The Carboxy-Terminal Domain of Glycoprotein N of Human Cytomegalovirus Is Required for Virion Morphogenesis

Abstract

Glycoproteins M and N (gM and gN, respectively) are among the few proteins that are conserved across the herpesvirus family. The function of the complex is largely unknown. Whereas deletion from most alphaherpesviruses has marginal effects on the replication of the respective viruses, both proteins are essential for replication of human cytomegalovirus (HCMV). We have constructed a series of mutants in gN to study the function of this protein. gN of HCMV is a type I glycoprotein containing a short carboxy-terminal domain of 14 amino acids, including two cysteine residues directly adjacent to the predicted transmembrane anchor at positions 125 and 126. Deletion of the entire carboxy-terminal domain as well as substitution with the corresponding region from alpha herpesviruses or mutations of both cysteine residues resulted in a replicationincompetent virus. Recombinant viruses containing point mutations of either cysteine residue could be generated. These viruses were profoundly defective for replication. Complex formation of the mutant gNs with gM and transport of the complex to the viral assembly compartment appeared unaltered compared to the wild type. However, in infected cells, large numbers of capsids accumulated in the cytoplasm that failed to acquire an envelope. Transiently expressed gN was shown to be modified by palmitic acid at both cysteine residues. In summary, our data suggest that the carboxy-terminal domain of gN plays a critical role in secondary envelopment of HCMV and that palmitoylation of gN appears to be essential for function in secondary envelopment of HCMV and virus replication.The formation of an infectious herpesvirus is exceedingly complex and requires the assembly of an enveloped particle containing Ͼ70 proteins. The morphogenesis of this particle is still not understood. A currently widely accepted view suggests that two separate envelopment stages occur within the infected host cell and have led to the proposed envelopment-de-envelopment-reenvelopment model (36). According to this model, a primary envelopment occurs when capsids bud at the inner nuclear membrane to become enveloped particles in the perinuclear space. These particles subsequently fuse with the outer nuclear membrane, leading to the release of capsids into the cytoplasm. The secondary envelopment occurs when capsids which have accumulated tegument proteins in the cytoplasm bud into the lumen of a compartment that carries markers of the late secretory pathway and the trans-Golgi network (TGN), operationally defined as the assembly compartment (49). It is here where herpesviruses acquire their complete set of envelope glycoproteins. It should be noted that this model is not universally accepted (31). In any case, the successful envelopment of the tegumented capsid requires that all envelope components be concentrated in the assembly compartment, a process that in itself also likely demands a high degree of structural and temporal coordination.Glycoproteins M and N (gM and gN, respectively) are among the few envelope gl...

Optimal Replication of Human Cytomegalovirus Correlates with Endocytosis of Glycoprotein gpUL132

“…Also, the signals were larger and had a doughnut shape, with larger vesicles in cells expressing gpUL132m1-4HA. Together with the change in intracellular gpUL132 distribution, the signal from the TGN was also altered, and in some of the cells that were infected with the gpUL132 mutant viruses, the TGN was hardly detectable, a phenomenon that is generally associated with HCMV infection in fibroblasts (22,29).…”

The m74 Gene Product of Murine Cytomegalovirus (MCMV) Is a Functional Homolog of Human CMV gO and Determines the Entry Pathway of MCMV

“…Deletion of the complete m74 reading frame was not possible, as the C-terminal end of the m74 ORF overlaps with the C-terminal end of the M73 ORF (gN). Deletion of the M73 3Ј end very likely would result in a replication-deficient virus as described for HCMV UL73 mutants (27). The stop was inserted at position 120 to exclude the usage of alternative internal start codons between positions 1 and 120.…”

Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway

“…The gM/gN complex is among the few envelope proteins that are conserved among herpesviruses, likely indicating an important role for viral pathogenesis (104). While mutagenesis studies of gM and gN indicate that they are essential for virus replication (105,106), and while the gM/gN complex is capable of binding to heparan sulfate proteoglycans in the first step in viral cell attachment prior to entry (Fig. 3A), the functions of gM and gN as virion glycoproteins are largely unknown (107,108).…”

“…3A), the functions of gM and gN as virion glycoproteins are largely unknown (107,108). Although no role in specific viral receptor attachment or membrane fusion has been reported for the gM/gN complex, antibodies targeting the complex are capable of neutralizing infection to an extent comparable to neutralization by anti-gB antibodies (105,(109)(110)(111). Thus, targeting the gM/gN complex may be an effective strategy to limit CMV infection and dissemination.…”