Objectives: Cytomegalovirus (CMV) infection causes extensive remodeling and expansion of intracellular membranes at the interface of early endosomes (EE), the endosomal recycling compartment (ERC), and the Golgi. The aim of this research was to identify cellular proteins at the EE and ERC interface that murine cytomegalovirus (MCMV) recruits in the early phase of infection. Additionally, we investigated how these proteins affect the viral replication cycle and the formation of the viral assembly compartment (pre-AC). Special attention was given to changes in the interactome of the protein Rab10, which is a marker of the pre-AC. Materials and methods: We used immunofluorescence microscopy and western blot analysis to monitor the expression of cellular and MCMV proteins. Pericentriolar accumulation of Rab10 served as a marker for the formation of the pre-AC. To identify proteins involved in the formation of the pre-AC, proteins at the EE-ERC interface were silenced by transfection with specific siRNA. We also used chemical inhibitors and transfection with dominant negative (DN) constructs for further investigation of the role of key proteins. The BioID2 method was used to analyze the Rab10 interactome, and for this purpose, we generated an NIH3T3 cell line stably expressing the Rab10-BioID2-HA fusion protein. Results: We discovered that most proteins at the EE-ERC interface accumulate in the pericentriolar area corresponding to the pre-AC, without significant changes in their quantity during infection. Knockdown the proteins SNX27, Vps35, SNX1, SNX4 and dynamin 2 significantly inhibited the formation of the pre-AC. Further investigation of SNX27 confirmed its key role in the formation of the inner part of the pre-AC, particularly in the synthesis of MCMV proteins during the delayed early and late phases of infection, as well as in virion synthesis. The dynamin inhibitor dynasore and the DN construct K44A Dyn2 EGFP also inhibited the formation of the pre-AC and the MCMV replication cycle. The Rab10 interactome in uninfected and MCMV-infected cells predominantly consisted of proteins EHBP1, MICAL-L1, and Rab11 FIP1. Conclusions: The SNX27:Retromer:ESCPE-1 complex (proteins SNX27, Vps35, SNX1) and dynamin 2 are required for the earliest events during pre-AC establishment in the early phase of MCMV infection. Furthermore, functional SNX27 (particularly its F3 FERM domain) and dynamin 2 are essential for the normal progression of the MCMV replication cycle. Finally, the Rab10 interactome does not change significantly in the early phase of MCMV infection, with Rab10 primarily interacting with EHBP1, MICAL-L1, and Rab11 FIP1 proteins.