Department of Microbiology, Tokyo Medical University, Tokyo, Japan1
Department of Tumor Virology, Division of Virology and Immunology, Medical Research Institute, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8510, Japan2
Department of Otolaryngology, Kyorin University School of Medicine, Tokyo, Japan3
Author for correspondence: Kanji Hirai. Fax +81 3 5803 0241. e-mail hirai.creg{at}mri.tmd.ac.jp
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Abstract |
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Since there is no in vitro natural permissive system for EBV replication, most studies of lytic virus gene expression have relied on semipermissive EBV-carrying Burkitts lymphoma cell lines or LCLs where the virus can be induced to enter the productive cycle by various means, such as exposure to tumour promoters (zur Hausen et al., 1978 ; Luka et al., 1979
), halogenated pyrimidine (Gerber, 1972
), antibodies to human immunoglobulin (Tovey et al., 1978
) and EBV-encoded trans-activator (Countrymann & Miller, 1985
). Among the EBV gene products of the replicative cycle, the protein product of the BZLF1 gene, named ZEBRA (BamHI Z fragment, EBV replication activator), acts as the switch from latency to the lytic cycle (Biggin et al., 1987
; Carey et al., 1992
). The late protein, viral capsid antigen (VCA), encoded by BcLF1 is expressed only in virus-producing cells and is closely associated with the capsid of virus particles (Vroman et al., 1985
).
Therefore, expression of both ZEBRA and VCA suggests that EBV-positive cells are undergoing a lytic cycle. Furthermore, DNADNA in situ hybridization (ISH) can detect cells productively infected with EBV, since this method can distinguish cells harbouring a low copy number of latent viral DNA from those with multiple copies of replicating viral DNA (Hironaka et al., 1993 ).
In contrast to the in vitro system, it is still a matter of dispute where the in vivo EBV replication site is. Pharyngeal epithelial cells and oral hairy leukoplakia (OHL) lesions appear to be naturally permissive, with virus replication in differentiating squamous cells leading to the release of infectious virions into buccal fluid (Young et al., 1991 ; De Souza et al., 1989
; Greenspan et al., 1985
; Sixbey et al., 1984
). Prang et al. (1997)
also detected lytic proteins and lytic gene transcripts of EBV in peripheral blood B lymphocytes by immunohistochemistry and RTPCR in infectious mononucleosis (IM) patients. The result suggests that EBV replication also occurs in lymphocytes. However, it remained unclear where the EBV replication sites are in normal (healthy) individuals. It has recently been shown that the tonsil is one of the candidates for the site of replication of EBV (Babcock et al., 1998
; Kobayashi et al., 1998
). We have previously found that cells with a high copy number of EBV DNA were located in the upper epithelial cell layers of non-neoplastic tonsil using DNADNA ISH and in situ PCR (Kobayashi et al., 1998
). In addition, Babcock et al. (1998)
could detect the linear and episomal forms of EBV DNA in tonsillar lymphocytes, while peripheral blood B cells contain only the episomal form. The presence of the linear form may indicate that viral DNA is replicating in the tonsillar lymphoid tissue of healthy, persistently infected individuals. These results suggest that EBV replication occurs in lymphocytes as well as epithelial cells in the tonsillar lymphoid tissue of normal individuals.
In the tonsils, the B cell population makes up 5070% of total lymphocytes (Yamanaka et al., 1983 ), much greater than in peripheral blood lymphocytes (PBL), where it is 723% (Reichert et al., 1991
). The frequency of EBV-infected cells in peripheral blood B lymphocytes was 5500 per 107 (Khan et al., 1996
), similar to that in tonsillar cells, 20100 per 107 (Babcock et al., 1998
). In addition, there was not much difference in the expression level of EBER1 between the right and left non-neoplastic tonsils of each patient with chronic tonsillitis, although the expression level varied among individuals (Takeuchi et al., 1996
), suggesting that the circulating EBV-infected cells were detected in the tonsils from both sides. Therefore, detection of EBV-infected cells may be easier in tonsillar tissues than in PBL. To find EBV replication sites, we examined tonsils by RTPCR, immunohistochemistry and ISH.
Fifteen tonsillar tissues from eight donors clinically diagnosed with chronic tonsillitis were taken from the Department of Otolaryngology, Kyorin University School of Medicine (Tokyo, Japan). Routine histological analysis showed no involvement of malignant lymphomas or other malignancies. These donors had no history of IM. The antibody titres of the donors to early antigen (EA), VCA and EBNA were examined by EBV-associated antigen detection kits (Kayaku, Tokyo) based on indirect immunofluorescent tests according to the manufacturers instructions. Twofold serial dilutions of the serum, from 1:10 to 1:2560, were analysed. These donors, except no. 5, had antibodies to EBNA (data not shown), indicating that they were not in an acute phase of EBV infection. Titres of donor no. 5 (anti-EA, -VCA and -EBNA) were less than 1:10.
To clarify the state of EBV infection in tonsils, RNA from mononuclear cells which were isolated from 15 tonsillar specimens was prepared using TRIzol (Life Technologies) (Ryon, 1999 ). Then, we tested for expression of EBV transcripts EBER1, BZLF1, EBNA2, LMP1 and LMP2a in these tonsillar mononuclear cells by RTPCR according to the protocols previously described (Tierney et al., 1994
; Chen et al., 1995
; Prang et al., 1997
). Southern hybridization was performed with locus-specific probes which were labelled with digoxigenin (Roche Diagnostics), after electrophoresis of PCR products. EBER1 transcripts were detected in 14 of 15 specimens (Fig. 1
). Since EBV-seronegative donor no. 5 did not have these transcripts, the donor may not be infected with EBV. There was little difference in the expression level of EBER1 between the right and left tonsils of each donor as reported previously (Takeuchi et al., 1996
). BZLF1 mRNA was also observed in six of 15 specimens. This transcript was detected in both of the tonsils isolated from each donor (nos 2, 3 and 7). However, LMP1, EBNA2 and LMP2a transcripts could not always be detected in both of the tonsils from each individual. Donor no. 3 had all of the transcripts (EBER1, EBNA2, LMP1, LMP2a and BZLF1).
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RNADNA ISH for the detection of EBER1 was also performed using the paraffin sections prepared from the same donors (nos 2, 3 and 7). Although EBER1 could be detected in lymphoid cells, the localization of these cells was different from ZEBRA- and VCA-positive areas (data not shown). This result suggests that EBV-producing cells down-regulated the expression of EBER1 as reported for OHL (Young et al., 1991 ). Table 1
summarizes the results of Figs 1
and 2
.
|
It is well known that the interleukin 10 (IL-10) coding sequence is highly homologous to the EBV open reading frame BCRF1 (Moore et al., 1990 ; Vieira et al., 1991
). BCRF1 protein, also termed viral IL-10 (vIL-10), inhibits the synthesis of T-helper 1 cytokines (Rousset et al., 1992
) and CTL activity (de Waal Malefyt et al., 1991
). Therefore, EBV-associated antigen-specific CTL activity might be down-regulated by vIL-10 in EBV-infected areas of the tonsil. Thus, EBV is much more likely to survive in the face of immune surveillance in the tonsils, suggesting that the immune response to EBV in tonsils may be different from that in PBL.
Our results demonstrate that tonsillar lymphocytes are one of the EBV replication sites and a reservoir for EBV in normal individuals.
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References |
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Received 31 August 1999;
accepted 26 January 2000.
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