Abstract text (incl. figure legends and references)

The human cytomegalovirus (HCMV) belongs to the family of herpesviruses. As all herpesviruses, its complex maturation pathway includes two distinct envelopment processes in the host cell: primary envelopment on the way from the nucleus into the cytoplasm, and secondary envelopment in the cytoplasmic viral assembly complex (cVAC), the process that provides the virions with their final envelope. HCMV glycoprotein M (gM) forms a protein complex with glycoprotein N (gN), whose precise function in viral morphogenesis is poorly understood. Both proteins are highly conserved among herpesviruses and presumably exert similar functions in assembly and egress, with the gM/gN complex being essential in HCMV.

Ultrastructural analyses using TEM imaging and STEM tomography of high-pressure frozen and freeze substitution were applied in combination with viral mutant and siRNA knockdown experiments to clarify at which step of secondary envelopment the gM/gN complex is involved.

In wild-type virus infected cells, most capsids at the cVAC are enveloped and only a few partially tegumented but naked capsids can be found. Furthermore, naked and enveloped capsids are homogeneously distributed throughout the entire cVAC area/volume. In contrast, TEM of a palmitoylation-deficient gN mutant virus (TB-gNmutC123S) showed few enveloped virions at the cVAC, however, many partially tegumented and not enveloped nucleocapsids accumulated in the outer region of the cVAC. The centre of the cVAC was devoid of capsids. Detailed analysis of the various stages of secondary envelopment revealed that most nucleocapsids in TB-gNmutC123S-infected cells either did not contact cellular membranes at all or, if they did, showed no sign of membrane curvature that marks the initiation of secondary envelopment. In addition, large protein aggregates were found in and adjacent to the cVAC, surrounded by numerous naked, partially tegumented capsids. This ultrastructural phenotype was accompanied with a pronounced release defect. A very similar phenotype concerning capsid distribution, defect in secondary envelopment, protein aggregates and release defect was found in wild-type virus infected cells transfected with siRNA against gM. Furthermore, wild-type virus infection after inhibition of palmitoylation by 2-BP resulted in an altered nucleocapsid distribution in the outer region of the cVAC, as observed in the TB-gNmutC123S mutant.

Taken together, our results show that the gM/gN complex and its palmitoylation are essential for the initiation of secondary envelopment of partially tegumented capsids at the cVAC.