Fig 1: Host VAMP3 is essential for release of infectious HCMV progeny.(A) Quantification of infectious units released from fibroblasts over-expressing (O/E) VAMP3 or GFP-FLAG (CTL) fusion proteins. Cells were infected with HCMV AD169 MOI = 3, and 5 DPI the supernatant was transferred to a reporter plate of uninfected cells, and IE1 assay conducted 24 HPI. (n = 3, bars = SEM, **p<0.01, Student’s t-test). (B) Quantification of infectious units released from siRNA-treated cells infected with HCMV strain Merlin (RCMV1158) MOI = 3. 7 DPI supernatant was transferred to a reporter plate of uninfected cells, and IE1 assay conducted 24 HPI (n = 3, bars = SEM, ****p<0.0001, Student’s t-test). (C) Quantitation of HCMV spread in ARPE-19 cells treated with siRNA. Cells were infected with Merlin (RCMV1120) at low MOI. At indicated time-points post infection, cells were fixed directly and IE1 positive nuclei quantified (n = 3, bars = SEM, *p<0.05, **p<0.01, Student’s t-test). (D) Quantitation of extracellular viral genomes in supernatant from siRNA treated cells infected with HCMV AD169, 5 DPI, MOI = 3. (n = 3, bars = SEM, **p<0.01, Student’s t-test). (E–G) Immunofluorescence staining of UL99, GM130, VAMP3, and gB in siRNA-treated cells infected with HCMV-GFP AD169, 4 DPI, MOI = 0.1. White arrows: peripheral gB puncta, Scale bars = 20 µm. (H) Quantification of peripheral gB-positive puncta (outside Golgi ring) in siRNA-treated or Opti-MEM control cells, infected with HCMV-GFP AD169, 4 DPI, MOI = 0.1. Between 44 and 65 cells were quantified in each condition (n = 3, bars = SD, ns: not significant, ****p<0.0001, one-way ANOVA with Tukey’s post-test). (I) Electron micrographs of VAMP3 siRNA and non-targeting control (NTC) treated cells infected with HCMV AD169, 5 DPI, MOI = 1. White arrows: maturing virions, Black arrows: enlarged empty vesicles, Scale bars = 2.0 µm.