Epstein-Barr virus (EBV) DNA structure and gene expression were analyzed in tissue specimens from oral hairy leukoplakia (HLP), a mucocutaneous lesion that develops in patients infected with human immunodeficiency virus (HIV). The structure of the terminal restriction enzyme fragments of EBV revealed that HLP is a permissive infection without a predominant, detectable population of EBV episomal DNA. In RNA preparations from this uniquely permissive infection, EBV replicative mRNAs could be identified by Northern analysis; however, the virally encoded small nuclear RNAs, the EBERs, were not detected in most HLP RNA preparations. In situ hybridization detected EBER expression in very rare cells. These data indicate that unlike other viral small nuclear RNAs, the EBERs are not expressed during viral replication and must participate in the complex maintenance of latent EBV infection.Oral hairy leukoplakia (HLP) is a newly recognized lesion, most often located on the tongue, that occurs almost exclusively in patients with human immunodeficiency virus (HIV) infection (1). Epstein-Barr virus (EBV), usually thought of as a lymphotrophic herpesvirus, is the etiologic agent of this mucocutaneous epithelial cell disorder. HLP is the only EBV-associated disease in which acyclovir, an antiviral drug that inhibits the replication of EBV, induces clinical remission (2). This response and the detection of masses of viral particles in the infected cells make these lesions unique in EBV biology and indicate that the virus is actively replicating (1,2).Replication is relatively unusual in EBV-infected cells. Although the virus replicates in oropharyngeal epithelial cells, the transforming infection of B lymphocytes, which is the hallmark of EBV infection, is largely nonpermissive (3-6). In the latently infected B-cell lines, the viral DNA is maintained as an episome (7,8). Permissive infection develops in a small subset of cells with the expression of multiple replicative RNAs and the generation of the linear, virion form of DNA. The structure of the EBV termini permits discrimination between the linear and episomal DNA forms and therefore between permissive and latent states (9, 10).In EBV-infected cells, the most abundantly expressed viral transcripts are the nonpolyadenylylated polymerase III transcripts, EBER1 and EBER2 (EBV-encoded small nuclear RNA), which are found in ribonucleoprotein particles (RNP) (11,12). Despite their abundance, the function ofthese RNAs is unknown. The EBERs can partially substitute for the similarly sized polymerase III virus-associated (VA) transcripts encoded by adenovirus that function during viral replication (13,14). This complementation suggested that perhaps, like the VA RNAs, the EBERs also function during lytic replication. This observation was supported by studies that revealed that the EBERs are more abundant in the partially permissive virus-producer cell line B95-8 than in a latently infected lymphoid line, IB4, established by infection with B95-8 virus (15, 16). However, in co...