Metformin suppressed human cytomegalovirus (hCMV) replication and its potential molecular mechanisms in human fibroblasts

Abstract Background Chronic hCMV infection may contribute to T-cell immunosenenscence and chronic inflammation, and is shown to be associated with frailty and mortality in old adults. Metformin is a first-line treatment for type 2 diabetes mellitus. Emerging data have shown its additional beneficial effects including cardiovascular protection as well as anti-inflammatory, anti-cancer, and anti-aging activities. The objective of this study was to investigate the effect of metformin on hCMV infection in human fibroblasts and its underlying mechanisms. Methods Metformin was added (at concentration of 0, 1mM, 3mM, or 5mM) 2 h before inoculation of hCMV (Towne strain) at multiplicity of infection (MOI) of 0.01 or 0.1 to 80% confluent MRC-5 culture. Cells were cultured for additional 24h to 96 h. Viral plaques were observed under microscope, replication measured by qPCR (primers targeted to hCMV UL123 gene), cell cycle effect determined using flow cytometer, and hCMV protein expression (IE1/IE2, pp28, ICP36) and signaling pathway molecules analyzed by Western blot. Results and discussion Metformin at 3mM potently suppressed hCMV viral plaque formation, replication, and protein production. While hCMV infection promoted MRC-5 cells from G2 to M and S phase, metformin treatment led to G0/G1 arrest. Metformin suppressed expression of cyclin D, CDK4, and p21. It had no effect on CK2 or CDk6 expression. While AMPK pathway antagonist compound C suppressed AMPKa phosphorylation from both hCMV and metformin, it did not antagonize suppressive effect of metformin on hCMV protein expression. Taken together, metformin may suppress hCMV infection in MRC-5 cells via cell cycle regulation and molecular mechanisms independent of AMPK pathway.