Fig. 3: HCMV UL44 interacts with L1 RNP and recruits ORF2p to the replication compartments.

a A schematic representation of identifying L1 RNP interacting with HCMV proteins. pBL1^ORF1p-FLAG U373MG were generated to express L1 with FLAG-tagged ORF1 upon dox treatment. b Average score from LC/MS analyses. Bars indicate human (black) and HCMV (red) proteins. The table shows the L1 RNP-interacting HCMV proteins and their function. c Co-IP assays using HCMV-infected pBL1^ORF1p-FLAG U373MG. IP mixture was eluted with either FLAG peptides or RNase, as indicated. Representative blot of n = 2 independent replicates. d Endogenous L1 ORF1p Co-IP assay of U373MG cells at 4 dpi showing interactions between L1 RNP and UL44. Given differences in detection efficiencies, long and short exposures of L1 ORF1p and L1 ORF2p blots are presented. Flow-through was saved before IP wash. Representative blot of n = 2 independent replicates. e Co-IP assays showing the interaction of L1 ORF2p with UL44 via its PIP-box plug residues. HeLa cells were transfected with L1 ORF2p-3×FLAG and/or HA-UL44 plasmids. Co-IP assays were performed using HA antibody. Representative blot of n = 2 independent replicates. f Structural modeling of UL44 with PCNA binding region of L1 ORF2p (403–419). UL44 hydrophobic crevice composed of Val⁵⁸, Val¹³⁶, Leu²⁵¹, and Phe²⁶⁶ (light blue) and L1 ORF2p hydrophobic plug Tyr⁴¹⁴ and Tyr⁴¹⁵ (red) are shown. g Co-IP assays using FLAG-UL44-transfected pBL1 HeLa with UL44 hydrophobic crevice mutations. Representative blot of n = 2 independent replicates. The band intensity of L1 ORF2p and L1 ORF1p are indicated below. Source data are provided as a Source Data file.