Proteins of purified Epstein-Barr virus

Abstract

Mature Epstein-Barr virus (EBV) was purified from the culture medium of infected lymphocytes made functionally conditional for Zta activation of lytic replication by an in-frame fusion with a mutant estrogen receptor. Proteins in purified virus preparations were separated by gradient gel electrophoresis and trypsin-digested; peptides were then analyzed by tandem hydrophobic chromatography, tandem MS sequencing, and MS scans. Potential peptides were matched with EBV and human gene ORFs. Mature EBV was mostly composed of homologues of proteins previously found in a herpes virion. However, EBV homologues to herpes simplex virus capsid-associated or tegument components UL7 (BBRF2), UL14 (BGLF3), and EBV BFRF1 were not significantly detected. Instead, probable tegument components included the EBV and gamma-herpesvirus-encoded BLRF2, BRRF2, BDLF2 and BKRF4 proteins. Actin was also a major tegument protein, and cofilin, tubulin, heat shock protein 90, and heat shock protein 70 were substantial components. EBV envelope glycoprotein gp350 was highly abundant, followed by glycoprotein gH, intact and furin-cleaved gB, gM, gp42, gL, gp78, gp150, and gN. BILF1 (gp64) and proteins associated with latent EBV infection were not detected in virions.

Fig. 1.

Induction of EBV lytic replication in ZHT-converted lymphoblasts. (A) Western blot of 4HT-dependent induction of lytic proteins in B95-8/ZHT cells. (B and C) Gp350 surface staining of 4HT-induced B95-8/ZHT lymphoblasts by fluorescence microscopy (B) and FACS analysis (C). (D) Electron microscopy with negative uranyl acetate staining performed on purified EB virions at ×12,000 magnification. (E and F) Electron microscopy of fixed and stained thin sections of EB virions (E) and NP-40-treated EB virions (F)at ×50,000 magnification.

Fig. 2.

Coomassie stain of SDS/PAGE 5-15% gel of purified EB virions. Arrows indicate gel slices from which the indicated proteins were found in peak abundance. Bands that were inconsistently observed in virion preparations and contained only low-abundance, non-EBV proteins are not labeled. Protein names and sizes are given in Table 1; abbreviations are explained in the text.

Fig. 3.

EB virion proteins before and after deenvelopment and virion phosphoproteins. (A) Purified EBV proteins were separated by SDS-polyacrylamide gels directly (lane E) or after treatment with NP-40 (lane N) or ND (lane ND) and stained with Sypro-Ruby. Molecular sizes are given in parenthesis. (B) Purified EB virion proteins were separated on SDS-polyacrylamide gels and stained with Sypro-Diamond to detect phosphoproteins (lane PP). The same gel was restained with Sypro-Ruby to visualize all virion proteins (lane P). BKRF4 is in small type because of its low abundance in virions and phosphopeptides from two host-encoded proteins (protein kinase A and neural-encoded death-determining protein 5) were also detected at 42 kDa (Table 6).

Fig. 4.

EB virion proteins. Proteins not previously detected in EBV are in bold. Proteins that were not detected are shown in italics outside the virion. Unique long (UL) designations refer to the EBV homologues of HSV tegument proteins as in Table 1.