Fig. 5: MENCs and fusion suppression stabilize inter-mitochondria contacts.

A Representative timelapses of mitochondria (OMP25) fusion in uninfected cells (top) and stable inter-mitochondria contact (IMC) in infected cells by 72 hpi (bottom) (ROI = 10 × 10 µm). B Quantification of IMC stability throughout HCMV replication. (Mock = 4, 48 hpi = 2, 24/72/96 hpi = 3 biological replicates; total of 400 events analyzed). C Quantification of MFN2 KO-induced IMC stability. ≥3 IMC per cell, ≥10 cells per condition, n = 3 biological replicates. Total of 182 IMCs. D Representative images of two mitochondria (GFP-OMP25, red) in MENCs associated with ER (mCh-Sec61β, cyan) forming a stable IMC (ROI = 4.76 ×4.57 µm). E Right: Cartoon representation of scoring IMC as arising from 0, 1 or 2 MENCs. Left: Quantification of whether 0, 1 or 2 mitochondria where in MENCs prior to IMC onset, binned by IMC temporal stability (10 cells per replicate; n = 3 biological replicates, 114 IMC across 30 cells). F Slice through a tomogram of HCMV-infected cell at 72 hpi showing a Mito-mito contact (whole volume shown in Supplementary Movie S8). Scale bar is 200 nm. Representative of one biological replicate with 23 cells across two grids being selected for FIB-milling. Out of these, 6 lamella sites were successfully polished and used for cryo-ET data acquisition. In total 34 tilt series were acquired of which 27 contained mitochondria and ER in close proximity. G Representative microscopy showing a MENC-IMC with a PTPIP51 enriched ER tubule running in between the contacting mtiochondria. Scale bar is 2 µm. H 3D reconstruction of a MENC-IMC. Top row shows a front view while the bottom row displays a side view, as indicated by the cartoons on the left. hpi hours post infection, mito mitochondria, ER endoplasmic reticulum, s seconds, M mock, MENC mitochondria–ER encapsulation, KO knockout, IMC inter-mitochondria contact. Data are presented as mean values with 95% confidence intervals. Black asterisks indicate statistical comparison to uninfected cells. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 using a Welch’s t test. Source data are provided as a Source Data file.