In this report, we show that HCMV gL and RhCMV gL are essential for HCMV and RhCMV infection, respectively. The loss of infectivity due to the deletion of RhCMV gL was rescued by expressing gL in trans or by treating gL-null virions with PEG. Expression of the viral late protein gB was similar in RhCMV- and RhCMVΔgL-infected cells, and virions produced from these cells were indistinguishable when examined by transmission electron microscopy. Finally, while HCMV and RhCMV gLs are highly homologous, they are unable to functionally complement replication of the heterologous deletion mutant.
HCMV and RhCMV gLs were essential for infectivity of their corresponding viruses. While this is the first time that gL deletion viruses have been constructed and characterized for a betaherpesvirus, other studies have characterized deletions of gL or gH in alpha- and gammaherpesviruses. gL−
HSV is not infectious, but partial infectivity is recovered when infection is performed in the presence of PEG (11
). Epstein-Barr virus and bovine herpesvirus 1 with gH deleted can bind to target cells but fail to enter the cells unless treated with PEG to induce membrane fusion (27
). Similarly, we found that PEG could restore infectivity of gL−
RhCMV, suggesting that RhCMV gL, and therefore the gH/gL complex, is important for an early step in virus entry. This conclusion is supported by the observation that gL−
virions produced in noncomplementing cells were indistinguishable from wild-type virions by transmission electron microscopy. This implies that gL (i.e., the gH/gL complex) plays little or no role in virus egress or virion morphogenesis. These findings are consistent with previous observations with HSV gL mutants (35
). Similarly, cells infected with gH−
pseudorabies virus have virions both in the cytoplasm and outside of cells but not in neighboring cells (31
Herpesvirus gH/gL complexes have been proposed to function as fusogens (6
). Coexpression of HCMV gH and gL by retroviral vectors induced syncytium formation in the absence of gB in fibroblasts (20
). In contrast, coexpression of HCMV gL and gH by adenovirus vectors in epithelial cells did not induce cell-to-cell fusion, but the combination of gL, gH, and gB did (42
). While HSV gH/gL has also been postulated to be a fusogen, the crystal structure of the gH/gL complex indicates that putative gH fusion peptides are buried in beta-hairpins located in multistranded sheets and thus unlikely to participate in fusion (5
). In addition, the crystal structure of HSV gH/gL does not resemble any known fusogen. Therefore, gH/gL, which binds gB, a known fusogen (13
), may regulate gB-induced fusion rather than function as a direct inducer of membrane fusion.
HCMV gL forms a tripartite complex with gH and gO in mammalian cells (16
), and expression of all three proteins is necessary for efficient infection of fibroblasts in cell culture (44
). In addition, HCMV gL forms a protein complex with gH, UL128, UL130, and UL131, which allows the virus to enter epithelial and endothelial cells (36
). The precise structure of this complex is unknown; however, HCMV gL binds to UL128 by noncovalent interactions and, while gL does not directly interact with UL131, UL131 does bind directly to gH in the presence of gL (36
). The HCMV gH/gL/UL128/UL130/UL131 complex is a target for neutralizing antibodies (26
) and should be considered when designing vaccines. Like HCMV, the entry of RhCMV into epithelial and endothelial cells is dependent on RhCMV UL128, UL130, and UL131, and it is assumed that RhCMV gH/gL/gO and RhCMV gH/gL/UL128/UL130/UL131 function in a manner similar to their HCMV orthologs (24
). Our data confirm the importance of gL, and therefore the gH/gL dimer, for entry of RhCMV and HCMV into fibroblasts. While the gL mutations were constructed in BACs lacking UL128/UL130/UL131, we have recently inserted the UL128/UL130/UL131 genes into RhCMVΔgL (unpublished results) and plan to use the latter virus in future studies.
Given the homology of the RhCMV and HCMV gLs we hypothesized that the two proteins might be able to complement each other. Heterologous gL proteins have been shown to complement gL function in part or in full. HSV-1 and HSV-2 gH and gL, which share 67% amino acid identity, can complement each other in cell-to-cell fusion assays (28
). In addition, while gL of varicella-zoster virus and Epstein-Barr virus have only 14% amino acid identity and 40% amino acid similarity, respectively, the two gLs can act as a chaperone for the heterologous gH to reach the cell surface (23
). We found that while RhCMV gL and HCMV gL share 53% amino acid identity, they were unable to complement replication of gL-null HCMV or gL-null RhCMV, respectively. This indicates that complementation of the gL function in CMV is highly specific for its corresponding gH, and it is unlikely that gL from other human herpesviruses will be able to complement HCMV gL-null mutants.
Currently, there is no licensed vaccine for prevention of HCMV infection. HCMV with gL deleted could function as a defective infectious single cycle (DISC) vaccine (reviewed in reference 9
) since the virus can infect cells that do not express gL and undergo a single cycle of replication, resulting in the release of mature, noninfectious viral particles. RhCMVΔgL, grown in complementing cells, can infect noncomplementing cells efficiently and produce levels of gB, the major target of neutralizing antibody to CMV, at levels similar to those seen in cells infected with wild-type virus. Unlike CMV glycoprotein subunit vaccines, CMV with gL deleted should present nearly all of the CMV proteins to the immune system in the context of MHC class I so that cellular immunity to a large number of viral proteins is induced. In addition, we report that naturally infected RhCMV-infected macaques do not produce detectable levels of antibody to gL, which suggests that loss of gL might have less of an effect on neutralizing antibody titers than deletion of gB. However, loss of gL might affect development of neutralizing antibodies to the gH/gL or gH/gL/UL128/UL130/UL131 complex. Since RhCMVΔgL and HCMVΔgL produced in complementing cells can infect noncomplementing cells, virus produced in complementing cells does contain functional gL complexes, which might be sufficient to elicit some neutralizing antibody to these complexes. Finally, the failure of the closely related HCMV or RhCMV gL to complement gL-null RhCMV or gL-null HCMV, respectively, suggests that gL encoded by human alpha- or gammaherpesviruses are unlikely to complement HCMV lacking gL. Thus, HCMV lacking gL is likely to be a safe vaccine candidate that might induce humoral and cellular immunity to nearly all of the viral proteins.