Initiation of human cytomegalovirus secondary envelopment requires the gM/gN glycoprotein complex and involves palmitoylation

Glycoprotein M (gM) of human cytomegalovirus (HCMV) forms a conserved protein complex with glycoprotein N (gN), whose precise function in viral morphogenesis is poorly understood. To elucidate the function of the gM/gN complex in secondary envelopment, we employed a combination of viral mutants, siRNA knockdown, and ultrastructural analyses. Ultrastructural examination of a mutant virus with a cysteine-to-serine mutation in the cytoplasmic tail of gN (TB-gN-C123S) showed a defect in the initiation of secondary envelopment as most capsids in TB-gN-C123S-infected cells were either not in contact with cytoplasmic membranes or, when near membranes, lacked signs of budding. The defect in secondary envelopment was associated with an accumulation of partially tegumented capsids in the peripheral region of the cytoplasmic viral assembly compartment (cVAC). Additionally, large protein aggregates were observed within and near the cVAC, often associated with non-enveloped capsids. A comparable ultrastructural phenotype was observed in wild-type virus-infected cells treated with siRNA against gM. Further evidence underscoring the role of the gM/gN glycoprotein complex in viral morphogenesis was obtained by investigating gM- and gN-null mutants, which displayed the same altered capsid distribution observed in TB-gN-C123S infections and after siRNA knockdown of gM. Finally, the inhibition of palmitoylation in wild-type virus-infected cells resulted in analogous defects, including an accumulation of partially tegumented capsids in the periphery of the cVAC and protein aggregates associated with capsids. In summary, our findings indicate a crucial role for the gM/gN complex in initiating secondary envelopment and highlight the involvement of palmitoylation in this process.IMPORTANCEHuman cytomegalovirus (HCMV) is a widespread herpesvirus that can cause severe illness in newborns and immunocompromised individuals. Like other herpesviruses, HCMV assembles its infectious particles through a complex process where the virus acquires its envelope through secondary envelopment. In this study, we investigated the role of glycoprotein M (gM) and glycoprotein N (gN), which form a conserved complex across herpesviruses. Using genetic mutants, RNA interference, and electron microscopy, we found that the gM/gN complex is crucial for initiating secondary envelopment. Disruption of gM or gN function, or palmitoylation inhibition, prevents capsids from budding into membranes, resulting in partially tegumented capsids that accumulated at the periphery of the cytoplasmic viral assembly compartment (cVAC). Our findings highlight the important role of the gM/gN complex and palmitoylation in HCMV assembly and suggest that the assembly occurs in a spatially organized manner within the cVAC, providing new insights into how herpesviruses produce infectious particles.