The role of autophagy in human cytomegalovirus IE2 expression.

Abstract

The purpose of this study was to determine whether autophagy regulates the expression of human cytomegalovirus (HCMV) immediately early two viral protein (IE2). Rapamycin and 3-methyladenine (3-MA) were used to stimulate or suppress autophagy during HCMV infection. UL122 recombinant plasmid was transfected to overexpress IE2 and small interference RNA against autophagy-related protein 3 (ATG3) was used to knockdown ATG3. Western blot was performed to measure the expression of viral proteins and autophagy levels. Immunofluorescence was used to detect the immediately early 1 viral protein (IE1) expression. In human embryonic lung fibroblasts, infection of HCMV promotes the lipidation of light chain 3 (LC3) at 6 and 24 hours post infection (hpi), which was accompanied by the increased expression of viral protein IE2. When only IE2 was overexpressed via UL122 recombinant plasmid transfection without HCMV infection, the autophagy hallmarks LC3II and ATG3 were upregulated. Furthermore, viral protein IE2 expression was reduced at 24 and 48 hpi either by the treatment of autophagy inducer rapamycin or by the inhibitor 3-MA before HCMV infection. At the same time, small interference ATG3 transient transfection, used to suppress autophagy, significantly inhibited IE2 expression. However, when 3-MA was used to regulate autophagy levels after HCMV infection, expression of IE2 and IE1 were both decreased, while autophagy inducer rapamycin treatment after HCMV infection increased IE2 expression slightly. IE2 was involved in autophagy induced by HCMV infection and blocking autophagy could inhibit the expression of HCMV viral protein IE2, which might be one way for autophagy to restrict HCMV replication.