Rhesus and human cytomegalovirus glycoprotein L are required for infection and cell-to-cell spread of virus but cannot complement each other.

Human cytomegalovirus (HCMV) is the causative agent of several life-threatening diseases in immunocompromised persons and is the leading viral cause of birth defects in the United States. Given the medical and economic burden of HCMV-related disease, the Institutes of Medicine has given the development of an HCMV vaccine “priority one” status (39). At present, the vaccine candidate that is farthest along in clinical trials, soluble HCMV glycoprotein B (gB), reduced the rate of CMV infection in healthy women by 50% (30). While promising, more effective vaccines, including those that might stimulate higher levels of cellular immune responses, would be desirable. With the exception of chimpanzee CMV, rhesus CMV (RhCMV), is the closest homolog to HCMV and has been used as a model for studies of pathogenesis and vaccine development (45). From 42 to 60% of RhCMV genes have functional and positional homologs in HCMV, depending on the strain of RhCMV analyzed and the criteria used for identifying open reading frames (12, 34). RhCMV encodes at least 21 homologs of HCMV glycoproteins, including gB, gH, gL, gM, gN, and gO and UL128, UL130, and UL131, all of which are essential for efficient replication of the virus in the host. All human herpesviruses encode a homolog of gL. While gL is thought to be essential for viral replication, all known functional properties of gL are directly associated with its dimerization with gH. The most extensively studied gH/gL complex is that of herpes simplex virus (HSV). Although gH and gL are expressed as separate open reading frames, maturation, transport, and function of both proteins are dependent on each other's expression; failure to express either gH or gL results in mislocalization and improper folding of the other (3, 8, 17, 32). Absence of gL, resulting in the lack of a functional gH, also results in a defect in virus entry (35) and an inability to initiate membrane fusion (7). Although gH/gL has been postulated to function as a fusogen (33, 40), the cocrystal structure of gH/gL does not resemble a classical type 1, 2, or 3 fusion protein (5). These results suggest that gH/gL does not act directly as a fusogen but rather modulates the function of gB, a known type 3 fusogen (13). The cocrystal structure also demonstrates the extensive contacts between gH and gL, highlighting the dependence of both glycoproteins on each other to maintain gH/gL structure and function (5). Not surprisingly, HSV gL deletion mutants are deficient for virus entry but can be propagated in cell lines that provide gL in trans (35). HCMV gH and gL, like their HSV homologs, interact extensively (15, 19, 38). In the absence of gL, HCMV gH does not fully mature and is not transported to the plasma membrane (19, 38). Unlike HSV, however, HCMV gH and gL form larger complexes with either gO or the UL128/UL130/UL131 proteins, the latter of which are required for entry into epithelial and endothelial cells (36, 44). While HCMV gL has been deleted from bacterial artificial chromosomes (BACs) containing the entire HCMV genome, virus was not recovered when the mutated BACs were transfected in human fibroblasts (10, 14). These data suggest that HCMV gL is essential for viral replication; however, since the mutant BACs were not complemented in trans, it was not proven that the failure to produce virus was not due to another mutation in the BAC or that the deletion of gL affected a neighboring gene that might be required for virus replication. To date, only one essential HCMV glycoprotein, gB, has been deleted and complemented in mammalian cells (18). We describe here the construction and characterization of RhCMV and HCMV gL deletion mutants that can be propagated in cells stably transduced to express the corresponding gL. Noncomplementing cells infected with the RhCMV gL deletion mutant express high levels of RhCMV gB and produce virions indistinguishable from wild-type virus by electron microscopy; however, these virions are not infectious. Despite their homology, cells expressing HCMV gL and RhCMV gL were not able to complement RhCMV with gL deleted or HCMV with gL deleted, respectively.