Figure 3. UL141 promotes endothelial cell tropism independently of the pentamer complex.

a, Representative images of UL141-dependent spread in endothelial cells. Fibroblasts and endothelial cells were infected with 50 or 100 genome equivalents/cell, respectively, of 141− or 141+ TB40 viruses produced by fibroblasts. Cells were stained for IE1 (green) and Hoechst (blue) at the indicated days post-infection to monitor viral spread. b, Low MOI (0.01 TCID50) multicycle viral growth kinetics in HUVEC that were infected with 141− or 141+ viruses up to 14 dpi. This graph is a compilation of 3 biological replicates. Lognormal data were logarithmically transformed to fit a Gaussian distribution prior to calculating statistical significance via 2-way ANOVA for 12 dpi data points. c, Non-reducing SDS-PAGE of HUVEC cell lysates and HUVEC-derived virions. Cells were infected with 141− and 141+ viruses to measure virion incorporation of known HCMV entry complexes, trimer (gH/gL/gO) and pentamer (gH/gL/128). HUVEC-derived virions were concentrated through a 20% sorbitol cushion at 12 dpi and 14 dpi prior to lysis. Whole cell lysates were collected at 14 dpi. Lysates were immunoblotted for gL to identify covalently linked entry complexes, major capsid protein (MCP) to measure virion abundance, and UL148 to assess the purity of the virion preparations. d, Quantification of band intensities for gH/gL/gO and gH/gL/128 abundance in HUVEC-derived virions from c. Band intensities of 141− and 141+ virions were normalized to MCP. e, Graphical summary illustrating the role of UL141 as an endotheliotropic factor that restores the ability of TB40 virions to subsequently infect endothelial cells.