Complex Formation by Human Cytomegalovirus Glycoproteins M (gpUL100) and N (gpUL73)

Abstract

The envelope glycoproteins of human cytomegalovirus (HCMV) virions are incompletely characterized. We have analyzed complex formation between glycoprotein M (gM or gpUL100) and a second glycoprotein. gM-homologous proteins are conserved throughout the herpesvirus family and represent type III membrane proteins containing multiple hydrophobic sequences. In extracellular HCMV particles, gM was found to be complexed through disulfide bonds to a second protein with an apparent molecular mass of 50 to 60 kDa. The 50-to 60-kDa protein was found to be derived from reading frame UL73 of HCMV strain AD169. UL73-homologous genes are also conserved within herpesviruses. When transiently expressed by itself, the UL73 gene product consisted of a protein of 18 kDa. However, in the presence of gM, the UL73 gene product was posttranslationally modified to the 50-to 60-kDa species. Thus, gM and the UL73 gene product, which represents the gN homolog of herpesviruses, form a disulfide-linked complex in HCMV virions. Transient expression of gM and gN followed by fluorescence imaging with monoclonal antibodies against either protein demonstrated that complex formation was required for transport of the proteins from the endoplasmic reticulum to the Golgi and trans-Golgi compartments. Finally, we tested the gM-gN complex for reactivity with sera from HCMV-seropositive donors. Whereas most sera failed to react with either gM or gN when expressed alone, 62% of sera were positive for the gM-gN complex. Because a murine monoclonal antibody reactive with gN in the gM-gN complex efficiently neutralizes infectious virus, the gM-gN complex may represent a major antigenic target of antiviral antibody responses.

Vaccine properties of a novel marker gene-free recombinant modified vaccinia Ankara expressing immunodominant CMV antigens pp65 and IE1

“…The example we used of CMV-gB, could only be made into a recombinant MVA when the TM region was deleted [28]. It is likely that other surface glycoproteins, including gM, gN [65,66], gL, gO and gH [67] are involved in virus neutralization, so it is incumbent to find structures that will allow MVA virus formation. If those practical issues can be overcome, then an MVA composed of a combination of strong humoral immunity inducing antigens, along with the antigens we have discussed in this report for stimulating cellular immunity might be useful in the setting of congenital infection.…”

Identification of Glycoprotein gpTRL10 as a Structural Component of Human Cytomegalovirus

“…All but one of the structural glycoproteins of HCMV which have been characterized thus far have been shown to form disulfide-linked complexes (2,3,26,28). It was therefore of interest to investigate potential complex formation of gpTRL10.…”

“…Two days later, the cover slips were washed and fixed in 2.0% paraformaldehyde in PBS. The fixed cells were permeabilized with NP-40-containing buffer and then blocked with 20% normal goat serum (28). Primary antibodies, including an anti-Myc monoclonal antibody, rabbit anticalreticulin (endoplasmic reticulum [ER] marker), and rabbit anti-gm130 (Golgi marker) were then added.…”

“…Images were collected using a Leitz Dialux fluorescence microscope fitted with a Photometrics charge-coupled device camera. Images were processed using Image Pro software and Adobe Photoshop as previously described (28).…”

“…The gCI complex is formed by disulfide-linked homodimers of glycoprotein B (gB; also called gpUL55) (2,3). The gCII complex is composed of gM (IMP; also called gpUL100) and gN (gpUL73), and large parts of this complex are also held together by disulfide bonds (28). Glycoproteins H (gH; also called gpUL75), L (gL; also called gpUL115) and O (gO; also called gpUL74), the constituents of gCIII, form a heterotrimeric, disulfide-linked complex (17,18,26).…”