Hairy leukoplakia: an unusual combination of transforming and permissive Epstein-Barr virus infections

Abstract

Human herpesviruses are characterized by distinct states of infection. Typically in permissive herpesvirus infection, abundant virus production results in cell lysis. In latent transforming Epstein-Barr virus (EBV) infection, viral proteins that induce cell growth are expressed. The immunodeficiency-associated hairy leukoplakia (HLP) lesion is the only pathologic manifestation of permissive EBV infection; however, within HLP, viral proteins characteristic of latent infection have also been detected. In this study, we further analyzed expression of EBV latent genes and investigated their contribution to the unique histologic phenotype of HLP. Coexpression of lytic and transforming viral proteins was detected simultaneously within individual HLP keratinocytes. LMP1 has now been shown to be uniformly expressed in the affected tissue, and it is associated and colocalizes with tumor necrosis factor receptor-associated factor (TRAF) signaling molecules. Effects induced by activated TRAF signaling that were detected in HLP included activation of NF-kappaB and c-Jun terminal kinase 1 (JNK1) and upregulated expression of epidermal growth factor receptor (EGFR), CD40, A20, and TRAFs. This study identifies a novel state of EBV infection with concurrent expression of replicative and transforming proteins. It is probable that both replicative and latent proteins contribute to HLP development and induce many of the histologic features of HLP, such as acanthosis and hyperproliferation. In contrast to other permissive herpesvirus infections, expression of EBV transforming proteins within the permissively infected HLP tissue enables epithelial cell survival and may enhance viral replication.

FIG. 1

(A) Nuclear expression of EBV-encoded proteins associated with permissive and transforming infection in HLP. Frozen sections of HLP were stained with a BRLF1 polyclonal antiserum and EBNA2 monoclonal antiserum. Rhodamine-conjugated anti-rabbit secondary antibody detected BRLF1 (red fluorescence) in the nuclei of cells within HLP (A1 and A3). FITC-conjugated anti-mouse secondary antibody detected EBNA2 (green fluorescence) in the nuclei and cytoplasm of keratinocytes within HLP (A2 and A4). (B) Cytoplasmic expression of EBV-encoded proteins associated with permissive and transforming infection in HLP. Frozen sections of HLP were stained with a BHRF1 monoclonal antibody and an LMP1 rabbit polyclonal antibody. Rhodamine-conjugated anti-rabbit secondary antibody detected LMP1 (red fluorescence) in the cytoplasm of cells within HLP (B1 and B3). Likewise, FITC-conjugated anti-mouse secondary antibody detected BHRF1 (green fluorescence) in the cytoplasm of keratinocytes within HLP (B2 and B4).

FIG. 2

Immunoblot detection of EBV proteins from tongue biopsy specimens. (A) EBNA2 was detected in the three HLP samples (HLP14, -15, and -16) and control lymphoblastoid cell line CB-B95-8. (B) EBNA-LP was detected at 44 kDa in the EBV-positive lymphoblastoid line X50-7, and 33- and 42-kDa proteins were detected in the HLP2 and -13 samples. (C) LMP1 was detected in HLP2, -11, and -13 samples and control lymphoblastoid cell line X50-7 but was not detected in PHLP. Locations of the viral proteins and the molecular mass markers (in kilodaltons) are indicated.

FIG. 3

LMP1 and EGFR expression in HLP. Images show immunofluorescence and immunoperoxidase-based staining of frozen and paraffin-embedded HIV-negative tongue sections (A, C, and E) or HLP (B, D, and F). Membrane and cytoplasmic staining of LMP1 with monoclonal antibody OT22C (1:20) was detected in suprabasal cells of HLP (staining of the upper stratum spinosum shown in panel B) and not in tongue specimens from HIV-negative individuals at a magnification of ×200 (A). Both immunoperoxidase-based staining and immunofluorescence detect significant suprabasal EGFR staining in HLP (staining of the lower to middle stratum spinosum shown in panels D and F), while staining of the control tissue was associated only with the basal layer of epithelial cells (C and E) (magnification, ×400).

FIG. 4

Expression of LMP1 and activated JNK1 and ERK1/2 in HLP. Protein lysates of HLP7, -9, and -10, of HIV-negative tongue specimens (CB-B95), and of EBV-negative cell lines (HeLa and H1299) were subjected to Western blot analysis. (Top) LMP1. (Middle and bottom) Blots probed with polyclonal antibodies specific for phosphorylated forms of MAPK and JNK detected active JNK1 and ERK1/2 in HLP and ERK1 in the HIV-negative control.

FIG. 5

LMP1 and TRAF molecules colocalize by confocal microscopy in HLP. Frozen sections of HLP and HIV-negative tongue specimens were stained with an LMP1 monoclonal antibody and rabbit polyclonal antibodies for TRAF1, TRAF2, and TRAF3. Rhodamine-conjugated secondary antibody detects TRAFs (red fluorescence), and FITC-conjugated secondary antibody detects LMP1 (green fluorescence) throughout the stratum spinosum of HLP. In panels A to C, colocalization of molecules in HLP is detected by yellow staining (magnification, ×400). (A) TRAF1-LMP1 colocalization; (B) colocalization of TRAF2 and LMP1; (C) colocalization of TRAF3 and LMP1. Staining was not detected in HLP stained with FITC or rhodamine alone (data not shown) or in normal tongue stained with LMP1, TRAF1, TRAF2, and TRAF3 and their secondary antibodies (D).

FIG. 6

Expression of TRAF1 and TRAF3 and detection of LMP1-TRAF complexes in an HLP biopsy specimen. Protein lysates from HLP and AIDS lymphoma specimens were prepared and either immunoblotted for TRAF1 (A) or TRAF3 (B) or immunoprecipitated (IP) with antibodies for TRAF3 (C) or TRAF1 (D) and analyzed for LMP1 by immunoblotting. TRAF1 and TRAF3 were detected in three of three HLP specimens (HLP12, -13, and -14) and in H1299 cells transfected with TRAF1 and TRAF3 by immunoblotting (A and B). LMP1 was detected in complexes immunoprecipitated for TRAF3 in the HLP (C) but not in complexes that contained TRAF1 (D). Direct loads of protein lysates from the LMP1-positive cell line X50-7 and the EBV-negative epithelial cell line H1299 were included as controls for LMP1 expression.

FIG. 7

Activated NF-κB is detected in HLP. A monoclonal antibody specific for the nuclear localization signal of the NF-κB subunit p65 was used to detect the activated form of the transcription factor in nuclei of several cells within HLP. The arrow in panel A indicates NF-κB-positive brown-staining nuclei in HLP tissue. This signal was not detected in the control specimens, in the HIV-negative control subject shown in panel B, or with secondary antibody alone (data not shown).