Epstein-Barr Virus Infection in Oral Hairy Leukoplakia: Virus Replication in the Absence of a Detectable Latent Phase

Abstract

Epstein-Barr virus (EBV) infects both B lymphocytes and oropharyngeal epithelium, and it has been argued that the true reservoir of virus persistence in vivo is the self-renewing basal epithelial compartment. The identification of oral hairy leukoplakia (HL) of AIDS patients as a clinically apparent focus of EBV replication in lingual epithelium therefore provides a means of studying the EBV-epithelial cell interaction in situ. Replicative EBV DNA and productive cycle antigens are restricted to the upper, more differentiated epithelial layers in HL, and here we have applied highly sensitive in situ hybridization and immunohistological methods to examine the lower basal/suprabasal layers for evidence of latent EBV infection. We could not detect EBV DNA in these layers using an in situ DNA hybridization protocol which, on reference B cell lines, detected 1 viral genome/cell. Likewise, using sensitive in situ RNA hybridization for both the small non-polyadenylated EBER RNAs (abundant transcripts seen in all known forms of EBV latency) and the latent membrane protein (LMP) mRNA (the most abundant viral mRNA in B lymphoblastoid cell lines), the basal/suprabasal cells in HL were consistently negative; immunohistological staining with specific monoclonal antibodies also gave no evidence of latently infected LMP-positive cells. When the biopsy extracts were analysed by immunoblotting with selected human antisera, in addition to abundant productive cycle antigens, a band of constant size (66K) was observed which also reacted with immunopurified antibodies monospecific for one of the latency-associated nuclear antigens, EBNA l; the cellular origin of this EBNA 1 could not be ascertained, but it is possible that in HL the protein is expressed during the productive cycle. The absence of demonstrable EBV latency in the basal/ suprabasal cells of HL suggests that this is purely a virus replicative lesion which is sustained by continual re-infection of the maturing epithelium, not by the maturation of latently infected cells from the basal compartment. IntroductionEpstein-Barr virus (EBV) is a human herpesvirus infecting more than 90~ of the population world-wide and persisting for life in the infected host. The best characterized target cell for EBV infection both in vivo and in vitro is the B lymphocyte; thus, when peripheral blood lymphocytes from virus carriers are placed in culture, EBV-immortalized B lymphoblastoid cell lines (LCLs) can be established by spontaneous outgrowth (Pope, 1967;Nilsson et al., 1971). Similarly, immortalization of B lymphocytes from previously uninfected individuals can be achieved by experimental EBV infection in vitro. Although a small subset of cells in LCLs may sustain EBV replication, the vast majority of cells remains non-productively (i.e. latently) infected. These latently infected B cells constitutively express a subset of viral genes encoding six EBV-encoded nuclear antigens (EBNA 1, 2, 3a, 3b, 3c and LP) and two membrane proteins, latent membrane protein (LMP) and terminal protein or ...

Epstein-Barr Virus Infection to Epstein-Barr Virus-Negative Nasopharyngeal Carcinoma Cell Line TW03 Enhances Its Tumorigenicity

“…There EBV turned to the lytic phase of infection. It is well known that EBV infection is lytic in oral hairy leukoplakia and in keratinizing cells in nasopharyngeal carcinoma (Niedobitek et al, 1991;Pathmanathan et al, 1995). However, there is no good experimental model for analyzing the relations between EBV and keratinizing differentiation.…”

Epstein‐Barr virus B95.8 produced in 293 cells shows marked tropism for differentiated primary epithelial cells and reveals interindividual variation in susceptibility to viral infection

“…1,9 EBV's genome has also been found in vivo in oral hairy leukoplakia (OHL), a benign epithelial lesion characterized by virus infection and replication in the differentiated layers of the tongue epithelium, in cells of the tongue epithelium at autopsies or in rare cells of the tonsillar epithelium. [10][11][12][13] EBV's infectious spectrum is therefore not limited to B cells but includes epithelial cells and T cells. 1 One possible interpretation of these data is that epithelial cell infection is not observed in healthy carriers and is only restricted to pathological states.…”

Expression of the recombination activating genes (RAG1 and RAG2) is not detectable in Epstein‐Barr virus‐associated human lymphomas

“…and sense RNA probes were generated by in vitro transcription using 35 S-labeled UTP as described. 14 …”

“…14 In brief, NALM6 cytospin preparations were air dried, fixed in 4% paraformaldehyde, de-hydrated through graded ethanols, air dried and stored at -80°C until use. Paraffin sections were de-waxed and re-hydrated through graded ethanols, treated with 0.2 N HCl for 20 min, digested with 0.5 mg/ml pronase (Boehringer, Mannheim, Germany) for 10 min, fixed in cold 4% paraformaldehyde for 20 min and acetylated using a freshly prepared 1:400 solution of acetic anhydride in 0.1 M triethanolamin, pH 8.0, for 10 min.…”