Cytomegalovirus Generates Assembly Compartment in the Early Phase of Infection by Perturbation of Host-Cell Factors Recruitment at the Early Endosome/Endosomal Recycling Compartment/Trans-Golgi Interface

Abstract

Beta-herpesviruses develop a unique structure within the infected cell known as an assembly compartment (AC). This structure, as large as the nucleus, is composed of host-cell-derived membranous elements. The biogenesis of the AC and its contribution to the final stages of beta-herpesvirus assembly are still unclear. In this study, we performed a spatial and temporal analysis of the AC in cells infected with murine CMV (MCMV), a member of the beta-herpesvirus family, using a panel of markers that characterize membranous organelle system. Out of 64 markers that were analyzed, 52 were cytosolic proteins that are recruited to membranes as components of membrane-shaping regulatory cascades. The analysis demonstrates that MCMV infection extensively reorganizes interface between early endosomes (EE), endosomal recycling compartment (ERC), and the trans-Golgi network (TGN), resulting in expansion of various EE-ERC-TGN intermediates that fill the broad area of the inner AC. These intermediates are displayed as over-recruitment of host-cell factors that control membrane flow at the EE-ERC-TGN interface. Most of the reorganization is accomplished in the early (E) phase of infection, indicating that the AC biogenesis is controlled by MCMV early genes. Although it is known that CMV infection affects the expression of a large number of host-cell factors that control membranous system, analysis of the host-cell transcriptome and protein expression in the E phase of infection demonstrated no sufficiently significant alteration in expression levels of analyzed markers. Thus, our study demonstrates that MCMV-encoded early phase function targets recruitment cascades of host cell-factors that control membranous flow at the EE-ERC-TGN interface in order to initiate the development of the AC.

Arf GTPases Are Required for the Establishment of the Pre-Assembly Compartment in the Early Phase of Cytomegalovirus Infection

“…Our previous study demonstrated the accumulation of Arf (Arf1–6) proteins at the AC in the late phase of MCMV infection (at 48 hpi) [ 6 ]. Thus, we first analyzed the expression level and intracellular recruitment of all Arf proteins in the early phase of infection at the pre-AC stage.…”

“…These data indicate that class I Arf proteins are rapidly upregulated in MCMV-infected cells, which can be a consequence of either increased synthesis or slowed degradation. The rapidity of upregulation together with the analysis of the host cell transcriptome, which demonstrated no significant alteration in any of the mRNAs of the Arf proteins in the early phase of infection [ 6 ], suggest that observed increases of the levels of Arf proteins are more likely due to altered mechanisms controlling endogenous class I Arf protein degradation rather than a consequence of altered synthesis.…”

“…The AC is a large and complex structure that is developed from the reorganized membranous organelles of the cell [ 5 ]. The onset of AC formation in murine MCMV-infected cells can be identified at 4–6 hpi as an extensive reorganization of the Golgi and the endosomal system [ 6 , 7 ]. A similar set of events was executed during HCMV infection with significantly delayed kinetics [ 8 , 9 , 10 , 11 ].…”

“…Many of these gene products embed into the reorganized membranous organelles and further drive the maturation of the AC. In general, we consider the reorganized structures that involve the Golgi and EE-ER-TGN-derived organelles to be present during the early phase of infection as pre-AC, whereas these structures that are introduced upon the integration of the late-gene products as AC [ 6 ].…”

“…A study of human CMV infection suggested impaired Arf6 function associated with the reprogramming of endosomal trafficking [ 52 ]. Our recent studies on murine CMV (MCMV) demonstrated the over-recruitment of all Arf isoforms within the AC in the late phase of infection, at 48 hpi [ 6 ]. However, the dynamics of Arf expression during the MCMV infection cycle, intracellular localization in the early phase of infection, and their role in pre-AC formation have not been examined in detail.…”