The BDLF2 protein of Epstein-Barr virus is a type II glycosylated envelope protein whose processing is dependent on coexpression with the BMRF2 protein

Abstract

Epstein-Barr virus has been documented to encode for ten envelope glycoproteins, gB, gH, gL, gM, gN, gp350, gp42, gp78, gp150 and BMRF2. The BDLF2 open reading frame is also predicted to encode a type II membrane protein but, although found in the virion, it has been described as a component of the tegument. We show here that, as predicted, it is the eleventh envelope glycoprotein of the virus. The full length 65 kDa glycoprotein formed a complex with BMRF2 and, as its homologs in other gammaherpesviruses, was dependent on BMRF2, for authentic processing and transport. Two cleavage products of BDLF2 were also identified in cells and in purified virion particles, one corresponding approximately to the aminoterminal half of the protein, that remained associated with the full length form, and one corresponding to the carboxyterminal glycosylated portion of the protein which did not.

Figure 1

Effect of BMRF2 on localization of BDLF2 in transfected cells. (A) Linear map of the predicted BDLF2 protein indicating positions of the transmembrane domain (black disk), the potential N-linked glycosylation sites (lollipops) and the sequences included in the GST fusion proteins (hatched rectangles) used to make antibodies to the aminoterminus (αBDLF2-N) or carboxyterminus (αBDLF2-C). (B) Indirect immunofluorescence of CV-1 cells transfected with pTM1-BDLF2, pTM1-BMRF2 or pTM1-BDLF2 and pTM1-BMRF2 as indicated. Cells were stained with antibodies to the aminoterminus of BDLF2 (αBDLF2-N) or to BMRF2 (αBMRF2).

Figure 2

Expression of BDLF2 and BMRF2 in the absence of other virus proteins. SDS-PAGE analysis of proteins immunoprecipitated by antibody to the aminoterminus of BDLF2 (αBDLF2-N) or antibody to BMRF2 (αBMRF2) from CV-1 cells labeled with [³H] leucine and transfected with pTM1-BDLF2, pTM1-BMRF2 or pTM1. Immunoprecipitated proteins were mock-digested, digested with endoglycosidase H (Endo H) or digested with PNGase F before analysis as indicated. The positions of the proteins discussed in the text are indicated by asterisks.

Figure 3

Reactivity of antibody to the carboxyterminus of BDLF2. SDS-PAGE analysis of proteins immunoprecipitated by antibody to the carboxyterminus of BDLF2 (αBDLF2-C) from CV-1 cells labeled with [³H] leucine and transfected with pTM1, pTM1-BDLF2 or pTM1-BMRF2. Immunoprecipitated proteins were mock-digested, digested with endoglycosidase H (Endo H) or digested with PNGase F before analysis as indicated. The positions of the proteins discussed in the text are indicated by asterisks.

Figure 4

Comparison by western blotting of BDLF2 expression in the presence and absence of BMRF2 with its expression in virus producing cells or purified virions. Left panel: western blot with antibody to the aminoterminus of BDLF2 (αBDLF2-N) of CV-1 cells transfected with pTM1-BDLF2, pTM1-BDLF2 and pTM1-BMRF2 or of induced Akata cells; Right two panels: western blot of purified B95-8 virus with antibody to the aminoterminus or carboxyterminus of BDLF2 (αBDLF2-C). Samples were mock digested or digested with endoglycosidase H (Endo H) or PNGase F as indicated. The positions of the proteins discussed in the text are indicated by asterisks.

Figure 5

Analysis of BDLF2 under reducing and non-reducing conditions. Western blot with antibody to the aminoterminus of BDLF2 (αBDLF2-N) of CV-1 cells transfected with pTM1-BDLF2 and pTM1-BMRF2 electrophoresed under (A) reducing or (B) non-reducing conditions. Proteins were mock digested or digested with PNGase F before analysis as indicated.

Figure 6

Coprecipitation of BMRF2 and BDLF2. SDS-PAGE analysis of proteins immunoprecipitated by antibodies to the BMRF2 (αBMRF2) from from CV-1 cells labeled with [³⁵S] methionine and transfected with pTM1-BMRF2, pTM1-BMRF2 and pTM1-BDLF2 or pTM1. Immunoprecipitated proteins were mock-digested or digested with PNGase F before analysis and immunoprecipitated with preimmune (PB) or immune (TB) antibodies as indicated. The positions of the proteins discussed in the text are indicated by asterisks.