Germline transmitted animals were bred in the Transgenic Mouse Model Core Facility of the National Core Facility System for Biotechnology (NCFPB), Academia Sinica, Taiwan

Germline transmitted animals were bred in the Transgenic Mouse Model Core Facility of the National Core Facility System for Biotechnology (NCFPB), Academia Sinica, Taiwan. timepoints after illness to measure viral titers. The remaining panels provide evidence of Uggt1 knockdown following siRNA treatment. (C) and (D) RD cells were transfected with 1, 2, or 4 g of pFlag-UGGT1 WASL or pFlag-vector, then infected 48 h later on with EVA71 at an MOI of 10. Plaque assays were performed to measure viral yields at 4 and 6 h post-infection. (E) and (F) EVD68 propagation in RD cells treated with NC or UGGT1 siRNA for 48 h and then infected with EVD68 at an MOI of 10 or 0.1. Viral titers were measured by plaque assays in the indicated time points post-infection, and the remaining panels confirm Uggt1 knockdown following siRNA treatment. ***P < 0.001, **P < 0.01, and *P < 0.05, as calculated by two-tailed unpaired College students t-test.(TIF) ppat.1006375.s003.tif (1.5M) GUID:?4E98E178-6226-4959-813A-62624D898295 S4 Fig: EVA71 viral yield in UGGT1 or UGGT1(mut) overexpressed cells. (A) RD cells were transfected with 4 g of pFLAG-vector or pFLAG-UGGT1 or pFLAG-UGGT1(mut) for 48 h, and then challenged with EVA71 at an MOI of 10. A plaque assay was performed to measure viral yields at 6 h post-infection. (B) UGGT1 was overexpressed by respectively transfecting 4 g of plasmid pFLAG-vector or pFLAG-UGGT1 or pFLAG-UGGT1(mut) to RD cells, and the panels display the corresponding increase in UGGT1 levels following overexpression, with actin offering as a loading control.(TIF) ppat.1006375.s004.tif (572K) GUID:?8869BC3D-8008-4AE1-B5C1-F0964ED8482F S5 Fig: UGGT1 associates with JEV polymerase NS5 and promotes JEV pathogenicity in suckling mice. (A) JEV-infected and mock-infected cells were harvested and subjected to immunoprecipitation assays. Immunoprecipitation assays were performed using an anti-UGGT1 antibody, and the precipitates were analyzed by Western blotting using an anti-NS5 antibody. Abacavir (B) 1-day-old WT or Uggt1 heterozygous knockout mice were injected with 104 PFU/mouse of JEV strain T1P1, and on Day time 7 after illness, JEV was extracted from mind cells and quantitated. ***P < 0.001 as calculated by two-tailed unpaired College students t-test.(TIF) ppat.1006375.s005.tif (845K) GUID:?06E14C3F-4E4E-415B-BBCB-F2ECE35E5C64 S6 Fig: EVA71 IRES activity in NC or UGGT1 siRNA-transfected cells. (A) RD cells were transfected with NC siRNA or Abacavir UGGT1 siRNA for 48 h, after which the Abacavir dicistronic construct, pRHF-EVA71, was transfected. After 48 h, the Renilla luciferase (RLuc) and FLuc activity in cell lysates was analyzed. Bars in the histogram represent Abacavir FLuc/RLuc activity percentages. Experiments were performed in triplicate to derive the pub graph.(TIF) ppat.1006375.s006.tif (576K) GUID:?7C555BB6-B3F9-4943-B1DC-B45620124B59 Data Availability StatementAll relevant data are within the paper and its Supporting Info files. Abstract Positive-strand RNA computer virus infections can induce the stress-related unfolded protein response (UPR) in sponsor cells. This study found that enterovirus A71 (EVA71) utilizes sponsor UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1), a key endoplasmic reticulum protein (ER) involved in UPR, to enhance viral replication and virulence. EVA71 forms replication complexes (RCs) on cellular membranes that contain a mix of sponsor and viral proteins to help viral replication, but the parts and processes involved in the assembly and function of RCs are not fully recognized. Using EVA71 like a model, this study found that sponsor UGGT1 and viral 3D polymerase co-precipitate along with other factors on membranous replication complexes to enhance viral replication. Improved UGGT1 levels elevated viral growth rates, while viral pathogenicity was observed to be reduced heterozygous knockout mice (Uggt1 +/- mice). These findings provide important insight on the part of UPR and sponsor UGGT1 in regulating RNA computer virus replication and pathogenicity. Author summary Positive-strand RNA viruses are adept at hijacking sponsor cell machinery to promote viral propagation, including the formation of RCs comprising viral and sponsor proteins on intracellular membranes to facilitate virion assembly and avoid detection by sponsor defense mechanisms. However, the processes by which RCs are put together, as well as the sponsor proteins involved, have not been fully elucidated as yet. Here, we display the endoplasmic reticulum (ER) Abacavir protein UGGT1, a key regulator of the UPR sponsor defense mechanism, co-precipitates with the 3D polymerase of EVA71 to facilitate RC formation, enhance viral RNA synthesis, and promote viral replication. Knockout of reduced viral pathogenicity in animal studies. These findings highlight the part to which viruses can hijack important sponsor proteins to promote viral replication, and may serve as the basis for the development of novel anti-viral strategies. Intro Positive-strand RNA viruses are capable.