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 ...

The BZLF1 protein of Epstein-Barr virus (EBV) is a key immediate-early protein which has been shown to disrupt virus latency in EBV-infected B cells. We have generated a monoclonal antibody, BZ1, to BZLF1 which reacts in immunohistology, immunoblotting, and immunoprecipitation and which recognizes both the active, dimeric form and the inactive, monomeric form of the protein. Biopsies of oral hairy leukoplakia, an AIDS-associated lesion characterized by high-level EBV replication, were examined by immunohistochemistry using the BZ1 monoclonal antibody. A differentiation-associated pattern of BZLF1 expression was observed, BZ1 reacting with nuclei of the upper spinous layer of the lesion. This finding suggests that the BZLF1 promoter may be regulated by the degree of squamous differentiation. A comparison of in situ hybridization to EBV DNA and viral capsid antigen staining with BZ1 reactivity suggested that BZLF1 expression precedes rampant virus replication. The inability to detect EBV in the lower epithelial layers of oral hairy leukoplakia raises questions concerning the nature of EBV latency and persistence in stratified squamous epithelium.

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