Journal: Journal of Virology

Article Title: Mutational Mapping of pUL131A of Human Cytomegalovirus Emphasizes Its Central Role for Endothelial Cell Tropism

doi: 10.1128/JVI.05354-11

Figure Lengend Snippet: Relative endothelial cell tropism of UL131ccta mutants. Fibroblasts (HFFs) and endothelial cells (HUVECs) were infected by various UL131ccta mutants at a multiplicity of infection of 2.5 infectious units/cell. One day after infection, viral immediate-early antigens were detected by indirect immunofluorescence staining (Cy3, red nuclear signals). Cell nuclei were counterstained with DAPI (blue nuclear signals). (A) Infection efficiency in HUVEC and HFF. One example for each phenotype category is shown: wild-type, HCMV-TB40-BAC4; no phenotype, HCMV-TB40-BAC4-UL131ccta54-58; intermediate phenotype, HCMV-TB40-BAC4-UL131ccta93-98; strong phenotype, HCMV-TB40-BAC4-UL131ccta116-121. (B) Relative EC tropism of all UL131ccta mutants, determined as the ratio of infection efficiency in HUVECs and infection efficiency in HFFs. Bars represent mean values from at least three experiments (standard error of the mean is indicated with each bar). Asterisks indicate a highly significant (***, P < 0.001), significant (**, P < 0.01) or marginally significant (*, P < 0.05) difference between the wild-type virus and the marked mutant. (C) Detection of UL128-131A proteins in virion particles of mutants with complete or partial reduction of EC tropism. Lysates of gradient-purified virions of HCMV-TB40-BAC4-UL131ccta64-69 and HCMV-TB40-BAC4-UL131ccta72-75 were analyzed by immunoblotting for the presence of pUL128, pUL130, and pUL131A. Anti-gB served as a control antibody. Wild-type HCMV-TB40-BAC4 and a mutant lacking the complete UL128-131A gene region (22) served as positive- and negative-control viruses, respectively.

Article Snippet: Human foreskin fibroblasts (HFFs) were cultured in minimal essential medium (MEM) (GIBCO/Invitrogen) containing 5% fetal calf serum (FCS), 2.4 mmol/liter glutamine, 100 μg/ml gentamicin, and 0.5 ng/ml basic fibroblast growth factor (bFGF) (GIBCO/Invitrogen).

Techniques: Infection, Immunofluorescence, Staining, Mutagenesis, Purification, Western Blot, Negative Control

Journal: Journal of Virology

Article Title: Mutational Mapping of pUL131A of Human Cytomegalovirus Emphasizes Its Central Role for Endothelial Cell Tropism

doi: 10.1128/JVI.05354-11

Figure Lengend Snippet: Phenotype of BAC4-UL131ccta72-75ccta93-98 dual mutant. (A) Fibroblasts (HFFs) and endothelial cells (HUVECs) were infected by cell-free preparations of the respective virus at a multiplicity of infection of 2.5 infectious units/cell. One day after infection, viral immediate-early antigens were detected by indirect immunofluorescence staining. The relative EC tropism is given as the ratio of infection efficiency in HUVECs and infection efficiency in HFFs. Bars represent mean values from at least three experiments (standard error of the mean is indicated with each bar). Asterisks indicate a significant (**, P < 0.01) or marginally significant (*, P < 0.05) difference between the respective viruses. (B) Infected fibroblasts were cocultured with an excess of uninfected ECs (ratio of infected HFFs and uninfected ECs, 1/500) for 5 days and then stained for viral immediate-early antigen. The focus expansion capacity in endothelial cell monolayers is given as the average number of infected cells per focus. Bars represent mean values from at least three experiments (standard error of the mean is indicated with each bar). Asterisks indicate a significant (**, P < 0.01) or marginally significant (*, P < 0.05) difference between the respective viruses.

Article Snippet: Human foreskin fibroblasts (HFFs) were cultured in minimal essential medium (MEM) (GIBCO/Invitrogen) containing 5% fetal calf serum (FCS), 2.4 mmol/liter glutamine, 100 μg/ml gentamicin, and 0.5 ng/ml basic fibroblast growth factor (bFGF) (GIBCO/Invitrogen).

Techniques: Mutagenesis, Infection, Immunofluorescence, Staining