Epstein-Barr Virus Lacking Glycoprotein gp42 Can Bind to B Cells but Is Not Able To Infect

The Epstein-Barr virus gH-gL complex includes a third glycoprotein, gp42, which is the product of the BZLF2 open reading frame (ORF). gp42 has been implicated as critical to infection of the B lymphocyte by virtue of its interaction with HLA class II on the B-cell surface. A neutralizing antibody that reacts with gp42 inhibits virus-cell fusion and blocks binding of gp42 to HLA class II; antibody to HLA class II can inhibit infection, and B cells that lack HLA class II can only be infected if HLA class II expression is restored. To confirm whether gp42 is an essential component of the virion, we derived a recombinant virus with a selectable marker inserted into the BZLF2 ORF to interrupt expression of the protein. A complex of gH and gL was expressed by the recombinant virus in the absence of gp42. Recombinant virus egressed from the cell normally and could bind to receptor-positive cells. It had, however, lost the ability to infect or transform B lymphocytes. Treatment with polyethylene glycol restored the infectivity of recombinant virus, confirming that gp42 is essential for penetration of the B-cell membrane.Entry of enveloped viruses into mammalian cells requires that the virion envelope fuse with the cell membrane after attachment to the cell surface. Herpesviruses require the functions of multiple protein species to mediate this event, and in keeping with the common origin and diverse habitats of these viruses, some of the proteins involved in penetration appear to be conserved throughout the family and some appear to be restricted to individual members or more closely related members with similar tropism. The two glycoproteins gH and gL fall into the first category of conserved proteins. Glycoprotein gH has been implicated as a major player in virus-cell fusion in many herpesviruses (8, 10, 11, 22, 28, 32, 34), and gL is an essential partner which is required for folding and transport of gH out of the endoplasmic reticulum (6, 19, 21, 27, 28, 35, 38, 45). The gH and gL homologs of Epstein-Barr virus (EBV) are gp85, the product of the BXLF2 open reading frame (ORF) (13, 31), and gp25, the product of the BKRF2 ORF (45), and these homologs appear to behave much as their counterparts in other herpesviruses do (45). However, a third glycoprotein, gp42, associates with the EBV gH-gL complex and falls into the second category of proteins, those with a more restricted distribution.Glycoprotein gp42 is the product of the BZLF2 ORF (26), and although there may be a functionally similar protein in cytomegalovirus (18, 24), it is not predicted to have a homolog in other human herpesviruses. It does, however, have a homolog in ORF51 of equine herpes virus 2 (43). Both EBV and equine herpes virus 2 infect B lymphocytes (1), and several lines of evidence suggest that, at least in the case of EBV, gp42 is critical to the infection of this cell type. A monoclonal antibody (MAb) called F-2-1 that reacts with gp42 has no affect on EBV attachment to its receptor, complement receptor type 2 (CR2) (CD21), but inhibits fusion of the virus with the B-cell membrane and neutralizes infection (29). Glycoprotein gp42 interacts with the β₁ domain of the HLA class II protein HLA-DR (39), and MAb F-2-1 interferes with this interaction (25). Like F-2-1, a MAb to HLA-DR or a soluble form of gp42 can block B-cell transformation, and B-cell lines which lack expression of HLA class II are not susceptible to superinfection with EBV unless expression of HLA class II is restored (25). Collectively these observations suggest that gp42, probably by virtue of its interaction with HLA class II, is essential to infection of the B lymphocyte. To answer directly the question of whether gp42 is an indispensable glycoprotein, we derived a virus that could be definitively shown to lack expression of the molecule and examined its ability to infect normal resting B lymphocytes. We report here that virus with expression of gp42 blocked can exit cells normally and can bind to receptor-positive target cells. However, it is unable to penetrate into cells and initiate infection.