV infection and replication,and furthered understandings of the infectious biology of HCMV.