Immunoprecipitation (with anti-Flag) and immunoblot analysis (with anti-Flag, anti-HA and anti-Tubulin) of HEK293T cells transfected with plasmids encoding HA-tagged NDRG1 and Flag-tagged ORF44 or ORF44 truncates for 48 h. Similarly, to explore the domains in ORF44 responsible for its interaction with NDRG1, we predicted the domains of ORF44 using the InterPro database and found that four P-loop-containing nucleoside triphosphate hydrolase domains are the major structural motifs in ORF44. mainly because mean SD, n = 3; **p 0.01; ***p 0.001; ****p 0.0001.(TIF) ppat.1009645.s002.tif (718K) GUID:?78082A6D-58EB-4B27-AEFA-7180B62D632A S3 Fig: NDRG1 increases ORF44 stability self-employed of Viperin. (A) HEK293T cells were transfected with siRNA as indicated for 48 h. The knockdown effectiveness of Viperin was determined by immunoblotting (remaining panel) and qPCR analysis (right panel). (B) HEK293T cells were transfected with indicated siRNA for 24 h, then the cells were cotransfected with plasmids encoding HA-tagged ORF44 and Flag-tagged NDRG1 or vacant vector for 48 h. The protein expression levels of ORF44, NDRG1 and Viperin were recognized by immunoblotting analysis. Data were demonstrated as mean SD, n = 3; *p 0.05; **p 0.01.(TIF) ppat.1009645.s003.tif (264K) GUID:?089976E2-872D-4229-AD84-551AD022829A S4 Fig: The protein abundance of ORF44 solitary lysine mutants. ODM-203 (A) Schematic diagram of the positions of all 34 lysine residues in ORF44. According to the lysine residue position, ORF44 is divided into five clusters, including A6, B6, C7, D6 and E9. (B-F) HEK293T cells were transfected with the indicated plasmids for 48 h, then the cells lysed and the protein abundance of these mutants were recognized by immunoblotting.(TIF) ppat.1009645.s004.tif (473K) GUID:?140982EF-D014-4902-BB72-6798DABB80C7 S1 Table: NDRG1 interacted with KSHV-encoded proteins identified in TAP-MS. (XLSX) ppat.1009645.s005.xlsx (19K) GUID:?131595E9-3A15-4B99-8020-670AC87E7959 S2 Table: Primers for PCR ODM-203 amplification and analysis. (XLSX) ppat.1009645.s006.xlsx ODM-203 (12K) GUID:?CCC9762C-CB74-45AE-9933-22A272A55A9D Data Availability StatementAll relevant data are within the manuscript and its Supporting Information documents. Abstract The presumed DNA helicase encoded by ORF44 of Kaposis sarcoma-associated herpesvirus (KSHV) takes on a crucial part in unwinding viral double-stranded DNA and initiating DNA replication during lytic reactivation. However, the regulatory mechanism of KSHV ORF44 has not been fully elucidated. In a earlier study, we recognized that N-Myc downstream controlled gene 1 (NDRG1), a host scaffold protein, facilitates viral genome replication by interacting Rabbit Polyclonal to CACNG7 with proliferating cell nuclear antigen (PCNA) and the latent viral protein latency-associated nuclear antigen (LANA) during viral latency. In the present study, we further shown that NDRG1 can interact with KSHV ORF44 during viral lytic replication. We also found that the mRNA and protein levels of NDRG1 were significantly improved by KSHV ORF50-encoded replication and transcription activator (RTA). Amazingly, knockdown of NDRG1 greatly decreased the protein level of ORF44 and impaired viral lytic replication. Interestingly, NDRG1 enhanced the stability of ORF44 and inhibited its ubiquitin-proteasome-mediated degradation by reducing the polyubiquitination of the lysine residues at positions 79 and 368 in ORF44. In summary, NDRG1 is definitely a novel binding partner of ORF44 and facilitates viral lytic replication by keeping the stability of ORF44. This study provides fresh insight into the mechanisms underlying KSHV lytic replication. Author summary During lytic replication, KSHV ORF44 unwinds viral DNA and initiates DNA replication. Here, we report the host protein NDRG1, a novel ORF44 binding partner, is definitely significantly upregulated during ODM-203 viral lytic replication and facilitates this process. Mechanistically, NDRG1 can increase the stability of ORF44, impairing the polyubiquitination of the lysine residues at positions 79 and 368 in ORF44, therefore inhibiting ubiquitin-proteasome-mediated degradation of ORF44. Our study demonstrates that NDRG1 takes on an important part in KSHV lytic replication and may thus constitute a promising therapeutic target for KSHV contamination. Introduction Kaposis sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8 and belonging to the human oncogenic herpesvirus family, is the etiological agent of several human malignancies, including the endothelial neoplasm Kaposis sarcoma (KS) and two B cell lymphoproliferative disorders: primary effusion lymphoma (PEL) and multicentric Castlemans disease (MCD) [1C3]. Similar to other herpesviruses, the life cycle of KSHV comprises two different phases: latency and lytic replication [4,5]. Extensive evidence has indicated that both the latent and lytic phases of the KSHV life cycle contribute prominently to viral tumorigenesis [6,7]. As a strategy to escape host immune surveillance, KSHV establishes latency for lifelong persistent contamination. During latency, KSHV expresses only a few viral genes, and latency-associated nuclear antigen.
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