EBV gp110, an envelope glycoprotein encoded by the BALF4 gene, shows marked structural homology to the gB proteins from other herpesviruses (8
). Both gp110 of EBV and gB of HSV-1 comprise a large extracellular domain, a hydrophobic domain of approximately 50 amino acids, and a short cytoplasmic tail (104 and 109 amino acids for gp110 and gB, respectively). Despite these genomic and structural similarities, gBs from different herpesviruses fail to complement the phenotype of EBV and HSV-1 gB deletion mutants (25
). This justifies independent studies of both gp110 and the other gB and their cognate cellular receptors.
Herein we report construction of a ΔBALF4 EBV mutant and the description of its phenotype. Virus lytic DNA replication and lytic protein synthesis were unaffected by the BALF4 deletion. Virus maturation and egress, monitored by electron microscopy, were indistinguishable for mutant and wild-type viruses; viral titers in supernatants from 293/ΔBALF4 cells, 293/ΔBALF4-C cells, and 293/EBV-wt cells, monitored by qPCR, were very similar and perfectly compatible with efficient virion production. ΔBALF4 virus infection was, however, strictly abortive in all tested target cell types. These included primary epithelial cells with squamous differentiation, primary B lymphocytes, and various cell lines of lymphoid and epithelial origin (Table ). Therefore, gp110 appears to be strictly required for target cell infection but not for maturation in 293 cells. These findings fit with our previous observation that overexpression of gp110 in 293/EBV-wt cells increases the efficiency of infection without altering viral titers (31
). We further attempted to identify the bottleneck in the ΔBALF4 virus infection pathway. We found strong evidence for normal binding, virus uptake, and internalization into cytoplasmic vesicles, demonstrating that the infection block was located further downstream, presumably during transport from the endosomal compartment to the nucleus (Fig. and ). Such an assumption would fit with reported evidence that gp110 is essential for virus-cell fusion. In such a context, our data would indicate that fusion with both the plasma membrane and the endosomal membrane absolutely requires gp110.
Our data are congruent with the phenotypic traits previously ascribed to another BALF4 null mutant constructed in an LCL and from which no infectious virus could be recovered upon induction of the lytic cycle (19
). There is, however, a discordance regarding the mechanisms that led to a loss of the infectious potential; the present work reports efficient production of ΔBALF4 viruses that are unable to complete a successful round of infection, whereas the authors of work describing the other BALF4 knockout suggested that gp110 plays an essential role during viral maturation (19
). In the absence of gp110, these authors found that the viral replicative cycle was abortive at the stage of capsid formation and assembly (26
). This discrepancy can reflect the different cellular backgrounds in which these experiments were conducted (293 cells versus B cells). Clearly, the very high level of virus production in 293 cells relative to that in LCL cells very easily allows identification and characterization of virion progeny. We further readdressed this issue by infecting primary B cells with the ΔBALF4 mutant or EBV-wt. The resulting LCL/ΔBALF4 cells or LCL/EBV-wt cells were induced to assess virus replication. Both LCLs were found to produce gp350 upon induction; their supernatants contained DNase I-resistant linear viral DNA in equal amounts as well as rare virions (Fig. ). These results concur to indicate that the BALF4 gene is required neither for virus maturation nor for viral egress. It should, however, be stated that replication in LCLs is usually very weak and that we had to enrich LCL/ΔBALF4 or LCL/EBV-wt cells for plasmids encoding BZLF1 in order to obtain a visible level of lytic replication; attempts to induce LCL/ΔBALF4 or LCL/EBV-wt cells with tetradecanoyl phorbol acetate and sodium butyrate proved unsuccessful (data not shown). Altogether, we could find only one or two viruses per section from pellets formed after centrifugation of 30-ml supernatants from LCL/EBV-wt and LCL/ΔBALF4 cells. Clearly, such a low level of replication prevents any meaningful comparison in the efficiency of virus replication between wild-type and ΔBALF4 viruses in B cells, and it is perfectly possible that, even if not absolutely required for virus maturation, gp110 plays an ancillary role during this process that could not be detected in our assays.
Further support for our results is provided by previous observations made with HSV-1 or pseudorabies viruses that showed that gB is not required for virus maturation (3
). However, it should be noted that HSV-1 and pseudorabies viruses appear to differ in that a double HSV-1 mutant lacking both gB or gH proteins shows impaired primary nuclear egress, whereas a pseudorabies virus carrying the same deletions displays perfectly normal virus maturation and production (9
). Furthermore, human herpesvirus 8 (HHV-8) gB has been found to be essential for virus egress at 293T cell plasma membranes (24
). HHV-8 gB, however, was not required for HHV-8 capsid assembly or nuclear egress. A similar role has been ascribed to the C-terminal sequences of varicella-zoster virus; mutant viruses lacking the C-terminal 36 amino acids show impaired virus egress into the extracellular milieu (18
). Despite a high level of genomic homology, gB from different herpesviruses seems to serve highly variable functions during virus maturation (including none), irrespective of its membership in the different HSV subfamilies.
We further found that the BALF4 gene is not required for effective virion antigen presentation by infected B cells. We therefore conclude that sequestration of the virion within the endosome does not preclude mounting of an efficient immune response against its components. These data fit with a model that posits that some virion antigens, such as gp350, gp110, or the tegument protein BNRF1, are processed within the endosome-lysosome before being associated with HLA class II molecules and presented to CD4+
T cells at the cell surface (1
). It will be interesting to determine whether the trapping of ΔBALF4 virions within the endosome as a result of a failed fusion might even enhance the amplitude of the T-cell response against capsid or core proteins that normally escape from the endosome to allow capsid migration toward the nucleus.
A potentially interesting consequence of the present work is that it is now in principle possible to induce a CD4+ EBV-specific cytotoxic T-cell response with a noninfectious viral mutant. Such an attenuated strain would fulfill the safety criteria expected for a live vaccine. Whether the elicited T-cell response would be sufficient to induce protective immunity, however, remains to be seen.