Adenovirus E3 14.7K protein functions in the absence of other adenovirus proteins to protect transfected cells from tumor necrosis factor cytolysis.

A 14,700-kDa protein (14.7K) encoded by the E3 region of adenovirus has been shown to protect adenovirus-infected mouse C3HA cells from lysis by tumor necrosis factor (TNF) (L. R. Gooding, L. W. Elmore, A. E. Tollefson, H. A. Brady, and W. S. M. Wold, Cell 53:341-346, 1988). These infected cells are sensitized to TNF by expression of the adenovirus E1A proteins (P. Duerksen-Hughes, W. S. M. Wold, and L. R. Gooding, J. Immunol. 143:4193-4200, 1989). In this study we show that 14.7K suppresses TNF cytolysis independently of adenovirus infection. Mouse C3HA and C127 cells were transfected with the 14.7K gene controlled by the mouse metallothionein promoter, and permanent 14.7K-expressing cell lines were tested for sensitivity to TNF cytolysis. Transfected cells which were sensitized to TNF either by inhibitors of protein synthesis, microfilament-destabilizing agents, or adenovirus infection were found to be resistant to TNF cytolysis. Two monoclonal antibodies were isolated and used to quantitate 14.7K in transfected and infected cells. Enzyme-linked immunosorbent assay (ELISA) analysis with these monoclonal antibodies and 14.7K immunoblots showed that 14.7K expression can be induced with cadmium in C3HA and C127 transfectants. The 14.7K induction correlated with a dose-dependent decrease in sensitivity to TNF cytotoxicity. The 14.7K protein does not substantially alter cell surface TNF receptor numbers or affinity on C3HA mouse fibroblasts, as determined by Scatchard analysis of 125I-TNF binding. The 14.7K protein also does not alter TNF signal transduction in general, because TNF induction of cell surface class I major histocompatibility complex molecules on 14.7K transfectants was unmodified. Our findings indicate that the adenovirus 14.7K protein functions as a specific inhibitor of TNF cytolysis in the absence of other adenovirus proteins and thus is a unique tool to study the mechanism of TNF cytotoxicity.     

The 10,400- and 14,500-dalton proteins encoded by region E3 of adenovirus form a complex and function together to down-regulate the epidermal growth factor receptor.

In adenovirus-infected cells, the epidermal growth factor receptor (EGF-R) is internalized from the cell surface via endosomes and is degraded, and the E3 10,400-dalton protein (10.4K protein) is required for this effect (C. R. Carlin, A. E. Tollefson, H. A. Brady, B. L. Hoffman, and W. S. M. Wold, Cell 57:135-144, 1989). We now report that both the E3 10.4K and E3 14.5K proteins are required for this down-regulation of EGF-R in adenovirus-infected cells. Down-regulation of cell surface EGF-R was demonstrated by results from several methods, namely the absence of EGF-R autophosphorylation in an immune complex kinase assay, the inability to iodinate EGF-R on the cell surface, the formation of endosomes containing EGF-R as detected by immunofluorescence, and the degradation of the metabolically [35S]Met-labeled fully processed 170K species of EGF-R. No effect on the initial synthesis of EGF-R was observed. This down-regulation was ascribed to the 10.4K and 14.5K proteins through the analysis of cells infected with rec700 (wild-type), dl748 (10.4K-, 14.5K+), or dl764 (10.4K+, 14.5K-) or coinfected with dl748 plus dl764. Further evidence that the 10.4K and 14.5K proteins function in concert was obtained by demonstrating that the 10.4K protein was coimmunoprecipitated with the 14.5K protein by using three different antisera to the 14.5K protein, strongly implying that the 10.4K and 14.5K proteins exist as a complex. Together, these results indicate that the 10.4K and 14.5K proteins function as a complex to stimulate endosome-mediated internalization and degradation of EGF-R in adenovirus-infected cells.     

The adenovirus E3-14.7K protein is a general inhibitor of tumor necrosis factor-mediated cytolysis

We have previously described a 14,700 m.w. protein (14.7K) encoded by the E3 region of adenovirus that prevents TNF-mediated cytolysis of adenovirus-infected C3HA mouse fibroblasts. In the studies described here we have extended our analysis of TNF cytolysis of C3HA cells and the circumstances under which 14.7K protects these cells from cytolysis. C3HA cells were killed by TNF in the presence of inhibitors of protein synthesis, in the presence of cytochalasin E (which disrupts the microfilaments), and when adenovirus E1A was expressed. As described for other cell types, pretreatment of C3HA cells with TNF prevented cytolysis by TNF plus cycloheximide or TNF plus cytochalasin E, indicating that TNF induces a response that protects against these treatments. Remarkably, when 14.7K was expressed in virus-infected cells, it also prevented TNF-induced lysis whether sensitivity to TNF was induced by inhibition of protein synthesis, disruption of the cytoskeleton by cytochalasin E, or expression of adenovirus E1A. The 14.7K protein also prevented TNF lysis of cells that are spontaneously sensitive to TNF lysis. Thus, 14.7K appears to be a general inhibitor of TNF cytolysis, and as such should be an important tool in unraveling the mechanism of TNF cytolysis. There was one exception; NCTC-929 cells were spontaneously sensitive to TNF lysis and that lysis was not affected by 14.7K even though the protein was made in large quantities and was metabolically stable in these cells. This suggests that there is heterogeneity among TNF-sensitive cell lines. The 14.7K protein was found in both the nuclear and cytosol fractions of TNF resistant as well as all spontaneously sensitive cells suggesting that 14.7K may have more than one site of action within the cell.     

A protein serologically and functionally related to the group C E3 14,700-kilodalton protein is found in multiple adenovirus serotypes.

A 14.7-kilodalton protein (14.7K protein) encoded by the E3 region of group C adenoviruses has been shown to protect virus-infected fibroblasts from lysis by tumor necrosis factor (TNF) (L.R. Gooding, L.W. Elmore, A.E. Tollefson, H.A. Brady, and W.S.M. Wold, Cell 53:341-346, 1988). In this study we show that adenoviruses of other groups are also protected from TNF-induced cytolysis. Representative serotypes of groups A, B, D, and E produce a protein analogous to the 14.7K protein found in human group C adenoviruses. Deletion of this protein in group C viruses permits virus infection to induce cellular susceptibility to TNF killing. As with group C adenoviruses, cells infected with wild-type adenoviruses of other serotypes are not killed by TNF and are protected from lysis induced by TNF plus cycloheximide. However, cells are susceptible to TNF-induced lysis when infected with adenovirus type 4 mutants from which the 14.7K gene has been deleted. Although all known adenovirus serotypes infect epithelial cells, adenoviruses cause several diseases with various degrees of pathogenesis. Our findings suggest that the 14.7K protein provides a function required for the in vivo cytotoxicity of many adenoviruses independent of the site of infection or degree of pathogenesis.     

An adenovirus mRNA which encodes a 14,700-Mr protein that maps to the last open reading frame of region E3 is expressed during infection.

The E3 regions of adenovirus types 2 and 5, respectively, are known to synthesize proteins of 19,000 Mr (19K) and 11.6K, but information regarding the identity and characterization of other potential E3 proteins encoded by the six remaining open reading frames (ORFs) is lacking. In this study, we show that the last ORF of region E3, which encodes a 14.7K protein, is expressed in adenovirus-infected cells. This information was largely derived from analysis of an E3 deletion mutant (H2dl801) in which an extensive deletion (1,939 base pairs) was found to eliminate all ORFs except for two proteins of 12.5K and 14.7K. The 14.7K protein was translated from RNA isolated from H2dl801-infected cells that had been hybridization selected to E3 DNA; hybridization-selected RNA from wild-type adenovirus type 5-infected cells translated both the 19K and the 14.7K proteins. Moreover, an antiserum directed against a bacterial 14.7K fusion protein (A. E. Tollefson and W. S. M. Wold, J. Virol. 62:33-39, 1988) immunoprecipitated the 14.7K translation product synthesized by wild-type and mutant H2dl801 adenovirus mRNAs.     

Adenovirus genes that modulate the sensitivity of virus-infected cells to lysis by TNF

TNF is a key inflammatory cytokine with antiviral properties. Human adenoviruses encode several intracellular proteins that mediate the effects of TNF. Expression of the adenovirus immediate early E1A proteins induces viral genes and a host of cellular genes, drives G₀ cells into S-phase, and induces apoptosis and susceptibility to TNF-induced apoptosis. The adenovirus E1B-19K protein inhibits both E1A- and TNF-induced apoptosis. The E3-14.7K protein and the E3-10.4K/14.5K complex of proteins inhibit TNF- but not E1A-induced apoptosis. The E3 14.7K and 10.4K/14.5K proteins inhibit TNF activation of cytosolic phospholipase A₂ (cPLA₂), which may explain how they inhibit TNF cytolysis. Since eicosinoids produced from arachidonic acid (the product of cPLA₂) are potent mediators of inflammation, the E3 proteins may block the inflammatory response to adenovirus infection. These adenovirus proteins should be novel tools to understand adenovirus pathogenesis, TNF signal transduction, and TNF cytolysis.     

The adenovirus E3 14.5-kilodalton protein, which is required for down-regulation of the epidermal growth factor receptor and prevention of tumor necrosis factor cytolysis, is an integral membrane protein oriented with its C terminus in the cytoplasm.

We previously reported that the adenovirus type 5 E3 14.5-kilodalton protein (14.5K) forms a complex with E3 10.4K and that both proteins are required to down-regulate the epidermal growth factor receptor in adenovirus-infected human cells. Both proteins are also required to prevent cytolysis by tumor necrosis factor of most mouse cell lines infected by adenovirus mutants that lack E3 14.7K. The E3 14.5K amino acid sequence suggests that 14.5K is an integral membrane protein with an N-terminal signal sequence for membrane insertion. Here we show that 14.5K was found exclusively in cytoplasmic membrane fractions. Radiochemical sequencing of 14.5K indicated that the N-terminal signal sequence is cleaved predominantly between Cys-18 and Ser-19. With a mutant that does not express 10.4K, cleavage occurs predominantly between Phe-17 and Cys-18, indicating that the presence or absence of 10.4K affects the signal cleavage site. 14.5K was extracted into the detergent phase with Triton X-114, it remained associated with membranes after extraction with Na2CO3 at pH 11.5, and it was partially protected by membranes from proteinase K digestion; these observations indicate that 14.5K is an integral membrane protein. Proteinase K digestion followed by immunoprecipitation with antipeptide antisera directed against the N or C terminus of mature 14.5K indicated that 14.5K is oriented in the membrane with its N terminus in the lumen and its C terminus in the cytoplasm. Thus, 14.5K is a type I bitopic membrane protein. Previous studies indicated that 10.4K is also an integral membrane protein oriented with its C terminus in the cytoplasm. Altogether, these findings suggest that cytoplasmic membranes are the site of action when 10.4K and 14.5K down-regulate the epidermal growth factor receptor and prevent tumor necrosis factor cytolysis.     

Mouse anti-adenovirus cytotoxic T lymphocytes. Inhibition of lysis by E3 gp19K but not E3 14.7K

Early region E3 of adenovirus (Ad) appears to encode proteins involved in the interaction of the virus with the host immune system. The E3 region 19-kDa glycoprotein (gp19K) binds to class I MHC Ag in the endoplasmic reticulum and inhibits their transport to the cell surface; it has been proposed that this protects virus infected cells from lysis by CTL. We have found that the E3 14.7-kDa protein (14.7K) inhibits lysis of infected cells by TNF, and here we show that it also protects cells from lysis by lymphotoxin, which has been implicated as a mediator of CTL lysis. We have developed a method for producing CTL specific for human Ad2 and Ad5 in mice, in order to test directly which of the genes in the E3 region protect infected cells from lysis by virus specific CTL. The presence of the E3 region inhibits both the induction of Ad-specific CTL in culture and the lysis of infected target cells by these CTL. The inhibition varies between different mouse strains, with almost complete inhibition in C57BL/10 (H-2b) mice, partial inhibition with BALB/c (H-2d) and little or no inhibition with C3H (H-2k); results were similar for Ad2 and Ad5. By using a panel of E3 deletion mutants, inhibition of target cell lysis by Ad5 specific CTL was mapped exclusively to the gp19K gene. The 14.7K gene had no effect on CTL lysis despite its ability to protect cells against lysis by lymphotoxin. gp19K was synthesized abundantly in mouse cells by mutants retaining the gp19K gene; some mutant forms of the protein were synthesized but were nonfunctional. These data support the hypothesis that gp19K can protect Ad infected cells against lysis by virus specific CTL.     

Characterization of mutants within the gene for the adenovirus E3 14.7-kilodalton protein which prevents cytolysis by tumor necrosis factor.

The 14,700-Da protein (14.7K protein) encoded by the E3 region of adenovirus has previously been shown to protect mouse cells from cytolysis by tumor necrosis factor (TNF). Delineating the sequences in the 14.7K protein that are required for this activity may provide insight into the mechanism of protection from TNF by 14.7K as well as the mechanism of TNF cytolysis. In the present study, we examined the ability of 14.7K mutants to protect cells from lysis by TNF. In-frame deletions as well as Cys-to-Ser mutations in the 14.7K gene were generated by site-directed mutagenesis and then built into the genome of a modified adenovirus type 5 (dl7001) that lacks all E3 genes. dl7001, which replicates to the same titers as does adenovirus type 5 in cultured cells, has the largest E3 deletion analyzed to date. 51Cr release was used to assay TNF cytolysis. Our results indicate that most mutations in the 14.7K gene result in a loss of function, suggesting that nearly the entire protein rather than a specific domain functions to prevent TNF cytolysis.     

Adenovirus proteins that regulate susceptibility to TNF also regulate the activity of PLA2

Expression of the adenovirus E1A protein induces susceptibility to TNF. To counteract this effect, the adenovirus encodes a number of gene products that preserve host cell resistance to TNF. The E1B-19K protein, the E3 14.7 K protein, and the E3 10.4/14.5 K dimer each function in different cell types to provide resistance to TNF. Expression of E1A also results in the activation of PLA₂ and the release of arachidonic acid from the dying cell. The E3 14.7 K protein can prevent the activation of PLA₂, suggesting that in addition to providing host cell resistance to TNF, this protein also functions in an anti-inflammatory manner.     

A 10,400-molecular-weight membrane protein is coded by region E3 of adenovirus.

Previous studies with adenovirus mutants have indicated that a 10,400-molecular-weight (10.4K) protein predicted to be coded by an open reading frame in region E3 of adenovirus functions to down regulate the epidermal growth factor receptor (C. R. Carlin, A. E. Tollefson, H. A. Brady, B. L. Hoffman, and W. S. M. Wold, Cell 57:135-144, 1989). We now demonstrate that the 10.4K protein is in fact synthesized in cells infected by group C adenoviruses. This was done by immunoprecipitation of 10.4K from cells infected by a variety of E3 mutants, using antisera against three different synthetic peptides corresponding to the predicted 10.4K sequence. The 10.4K protein was translated primarily from E3 mRNA f, as indicated by cell-free translation of mRNA purified by hybridization from cells infected with an RNA processing mutant that synthesizes predominantly mRNA f. The 10.4K protein was overproduced or underproduced in vivo, respectively, by mutants that overproduce or underproduce E3 mRNA f, also indicating that the 10.4K protein is translated primarily from mRNA f. The 10.4K protein migrated as two bands with apparent molecular weights of 16,000 and 11,000 (10 to 18% gradient gels); both bands contained 10.4K epitopes, as shown by Western blot (immunoblot). Only the 16K band was obtained by cell-free translation, suggesting that the 16K protein is the precursor to the 11K protein. The 10.4K protein is a membrane protein, as shown by cell fractionation experiments and as predicted from its sequence. The predicted 10.4K sequence as well as a putative N-terminal signal sequence and 30-residue transmembrane domain are conserved in adenovirus types 2 and 5 (group C) and in types 3, 7, and 35 (group B).     

The amino-terminal portion of CD1 of the adenovirus E1A proteins is required to induce susceptibility to tumor necrosis factor cytolysis in adenovirus-infected mouse cells.

Previous work by our laboratory and others has shown that mouse cells normally resistant to tumor necrosis factor can be made sensitive to the cytokine by the expression of adenovirus E1A. The E1A gene can be introduced by either infection or transfection, and either of the two major E1A proteins, 289R or 243R, can induce this sensitivity. The E1A proteins are multifunctional and modular, with specific domains associated with specific functions. Here, we report that the CD1 domain of E1A is required to induce susceptibility to tumor necrosis factor cytolysis in adenovirus-infected mouse C3HA fibroblasts. Amino acids C terminal to residue 60 and N terminal to residue 36 are not necessary for this function. This conclusion is based on 51Cr-release assays for cytolysis in cells infected with adenovirus mutants with deletions in various portions of E1A. These E1A mutants are all in an H5dl309 background and therefore they lack the tumor necrosis factor protection function provided by the 14.7-kilodalton (14.7K) protein encoded by region E3. Western blot (immunoblot) analysis indicated that most of the mutant E1A proteins were stable in infected C3HA cells, although with certain large deletions the E1A proteins were unstable. The region between residues 36 and 60 is included within but does not precisely correlate with domains in E1A that have been implicated in nuclear localization, enhancer repression, cellular immortalization, cell transformation in cooperation with ras, induction of cellular DNA synthesis and proliferation, induction of DNA degradation, and binding to the 300K protein and the 105K retinoblastoma protein.     

The adenovirus E3 10.4K and 14.5K proteins, which function to prevent cytolysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor, are localized in the plasma membrane.

The adenovirus type 2 and 5 E3 10,400- and 14,500-molecular-weight (10.4K and 14.5K) proteins are both required to protect some cell lines from lysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor. We have shown previously that both 10.4K and 14.5K are integral membrane proteins and that 14.5K is phosphorylated and O glycosylated. The 10.4K protein coimmunoprecipitates with 14.5K, indicating that the two proteins function as a complex. Here we show, using immunofluorescence and two different cell surface-labeling techniques, that both proteins are localized in the plasma membrane. In addition, we show that trafficking of each protein to the plasma membrane depends on concomitant expression of the other protein. Finally, neither protein could be immunoprecipitated from conditioned media, indicating that neither is secreted. Taken together, these results suggest that the plasma membrane is the site at which 10.4K and 14.5K function to inhibit cytolysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor.     

Inhibition of TRAIL-induced apoptosis and forced internalization of TRAIL receptor 1 by adenovirus proteins

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis through two receptors, TRAIL-R1 (also known as death receptor 4) and TRAIL-R2 (also known as death receptor 5), that are members of the TNF receptor superfamily of death domain-containing receptors. We show that human adenovirus type 5 encodes three proteins, named RID (previously named E3-10.4K/14.5K), E3-14.7K, and E1B-19K, that independently inhibit TRAIL-induced apoptosis of infected human cells. This conclusion was derived from studies using wild-type adenovirus, adenovirus replication-competent mutants that lack one or more of the RID, E3-14.7K, and E1B-19K genes, and adenovirus E1-minus replication-defective vectors that express all E3 genes, RID plus E3-14.7K only, RID only, or E3-14.7K only. RID inhibits TRAIL-induced apoptosis when cells are sensitized to TRAIL either by adenovirus infection or treatment with cycloheximide. RID induces the internalization of TRAIL-R1 from the cell surface, as shown by flow cytometry and indirect immunofluorescence for TRAIL-R1. TRAIL-R1 was internalized in distinct vesicles which are very likely to be endosomes and lysosomes. TRAIL-R1 is degraded, as indicated by the disappearance of the TRAIL-R1 immunofluorescence signal. Degradation was inhibited by bafilomycin A1, a drug that prevents acidification of vesicles and the sorting of receptors from late endosomes to lysosomes, implying that degradation occurs in lysosomes. RID was also shown previously to internalize and degrade another death domain receptor, Fas, and to prevent apoptosis through Fas and the TNF receptor. RID was shown previously to force the internalization and degradation of the epidermal growth factor receptor. E1B-19K was shown previously to block apoptosis through Fas, and both E1B-19K and E3-14.7K were found to prevent apoptosis through the TNF receptor. These findings suggest that the receptors for TRAIL, Fas ligand, and TNF play a role in limiting virus infections. The ability of adenovirus to inhibit killing through these receptors may prolong acute and persistent infections.     

The adenovirus E3-10.4K/14.5K complex mediates loss of cell surface Fas (CD95) and resistance to Fas-induced apoptosis.

Cytotoxic T cells use Fas (CD95), a member of the tumor necrosis factor (TNF) receptor superfamily, to eliminate virus-infected cells by activation of the apoptotic pathway for cell death. The adenovirus E3 region encodes several proteins that modify immune defenses, including TNF-dependent cell death, which may allow this virus to establish a persistent infection. Here we show that, as an early event during infection, the adenovirus E3-10.4K/14.5K complex selectively induces loss of Fas surface expression and blocks Fas-induced apoptosis of virus-infected cells. Loss of surface Fas occurs within the first 4 h postinfection and is not due to decreased production of Fas protein. The decrease in surface Fas is distinct from the 10.4K/14.5K-mediated loss of the epidermal growth factor receptor on the same cells, because intracellular stores of Fas are not affected. Further, 10.4K/14.5K, which was previously shown to protect against TNF cytolysis, does not induce a loss of TNF receptor, indicating that this complex mediates more than one function to block host defense mechanisms. These results suggest yet another mechanism by which adenovirus modulates host cytotoxic responses that may contribute to persistent infection by human adenoviruses.     

The adenovirus E3 14.7-kilodalton protein which inhibits cytolysis by tumor necrosis factor increases the virulence of vaccinia virus in a murine pneumonia model.

The 14.7-kilodalton protein (14.7K protein) encoded by the adenovirus (Ad) E3 region inhibits tumor necrosis factor alpha (TNF-alpha)-mediated lysis of cells in tissue culture experiments, but the relevance of this effect in vivo is incompletely understood. To examine the effect of the ability of the Ad 14.7K protein to block TNF lysis upon viral pathogenesis in a murine model, we cloned the 14.7K protein-encoding gene into vaccinia virus (VV), permitting its study in isolation from other Ad E3 immunomodulatory proteins. The gene for murine TNF-alpha was inserted into the same VV containing the 14.7K gene to ensure that each cell infected with the VV recombinant would express both the agonist (TNF) and its antagonist (14.7K). VV was utilized as the vector because it accommodates large and multiple inserts of foreign DNA with faithful, high-level expression of the protein products. In addition, infection of mice with VV induces disease with quantifiable morbidity, mortality, and virus replication. The results of intranasal infections of BALB/c mice with these VV recombinants indicate that the Ad 14.7K protein increases the virulence of VV carrying the TNF-alpha gene by reversing the attenuating effect of TNF-alpha on VV pathogenicity. This was demonstrated by increased mortality, pulmonary pathology, and viral titers in lung tissue following infection with VV coexpressing the 14.7K protein and TNF-alpha, compared with the control virus expressing TNF-alpha alone. These results suggest that the 14.7K protein, which is nonessential for Ad replication in tissue culture, is an immunoregulatory protein which functions in vivo to help counteract the antiviral effects of TNF-alpha.     

The adenovirus E3 RID complex protects some cultured human T and B lymphocytes from Fas-induced apoptosis

Human group C adenoviruses cause an acute infection in respiratory epithelia and establish a long-term or persistent infection, possibly in lymphocytes. The mechanism by which this persistence is maintained is unknown; however, it would require that persistently infected lymphocytes not be deleted. The adenovirus genome encodes proteins that prevent the immune system from eliminating the virus-infected cell, including the E3 receptor internalization and degradation (RID) complex. The RID complex prevents death of infected cells by blocking apoptosis initiated through death domain-containing receptors of the tumor necrosis factor receptor (TNFR) superfamily, including TNFR1 (L. R. Gooding, T. S. Ranheim, A. E. Tollefson, L. Aquino, P. Duerksen-Hughes, T. M. Horton, and W. S. Wold, J. Virol. 65:4114-4123, 1991), TNF-related apoptosis-inducing ligand receptors (TRAIL-R1 and -R2) (C. A. Benedict, P. S. Norris, T. I. Prigozy, J. L. Bodmer, J. A. Mahr, C. T. Garnett, F. Martinon, J. Tschopp, L. R. Gooding, and C. F. Ware, J. Biol. Chem. 276:3270-3278, 2001; A. E. Tollefson, K. Toth, K. Doronin, M. Kuppuswamy, O. A. Doronina, D. L. Lichtenstein, T. W. Hermiston, C. A. Smith, and W. S. Wold, J. Virol. 75:8875-8887, 2001), and Fas (J. Shisler, C. Yang, B. Walter, C. F. Ware, and L. R. Gooding, J. Virol. 71:8299-8306, 1997). Here, we test the ability of RID to protect human lymphocytes from apoptosis induced by ligation of Fas, a mechanism important for regulating lymphocyte populations. Using a retrovirus expressing RID to infect six human lymphocyte cell lines, we found that RID functions in the absence of other viral proteins to downregulate surface Fas on some, but not all, cell lines. Total cellular levels of Fas decrease as measured by Western blotting, and this loss of Fas correlates with protection from apoptosis induced by ligation of Fas in every cell line tested. Although in some cases, RID causes loss of only a fraction of surface Fas, the presence of RID completely blocks the immediate events downstream of Fas ligation (i.e., Fas-FADD association and caspase-8 cleavage) in susceptible cell lines. Nonetheless, the ability of RID to block Fas signaling is independent of the Fas signaling pathway used (type I or type II). Interestingly, among the four T-cell lines tested, RID caused loss of Fas in the two T-cell lines bearing a relatively immature phenotype, while having no activity in T cells with mature phenotypes. Collectively, these data suggest that RID functions to prevent apoptosis of some human lymphocytes by internalizing surface Fas receptors. It is possible that the expression of RID facilitates long-term infection by preventing Fas-mediated deletion of persistently infected lymphocytes.