Abstract
A prerequisite for understanding the molecular function of the human cytomegalovirus (HCMV) gH (UL75)-gL (UL115) complex is a detailed knowledge of the structure of this complex in its functional form, as it is present in mature virions. The gH protein is known to be a component of a 240-kDa envelope complex designated as gCIII (D. R. Gretch, B. Kari, L. Rasmussen, R. C. Gehrz, and M. F. Stinski, J. Virol. 62:875-881, 1988). However, the exact composition of the gCIII complex remains unknown. In this report, we attempted reconstitution of the gCIII complex by coexpression of gH and gL in the baculovirus expression system. Formation of recombinant gH-gL complexes of approximately 115 kDa was demonstrated; however, no higher-molecular-mass (approximately 240-kDa) recombinant gH-gL complexes were detected, suggesting that the presence of gH and gL alone is not sufficient for reconstitution of the gCIII complex. To identify other mammalian and/or HCMV factors which may be necessary for gCIII formation, immunoprecipitates of gH and gL from HCMV-infected fibroblasts and purified HCMV virions were examined. This analysis did reveal a number of coprecipitating proteins which associate either transiently or integrally with gH and gL. One coprecipitating protein of 145 kDa was shown to be an integral component of gCIII, along with gH and gL. Characterization of the 145-kDa protein demonstrates that it is structurally and antigenically unrelated to gH and gL and that it appears to be virally encoded. Together, these data indicate that the 145-kDa protein is a third novel component of the mature HCMV gH-gL complex.
Highlights
In all human herpesviruses studied to date, homologs of glycoprotein H and glycoprotein L have been found
It has been postulated that the function of the human cytomegalovirus (HCMV) glycoprotein H (gH)-glycoprotein L (gL) complex in the entry of HCMV is mediated through a 92.5-kDa cellular membrane protein, but whether this cellular protein acts as a receptor remains to be verified [19]
To express the recombinant proteins (Fig. 1), monolayer cultures of Tn-5 cells either were infected with each of the three recombinant baculoviruses (BV-gH743, BV-gH719, and BV-gL-ST) singly or were dually infected simultaneously with gH and gL baculoviruses (BV-gH743 plus BV-rgL-ST or BV-gH719 plus BV-gL-ST). Lysates of these infected insect cells were analyzed for reactivity with gH- and gL-specific antibodies in immunoblots
Summary
In all human herpesviruses studied to date, homologs of glycoprotein H (gH) and glycoprotein L (gL) have been found. Both HCMV-infected cells and mature virions and associates with gH and gL to comprise the gCIII complex. These data indicate that a novel viral gene product of 145 kDa represents a third distinct member of the mature HCMV gH-gL envelope complex.
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