African swine fever virus (ASFV) infection leads to a mortality rate of up to 100%, causing devastating disasters to the pig industry. Understanding the ASFV infection and replication is therefore of great importance. ASFV has more than 150 open reading frames, among which the inner coat protein p17 encoded by the D117L gene is involved in the formation of the icosahedral structure of the virus. However, little is known about the mechanism how p17 regulates host cell function. In this study, the potential host proteins interacting with ASFV p17 were screened by immunoprecipitation technique combined with protein profiling analysis. The interactions of p17 with mitochondrial membrane protein TOMM70 and heat shock protein HSPA8 were confirmed by co-immunoprecipitation technique and laser confocal experiments. This study provides important information for further exploring the function of p17 during ASFV infection.
African Swine Fever ; African Swine Fever Virus/metabolism* ; Animals ; Open Reading Frames ; Swine ; Viral Proteins/metabolism*

Objective: To investigate the impact of homocysteine inducible endoplasmic reticulum(ER) protein with ubiquitin like domain 1 protein (Herpud1) in the homocysteine (Hcy) -induced phenotypic switching of vascular smooth muscle cells (VSMCs). Methods: VSMCs were derived from thoracic aortic artery of male Sprague Dawley rats and cultured VSMCs (4-7 passage) were treated with various concentrations of Hcy (0, 100, 500 and 1 000 μmol/L) and applied to immunofluorescence to observe the morphological changes of VSMCs via SM-actin staining. Western blot was used to detect the expression of VSMCs phenotypic markers, including Osteopontin, Calponin and smooth muscle myosin heavy chain (SM-MHC) and the expression of endoplasmic reticulum stress (ERS) related proteins, including C/EBP-homologous protein (CHOP), inositol-requiring kinase 1 (IRE-1) and glucose regulating protein 78 (GRP78) in the absence and presence of non-selective inhibitor of ERS, 4-phenylbutyric acid (4-PBA, 2 mg/ml). The Herpud1 mRNA and protein levels were determined in Hcy-stimulated VSMCs treated with 4-PBA or transfected with specific siRNA targeting Herpud1. Results: Compared with the control group, SM-actin staining results showed that the shape of VSMCs treated with different concentrations of Hcy for 24 hours changed from long fusiform into round form, arrangement of myofilament became irregular and the most significant alteration was found in the 500 μmol/L Hcy group. After intervention of 24 hours, various concentration of Hcy increased protein expression of Osteopontin, and reduced Calponin and SM-MHC protein expressions in VSMCs (all P<0.05). In addition, the results showed that Hcy increased the expression of CHOP, IRE-1 and GRP78 in a dose-dependent manner, which could be reversed by 4-PBA treatment (all P<0.05). However, 4-PBA inhibited Hcy induced upregulation of Osteopontin and downregulation of Calponin and SM-MHC, suggesting that ERS was involved in Hcy-induced phenotypic switching of VSMCs. Herpud1 protein was mostly expressed in the cytoplasm and was also expressed in the nucli, both in the control, Hcy and Hcy+4-PBA groups. Moreover, Hcy increased mRNA and protein levels of Herpud1 (P<0.05), whereas treatment with 4-PBA could significantly reduce Hcy-induced upregulation of Herpud1 (P<0.05). Furthermore, knockdown of Herpud1 abrogated the effects of Hcy on VSMCs phenotype markers. Conclusion Herpud1 plays an important role in Hcy-induced phenotypic switching of VSMCs.