Epstein-Barr virus lacking glycoprotein gp42 can bind to B cells but is not able to infect

Abstract

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.

FIG. 1

(A) Diagram of the positions of the EcoRI and HindIII sites, numbered according to the B95-8 sequence, surrounding the SpeI/SalI fragment targeted for homologous recombination. The boxes above the line indicate the position of the SpeI/SalI fragment used as a probe and the insertion of the Neo^r gene at bp 101934. (B) Sizes of fragments expected from DNA from cells harboring wild-type episomes, a mixture of wild-type and recombinant episomes, or pure recombinant episomes after digestion with EcoRI and HindIII and probing with the 5.7-kb SpeI/SalI fragment.

FIG. 2

Southern blot analysis of DNA extracted from Akata cells harboring wild-type episomes (Wt), a parental clone of Akata cells harboring a mixture of wild-type and recombinant episomes (Wt + Rc), and a clone derived from the parental clone which contains only recombinant episomes (Rc). DNA was digested with EcoRI and HindIII, and the two identical halves of the membrane were cut apart and probed either with the SpeI/SalI fragment of EBV or the XmnI/HincII fragment containing the Neo^r gene as indicated. Sizes in kilobases are indicated by the arrows.

FIG. 3

Electrophoretic analysis of proteins immunoprecipitated by MAb 72A1 to gp350, MAb F-2-1 to gp42, or rabbit anti-peptide antibodies to gp150 or gp25 from Akata cells harboring wild-type or recombinant episomes. The cells were induced with anti-human immunoglobulin and labeled with [³H]glucosamine. The arrows and numbers at the right indicate the masses of the immunoprecipitated proteins in kilodaltons.

FIG. 4

Indirect immunofluorescence staining of Akata cells harboring only recombinant episomes (A) or wild-type episomes (B). Cells were induced with anti-human immunoglobulin for 48 h, fixed in 0.1% paraformaldehyde, and reacted with MAb E1D1 to the gH-gL complex followed by anti-mouse immunoglobulin conjugated to fluorescein.

FIG. 5

Induction of EBNA 1 in T-cell-depleted peripheral blood leukocytes (A) or EBV-negative Akata cells (B) mock infected (0) or infected 5 days previously with wild-type (Wt) or recombinant (Rc) viruses at the dilutions indicated. Western blots were reacted with human serum containing antibody to EBNA 1 and with goat anti-human immunoglobulin conjugated to alkaline phosphatase.

FIG. 6

Infection and transformation of T-cell-depleted peripheral blood leukocytes with recombinant virus in the presence of polyethylene glycol. (A) Western blot analysis of Akata cells containing wild-type virus (Wt); a B-cell line derived from peripheral blood leukocytes by infection with recombinant virus in the presence of polyethylene glycol (PBL/Rc); or freshly isolated leukocytes harvested 14 days after mock infection (Mock), treatment with polyethylene glycol alone (PEG), infection with recombinant virus (Rc), or infection with recombinant virus in the presence of polyethylene glycol (PEG+Rc). Western blots were reacted with human serum containing antibody to EBNA 1 and with goat anti-human immunoglobulin conjugated to alkaline phosphatase. (B) Southern blot analysis of DNA extracted from Akata cells containing wild-type virus (Wt), a parental clone containing a mixture of recombinant and wild-type virus (Wt+Rc), Akata cells containing pure recombinant virus (Rc), and the B-cell line derived from peripheral blood leukocytes by infection with recombinant virus in the presence of polyethylene glycol (PBL/RC). DNA was digested with EcoRI and HindIII. The blot (left) was probed with the SpeI/SalI fragment of EBV, and then (right) stripped and reprobed with the 1.5-kb XmnI/SalI fragment containing the Neo^r gene. Sizes in kilobases are indicated by the arrows.