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Region E3 of adenovirus: A cassette of genes involved in host immunosurveillance and virus-cell interactions
VlkOLOGY
184,
l-8
(1991)
MINIREVIEW Region E3 of Adenovirus: A Cassette of Genes Involved in Host Immunos Virus-Cell Interactions WILLIAM S. M. WOLD**’
AND
and
LINDA R. GOODlNGt
*institute for Molecular Virology, St. Louis University School of Medicine, 368 1 Park Avenue, St. Louis, Missouri 63 110; and Wapartment Microbiology and Immunology, 3 107 Rollins Research Center, Emory University School of Medicine, Atlanta, Georgia 30322 Received April 4, 199 1; accepted
INTRODUCTION A great deal has been learned about the molecular biology of viruses. However, much less is known at the molecular level about how viruses usurp the infected cell and how they interact with the host’s antiviral defenses. In this review, we will discuss several novel human adenovirus (Ad) proteins, coded mainly within the E3 transcription unit, that may allow adenovirus to evade immunosurveillance and that may prepare the infected cell for efficient virus replication. Several recent reviews are related to this topic (PBBbo et al., 1989; Wold and Gooding, 1989; Gooding and Wold, 1990; Horwitz, 1QQOa). There are 47 known human adenovirus serotypes (Ad1 to Ad47) that form six distinct groups (A to F). Although adenoviruses in general infect epithelial surfaces of the respiratory and GI tracts, the serotypes and groups can differ markedly in tissue specificity and virulence (Horwitz, 1990b). Ad2 and Ad5 (group C), the most extensively studied, commonly cause respiratory infections in infants and young children. Infections occasionally persist for months or even years; lymphoid cells may be the reservoir for these persistent infections. Adenoviruses are nonenveloped viruses with an inner DNA protein core enclosed by a protein capsid. The genome (Fig. 1) is expressed in the cell nucleus in a temporaify regulated manner beginning with the “immediate early” genes in the El A transcription unit. The El A 289R (289 amino acids) protein then transactivates the “delayed early” transcription units, El B, E2, E3, E4, and Ll (early). These early proteins carry out a variety of functions that prepare the cell for efficient virus replication. Viral DNA replication begins at about ’ To whom
requests
for reprints
should
be addressed.
of
May 24, 199 1
7 hr postinfection, and this initiates the late phase. Most late genes fall into five families, Ll to L5, coded within the major late transcript&n unit. Late genes encode mainly virion structural pro&&s. Host DNA, mRNA, and protein synthesis are shut off in I stages of infection. At about 24 hr postin%ction, &ions assemble in the cell nucleus. Region E3 (Fig. 2) has been s~q~~~~ in Ad2 (H&is& et al., 1980; H&is& and GaE%ert, 1981) Ad5 (Cladaras and Wold, 19853, Ad3 (Sis & al., 1986) and partially in Ad7 (Hong et al., 198 menberg eta/., 1988). Ad3, Ad7, and adenoviruses. About nine overt are expressed by alternative Proust & a common pre-mRNA that initiates from thetE3 al., 1979; Cladaras eta/., 1985). Gen&?&c indicated that specific mechan&ms mine the relative abundance of the E3 m&NAs (see Bhat and Wold, 1987; Brady and W&d, lQ88). The E3 promoter is unique among the ~~~~ promoters in that it contains binding sites for the NF& Bransoription factor; this allows for El A-indepen ~~~~~p~~on of E3 in lymphoid cells (Williams eta!., 19%3). Since some of the E3 proteins counteract immun~~~i~nce, this could explain why group C adenoviruses persist in lymphoid tissue. There are nine predicted E3 pr~te&rs in 9ruup C adenoviruses, six of which have bean iti cells by immunopreci These are 6.7K (6700 gpl QK (also called El 9 &Void et al., 1984) 14.5K (Tollefson et al., 1998b) and Weld, 1988; Wang et&., 19 except perhaps 6.7K, are viruses. Group B adenovi E3 proteins, 20.1 K and 20.4K, that are not found in group C adenoviruses (Signas et al., 1986). 0042-682219
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FIG. 1. Schematic of the genome of human Ad2 The parallel lines indicate the linear duplex DNA genome of 36 kbp; rand /refer to rightward and leftward transcription, respectively. 289R 13.6K, Illa, etc. refer to proteins. El B-l 75R is also called El B-l 9K. The split arrows indicate the spliced structures of the mRNAs. El A, E3, etc. refer to transcription units. Most late genes are in the “major late transcription unit” which initiates at map position 16 of rstrand, and which includes the “ 1, ” “2.” and “3” leaders as well as the Ll , L2, L3, L4, and L5 families of mRNAs.
None of the E3 proteins is required for adenovirus replication in cultured cells or in acute infections of the lungs of hamsters (Morin et al., 1987) or cotton rats (Ginsberg eta/., 1989). Despite this, E3 is always maintained in natural isolates of adenoviruses (Adrian et a/., 1989a,b), indicating that these genes are important for natural infections of humans. Functions associated with the gpl9K, 10.4K, 14.5K, and 14.7K proteins are discussed below. At present, nothing is known about the functions of the 6.7K and 11.6K proteins. Many viable virus mutants have been constructed with mutations throughout E3 (e.g., see Gooding et a/., 1988; Carlin et a/., 1989); these mutants have been invaluable in characterizing the E3 proteins. Gpl9K prevents lysis of adenovirus-infected cytotoxic T-lymphocytes
cells by
Gpl9K of Ad2 is an abundant transmembrane glycoprotein of 142 amino acids (aa) following cleavage of the N-terminal signal sequence which directs gpl9K into the membrane of the endoplasmic reticulum (ER). Gpl9K has a 20 to 24 aa transmembrane domain followed by a 15 aa polar cytoplasmic domain at the Cterminus (Persson et al., 1980). There are two Asnlinked glycosylation sites in the N-terminal lumenal domain which are modified with exclusively high-man-
nose oligosaccharides (Kornfeld and Wold, 1981). The C-terminal 15 aa of gp 19K contain the signal for retention of gpl9K in the membrane of the ER (Paabo et al., 1987). The nature of this signal is controversial. One group claims that it is a linear sequence consisting of the C-terminal six or so residues (Nilsson et al., 1989) and containing a lysine at position -3 and another at positions -2, -4, or -5 (Jackson et a/., 1990). Another group suggests that it is a noncontiguous sequence with a complex spatial arrangement and that the lysines at positions -3 and -4 are of little importance (Gabathuler and Kvist, 1990). Binding of this signal to microtubules may be important for retention of gpl9K in the ER (Dahllof et al., 1991). The first clue to the function of gpl9K came when it was shown to bind to Class I antigens of the major histocompatibility complex (MHC) in Ad2-transformed rat cells (Kvist et al., 1978). A variety of subsequent studies showed that gpl9K binding to Class I antigens is an intrinsic property of the protein, is noncovalent, does not require oligosaccharides, does not require other adenovirus proteins, occurs when /3,-microglobulin is complexed with Class I antigens, and is observed with Class I antigens from human, rat, and mouse. Gpl9K from serotypes in adenovirus groups B, C, D, and E, but not in group A, binds Class I antigens (Paabo et al., 1986a).
ADENOVIRUS
REGION E3 AND IMMUNOSURVEILLANCE
E3A 3.6 K @ a ~~~.~~~. 291
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FIG. 2. Schematic illustration of region E3 of rec700. Arrows indicate the spliced structures of the mRNAs; the.thickneas of the arrow r&ecta the relative abundance of the mRNAs, and the dashed lines indicate introns. E3A and E3B are polyadenylabn sites. Bars above the arrows indicate proteins; hatched bars are proteins that have been identified. and stippled bars are proteins that are proposed2oF3xi~t. PluckWide +l is the transcription initiation site. rec700 is an Ad5-Ad2-Ad5 recombinant that has Ad2 sequences from nucleotide -236 to 2437 in the E3 transcription unit.
The net effect of gpl9K being retained in the ER and binding to Class I antigens is to block the transport of Class I antigens to the cell surface. This has been shown by FACS analysis and by the differential sensitivity of the oiigosaccharides on Class I antigens to cleavage with endo H (endo H cleaves high mannose but not complex oligosaccharides and thus can distinguish pre-Golgi from post-Golgi molecules) (Andersson et al., 1985; Burgert and Kvist, 1985). Cell surface expression of Class I antigens complexed with small viral peptide antigens is required for recognition and lysis of virus-infected cells by virus-specific CTL (reviewed in Townsend and Bodmer, 1989). If gpl9K blocks Class I antigens in the ER, then gpl9K should protect cells from lysis by CTL. Consistent with this, cells expressing gpl9K are less sensitive to lysis by alloreactive CTL (Andersson et a/., 1987; Burgert et a/., 1987). Also, co-infection with SV40 and adenovirus decreases sensitivity to anti-SV40 CTL (Tanaka and Tevethia, 1988), and infaction with a vaccinia virus vector expressing gpl9K decreases CTL killing (Cox et al., 1990). And
most important, gpl9K alone among E3 proteins prevents lysis of adenovirusMect& c&s by adenuvirusspecific CTL (Rawk et al., 1989). Thus, a masonable hypothesis is that gp19K functions in viGfo to protect adenouirus-infected cells against lysis by adenovlrusspecific CTL. Gpl9K has different affinities for different Class I antigens. Studies in the mouse system ~~i~g coimmunoprecipitation, sensitivity of Ct&ss i antigens to endo H, and prevention of ~yloiysks by CTL, imply that gpl9K interacts w&l with K”, L*, and Db, m&My with Dd, and not with Dk, Kk, or Kb C&ss 1 anti and Kvist, 1987; Tanaka and T@v&thia, 1 al., 1989; Cox et a/., 1990). Gpl Qk binds more strongly to HLA-A2 than to HLA-B7 human Cl&ss I antigens (Severinsson et al., 1986). These the possibility that suscaptW&y to tion in humans may be relisted to the ef#nity of an individual’s Class I antigens to gpl9K. The lumenal domain of gpt9K is st&oiit for binding to Class I antigens, and this binding does not re-
4
WOLD AND GOODING
quire p,-microglobulin (Paabo eta/., 1986b; Gabathuler et a/., 1990). The CX,and (Y* domains (the peptide antigen binding domains) in mouse Class I antigens are involved in binding to gpl9K (Burger-t and Kvist, 1987). It is conceivable that gp19K binding to the a, and (Ye domains could prevent peptide binding, which could in turn preclude transport of Class I antigens to the cell surface, because association with peptide is required for this transport (Townsend et al., 1989). If the above hypothesis for gp19K function is true, then one would predict that mutants lacking gpl9K would be more readily destroyed in vivo and hence would be less pathogenic. Surprisingly, however, deletion of the gpl9K gene resulted in markedly increased pathogenicity and a dramatic increase in the lymphocyte and macrophage inflammatory response with no effect on virus replication in the lungs of adenovirus-infected cotton rats (Ginsberg et al., 1989). Since this pathogenic@ occurred within 3 days, a time probably too short for adenovirus-specific CTL to form, it was suggested that gpl9K may directly or indirectly suppress cytokines which attract lymphocytes. This finding underscores our lack of understanding of the processes involved in viral pathogenesis. ElA proteins render cells susceptible to cytolysis by TNF and, depending on the cell type, the E3-14.7K, E3-10.4W14.5K, and El B-l 9K proteins protect the cells against cytolysis by TNF Although CTLare one means bywhich the hosteliminates virus-infected cells, tumor necrosis factor (TNF) may be another. TNF is a multifunctional cytokine that is secreted by activated macrophages and lymphocytes and that regulates a wide variety of aspects of the immune system and the inflammatory response (reviewed in Beutler and Cerami, 1989). A notable feature of TNF is that it is cytotoxic or cytostatic to certain tumor cells. The mechanism underlying cell lysis by TNF is largely unknown, but appears to occur in three steps: binding of TNF to its cell surface receptor followed by internalization and degradation of the receptor-ligand complex, intracellular signaling, and the lytic event itself, which is unknown. TNF also lyses cells infected with certain viruses, it inhibits the replication in cultured cells of a variety of DNA and RNA viruses, and it is released during infections by influenza virus (reviewed in Gooding et a/., 1991 a). Thus, TNF may be an antiviral agent in vivo. Gooding et al. (1988) showed that mouse CBHA fibroblasts are lysed by TNF when infected by adenovirus mutants that lack region E3. Uninfected cells are not lysed by TNF, nor are cells infected by wild-type adenovirus. This experiment shows two things: that
adenovirus infection induces susceptibility to TNF lysis and that a product(s) in region E3 prevents TNF cytolysis. It was subsequently shown, using adenovirus mutants that lack E3 as well as other early regions, that either the El A-289R or El A-243R protein is able to induce TNF susceptibility (Duerksen-Hughes et al., 1989). Stable El A-transfected cell lines also become spontaneously susceptible to cytolysis by TNF; this has been shown for mouse (Chen et a/., 1987; Duerksen-Hughes et al., 1989; Vanhaesebroeck et al., 1990) and rat (Kenyon et al., 1991) cell lines. The El A proteins are multifunctional proteins with a modular structure (Moran and Mathews, 1987). There are three regions in the El A proteins that are conserved among the adenovirus serotypes, these are conserved regions 1 (CRl), CR2, and CR3. Deletion of a portion of CR1 abrogates ElA induction of TNF susceptibility in adenovirus-infected cells (DuerksenHughes et a/., 1991). Functions associated with CR1 include immortalization of primary cells, cell transformation in cooperation with Ha-ras, induction of DNA synthesis, enhancer repression, and induction of TNFindependent cytotoxicity (see Gooding and Wold, 1990; White et a/., 1991). It will be of interest to determine which, if any, of these properties is linked to the ability of ElA to induce susceptibility to TNF. Whereas ElA induces susceptibility to TNF lysis in mouse C3HA cells, region E3 is required to prevent TNF cytolysis. By infecting C3HA cells with virus mutants having deletions throughout E3, it was determined that the E3-14.7K protein is required to prevent TNF lysis of C3HA cells (Gooding et al., 1988). Cells can be sensitized to cytolysis by TNF not only by expression of El A, but also by inhibition of protein synthesis or by treatment with cytochalasin E, an agent that disrupts microfilaments; 14.7K prevents TNF cytolysis in adenovirus-infected C3HA cells sensitized to TNF by these treatments (Gooding et a/., 1990). Some cell lines are spontaneously sensitive to TNF; 14.7K prevented TNF lysis in two of three of these cell lines infected by adenovirus mutants (Gooding et al., 1990). To date, 14 of 15 mouse cell lines infected with adenovirus mutants have been shown to be protected against TNF cytolysis by 14.7K (Gooding et a/., 1990, 1991 b). Thus, 14.7K appears to be a general inhibitor of TNF cytolysis. C3HA cells infected with representative adenovirus serotypes in groups A, B, D, and E are not killed by TNF when the cells are sensitized to TNF either by expression of ElA or by inhibition of protein synthesis; thus, this anti-TNF function seems to be a general property of adenoviruses in groups A through E (Horton et a/., 1990). These representative serotypes express a pro-
ADENOVIRUSREGION E3 AND IMMUNOSURVEILIANCE
tein serologically
related to 14.7K of group C adenovi-
ruses. StablytransfectedmouseC3HAandCl 27 cellsexpressing14.7Kfrom a bovinepapillomavirus vector (BPV)are protected from TNF cytolysis regardless of whether they are sensitized to TNF by infection with adenovirus mutants that lack 14.7K, by inhibition of protein synthesis, or by treatment with cytochalasin E (Horton et al., 1991). Thus, 14.7K can prevent TNF cytolysis in the absence of other adenovirus proteins. 14.7K is the only adenovirus protein that prevents TNF cytolysis of adenovirus-infected C3HA cells (Gooding et al., 1988). However, in most other mouse cell lines including C127, the E3-10.4K and E3-14.5K proteins, which appear to exist as a complex (Tollefson et al., 1991) can also prevent TNF cytolysis (Gooding et a/., 1991 b). Specifically, Cl 27 cells are not lysed by TNF when infected with mutants that express either 14.7K or 10.4W14.5K, but they are lysed when infected with mutants that lack 14.7K and either 10.4K or 14.5K (Gooding et al., 1991 b). 10.4W14.5K also suppress TNF killing when cells are sensitized to TNF by inhibition of protein synthesis. Of 15 mouse cell lines tested, 10.4KJ14.5K were found to suppress TNF killing in 1 1, including both transformed and normal cells. Thus, for most mouse cell lines, there are two independent functions in E3 that prevent TNF cytolysis, 14.7K and 10.4W14.5K. Human cells are even more complicated in their response to adenovirus infection and TNF. HEL-299 untransformed human embryo fibroblasts and ME-180 cervical carcinoma cells are not lysed by TNF when infected with adenovirus mutants that lack region E3 (Gooding eta/., 1991a). Using a series of mutants lacking E3 as well as another region of adenovirus, it was determined that the El B-l 9K protein (El B-l 75R in Fig. 1) protects human cells against TNF lysis (Gooding et al., 199 1 a). Identical results were obtained with a panel of six human cell lines of both tumor and normal tissue origin. In common with 14.7K and 10.4W14.5K, El B19K prevents TNF cytolysis when cells are sensitized to TNF either by expression of El A or by inhibition of protein synthesis. It is not clear why El B-l 9K prevents TNF lysis in human cells but not in mouse cells. If El B-19K prevents TNF lysis of human cells, do 14.7K and/or 10.4W14.5K also have this function? The answer to this is probably. HEL-299 and ME-180 human cells are not killed by TNF when infected with H2d/250, a mutant that lacks El B-l 9K but retains E3 (Gooding et a/., 199 1 a). Therefore, one or more E3 proteins can protect human cells against TNF lysis in the absence of ElB-19K. Human cells infected with H5dll 1 1 are killed by TNF; H5dllll lacks El B-l 9K, it lacks E3-10.4K, E3-14.5K, and E3-14.7K, but it retains
5
the other E3 genes. This suggests that 14.7K and/or
10.4W14.5K canpreventTNFcytolysisof humancells. If so,thenadenovirus hasthreeindependent functions that preventTNFcytolysisof humancells. Little is known about the mechanism of action of these TNF-protecting proteins. As judged by its predicted sequence (Tollefson and Weld, 1988) 14.7K is a hydrophilic protein without apular domains that normally are associated with integral membrane proteins. One possibility is that 14.7K might down-regulate the TNF receptor(s) and thereby preclude TNF signal transduction. However, 14.7K does not substantially alter the cell surface TNF receptor number or affinity in stably transfected C3HA cells (Horton et al., 1991). Although 14.7K could function at a downstream point in TNF signal transduction, it is not a general inhibitor of TNF signal transduction because TNFinduction of cell surface Class I MHC antigens in 14.7K transfectants is not inhibited (Horton et a/., 1991). 10.4K and 14.5K are both integral cytoptasmic membrane proteins (Tollefson et al., 199Ua,b; Hoffman et a/., 1990; unpublished results), Little else is known about these proteins except their role in down-regulating the epidermal growth factor receptor (EGF-R) (see below). E 1B- 19K is an interesting multifunctional protein (reviewed in Gooding et a/., 1990a). It is localized to the nuclear membrane, it binds to and disrupts the nuclear lamina and vimentin-containing intermediate fiiaments, it cooperates with E 1A in adenovirus transformation of rodent cells, and it prevents El A-induced DNA degradation and cytotoxicity. It will be of great interest to determine which, if any, of these properties are linked to the ability of El B-l 9K to prevent TNF cytolysis. Why should three “sets” of proteins have evolved in adenovirus to prevent TNF cytolysis, and why should the proteins be so different? Perhaps these proteins function in different tissues of the host or perhaps at different steps in the TNF cytolysis pathway(s). It is remarkable that of the 25-30 adenovirus early genes, 4 of them function in preventing TNF cytolysis. This supports the idea that TNF is an important antiviral defense of the host. Aside from the soluble form of the TNF receptor (see Nopha et al., 1990, and references therein) and manganese superoxide dismutase (Wang et al., 1989), these adenovirus proteins are the only known inhibitors of TNF cytoiysis. Thus, they should be useful tools to understand the mechanism of TNF cytolysis. The E3-10.4K and E3-14.5K pro@ins function in concert to down-reguk%e tb EGF receptor in adenovirus-infected celJs EGF-R is a member of the protein tyrosine kinase class of plasma membrane receptors (reviewed in
6
WOLD AND GOODING
Ullrich and Schlessinger, 1990). Binding of the polypeptide growth factor EGF to EGF-R stimulates the kinase activity of EGF-R, resulting in autophosphorylation of EGF-R on Tyr residues as well as Tyr phosphorylation of a variety of cellular proteins. The EGWEGF-R complex clusters in clathrin-coated pits which are internalized into endosomes and degraded in lysosomes. Stimulation of the kinase activity of EGF-R results in the activation of cellular metabolism and eventually in the induction of DNA synthesis and mitosis. Adenovirus infection mimics EGF in that EGF-R is internalized via endosomes and is degraded (Carlin et a/., 1989). Using a panel of virus mutants, it was shown that E3-10.4K and E3-14.5K are both required for this down-regulation of EGF-R (Carlin eta/., 1989; Tollefson et a/., 1991). 10.4K is efficiently coimmunoprecipitated with 14.5K, strongly suggesting that the proteins exist as a complex in viva and consistent with the genetic data indicating that they function in concert (Tollefson et al., 1991). Although both 10.4K and 14.5K are required to down-regulate EGF-R in adenovirus-infected cells, 10.4K alone expressed from a retrovirus vector is sufficient to down-regulate EGF-R in mouse HERc cells (cells stably transfected with human EGF-R) (Hoffman et a/., 1990). It is not known why adenovirus-infected cells differ in this respect from the 10.4K-retrovirus-infected cells. We know little about the mechanism of action of 10.4W14.5K. Both are cytoplasmic membrane proteins, and it seems likely that they localize to the plasma membrane where they interact with EGF-R. However, this remains to be demonstrated. There are now three classes of viral proteins that act on EGF-R, vaccinia virus growth factor (VGF), the E5 transforming protein of BPV, and 10.4W14.5K. VGF is a secreted protein, related to EGF, which appears to mimic EGF in stimulating EGF signal transduction and which is responsible for the cellular proliferative response to VGF (Buller eta/., 1988). Thus, VGF probably serves to activate the infected and neighboring cells so that they become efficient factories for virus replication. The BPV E5 protein may similarly activate the infected cell by reducing down-regulation of EGF-R (Martin et a/., 1989). By analogy with VGF and E5, 10.4W 14.5K probably activate the infected cell. In common with EGF, 10.4W14.5K would initially activate the protein kinase activity of EGF-R (which has not been tested), and this would be followed by internalization and degradation of EGF-R (which has been demonstrated). As noted above, 10.4W14.5K also function to protect most mouse cell lines examined against lysis by TNF. A key unanswered question is whether the ability
TABLE 1 FUNCTIONSOFADENOVIRUSPROTEINS Protein
Function in vitro
E3-gpl9K El A-289R and 243R
Prevents cytolysis by CTL. Induce cellular susceptibility to TNF cytolysis. Prevents cytolysis of mouse cells by TNF. Prevent cytolysis of mouse cells by TNF. Down-regulate EGF receptor. Prevents cytolysis of human but not mouse cells by TNF. E3-14.7K and/or E3-10.4K/14.5K also probably prevent cytolysis of human cells by TNF.
E3-14.7K E3-10.4iV14.5K ElB-19K
of 10.4W14.5K to act on EGF-R is related to their ability to prevent TNF cytolysis. Is region E3 a cassette of functionally
related genes?
Adenovirus transcription units tend to encode genes with related functions. Regions El A and El B encode proteins that function in gene regulation and cell transformation, E2 proteins function in DNA replication, and the genes in the major late transcription unit encode mainly virion structural proteins. Of the six known E3 proteins, four function in counteracting the immune system, gpl9K preventing cytolysis by CTL, and 14.7K and 10.4W14.5K preventing cytolysis by TNF (see Table 1). Thus, E3 may be a cassette of genes, at least in part, that helps the virus to evade immunosurveillance. 10.4W14.5K also act on EGF-R. Thus, another possible unifying theme for E3 proteins is that they interdict signal transduction, 14.7K and 10.4K/14.5K for TNF, and 10.4W14.5K for EGF. ACKNOWLEDGMENTS Research from the authors’ laboratories that is discussed in this report was supported by grants CA24710, A129492, CA49540, CA4821 9, and Al26035 from the National Institutes of Health.
REFERENCES ADRIAN,T., BEST, B., HIERHOLZER,J. C., and WIGAND. R. (1989a). Molecular epidemiology and restriction site mapping of adenovirus type 3 genome types. J. Clin. Microbial. 27. 1329-l 334. ADRIAN,T., BECKER,M., HIERHOLZER,J. C., and WIGAND, R. (1989b). Molecular epidemiology and restriction site mapping of adenovirus 7 genome types. Arch. Virol. 106, 73-84. ANDERSSON,M., MCMICHAEL, A., and PETERSON,P. A. (1987). Reduced allorecognition of adenovirus 2 infected cells. /. lmmunol. 138,3960-3966. ANDERSSON,M., PUao, S., NILSSON,T., and PETERSON.P. A. (1985). Impaired intracellular transport of class I MHC antigens as a possi-
ADENOVIRUS
ble means
for adenoviruses
to evade
immune
REGION
surveillance.
E3 AND
Cell
43,2 15-222. B., and CERAMI, A. (1989). The biology of cachectin/rNFA primary mediator of the host response, Annu. Rev. Immunol. 7, 625-711. BHAT, B. M., and WOLD, W. S. M. (1987). A small deletion distant from a splice or polyadenylation site dramatically alters pre-mRNA processing in region E3 of adenovirus. J. Viral. 61, 3938-3945. BRADY, H. A., and WOLD, W. S. M. (1988). Competition between splicing and polyadenylation reactions determines which adenovirus region E3 mRNAs are synthesized. Mol. Cell. B/o/. 8, 32913297. BULLER, R. M. L., CHAKRABARTI, S., Moss, B., and FREDRICKSON, T. (1988). Cell proliferative response to vaccinia virus is mediated by VGF. Virology 164, 182-l 92. BURGERT. H.-G., and KVIST, S. (1985). An adenovirus type 2 glycoprotein blocks cell surface expression of human histocompatibility class I antigens. Cell 41, 987-997. BURGERT, H.-G., and KVIST, S. (1987). The E3/19K protein of adenovirus type 2 binds to the domains of histocompatibility antigens required for CTL recognition. ElMi /. 6, 201 g-2026. BURGERT, H.-G., MARYANSKI, J. L., and KVIST, S. (1987). “E3/19K” protein of adenovirus type 2 inhibits lysis of cytolytic T lymphocytes by blocking cell-surface expression of histocompatibility class I antigens. Proc. Nat/. Acad. Sci. USA 84, 1356-l 360. CARLIN, C. R., TOLLEFSON, A. E., BRADY, H. A., HOFFMAN, B. L., and WOLD, W. S. M. (1989). Epidermal growth factor receptor is downregulated by a 10,400 MW protein encoded by the E3 region of adenovlrus. Cell 57, 135-l 44. CHEN, M.-J., HOLSKIN, B.. STRICKLER, J., GORNIAK, J.. CLARK, M. A., JOHNSON, P. J., MITCHO, M., and SHALLOWAY, D. (1987). Induction by El A oncogene expression of cellular susceptibility to lysis by TNF. Nature 330, 581-583. CHOW, L. T., BROKER, T. R., and LEWIS, J. B. (1979). Complex splicing patterns of RNAs from the early regions of adenovirus-2. J. Mol. Biol. 134, 265-303. CLADARAS. C., and WOLD, W. S. M. (1985). DNA sequence of the E3 transcription unit of adenovirus 5. Virology 140, 28-43. CLADARAS, C., BHAT, B. M., and WOLD, W. S. M. (1985). Mapping the 5’ ends, 3’ ends, and splice sites of mRNAs from the E3 transcription unit of adenovirus 5. Virology 140, 44-54. Cox. J. H., YEWDELL, J. W., EISENLOHR, L. C., JOHNSON, P. R., and BENNINK, J. R. (1990). Antigen presentation requires transport of MHC class I molecules from the endoplasmic reticulum. Science 247, 715-718. D~~HLLOF, B., WALLIN, M., and KVIST, S. (199 1). Theendoplasmic reticulum retention signal of the E3/19K protein of adenovirus-2 is microtubule binding. /. Biol. Chem. 266, 1804-l 808. DUERKSEN-HUGHES, P. J., HERMISTON, T. W., WOLD, W. S. M., and GOODING, L. R. (1991). The amino-terminal portion of CD1 of the adenovtrus ElA proteins is required to induce susceptibility to tumor necrosis factor cytolysis in adenovirus-infected mouse cells. J. Virol. 65, 1236-1244. DUERKSEN-HUGHES, P., WOLD, W. S. M., and GOODING, L. R. (1989). Adenovirus ElA renders infected cells sensitive to cytolysis by tumor necrosis factor. /. Immunol. 143, 4193-4200. FLOMENBERG, P. R., CHEN, M., and HORWITZ, M. S. (1988). Sequence and genetic organization of adenovirus type 35 early region 3. J. Viral. 62, 443 1-4437. GABATHULER, R., and KVIST, S. (1990). The endoplasmic reticulum retention signal of the E3/19K protein of adenovirus type 2 consists of three separate amino acid segments at the carboxy terminus. J. Cell Biol. 111, 1803-l 810. BEUTLER,
IMMUNOSURVEILLANCE
GABATHIJLER,
7
R., LEVY,F., and KVIST, S. (1990). Requirementsfor the
associationof adenovirustype 2 E3/19Kwild-typeand mutant proteins with HLA antigens. /. viral. 64, 3679-3685, GINSBERG, H. S., LUNDHOLM-BEAUCHAMP, U., Ho~s~~oo,o, R. L.. PERNIS, B., WOLD, W. S. M., CHANOCK, R. M., and PRINCE, G. A. (1989). Role of early region 3 (E3) in pathogenesis of adenovirus disease. Proc. Natl. Acad. Sci. USA 86, 3823-3827. GOODING, L. R., AQUINO, L., DUERKSEN-HUGHES, P. J., DAY, D., HORTON, T. M., YEI, S., and WOLD, W. S. M. (199la). The ElB 19,000-molecular-weight protein of group C adenovirusea prevents tumor necrosis factor cytolysis of human cells but not of mouse cells. J. Viral., 85, 3083-3094. GOODING, L. R., ELMORE, L. W., TOLLEFSON, A. E., BRADY, H. A., and WOLD, W. S. M. (1988). A 14,700 MW protein from the E3 region of adenovirus inhibits cytolysis by tumor necrosis factor. Cell 53, 341-346. GOOOING, L. R.. RANHEIM, T. S., TOLLEFSON, A. E., ACXJINO, L.. DUERKSEN-HUGHES, P., HORTON, T. M., and Woto, W. S. M. (1991 b). The 10.400and 14,500-dalton proteins encoded by region E3 of adenovirus function together to protect many but not all mouse cell lines against lysis by tumor necrosis factor. / Viro/., 65, Issue no. 8, in press. GOODING. L. R., SOFOLA, I. O., TOLLEFSON, A. E., DUERKSEN-HUGHES, P., and WOLD, W. S. M. (1990). The adenovirus E3-14.7K protein is a general inhibitor of tumor necrosis factor-mediated cytolysis. /. Immunol. 145, 3080-3086. GOOOING, L. R., and WOLD, W. S. M. (1990). Molecular mechanisms by which adenovlruses counteract antiviral immune defenses. CRC Crit. Rev. Immunol. IO, 53-7 1. H~RISS~. J., and GALIBERT, F. (1981). Nucleotide sequence of the EcoRl fragment of adenovirus 2 genome. Nucleic Acids Res. 9, 1229-1240. H~RISS~. J., COURTOIS. G., and GALIBERT, F. (1980). Nucleotide sequence of the EcoRl D fragment of adenovirus 2 genome. Nucleic Acids Res. 8, 2173-2192. HOFFMAN, B. L., ULLRICH, A., WOLD, W. S. M., and CARLIN, C. R. (1990). Retrovirus-mediated transfer of an adenovirus gene encoding an integral membrane protein is sufficient to down-regulate the receptor for epidermal growth factor. Mol. Cell. Biol. 10,552 l5524. HONG, J. S., MULLIS. K. G., and ENGLER, J. A. (1988). Characterization of the early region 3 and fiber genes of Ad7. Virology 167, 54% 553. HORTON. T. M., RANHEIM, T. S., AQUINO, L., KUSHER, D. I., SAHA, S. K.. WARE, C. F., WOLD, W. S. M., and Goo~~ffi, L. R. (1991). Adenowrus E3 14.7K protein functions in the absence of other adenovirus proteins to protect transfected cells from tumor necrosis factor cytolysis. J. Viral. 65, 2629-2639. HORTON, T. M., TOLLEFSON, A. E., WOLD, W. S. M., and GOODING, L. R. (1990). A protein serologically and functionally related to group C E3 14,700-kilodalton protein is found in multiple adenovirus serotypes. J. Viral. 64, 1250-l 255. HORWITZ, M. S. (1990a). Adenoviridae and their replication. In “Virology” (B. N. Fields, Ed.), Vol. 2, pp. 1679-l 721. Raven Press, New York. HORWITZ, M. S. (1990b). Adenoviruses. ln “Virology” (B. N. Fields, Ed.), Vol. 2, pp. 1723-l 740. Raven Press, New York. JACKSON, M. R., NILSSON, T., and PETERSON, P. A. (1990). Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum. EMBO i. 9, 3 i 53--3162. KENYON. D. J., DOUGHERP/, J., and RA%A, K., JR. (1991). Tumorigenicity of adenovirus-transformed cells and their sensitivity to tumor
8
WOLD AND GOODING
necrosis factor LYand NWIAK cell cytolysis. Virology 180, 818821. KORNFELD,R., and WOLD, W. S. M. (1981). Structures of the oligosaccharides of the glycoprotein coded by early region E3 of adenovirus 2. J. Virol. 40, 440-449. KVIST, S., OSTBERG, L., PERSSON,H., PHILIPSON,L., and PETERSON, P. A. (1978). Molecular association between transplantation antigens and cell surface antigen in adenovirus-transformed cell line. Proc. Natl. Acad. Sci. USA 75, 5674-5678. MARTIN, P., VASS, W. C., SCHILLER,J. T., Lowv, D. R., and VELU, T. J. (1989). The bovine papillomavirus E5 transforming protein can stimulate the transforming activity of EGF and CSF-1 receptors. Cell 59, 21-32. MORAN, E., and MATHEWS,M. B. (1987). Multiple functional domains in the adenovirus El A gene. Cell 48, 177-l 78. MORIN, J. E., LUBECK,M. D., BARTON,J. E., CONLEY,A. J., DAVIS,A. R., and HUNG, P. P. (1987). Recombinant adenovirus induces antibody response to hepatitis B virus surface antigen in hamsters. Proc. Nat/. Acad. Sci. USA 84, 4626-4630. NILSSON,T., JACKSON,M., and PETERSON,P. A. (1989). Short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum. Cell 58, 707-718. NOPHA, Y., KEMPER,O., BRAKEBUSCH,C., ENGELMANN.H., ZWANG, R., ADERKA,D., HOLTMANN,H., and WALLACH, D. (1990). Soluble forms of tumor necrosis factor receptors (TNF-Rs). The cDNA for the type I TNF-R, cloned using amino acid sequence data of its soluble form, encodes both the cell surface and a soluble form of the receptor. EMBO 1. 9, 3269-3278. Pii2i~0, S., BHAT, B. M., WOLD, W. S. M., and PETERSON,P. A. (1987). A short sequence in the COOH-terminus makes an adenovirus membrane glycoprotein a resident of the endoplasmic reticulum. Cell50,311-317. P&&IO, S., NILSSON,T., and PETERSON,P. A. (1986a). Adenovirusesof subgenera B, C, D, and E modulate cell-surface expression of major histocompatibility complex class I antigens. Proc. Nat/. Acad. Sci. USA 83,9665-9669. Pb;ii~o, S., SEVERINSSON,L., ANDERSSON,M., MARTENS,I., NILSSON,T., and PETERSON,P. A. (1989). Adenovirus proteins and MHC expression. Adv. Cancer Res. 52, 151-l 63. Pii;lso, S., WEBER, F., NILSSON, T., SCHAFFNER,W., and PETERSON, P. A. (1986b). Structural and functional dissection of an MHC class I antigen-binding adenovirus glycoprotein. EMBOJ. 5, 19211927. PERSSON,H., JORNVALL,H., and ZABIELSKI,1. (1980). Multiple mRNA species for the precursor to an adenovirus-encoded glycoprotein: Identification and structure of the signal sequence. Proc. Narl. Acad. Sci. USA 77,6349-6353. RAWLE,F. C., TOLLEFSON,A. E., WOLD, W. S. M., and GOODING,L. R. (1989). Mouse anti-adenovirus cytotoxic T lymphocytes. Inhibition of lysis by E3 gp19K but not E3 14.7K. J. lmmunol. 143, 20312037. SEVERINSSON,L., MARTENS,I., and PETERSON,P. A. (1986). Differential association between two human MHC class I antigens and an adenoviral glycoprotein. 1. Immunol. 137, 1003-l 009. SIGN&, C.. AKUSJARVI.G., and PETERSSON.U. (1986). Region E3 of human adenoviruses: Differences between the oncogenic adenovirus-3 and the nononcogenic adenovirus-2. Gene 50, 173-l 84.
TANAKA, Y., and TEVETHIA,S. S. (1988). Differential effect of adenovirus 2 E3/19K glycoprotein on the expression of H-2Kb and H-2Db class I antigens and H-2Kb- and H-2Db-restricted SV40-specific CTL-mediated lysis. Virology 165, 357-366. TOLLEFSON,A. E., KR/USCI,P., PURSLEY,M. H., GOODING, L. R., and WOLD, W. S. M. (1990b). A 14,500 MW protein is coded by region E3 of group C human adenoviruses. Virology 175, 19-29. TOLLEFSON, A. E., KRAJSCI,P., YEI, S.. CARLIN, C. R., and WOLD, W. S. M. (1990a). A 10,400-molecular-weight membrane protein is coded by region E3 of adenovirus. J. Viral. 64, 794-801. TOLLEFSON,A. E., STEWART,A. R., YEI, S.. SAHA, S. K., and WOLD. W. S. M. (199 1). The 10,400- and 14,500-daiton proteins encoded by region E3 of adenovirus form a complex and function together to down-regulate the epidermal growth factor receptor. 1. Viral. 65, 3095-3105. TOLLEFSON,A. E., and WOLD, W. S. M. (1988). Identification and gene mapping of a 14,700-molecular-weight protein encoded by region E3 of group C adenoviruses. J. Viral. 62, 33-39. TOWNSEND,A., and BODMER,H. (1989). Antigen recognition by class l-restricted T lymphocytes. Annu. Rev. Immunol. 7, 601-624. TOWNSEND,A., OHLEN, C., BASTIN,J.. LJUNGGREN,H.-G., FOSTER,L., and KARRE,K. (1989). Association of class I major histocompatibility heavy and light chains induced by viral peptides. Nature 340, 443-448. ULLRICH,A., and SCHLESSINGER,1. (1990). Signal transduction by receptors with tyrosine kinase activity. Cell 61, 203-212. VANHAESEBROECK,B., TIMMERS,H. T. M., PRONK, G. J., VAN ROY, F., VAN DEREB, A. J., and FIERS,W. (1990). Modulation of cellular susceptibility to the cytotoxic/cytostatic action of tumor necrosis factor by adenovirus El gene expression is cell type-dependent. Virology 176,362-368. WANG, E. W., Scorr, M. O., and RICCIARDI,R. P. (1988). An adenovirus mRNA which encodes a 14,700-M, protein that maps to the last open reading frame in region E3 is expressed during infection. J. Virol. 62, 1456-1459. WHITE, E., CIPRIANI,R., SABBATINI,P., and DENTON, A. (1991). The adenovirus El B 19kD protein overcomes cytotoxicity of the El A proteins. J. Viral., in press. WILLIAMS, J. L., GARCIA, J., HARRICH.D., PEARSON,L., Wu, F., and Gaynor, R. (1990). Lymphoid specific gene expression of the adenovirus early region 3 promoter is mediated by NF-KB binding motifs. EMBO J. 9, 4435-4442. WILSON-RAWLS,J.. SAHA, S. K., KRA~CSI,P., TOLLEFSON,A. E., GOODING, L. R., and WOLD, W. S. M. (1990). A 6700 MW membrane protein is encoded by region E3 of adenovirus type 2. virology 178, 204-212. WOLD, W. S. M., and GOODING, L. R. (1989). Adenovirus region E3 proteins that prevent cytolysis by cytotoxic T cells and tumor necrosis factor. Mol. Biol. Med. 6, 433-452. WOLD, W. S. M., CLADARAS,C., MAGIE, S. C., and YACOUB,N. (1984). Mapping a new gene that encodes and 11,600-molecular-weight protein in the E3 transcription unit of adenovirus 2. 1. Viral. 52, 307-313. WONG, G. H. W., ELWELL, J. H., OBERLEY,L. W., and GOEDDEL, D. V. (1989). Manganous superoxide dismutase is essential for cellular resistance to cytotoxicity of tumor necrosis factor. Ce// 58, 923931.
184,
l-8
(1991)
MINIREVIEW Region E3 of Adenovirus: A Cassette of Genes Involved in Host Immunos Virus-Cell Interactions WILLIAM S. M. WOLD**’
AND
and
LINDA R. GOODlNGt
*institute for Molecular Virology, St. Louis University School of Medicine, 368 1 Park Avenue, St. Louis, Missouri 63 110; and Wapartment Microbiology and Immunology, 3 107 Rollins Research Center, Emory University School of Medicine, Atlanta, Georgia 30322 Received April 4, 199 1; accepted
INTRODUCTION A great deal has been learned about the molecular biology of viruses. However, much less is known at the molecular level about how viruses usurp the infected cell and how they interact with the host’s antiviral defenses. In this review, we will discuss several novel human adenovirus (Ad) proteins, coded mainly within the E3 transcription unit, that may allow adenovirus to evade immunosurveillance and that may prepare the infected cell for efficient virus replication. Several recent reviews are related to this topic (PBBbo et al., 1989; Wold and Gooding, 1989; Gooding and Wold, 1990; Horwitz, 1QQOa). There are 47 known human adenovirus serotypes (Ad1 to Ad47) that form six distinct groups (A to F). Although adenoviruses in general infect epithelial surfaces of the respiratory and GI tracts, the serotypes and groups can differ markedly in tissue specificity and virulence (Horwitz, 1990b). Ad2 and Ad5 (group C), the most extensively studied, commonly cause respiratory infections in infants and young children. Infections occasionally persist for months or even years; lymphoid cells may be the reservoir for these persistent infections. Adenoviruses are nonenveloped viruses with an inner DNA protein core enclosed by a protein capsid. The genome (Fig. 1) is expressed in the cell nucleus in a temporaify regulated manner beginning with the “immediate early” genes in the El A transcription unit. The El A 289R (289 amino acids) protein then transactivates the “delayed early” transcription units, El B, E2, E3, E4, and Ll (early). These early proteins carry out a variety of functions that prepare the cell for efficient virus replication. Viral DNA replication begins at about ’ To whom
requests
for reprints
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of
May 24, 199 1
7 hr postinfection, and this initiates the late phase. Most late genes fall into five families, Ll to L5, coded within the major late transcript&n unit. Late genes encode mainly virion structural pro&&s. Host DNA, mRNA, and protein synthesis are shut off in I stages of infection. At about 24 hr postin%ction, &ions assemble in the cell nucleus. Region E3 (Fig. 2) has been s~q~~~~ in Ad2 (H&is& et al., 1980; H&is& and GaE%ert, 1981) Ad5 (Cladaras and Wold, 19853, Ad3 (Sis & al., 1986) and partially in Ad7 (Hong et al., 198 menberg eta/., 1988). Ad3, Ad7, and adenoviruses. About nine overt are expressed by alternative Proust & a common pre-mRNA that initiates from thetE3 al., 1979; Cladaras eta/., 1985). Gen&?&c indicated that specific mechan&ms mine the relative abundance of the E3 m&NAs (see Bhat and Wold, 1987; Brady and W&d, lQ88). The E3 promoter is unique among the ~~~~ promoters in that it contains binding sites for the NF& Bransoription factor; this allows for El A-indepen ~~~~~p~~on of E3 in lymphoid cells (Williams eta!., 19%3). Since some of the E3 proteins counteract immun~~~i~nce, this could explain why group C adenoviruses persist in lymphoid tissue. There are nine predicted E3 pr~te&rs in 9ruup C adenoviruses, six of which have bean iti cells by immunopreci These are 6.7K (6700 gpl QK (also called El 9 &Void et al., 1984) 14.5K (Tollefson et al., 1998b) and Weld, 1988; Wang et&., 19 except perhaps 6.7K, are viruses. Group B adenovi E3 proteins, 20.1 K and 20.4K, that are not found in group C adenoviruses (Signas et al., 1986). 0042-682219
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FIG. 1. Schematic of the genome of human Ad2 The parallel lines indicate the linear duplex DNA genome of 36 kbp; rand /refer to rightward and leftward transcription, respectively. 289R 13.6K, Illa, etc. refer to proteins. El B-l 75R is also called El B-l 9K. The split arrows indicate the spliced structures of the mRNAs. El A, E3, etc. refer to transcription units. Most late genes are in the “major late transcription unit” which initiates at map position 16 of rstrand, and which includes the “ 1, ” “2.” and “3” leaders as well as the Ll , L2, L3, L4, and L5 families of mRNAs.
None of the E3 proteins is required for adenovirus replication in cultured cells or in acute infections of the lungs of hamsters (Morin et al., 1987) or cotton rats (Ginsberg eta/., 1989). Despite this, E3 is always maintained in natural isolates of adenoviruses (Adrian et a/., 1989a,b), indicating that these genes are important for natural infections of humans. Functions associated with the gpl9K, 10.4K, 14.5K, and 14.7K proteins are discussed below. At present, nothing is known about the functions of the 6.7K and 11.6K proteins. Many viable virus mutants have been constructed with mutations throughout E3 (e.g., see Gooding et a/., 1988; Carlin et a/., 1989); these mutants have been invaluable in characterizing the E3 proteins. Gpl9K prevents lysis of adenovirus-infected cytotoxic T-lymphocytes
cells by
Gpl9K of Ad2 is an abundant transmembrane glycoprotein of 142 amino acids (aa) following cleavage of the N-terminal signal sequence which directs gpl9K into the membrane of the endoplasmic reticulum (ER). Gpl9K has a 20 to 24 aa transmembrane domain followed by a 15 aa polar cytoplasmic domain at the Cterminus (Persson et al., 1980). There are two Asnlinked glycosylation sites in the N-terminal lumenal domain which are modified with exclusively high-man-
nose oligosaccharides (Kornfeld and Wold, 1981). The C-terminal 15 aa of gp 19K contain the signal for retention of gpl9K in the membrane of the ER (Paabo et al., 1987). The nature of this signal is controversial. One group claims that it is a linear sequence consisting of the C-terminal six or so residues (Nilsson et al., 1989) and containing a lysine at position -3 and another at positions -2, -4, or -5 (Jackson et a/., 1990). Another group suggests that it is a noncontiguous sequence with a complex spatial arrangement and that the lysines at positions -3 and -4 are of little importance (Gabathuler and Kvist, 1990). Binding of this signal to microtubules may be important for retention of gpl9K in the ER (Dahllof et al., 1991). The first clue to the function of gpl9K came when it was shown to bind to Class I antigens of the major histocompatibility complex (MHC) in Ad2-transformed rat cells (Kvist et al., 1978). A variety of subsequent studies showed that gpl9K binding to Class I antigens is an intrinsic property of the protein, is noncovalent, does not require oligosaccharides, does not require other adenovirus proteins, occurs when /3,-microglobulin is complexed with Class I antigens, and is observed with Class I antigens from human, rat, and mouse. Gpl9K from serotypes in adenovirus groups B, C, D, and E, but not in group A, binds Class I antigens (Paabo et al., 1986a).
ADENOVIRUS
REGION E3 AND IMMUNOSURVEILLANCE
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FIG. 2. Schematic illustration of region E3 of rec700. Arrows indicate the spliced structures of the mRNAs; the.thickneas of the arrow r&ecta the relative abundance of the mRNAs, and the dashed lines indicate introns. E3A and E3B are polyadenylabn sites. Bars above the arrows indicate proteins; hatched bars are proteins that have been identified. and stippled bars are proteins that are proposed2oF3xi~t. PluckWide +l is the transcription initiation site. rec700 is an Ad5-Ad2-Ad5 recombinant that has Ad2 sequences from nucleotide -236 to 2437 in the E3 transcription unit.
The net effect of gpl9K being retained in the ER and binding to Class I antigens is to block the transport of Class I antigens to the cell surface. This has been shown by FACS analysis and by the differential sensitivity of the oiigosaccharides on Class I antigens to cleavage with endo H (endo H cleaves high mannose but not complex oligosaccharides and thus can distinguish pre-Golgi from post-Golgi molecules) (Andersson et al., 1985; Burgert and Kvist, 1985). Cell surface expression of Class I antigens complexed with small viral peptide antigens is required for recognition and lysis of virus-infected cells by virus-specific CTL (reviewed in Townsend and Bodmer, 1989). If gpl9K blocks Class I antigens in the ER, then gpl9K should protect cells from lysis by CTL. Consistent with this, cells expressing gpl9K are less sensitive to lysis by alloreactive CTL (Andersson et a/., 1987; Burgert et a/., 1987). Also, co-infection with SV40 and adenovirus decreases sensitivity to anti-SV40 CTL (Tanaka and Tevethia, 1988), and infaction with a vaccinia virus vector expressing gpl9K decreases CTL killing (Cox et al., 1990). And
most important, gpl9K alone among E3 proteins prevents lysis of adenovirusMect& c&s by adenuvirusspecific CTL (Rawk et al., 1989). Thus, a masonable hypothesis is that gp19K functions in viGfo to protect adenouirus-infected cells against lysis by adenovlrusspecific CTL. Gpl9K has different affinities for different Class I antigens. Studies in the mouse system ~~i~g coimmunoprecipitation, sensitivity of Ct&ss i antigens to endo H, and prevention of ~yloiysks by CTL, imply that gpl9K interacts w&l with K”, L*, and Db, m&My with Dd, and not with Dk, Kk, or Kb C&ss 1 anti and Kvist, 1987; Tanaka and T@v&thia, 1 al., 1989; Cox et a/., 1990). Gpl Qk binds more strongly to HLA-A2 than to HLA-B7 human Cl&ss I antigens (Severinsson et al., 1986). These the possibility that suscaptW&y to tion in humans may be relisted to the ef#nity of an individual’s Class I antigens to gpl9K. The lumenal domain of gpt9K is st&oiit for binding to Class I antigens, and this binding does not re-
4
WOLD AND GOODING
quire p,-microglobulin (Paabo eta/., 1986b; Gabathuler et a/., 1990). The CX,and (Y* domains (the peptide antigen binding domains) in mouse Class I antigens are involved in binding to gpl9K (Burger-t and Kvist, 1987). It is conceivable that gp19K binding to the a, and (Ye domains could prevent peptide binding, which could in turn preclude transport of Class I antigens to the cell surface, because association with peptide is required for this transport (Townsend et al., 1989). If the above hypothesis for gp19K function is true, then one would predict that mutants lacking gpl9K would be more readily destroyed in vivo and hence would be less pathogenic. Surprisingly, however, deletion of the gpl9K gene resulted in markedly increased pathogenicity and a dramatic increase in the lymphocyte and macrophage inflammatory response with no effect on virus replication in the lungs of adenovirus-infected cotton rats (Ginsberg et al., 1989). Since this pathogenic@ occurred within 3 days, a time probably too short for adenovirus-specific CTL to form, it was suggested that gpl9K may directly or indirectly suppress cytokines which attract lymphocytes. This finding underscores our lack of understanding of the processes involved in viral pathogenesis. ElA proteins render cells susceptible to cytolysis by TNF and, depending on the cell type, the E3-14.7K, E3-10.4W14.5K, and El B-l 9K proteins protect the cells against cytolysis by TNF Although CTLare one means bywhich the hosteliminates virus-infected cells, tumor necrosis factor (TNF) may be another. TNF is a multifunctional cytokine that is secreted by activated macrophages and lymphocytes and that regulates a wide variety of aspects of the immune system and the inflammatory response (reviewed in Beutler and Cerami, 1989). A notable feature of TNF is that it is cytotoxic or cytostatic to certain tumor cells. The mechanism underlying cell lysis by TNF is largely unknown, but appears to occur in three steps: binding of TNF to its cell surface receptor followed by internalization and degradation of the receptor-ligand complex, intracellular signaling, and the lytic event itself, which is unknown. TNF also lyses cells infected with certain viruses, it inhibits the replication in cultured cells of a variety of DNA and RNA viruses, and it is released during infections by influenza virus (reviewed in Gooding et a/., 1991 a). Thus, TNF may be an antiviral agent in vivo. Gooding et al. (1988) showed that mouse CBHA fibroblasts are lysed by TNF when infected by adenovirus mutants that lack region E3. Uninfected cells are not lysed by TNF, nor are cells infected by wild-type adenovirus. This experiment shows two things: that
adenovirus infection induces susceptibility to TNF lysis and that a product(s) in region E3 prevents TNF cytolysis. It was subsequently shown, using adenovirus mutants that lack E3 as well as other early regions, that either the El A-289R or El A-243R protein is able to induce TNF susceptibility (Duerksen-Hughes et al., 1989). Stable El A-transfected cell lines also become spontaneously susceptible to cytolysis by TNF; this has been shown for mouse (Chen et a/., 1987; Duerksen-Hughes et al., 1989; Vanhaesebroeck et al., 1990) and rat (Kenyon et al., 1991) cell lines. The El A proteins are multifunctional proteins with a modular structure (Moran and Mathews, 1987). There are three regions in the El A proteins that are conserved among the adenovirus serotypes, these are conserved regions 1 (CRl), CR2, and CR3. Deletion of a portion of CR1 abrogates ElA induction of TNF susceptibility in adenovirus-infected cells (DuerksenHughes et a/., 1991). Functions associated with CR1 include immortalization of primary cells, cell transformation in cooperation with Ha-ras, induction of DNA synthesis, enhancer repression, and induction of TNFindependent cytotoxicity (see Gooding and Wold, 1990; White et a/., 1991). It will be of interest to determine which, if any, of these properties is linked to the ability of ElA to induce susceptibility to TNF. Whereas ElA induces susceptibility to TNF lysis in mouse C3HA cells, region E3 is required to prevent TNF cytolysis. By infecting C3HA cells with virus mutants having deletions throughout E3, it was determined that the E3-14.7K protein is required to prevent TNF lysis of C3HA cells (Gooding et al., 1988). Cells can be sensitized to cytolysis by TNF not only by expression of El A, but also by inhibition of protein synthesis or by treatment with cytochalasin E, an agent that disrupts microfilaments; 14.7K prevents TNF cytolysis in adenovirus-infected C3HA cells sensitized to TNF by these treatments (Gooding et a/., 1990). Some cell lines are spontaneously sensitive to TNF; 14.7K prevented TNF lysis in two of three of these cell lines infected by adenovirus mutants (Gooding et al., 1990). To date, 14 of 15 mouse cell lines infected with adenovirus mutants have been shown to be protected against TNF cytolysis by 14.7K (Gooding et a/., 1990, 1991 b). Thus, 14.7K appears to be a general inhibitor of TNF cytolysis. C3HA cells infected with representative adenovirus serotypes in groups A, B, D, and E are not killed by TNF when the cells are sensitized to TNF either by expression of ElA or by inhibition of protein synthesis; thus, this anti-TNF function seems to be a general property of adenoviruses in groups A through E (Horton et a/., 1990). These representative serotypes express a pro-
ADENOVIRUSREGION E3 AND IMMUNOSURVEILIANCE
tein serologically
related to 14.7K of group C adenovi-
ruses. StablytransfectedmouseC3HAandCl 27 cellsexpressing14.7Kfrom a bovinepapillomavirus vector (BPV)are protected from TNF cytolysis regardless of whether they are sensitized to TNF by infection with adenovirus mutants that lack 14.7K, by inhibition of protein synthesis, or by treatment with cytochalasin E (Horton et al., 1991). Thus, 14.7K can prevent TNF cytolysis in the absence of other adenovirus proteins. 14.7K is the only adenovirus protein that prevents TNF cytolysis of adenovirus-infected C3HA cells (Gooding et al., 1988). However, in most other mouse cell lines including C127, the E3-10.4K and E3-14.5K proteins, which appear to exist as a complex (Tollefson et al., 1991) can also prevent TNF cytolysis (Gooding et a/., 1991 b). Specifically, Cl 27 cells are not lysed by TNF when infected with mutants that express either 14.7K or 10.4W14.5K, but they are lysed when infected with mutants that lack 14.7K and either 10.4K or 14.5K (Gooding et al., 1991 b). 10.4W14.5K also suppress TNF killing when cells are sensitized to TNF by inhibition of protein synthesis. Of 15 mouse cell lines tested, 10.4KJ14.5K were found to suppress TNF killing in 1 1, including both transformed and normal cells. Thus, for most mouse cell lines, there are two independent functions in E3 that prevent TNF cytolysis, 14.7K and 10.4W14.5K. Human cells are even more complicated in their response to adenovirus infection and TNF. HEL-299 untransformed human embryo fibroblasts and ME-180 cervical carcinoma cells are not lysed by TNF when infected with adenovirus mutants that lack region E3 (Gooding eta/., 1991a). Using a series of mutants lacking E3 as well as another region of adenovirus, it was determined that the El B-l 9K protein (El B-l 75R in Fig. 1) protects human cells against TNF lysis (Gooding et al., 199 1 a). Identical results were obtained with a panel of six human cell lines of both tumor and normal tissue origin. In common with 14.7K and 10.4W14.5K, El B19K prevents TNF cytolysis when cells are sensitized to TNF either by expression of El A or by inhibition of protein synthesis. It is not clear why El B-l 9K prevents TNF lysis in human cells but not in mouse cells. If El B-19K prevents TNF lysis of human cells, do 14.7K and/or 10.4W14.5K also have this function? The answer to this is probably. HEL-299 and ME-180 human cells are not killed by TNF when infected with H2d/250, a mutant that lacks El B-l 9K but retains E3 (Gooding et a/., 199 1 a). Therefore, one or more E3 proteins can protect human cells against TNF lysis in the absence of ElB-19K. Human cells infected with H5dll 1 1 are killed by TNF; H5dllll lacks El B-l 9K, it lacks E3-10.4K, E3-14.5K, and E3-14.7K, but it retains
5
the other E3 genes. This suggests that 14.7K and/or
10.4W14.5K canpreventTNFcytolysisof humancells. If so,thenadenovirus hasthreeindependent functions that preventTNFcytolysisof humancells. Little is known about the mechanism of action of these TNF-protecting proteins. As judged by its predicted sequence (Tollefson and Weld, 1988) 14.7K is a hydrophilic protein without apular domains that normally are associated with integral membrane proteins. One possibility is that 14.7K might down-regulate the TNF receptor(s) and thereby preclude TNF signal transduction. However, 14.7K does not substantially alter the cell surface TNF receptor number or affinity in stably transfected C3HA cells (Horton et al., 1991). Although 14.7K could function at a downstream point in TNF signal transduction, it is not a general inhibitor of TNF signal transduction because TNFinduction of cell surface Class I MHC antigens in 14.7K transfectants is not inhibited (Horton et a/., 1991). 10.4K and 14.5K are both integral cytoptasmic membrane proteins (Tollefson et al., 199Ua,b; Hoffman et a/., 1990; unpublished results), Little else is known about these proteins except their role in down-regulating the epidermal growth factor receptor (EGF-R) (see below). E 1B- 19K is an interesting multifunctional protein (reviewed in Gooding et a/., 1990a). It is localized to the nuclear membrane, it binds to and disrupts the nuclear lamina and vimentin-containing intermediate fiiaments, it cooperates with E 1A in adenovirus transformation of rodent cells, and it prevents El A-induced DNA degradation and cytotoxicity. It will be of great interest to determine which, if any, of these properties are linked to the ability of El B-l 9K to prevent TNF cytolysis. Why should three “sets” of proteins have evolved in adenovirus to prevent TNF cytolysis, and why should the proteins be so different? Perhaps these proteins function in different tissues of the host or perhaps at different steps in the TNF cytolysis pathway(s). It is remarkable that of the 25-30 adenovirus early genes, 4 of them function in preventing TNF cytolysis. This supports the idea that TNF is an important antiviral defense of the host. Aside from the soluble form of the TNF receptor (see Nopha et al., 1990, and references therein) and manganese superoxide dismutase (Wang et al., 1989), these adenovirus proteins are the only known inhibitors of TNF cytoiysis. Thus, they should be useful tools to understand the mechanism of TNF cytolysis. The E3-10.4K and E3-14.5K pro@ins function in concert to down-reguk%e tb EGF receptor in adenovirus-infected celJs EGF-R is a member of the protein tyrosine kinase class of plasma membrane receptors (reviewed in
6
WOLD AND GOODING
Ullrich and Schlessinger, 1990). Binding of the polypeptide growth factor EGF to EGF-R stimulates the kinase activity of EGF-R, resulting in autophosphorylation of EGF-R on Tyr residues as well as Tyr phosphorylation of a variety of cellular proteins. The EGWEGF-R complex clusters in clathrin-coated pits which are internalized into endosomes and degraded in lysosomes. Stimulation of the kinase activity of EGF-R results in the activation of cellular metabolism and eventually in the induction of DNA synthesis and mitosis. Adenovirus infection mimics EGF in that EGF-R is internalized via endosomes and is degraded (Carlin et a/., 1989). Using a panel of virus mutants, it was shown that E3-10.4K and E3-14.5K are both required for this down-regulation of EGF-R (Carlin eta/., 1989; Tollefson et a/., 1991). 10.4K is efficiently coimmunoprecipitated with 14.5K, strongly suggesting that the proteins exist as a complex in viva and consistent with the genetic data indicating that they function in concert (Tollefson et al., 1991). Although both 10.4K and 14.5K are required to down-regulate EGF-R in adenovirus-infected cells, 10.4K alone expressed from a retrovirus vector is sufficient to down-regulate EGF-R in mouse HERc cells (cells stably transfected with human EGF-R) (Hoffman et a/., 1990). It is not known why adenovirus-infected cells differ in this respect from the 10.4K-retrovirus-infected cells. We know little about the mechanism of action of 10.4W14.5K. Both are cytoplasmic membrane proteins, and it seems likely that they localize to the plasma membrane where they interact with EGF-R. However, this remains to be demonstrated. There are now three classes of viral proteins that act on EGF-R, vaccinia virus growth factor (VGF), the E5 transforming protein of BPV, and 10.4W14.5K. VGF is a secreted protein, related to EGF, which appears to mimic EGF in stimulating EGF signal transduction and which is responsible for the cellular proliferative response to VGF (Buller eta/., 1988). Thus, VGF probably serves to activate the infected and neighboring cells so that they become efficient factories for virus replication. The BPV E5 protein may similarly activate the infected cell by reducing down-regulation of EGF-R (Martin et a/., 1989). By analogy with VGF and E5, 10.4W 14.5K probably activate the infected cell. In common with EGF, 10.4W14.5K would initially activate the protein kinase activity of EGF-R (which has not been tested), and this would be followed by internalization and degradation of EGF-R (which has been demonstrated). As noted above, 10.4W14.5K also function to protect most mouse cell lines examined against lysis by TNF. A key unanswered question is whether the ability
TABLE 1 FUNCTIONSOFADENOVIRUSPROTEINS Protein
Function in vitro
E3-gpl9K El A-289R and 243R
Prevents cytolysis by CTL. Induce cellular susceptibility to TNF cytolysis. Prevents cytolysis of mouse cells by TNF. Prevent cytolysis of mouse cells by TNF. Down-regulate EGF receptor. Prevents cytolysis of human but not mouse cells by TNF. E3-14.7K and/or E3-10.4K/14.5K also probably prevent cytolysis of human cells by TNF.
E3-14.7K E3-10.4iV14.5K ElB-19K
of 10.4W14.5K to act on EGF-R is related to their ability to prevent TNF cytolysis. Is region E3 a cassette of functionally
related genes?
Adenovirus transcription units tend to encode genes with related functions. Regions El A and El B encode proteins that function in gene regulation and cell transformation, E2 proteins function in DNA replication, and the genes in the major late transcription unit encode mainly virion structural proteins. Of the six known E3 proteins, four function in counteracting the immune system, gpl9K preventing cytolysis by CTL, and 14.7K and 10.4W14.5K preventing cytolysis by TNF (see Table 1). Thus, E3 may be a cassette of genes, at least in part, that helps the virus to evade immunosurveillance. 10.4W14.5K also act on EGF-R. Thus, another possible unifying theme for E3 proteins is that they interdict signal transduction, 14.7K and 10.4K/14.5K for TNF, and 10.4W14.5K for EGF. ACKNOWLEDGMENTS Research from the authors’ laboratories that is discussed in this report was supported by grants CA24710, A129492, CA49540, CA4821 9, and Al26035 from the National Institutes of Health.
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