NDRG1 Facilitates Lytic Replication Of Kaposi's Sarcoma-associated Herpesvirus By Maintaining The Stability Of KSHV Helicase

Kaposi's Sarcoma-associated herpesvirus(KSHV),also referred to as human herpesvirus 8,belonging to the human oncogenic linear double-stranded DNA gammaherpesvirus family.Epidemiological studies have shown that KSHV infection may cause three human malignancies,including Kaposi's sarcoma(KS)and two lymphoproliferative disorders: primary effusion lymphoma(PEL)and multicentric Castelman's disease(MCD).Similar to other herpesvirus,KSHV also establishes two different phases in the life cycle,the latency and lytic replication.Extensive evidence has indicated that both KSHV latent and lytic phases contribute to the viral oncogenesis.Therefore,exploring the molecular mechanisms of viral lytic replication phase is crucial to elucidating the viral pathogenesis and may provide potential therapeutic strategies for KSHV-related diseases.During viral lytic reactivation,KSHV genome synthesis is essential for the production of mature progeny viruses.KSHV encodes a set of core DNA replication proteins,including ORF6(ss DNA binding protein),ORF9(DNA polymerase),ORF40-41(primase-associated factor),ORF44(helicase),ORF56(primase)and ORF59(polymerase processivity factor).KSHV helicase ORF44,one of the six core DNA replication proteins,plays an important role in unwinding viral double-stranded DNA and initiating DNA replication,but the regulatory mechanism of this gene has not been fully understood.In our previous study,we have identified a novel host protein N-Myc downstream regulated gene 1(NDRG1),as a scaffold protein,which forms a complex with proliferating cell nuclear antigen(PCNA)and latency-associated nuclear antigen(LANA),thereby assisting LANA to recruit PCNA onto the viral genome and facilitating viral genome replication and episome persistence in the viral latency.Interestingly,we found that 3 KSHV proteins were enriched in the NDRG1 potential interacting proteins by mass spectrometry,and KSHV helicase is ranked the first among these viral proteins.Firstly,the interaction between NDRG1 and ORF44 was authenticated by immunoprecipitation and immunofluorescence experiments,and the N-terminal domain of NDRG1 bound to the N-terminal region of ORF44.Then,the expression kinetic analysis of NDRG1 and corresponding double luciferase reporter gene experiments demonstrated that the replication and transcription activator(RTA)encoded by KSHV ORF50,together with the recombination signal sequence-binding protein-J?(RBP-J?)enhance the transcription level of NDRG1,followed by promoting the protein expression of NDRG1.Furthermore,knockdown of NDRG1 gene in i SLK.RGB cells did not affect the viral gene transcription level,but markedly reduced the protein level of ORF44 and impaired viral lytic replication.In addition,cycloheximide(CHX)half-life assay showed that NDRG1 increased the stability of ORF44,and subsequent ubiquitination assay and protein abundance assay of ORF44 lysine mutants demonstrated that NDRG1 inhibits ubiquitination proteasomal pathway mediated ORF44 degradation by reducing the polyubiquitination of lysine residues at positions 79 and 368 in ORF44.Taken together,our findings show that NDRG1 is a new binding partner of ORF44 and NDRG1 can maintain the stability of ORF44 by inhibiting ORF44 polyubiquitination to promote viral lytic replication,providing new clues for understanding the underlying mechanisms of KSHV lytic replication.Moreover,NDRG1 may serve as a potential antiviral target,providing a new perspective for antiviral research.