Journal: Journal of Virology

Article Title: Host MicroRNA Regulation of Human Cytomegalovirus Immediate Early Protein Translation Promotes Viral Latency

doi: 10.1128/JVI.00481-14

Figure Lengend Snippet: Cellular hsa-miR-200 inhibits wild-type infection but not infection with an UL122 (IE2) 3′ UTR mutant virus. (A) Primary human embryonic lung fibroblasts (MRC5 cells) were stably transduced with either a control retrovirus or one that overexpressed the C1 cluster of the hsa-miR-200 family. In each cell type, levels of hsa-miR-200b (light gray bars) or hsa-miR-200c (dark gray bars) were assessed by qPCR. Samples were normalized to those of cellular RNU44 and analyzed in triplicate. (B, C) MRC5 cells stably transduced with a C1-expressing lentivirus (white bars) or an empty control (gray bars) were then infected with either wild-type FixBACgfp virus (B) or FixBACgfpIE2cisΔ virus (C) at a multiplicity of 0.5 PFU/cell for 4 days. The titer of cell-free virus was then determined by a modified immunofluorescence assay for IE1. Samples were analyzed in triplicate. (D) MRC5 cells transduced with either the C1-expressing or control lentivirus were infected with either wild-type FixBACgfp virus or FixBACgfpIE2cisΔ virus at a multiplicity of 1 PFU/cell. Cell lysates were harvested at the indicated time points (hpi, hours postinfection), and IE2 levels were assessed using a monoclonal antibody (clone 3A9). α-Tubulin was used as a control.

Article Snippet: Primary newborn human fibroblasts (NUFF-1 cells; GlobalStem) or primary human embryonic lung fibroblasts (MRC5 cells) were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% FBS, 2 mM l -glutamine, 0.1 mM nonessential amino acids, and 100 U/ml each penicillin and streptomycin.

Techniques: Infection, Mutagenesis, Virus, Stable Transfection, Transduction, Control, Expressing, Modification, Immunofluorescence