Extensive reorganization of infected cells and the formation of large structures known as the nuclear replication compartment (RC) and cytoplasmic assembly compartment (AC) is a hallmark of beta-herpesvirus infection. These restructurings rely on extensive compartmentalization of the processes that make up the virus manufacturing chain. Compartmentalization of the nuclear processes during murine cytomegalovirus (MCMV) infection is not well described. In this study, we visualized five viral proteins (pIE1, pE1, pM25, pm48.2, and pM57) and replicated viral DNA to reveal the nuclear events during MCMV infection. As expected, these events can be matched with those described for other beta and alpha herpesviruses and contribute to the overall picture of herpesvirus assembly. Imaging showed that four viral proteins (pE1, pM25, pm48.2, and pM57) and replicated viral DNA condense in the nucleus into membraneless assemblies (MLAs) that undergo a maturation sequence to form the RC. One of these proteins (pM25), which is also expressed in a cytoplasmic form (pM25l), showed similar MLAs in the AC. Bioinformatics tools for predicting biomolecular condensates showed that four of the five proteins had a high propensity for liquid–liquid phase separation (LLPS), suggesting that LLPS may be a mechanism for compartmentalization within RC and AC. Examination of the physical properties of MLAs formed during the early phase of infection by 1,6-hexanediol treatment in vivo revealed liquid-like properties of pE1 MLAs and more solid-like properties of pM25 MLAs, indicating heterogeneity of mechanisms in the formation of virus-induced MLAs. Analysis of the five viral proteins and replicated viral DNA shows that the maturation sequence of RC and AC is not completed in many cells, suggesting that virus production and release is carried out by a rather limited number of cells. This study thus lays the groundwork for further investigation of the replication cycle of beta-herpesviruses, and the results should be incorporated into plans for high-throughput and single-cell analytic approaches.

Background/Objectives: Cytomegalovirus (CMV) infection expands early endosomes (EEs) into tubular extensions that may contribute to the control of virus replication and virion assembly. Sequential recruitment of protein coats and sorting nexins (SNXs) creates membrane zones at the EEs that serve as scaffolds for membrane tubulation and retrieval of cargo proteins, including host cell signaling proteins and viral glycoproteins. This study aims to investigate whether the SNX3-dependent zone of EEs contributes to CMV replication and assembly. Methods: Protein localization was analyzed by confocal imaging and expression by Western blot. The contribution of SNX3 to murine CMV (MCMV) replication, assembly compartment (AC) formation, and virion release was analyzed by siRNA and shRNA depletion. The impact of other downstream SNXs that act in EE tubulation was investigated by combined siRNA knockdowns of SNX1, SNX2, SNX4, SNX17, and SNX27 on cell lines expressing shRNA for SNX3. Results: The SNX3-162 isoform acting at EEs was efficiently knocked down by siRNA and shRNA. The SNX3-dependent EE zone recruited SNX27 and contributed to Rab10-dependent tubulation within the pre-AC. SNX3 was not essential for MCMV replication but contributed to the SNX27-, SNX17- and SNX4-dependent release of virions. Silencing SNX3 further reduced the release of virions after silencing SNX27, SNX4, and SNX17, three SNXs that control recycling to the plasma membrane. Conclusions: SNX3 contributes to the formation of pre-AC and MCMV assembly. It acts sequentially with SNX27, SNX4, and SNX17 along the recycling pathway in the process of the production and release of infection virions, suggesting that multiple membrane sources may contribute to the secondary envelopment of MCMV virions.

Background/Objectives: Cytomegalovirus (CMV) infection expands early endosomes (EEs) into tubular extensions that may contribute to the control of virus replication and virion assembly. Sequential recruitment of protein coats and sorting nexins (SNXs) creates membrane zones at the EEs that serve as scaffolds for membrane tubulation and retrieval of cargo proteins, including host cell signaling proteins and viral glycoproteins. This study aims to investigate whether the SNX3-dependent zone of EEs contributes to CMV replication and assembly. Methods: Protein localization was analyzed by confocal imaging and expression by Western blot. The contribution of SNX3 to murine CMV (MCMV) replication, assembly compartment (AC) formation and virion release was analyzed by siRNA and shRNA depletion. The impact of other downstream SNXs that act in EE tubulation was investigated by combined siRNA knockdowns of SNX1, SNX2, SNX4, SNX17, and SNX27 on cell lines expressing shRNA for SNX3. Results: The SNX3-162 isoform acting at EEs was efficiently knocked down by siRNA and shRNA. The SNX3-dependent EE zone recruited SNX27 and contributed to Rab10-dependent tubulation within the pre-AC. SNX3 was not essential for MCMV replication but contributed to SNX27-, SNX17- and SNX4-dependent release of virions. Silencing SNX3 further reduced the release of virions after silencing SNX27, SNX4, and SNX17, three SNXs that control recycling to the plasma membrane. Conclusions: The scaffold zone formed by SNX3 contributes to the formation of pre-AC and to the virion release dependent on SNX27-, SNX4-, and SNX17-controlled recycling membranes, suggesting that multiple membrane sources may contribute to the control of MCMV assembly.