Initiation of human cytomegalovirus secondary envelopment requires the gM/gN glycoprotein complex and involves palmitoylation
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
Glycoprotein M (gM) of human cytomegalovirus (HCMV) forms a protein complex with glycoprotein N (gN), whose precise function in viral morphogenesis is poorly understood. Both proteins are highly conserved across herpesviruses and likely perform similar functions in virion assembly and egress, with the gM/gN complex being essential for HCMV morphogenesis. To elucidate the function of the gM/gN complex in secondary envelopment, we employed a combination of viral mutants, siRNA knockdown experiments, and ultrastructural analyses.
A detailed examination of the virion morphogenesis 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 initiation of secondary envelopment was associated with an accumulation of partially tegumented capsids in the peripheral region of the cytoplasmic viral assembly compartment (cVAC), whereas wild-type virus-infected cells predominantly accumulated enveloped capsids in the central area of the cVAC. Additionally, large protein aggregates were observed within and near the cVAC, often associated with non-enveloped capsids. A comparable ultrastructural phenotype, including altered capsid distribution, envelopment defect, and protein aggregation, was observed in wild-type virus-infected cells treated with siRNA against gM. Indirect immunofluorescence analysis confirmed the altered distribution of capsids in the cVAC in wild-type virus-infected cells with reduced or undetectable gM levels, resembling the pattern observed in TB-gN-C123S-infected cells. 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 using 2-bromopalmitate (2-BP) 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 the secondary envelopment of partially tegumented capsids and highlight the involvement of palmitoylation in this process. Defects in the initiation of secondary envelopment are associated with an altered spatial distribution of capsids within the cVAC.
AUTHORS SUMMARY
Human 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 that includes a final step called “secondary envelopment,” in which the virus acquires its envelope. In this study, we investigated the role of two viral proteins, 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. When the function of gM or gN was disrupted, or when palmitoylation was chemically blocked, the capsids were unable to initiate budding into membranes, resulting in partially tegumented capsids that accumulated in the outer area 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 assembly occurs in a spatially organized manner within the cVAC, offering new insights into how herpesviruses produce infectious particles.
