Introduction

1958- first description of Burkitt's lymphoma in the malaria belt of east Africa.
1964- Epstein and Barr isolate continuous cell lines from Burkitt lymphoma cells; initial description of a herpesvirus present in these cells.
1968- EBV demonstrated as the etiological agent of infectious mononucleosis.
1969- EBV shown to immortalize lymphocytes in culture; EBV can immortalize marmoset cells and cause tumors in non-human primates.

Primary infection of Epstein-Barr virus (EBV), which is primarily transmitted by saliva, actively replicates in the epithelial cells of the oropharynx and can subsequently infect recirculating B lymphocytes which may lead to acute infectious mononucleosis (glandular fever).

Infectious mononucleosis is a benign lymphoproliferative disease that is usually seen in children, although most cases of EBV infection occur in early childhood and have no symptoms. It is characterised by transient immunosuppression and an unusual expansion of atypical lymphocytes, the majority of which are not B cells but CD8⁺ T cells.

Instead, in these cells EBV establishes a latent infection that persists for life during which only a few viral genes are expressed. Latent infection is a common feature to other herpesviruses (Cayrol and Flemington 1995; Cooper 1994). EBV has a narrow tissue tropism limited to B lymphocytes, T lymphocytes and epithelial cells of primate origin. By the age of twenty, greater that 90% of humans are seropositive, demonstrating previous exposure to EBV. EBV is potentially oncogenic and has been linked to Burkitt's lymphoma, nasopharyngeal carcinoma, B cell lymphoma, X-linked lymphoproliferative disorder (Miller 1990; Jabs 1996), and more recently to other neoplasia, including Hodgkin's disease, peripheral T cell tumours, and gastric cancer (Yoshiyama et al., 1995).

EBV is also a significant problem in AIDS patients where it is associated with diffuse polyclonal lymphomas, lymphocytic interstitial pneumonitis and oral hairy leukoplakia of the tongue. EBV positive Burkitt's lymphomas are observed with increasing frequency in transplant recipients receiving immunosuppressive therapy.

In normal individuals, latent EBV infection is controlled by humoral immunity, cytotoxic T cells, and the interferon (IFN) system (Jabs et al., 1996).

Epstein-Barr Virus is grouped as a member of the Herpesviridae family, subfamily gammmaherpesvirinae, genus lymphocryptovirus. The Herpesviridae family contain viruses grouped together based on the architecture of their virion (see figure 1).

A typical herpesvirion consists of a core containing a linear, double stranded DNA; an icosahedral capsid, approximately 100-110 nm in diameter, containing 162 capsomeres with a hole running down the long axis; an amorphous, sometimes asymmetric material that surrounds the capsid, designated as the tegument; and an envelope containing viral glycoprotein spikes on its surface. (Roizman 1990)

The subfamily Alphaherpesvirinae are grouped together on the basis of a variable host range, relatively short reproductive cycle, rapid spread in culture, efficient destruction of infected cells, and capacity to establish latent infections primarily, but not exclusively, in sensory ganglia.

The Alphaherpesvirinae contains the genera Simplexvirus (HSV-1, HSV-2, circopithecine herpesvirus 1, bovine mamillitis virus) and Varicellovirus (VZV, pseudorabies virus, and equine herpesvirus 1). (Roizman 1990)

The Betaherpesvirinae subfamily conatins the genera Cytomegalovirus (HCMV) and Muromegalovirus (murine cytomegalovirus). These viruses have a long reproductive cycle, and the infection progresses slowly in culture. The reproductive cycle is long, and the infection progresses slowly in culture. The infected cells frequently become enlarged (cytomegalia), and carrier cultures are readily established. The virus can be maintained in latent form in secretory glands, lymphoreticular cells, kidneys, and other tissues. A nonexclusive characteristic of the members of this subfamily is a restricted host range. (Roizman 1990)

The experimental host range of the members of the Gammaherpesvirinae subfamily is limited to the family or order to which the natural host belongs. In vitro, all known members replicate in lymphoblastoid cells, and are specific and some also cause lytic infections in some types of epithelioid and fibroblastic cells. Viruses in this group are specific for either T or B lymphocytes.

In the lymphocyte, infection is frequently either at a pre-lytic or lytic stage, but without production of infectious progeny and latent virus is frequently demonstrated in lymphoid tissue. Gammaherpesvirinae contains the genus Lymphocryptovirus, including EBV, and Rhadinovirus (herpesvirus ateles and herpesvirus saimiri). (Cooper 1994; Roizman 1990)

EBV is a very intriguing virus as it has been shown to interact with many cellular proteins and control the expression of several cellular genes. It is also capable of transforming cells, linking it to production of several different malignancies especially those in B and T lymphocytes.

This paper reviews the functions of EBV latent proteins and their roles in cellular and viral regulation, the cellular and viral interactions involved in the transactivation of lytic infection, the methods of EBV entry into cells, and insights into evolution and malignancies by the comparision of EBV with Human Herpesvirus 6 (HHV-6).

EBV Morphology

The EBV genome isolated from virus particles is a linear, double stranded DNA molecule of about 175 kilobase pairs (kbp). It is characterised by a number of different repetitions. The termini consist of tandem repeats of approximately 540 base pairs (bp). A variable number of large internal repeats of about 3.1 kbp joins a short and a long unique region. Several different other repeats are interspersed in the genome.

Two clusters of small tandem repeats of 125 and 102 bp show partial homology and have the same orientation on the genome. Each cluster is flanked by a highly conserved region of about 1 kbp. These left and right duplicated regions, which are denoted DL and DR respectively, are located about 100 kbp apart from each other in the viral genome (Keiff and Leibowitz 1990; Laux et al., 1985; Miller 1990).

The entire genome persists in the proliferating lymphocytes as linear-integrated and covalently closed circular episomal DNA (Fennewald et al., 1984; Keiff and Leibowitz 1990). The EBV genome has been mapped using Bam HI restriction endonuclease to produce fragments which have subsequently been denoted with a letter to identify specific fragments (Sample et al., 1984).

The term tegument, which is common to all herpesviruses, was introduced to describe the structures between the capsid and envelope. These structures have no distinctive features in thin sections, but they may appear to be fibrous on negative staining. The available evidence suggests that the amount of tegument is more likely to be determined by the virus than by the host. The tegument is frequently distributed asymmetrically. (Roizman 1990)

The most abundant EBV envelope proteins are pg350 and gp220, which are encoded by the same viral gene. Additional viral envelope proteins expressed at much lower levels include gp85, gp25, gp42/38, gp43, and gp78/55 (Cooper 1994; Li et al., 1995)

Glycoproteins Involved in Attachment and Entry into Cells

EBV infection of B lymphocytes is initiated by the binding of the virus-encoded glycoprotein gp350/220, which dominates the external viral envelope, to CD21 (Tanner et al., 1987; Nemerow 1987). CD21, also known as the C3dg receptor, and CR2, is a B lymphocyte receptor and is part of a multimeric signal transduction complex comprising CD21, CD19, TAPA-1, and Leu-13 (Sinclair and Farrell 1995). CD21 is composed of an extracellular domain of 15 or 16 short consensus repeat elements of 60 to 75 amino acids that is followed by a transmembrane domain and a short cytoplasmic tail and is the only B cell membrane protein that binds to gp350/220 (Moore et al., 1991; Tanner et al., 1987). EBV coated with gp350/220 adsorb to B lymphocytes, cap with CD21, become endocytosed into vesicles, and are subsequently released into the cytoplasm.

Cap formation, characterised by the consequence of cross-linking surface proteins, is generally associated with activation of the cell following its induction. Interestingly, the capping of CD21 in response to gp350/220 is associated with cocapping of surface immunoglobulins (Tanner et al., 1987). This suggests that EBV may sequester and activate pathways utilised during normal B-cell receptor activation. gp350/220 has two short regions that are homologous with C3dg, the natural ligand for CD21 (Nemerow et al., 1987). The first primary sequence similarity, located at or near the amino terminus of gp350/220, overlaps the sequence in C3dg which has been implicated in C3dg's binding to CD21.

The other region of similarity corresponds to a area in C3dg that is involved in an unusual thiolester bond. Experimental evidence indicates that the first sequence similarity, a 9 amino acid sequence EDPGFFNVE, mediates EBV binding to CD21. Slight changes in sequence will also abrogate binding (Nemerow et al., 1989).

Epithelial cells and T cells, as well as B cells, have also been shown to be infectible with EBV, since the viral genome can be found in the epithelial tumour cells of nasopharyngeal carcinoma, epithelial lesions in oral hairy leukoplakia, Hodgkin's disease, and peripherial T-cell tumours (Cooper 1994; Nonoyama et al., 1973; Yoshiyama et al., 1995). Although low levels of authentic CD21 have been identified in both epithelial cell lines and some T cell lines, the EBV receptor on these non-B cells that initiates infection has not been clearly characterised.

Interestingly, T cell lines that lack CD21 have been reported to bind to EBV and are infectible. This may be due to an EBV-binding moiety unrelated to CD21 which has been reported on some normal T cells. Infection of T cells may also occur in immature thymocytes which bear CD21. However, it remains to be determined whether CD21 generally mediates EBV absorption to, and infection of, epithelial cells, since non-B cells lack detectable CD21. Epithelial cells transfected with CD21 cDNA are readily infectible with EBV, demonstrating that CD21 can serve as a functional EBV receptor if expressed on epithelial cells (Cooper 1994).

Recent studies by Li et al. (1995) indicate that the penetration of epithelial cells and B cells by EBV involves the glycoproteins gp25, and gp42/38, in a complex with gp85. The ability of monoclonal antibodies against gp85 to inhibit fusion but not attachment of EBV with B cell membranes suggest a direct function of gp85 as a fusion protein (Haddad and Hutt-Fletcher 1989; Miller and Hutt-Fletcher 1988). gp85, encoded by the open reading frame BXLF2, is the homologue of a herpes simplex virus glycoprotein (gH) involved in virus penetration. gp25, the homologue of the herpes simplex virus glycoprotein gL, is the product of the BKRF2 open reading frame which associates with gp85.

Unlike the gp85 homologues of other human herpesviruses, EBV gp85 also complexes with the two additional glycoproteins gp42 and gp38 (Li et al., 1995). These two proteins are alternatively processed forms of the BZLF2 gene product and have no apparent homologues in any other known human herpesviruses. Li et al. (1995) suggest that EBV has added a unique glycoprotein to its gp85-gp25 complex and that the gp85-gp25-gp42/38 complex interacts differently with lymphocytes and epithelial cells.

This implies that the BZLF2 gene product may have evolved as a unique adaptation to infection of B lymphocytes by EBV and may explain the difference in infection between B lymphocytes and non-B cells.

Epstein-Barr Virus enters B Cells and epithelial cells by different routes. Electron microscopic studies show that EBV enters normal B cells by endocytosis into large thin-walled non-clathrin coated vesicles, followed by fusion of the viral envelope with the vesicle membrane, and entry of the nucleocapsid into the cytoplasm (Miller and Hutt-Fletcher 1992). CD21 is also internalised during endocytosis (Tedder et al., 1986).

Most evidence suggests that EBV may be incapable of fusing with normal B cells unless it has first been endocytosed. EBV enters epithelial cells by direct fusion at the cell surface, and in both methods of entry pH appears to be irrelevant (Miller and Hutt-Fletcher 1992; Nemerow and Cooper 1984). The reasons for this fundamental difference with the mechanism of entry into normal B cells is not known, but it may somewhat account for the differences in infection of the different cell lines. In conclusion EBV endocytosis is mediated by the interaction of CD21 with gp350/220 and fusion is mediated by the gp85-gp25-gp42/38 complex.

Latent Infection

Like other herpesviruses, Epstein-Barr virus persists in its hosts through its ability to establish a latent infection that periodically reactivates. EBV establishes latent infection as a self-replicating extrachromosomal nucleic acid (an episome). EBV has shown to have three transcriptionally distinct forms of latency. These are known as latency I, II, and III (Rowe et al., 1992; Brooks et al., 1993). The first two different forms of latency to be identified were latency I and latency III, and were detected in phenotypically distinct human B cell lines. All of the latent proteins, comprising of six EBV-specified nuclear antigens (EBNAs 1, 2, 3A, 3B, 3C, and leader protein, EBNA-LP) and three latent membrane proteins (LMPs 1, 2A and 2B), are expressed in latency III which was the first form of latency to be detected. The EBNAs are all polymorphic, that is, their structure differs from one strain to the next (Miller 1990).

The different EBNAs are encoded by individual mRNAs that are all generated by alternative splicing of the same long transcripts expressed from the BamHI C promoter or adjacent BamHI W promoter. LMP1 is expressed leftward and LMP2B is expressed rightward from a bidirectional promoter in the BamHI N region. LMP2A, the longer form of the LMP2, is encoded by a transcript originating from a separate promoter and containing a unique first exon not found in the LMP2B mRNA. Latency III has been identified in group III Burkitt's lymphoma cell lines and EBV transformed lymphoblastoid cell lines (Kerr et al., 1992; Yoshiyama et al., 1995).

Latency I was subsequently identified in Burkitt's lymphoma (BL) cell lines which stably retained their original tumour biopsy group I phenotype on serial passage and were all positive for the nuclear antigen EBNA1. Many BL cell lines are phenotypically unstable on serial passage and progress to a type three latency. This is accompanied by promoter switching and expression of the group III lymphoblastoid cell line-like surface phenotype (Kerr et al., 1992; Rowe et al., 1987; Sample et al., 1992). EBNA1, which is translated from a 2.5kb mRNA with a unique BamHI-Q/U/K splice structure, is the only latent protein synthetised in latency I. EBNA1, originally thought to be driven by a novel promoter in the BamHI F region (Lear et al., 1992), is driven by a promoter which is presently undefined (Schaefer et al., 1995).

The more recently identified form of latency, latency II, is characterised by the expression of all the LMPs and EBNA1. Latency II has been recognised in non-B cell tumours, such as nasopharyngeal carcinomas, T cell tumours, and Hodgkin's disease (Minarovits et al., 1994; Yoshiyama et al., 1995; Brooks et al., 1993). Only EBNA1, LMP1 and LMP2 are consistently detected in latently infected B cells and EBV associated disease tissue.

This suggests that these proteins may have important roles in the persistence of latent infection, viral replication, and EBV-associated diseases. One feature common to all forms of latency, however, is the abundant transcription of the small nonployadenylated EBER RNAs, noncoding nuclear species whose function remains unknown (Brooks et al., 1993). The plasmid maintenance origin, or oriP, is the region of DNA that supports the stable maintenance of plasmids in the presence of EBNA1 (see section 4.1). The majority of latent EBV DNA plasmids have been shown to replicate once per cell division during the S phase and replication molecules are efficiently segregated to daughter cells (Adams 1987; Mackey et al., 1995; Yates and Guan et al., 1991).

There have presently been several cellular genes which encode RNAs that are up to 100 times more abundant in latent EBV infected cells than in uninfected cells. These genes encode CD21, vimentin, cathepsin H, annexin VI (p68), serglycin proteoglycan core protein, CD44, myristylated alanine-rich protein kinase C substrate (MARCKS), and three EBV-induced genes (EBI 1, EBI2, and EBI3) (Birkenbach et al., 1993; Devergne et al., 1996).

EBI 1 and EBI 2 are predicted to encode G protein-coupled peptide receptors. EBI1, whose expression has only been identified in B and T lymphocyte cell lines and in lymphoid tissues, is highly homologous to the interleukin 8 receptors. EBI2 is most closely related to the thrombin receptor and is expressed in B lymphocyte cell lines and in lymphoid tissues but not in T cells. Both EBI1 and EBI2 are expressed at low or undetectable levels in EBV negative cells. These predicted G protein coupled peptide receptors are more likely to be mediators of EBV effects on B lymphocytes or of normal lymphocyte functions than are genes previously known to be up-regulated by EBV infection (Birkenbach et al., 1993). EBI3 encodes a soluble haematopoietin receptor related to the ciliary neurotrophic factor receptor (CNTFR) and the p40 subunit of interleukin-12.

EBI3 is expressed normally in some EBV-negative cells including cells associated with sinusoids in perifollicular areas of spleen tissue, disseminated cells in interfollicular zones of tonsil tissue, and at very high levels by placental syncytiotrophoblasts. The function of the EBI3 protein in normal or EBV-positive cells is not known. EBI3 is located at chromosome 19, band p13.3 which is a region that encodes several other proteins involved in haematopoietic cell growth and differentiation. Due to the EBI3 protein similarities to interleukin-12 p40, EBI3 has been implicated in regulating cell-mediated immune responses (Devergne et al., 1996).

EBNA1

EBNA1 is the only protein synthetised in all three types of latency, therefore it would be assumed that the EBNA1 has a critical role in maintaining latency. EBNA1 is a phosphoprotein that is separated into two separate domains, the N-terminal and C-terminal domains, joined by internal glycine/alanine rich short repeat sequences.

The size of EBNA1 varies considerably in individual cell populations (from approximately 69 to 94 kD), depending on the amount of the internal repeats (Kirchmaier and Sugden 1995; Mackey et al., 1995). EBNA1 binds DNA as a homodimer, and both DNA binding and dimerisation domains occur at the C-terminal domain (Wilson et al., 1996). EBV ENBA1 binds specifically to two clusters of sites within the EBV plasmid origin of latent DNA replication, termed oriP. EBNA1 and oriP, a 1.7 kbp cis-acting region of the EBV genome, are the only viral contributors needed to mediate viral DNA replication in latently infected cells (Kirchmaier and Sugden 1995; Mackey et al., 1995; Yates et al., 1984). oriP is composed of two clusters of EBNA1 binding sites known as, the family of repeats (FR) and the dyad symmetry elements (DS).

Both of these elements are composed of multiple, high affinity binding sites, 4 for the DS and 21 for the FR. FR is the site at which replication forks emerge as well as the site at which they terminate. The other element of oriP, DS, is about 1 kbp away from FR, and, is the site or near the site at which replication initiates. EBNA1 has been shown to link FR and DS, looping out the intervening sequence. Replication initiates in a manner largely independent of the spacing or orientation of FR and DS. The regions of EBNA1 that are required for increased plasmid retention overlap with those required to activate replication of transcription and replication (Middleton and Sugden 1994).

The mechanisms by which EBV EBNA1 activates DNA replication mediated by oriP, transcription, and retention of DNA in cells has presently not been revealed (Kirchmaier and Sugden 1995; Mackey et al., 1995). There are a further two consensus binding sites in the BamHI Q region that bind EBNA1 with weaker affinity. EBNA1 also binds to specific RNA sequences, including EBER1. Experimental data suggests that EBNA1 may play a role in EBV associated tumours through viral and/or cellular transcription or post-transcriptional control (Jones et al., 1989; Snudden et al., 1994).

EBNA1 has been demonstrated to be oncogenic in vivo and suggested that the gene product may play a direct role in the pathogenesis of Burkitt's lymphoma and possibly other EBV-associated malignancies (Wilson et al., 1996).

EBNA2

EBNA2 is one of the first two viral genes expressed and is a key regulator of viral and cellular gene transcription. EBNA2 has shown to transactivate EBV-encoded as well as target cell-encoded genes.

EBNA2 is essential for B lymphocyte growth transformation and transactivates expression of the EBNA1, EBNA3s, and the LMPs (Abbot et al., 1990; Wang et al., 1990b; Robertson et al., 1995). Although in latency II infection, the LMPs can be expressed independently of ENBA 2 mediated transactivation (Yoshiyama et al., 1995). EBNA2 also up-regulates transcription of cellular genes including CD21, and c-fgr, and with LMP1 cooperatively induces expression of CD23 (Cohen et al., 1991; Wang et al., 1990a; Wang et al., 1991).

Mutational analysis indicates that at least four separate EBNA2 domains are essential for lymphocyte transformation and two other domains that are necessary for the full transforming activity. Mutations which diminish or abolish lymphocyte transformation also diminish or abolish LMP1 transactivation, respectively. These results indicate that the transformation and transactivation functions of EBNA2 may not be separate (Cohen et al., 1991). EBNA2, an acidic-type transactivator, recruits cellular transcription factors to specific promoters via its interaction with cellular sequence specific DNA binding proteins, including Jk and PU.1.

The binding of EBNA2 to Jk appears to be an important interaction in the transforming of primary B lymphocytes (Robertson et al., 1995). Due to its ability to activate viral and cellular genes, EBNA2 may have a presently undefined role in the expression of other cellular genes, including the EBV-induced genes, EBI1, EBI2, and EBI3.

EBNA3

Until recently almost nothing was known about the functions of the EBNA3 proteins. The EBNA3 family of proteins, EBNA3A, EBNA3B, and EBNA3C, have now shown to have a function in modulating LMP1 and LMP2 transcription by preventing EBNA2 transactivation of the LMP1 and LMP2 promoters (Longnecker and Miller 96).

EBNA3C has shown to partially overlap the functions of EBNA2, as it also regulates some viral and cellular genes that are regulated by EBNA2, including C21, and LMP1 (Birkenbach et al., 1993; Robertson et al., 1995). EBNA3C also binds to Jk causing Jk not to bind to DNA or EBNA2 (Robertson et al., 1995). Therefore, it appears that EBNA3C acts as a feedback down regulator of EBNA2 mediated transactivation

EBNA-LP

EBNA-LP, encoded by the leader of EBNA mRNA (Wang et al., 1987), is one of the most least studied EBV latent proteins. EBNA-LP does have a strong role in B lymphocyte growth transactivation, as recombinant Epstein-Barr viruses encoding a mutant EBNA-LP have significantly reduced abilities to transform B lymphocytes.

It has been postulated that EBNA-LP may be important in regulation EBNA expression or the regulation of virus or cell gene expression mediated by EBNAs. Experimental data has suggested that EBNA-LP affects expression of a B lymphocyte gene which is a mediator of cell growth or differentiation and may induce transition from G0 to G1 in resting B cells (Mannik et al., 1991; Longnecker and Miller 1996 ).

The LMP1, LMP2A, & LMP2B

The latent membrane proteins (LMPs) are expressed in all forms of latency. Therefore it would be expected that these proteins would play a major role in the maintenance of transformed cells. The LMPs may play a role in viral target antigen (LYMDA) recognised by those immune T lymphocytes which specifically kill latently infected B lymphocytes (Leibowitz et al., 1986).

A significant feature of LMP1 in EBV transformed lymphocytes is its association with the vimentin cytoskeletal network (Wang et al., 1988). LMP1 has been shown to upregulate the expression of the cellular oncogene bcl-2 (Henderson et al., 1991).

The interaction between EBV infection and expression of this cellular oncogene has important implications for virus persistence and for the pathogenesis of virus associated malignant disease. LMP1 transforms immortalised rodent fibroblasts and induces vimentin, bcl-2, and many of the activation markers and adhesion molecules that EBV induces in BL cells or primary B lymphocytes (Birkenbach et al., 1993), giving further evidence that LMP1 is critical in cellular transformation.

LMP1 interacts with the novel human proteins, LMP1 associated protein 1 and EBV-induced gene 6, which are related to the murine tumour-necrosis factor receptor associated factors, TRAF1 and TRAF2 (Longnecker and Miller 96).

The two forms of LMP2, LMP2A, and LMP2B, are encoded by two mRNAs that have different 5' exons followed by eight common exons. The LMP2 proteins consists of 12 hydrophobic membrane spanning domains, which are critical for the aggregation of the protein in the plasma membrane and for blocking B cell receptor mediated signal transduction. LMP2 localises to a peripheral membrane patch in transfected B lymphoma cells and colocalises with many of the cellular tyrosine phosporylated proteins (Longnecker et al., 1991).

LMP2A has a major role in preventing the activation of lytic EBV replication via cell surface mediated signal transduction. This action of LMP2A would appear to be important in the prevention of lytic replication in latently infected B cells as they circulate in the peripheral blood, bone marrow or lymphatic tissue, where they might encounter antigens, superantigens, or other ligands that could engage B cell receptors and activate EBV lytic replication. LMP2A contains a receptor-like amino tail which, in plasma membrane aggregates, cross link and become tyrosine phosphorylated.

Cellular proteins that contain noncatalytic domains conserved among cytoplasmic signalling proteins which bind tryosine phosphorylated proteins, known as Scr homology 2 (SH2) domians, have been suggested to bind to these LMP2A phospho-tyrosines. The LMP2A complexed with proteins containing SH2 domains blocks the signal transduction through the B cell receptor, preventing activation to lytic replication. LMP2B, lacking the receptor-like amino terminal domain of LMP2A, may downregulate the activity of LMP2A by increasing the spacing between the receptor-like tail domains of individual LMP2A molecules of the plasma membrane LMP2 aggregates.

LMP2A and LMP2B are controlled by two separate promoters and may be differentially expressed depending on the environment of the latently infected lymphocyte (Longnecker and Miller 1996). Thus, the differential expression may cause either lytic activation or maintenance of latent infection.

EBER1 and EBER2

The genome of EBV encodes two Epstein-Barr virus-associated small RNAs, EBER1 (166 bases) and EBER2 (172 bases), which are expressed in all forms of latency. Both RNAs are transcribed by RNA polymerase III and are synthesised in large quantities. The EBERs exist as ribonucleoprotein particles complexed with the cellular La antigen.

Because of the relative abundance of EBER1 and EBER2 (approximately 10⁷ copies per cell) and because they are two of a the few EBV latent gene products, it would be expected that one or both of the EBERs are important in B cell transformation and/or maintenance of the latent state (Glickman et al., 1988). The EBERs have been shown to be capable of substituting for the VAI RNA function in adenovirus infected cells. Adenovirus VAI RNA is essential for the efficient initiation of viral mRNAs in the late phase of infection (Bhat and Thimmappaya 1985).

Therefore it can be predicted that the EBERs play major a role in the efficient initiation of EBV viral mRNAs. There is still very little data on the functions of the EBERs presently available, although studies suggest that the EBERs may work at the level of replication, transcription, or RNA processing (Glickman et al., 1988).

Lytic Activation

Epstein-Barr virus, as in other herpes viruses, can follow either a productive lytic infection or a latent cycle of infection.

Infection of B-lymphocytes is predominantly latent and results in lymphoproliferation. Infection of epithelial cells can be latent, as in nasopharyngeal carcinoma, but usually leads to complete viral replication resulting in cytolysis (Sista et al., 1995). The EBV encoded protein, Z, plays a dominant role in the switch from latent cycle to productive infection, and is transcribed from the immediate early gene, BZLF1 (Flemington and Speck 1990; Lieberman and Berk 1990).

Z has also been referred to as ZEBRA, Zta, and the BZLF1 protein. Z is a 34 to 38kD DNA-binding protein related to the basic leucine-zipper family of transcription factors. The basic region of Z also has sequence similarity with the DNA binding domain of members of the AP-1 family of the transcription factors. Z transactivates the viral early promoters by binding to upstream binding sites known as Z response elements which have high degrees of homology to AP-1 sites. Z acts as a trigger to disrupt latency by transactivating the genes encoding two other regulatory proteins BRLF1 and BMLF1.

Together these three proteins initiate a cascade of expression of an estimated 100 or more viral replication associated genes, including early proteins, viral capsid antigens, and membrane antigens (Cayrol and Flemington 1995; Farrell et al., 1989; Sista et al., 1995). A distinct element of the EBV genome, ori-lyt, supports the amplification of recombinant plasmids in cells that are supporting lytic replication of EBV and requires the EBV encoded DNA polymerase (Yates and Guan 1991).

The amino terminal domain of Z has been shown to play a role in activation of transcription and to be essential for in vitro association with the TATA box-binding protein TBP and the general transcription factor TFIIA. Recent evidence suggests that Z may also be involved in altering the host cell environment through autocrine or paracrine pathways (Cayrol and Flemington 1995). Interaction of cellular or viral factors can result in altering the action of Z to produce synergistic activation or repression of early promoters.

For instance, BZLF1 and the cellular factor, c-myb can associate with Z to transactivate the early promoter, while the cellular proteins p53 and p65, as well as the viral-encoded protein RAZ have been shown to interact with Z and repress Z mediated transactivation (Sista et al., 1995). Thus, there are several cellular regulators of which some appear to help sustain the latent infection by impeding the activation of lytic infection. Characterisation of cellular Z responsive genes is important to fully understand EBV host cell interactions.

For example, modification of cellular gene expression by Z could influence spreading of infected cells by the immune system. Recently the LMP2 proteins have also shown to play a hypothesised major role in lytic activation (see section 4.5). The mechanisms for lytic activation of EBV are still not fully understood.

Insights in Evolution and Malignancies by comparison of EBV with HHV-6

By investigating a recently identified herpesvirus, human herpes virus 6 (HHV-6), a better insight on the evolution of herpesviruses and why EBV causes malignancies may be elucidated. Human herpesvirus type 6 was originally isolated from peripheral blood lymphocytes of six patients with lymphoproliferative disorders, two of whom were infected with human immunodeficiency virus type 1 (HIV-1).

HHV-6 infection causes exanthem subitum (roseola infantum) and has been linked to meningoencephalitis as well as pneumonitis. HHV-6 has also been proposed to be a cofactor in the progression of AIDS, because HHV-6 and HIV-1 have been demonstrated to coinfect CD4⁺ human T cells and accelerate cytopathic effects (Kashanchi et al., 1994). Studies suggest that HHV-6 might also contain a gene which could affect transforming activity (Araujo et al., 1995). HHV-6A was shown to contain a 1 473 bp functional transformation suppressor gene (ts).

Furthermore, HHV-6 DNA sequences have been detected in a number of human malignancies, including Burkitt's lymphoma, Hodgkin's lymphomas, and Epstein-Barr virus-negative B-cell lymphomas as well as AIDS-related malignancies (Araujo et al., 1995). Thus, HHV-6 may be a cofactor in AIDS related, as well as non-AIDS related malignancies including EBV related malignancies.

Recent reports show that HHV-6 is expressed to an unusual degree in the oligodendrocytes of multiple sclerosis patients, and these studies suggest an association of HHV6 with the etiology or pathogenisis of multiple sclerosis (Challoner et al., 1995).

The human herpesvirus 6 genome consists of a unique region of approximately 140 kb and a pair of 10 to 13 kb terminal elements. There has been much debate over which subfamily HHV-6 should be included. HHV-6 nucleotide and encoded protein sequences demonstrate that these viruses are genetically collinear with human cytomegalovirus (HCMV) over much of their genomes, but also that there are significant differences with HCMV with respect to genome size, G + C content, and the existence of unique genes.

On the basis of this information, HHV-6 is classified in the beta herpesvirus subfamily (Inoue and Pellett 1995; Josephs et al., 1991). HHV-6B has shown to encode a homologue of the herpes virus type 1 origin-binding protein. The binding sites for this protein has sequences and overall arrangements similar to those of the replication origins of several alphaviruses. Therefore, HHV-6B DNA replication may resemble that of alphaherpesviruses (Inoue and Pellett 1995).

An examination of the relative order of conserved genes in the sequenced herpesviruses can suggest some of the major events contributing to the divergence in each lineage. In terms of compactness and organisation of the conserved genes, it has been suggested that HHV-6 is the smallest herpesviruses, with few gaps between the gene blocks. Recent studies on available herpesvirus nucleotide sequences have proposed a HHV-6 like virus as a precursor herpesvirus from which the three herpesvirus subfamilies arose.

The simplest model to account for the genetic rearrangements observed between alpha, beta, and gammaherpesvirus lineages, is that betaherpesviruses gave rise to the alpha and gammaherpesviruses and that A + T rich version in each lineage are earlier forms (Gomples et al., 1995).

Potential Therapeutic Treatments

Several potential antiviral treatments have been shown to inhibit some aspects of EBV replication in vivo but so far they have had only a very limited success. Acyclovir (acycloguanosine) inhibits viral DNA synthesis in lytic infection but not latent infection. Acyclovir given to patients with infectious mononucleosis, causes a reduction in the level of oropharyngeal viral replication but after the treatment is stopped the replication returns to the pre-acyclovir treatment levels. Alpha interferon (IFN-a ), by blocking the capping of EBV-CD21 complexes, has had some significant success in reducing virus output in EBV infected patients (Delcayre et al., 1993; Miller 1990). Soluble forms of truncated CD2 proteins may have potential therapeutic value in the treatment of EBV-induced lymphoproliferative disorders in humans that involve viral replication (Moore et al., 1991)

Recently, interleukin 6 (IL-6) has been demonstrated to be a paracrine or autocrine growth factor for EBV-immortalised B cells, resulting in increased immunoglobulin production and B-cell immortalisation.

In vitro EBV-immortalised B cells secrete IL-6 into culture supernatant, express the IL-6 receptor, and can use IL-6 as an autocrine or paracrine growth factor. If IL-6 is expressed at high levels in EBV- immortalised cells, it will promote tumour formation by impairing the activity of natural killer cells (Tanner et al., 1996).

Therefore if IL-6 production in EBV-immortalised cells could be reduced, or the action of IL-6 in promoting tumour formation could be blocked, the detrimental effects of tumours could be reduced.

Conclusion

The attachment and entry of EBV into host cells is initiated by the EBV envelope glycoproteins gp350/220, gp25, gp85, and gp42/38.

The differences in cell tropism may be due to the differences in the interaction between these proteins and cellular receptors. EBV produces three different types of latency which are characterised by different expression patterns of a possible nine latent proteins. EBNA1 and the LMPs are the only latent proteins that are expressed in all forms of latency.

Data reinforces the assumption that EBNA1 and the LMPs are the most important EBV-encoded proteins in the maintenance of latently infected cells. Most of the latent proteins have shown to play possible roles in the immortalisation of EBV infected cells. Replication of episomes in EBV latent infections is mediated by EBNA1, initiated at oriP, and usually occurs only once per cell division.

An exciting feature of EBNA2 is its abilty to bind to cellular DNA and transactivate cellular promoters. This activation by EBNA2 may cause the expression of three previously uncharacterised cellular proteins. Lytic infection is activated by Z, which has also shown to be regulated by cellular as well as viral factors. Presently there have been no therapeutic treatments that have shown a high degree of success.

HHV-6 may be a cofactor in EBV related malignancies, and it has been proposed that the three subfamilies of herpesviruses arose from a HHV-6 like progenitor. During the process of evolution it appears that EBV may have utilised many cellular functions and may have happened on what appears to be an easier and simpler process to achieve the reproduction of their genome by causing a latent infection.

EBV may be regarded as a successful virus as it has an effective mode of transmission; a very high incidence of infection throughout its susceptible hosts causing few fatalities; and ensures the continuance of its genome by producing in B cell lymphocytes that are latently infected for the life of the host. These mechanisms of EBV suggest that EBV has evolved in the human host for a very long time.

EBV has shown to have many interwoven cellular and viral functions, many of which are still not understood. Thus, to understand these processes there needs to be more research into both viral and cellular mechanisms. The information known about viruses is increasing rapidly and it appears that EBV may soon be understood to the extent of our present knowledge of other much simpler viruses such as Phage l .

As this research broadens our knowledge of viruses it also unravels many problems and produces many beneficial medicinal techniques that are not directly viral-related, such as the discovery that binding molecules to the Epstein-Barr virus receptor, CD21, may be used as a novel immunotoxin delivery system (Tedder et al., 1986). There is still much unknown about EBV, and realistically, we can presently only say that we are just beginning to understand it.

References

Abbot, S. D., Rowe, M., Cadwallader, K., Ricksten, A., Gordon, J., Wang, F., Rymo, L. & Rickinson, A. B. (1990). Epstein-Barr Virus Nuclear Antigen 2 Induces Expression of the Virus-Encoded Latent Membrane Protein. Journal of Virology 64, No. 5, 2126-2134.

Adams, A. (1987). Replication of Latent Epstein-Barr Virus Genomes in Raji Cells. Journal of Virology 61, No. 5, 1743-1746.

Araijo, J. C., Doniger, J., Kashanchi, F., Hermonat, P. L., Thompson, J. & Rosenthal, L. J. (1995). Human Herpesvirus 6A ts Suppresses both Transformation by H-ras and Transcription by the H-ras and Human Immunodeficiency Virus Type 1 Promoters. Journal of Virology 69, No. 8, 4933-4940.

Bhat, R. A. & Thimmappaya, B. (1985). Construction and analysis of Additional Adenovirus Substitution Mutants Confirm the Complementation of VAI RNA Function by Two Small RNAs Encoded by Epstein-Barr Virus. Journal of Virology 56, No. 3, 750-756.

Birkenbach, M., Josefsen, K., Yalamanchili, R., Lenoir, G. & Kieff, E. (1993). Epstein-Barr Virus-Induced Genes: First Lymphocyte-Specific G Protein-Coupled Peptide Receptors. Journal of Virology 67, No. 4, 2209-2220.

Brooks, L. A., Lear, A. L., Young, L. S. & Rickinson, A. B. (1993). Transcripts from the Epstein-Barr Virus BamHI A Fragment Are Detectable in All Three Forms of Virus Latency. Journal of Virology 67, No. 6, 3182-3190.

Cayrol, C. & Flemington, E. K. (1995). Identification of Cellular Target Genes of the Epstein-Barr Virus Transactivator Zta: Activation of Transforming Growth Factor b igh3 (TGF-b igh3) and TGF-b 1. Journal of Virology 69, No. 7, 4206-4212.

Challoner, P. B., Smith, K. T., Parker, J. D., MacLeod, D. L., Coulter, S. N., Rose, T. M., Schultz, E. R., Bennett, J. L., Garber, R. L., Chang, M., Schad, P. A. Stewart, P. M., Nowinski, R. C., Brown, J. P. & Burmer, G. C. (1995). Plaque-associated expression of human herpesvirus 6 in multiple sclerosis. Proceedings from the National Academy of Science 92, 7440-7444.

Cohen, J. I., Wang, F. & Kieff, E. (1991). Epstein-Barr Virus Nuclear Protein 2 Mutations Define Essential Domains for Transformation and Transactivation. Journal of Virology 65, No. 5, 2545-2554.

Cooper, N.R. (1994). Early Events in Human Herpesvirus Infection of Cells: In Cellular Receptors for Animal Viruses. ed. E. Wimmer, 365-388. Cold Spring Harbor Laboratory Press, New York.

Delcayre, A. X., Lotz, M. & Lernhardt, W. (1993). Inhibition of Epstein-Barr Virus-Mediated Capping of CD21/CR2 by Alpha Interferon (IFN-a ): Immediate Antiviral Activity of IFN-a during the Early Phase of Infection. Journal of Virology 67, No. 5, 2918-2921.

Devergne, O., Hummel, M., Koeppen, H., Le Beau, M. M., Nathanson, E. C., Keiff, E. & Birkenbach, M. (1996). A Novel Interleukin-12 p40-Related Protein Induced by Latent Epstein-Barr Virus Infection in B Lymphocytes. Journal of Virology 70, No. 2, 1143-1153.

Farrell, P. J., Rowe, D. T., Rooney, C. M. & Kouzarides, T. (1989). Epstein-Barr virus trans-activator specifically binds to a consensus AP-1 site is related to c-fos. The EMBO Journal 8, No. 1, 127-132.

Fennewald, S., Van Santen, V. & E. Kieff. (1984). Nucleotide Sequence of an mRNA Transcribed in Latent Growth-Transforming Virus Infection Indicates That It May Encode a Membrane Protein. Journal of Virology 51, No. 2, 411-419.

Flemington, E. & Speck, S. H. (1990). Autoregulation of Epstein-Barr Virus Putative Lytic Switch Gene BZLF1. Journal of Virology 64, No. 3, 1227-1232.

Glickman, J. N., Howe, J. G. & Steitz, J. A. (1988). Structural Analyses of EBER1 and EBER2 Ribonucleoprotein Particles Present in Epstein-Barr Virus-Infected Cells. Journal of Virology 62, No. 3, 902-911.

Gompels, U. A., Nicholas, J., Lawrence, G., Jones, M., Thomson, B. J., Martin, M. E. D., Efstathiou, S., Craxton, M. & Macaulay, H. A. (1995). The DNA Sequence of Human Herpesvirus-6: Structure, Coding Content, and Genome Evolution. Virology 209, 29-51.

Henderson, S., Rowe, M., Gregory, C., Croom-Carter, D., Wang, F., Longnecker, R., Kieff, E. & Rickinson, A. (1991). Induction of bcl-2 Expression by Epatein-Barr Virus Latent Membrane Protein 1 Protects Infected B cells from Programmed Cell Death. Cell 65, 1107-115.

Huddad, R. & Hutt-Fletcher, L. M. (1989). Depletion of Glycoprotein gp85 from Virosomes Made with Epstein-Barr Virus Proteins Abolishes Their Ability To Fuse with Virus Receptor-Bearing Cells. Journal of Virology 63, No. 12, 4998-5005.

Inoue, N. & Pellett, P. E. (1995). Human Herpesvirus 6B Origin-Binding Protein: DNA-Binding Domain and Consensus Binding Sequence. Journal of Virology 69, No. 8, 4619-4627.

Jabs, W. J., Wagner, H. J., Neustock, P., Kircher, H. (1996). Immunologic Properties of Epstein-Barr Virus-Seronegative Adults. The Journal of Infectious Diseases 173, 1248-1251.

Jones, C. H., Hayward, D. & Rawlins, D. R. (1989). Interaction of the Lymphocyte-Derived Epstein-Barr Virus Nuclear Antigen EBNA-1 with Its DNA-Binding Sites. Journal of Virology 63, 101-110.

Josephs, S. F., Ablashi, D. V., Salahuddin, S. Z., Jagodzinski, L. L., Wong-Staal, F. & Gallo, R. C. (1991). Identification of the Human Herpesvirus 6 Glycoprotein H and Putative Large Tegumant Protein Genes. Journal of Virology 65, No. 10, 5597-5604.

Kashanichi, F., Thompson, J., Sadaie, M. R., Doniger, J., Duvall, J., Brady, J. N. & Rosenthal, L. J. (1994). Transcriptional Activation of Minimal HIV-1 Promoter by ORF-1 Proterin Expression from the SalI-L Fragment of Human Herpesvirus 6. Virology 201, 95-106.

Keiff, E. & Leibowitz, D. (1990). Epstein-Barr Virus and Its Replication. In Virology, 2^nd., eds, B. N. Fields & D. M. Knipe, 1889-1920. Raven Press, New York.

Kerr, B, M., Lear, A. L., Rowe, M., Croom-Carter, D., Young, L. S., Rookes, S. M., Gallimore, P. H. & Rickinson, A. B. (1992). Three Transcriptionally Distinct Forms of Epstein-Barr Virus Latency in Somatic Cell Hybrids: Cell Phenotype Dependence of Virus Promoter Usage. Virology 187, 189-201.

Kirchmaier, A. L., & Sugden, B. (1995). Plasmid Maintence of Derivatives of oriP of Epstein-Barr Virus. Journal of Virology 69, No. 2, 1280-1283.

Lear, A. L., Rowe, M., Kurilla, M. G., Lee, S., Henderson, S., Kieff, E. & Rickinson, A. B. (1992). The Epstein-Barr Virus (EBV) Nuclear Antigen 1 BamHI F Promoter Is Activated on Entry of EBV-Transformed B Cells into the Lytic Cycle. Journal of Virology 66, No. 12, 7461-7468.

Leibowitz, D., Wang, D. & Kieff, E. (1986). Orientation and Patching of the Latent Infection Membrane Protein Encoded by Epstein-Barr Virus. Journal of Virology 58, No. 1, 233-237.

Li, Q., Turk, S. M. & Hutt-Fletcher, L. M. (1995). The Epstein-Barr Virus (EBV) BZLF2 Gene Product Associates with the gH and gL Homologs of EBV and Carries an Epitope Critical to Infection of B cells but Not of Epithelial Cells. Journal of Virology 69, No. 7, 3987-3944.

Lieberman, P. M. & Berk, A. J. (1990). In Vitro Activation, Dimerization, and DNA-Binding Specificity of the Epstein-Barr Virus Zta Protein. Journal of Virology 64, No. 6, 2560-2568.

Longnecker, R. & Miller, C. L. (1996). Regulation of Epsetin-Barr virus latency by latent membrane protein 2. Trends in Microbiology 4, No. 1, 38-42.

Longnecker, R., Drunker, B., Roberts, T. M. & Keiff, E. (1991). An Epstein-Barr Virus Protein Associated with Cell Growth Transformation Interacts with a Tyrosine Kinase. Journal of Virology 65, No. 7, 3681-3692.

Mackey, D., Middleton, T. & Sugden, B. (1995). Multiple Regions within EBNA1 Can Link DNAs. Journal of Virology 69, No. 10, 6199-6208.

Mannick, J. B., Cohen, J. I., Birkenbach, M., Marchini, A. & Kieff, E. (1991). The Epstein-Barr Virus Nuclear Protein Encoded by the Leader of the EBNA RNAs Is Importnat in B-Lymphocyte Transformation. Journal of Virology 65, No. 12, 6826-6837.

Middleton, T. & Sugden, B. (1994). Retention of Plasmid DNA in Mammalian Cells Is Enhanced by Binding of the Epstein-Barr Virus Replication Protein EBNA1. Journal of Virology 68, No. 6, 4067-4071.

Miller, M. (1990). Epstein-Barr Virus Biology, Pathogenesis, and Medical Aspects. In Virology, 2^nd., eds. B. N. Fields & D. M. Knipe, 1921-1958. Raven Press, New York.

Miller, N. & Hutt-Fletcher, L. M. (1988). A Monoclonal Antibody to Glycoprotein gp85 Inhibits Fusion but Not Attachment of Epstein-Barr Virus. Journal of Virology 62, No. 7, 2366-2372.

Miller, N. & Hutt-Fletcher, L. M. (1992). Epstein-Barr Virus Enter B Cells and Epithelial Cells by Different Routes. Journal of Virology 66, No. 6, 3409-3414.

Minarovits, J., Hu, L. -F., Imai, S., Harabuchi, Y., Kataura, A., Minarovits-Kormuta, S., Osato, T. & Klein, G. (1994). Clonality, expression and methylation patterns of the Epstein-Barr virus genomes in lethal midline granulomas classified as peripheral angiocentric T cell lymphomas. Journal of General Virology 75, 77-84.

Moore, M. D., Cannon, M. J., Sewall, A. Finlayson, M., Okimoto, M. & Nemerow, G. R. (1991). Inhibition of Epstein-Barr Virus In Vitro and In Vivo by Soluble CR2 (CD21) Containing Two Short Consensus Repeats. Journal of Virology 65, No. 7, 3559-3565.

Nemerow, G. R. & Cooper, N. R. (1984). Early Events in the Infection of Human B Lymphocytes by Epstein-Barr Virus: The Internalization Process. Virology 132, 186-198.

Nemerow, G. R., Houghten, R. A., Moore, M. D. & Cooper, N. R. (1989). Identification of an Epitope in the Major Envelope Protein of Epstein-Barr Virus That Mediates Viral Binding to the B Lymphocyte EBV Receptor (CR2). Cell 56, 369-377.

Nemerow, G. R., Mold, C., Schwend, V. K., Tollefson, V. & Cooper, N. R. (1987). Identification of gp350 as the Viral Glycoprotein Mediating Attachment of Epstein-Barr Virus (EBV) to the EBV/C3d Receptor of B Cells: Sequence Homology of gp350 and C3 Complement Fragment C3d. Journal of Virology 61, No. 5, 1416-1420.

Nonoyama, M., Huang, C. H., Pagano, J. S., Klein, G. & Singh, S. (1973). DNA of Epstein-Barr Virus Detected in Tissue of Burkitt's Lymphoma and Nasopharyngeal Carcinoma. Proceedings from the National Academy of Science 70, No. 11, 3265-3268.

Robertson, E. S., Grossman, S., Johannsen, E., Miller, C., Lin, J., Tomkinson, B. & Keiff, E. (1995). Epstein-Barr Virus Nuclear Protein 3C Modulates Transcription through Interaction with the Sequence-Specific DNA-Binding Protein Jk . Journal of Virology 69, No. 5, 3108-3116.

Roizman, B. (1990). Herpesviridae: A Brief Introduction. In Virology, 2^nd., eds. B. N. Fields & D. M. Knipe, 1787-1793. Raven Press, New York.

Rowe, M., Lear, A. L., Croom-Carter, D., Davies, A. H. & Rickinson, A. B. (1992). Three Pathways of Epstein-Barr Virus Gene Activation from EBNA1-Positive Latency in B Lymphocytes. Journal of Virology 66, No. 1, 122-131.

Rowe, M., Rowe, D. T., Gregory, C. D., Young, L. S., Farrell, P. J., Rupani, H. & Rickinson, A. B. (1987). Differences in B cell growth phenotype reflect novel patterns of Epstein-Barr virus latent gene expression in Burkitt's lymphoma cells. The EMBO Journal 6, No. 9, 2743-2751.

Sample, J., Henson, D. & Sample, C. (1992). The Epstein-Barr Virus Nuclear Protein 1 Promoter Active in Type I Latency Is Autoregulated. Journal of Virology 66, No. 8, 4654-4661.

Sample, J., Lancz, G. & Nonoyama, M. (1984). Use of Cloned Epstein-Barr Virus DNA to Identify Genes that Determine the Fate of Viral Infection. In VIRAL MESSENGER RNA Transcription, Processing, Splicing and Molecular Structure. Ed. Y. Becker, 127-146.

Schaefer, B. C., Strominger, J. L. & Speck, S. H. (1995). The Epstein-Barr Virus BamHI F Promoter Is an Early Lytic Promoter: Lack of Correlation with EBNA1 Gene Transcription in Group 1 Burkitt's Lymphoma Cell Lines. Journal of Virology 69, No. 8, 5039-5047.

Sinclair, A. J. & Farrell, P. J. (1995). Host Cell Requirements for Efficient Infection of Quiescent Primary B Lymphocytes by Epstein-Barr Virus. Journal of Virology 69, No. 9, 5461-5468.

Sista, N. D., Barry, C., Sampson, K. & Pangano, J. (1995). Physical and functional interaction of the Epstein-Barr virus BZLF1 transactivator with the retinoic acid receptors RARa and RXRa. Nucleic Acids Research 23, No. 10, 1729-1736.

Snudden, D. K., Hearing, J., Smith, P. R., Grasser, F. A. & Griffen, B. E. (1994). EBNA-1, the major nuclear antigen of Epstein-Barr virus, resembles 'RGG' RNA binding protein. The EMBO Journal 13, No. 20, 4840-4874.

Tanner, J., Weis, J., Fearon, D., Whang, Y. & Kieff, E. (1987). Epstein-Barr Virus gp350/220 Binding to the B Lymphocyte C3d Receptor Mediates Adsorption, Capping, and Endocytosis. Cell 50, 203-213.

Tedder, T. F., Goldmacher, V. S., Lambert, J. M. & Schlossman, S. F. (1986). Epstein Barr Virus Internalization of the C3d Receptor: A Novel Immunotoxin Delivery System. Journal of Immunology 137, No. 4, 1387-1391.

Wang, D., Leibowitz, D. Kieff, E. (1988). The Truncated Form of the Epstein-Barr Virus Latent-Infection Membrane Protein Expressed in Virus Replication Does Not Transform Rodent Fibroblasts. Journal of Virology 62, No. 7, 2337-2346.

Wang, F., Gregory, C., Sample, C., Rowe, M., Leibowitz, D., Marray, R., Rickinson, A. & Kieff, E. (1990a). Epstein-Barr Virus Latent Membrane Protein (LMP1) and Nuclear Proteins 2 and 3C Are Effectors of Phenotypic Changes in B Lymphocytes: EBNA-2 and LMP1 Cooperatively Induce CD23. Journal of Virology 64, No. 5, 2309-2318.a

Wang, F., Kikutani, H., Tsang, S., Kishimoto, T. & Kieff, E. (1991). Epstein-Barr Virus Nuclear Protein 2 Transactivates a cis-Acting CD23 DNA Element. Journal of Virology 65, No. 8,4101-4106.

Wang, F., Petti, L., Braun, D., Seung, S. & Kieff, E. (1987). A Biscistronic Epstein-Barr Virus mRNA Encodes Two Nuclear Proteins in Latently Infected, Growth-Transformed Lymphocytes. Journal of Virology 61, No. 4, 945-954.

Wang, F., Tsang, S., Kurilla, M. G., Cohen, J. I. & Keiff, E. (1990b). Epstein-Barr Nuclear Antigen 2 Transactivates Latent Membrane Protein LMP1. Journal of Virology 64, No. 7, 3407-2416.

Wilson, J. B., Bell, J. L. & Levine, A. J. (1996). Expression of Epstein-Barr virus nuclear antigen-1 induces B cell neoplasia in transgenic mice. The EMBO Journal 15, No. 12, 3117-3126.

Yates, J. L. & Guan, N. (1991). Epstein-Barr Virus-Derived Plasmids Replicate Only Once per Cell Cycle and Are Not Amplified after Entry into Cells. Journal of Virology 65, No. 1, 483-488.

Yates, J., Warren, N., Reisman, D. & Sugden, B. (1984). A cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Proceeding from the National Academy of Science 81, 3806-3810.

Yoshiyama, H., Shimizu, N. & Takada, K. (1995). Persistent Epstein-Barr virus infection in a human T-cell line: unique program of latent virus expression. The EMBO Journal 14, No. 15, 3706-3711.