The Molecular Basis of Cell and Tissue Organisation Glycoprotein spikes

Envelope

Tegument

Envelope

Tegument

Core

Figure 7 Schematic structure of a herpesvirus. (From IARC, 1997, p. 34.)

DNA-associated proteins

Core

Capsid

Figure 7 Schematic structure of a herpesvirus. (From IARC, 1997, p. 34.)

when dealing with a large and complicated genome. Out of the 100 or so genes encoded by EBV, many of which are still poorly characterized, latent functions, as derived from data mainly drawn from EBV gene expression in B lymphocytes, consist of a small number of species only. These include six discrete EBV nuclear antigens (EBNAs), three discrete membrane antigens (LMPs) and two small RNAs (EBERs), of as yet unknown function. Interestingly, among other viruses studied in detail, only adenovirus encodes similar small RNAs (VA I and II) that structurally resemble EBERS, and although themselves not fully functionally characterized, are thought to modulate translation of viral proteins. For EBV, the EBERs are mainly localized in the nucleus and thus they may play alternative roles. Because of their very high levels of expression, EBERs have proved useful for detecting the presence of EBV in cells although, notably, they are apparently not expressed in all cells. For example, they are not found in a nonmalignant pathology associated with immunosuppression, oral hairy leucoplakia (OHL), where infected cells are frequently undergoing lytic replication. The nomenclatures used for the latent antigens in the EBV field are given in Table 3. The rest of the viral genes have been categorically designated as lytic, or lyti-cally related. This distinct dichotomy into latent and lytic gene expression may be reassessed with time, since many EBV-associated tumours have recently been shown to express genes now designated as immediate early (or lyti-cally related). Some of these, which may play initiating roles in the viral lytic cycle, may have other roles in tumours (discussed by Griffin and Xue, 1998). Alternatively, as proposed for KSHV (Ganem, 1998), a small amount of replication and thereby re-infection may be relevant to, and essential for, tumour growth.

These latent functions, on the assumption that most or all may play roles in the alteration of cell growth induced

Table 3 Nomenclature of latent EBV gene products

Adopted terminology3

Alternative nomenclature3

Table 3 Nomenclature of latent EBV gene products

EBNA-l

EBNA-l

EBNA-l

EBNA-2

EBNA-2

EBNA-2

EBNA-3A

EBNA-3

EBNA-3A

EBNA-3B

EBNA-4

EBNA-3B

EBNA-3C

EBNA-6

EBNA-3C

EBNA-LP

EBNA-5

EBNA-4

LMP-l

LMP-2A

TP-l

LMP-2B

TP-2

EBER-l

EBER-2

aEBNA, EBV nuclear antigen; LMP, latent membrane protein; EBER, EBV-encoded RNA; TP, terminal protein. (From IARC, 1997, p. 53.)

aEBNA, EBV nuclear antigen; LMP, latent membrane protein; EBER, EBV-encoded RNA; TP, terminal protein. (From IARC, 1997, p. 53.)

by EBV, at least for B lymphocytes in culture, are briefly defined as follows (IARC, 1997).

EBNA-1: a DNA-binding protein identified in all EBV-infected cells and responsible for EBV genome replication in latently infected cells. EBNA-1 is not recognized by the host cellular immune system, probably as a consequence of the glycine-alanine-rich repetitive (IR3) sequence within the protein. In transgenic mice, it is tumorigenic. This antigen and its pivotal function in EBV latency has recently been reviewed (Leight and Sugden, 2000)

EBNA-2: a transactivator both of other viral and cellular functions, and a key protein in B cell immortalization in culture. It is not generally expressed in EBV-associated tumours, although this does not rule out an early role in tumour induction. It is expressed in post-transplant lymphoproliferative disorders and in infectious mononucleosis.

EBNA-LP: appears to be important for the stimulation of B cell growth in culture and, like EBNA-2, be a contributing factor in post-transplant lymphoproliferative disorders and infectious mononucleosis. EBNA-2 and EBNA-LP are the first two proteins to be identified following cellular infection with the virus.

EBNA-3A, 3B and 3C: often considered together because they are derived from adjacent regions of the viral genome. EBNA-3A and 3C, but possibly not 3B, are involved in growth stimulation of B cells, but all three may have regulatory roles in the transcriptional control of other key viral functions. EBNA-3C has been compared in its properties to HPV E7 and adenovirus E1A proteins, both associated with cell growth alterations induced by their respective viruses.

LMP-1: often found expressed in EBV-associated tumours. In in vitro assays using heterologous promoters like SV40 LT, it is capable of inducing tumorigenic transformation of rodent fibroblasts in culture. It alters cytokeratin expression and inhibits cell differentiation.

This transmembrane antigen may recruit signalling antibodies and is absolutely required for both the initiation and maintenance of B cell growth in culture. In transgenic mice, LMP-1 produces a pathological response in kerati-nocytes, which has not been fully characterized.

LMP-2A and 2B: map across the terminal junctions of the viral DNA and therefore can only be expressed in latently infected cells, where the genome is circular. They do not appear to be directly involved in the in vitro growth stimulation of B cells, but may be important for the maintenance of latency. LMP-2A is a phosphoprotein, stably phosphorylated on tyrosine, and thus may have other unidentified functions.

Two other genes, BARF1 and BARFO: more recently identified, both of which may play key roles, particularly in epithelial cell growth regulation. Their importance to B lymphocyte growth stimulation in vivo is less clear. The BARF1 gene, like LMP-1, is fully competent for inducing tumorigenic cellular transformation of rodent cells, and even B lymphocytes in culture, when expressed under a strong, heterologous promoter. It has some homology with the human intercellular cell adhesion molecule 1 (ICAM-1). In limited studies carried out to date, BARF1 has been found expressed in most EBV-associated nasopharyngeal carcinomas (NPCs) examined. Its activities remain to be fully characterized. The second gene comes from BamHI I/A transcript, also called complementary strand transcripts (CSTs), or BARFO gene. Primary CSTs extend over about 25 kbp of the viral genome (Figure 6) and spliced variants of it make up the major transcripts in NPCs. They were first identified in 1989 (Hitt et al, 1989) as a family of processed, multiply spliced polyadenylated RNAs and were subsequenty designated as 'complementary' in recognition of the fact that they were generated from the DNA strand with opposite polarity to that specifying numerous previously known viral genes. Each of the ORFs in the polycistronic CSTs, created by splicing events, overlap genes on the opposite strand, most of which are associated with lytic replication, which has led to the speculation that they may be involved in the maintenance of viral latency. CSTs are expressed also in BLs and other EBV-associated tumours, but at lower levels. They are often designated as latent functions as a consequence of their ubiquitous expression in tumours, but have also been found in lytically infected cells. A protein first described as a product of BARFO, the largest and terminal (3' end of the gene, with its termination codon in the polyadenylation signal of the message) of the CST ORFs, was later identified in uninfected cells, casting doubt on its authenticity. BARF1 and CST expression and function(s) in EBV infected cells are key targets for future research.

The locations of some of the genes described on the physical map of EBV are given in Figure 6, and their designations and functions, where known, are summarized in Table 4. A unique working nomenclature has been established for EBV genes, where B stands for the BamHI

restriction DNA fragment containing a particular gene, a letter represents fragment size relative to the other BamHI products (A being the largest and g the smallest in the sequenced B95-8 EBV genome; Baer et al., 1984), R (right) or L (left) denotes its direction (and polarity) on the conventional physical map of the genome, and a number denotes which reading frame is represented within a particular fragment. Thus, BARF1, above, is the first right-wardly expressed ORF in the BamHI A fragment. BARFO was not predicted by the DNA sequence, so it carries an aberrant designation. The differential expression of these genes in various EBV-associated tumours, or in lympho-blastoid cell lines (LCLs) generated by infecting B lymphocytes with the virus, have now led to subclassifications of viral latency, as simplistically illustrated for EBNAs and LMPs in Figure 8, and given in detail in Table 5.

The EBV genome also includes two other genes with interesting homologies to human genes: BCRF1 and BHRF1, IL-10 and Bcl-2 homologs, respectively. Their roles in the virus have not been defined.

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