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JVI Accepted Manuscript Posted Online 19 October 2016 J. Virol. doi:10.1128/jvi.01677-16 Copyright 2016, American Society for Microbiology. All Rights Reserved. 1 2 3 4 5 6 7 8 9 10 The long and complicated relationship between Epstein-Barr virus and epithelial cells Lindsey Hutt-Fletcher Department of Microbiology and Immunology, Center for Molecular and Tumor Virology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana, USA Downloaded from http://jvi.asm.org/ on November 3, 2018 by guest

11 12 ABSTRACT The role of epithelial cells in infection and persistence of Epstein-Barr virus (EBV) has 13 long been difficult to resolve. Recent developments have, however, reinforced both the 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 conclusion that they are a major site of virus replication and raised the possibility that, like papillomaviruses, EBV has evolved to take advantage of epithelial differentiation to ensure both survival persistence and spread. INTRODUCTION Epstein-Barr virus (EBV) is well known as a human lymphotropic herpesvirus isolated from a Burkitt s lymphoma in the early 1960 s (1). It is well described as a causal agent of infectious mononucleosis and an important player in the development of lymphoid tumors. It is well used as a tool for immortalizing human B cells. Soon after its discovery its additional association with epithelial malignancies was revealed (2) and the presence of the virus was confirmed within the epithelial cells of nasopharyngeal carcinoma (3). And yet as late as 2005 a distinguished speaker at an international herpesvirus meeting declared that epithelial cells were not relevant to the general biology of EBV. How was that possible and where are we today? EARLY WORK Early work described EBV DNA and RNA in squamous epithelial cells shed in the oral cavity during acute infectious mononucleosis (4, 5). Thus the model of B cells as the reservoir of latent EBV and epithelial cells as the site of productive lytic replication in vivo began to be developed. However, additional support for the routine involvement of epithelial cells in primary or persistent infection was, for many years, quite elusive. In the absence of any obvious lesions, such as are seen in herpes simplex infections, finding an infected cell in the oral cavity was a lot like looking for the proverbial needle in the haystack. Compounding the problem was the difficulty found in infecting epithelial cells in vitro. With the advent of the AIDS epidemic, oral hairy leukoplakia appeared, the first and so far only disease caused entirely by productive

37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 replication of EBV. Oral hairy leukoplakia, which is an epithelial hyperplasia, typically found on the lateral tongue (6), is full of and driven by EBV lytic replication (7, 8). It bolstered the case for epithelial cells in the oral cavity being responsible for production of cell-free virus in saliva, but still, only in individuals coinfected with the human immunodeficiency virus were normal epithelial cells readily found to contain virus (9). For a while the model of persistence pivoted to towards the idea that, in the absence of malignancy or other underlying disease, B cells alone were involved in infection (10). Three sets of observations subsequently helped spur a return to the yin and yang of both epithelial and B cell infection. Despite the uncertainty about the role of epithelial cells in EBV biology its presence in epithelial cells of nasopharyngeal carcinoma and gastric cancer indicated that it remained important to understand how the virus accessed both cell types. The fusion machinery used for the two cell types, glycoproteins gb and a complex of gh, gl and gp42 for B cells and gb, and a complex of gh and gl alone for epithelial cells (11), was different as were the cellular partners responsible for triggering the event. B cell fusion was triggered by an interaction between gp42 and HLA class II (12) and epithelial fusion was triggered by an interaction between gh and what ultimately turned out to be any one of three alpha v integrins (13, 14). Virus replicating in a B cell lost some gp42 containing complexes to the HLA class II processing pathway, which delivered them to the peptide loading compartment and its proteases. This did not happen in an HLA class II-negative epithelial cell (15). The gp42 enriched virus made in an epithelial cell was as much as two logs more infectious for a B cell than virus made in a B cell. In contrast, virus made in a B cell was slightly more infectious for an epithelial cell, about fivefold more than virus made in an epithelial cell, because the presence of gp42 impedes access of gh to an integrin. This generated the hypothesis that a switch in tropism occurred during infection in vivo which led naturally to alternate replication in the two cell types. Beyond this, the change in glycoprotein composition of B cell derived and epithelial 62 derived virus made it possible to determine the most likely source of virus in saliva. A

63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 comparison of virus shed in saliva and virus made in B cells from the same donor transformed by the virus shed in saliva revealed that virus in saliva had a much higher ratio of gp42 to gh than virus produced in a B cell, implicating it as being derived from an epithelial source (16). The third set of observations was made by Thorley-lawson and colleagues who measured the rate at which virus is shed in saliva. They concluded that the most likely explanation for its unexpectedly high value was that virus reactivated from B cells is amplified in epithelial tissues (17). NOW AND THE FUTURE The majority of textbooks now describe B cells as the reservoir of latent EBV and epithelial cells as the site of productive lytic replication (18) and several recent important observations have made sense of the long lasting inability to demonstrate productive replication of virus in vitro in monotypic cultures. Normal human epithelial cells, when grown in airinterface organotypic raft cultures, differentiate to form stratified epithelium. Such cells infected from the apical surface with EBV support complete lytic replication and produce new virus particles (19). In B cells the viral lytic cascade is induced by the two immediate early genes BZLF1 and BRLF1 which cooperate to induce expression of all downstream lytic genes (20). In at least some epithelial cells, BRLF1, whose expression is dependent on a different set of cellular transcription factors than those required by BZLF1, is uniquely required (21). Expression of BRLF1 is enhanced by B-lymphocyte-induced maturation protein-1 (BLIMP1) and KLF4, both of which are expressed in epithelial cells in a differentiation-dependent manner. Thus when telomerase immortalized normal oral keratinocytes are latently infected with EBV carrying a drug-resistance marker for selection and differentiated in air-interface organotypic raft 85 cultures, the virus also undergoes lytic replication (22). The differentiation status of the 86 87 epithelial cell is thus critical to its ability to support EBV replication. The question now arises, is EBV in an epithelial cell in a persistently infected host 88 always undergoing lytic replication or is it ever latent? Although virus is clearly latent in

89 epithelial tumors the assumption has been made that that epithelial latency is dependent on 90 some malignant or premalignant state existing in the infected cell (23). Overexpression of 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 cyclin D1 has for example been reported as being required for persistence of latent EBV in the absence of a drug-resistance marker in telomerase-immortalized nasopharyngeal epithelial cells and loss of the tumor suppressor p16, which often occurs in nasopharyngeal tumors or premalignant lesions, is a negative regulator of cyclin D1 signaling (24). In addition, no evidence was found for the presence of latent EBV in the organotypic raft cultures infected from the apical surface (19). However, there is some evidence for the presence of EBV in basal epithelial cells in vivo. The original claim, based on in situ hybridization data, was that in oral hairy leukoplakia only the more differentiated layers of cells carry EBV and carry virus only in its lytic replication cycle. The basal layers were said to lack virus altogether (25). Revisiting this question with a more sensitive reverse transcriptase real time PCR analysis of basal cells, isolated from formalin-fixed paraffin-embedded sections by laser capture microdissection, challenged this conclusion by demonstrating EBER RNA transcripts in the absence of lytic gene expression (22). The same type of analysis also found evidence for latent infection in basal epithelial cells taken from sections of normal tonsil. Many years ago it was suggested that EBV might behave in epithelial cells in the same way as does a papillomaviruses, establishing latency in basal layers of epithelium and relying on differentiation of the cell to move the virus into productive replication (26). Although this hypothesis was later discarded, perhaps it is time to revisit the possibility. Reactivation of virus as latently infected B cells differentiate into plasmablasts in Waldeyer s ring, the lymphatic tissue of tonsils and adenoids surrounding the oropharynx, would produce an epitheliotropic virus This virus would be well positioned to infect basal layers of epithelium which express the integrins needed to trigger virus and cell fusion. If the initial infection of this basal layer established latency which was only followed by reactivation as cells differentiated upward, virus would be released into the oropharynx. If instead it were to replicate

115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 immediately upon epithelial infection the virus released would be highly lymphotropic and return to the lymphoid compartment, good for establishing infection but not for person to person spread (Figure 1). This scenario does of course beg the question of how virus access B cells in the first place if, as seems plausible, in the absence of epithelial wounding, epithelial cells provide something of an initial barrier. Virus shed in saliva is high in g42 and thus not ideally suited to infect epithelial cells. However, the difference in tropism in this direction is relatively small, only fivefold. It also remains uncertain whether reactivating lymphocytes may also be transmitted in saliva and contribute to infection, or whether the abundant cell-free virus in saliva attaches to uninfected B cells that may also be present and is thus enhanced for epithelial infection by socalled transfer-infection (27). In contrast, virus emerging from replication in an epithelial cell is considerably more lymphotropic and driven towards the B cell compartment. By switching its tropism, but in a very unequal manner, EBV may have managed to attain the best of both worlds. REFERENCES 1. Epstein, M. A., B. G. Achong, and Y. M. Barr. 1964. Virus particles in cultured lymphoblasts from Burkitt's lymphoma. Lancet 1:702-703. 2. Henle, W., G. Henle, H. G. Ho, P. Burtin, Y. Cachin, P. Clifford, A. de Schryver, G. de The, V. Diehl, and G. Klein. 1970. Antibodies to Epstein-Barr virus in nasopharyngeal carcinoma, other head and neck cancers and control groups. J.N.C.I. 44:225-231. 3. Desgranges, C., H. Wolf, g. de The, K. Shanmugaratnam, N. Cammoun, R. Ellouz, G. Klein, K. Lennert, N. Munoz, and H. zur Hausen. 1975. Nasopharyngeal carcinoma. X. Presence of Epstein-Barr virus genomes in separated epithelial cells of tumors in patients from Singampore, Tunisia and Kenya. Int. J. Cancer 16:7-15.

141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 4. Lemon, S. M., L. M. Hutt, J. E. Shaw, J.-L. H. Li, and J. S. Pagano. 1977. Replication of EBV in epithelial cells during infectious mononucleosis. Nature 268:268-270. 5. Sixbey, J. W., J. G. Nedrud, N. Raab-Traub, R. A. Hanes, and J. S. Pagano. 1984. Epstein-Barr virus replication in oropharyngeal epithelial cells. New Engl. J. Med. 310:1225-1230. 6. Walling, D. M., N. M. Clark, D. M. Markovitz, T. S. Frank, B. D.K., E. Eisenberg, D. J. Krutchkoff, D. H. Felix, and N. Raab-Traub. 1995. Epstein-Barr virus coinfection and recombination in non-human immunodeficiency virus-associated oral hairy leukoplakia. J. Inf. Dis. 171:1122-1130. 7. Greenspan, D., Y. G. De Souza, M. A. Conant, H. Hollander, S. K. Chapman, E. T. Lennette, V. Petersen, and J. S. Greenspan. 1990. Efficacy of desciclovir in the treatment of Epstein-Barr virus infection in oral hairy leukoplakia. J. AIDS 3:571-578. 8. Greenspan, J. S., D. Greenspan, E. T. Lennette, D. I. Abrams, M. A. Conant, V. Petersen, and U. K. Freese. 1985. Replication of Epstein-Barr virus within the epithelial cells of oral "hairy" leukoplakia, an AIDS-associated lesion. New Engl. J. Med. 313:1564-1571. 9. Walling, D. M., C. M. Flaitz, C. M. Nichols, S. D. Hudnall, and K. Adler-Storthz. 2001. Persistent productive Epstein-Barr virus replication in normal epithelial cells in vivo. J. Inf. Dis. 184:1499-1507. 10. Thorley-Lawson, D. A., E. M. Miyashita, and G. Khan. 1996. Epstein-Barr virus and the B cell: that's all it takes. Trends Microbiol. 4:204-208. 11. Hutt-Fletcher, L. M. 2007. Epstein-Barr virus entry. J. Virol. 81:7825-7832. 12. Li, Q. X., M. K. Spriggs, S. Kovats, S. M. Turk, M. R. Comeau, B. Nepom, and L. M. Hutt-Fletcher. 1997. Epstein-Barr virus uses HLA class II as a cofactor for infection of B lymphocytes. J. Virol. 71:4657-4662.

166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 13. Chesnokova, L. S., and L. M. Hutt-Fletcher. 2011. Fusion of EBV with epithelial cells can be triggered by αvβ5 in addition to αvβ6 and αvβ8 and integrin binding triggers a conformational change in ghgl J. Virol. 85:13214-13223. 14. Chesnokova, L. S., S. Nishimura, and L. Hutt-Fletcher. 2009. Fusion of epithelial cells by Epstein-Barr virus proteins is triggered by binding of viral proteins ghgl to integrins avb6 or avb8. Proc. Natl. Acad. Sci. USA 106:20464-20469. 15. Borza, C. M., and L. M. Hutt-Fletcher. 2002. Alternate replication in B cells and epithelial cells switches tropism of Epstein-Barr virus. Nature Med. 8:594-599. 16. Jiang, R., R. S. Scott, and L. M. Hutt-Fletcher. 2006. Epstein-Barr virus shed in saliva is high in B cell tropic gp42. J. Virol. 80:7281-7283. 17. Hadinoto, V., M. Shapiro, C. C. Sun, and D. A. Thorley-Lawson. 2009. The dynamics of EBV shedding implicate a central role for epithelial cells in amplifying viral output. PLoS Pathog. 7:e10000496. 18. Longnecker, R. M., E. Kieff, and J. I. Cohen. 2013. Epstein-Barr Virus, p. 1898-1959. In D. M. Knipe and P. M. Howley (ed.), Fields Virology, vol. 2. Lippincott Williams & Wilkins, Philadelphia. 19. Temple, R. M., J. Zhu, L. R. Budgeon, N. D. Christensen, C. Meyers, and C. E. Sample. 2014. Efficient replication of Epstein-Barr virus in stratified epithelium in vitro. Proc. Natl. Acad. Sci. USA 111:16544-16549. 20. Kenney, S. C., and J. E. Mertz. 2014. Regulation of the latent-lytic switch in Epstein- Barr virus. Semin. Cancer Biol. 26:60-68. 21. Wille, C. K., D. M. Nawandar, A. R. Panfil, M. M. Ko, S. R. Hagemeier, and S. C. Kenney. 2013. Viral genome methylation differentially affects the ability of BZLF1 versus BRLF1 to activate Epstein-Barr virus lytic gene expression and viral replication. J. Virol. 87:935-950.

191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 22. Nawandar, D. M., A. Wang, K. Makeilski, D. Lee, S. Ma, E. Barlow, J. Reusch, R. Jiang, C. K. Wille, D. Greeenspan, J. S. Greenspan, J. E. Mertz, L. M. Hutt-Fletcher, E. C. Johannsen, P. F. Lambert, and S. C. Kenney. 2015. Differentiation-dependent KLF4 expression promotes lytic Epstein-Barr virus infection in epithelial cells. PLoS Pathog. 11:e1005195. 23. Lo, K. W., G. T. Chung, and K. F. To. 2012. Deciphering the molecular genetic basis of NPC through molecular, cytogenetic and epigenetic approaches. Semin. Cancer Biol. 22:79-86. 24. Tsang, C. M., Y. L. Yip, K. W. Lo, W. Deng, K. F. To, P. M. Hau, V. M. Lau, K. Takada, V. W. Lui, M. L. Lung, H. Chen, M. S. Zeng, J. Middeldorp, A. L. Cheung, and S. W. Tsao. 2012. Cyclin D1 overexpression supports stable EBV infection in nasophyryngeal epithelial cells. Proc. Natl. Acad. Sci. USA 109:E3473-E3482. 25. Niedobitek, G., L. S. Young, R. Lau, L. Brooks, D. Greenspan, J. S. Greenspan, and A. B. Rickinson. 1991. Epstein-Barr virus infection in oral hairy leukoplakia: virus replication in the absence of a detectable latent phase. J. Gen. Virol. 72:3035-3146. 26. Sixbey, J. W. 1989. Epstein-Barr virus and epithelial cells. Adv. Viral Oncol. 8:187-202. 27. Shannon-Lowe, C. D., B. Neuhierl, G. Baldwin, A. B. Rickinson, and H.-J. Delecluse. 2006. Resting B cells as a transfer vehicle for Epstein-Barr virus infection of epithelial cells. Proc. Natl. Acad. Sci. USA 103:7065-7070. FIGURE LEGEND Figure 1. Possible routes for infection and spread of EBV in vivo. 1. Cell-free virus, virus produced by a transferred B cell or virus using an uninfected B cell as a transfer vehicle infects the apical surface of the epithelium from which point it spreads through the epithelium to emerge as highly lymphotropic virus. 2. Virus infects B cells and establishes latency in the memory B cell compartment. 3. Terminal differentiation of a B cell into a plasmablast induces

217 218 219 220 reactivation and production of virus which is epitheliotropic, infects basal epithelial cells and establishes latency. 4. As basal cells differentiate they become capable of supporting productive replication and shed virus into saliva for transmission to a new host or replenishment of the reservoir in the existing host.