Mouse Adenovirus Type 1 Replication in Vitro Is Resistant to Interferon

Virology 274, 213–219 (2000) doi:10.1006/viro.2000.0459, available online at http://www.idealibrary.com on

Mouse Adenovirus Type 1 Replication in Vitro Is Resistant to Interferon Adriana E. Kajon 1 and Katherine R. Spindler 2 Department of Genetics, University of Georgia, Athens, Georgia 30602 Received March 16, 2000; returned to author for revision April 18, 2000; accepted June 7, 2000 The effects of mouse interferon (IFN)-␣/␤ and recombinant IFN-␥ on mouse adenovirus type 1 (MAV-1) replication were investigated in single-cycle infectious virus yield reduction assays on mouse L929 cells. Viral yields at 3 days postinfection indicated that wt MAV-1 and pmE314, an early region 3 null mutant, were relatively insensitive to both IFN-␣/␤ and IFN-␥, whereas early region 1A (E1A) mutants pmE109 (null), dlE105 (conserved region 1 deletion, CR1⌬), dlE102 (CR2⌬), and dlE106 (CR3⌬) were sensitive. MAV-1 E1A that was inducibly expressed in mouse fibroblast 37.1 cells rescued vesicular stomatitis virus from the antiviral effect of IFN-␣/␤ but not from the antiviral effect of IFN-␥. Interferon-inducible gene expression was reduced in 37.1 cells as compared to the parental 3T6 cell line. Steady-state levels of IFN-inducible gene mRNAs were also reduced in 3T6 cells infected with the wild-type virus and pmE314 but not in cells infected with pmE109. These results suggest that the MAV-1 E1A gene product is capable of interfering with the signaling pathways of both types of IFN, although modulation of IFN-␣/␤ antiviral activity was more pronounced. © 2000 Academic Press

kinase R (PKR) (Chang et al., 1992), an IFN-inducible enzyme that plays a central role in the establishment of an antiviral state. In human adenoviruses, both the virusassociated (VA) RNA (reviewed in Mathews and Shenk, 1991) and E1A gene products (Anderson and Fennie, 1987) have been shown to antagonize the IFN response in infected cells. The VA RNAs inhibit activation of the IFN-induced PKR (Kitajewski et al., 1986; O’Malley et al., 1986). E1A has been shown to block IFN-␣ and IFN-␥ signaling by reducing the functional levels of both p48, a DNA-binding protein member of the IFN regulatory factor family, and the signal transducer and activator of transcription 1 (STAT1) (Ackrill et al., 1991; Gutch and Reich, 1991; Kalvakolanu et al., 1991; Leonard and Sen, 1996). Human adenovirus E1A thus interferes with the formation of the IFN-␣/␤-induced transcription complex, the trimeric interferon-stimulated gene factor 3 (ISGF3) composed of STAT1, STAT2, and p48. It also reduces the formation of the IFN-␥-induced gamma activating factor, GAF, consisting of a tyr-phosphorylated STAT1 homodimer. Despite many similarities between human adenoviruses and MAV-1, there are several significant differences that could have an impact on the response of MAV-1 to interferon. For example, the MAV-1 genome lacks a VA RNA gene (Meissner et al., 1997) and the E1A gene product is different from its human counterpart at both the sequence and the functional level (Ball et al., 1988, 1999; Ying et al., 1998). Therefore it was of interest to determine whether MAV-1 would be sensitive, or like human adenoviruses, be resistant to the antiviral effects of IFN. We examined the effects of IFN-␣/␤ and IFN-␥ on MAV-1 replication in vitro and assayed the steady-state

INTRODUCTION Interferons are cytokines with potent antiviral and immunomodulatory activities that play a primary physiological role in the host defense against viral infection (reviewed in Vilcˇek and Sen, 1996). This family of related proteins comprises two major types, I and II. The type I IFNs are induced by virus infection and include the ␣ (leukocyte), ␤ (fibroblast), and ␻ (trophoblast) interferons. The type II or immune interferon (␥) is unrelated to the type I IFNs and is induced by stimulation of T lymphocytes and natural killer cells. IFN-␣/␤ and IFN-␥ utilize different receptors to induce rapid activation of gene expression through the JAK-STAT signal transduction pathway following binding to cell surface receptors. The IFN-stimulated genes (ISGs) that mediate antiviral activity are regulated primarily at the level of transcription. Diverse mechanisms of resistance to IFN effects have been identified in different virus families. Replication of many DNA viruses is only marginally inhibited in cells treated with IFN. Certain large DNA viruses such as poxviruses and the Kaposi sarcoma-associated herpesvirus encode IFN-regulatory transcription factors (Moore et al., 1995) or IFN receptor analogs (Alcamı´ and Smith, 1995; Symons et al., 1995). The vaccinia virus E3L gene encodes an inhibitor of the dsRNA-dependent protein 1

Present address: Asthma and Pulmonary Immunology Group, Lovelace Respiratory Research Institute, P.O. Box 5890, Albuquerque, NM 87185. 2 To whom correspondence and reprint requests should be addressed at Department of Genetics, University of Georgia, Life Sciences Bldg., Athens, GA 30602-7223. Fax: (706) 542-3910. E-mail: [email protected]. 213

0042-6822/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1. Single-cycle infectious virus yield reduction assays on L929 cells. Cells were pretreated with 50 to 4000 IU/ml of IFN-␣/␤ or IFN-␥ as indicated for 24 h and then infected with wt MAV-1 or viral mutants pmE314 (E3 null), pmE109 (E1A null), dlE105 (E1A ⌬CR1), dlE102 (E1A ⌬CR2), and dlE106 (E1A ⌬CR3). Standard plaque assays were used to determine MAV-1 or VSV yields at 72 or 24 h postinfection, respectively. The percentage virus yield (compared to no IFN treatment) was calculated for each independent IFN treatment (3–6 replicates per virus strain). The mean and standard error of the log-transformed values of the virus yields are shown. The 100% yield values for wt and mutant MAV-1 viruses, when assayed in the same IFN treatment experiment, were within one order of magnitude. This is consistent with published findings that these MAV-1 E1A and E3 mutant viruses do not show growth differences from wt virus in cultured cells (Cauthen et al., 1999; Ying et al., 1998). The average of the log-transformed 100% yields of the wt virus (n ⫽ 7) was 5.66 ⫻ 10 6 PFU/ml.

levels of mRNA of two mouse genes stimulated by IFN. Our results showed that MAV-1 E1A was a major antagonist of the IFN response in infected cells and that this effect was at least in part mediated by an inhibition of IFN-inducible gene expression. RESULTS Effects of IFN-␣/␤ and rIFN-␥ on wt and mutant MAV-1 replication To determine whether MAV-1 is sensitive or resistant to the antiviral effects of IFN, we assayed the effects of IFN on viral replication. Single-cycle infectious virus yield reduction assays were carried out on L929 cells pretreated with mouse ␣ and ␤ fibroblast IFN (IFN-␣/␤) or recombinant mouse IFN-␥ (rIFN-␥) at concentrations of 50, 250, 1000, and 4000 IU/ml. Vesicular stomatitis virus (VSV), known for its relatively high sensitivity to IFN compared to adenovirus (Anderson and Fennie, 1987), was used as a positive control. Viral yields were determined by plaque assay at 3 days postinfection and indicated that wt MAV-1 and pmE314, an E3 null mutant (Cauthen et al., 1999), were relatively insensitive to both IFN-␣/␤ and IFN-␥ (Fig. 1). E1A mutants pmE109 (null), dlE105 (CR1⌬), dlE102 (CR2⌬), and dlE106 (CR3⌬) (Ying et al., 1998) were sensitive to both types of IFN, with infectious virus titers at 4000 IU/ml of IFN reduced by 2.5

to 3 log units relative to those obtained in untreated cells. A marked 4-log-unit reduction in the infectious VSV yields was observed at 4000 IU/ml of both IFN types. Yields of wt, E1A-, and E3-mutant viruses in the absence of IFN (100% values) were comparable, consistent with findings that MAV-1 E1A- and E3-mutant viruses are not defective for growth in fibroblasts in culture (Cauthen et al., 1999; Ying et al., 1998). Thus MAV-1 appears to be relatively resistant to IFN and to require E1A function for this resistance. Rescue of VSV replication in IFN-treated 37.1 cells The role of E1A in the modulation of IFN antiviral activity was further investigated by examining the effect of IFN pretreatment on VSV replication in 37.1 cells, which contain the MAV-1 E1A gene under the control of the mouse mammary tumor virus promoter (Smith et al., 1996). VSV replication in the 37.1 cells was compared to the control parental 3T6 cell line. MAV-1 E1A is produced constitutively at low levels in the 37.1 cells and at levels 10- to 100-fold higher after induction by dexamethasone (A. N. Cauthen and K. R. Spindler, unpublished). The induced level of E1A in 37.1 cells is comparable to that seen during the early phase of MAV-1 infection of 3T6 cells. As shown in Fig. 2, IFN-␣/␤-mediated inhibition of VSV replication was reduced in both untreated and dex-

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FIG. 2. Single cycle infectious VSV yield reduction assays on 37.1 and 3T6 cells. Cells were pretreated with 50 to 4000 IU/ml of IFN-␣/␤ or IFN-␥ as indicated for 24 h and then infected with VSV at an m.o.i. of 1. Virus yields at 24 h postinfection were determined by plaque assays. ⫹dex, dexamethasone was added to the growth medium at a concentration of 10 ⫺5 M; ⫺dex, dexamethasone was not present in the growth medium. The percentage virus yield (compared to no IFN treatment) was calculated for each independent IFN treatment (4–7 replicates per cell type). The mean and standard error of the log-transformed values of the virus yields are shown.

amethasone-treated 37.1 cells. This suggests that even the low level of E1A expressed in uninduced 37.1 cells is sufficient to rescue VSV replication from the antiviral effects of IFN-␣/␤. Furthermore, the data suggest that there is a threshold amount of E1A necessary for rescue of VSV, but higher levels of E1A (produced in the induced 37.1 cells) do not increase the rescue of VSV. In contrast to the IFN-␣/␤ results, VSV replication was not rescued from the antiviral effects of rIFN-␥ under any of the experimental conditions allowing for E1A expression. IFN-inducible gene expression in 37.1 and 3T6 cells The effect of E1A on gene expression in response to IFN-␣/␤ and rIFN-␥ was evaluated in 37.1 and 3T6 cells using mouse ISGs crg-2 and isg-56K as test genes. Crg-2 is a mouse homolog of human IP-10 and encodes a CXC chemokine that is strongly induced by both types of interferon (Smith and Herschman, 1996). Crg-2 has been recently shown to be differentially induced by MAV-1 infection in the central nervous system of susceptible and resistant strains of mice (Charles et al., 1999). Isg56K is strongly induced by IFN-␣/␤ in mouse cells (Smith and Herschman, 1996) and is a homolog of human ISG56K (p56). ISG-56K is the most abundant human protein induced by IFN but its function is unknown (Chebath et al., 1983; Guo et al., 2000; Kusari and Sen, 1986). Steadystate levels of ISG-56K are reduced by expression of the human adenovirus E1A product (Reich et al., 1988). To determine whether MAV-1 E1A also affects ISG mRNA

expression, we treated 37.1 cells with 1000 IU/ml of either IFN-␣/␤ or IFN-␥ and analyzed ISG mRNA levels by multiple Northern blots. As shown in a representative blot in Fig. 3A, a dramatic reduction of the steady-state levels of mouse isg-56K and crg-2 was observed in IFN-␣/␤-treated cells expressing E1A (37.1) compared to cells not expressing E1A (3T6) (compare lanes 5 and 6 with lanes 1 and 2). While the steady-state levels of the isg-56K message were markedly decreased in rIFN-␥treated cells expressing E1A (37.1) compared to non E1A-expressing 3T6 cells, levels of crg-2 were only partially reduced (Fig. 3B, lanes 5 and 6 vs lanes 1 and 2). ISG expression in wt or mutant MAV-1-infected 3T6 cells The effect of virus-encoded E1A expression on steadystate levels of ISGs was also studied in virus-infected 3T6 cells. Cells were infected with wt and mutant MAV-1 at an m.o.i. of 5 for 24 or 48 h prior to induction with 1000 IU/ml of IFN-␣/␤ or rIFN-␥. Total RNA was extracted 4 h postinduction and analyzed by Northern blot in multiple experiments. Changes in crg-2 expression in infected cells treated with IFN-␣/␤ or -␥ were variable from experiment to experiment and between 24 and 48 h time points within experiments. No differences were seen between wt and mutant virus infections with respect to crg-2 expression (data not shown). Under our experimental conditions, IFN-␥ did not induce changes in the steady-state levels of isg-56K (Fig. 4). However, we ob-

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served consistent and reproducible effects of MAV-1 on isg-56K in IFN-␣/␤-treated cells. A typical experiment is shown in Fig. 4A. A reduction in the steady-state levels of the IFN ␣/␤-induced isg-56K message was observed in cells infected with the wt virus and in cells infected with the E3 null mutant pmE314 for 24 and 48 h prior to induction (Figs. 4A and 4B, compare lanes 5 and 11 with lanes 2). In contrast, the levels of isg-56K in cells infected with the E1A null mutant pmE109 did not differ greatly from those in the uninfected controls (Figs. 4A and 4B, compare lanes 8 to lanes 2). Thus both human adenovirus and MAV-1 E1A products down-regulate expression of ISG-56K. DISCUSSION

FIG. 3. Northern blot analysis of interferon-inducible gene expression in 37.1 and non-E1A expressing 3T6 cells at 4 h postinduction with IFN-␣/␤ (A) or rIFN-␥ (B). Twenty micrograms of total RNA from 3T6 or 37.1 infected cells was loaded per lane. RNAs were fractionated in a 1.2% agarose-formaldehyde gel, transferred to a positively charged nylon membrane, and hybridized to garg-10, garg-16, and ␤-actin or 28S RNA probes as described under Materials and Methods. The blots were visualized and quantitated by phosphorimager. Graphs in the lower portion of the figure represent crg-2 or isg-56K band intensities normalized to ␤-actin. Lane numbers for the gels and quantitations are indicated at the bottom.

The VA RNAs and E1A are important for the ability of human adenoviruses to counteract the IFN response. One feature that distinguishes MAV-1 from the human adenoviruses is the absence of a VA RNA encoded by the viral genome (Meissner et al., 1997). In addition the MAV-1 E1A has structural and functional differences from human adenovirus DNA (Ball et al., 1988, 1999; Ying et al., 1998). For example, unlike human adenovirus E1As, MAV-1 E1A does not appear to increase steady-state levels of the viral early mRNAs during infection (Ying et al., 1998). Thus it was of interest to determine whether MAV-1 is sensitive to IFN. Our experiments showed that replication of wt MAV-1 and an E3 null mutant were only marginally inhibited by pretreatment of the host cells with IFN-␣/␤ or rIFN-␥. The extent of the inhibition of viral replication (about 10-fold) was similar to that observed with H5dl331, a human adenovirus mutant lacking VA RNA, relative to H5 wild-type virus (Kitajewski et al.,

FIG. 4. Interferon-inducible gene expression in 3T6 cells infected with wt and mutant MAV-1. Cells were infected with the indicated viruses or mock-infected for 24 h (A) or 48 h (B). They were then treated with IFN-␣/␤ (␣/␤) or rIFN-␥ (␥) or untreated (⫺). Total RNA was prepared from infected 3T6 cells and analyzed by Northern blot as described in Fig. 3.

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1986). In contrast, a dramatic drop in infectious virus yields in IFN-treated cells was observed with MAV-1 E1A mutants, suggesting a role for the MAV-1 E1A product in antagonizing the antiviral effects of IFN. E1A of subgenus C human adenoviruses inhibits the cellular response to IFN (Ackrill et al., 1991; Anderson and Fennie, 1987; Gutch and Reich, 1991; Kalvakolanu et al., 1991; Leonard and Sen, 1996; Reich et al., 1988). The inhibitory effect of E1A on IFN-inducible gene expression is dependent on sequences mapping to CR1 of the 289-aa protein (Ackrill et al., 1991). MAV-1 encodes a 200-aa E1A protein (Ball et al., 1988) that has sequence similarity with human adenovirus E1A in the three conserved regions of the protein, CR1, CR2, and CR3 (Moran and Mathews, 1987). The IFN resistance-conferring activity of MAV-1 E1A was lost upon deletion of the CR1, CR2, or CR3 domains (Fig. 1). All three E1A mutants tested, dlE105 (CR1⌬), dlE102 (CR2⌬), and dlE106 (CR3⌬), exhibited similar responses. These results suggest that in contrast with human adenovirus E1A, all three regions of MAV-1 E1A are required for biological activity in the resistance to IFN. This could be due to the contribution of all three regions to protein conformation or to stabilization of the complexes that MAV-1 E1A forms with cellular proteins. Our experiments also showed that MAV-1 E1A, in the absence of other MAV-1 proteins, rescued VSV from the antiviral effects of IFN-␣/␤ pretreatment. In 37.1 cells expressing E1A there was a substantial increase in the VSV yields after pretreatment with IFN-␣/␤ as compared to the parental 3T6 controls (Fig. 2). This coincided with a marked reduction in the IFN-␣/␤-induced levels of the messages for two mouse ISGs, isg-56K and the chemokine crg-2 (Fig. 3). Interestingly, the antiviral effects of rIFN-␥ on VSV replication were not abrogated by overexpression of E1A, although there was an observable decrease in the levels of IFN ␥-inducible expression of crg-2 and isg-56K (Figs. 2 and 3). This suggests that reduction in levels of these two ISGs is not reflective of the entire anti-VSV state induced by IFN-␥. IFN-␣/␤ and IFN-␥ differentially regulate gene expression, inducing mechanistically distinct antiviral states (Samuel and Knutson, 1983). Our observations are consistent with MAV-1 E1A expression having an effect only on the anti-VSV response induced by IFN-␣/␤. The fact that MAV-1 E1A provides a defense against both IFN types for MAV-1, but is only sufficient to rescue VSV from the effects of IFN-␣/␤, is likely related to inherent differences in the two viruses and their response to different antiviral states induced by the two IFN types. In addition, the effects of E1A on MAV-1 replication were measured in the context of the viral infection (Fig. 1) whereas the effects on rescue of VSV were measured with just E1A alone expressed (Fig. 2). Interference with the activation of transcription factors and disruption of signal transduction pathways is a strat-

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egy for counteracting the IFN response that is conserved among the small DNA tumor viruses. Barnard and McMillan (1999) showed that the human papillomavirus 16 E7 oncoprotein disrupts ISGF3 transcription complex formation via an interaction with p48. Polyoma virus large T antigen can inactivate signaling through IFN receptors by binding to JAK (Weihua et al., 1998). Human adenovirus E1A interferes with IFN-mediated STAT-dependent gene expression through a direct interaction with the carboxyl-terminal transactivation domain of the STAT1 protein itself (Look et al., 1998). Human adenovirus E1A also sequesters the transcriptional adaptors p300/CBP that interact specifically with STAT2 (Bhattacharya et al., 1996; Horvai et al., 1997; Zhang et al., 1996). In addition, human adenovirus E1A has been shown to reduce the functional levels of p48 (Leonard and Sen, 1996). Our data suggest that MAV-1 E1A counteracts the IFN response by reducing steady-state levels of at least some ISG mRNAs. The experiments presented here do not distinguish among transcriptional and nontranscriptional mechanisms for this reduction. For example, E1A could be affecting ISG mRNA stability. We have preliminary Western blot data suggesting that 37.1 cells, which express MAV-1 E1A, have reduced levels of STAT1␣ protein compared to the parental 3T6 cells (A. E. Kajon and K. R. Spindler, unpublished). Since STAT1 is a component of both ISGF3 and GAF transcription complexes required for IFN-␣/␤- and -␥-induced gene expression, a lower cellular abundance of STAT1␣ could impair the formation of both active transcription complexes, with a subsequent decrease in IFN-␣/␤- and IFN-␥-mediated transcriptional induction. The mechanism by which MAV-1 E1A decreases STAT1 levels has not been determined, nor have protein or phosphorylation levels of other components of the IFN signaling pathways been determined. E1A effects on these other components might also (or instead) be responsible for decreases in steady-state levels of ISGs. In preliminary experiments we have been unable to demonstrate an association of MAV-1 E1A and p300 (K. Smith and K. R. Spindler, unpublished). The ability of MAV-1 E1A to bind p48 or to interfere with expression of other ISGs has not been investigated. Although the possibility that other regions of the viral genome might be involved in resistance to IFNs cannot be eliminated, MAV-1 E1A appears to be a major modulator of the responses induced by these cytokines and possibly by other extracellular proteins that signal through the JAK-STAT pathway (Darnell et al., 1994). Despite the low homology between the MAV-1 E1A product and its human adenovirus counterparts, this very important function of E1A is clearly conserved. Like the human adenoviruses and viruses of the papovaviridae family, MAV-1 uses a strategy to inhibit expression of interferon stimulated genes to modulate the IFN response.

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MATERIALS AND METHODS Cells and virus. Mouse 3T6 and L929 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% heat-inactivated calf serum. 37.1 cells, obtained by transfection of 3T6 cells with a plasmid expressing MAV-1 E1A under the control of the MMTV promoter (Smith et al., 1996), were maintained in DMEM containing 5% heat-inactivated calf serum and 200 ␮g/ml G418. For virus propagation, IFN assays, and plaque assays, cells were passed once in the absence of G418. When required, 10 ⫺5 M dexamethasone was added to induce E1A expression. The E1A initiator methionine mutant pmE109 and deletion mutants dlE105 (E1A ⌬CR1), dlE102 (E1A ⌬CR2), and dlE106 (E1A ⌬CR3) (Ying et al., 1998) were propagated in monolayers of 37.1 cells. Mutant pmE314, which does not express any of the proteins encoded in E3, was propagated in monolayers of IE3.3 cells as previously described (Cauthen et al., 1999). VSV Indiana was plaque-purified twice and a stock was prepared in L929 cells. Interferon and infectious virus yield reduction assays. IFN-␣/␤ produced in mouse L929 cell cultures and rIFN-␥ were purchased from Sigma (St. Louis, MO) and handled as recommended by the supplier. Interferon activity against MAV-1 was quantitated by single-cycle infectious virus yield reduction assays. IFN at concentrations of 50, 250, 1000, and 4000 IU/ml was applied to monolayers of L929 cells for 24 h. Cells were infected with MAV-1 or VSV at an m.o.i. of 1 PFU/cell. Cells for assay of infectious viral yield were harvested 72 or 24 h postinfection for MAV-1 or VSV, respectively. Infected cells were frozen and thawed and clarified suspensions were serially diluted in PBS in 10-fold increments for plaque assays on monolayers of 3T6 cells or 37.1 cells (Cauthen and Spindler, 1999). RNA extraction and Northern blot analysis. Total cellular RNA was extracted by Nonidet P-40 lysis and phenol/chloroform extraction (Berk et al., 1979). Total RNA (20 ␮g) from each experimental sample was electrophoresed on a denaturing 1.2% agarose/formaldehyde gel. The RNA was then blotted to positively charged nylon membranes (Boehringer Mannheim) and crosslinked using an UV crosslinker (FB-UVXL-1000, Fisher Scientific). For random hexamer-labeled probes, blots were incubated in a solution containing 50% formamide, 5⫻ SSC, 50 mM NaPO 4, pH 7, 100 ␮g/ml denatured DNA, 1⫻ Denhardt’s solution, and 0.1% SDS for 2 h at 42°C. Fresh hybridization solution and probe were added and then incubated overnight at 42°C. Membranes were washed three times for 5 min at room temperature in 2⫻ SSC, 0.1% SDS, followed by two washes for 15 min each at 50°C in 0.1⫻ SSC, 0.2% SDS, and then exposed to a phosphorimager screen (Molecular Dynamics). For oligonucleotide probes, blots were incubated in a hybridization solution containing 6⫻ SSPE (0.9 M NaCl,

0.06 M NaH 2PO 4, 0.06 M EDTA, pH 7.4), 0.1% SDS, and 5⫻ Denhardt’s solution for 2 h at 37°C. Hybridization with the probe was subsequently carried out in 6⫻ SSPE, 0.1% SDS, 1⫻ Denhardt’s solution, and 200 ␮g/ml tRNA. Membranes were washed twice for 15 min at room temperature and twice for 15 min at 40°C in 6⫻ SSPE, 0.1% SDS, and then exposed as described above. For the study of ISG expression, plasmids garg-10 and garg-16, containing the cDNA sequences of mouse ISGs crg-2 and isg-56K, respectively (Smith and Herschman, 1996), were used to produce 32P-labeled probes by the random hexamer-primed method (Feinberg and Vogelstein, 1983). Detection of actin and 28S ribosomal RNA was carried out to control for sample loading efficiency. A plasmid containing full-length mouse ␤-actin cDNA (pSP6-␤-actin, Ambion, Inc.) was labeled by the random hexamer-primed method. A 28S rRNA-specific oligonucleotide (5⬘-AACGATCAGAGTAGTGGTATTTCACC-3⬘) was end-labeled with [␥- 32P]ATP using polynucleotide kinase. Relative amounts (normalized to actin or 28S rRNA) of each message were quantitated by phosphorimager analysis using ImageQuant software (Molecular Dynamics). ACKNOWLEDGMENTS The authors thank Elizabeth Howerth for the VSV stock, Harvey Herschman for the crg-2 and isg-56K probes (Garg-10 and Garg-16, respectively), Gwen Hirsch for technical assistance, and Mary Bedell and Nickie Cauthen for comments on the manuscript. We thank Daniel Promislow for statistical advice. A.E.K. is a recipient of a postdoctoral fellowship award from the National Multiple Sclerosis Society. This work was supported by NIH AI 23762 to K.R.S.

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