Proc. Natl. Acad. Sci. USA Vol. 93, pp. 383-387, January 1996 Immunology

Inhibition of human immunodeficiency type 1 and virus infection by a dominant negative factor of the interferon regulatory factor family expressed in monocytic cells (antiviral activity/interferon-stimulated response element/transcription factor ICSBP/DNA-binding domain/monocyte) ANGELA M. THORNTON*t, R. MARK L. BULLERt, ANTHONY L. DEVICO§, I-MING WANG*, AND KEIKO OZATO*¶ *Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; tDepartment of Microbiology and Immunology, George Washington University, Washington, DC 20052; tDepartment of Molecular Microbiology and Immunology, St. Louis University, School of Medicine, St. Louis, MO 63104; and §Advanced BioScience Laboratories, Kensington, MD 20852 Communicated by Igor B. Dawid, National Institutes of Health, Bethesda, MD, September 18, 1995

ABSTRACT ICSBP is a member of the interferon (IFN) communication we show that infection with two , VV regulatory factor (IRF) family that regulates expression of and HIV, is strongly inhibited in cells that express the trun- type I interferon (IFN) and IFN-regulated genes. To study the cated ICSBP. The genomes of these viruses are found to have role of the IRF family in viral infection, a cDNA for the ISRE-like sequences that could bind IRF family . Our DNA-binding domain (DBD) of ICSBP was stably transfected results may illustrate a novel host-virus interaction in which into U937 human monocytic cells. Clones that expressed DBD infectivity of some viruses is critically controlled by IRF family exhibited a dominant negative phenotype and did not elicit proteins through the ISRE. antiviral activity against vesicular stomatitis virus (VSV) infection upon IFN treatment. Most notably, cells expressing MATERIALS AND METHODS DBD were refractory to infection by vaccinia virus (W) and human immunodeficiency virus type 1 (HIV-1). The inhibition Plasmids. A full-length ICSBP cDNA and the DBD cDNA of W infection was attributed to defective virion assembly, (13) were cloned into the expression vector pCXN. The and that ofHIV-1 to low CD4 expression and inhibition ofviral ISRE3LdLuc reporter was constructed by cloning three copies transcription in DBD clones. HIV-1 and W were found to have of the ISRE from the IFN-responsive GBP gene (nt -135 to sequences in their regulatory regions similar to the IFN- -112; ref. 14) fused to the 40-bp basal promoter of the Ld gene stimulated response element (ISRE) to which IRF family into the pGL2 luciferase plasmid (Promega). As a control, the proteins bind. Accordingly, these viral sequences and a cel- LdLuc reporter without ISRE was also used. lular ISRE bound a shared factor(s) expressed in U937 cells. Transfection. U937 human monocytic cells cultured in These observations suggest a novel host-virus relationship in RPMI.1640 supplemented with 10% fetal bovine serum were which the productive infection of some viruses is regulated by stably transfected with DBD or ICSBP cDNA or a control the IRF-dependent transcription pathway through the ISRE. pCXN2 without insert by electroporation with a Cell-Porator (Life Technologies, Gaithersburg, MD). Cells were selected in Transcription factors of the interferon regulatory factor (IRF) medium with G418 at 400 ,g/ml for 14 days and were cloned family (IRF-1, IRF-2, ISGF3y, and ICSBP) bind to the inter- by limiting dilution. Expression of DBD and ICSBP was feron-stimulated element and similar ele- monitored by immunoblot analysis with rabbit anti-ICSBP response (ISRE) antibody. For luciferase reporter assays 107 cells were cotrans- ments and regulate interferon (IFN) and IFN-responsive genes fected with 10 jig of control reporter (LdLuc) or ISRE3LdLuc (1-4) and thus are thought to be involved in the defense and 10 jig of pAct-1, which encodes IRF-1, or control without against viral infection. However, the role for the IRF family of insert. Cells were incubated for 20 hr until harvest. proteins in viral infection is likely to be complex, since antiviral Viral Infection. Vesicular stomatitis virus (VSV). Cells (2.5 x activities of IFN are diverse and vary among viruses (5). To add 105) were incubated with the Indiana strain of VSV at 10 to the complexity, some viruses have evolved mechanisms to plaque-forming units (pfu) per cell for 1 hr at 37°C, washed, and counteract host defenses, particularly against IFNs (6-9). For cultured in complete medium for the indicated times, and viral example, the double-stranded RNA (dsRNA)-dependent pro- yields from supematants were measured by plaque assay (14). tein kinase induced by IFNs is inhibited by a variety of viruses, When indicated, cells were pretreated with recombinant human including vaccinia virus (VV) and human immunodeficiency IFNct2A (Lee Biomolecular Laboratories, San Diego) at 500 virus (HIV). Although the complex host-virus interplay makes units/ml overnight and cultured with IFN after infection. it difficult to study, the role of IRF proteins in viral infection W. Cells (106) were infected with VV (WR strain) at 2 pfu has been addressed by several approaches (10, 11). Our per cell for 1 hr at 37°C and cultured for 48 hr. VV yield was approach has been to study the function of a truncated ICSBP determined by plaque assay on BS-C-1 cells (15). To study VV construct that retains the DNA-binding domain (DBD) but and DNA synthesis, 106 cells were infected with VV at lacks the regulatory domain. The truncated ICSBP expressed 20 pfu per cell and immunoblot analysis was performed with in monocytic cells was found to act as a dominant negative 30 of extract protein and rabbit anti-E3L antibody (1:500 factor for the IRF family. The dominant negative activity is jig because the truncated occu- elicited, presumably protein, by Abbreviations: DBD, DNA-binding domain; dsRNA, double-stranded pying the ISRE, interferes with the function of other IRF RNA; EMSA, electrophoretic mobility-shift assay; HIV, human im- family proteins. Dominant negative approaches with similar munodeficiency virus; IFN, interferon; IRF, IFN regulatory factor; reasoning have been successfully employed to elucidate activ- ISRE, IFN-stimulated response element; pfu, plaque-forming unit(s); ities of transcription factors and other proteins (12). In this TCID5o, 50% tissue culture infectious dose(s); VSV, vesicular stoma- titis virus; VV, vaccinia virus. STo whom reprint requests should be addressed at: Laboratory of The publication costs of this article were defrayed in part by page charge Molecular Growth Regulation, Building 6, Room 2A01, National payment. This article must therefore be hereby marked "advertisement" in Institute of Child Health and Human Development, National Insti- accordance with 18 U.S.C. §1734 solely to indicate this fact. tutes of Health, Bethesda, MD 20892-2753. 383 Downloaded by guest on October 1, 2021 384 Immunology: Thornton et al. Proc. Natl. Acad. Sci. USA 93 (1996) dilution) (7) or hybridoma supernatant containing rat monoclo- fected into U937 cells and clones expressing the DBD peptide nal anti-p37 antibody (1:2 dilution) (16). Bound antibody was were established. As controls, clones transfected with a vector detected with peroxidase-coupled anti-rabbit or anti-rat IgG and without insert and clones expressing the intact ICSBP were enhanced chemiluminescence (ECL; Amersham). Total DNA also established (A.M.T., B. Z. Levi, A. Dent, L. Staudt, J. E. from 105 VV-infected cells was transferred to Hybond N+ nylon Coligan, V. Ogryzko, and K.O., unpublished work). Three membrane (Amersham) and hybridized with a 32P-labeled KlL clones from each group were tested for ISRE-dependent probe (17). Electron microscopy (EM) analysis was performed promoter activity in a transient reporter assay. Promoter with cells infected with VY at 20 pfu/cell and incubated for 48 hr. activation was assessed by cotransfection of IRF-1, which acts Cells were fixed with 2.5% glutaraldehyde in Millonig's sodium as an activator (2). The activity of the ISRE reporter was phosphate buffer and processed for transmission EM by JFE increased by >20-fold in control clones upon cotransfection of Enterprises (Brookeville, MD). IRF-1 (Fig. 1B). However, reporter activation in DBD clones HIV. Cells (106) were infected with HIV-lI1B at 500 TCID50 was very low, <3-fold. Reporter activation by IRF-1 was also (50% tissue culture infectious doses) for 1 hr and cultured for lower in ICSBP clones than in control clones, as expected of 8 days. Supernatants were assessed for viral production by p24 the repressor (13), although not as low as that in DBD clones. antigen-capture ELISA (Organon Teknika). CD4-bound However, an unrelated retinoic acid-responsive reporter was gp120 was detected by flow cytometry analysis with monoclo- activated in DBD clones (data not shown). These data indicate nal antibody 8F101 (18). HIV-1 gag DNA was detected from that the DBD elicits a dominant negative activity and inhibits 2 x 106 cells infected with 2000 TCID50 of HIV-lIIIb for 1 hr ISRE-mediated transcription. Further supporting the domi- and incubated for indicated periods of time. Cellular DNA from nant negative property of the DBD, these clones failed to 2.5 x 105 cells was extracted at the indicated times and a 115-bp induce mRNA for several IFN-inducible genes upon IFN fragment of the gag gene was amplified by PCR using Perkin- treatment (A.M.T. et al., unpublished work). Elmer primers SK38 and SK39. The amplified fragment was IFN Fails to Elicit Antiviral Activity in VSV-Infected DBD detected by oligomer hybridization with the SK19 probe (19). To Clones. VSV infects many types of cells (25), and type I IFN detect gag transcripts, 105 HIV-infected cells as above were inhibits VSV infection in these cells (26). We examined resuspended in NASBA lysis buffer and sent to Advanced Bio- whether the antiviral activity of type I IFN is altered in DBD Science Laboratories for NASBA quantitation (20). clones. Three clones from each group were infected with VSV Electrophoretic Mobility-Shift Assay (EMSA). Samples (8 ,ug) with and without IFN and the viral titer was determined at ofwhole-cell or nuclear extracts prepared from U937 clones were various times following infection. In the absence of IFN, all analyzed by EMSA (14, 21) using the following probes: ISG15 clones were susceptible to VSV infection, yielding a 200- to ISRE (21) (5'-GATCCTCGGGAAAGGGAAACCGAAACT- 700-fold increase in viral titer by 16 hr postinfection (Fig. 1C). GAAGCC-3'), HIV DBF1 (22) (5'-AGGGACTTGAAAGC- The clones were then pretreated with IFNa prior to challenge GAAAGGGAAACCAGAG-3'; nt +195 to +224), CD4 (23) with VSV. Viral titers in IFN-treated control and ICSBP (5'-GACTTTGGCATTTTCACTTTGACATGTTCCC-3'; nt clones did not increase over 40 hr following infection, con- -135 to -105), VV F7L (24) 5'-AATGTGAACTTTTCAC- firming that IFN protects cells from VSV infection. In con- TTTCTTGCCGGTT-3', nt -99 to -71), and UCR, which was trast, VSV multiplied proficiently in IFN-treated DBD clones, used as a control (21). yielding a 3000-fold increase in viral titer after 24 hr. These results show that DBD clones fail to elicit antiviral activity against VSV in response to IFN. This failure was not due to a RESULTS loss of IFN receptors or a defect in signal transduction, since Dominant Negative Activity of DBD. The DNA-binding and IFN treatment led to the formation of ISGF3 in the DBD regulatory domains of the IRF proteins are distinct and clones as detected by EMSA (data not shown). Rather, the functionally separable; thus, a truncated ICSBP construct that failure of IFN to confer protection against VSV was most likely contains the conserved DBD (13) (Fig. 1A) was stably trans- due to the repression of IFN-inducible antiviral genes. Lack ofW Infection in DBD Clones. The infectivity of DBD

ICSBP 424 a.a. clones was examined for two other classes of viruses, VV and A I C 10B' -IFN HIV-1. The former, a DNA virus belonging to the poxvirus family, is relatively resistant to IFN (27, 28) and possesses two 106 io' O ICSBP proteins, encoded by the E3L and K3L genes, that interfere 110 a.a. D 104' with the activity of dsRNA-dependent protein kinase, an LL (Li 106103' *- < IFN-induced antiviral protein (28). Three clones from each B 25 1 .CCONT group were infected with VV and viral recovery was measured w *+IFN DBD 48 hr postinfection (Fig. 2A) (15). The viral titer increased by z 20 CONTROL >200-fold in both control and ICSBP clones; however, the titer S 15. 0 ICSBP increased <10-fold in DBD clones, indicating that expression 10. DBD i104' of DBD inhibits VV infection in these cells. Similarly, DBD 10' CONT clones were not readily infected by ectromelia virus, another 0. 0 10 20 30 40 poxvirus (data not shown), indicating that expression of DBD Ld LUC ISRE3Ld LUC HOUJRS POST INFECTION interferes with a critical step of viral growth that is shared among poxviruses. FIG. 1. (A) Schematic representation of ICSBP and DBD. The To determine the mechanism of the inhibition of VV N-terminal 110-aa sequence (hatched) represents the DBD. (B) infection in DBD clones, we examined the expression of E3L, Inhibition of ISRE promoter activation in DBD clones. U937 clones an early VV protein, and p37, a late VV protein, as well as viral were transiently transfected with 10 jig of luciferase reporter and 10 DNA synthesis. Immunoblot analysis (Fig. 2B) showed that jug of IRF-1 expression vector (or control vector). Fold activation both E3L and p37 were produced in clones of all groups, but represents the average of three experiments in which three clones from levels of these proteins were higher in DBD clones than in each group were independently tested. (C) Absence of IFN protection of the against VSV infection in DBD clones. Cells (2.5 x 105) of clones from other clones. Cytosine arabinoside inhibited production each group were infected with VSV at 10 pfu per cell and the viral titers late protein, but not the , as expected. DNA dot in supernatants were determined at the indicated times. Values blot analysis performed with a probe for the KlL gene (17) represent the average of three experiments in which three clones from showed that VV DNA replicated in DBD clones slightly more each group were independently tested. efficiently than in the clones of other groups (Fig. 2C). These Downloaded by guest on October 1, 2021 Immunology: Thornton et al. Proc. Natl. Acad. Sci. USA 93 (1996) 385 A 107 D a 106' LLU- g3 CONT 0- O ICSBP 0 105- -j w d DBD 104-

HOURS POST INFECTION

mock 3 hr 8 hr 8 hr + araC S o oa Z ocoZc

E3L (early) t ''

p37 (late)

II III IV- VI C 0 hr 10 hr = CONT # particles at each CONT 46 50 79 183 ICSBP stage per 21 cells DBD DBD 46* - 50*

FIG. 2. (A) Inhibition of VV infection in DBD clones. Cells (106) of clones from each group were infected with VV at 2 pfu per cell and the viral titers were determined 48 hr later. CONT, control. (B) Expression of VV proteins E3L and p37 assessed by immunoblot assays with cells infected with VV at 20 pfu per cell. Cells in the 8 hr + araC group were incubated in the presence of cytosine arabinoside (40 ,Lg/ml). (C) VV DNA synthesis was assessed by dot blot hybridization with the KIL probe (17). (D) EM analysis of VV virion assembly. (Upper) VV-infected control clone. Shown is a composite from several fields to show normal virions at various stages of assembly (I through IV). (Lower) VV-infected DBD clone. Note virions with abnormal morphology. Magnification was 20,000 fold. At the bottom, the number of virions at each stage in a total of 21 cells counted from each group. *, Abnormal morphology.

results show that the poor VV infectivity in DBD clones was supernatants were monitored by ELISA (Fig. 3A). Control and not due to the global inhibition of or DNA ICSBP clones produced similar levels of p24 antigen (400-800 synthesis. The viral assembly process in DBD clones was then pg/ml) by 8 days postinfection. The levels of p24 declined in examined by EM analysis. Virion assembly is known to follow control and ICSBP clones after day 14 (data not shown), as can six distinct stages (27, 29). The first structure seen after be the case for acute infection in U937 cells. In contrast, all infection is the crescent consisting of a lipoprotein bilayer (I), three DBD clones failed to produce detectable levels of p24 on which gives rise to a vesicle filled with granular matter (II). The day 8 (Fig. 3A) and even through 30 days postinfection (data vesicle then becomes an immature particle with a nucleoid core not shown). (III), followed by the formation of mature particle (IV). Virion HIV binds to CD4 receptors through an interaction with the morphogenesis continues further to produce an enveloped viral gp120 envelope protein (33, 34). Therefore, we tested the mature virion (stages V and VI not present in Fig. 2D). The ability of HIV gpl20 to bind to CD4 receptors expressed on these cytoplasm of the control clone (Fig. 2D Upper) harbored a clones. Flow cytometry analysis was performed with a newly heterogenous population of viral particles at stages I through developed monoclonal antibody specific for a gpl20 epitope that IV. In contrast, virion morphology in the DBD clone was is exposed upon binding to CD4 (18). Antibody binding was clearly abnormal, although the number of virions was compa- detected with DBD clones, but at much lower levels than with the rable to that of the control clone (Fig. 2D Lower). In DBD control clones (Fig. 3B). In agreement, levels of anti-CD4 mono- clones, the vesicles did not contain the granular matter char- clonal antibody (OKT4) binding to DBD clones were also much acteristic of a stage I particle; instead, they contained a dense less than those of control clones (data not shown). Thus, CD4 irregular mass. The DBD clone was completely devoid of the expression is strongly inhibited in DBD clones. The reduced CD4 well-developed immature particles and mature virions of stage expression could have led to a significant reduction in HIV entry IV (quantitation in Fig. 2D). Thus, VV assembly is severely into DBD clones; however, since some receptor binding was impaired in DBD clones. evident with DBD clones, HIV entry remained possible. There- Lack of HIV-1 Infection in DBD Clones. IFN plays a fore, we tested for the presence of a HIV-1 genomic sequence in complex, sometime paradoxical role during HIV infection the infected DBD clones. The HIV-1 gag gene sequence was (30). Although IFN inhibits HIV growth in vitro (31), the virus amplified semiquantitatively by PCR from cellular DNA of does have mechanisms to counteract IFN: HIV has been infected clones and then detected with a labeled oligonucleotide shown to inhibit IFNa gene expression in monocytes/ (19). Approximately 104 copies of the gag sequence were detected macrophages (32), and the HIV Tat protein and TAR RNA in 105 control cells 5 days after infection, whereas 3 x 102 gag gene inactivate the IFN-induced, dsRNA-dependent protein kinase copies were found in DBD clones on day 5 (Fig. 3C, lanes 3 and (8, 9). Clones from each group were incubated with HIV-lIIlb 4), indicating HIV-1 entry into DBD clones. The number of gene and the levels of HIV-1 gag-encoded p24 antigen in the copies in DBD subsequently declined (day 8, lanes 5 and 6). Downloaded by guest on October 1, 2021 386 Immunology: Thornton et al. Proc. Nati. Acad. Sci. USA 93 (1996)

A creased, in agreement with a decrease in p24 (Fig. 3A). In contrast, no transcripts were detected in DBD clones. Even 800 though the amount of HIV genome was 30-fold less in DBD clones, DBD clones would have given a measurable signal of 600 -4 x 104i, had the HIV gag gene been transcribed. These data coJ 400 indicate that both the CD4 expression and HIV transcription are inhibited in DBD clones. 200 ISRE-Like Sequences in the HIV and W Genes: Reduced Binding Activity in DBD Clones. The above data raised the 0 possibility that HIV and VV have ISRE (or ISRE-like) sequences in their genome and that their infectivity is, to some B 300 degree, governed by the IRF family proteins that bind to the CONTROL ISRE. In support of this idea, El Kharroubi and Verdin (22) 225 have shown that the HIV genome has a regulatory sequence gpl2O/CD4. in the R-U5 region, designated DBF1, that is similar to the cc w ISRE and is occupied in vivo. An ISRE-like sequence is also 75\ present in the upstream region of the VV F7L gene, although z 300 its function has not been understood (24). Furthermore, an -J ISRE-like sequence is present in the promoter region of the w 225 DBD 0 human and mouse CD4 genes (23), whose expression was ~~~gpl20/CD4 found to be reduced (Fig. 3B). To test whether these sequences 75 ; bind factors expressed in U937 clones, EMSA analysis was performed with HIV DBF1, VV F7L, and the CD4 sequence as probes for extracts from clones of each group. As a control, 1 10 100 LOG FLUORESCENCE a canonical ISRE from the ISG15 gene (21) was also tested. HIV DBF1 and CD4 probes generated a gel shift pattern very . j- similar to that of the control ISG15 probe (Fig. 4A). Experi- ments with specific antibodies indicated that these probes bind to IRF-2 and ICSBP expressed in U937 cells (A.M.T. et al., unpublished work). The intensity of some bands was increased C in ICSBP clones over control clones, due to increased levels of ICSBP/IRF-2 complexes; however, factor binding was greatly reduced in DBD clones. In addition, DBD clones gave an additional fast migrating band, most likely representing bind- 1 2 3 4 5 6 ing of DBD (Fig. 4A, star). Extracts from these clones gave

D ISG15 DBFl F7L CD4

cn

-4 1.5x

0 A U, 1.0x 106 CL) 0~ 1.5x 105 B. - C/)

0 8 15 22 DAYS L~~~~~~~ FIG. 3. (A) Inhibition of HIV-1 infection in DBD clones. Clones from each group were infected with HIV-IIIIB at 500 TCID5o and production of p24 antigen was measured by ELISA. Values represent the average of three experiments. (B) gpl2O-CD4 complexes on DBD clones. The complexes were detected with antibody 8F101 and fluo- rescein isothiocyanate-labeled anti-mouse F(ab')2 (18). Upper and Lower represent FACScan (Becton Dickinson) profiles of a control and DBD clone, respectively. (C) Presence of the HIV-1 gag sequence in DBD clones. The sequence was amplified by PCR and detected with oligomer hybridization (19). Lanes 1 and 2, uninfected cells; lanes 3 and 4, cells 5 days postinfection; lanes 5 and 6, cells 8 days postinfec- tion. (D) Absence of HIV-1 gag RNA in DBD clones. The number of FiG. 4. (A) EMSA analysis of viral ISRE-like sequences was gag transcripts in control and DBD clones was quantitated by NASBA performed with 32p-labeled ISG15 ISRE (21), HIV DBFI (22), or (20). CD4 promoter (23) with 8 ji~g of nuclear extract or whole-cell extract for VV F7L (24). CONT, control. (B) Competition assay. Extracts We then tested whether the HIV gag gene was transcribed from a control clone were incubated with a 100-fold molar excess of in these clones, using a single-tube quantitative RNA detection unlabeled oligomers for ISREs from the ISG15, ISG54, 6-16, and assay (NASBA; ref. 20) (Fig. 3D). About 1.4 x 106 copies of IFN,B (positive regulatory domain I, PRDI) genes or a control the HIV gag transcript were detected in 105 control clones 8 competitor, pIRE (14). EMSA was performed with 32P-labeled HIV days postinfection. The levels of transcripts subsequently de- DBF1 probe. Downloaded by guest on October 1, 2021 Immunology: Thornton et al. Proc. Natl. Acad. Sci. USA 93 (1996) 387 comparable binding activities for an unrelated probe, UCR proteins of the IRF family. It would be of interest to study (21), confirming the specificity of the altered binding (data not whether DBD expressed in other cell types also results in a shown). Competition experiments were then performed with similar inhibition of viral infection. ISREs from the ISG15, ISG54, 6-16, and IFN,B (positive regulatory domain I, PRDI) genes (14). All ISRE competitors We thank M. Merchlinsky, R. Pal, and members of the Ozato lab for tested eliminated bands produced by the HIV DBF1 probe their discussions and/or reagents. The studies were performed by (Fig. 4B) as well as those by the CD4 probe (data not shown), A.M.T. as part of the Ph.D. requirements in Microbiology and while formation of the labeled complex with the VV F7L probe Immunology of The George Washington University. was inhibited by only ISG54 and 6-16 (data not shown). 1. Miyamoto, M., Fujita, T., Kimura, Y., Maruyama, M., Harada, H., However, the non-ISRE competitor, the IFN-y activation Sudo, Y., Miyata, T. & Taniguchi, T. (1988) Cell 54, 903-913. site-like pIRE sequence (14) did not compete for binding, 2. Harada, H., Fujita, T., Miyamoto, M., Kimura, Y., Maruyama, M., verifying specificity of competition. These results show that Furia, A., Miyata, T. & Taniguchi, T. (1989) Cell 58, 729-739. HIV DBF1 CD4 bind factors that also bind to the 3. Driggers, P. H., Ennist, D. L., Gleason, S. L., Mak, W.-H., Marks, and probes M. S., Levi, B.-Z., Flanagan, J. R., Appella, E. & Ozato, K. (1990) canonical ISRE. Proc. Natl. Acad. Sci. USA 87, 3743-3787. 4. Veals, S. A., Schindler, C., Leonard, D., Fu, X.-Y., Aebersold, R., Darnell, J. E., Jr., & Levy, D. E. (1992) Mol. Cell. Biol. 12,3315-3324. DISCUSSION 5. De Meayer, E. & De Meayer-Guignard, J. (1988) Interferons and Other Regulatory Cytokines (Wiley, New York). The results demonstrate that U937 cells expressing DBD 6. Smith, G. L. (1994) Trends Microbiol. 2, 81-88. exhibit a classical dominant negative phenotype, as (i) IRF-1 7. Chang, H.-W., Watson, J. C. & Jacobs, B. L. (1992) Proc. Natl. Acad. Sci. USA 89, 4825-4829. stimulation of ISRE promoter activity was strongly inhibited 8. Gunnery, S., Rice, A. P., Robertson, H. D. & Mathews, M. B. (1990) and (ii) IFN did not elicit antiviral activity against VSV in DBD Proc. Natl. Acad. Sci. USA 87, 8687-8691. clones. In accordance, EMSA analysis showed that ISRE 9. Roy, S., Katze, M. G., Parkin, N. T., Edery, I., Hovanessian, A. G. & Sonenberg, N. (1990) Science 247, 1216-1219. binding activities were strongly reduced in DBD clones relative 10. Kimura, T., Nakayama, K., Penninger, J., Kitagawa, M., Harada, H., to those in control clones. It is of note that clones overex- Matsuyama, T., Tanaka, N., Kamijo, R., Vilcek, J., Mak, T. W. & pressing full-length ICSBP were protected from VSV infection Taniguchi, T. (1994) Science 264, 1921-1924. after even ICSBP functions as a 11. Pine, R. (1992) J. Virol. 66, 4470-4478. IFNa treatment, though 12. Espeseth, A. S., Darrow, A. L. & Linney, E. (1993) Mol. Cell. Differ. repressor (13). These data indicate that the action of DBD is 1, 111-161. distinct from that of a repressor IRF protein, underscoring a 13. Nelson, N., Marks, M. S., Driggers, P. H. & Ozato, K. (1993) Mol. Cell. merit of a dominant negative approach. Biol. 13, 588-599. 14. Bovolenta, C., Lou, J., Kanno, Y., Park, B.-K., Thornton, A. M., The most striking observation is that DBD clones were Coligan, J. E., Schubert, M. & Ozato, K. (1995) J. Virol. 69, 4173- nonpermissive to VV and HIV infection whereas control 4181. clones and those overexpressing full length ICSBP were per- 15. Karupia, G., Xie, Q., Buller, R. M. L., Nathan, C., Duarte, C. & MacMicking, J. D. (1993) Science 261, 1445-1448. missive for both VV and HIV. Although infection of the two 16. Schmelz, M., Sodeik, B., Ericsson, M., Wolffe, E. J., Shida, H., Hiller, viruses was blocked at different levels in DBD clones, EMSA G. & Griffiths, G. (1994) J. Virol. 68, 130-147. analysis (Fig. 4) demonstrated that both HIV and VV harbor 17. Chen, W., Drillien, R., Spehner, D. & Buller, R. M. L. (1993) Virology 196, 682-693. ISRE (or ISRE-like) sequences that bind factors specific for 18. DeVico, A. L., Rahman, R., Welch, J., Crowley, R., Lusso, P., the canonical ISRE. A simple interpretation of these results is Sarngadharan, M. G. & Pal, R. (1995) Virology 211, 583-588. that infection of some viruses is, at least in part, governed by 19. Kellogg, D. E. & Kwok, S. (1990) in PCR Protocols, eds. Innis, M. A., the IRF-dependent transcriptional pathway through the ISRE. Gelfand, D. H., Sninsky, J. J. & White, T. J. (Academic, San Diego), pp. 337-347. EMSA data on HIV DBF1 and the report that HIV DBF1 has 20. van Gemen, B., van Beuningen, R., Nabbe, A., van Strijp, D., a regulatory function (22) support direct involvement of the Jurriaans, S., Lens, P. & Kievits, T. (1994) J. Virol. Methods 49, IRF family proteins in HIV gene regulation. Recruitment of 157-168. 21. Bovolenta, C., Driggers, P. H., Marks, M. S., Medin, J. A., Politis, IRF family proteins for the regulation of viral gene expression A. D., Vogel, S. N., Levy, D. E., Sakaguchi, K., Appella, E., Coligan, could be advantageous for the virus, since IRF proteins are J. E. & Ozato, K. (1994) Proc. Natl. Acad. Sci. USA 91, 5046-5050. activated upon viral infection (1-4). In support of the shared 22. El Kharroubi, A. & Verdin, E. (1994)J. Biol. Chem. 269, 19916-19924. 23. Salmon, P., Giovane, A., Wasylyk, B. & Klatzmann, D. (1993) Proc. use of cellular transcription factors by a virus, HIV-1 tran- Natl. Acad. Sci. USA 90, 7739-7743. scription is known to be dependent on Sp-1, NF-KB, and TFIID 24. Roseman, N. A. & Slabaugh, M. B. (1990) Virology 178, 410-418. (35). The role of IRF family proteins in the VV F7L gene 25. Wagner, R. R. (1987) in The Rhabdoviruses, ed. Wagner, R. R. on is not (Plenum, New York), pp. 1-74. expression is, the other hand, less clear. The F7L gene 26. Marcus, P. I., Engelhardt, D. L., Hunt, J. M. & Sekellick, M. J. (1971) an essential gene for VV infection in various cell types, Science 174, 593-598. although it may have an important role in monocytic cells. The 27. Buller, R. M. L. & Palumbo, G. J. (1991) Microbiol. Rev. 55, 80-122. possibility that cellular transcription factors are involved in VV 28. Moss, B. (1989) in Virology, eds. Fields, B. N., Knipe, D. M., Chanock, R. M., Hirsch, M. S. & Melnick, J. (Raven, New York), pp. 2079- gene expression warrants further examination, since VV re- 2122. cruits components of the host cell nucleus, including RNA 29. de Harven, E. & Yohn, D. S. (1966) Cancer Res. 26, 995-1008. polymerase II, to develop a mature virus (36). 30. Pomerantz, R. J. & Hirsch, M. S. (1987) Interferon 9, 113-127. 31. Sperber, S. J., Gocke, D. J., Haberzettl, C., Kuk, R., Schwartz, B. & In addition to a direct action, DBD may indirectly affect Pestka, S. (1992) J. Interfer. Res. 12, 363-368. viral infectivity by altering expression of cellular genes whose 32. Gendelman, H. E., Friedman, R. M., Joe, S., Baca, L. M., Turpin, expression is regulated by IRF family proteins. The reduced J. A., Dveksler, G., Meltzer, M. S. & Dieffenbach, C. (1990) J. Exp. Med. 172, 1433-1442. expression of CD4 molecules in DBD clones may be such an 33. Dalgleish, A. C., Beverley, P. C. L., Clapham, P. R., Crawford, D. H., example, as the CD4 promoter has an ISRE-like sequence Greaves, M. F. & Weiss, R. A. (1984) Nature (London) 312, 763-766. which bound a factor(s) that also binds to the known ISREs. 34. Klatzmann, D., Champagne, E., Chamaret, S., Gruest, J., Guetard, D., In our observations to a Hercend, T., Gluckman, J.-C. & Montagnier, L. (1984) Nature summary, may begin clarify previ- (London) 312, 767. ously obscure aspect of the complex interactions between host 35. Cullen, B. R. (1991) Annu. Rev. Microbiol. 45, 219-250. and viruses which is mediated through the ISRE and the 36. Moyer, R. W. (1987) Virus Res. 8, 173-191. Downloaded by guest on October 1, 2021