Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Minireviews
    • JVI Classic Spotlights
    • Archive
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JVI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Journal of Virology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Minireviews
    • JVI Classic Spotlights
    • Archive
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JVI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Pathogenesis and Immunity

The Zinc Finger Antiviral Protein Acts Synergistically with an Interferon-Induced Factor for Maximal Activity against Alphaviruses

Margaret R. MacDonald, Erica S. Machlin, Owen R. Albin, David E. Levy
Margaret R. MacDonald
1Laboratory of Virology and Infectious Disease, The Rockefeller University, 1230 York Avenue, New York, New York 10065
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: macdonm@rockefeller.edu
Erica S. Machlin
1Laboratory of Virology and Infectious Disease, The Rockefeller University, 1230 York Avenue, New York, New York 10065
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Owen R. Albin
1Laboratory of Virology and Infectious Disease, The Rockefeller University, 1230 York Avenue, New York, New York 10065
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
David E. Levy
2Department of Pathology and New York University Cancer Institute, New York University School of Medicine, New York, New York 10016
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/JVI.00402-07
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

Type I interferons (IFNs) signal through specific receptors to mediate expression of genes, which together confer a cellular antiviral state. Overexpression of the zinc finger antiviral protein (ZAP) imparts a cellular antiviral state against Retroviridae, Togaviridae, and Filoviridae virus family members. Since ZAP expression is induced by IFN, we utilized Sindbis virus (SINV) to investigate the role of other IFN-induced factors in ZAP's inhibitory potential. Overexpressed ZAP did not inhibit virion production or SINV-induced cell death in BHK cells deficient in IFN production (and thus IFN signaling), suggesting a role for an IFN-induced factor in ZAP's activity. IFN pretreatment in the presence of ZAP resulted in greater inhibition than IFN alone. Using mouse embryo fibroblast (MEF) cells deficient in Stat1, we showed that signaling through the IFN receptor is necessary for IFN′s enhancement of ZAP activity. Unlike in BHK cells, however, overexpressed ZAP exhibited antiviral activity in the absence of IFN. In wild-type MEFs with an intact Stat1 gene, IFN pretreatment synergized with ZAP to generate a potent antiviral response. Despite failing to inhibit SINV virion production and virus-induced cell death in BHK cells, ZAP inhibited translation of the incoming viral RNA. IFN pretreatment synergized with ZAP to further block protein expression from the incoming viral genome. We further show that silencing of IFN-induced ZAP reduces IFN efficacy. Our findings demonstrate that ZAP can synergize with another IFN-induced factor(s) for maximal antiviral activity and that ZAP's intrinsic antiviral activity on virion production and cell survival can have cell-type-specific outcomes.

Alphaviruses are positive-sense RNA viruses in the family Togaviridae, whose members cause significant disease to livestock and humans (reviewed in reference 10). Cycling between mosquito vectors and vertebrate hosts, New World members of this genus are responsible for summertime epidemics of equine encephalitis. Human infection can also result in encephalitis for which there is currently no specific therapy. Similarly, summertime epidemics of polyarthritis, fever, and rash can occur upon human infection with Old World alphavirus members.

The alphaviruses share a common replication strategy (reviewed in references 14 and 28) that has been extensively studied in, among other viruses, Sindbis virus (SINV). After virus entry via receptor-mediated endocytosis, fusion of the virion membrane with the endosomal membrane occurs, releasing the nucleocapsid into the cytoplasm. After uncoating of the RNA, the 5′ two-thirds of the SINV genome of ∼11,700 nucleotides is translated to generate a polyprotein that is co- and posttranslationally processed to form the nonstructural proteins (nsPs), nsP1, nsP2, nsP3 as well as nsP4, the RNA-dependent RNA polymerase. Together with host-derived factors, the nsPs form a replicase complex, which produces new genome RNA through a negative-strand intermediate. A subgenomic RNA, also produced from the negative-strand intermediate and representing the 3′ one-third of the genome, is translated to generate the structural proteins, which include the capsid protein as well as two membrane glycoproteins. Progeny enveloped virions are generated after encapsidated genomic RNA buds through the plasma membrane.

Viral infection of cells initiates a cascade of events (reviewed in references 18 and 29) resulting in the interferon (IFN) regulatory factor 3 (IRF3)-dependent production of IFN-β and IFN-α4. Binding of these IFNs to type I IFN receptors on the cell surface initiates the JAK/STAT (Janus protein tyrosine kinase/signal transducers and activators of transcription) signaling cascade and results in the induction of IRF7 and a number of IFN-stimulated genes (ISGs) that confer an antiviral state upon the cell. IRF7 activation leads to expression of a family of related IFN-α species, which, when secreted, amplify the IFN response. Signaling through the type I IFN receptor and expression of the resulting ISGs confer an antiviral state upon neighboring cells.

Previously we demonstrated that expression of the rat zinc finger antiviral protein (ZAP) results in dramatic inhibition of multiple Alphavirus genus members and established, using SINV, that rat ZAP prevents translation of the incoming genomic RNA (2). Our studies indicate that ZAP can bind to viral RNA sequences, that the CCCH-type zinc finger motifs are important for ZAP-mediated inhibition, and that the presence of specific viral sequences in a reporter RNA results in reduced steady-state levels of the RNA in cells expressing ZAP (11). Recently, it was reported that ZAP recruits the exosome to mediate mRNA degradation (12). Rat ZAP, or the amino terminal 254 amino acids of ZAP fused to the zeocin resistance gene (NZAP-Zeo), exhibits antiviral function in cells of rat (Rat2, fibroblast) as well as human (TREx-293, kidney epithelial) origin (2; also unpublished data). Although not active against a number of viruses, including vesicular stomatitis virus (VSV), yellow fever virus, and herpes simplex virus (2), rat ZAP exhibits antiviral activity against diverse viruses including, in addition to alphaviruses, Moloney murine leukemia virus (MLV) (9) and Ebola virus (23). This suggests that it may have evolved to protect cells against specific viral pathogens. Previous studies demonstrated that endogenous ZAP expression is induced upon stimulation of murine dendritic (25) and human hepatic (20) cells with type I IFNs. Furthermore, infection of primary cells with SINV (25) or human cytomegalovirus (CMV) (5) results in up-regulation of ZAP expression, which is dependent on the type I IFN receptor or IRF3, respectively. Taken together, these suggest that ZAP is an ISG that mediates antiviral activity against viruses from divergent families.

Although a number of ISGs have been identified (6), the mechanisms by which the gene products mediate virus inhibition are poorly understood. ISG products likely exhibit cellular activities to which specific viruses display susceptibility. We hypothesized that maximal antiviral activity against SINV might require additional IFN-induced factors and would, therefore, require functional IFN signaling pathways. However, overexpression of rat ZAP is able to mediate significant antiviral activity in the absence of IFN treatment of cells (2). Recent studies have implicated constitutive, low-level IFN signaling in preparing cells for maximal antiviral responses (reviewed in reference 29). A low level of expression of ISGs might therefore complement the vector-expressed ZAP to mediate maximal antiviral activity. Here, we investigate rat ZAP's anti-SINV activity in cells defective in IFN production or signaling. Our studies suggest that ZAP inhibition of SINV replication occurs maximally in the presence of another IFN-induced factor(s) and that the effect of ZAP on preventing SINV genome translation renders cells less permissive to virion production in a cell-type-specific manner.

MATERIALS AND METHODS

Cell lines.BHK-21 and Rat2 cell lines transduced with vectors pBabe-HAZ or pBabe-NZAP-Zeo (9) and designated here Rat2/HA-Zeo and Rat2/NZAP-Zeo cells were maintained as previously described (2). The retroviral vector pBabe-HAZ (9) is an MLV-based vector expressing a hemagglutinin (HA)-tagged zeocin resistance gene product under the control of the Simian virus 40 early promoter. Vector pBabe-NZAP-Zeo expresses the amino terminal 254 amino acids of rat ZAP fused in frame with the zeocin resistance gene (9). Immortalized mouse embryo fibroblast (MEF) cells derived from C57BL/6 wild-type (wt) and Stat1−/− mice (21) were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). wt and Stat1−/− MEF cells stably transduced (see below) with pBabe-HAZ or pBabe-NZAP-Zeo (wt MEF/HA-Zeo, wt MEF/NZAP-Zeo, Stat1−/− MEF/HA-Zeo, and Stat1−/− MEF/NZAP-Zeo) were maintained in DMEM containing 10% FBS and 200 μg/ml zeocin. wt MEF/HA-Zeo cells transduced with short hairpin RNA (shRNA)-expressing defective lentiviruses (see below) were maintained in DMEM containing 10% FBS, 200 μg/ml zeocin, and 5 μg/ml blasticidin. BHK-21 cells stably transduced with pBabe-HAZ or pBabe-NZAP-Zeo (BHK/HA-Zeo and BHK/NZAP-Zeo) were maintained in minimal essential medium supplemented with 7.5% FBS and 200 μg/ml zeocin.

Production of VSV-G-pseudotyped retroviral particles and cell transductions.MLV particles pseudotyped with VSV protein G (VSV-G) were generated by cotransfection of 293T cells with pBabe-HAZ or pBabe-NZAP-Zeo DNA and DNAs encoding MLV Gag-Pol and VSV-G envelope proteins as described previously (9). Bulk transduced cell lines were obtained by infection of the cells with the pseudotyped viral particles and selection in the presence of 200 μg/ml zeocin. Human immunodeficiency virus particles pseudotyped with VSV-G were obtained by cotransfection of 293T cells with shRNA-expressing derivatives of pLenti-3′-U6-EC-EP7 (see “Silencing of endogenous murine ZAP,” below) and DNAs encoding human immunodeficiency virus Gag-Pol and VSV-G. Bulk transduced cell lines were obtained by infection with the pseudotyped viral particles and selection in the presence of 5 μg/ml blasticidin.

Silencing of endogenous murine ZAP.The pLenti-3′-U6-EC-EP7 plasmid (provided by Daniel Boden, Aaron Diamond AIDS Research Center, New York, NY) was engineered to express an irrelevant shRNA or shRNAs targeting murine ZAP. The shRNA expression vector pLenti-3′-U6-EC-EP7 is a derivative of vector pLenti6/V5-D-TOPO (Invitrogen) and was generated as follows (D. Boden, personal communication): briefly, the Topo binding sites and the CMV promoter of pLenti6/V5-D-TOPO were removed, and a BamHI/HpaI polylinker was introduced into the lentiviral 3′ long terminal repeat. These two restriction sites were used to insert an shRNA expression cassette containing the U6 polymerase III promoter preceded by the enhancer of the CMV immediate-early promoter. Two restriction sites (EcoRV and PstI) downstream of the U6 promoter allow for the introduction of short hairpin DNA sequences. Two repeated stretches of six thymidine residues following the PstI site serve as a polymerase III transcription terminator site. The pLenti-3′-U6-EC-EP7 defective lentiviral vector also expresses the blasticidin resistance gene. The murine ZAP mRNA sequence (NM_028864) was analyzed using a Dharmacon Custom small interfering RNA design tool (www.dharmacon.com ) to locate potential sites for targeting. Sense and antisense oligonucleotides (W. M. Kech Oligonucleotide Synthesis Facility, Yale University) containing inverted repeats of the 23-mer sites to be targeted were annealed and cloned into EcoRV- and PstI-digested pLenti-3′-U6-EC-EP7. The PmeI-NotI fragment of pcDNA4/TO/myc-mouseZAP995, which was provided by Guangxia Gao (Chinese Academy of Sciences, Beijing, China) and contains the murine ZAP cDNA, was cloned into the 3′ untranslated region of the Renilla luciferase gene in the psiCHECK-2 vector (Promega). Efficiency of silencing was assessed as recommended by the manufacturer by cotransfection of the murine ZAP-containing psiCHECK derivative into 293T cells with the various shRNA-expressing plasmids. The two plasmids showing the lowest normalized Renilla luciferase activity, as well as plasmid pLenti-3′-irrelevant-shRNA (obtained from Thomas von Hahn, Rockefeller University, New York, NY), were utilized to transduce wt MEF/HA-Zeo cells to generate constitutively silenced cell lines. Sequences of the sense and antisense oligonucleotides utilized for generation of the two silenced cell lines (wt MEF/HA-Zeo/shRNA-ZAP-1 and wt MEF/HA-Zeo/shRNA-ZAP-5, respectively) were as follows, with the target sequence shown in uppercase letters: ZAP-1 sense, 5′-atcAAGAGAAGAGGTCCAGAGTAAGTaggatcACTTACTCTGGACCTCTTCTCTTttttttctgca-3′; ZAP-1 antisense, 5′-gaaaaaaAAGAGAAGAGGTCCAGAGTAAGTgatcctACTTACTCTGGACCTCTTCTCTTgat-3′; ZAP-5 sense, 5′-atcCACGGCATTCCTGCATAGTAAATaggatcATTTACTATGCAGGAATGCCGTGttttttctgca-3′; and ZAP-5 antisense, 5′-gaaaaaaCACGGCATTCCTGCATAGTAAATgatcctATTTACTATGCAGGAATGCCGTGgat-3′. ZAP-1 and ZAP-5 target sequences start at nucleotides 2731 and 1731 of the murine ZAP mRNA, respectively. Sequences of the sense and antisense oligonucleotides utilized to generate pLenti-3′-irrelevant-shRNA were as follows (target sequences are shown in uppercase letters): sense, 5′-atcAATAGCGACTAAACACATCAATTaggatcAATTGATGTGTTTAGTCGCTATTttttttctgca-3′; and antisense, 5′-gaaaaaaAATAGCGACTAAACACATCAATTgatcctAATTGATGTGTTTAGTCGCTATTgat-3′. pLenti-3′-irrelevant-shRNA, which is not predicted to target any human or murine sequences, was used to generate the control cell line (wt MEF/HA-Zeo/shRNA-irrel).

Real-time quantitative RT-PCR.Total RNA was harvested from cells using an RNeasy Mini Kit (Qiagen) according to the manufacturer's directions. One microgram of total RNA was transcribed into cDNA using a SuperScript III First-Strand Synthesis System for reverse transcription-PCR (RT-PCR) (Invitrogen) according to the manufacturer's instructions, using random hexamers as primers. cDNAs diluted 1:20 were then amplified with a QuantiTect SYBR Green PCR kit (Qiagen) and detected with a LightCycler 480 (Roche). Enzyme activation occurred at 95° for 15 min, followed by 40 cycles of 94° for 15 s, 55° for 20 s, and 72° for 20 s. Primers for amplification of murine ZAP were obtained from Qiagen (QuantiTect primer assay, QT00107100). Samples were normalized based on the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA present, as determined by real-time PCR using Qiagen QuantiTect Primers (QT00309099). Relative levels were determined using samples from duplicate wells, each assayed in triplicate PCRs, using the comparative threshold cycle method (1a). The efficiency of ZAP and GAPDH amplification was tested and found to be approximately equal in the cDNA dilution range utilized in the assay.

IFN treatment.Universal type I IFN (PBL Biomedical Laboratories), a recombinant human IFN-αA and IFN-αD chimeric protein with activity for type I IFN receptors from multiple mammalian species, including hamster and rat, was diluted in medium appropriate for the cell type and utilized for all IFN-α treatments. Duration of treatment is indicated in the figure legends.

Viruses, infections, and titrations.Stocks of wt SINV (Toto1101), SINV expressing firefly luciferase as a fusion with nsP3 (Toto1101/Luc), a temperature-sensitive mutant SINV expressing luciferase (Toto1101/Luc:ts110), and VSV (San Juan strain) were prepared, and titers were determined on BHK-21 cells as described previously (2). Multiplicity of infection (MOI) was determined based on BHK-21-derived titers. Viral infections, growth curves, and virus titrations were performed as previously described (2). The temperature sensitivity property of the Toto1101/Luc:ts110 virus stock was verified by plaque assay titration on BHK/HA-Zeo cells; no plaques were detected at any dilution (including undiluted stock) at 40°C, while the stock titer, determined at 28°C was >108/ml.

Luciferase assays.Cell monolayers were washed with phosphate-buffered saline, and lysates were prepared and measured using 1× passive lysis buffer and a luciferase assay system (Promega) according to the manufacturer's recommendations. Luciferase activity was measured using a Berthold LB960 luminometer.

Antibody production.A bacterial expression plasmid (pGEX-NZAP) producing glutathione-S-transferase (GST) with the amino-terminal 254 residues of rat ZAP (NZAP) fused to the carboxy terminus was generated in plasmid pGEX-6P-2 (GE Healthcare) using standard techniques. Details of the construction and the sequence of the plasmid are available upon request. Bacterially expressed GST-NZAP was purified on a GSTrap FF (GE Healthcare) column according to the manufacturer's recommendations. For generation of NZAP without the GST tag, GST-NZAP was dialyzed overnight in the presence of PreSission Protease (GE Healthcare), and the GST and protease were removed by passage through the GSTrap FF column. GST-NZAP and NZAP were utilized as the antigen in an alternating schedule to immunize BALB/c mice, and monoclonal antibodies recognizing NZAP were generated by the Monoclonal Antibody Core Facility of Memorial Sloan Kettering Cancer Center using their standard protocols. Antibodies that react with both rat NZAP and full-length rat ZAP were purified from culture supernatants of clone 13B10.1 using a protein A Sepharose CL-4B (GE Healthcare) column.

Antibodies used.Anti-NZAP monoclonal antibody from clone 13B10.1 was utilized for immunofluorescence analysis at 0.5 μg/ml and for Western analysis at 0.26 μg/ml. Anti-β-actin mouse monoclonal antibody AC-15 (Sigma A5441) was utilized at a 1:5,000 dilution. AlexaFluor 594-conjugated goat anti-mouse immunoglobulin G (Molecular Probes) was utilized at a 1:1,000 dilution. Horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin G (Pierce) was utilized at a 1:10,000 dilution.

Immunofluorescence and image acquisition.Cell were fixed and analyzed by indirect immunofluorescence as previously described (4) using a Nikon Eclipse TE300 inverted microscope with a transmission electron-fluorescence microscopy epifluorescence attachment and mercury lamp power supply. Images were acquired with a 60× oil immersion objective using a Spot RT camera (model 2.2.1; Diagnostic Instruments) using the Spot software. Phase-contrast images were obtained using automatic exposure with either the Spot RT camera or with a Nikon Eclipse TS100 microscope equipped with a Nikon CoolPix 995 camera. Adobe Photoshop or the Spot software was utilized for brightness and contrast adjustment using identical changes to all images within an experiment.

Western blotting.Cells were lysed in 2× Laemmli sample buffer, and the proteins in the lysate were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to Hybond ECL (enhanced chemiluminescence) nitrocellulose membranes (GE Healthcare), and incubated with primary and secondary antibodies as previously described (19). Enhanced chemiluminescence detection was performed with SuperSignal West Pico chemiluminescent substrate (Pierce) according to the manufacturer's recommendation.

RESULTS

Rat ZAP fails to inhibit SINV production in cells defective in type I IFN production.BHK-21 cells (hamster kidney) are frequently utilized for the propagation of a variety of viruses, including SINV. The robust and often persistent growth of various viruses is likely due to a deficiency in IFN production by these cells (1, 3, 24, 27, 30), although the deficiency has not been fully characterized, nor has the molecular mechanism(s) of the defect been elucidated. We utilized these cells to test whether rat ZAP is able to mediate its inhibitory effects in cells with a deficiency in IFN production (and thus likely also with little to no IFN signaling). Using VSV-G-pseudotyped MLV vectors pBabe-HAZ and pBabe-NZAP-Zeo (9), we generated stably transduced BHK-21 cell populations expressing the zeocin resistance gene (BHK/HA-Zeo) or NZAP fused to the zeocin resistance gene (BHK/NZAP-Zeo) and examined the growth of SINV. Virus production at both low (Fig. 1A) and high (Fig. 1B) MOIs was found to be virtually indistinguishable in the two cell lines. SINV-mediated cell death was also similar, although a possible slight delay in cytopathic effect (CPE) was noted in the cells expressing NZAP (Fig. 1C).

FIG. 1.
  • Open in new tab
  • Download powerpoint
FIG. 1.

ZAP inhibition of SINV replication and virus-induced cell death in BHK cells. (A and B) BHK/HA-Zeo and BHK/NZAP-Zeo cells seeded at 7 × 105 cells per 35-mm dish the day prior were infected with SINV Toto1101 at low (A, 0.01) and high (B, 5) MOIs, and the amount of virus present in the medium was determined at various times after infection by titration on permissive BHK-21 cells. A separate, single well was utilized for each time point; symbols represent the mean of duplicate titrations. (C) Photographs of mock- or SINV-infected BHK/HA-Zeo and BHK/NZAP-Zeo monolayers (MOI of 5) at the indicated times after infection are shown.

Possible reasons for the failure of ZAP to inhibit SINV growth and virus-mediated CPE in BHK cells include a species incompatibility, the lack of a necessary factor(s), the presence of a factor(s) that interferes with ZAP's action, inadequate levels of ZAP expression from the transducing retroviral vector, or an altered subcellular distribution of ZAP in BHK cells. We felt a species incompatibility was unlikely, given the high similarity in ZAP's amino terminal sequences among different mammalian species (rat NZAP shares 93% and 80% amino acid identity and 94% and 89% similarity with murine and human NZAP, respectively). Additionally, since rat ZAP is able to function in human cells, we felt it was likely to function in the more closely related hamster cells. We tested expression of the NZAP-Zeo protein in the BHK/NZAP-Zeo cells by indirect immunofluorescence (Fig. 2) and Western blotting (see Fig. 5) and found that levels were similar to the level seen in the highly SINV-nonpermissive Rat2/NZAP-Zeo cells (2). Moreover, the diffusely cytoplasmic subcellular distribution (Fig. 2) was similar to that seen in Rat2/NZAP-Zeo cells (not shown).

FIG. 2.
  • Open in new tab
  • Download powerpoint
FIG. 2.

Immunofluorescence analysis of NZAP-Zeo expression in BHK/NZAP-Zeo cells. BHK/HA-Zeo and BHK/NZAP-Zeo cells were analyzed by indirect immunofluorescence for the presence of NZAP protein (red) using anti-NZAP monoclonal antibody 13B10.1 and AlexaFluor 594 goat anti-mouse secondary antibody as described in Materials and Methods. The two images shown were acquired with identical exposure conditions under a 60× oil immersion lens. Nuclei are shown in blue.

Pretreatment with type I IFN restores ZAP's inhibitory activity in BHK cells.If, due to defective IFN production (and thus also with reduced constitutive low-level IFN signaling), BHK cells lack an ISG factor necessary for ZAP's function, then treatment of cells with IFN might restore ZAP's inhibitory activity in these cells. We pretreated BHK/HA-Zeo and BHK/NZAP-Zeo cells with various doses of IFN-α and assessed the ability of the cells to support SINV growth (Fig. 3A). In BHK cells expressing vector alone, pretreatment with IFN-α had little effect on SINV growth, with only a mild (less than 1 log) reduction in virion production seen at the highest IFN dose tested (100 U/ml) for both low and high MOIs. In contrast, IFN-α pretreatment of BHK/NZAP-Zeo cells resulted in a dose-dependent reduction of SINV growth at both low and high MOIs. Virion production 24 h after infection at a low MOI was reduced by >2 or >4 logs after pretreatment with 10 and 100 U/ml IFN-α, respectively. Similarly, virion production 12 h after infection at a high MOI was reduced by >1 or >2 logs after pretreatment with 10 and 100 U/ml IFN-α, respectively. IFN-α-treated BHK/NZAP-Zeo cells were also protected from SINV-mediated cell death to a greater degree than BHK/HA-Zeo cells (Fig. 3B).

FIG. 3.
  • Open in new tab
  • Download powerpoint
FIG. 3.

Restoration of ZAP's inhibitory function in BHK cells by IFN-α pretreatment. (A) BHK/HA-Zeo and BHK/NZAP-Zeo cells seeded at 3 × 105 (low MOI) or 2.5 × 105 (high MOI) cells per 35-mm dish were allowed to adhere and were then incubated overnight (12 to 14 h) with medium containing the indicated concentration of IFN-α. After infection the next day with SINV Toto1101 at the indicated MOI, the cells were washed, and the medium containing the same IFN-α concentration was added back. The amount of virus present in the medium was determined at the indicated times after infection by titration in duplicate on permissive BHK-21 cells. Two separate wells were utilized for each time point. Symbols represent the mean log titers; error bars, often obscured by the symbol, indicate the SEM. (B) Photographs of the BHK/HA-Zeo and BHK/NZAP-Zeo monolayers from panel A, treated with the indicated concentrations of IFN-α and infected with SINV (MOI of 0.01) for 49 h are shown.

One possible explanation for the SINV inhibition seen in the NZAP-Zeo-expressing cells treated with IFN is that these cells are inherently more responsive to IFN. To test this, we examined the effects of IFN-α pretreatment of BHK/HA-Zeo and BHK/NZAP-Zeo cells on the growth of VSV, a highly IFN-sensitive virus, whose growth is unaffected by ZAP expression (2). The susceptibility of IFN-α-treated BHK/HA-Zeo and BHK/NZAP-Zeo cells to VSV-mediated CPE was essentially identical (Fig. 4). Over multiple experiments, the BHK/NZAP-Zeo responsiveness to IFN′s effects was always equal to or less than that of the BHK/HA-Zeo cells (not shown). Furthermore, IFN-α pretreatment led to similar reductions in VSV virion production in the two lines, with a slightly reduced effect of IFN-α on VSV growth in the BHK/NZAP-Zeo cells (Table 1). Therefore, the increased ability of IFN-α treatment to inhibit SINV replication in the BHK/NZAP-Zeo cells is not due to an inherent increase in IFN sensitivity.

FIG. 4.
  • Open in new tab
  • Download powerpoint
FIG. 4.

IFN-α sensitivity of BHK/HA-Zeo and BHK/NZAP-Zeo cells. BHK/HA-Zeo and BHK/NZAP-Zeo cells were seeded at 1 × 105 cells per well in a 96-well plate in the presence of twofold serial dilutions (500 U/ml to 1 U/ml) of IFN-α as indicated. The next day (28 h later) cells were infected as indicated with VSV (MOI of 0.025), and after 24 h the monolayers were stained with crystal violet as described in Materials and Methods.

View this table:
  • View inline
  • View popup
TABLE 1.

VSV growth in BHK/HA-Zeo and BHK/NZAP-Zeo cells treated with IFN-αa

Another possible explanation for enhanced SINV inhibition in the BHK/NZAP-Zeo cells is that IFN-α treatment could increase NZAP-Zeo expression from the transducing vector and thus potentially increase the inhibitory activity. To test this, we evaluated NZAP-Zeo levels after IFN-α treatment by Western blotting and found that levels were not altered and were similar to the level seen in Rat2/NZAP-Zeo cells (Fig. 5). Together with the VSV results above, this suggests that the ability of the IFN-α treatment to result in enhanced SINV inhibition in the BHK/NZAP-Zeo cells compared to control cells is due to the expression of NZAP-Zeo, which likely functions in concert with an IFN-induced factor(s).

FIG. 5.
  • Open in new tab
  • Download powerpoint
FIG. 5.

Western blot analysis of NZAP-Zeo levels in IFN-α-treated cells. The indicated cell lines were seeded at 7 × 105 cells per 35-mm dish and allowed to adhere. Cells were then treated overnight as indicated with 500 U/ml IFN-α before harvesting. Equal volumes of lysate were loaded onto 10% polyacrylamide gels and subjected to Western blot analysis using anti-NZAP monoclonal antibody 13B10.1 (top panel). A duplicate blot was probed with anti-β-actin antibodies as a loading control (bottom panel). Similar results were obtained in one other independent experiment.

Enhanced activity of ZAP requires signaling through the type I IFN receptor.If ZAP inhibitory activity requires the presence of an IFN-induced factor, then inhibition of IFN signaling should abrogate the ability of IFN-α to restore ZAP-mediated inhibition of SINV. To examine IFN-α effects on ZAP function in the absence of IFN signaling, we utilized immortalized MEFs generated from mice with a targeted disruption of the Stat1 gene (8) (Stat1−/− mice). Mice deficient in Stat1 have previously been shown to lack responsiveness to IFN-α/β and to be highly susceptible to viral pathogens (8, 22). Using immortalized MEFs from wt mice as well as Stat1−/− mice, we generated cell lines expressing HA-Zeo (wt MEF/HA-Zeo and Stat1−/− MEF/HA-Zeo, respectively) or expressing NZAP-Zeo (wt MEF/NZAP-Zeo and Stat1−/− MEF/NZAP-Zeo, respectively) to examine ZAP's ability to inhibit SINV in the presence or absence of IFN signaling.

As expected, due to the lack of IFN-α/β responsiveness, IFN pretreatment of Stat1−/− MEFs expressing either HA-Zeo or NZAP-Zeo had no effect on SINV growth (Fig. 6, top). Surprisingly, however, in contrast to the results obtained in BHK cells defective in IFN production, NZAP-Zeo expression resulted in a SINV growth inhibition despite the inability of these cells to respond to IFN. SINV titers were consistently ∼2 logs lower in the NZAP-Zeo-expressing cells for the first 24 h after infection. This suggests that in some cell types, ZAP is able to mediate SINV growth inhibition in the absence of additional IFN-induced factors.

FIG. 6.
  • Open in new tab
  • Download powerpoint
FIG. 6.

ZAP-mediated inhibition in Stat1−/− and wt MEFs. Stat1−/− or wt MEFs expressing HA-Zeo or NZAP-Zeo were seeded at 3 × 105 (Stat1−/−) or 1.2 × 105 (wt) cells per 35-mm dish and were allowed to adhere and then were incubated overnight (15 h) with normal medium (filled symbols) or medium containing 100 U/ml IFN-α (open symbols). The cells were then infected with SINV Toto1101 (MOI of 5), and medium (without IFN-α) was added back. Two separate wells were utilized for each condition, and the medium in each was harvested and replaced at each time point. The amount of virus present in each sample was determined by titration in duplicate on permissive BHK-21 cells, and the cumulative total for each time point is presented. Symbols represent the mean log titers of the duplicate dishes; error bars, often obscured by the symbol, indicate the SEM.

To test whether an IFN-induced factor(s) could further enhance ZAP's inhibitory activity in these cells, we examined the effect of IFN-α on SINV growth in wild-type (IFN-α/β responsive) MEFs expressing HA-Zeo or NZAP-Zeo (Fig. 6, bottom). The growth of SINV in the wt MEF/HA-Zeo cells, which peaked at ∼108 PFU/ml, was reduced compared to the >109 PFU/ml seen in the Stat1−/− MEF/HA-Zeo cells, presumably due to endogenous IFN production and signaling in the wt cells. As expected, pretreatment with IFN-α resulted in a reduction of SINV growth, with a ∼2 log reduction seen from 6 to 48 h. In the absence of exogenous IFN treatment, the expression of NZAP-Zeo resulted in a ∼2 log reduction of SINV growth from 6 to 48 h. IFN-α pretreatment of wt MEF/NZAP-Zeo cells resulted in enhanced SINV growth inhibition, with a ∼4 log reduction in titers after 12 h compared to that seen in the untreated wt MEF/HA-Zeo cells. Thus, ZAP and an IFN-induced factor(s) are able to mediate synergistic antiviral effects against SINV.

The synergistic antiviral effect of ZAP and IFN-α requires new gene transcription.To further support the idea that the effects of IFN-α on ZAP-mediated SINV inhibition are likely due to production of an ISG factor(s), we examined the effect of transcription inhibition (Fig. 7) on SINV inhibition in wt (IFN responsive) MEFs. Cells expressing HA-Zeo or NZAP-Zeo treated with medium containing vehicle alone (ethanol) showed similar results to the result seen previously (Fig. 6), with a 2-log reduction in viral titers mediated by NZAP expression. Treatment with the cellular transcription inhibitor actinomycin D (ActD) alone had minimal effect on ZAP's inhibitory effect. As expected, treatment of wt MEF/NZAP-Zeo cells with IFN-α resulted in synergistic inhibition of SINV growth (∼3 logs at 24 h compared to that seen in the wt MEF/HA-Zeo cells treated with vehicle alone). The synergistic effect of IFN-α upon ZAP-mediated SINV inhibition was completely abrogated when IFN-α treatment was performed in the presence of ActD. The reduced inhibition seen in IFN-α-treated wt MEF/NZAP-Zeo cells in this experiment (3 logs) compared with the data presented in Fig. 6 (4 logs) is likely due to the short duration of IFN treatment (2 versus 15 h), which was necessary due to toxic effects of ActD (not shown). In the absence of ActD, the effects of IFN-α were time dependent, with a maximal effect requiring 4 to 6 h of treatment in these cells (not shown). Thus, the synergistic inhibitory effect of IFN-α with ZAP on SINV takes time and is dependent on new cellular gene transcription, similar to the production of ISG factors after IFN-α stimulation.

FIG. 7.
  • Open in new tab
  • Download powerpoint
FIG. 7.

Effect of transcription inhibition on the ZAP-IFN synergy. wt MEFs expressing HA-Zeo or NZAP-Zeo were seeded at 1.5 × 105 cells per 35-mm dish the day prior to treatment for 2 h with medium containing vehicle alone or with medium containing 500 U/ml IFN-α in the presence or absence of 1 μg/ml ActD. The cells were then washed and infected with SINV Toto1101 (MOI of 5), and medium without additives was added back. Two separate wells were utilized for each condition, and the medium in each was harvested and replaced at each time point. The amount of virus present in each sample was determined by titration in duplicate on permissive BHK-21 cells, and the cumulative total for each time point is presented. Symbols represent the mean log titers of the duplicate dishes; error bars representing the SEM are obscured by the symbols.

IFN-independent ZAP inhibition of SINV replication has cell-type-specific outcomes on virus production.That ZAP mediates a 2-log SINV reduction in Stat1−/− MEFs incapable of responding to IFN (Fig. 6) suggests that ZAP has intrinsic antiviral activity which functions independently of the activity of other ISGs. In wt MEFs, the synergistic activities of ZAP and an ISG factor have profound effects on virus production. However, in BHK cells ZAP fails to mediate inhibition of SINV growth in the absence of IFN-α treatment. We wondered, therefore, whether the ISG-independent activity was unable to function in the BHK cellular environment or, alternatively, whether the activity was functional but insufficient to result in significant inhibition of virus production.

Previously, using Rat2 fibroblast cells, we found that overexpression of rat NZAP-Zeo dramatically inhibits SINV virion production (∼6 logs at 24 h after infection at a low MOI) and found that ZAP mediates its inhibition after entry and at or before translation of the incoming genomic RNA (2). The inhibitory effect did not require pretreatment with IFN. Here, we utilized SINV Toto1101/Luc, which expresses luciferase as an in-frame fusion with the nsP3 protein, and monitored luciferase activity after 2 h of infection in order to investigate ZAP's effect on early translation and replication events in MEF and BHK cells. In wt MEFs, either expression of NZAP-Zeo or IFN-α pretreatment (100 U/ml) resulted in a >99% reduction in early replication events, as measured by luciferase activity after 2 h of infection (data not shown). This is consistent with the ability of ZAP or IFN-α to significantly reduce virion production, as seen in Fig. 6. Since the luciferase values obtained in the MEFs with NZAP-Zeo expression or IFN-α pretreatment were at background levels, we were unable to assess for synergistic inhibition of the early viral translation and replication events.

Surprisingly, despite a failure to inhibit SINV production in the BHK cellular environment in the absence of IFN (Fig. 1 and 3), expression of NZAP-Zeo was able to significantly decrease early replication events (Fig. 8), even without IFN-α pretreatment. In nine experiments measuring luciferase activity after 2 to 3 h of infection, expression of NZAP-Zeo resulted in a 92% (standard error of the meant [SEM], 1%) reduction in early replication relative to BHK cells expressing HA-Zeo (data not shown). Pretreatment with IFN-α resulted in a dose-dependent decrease in early viral replication in BHK/NZAP-Zeo cells, with higher doses resulting in complete inhibition, similar to the inhibition seen in the highly nonpermissive Rat2/NZAP-Zeo cells, where inhibition is not dependent on exogenous IFN treatment (Fig. 8). While IFN treatment did not significantly alter the levels of early viral replication in Rat2 cells expressing HA-Zeo, a mild reduction was seen in the BHK/HA-Zeo cells. These findings suggest that, in the absence of additional ISGs, ZAP mediates an antiviral effect on early SINV replication events but that the magnitude of the inhibition in some cell types (BHK) is insufficient to alter virus production, while in other cell types (MEFs and Rat2) virion production is decreased. Amplification of the early inhibition, mediated by the synergistic effects of an ISG factor(s), increases the extent of inhibition such that virion production is reduced.

FIG. 8.
  • Open in new tab
  • Download powerpoint
FIG. 8.

Effect of IFN-α pretreatment on ZAP-mediated inhibition of early replication events. The indicated cells were seeded at 5 × 104 cells per well in 96-well plates in the presence of twofold serial dilutions (500 U/ml to 0.06 U/ml) of IFN-α as indicated. The next day (24 h later) cells were infected with SINV Toto1101/Luc (MOI of 5), after which medium was added back. The cells were lysed after 2 h, and firefly luciferase activity was determined. Symbols represent the mean value of triplicate wells; error bars represent the standard deviations. The dashed line represents the mean luciferase value obtained from uninfected cells. RLU, relative light units. Similar results were obtained in four additional independent experiments.

Like ZAP, the IFN-induced factor(s) targets an early step in the SINV life cycle.Since SINV Toto1101/Luc is replication competent, luciferase activity would be expected to increase upon translation of any newly replicated viral RNA. Therefore, reductions in luciferase activity with IFN pretreatment (Fig. 8) could be due to inhibition at any one of several steps of the virus life cycle. We previously determined that ZAP mediates its inhibitory step at or prior to translation of the incoming viral RNA (2). To test if the IFN-induced factor(s) was also affecting a similar step, we infected IFN-α-treated cells with a temperature-sensitive derivative of Toto1101/Luc, Toto1101/Luc:ts110 (2), and measured the luciferase activity under nonpermissive conditions where RNA replication is blocked (Fig. 9). An IFN-α dose-dependent reduction of SINV genome translation was apparent in BHK cells expressing NZAP-Zeo, suggesting that the IFN-induced factor(s) is also inhibiting early events in the SINV life cycle. Since RNA replication and subsequent virus life cycle steps are blocked at the nonpermissive temperature, these results demonstrate that the IFN-induced factor(s) and ZAP both act at or before translation of the incoming viral RNA genome. Additional IFN-induced blocks at later life cycle steps might contribute to the overall inhibition seen in ZAP-expressing cells infected with replication-competent SINV.

FIG. 9.
  • Open in new tab
  • Download powerpoint
FIG. 9.

The IFN-α-induced factor(s) blocks translation of incoming SINV RNA. The indicated cells were seeded at 5 × 104 cells per well in 96-well plates in the presence of twofold serial dilutions (500 U/ml to 0.06 U/ml) of IFN-α as indicated. The next day (∼17 h later) cells were infected at 40°C with SINV Toto1101/Luc:ts110 (MOI of 17), after which medium was added back. The cells were incubated at 40°C for 5 h, and firefly luciferase activity was determined. Symbols represent the mean value of triplicate wells; error bars represent the standard deviations. The dashed line represents the mean luciferase value obtained from uninfected cells. RLU, relative light units. Similar results were obtained in one other independent experiment.

Silencing of endogenous ZAP reduces the antiviral efficacy of IFN-α.While our studies utilizing overexpressed ZAP demonstrate anti-SINV activity, we wondered if ZAP induced by IFN-α treatment might contribute to the antiviral state of the cell. To examine ZAP's role in the effects of IFN-α, we generated derivatives of wt MEF/HA-Zeo cells that stably express ZAP-specific shRNAs (wt MEF/HA-Zeo/shRNA-ZAP-1 or -5) or an irrelevant (wt MEF/HA-Zeo/shRNA-irrel) shRNA. Treatment of wt MEF/HA-Zeo/shRNA-irrel cells with IFN-α (100 U/ml for 6 h) resulted in a 4.7-fold increase in ZAP RNA levels (range, 4.6 to 4.8 in duplicate samples), consistent with previous work showing that ZAP is an ISG (20, 25, 31). Treatment of cells expressing ZAP-specific shRNAs with various doses of IFN-α revealed a decreased IFN-mediated antiviral efficacy (Fig. 10). While treatment with a low dose (6.25 U/ml) of IFN-α reduced SINV Toto1101/Luc replication in cells expressing the irrelevant shRNA to less than 5% of that seen in untreated cells, significant replication (∼30% of that seen in untreated cells) was apparent in the two cell lines expressing ZAP-specific shRNAs. The level of ZAP RNA induced by treatment of cells expressing the irrelevant shRNA with 6.25 U/ml IFN-α was assessed by quantitative RT-PCR and showed a ∼1.7-fold induction (range, 1.67 to 1.76 in duplicate samples) compared to untreated cells. RT-PCR analysis confirmed that silencing of ZAP in the ZAP-1 and ZAP-5 shRNA-expressing cells was efficient. Compared to untreated wt MEF/HA-Zeo/shRNA-irrel cells, the levels of ZAP RNA in IFN-treated wt MEF/HA-Zeo/shRNA-ZAP-1 and MEF/HA-Zeo/shRNA-ZAP-5 cells were reduced, with relative levels of 0.8 (range, 0.71 to 0.84) and 0.2 (range, 0.22 to 0.23), respectively. Even at higher doses of IFN-α, the effect of ZAP silencing was apparent, with higher levels of viral replication occurring in the cells silenced for ZAP expression than in cells expressing the irrelevant shRNA after treatment with 12.5 or 25 U/ml IFN-α (Fig. 10). Treatment with 50 U/ml resulted in minimal virus replication in all three lines, likely due to the induction of multiple ISGs with antiviral activity. That silencing of endogenous ZAP reduces the anti-SINV efficacy of IFN treatment was also recently reported by others (31).

FIG. 10.
  • Open in new tab
  • Download powerpoint
FIG. 10.

Effect of ZAP silencing on antiviral activity of IFN. The indicated cells were seeded at 2.4 × 105 cells per well in 12-well plates the day prior to addition of twofold serial dilutions (50 U/ml to 6.25 U/ml) of IFN-α as indicated. After 6 h, cells were infected with SINV Toto1101/Luc (MOI of 10), after which medium (without IFN-α) was added back. The cells were lysed after 3 h, and firefly luciferase activity was determined. For each cell type, mean luciferase values obtained from mock-infected cells were subtracted from values of infected samples. The bars indicate the relative luciferase activity from IFN-treated cells as a percentage of the mean luciferase values obtained from infected cells without IFN pretreatment (set at 100%). Error bars represent the range of values from duplicate wells.

DISCUSSION

Cellular responses to viral infection include activation of signaling pathways, which ultimately lead to the production of type 1 IFNs. IFNs have long been known to confer cellular resistance to viral infection through the induction of antiviral proteins such as the RNA-dependent protein kinase, 2′,5′-oligoadenylate synthetase, RNase L, and the Mx family proteins (reviewed in reference 26). IFN treatment of cells, however, induces the expression of a large number of genes (6, 7) whose individual contributions to the cellular antiviral response are poorly understood. While for some proteins, such as human MxA, an intrinsic antiviral effect in the absence of IFN-induced ISG expression has been demonstrated (13), for most ISGs this has not been investigated. ISG20, ISG56, ISG15, ZAP, and viperin genes have been recently implicated in mediating IFN-α's anti-SINV effects (31). Elucidating whether individual factors exhibit intrinsic antiviral activity or require coexpressed factors is necessary for a full understanding of the IFN-mediated antiviral state. The present study underscores some of the complexities associated with the understanding of the IFN response and in deciphering antiviral mechanisms.

This study focused on the anti-alphaviral activity of ZAP, which, when overexpressed in rat and human fibroblast cell lines, potently inhibits SINV by blocking viral gene expression from the incoming genomic RNA. Similar to findings recently reported (31), we confirmed in this study that ZAP is induced by IFN-α treatment and contributes to the overall antiviral state of the cell (Fig. 10). Since ZAP is induced by IFN, we considered the possibility that the antiviral effect might require the activity of additional IFN-induced factors. Our findings clearly demonstrate that maximal ZAP antiviral activity against SINV occurs in the context of an intact IFN pathway. Overexpression of ZAP fails to inhibit SINV virion production and fails to protect from virus-induced cellular death in BHK cells defective in IFN production (Fig. 1); this failure is overcome by pretreatment of the cells with IFN (Fig. 3) and is not due to low levels of ZAP expression (Fig. 2 and 5). The synergism between IFN and ZAP is not due to a general enhancement of IFN action by ZAP (Fig. 4 and Table 1). Based on this one might conclude that ZAP requires coexpression of an additional IFN-induced factor(s) for its antiviral effect. Surprisingly, however, ZAP exhibits antiviral activity in MEFs derived from mice with a targeted disruption of the Stat1 gene and thus lacking Stat1-dependent IFN-induced ISG expression (Fig. 6). ISGs induced in a Stat1-independent manner, for example, through signaling mediated by Stat2 or Stat3, are also not likely required for ZAP's inhibitory effect; no additional antiviral effect was observed when the Stat1−/− MEFs were pretreated with IFN.

The finding of ZAP antiviral activity in some cell types (MEFs) but not apparently in others (BHK) could be due to differences in the intracellular environments of those cell types, for example, in the levels of constitutively expressed proteins. These might be host proteins typically thought of as antiviral (such as many of the ISGs) but also might be factors involved in ongoing cellular processes which the virus subverts or with which it interacts to mediate its life cycle. In addition, however, the amount of antiviral activity attributed to an antiviral factor could depend on the assay utilized to measure antiviral activity. Indeed, we surprisingly found that ZAP exhibits antiviral activity in BHK cells if early gene expression rather than virion production is assessed (Fig. 8 and 9). That this ∼1-log decrease in gene expression fails to impact the production of extracellular virus suggests that in BHK cells this step (translation of the incoming viral RNA) is not rate-limiting to SINV production. Pretreatment with IFN, however, results in increased inhibition of early gene expression to levels sufficient to impact virus production and cell survival (Fig. 3 and 8). Furthermore, through the use of a replication-defective SINV mutant, we showed that the IFN-induced factor is synergizing with ZAP's inhibition of this life cycle step (Fig. 9). Thus, the ultimate outcome of cellular infection with SINV, and likely other viruses, is affected by the constellation of host cellular proteins present, which influence not only cellular antiviral pathways but also the robustness of cellular processes required for each step of the virus life cycle.

In this study we determined that an IFN-induced factor or factors are able to synergize with ZAP to mediate cell-type-specific viral inhibition. While the factor(s) is able to limit a SINV life cycle step at or before that targeted by ZAP, the nature of the factor is unknown. Given that several attempts using a library screening approach have so far been unsuccessful at identifying the responsible ISG product (data not shown), it is possible that IFN treatment reduces or eliminates expression of a factor inhibitory to ZAP's activity. Alternatively, multiple genes might work in concert to synergize with ZAP. Recently, the IFN-stimulated gene ISG15 was demonstrated to exhibit antiviral effects against several viruses, including SINV (16, 17, 31). IFN treatment is known to induce protein ISGylation (reviewed in reference 15) where, similar to the ubiquitin system, ISG15 protein is conjugated to a variety of proteins. Since in addition to ISG15, IFN-induced activation and conjugation enzymes are necessary for the ISGylation process, multiple gene products would be necessary if IFN′s synergistic effect with ZAP involved ISGylation. Although ZAP has been identified as a possible target for ISGylation in one study (32), IFN treatment of BHK cells does not alter the mobility of ZAP in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 5), and anti-ISG15 Western blot analysis did not detect any bands specific to the IFN-treated BHK/NZAP-Zeo cells (not shown). Whether ISGylation of ZAP or other cellular proteins plays a role in IFN′s enhancement of ZAP antiviral activity requires further study.

Our study utilized SINV to investigate the role of additional ISGs in ZAP's inhibitory effect. Since members of the Alphavirus genus share common genomic organization and replication strategies, it is possible that ISGs important for maximal ZAP-mediated anti-SINV activity will also play a role in ZAP's inhibition of other members of this genus. Although not active against all viruses (2), ZAP also exhibits inhibitory activity towards MLV (9) and both Marburg and Ebola viruses (23). Since these viruses have distinct genomic organizations and replication strategies, it is likely that the cellular requirements for the maximal ZAP-mediated inhibitory effect will be unique to each virus. In fact, we found no evidence for any synergistic effect of IFN pretreatment of BHK cells on ZAP's inhibitory effect against a luciferase-expressing MLV (not shown). This suggests that ZAP's inhibitory mechanisms may be both cell type and virus specific.

ACKNOWLEDGMENTS

We thank Daniel Boden for providing plasmid pLenti-3′-U6-EC-EP7, Thomas von Hahn for pLenti-3′-irrelevant-shRNA and helpful advice, and Steve Goff and Guangxia Gao for sharing numerous ZAP-related reagents. We thank Matthew Bick for technical assistance early on in the project, John-William Carroll and Frances Weiss-Garcia for assistance in the generation of anti-NZAP monoclonal antibodies, and Lindsey Sperzel for assistance in retroviral packaging. We thank Randy Longman and Charlie Rice for critical reading of the manuscript and helpful discussions.

This work was supported by Public Health Service Grant AI-057905 from the National Institute of Allergy and Infectious Diseases (M.R.M.) and by the Greenberg Medical Research Institute.

FOOTNOTES

    • Received 25 February 2007.
    • Accepted 27 September 2007.
  • Copyright © 2007 American Society for Microbiology

REFERENCES

  1. 1.↵
    Andzhaparidze, O. G., N. N. Bogomolova, Y. S. Boriskin, M. S. Bektemirova, and I. D. Drynov. 1981. Comparative study of rabies virus persistence in human and hamster cell lines. J. Virol.37:1-6.
    OpenUrlAbstract/FREE Full Text
  2. 1a.↵
    Applied Biosystems. 1997. User bulletin 2. ABI Prism 7700 sequence detection system: relative quantification of gene expression. Applied Biosystems, Foster City, CA.
  3. 2.↵
    Bick, M. J., J.-W. N. Carroll, G. Gao, S. P. Goff, C. M. Rice, and M. R. MacDonald. 2003. Expression of the zinc-finger antiviral protein inhibits alphavirus replication. J. Virol.77:11555-11562.
    OpenUrlAbstract/FREE Full Text
  4. 3.↵
    Clarke, J. B., and R. E. Spier. 1983. An investigation into causes of resistance of a cloned line of BHK cells to a strain of foot-and-mouth disease virus. Vet. Microbiol.8:259-270.
    OpenUrlCrossRefPubMed
  5. 4.↵
    Cristea, I. M., J.-W. N. Carroll, M. P. Rout, C. M. Rice, B. T. Chait, and M. R. MacDonald. 2006. Tracking and elucidating alphavirus-host protein interactions. J. Biol. Chem.281:30269-30278.
    OpenUrlAbstract/FREE Full Text
  6. 5.↵
    DeFilippis, V. R., B. Robinson, T. M. Keck, S. G. Hansen, J. A. Nelson, and K. J. Fruh. 2006. Interferon regulatory factor 3 is necessary for induction of antiviral genes during human cytomegalovirus infection. J. Virol.80:1032-1037.
    OpenUrlAbstract/FREE Full Text
  7. 6.↵
    Der, S. D., A. Zhou, B. R. Williams, and R. H. Silverman. 1998. Identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays. Proc. Natl. Acad. Sci. USA95:15623-15628.
    OpenUrlAbstract/FREE Full Text
  8. 7.↵
    de Veer, M. J., M. Holko, M. Frevel, E. Walker, S. Der, J. M. Paranjape, R. H. Silverman, and B. R. Williams. 2001. Functional classification of interferon-stimulated genes identified using microarrays. J. Leukoc. Biol.69:912-920.
    OpenUrlCrossRefPubMedWeb of Science
  9. 8.↵
    Durbin, J. E., R. Hackenmiller, M. C. Simon, and D. E. Levy. 1996. Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell84:443-450.
    OpenUrlCrossRefPubMedWeb of Science
  10. 9.↵
    Gao, G., X. Guo, and S. P. Goff. 2002. Inhibition of retroviral RNA production by ZAP, a CCCH-type zinc finger protein. Science297:1703-1706.
    OpenUrlAbstract/FREE Full Text
  11. 10.↵
    Griffin, D. E. 2001. Alphaviruses, p. 917-962. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman, and S. E. Straus (ed.), Fields virology, 4th ed., vol. 1. Lippincott Williams and Wilkins, Philadelphia, PA.
    OpenUrl
  12. 11.↵
    Guo, X., J.-W. N. Carroll, M. R. MacDonald, S. P. Goff, and G. Gao. 2004. The zinc finger antiviral protein directly binds to specific viral mRNAs through the CCCH zinc finger motifs. J. Virol.78:12781-12787.
    OpenUrlAbstract/FREE Full Text
  13. 12.↵
    Guo, X., J. Ma, J. Sun, and G. Gao. 2007. The zinc-finger antiviral protein recruits the RNA processing exosome to degrade the target mRNA. Proc. Natl. Acad. Sci. USA104:151-156.
    OpenUrlAbstract/FREE Full Text
  14. 13.↵
    Hefti, H. P., M. Frese, H. Landis, C. Di Paolo, A. Aguzzi, O. Haller, and J. Pavlovic. 1999. Human MxA protein protects mice lacking a functional alpha/beta interferon system against La Crosse virus and other lethal viral infections. J. Virol.73:6984-6991.
    OpenUrlAbstract/FREE Full Text
  15. 14.↵
    Kääriäinen, L., and T. Ahola. 2002. Functions of alphavirus nonstructural proteins in RNA replication. Prog. Nucleic Acid Res. Mol. Biol.71:187-222.
    OpenUrlCrossRefPubMed
  16. 15.↵
    Kim, K. I., and D. E. Zhang. 2003. ISG15, not just another ubiquitin-like protein. Biochem. Biophys. Res. Commun.307:431-434.
    OpenUrlCrossRefPubMedWeb of Science
  17. 16.↵
    Lenschow, D. J., N. V. Giannakopoulos, L. J. Gunn, C. Johnston, A. K. O'Guin, R. E. Schmidt, B. Levine, and H. W. Virgin IV. 2005. Identification of interferon-stimulated gene 15 as an antiviral molecule during Sindbis virus infection in vivo. J. Virol.79:13974-13983.
    OpenUrlAbstract/FREE Full Text
  18. 17.↵
    Lenschow, D. J., C. Lai, N. Frias-Staheli, N. V. Giannakopoulos, A. Lutz, T. Wolff, A. Osiak, B. Levine, R. E. Schmidt, A. Garcia-Sastre, D. A. Leib, A. Pekosz, K. P. Knobeloch, I. Horak, and H. W. Virgin, IV. 2007. IFN-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and Sindbis viruses. Proc. Natl. Acad. Sci. USA104:1371-1376.
    OpenUrlAbstract/FREE Full Text
  19. 18.↵
    Levy, D. E., I. Marie, and A. Prakash. 2003. Ringing the interferon alarm: differential regulation of gene expression at the interface between innate and adaptive immunity. Curr. Opin. Immunol.15:52-58.
    OpenUrlCrossRefPubMedWeb of Science
  20. 19.↵
    MacDonald, M. R., M. W. Burney, S. B. Resnick, and H. W. Virgin IV. 1999. Spliced mRNA encoding the murine cytomegalovirus chemokine homolog predicts a beta chemokine of novel structure. J. Virol.73:3682-3691.
    OpenUrlCrossRefPubMedWeb of Science
  21. 20.↵
    Marcello, T., A. Grakoui, G. Barba-Spaeth, E. S. Machlin, S. V. Kotenko, M. R. MacDonald, and C. M. Rice. 2006. Interferons alpha and lambda inhibit hepatitis C virus replication with distinct signal transduction and gene regulation kinetics. Gastroenterology131:1887-1898.
    OpenUrlCrossRefPubMedWeb of Science
  22. 21.↵
    Marie, I., J. E. Durbin, and D. E. Levy. 1998. Differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7. EMBO J.17:6660-6669.
    OpenUrlAbstract
  23. 22.↵
    Meraz, M. A., J. M. White, K. C. Sheehan, E. A. Bach, S. J. Rodig, A. S. Dighe, D. H. Kaplan, J. K. Riley, A. C. Greenlund, D. Campbell, K. Carver-Moore, R. N. DuBois, R. Clark, M. Aguet, and R. D. Schreiber. 1996. Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell84:431-442.
    OpenUrlCrossRefPubMedWeb of Science
  24. 23.↵
    Müller, S., P. Möller, M. J. Bick, S. Wurr, S. Becker, S. Günther, and B. M. Kümmerer. 2007. Inhibition of filovirus replication by the zinc finger antiviral protein. J. Virol.81:2391-2400.
    OpenUrlAbstract/FREE Full Text
  25. 24.↵
    Otsuki, K., J. Maeda, H. Yamamoto, and M. Tsubokura. 1979. Studies on avian infectious bronchitis virus (IBV). III. Interferon induction by and sensitivity to interferon of IBV. Arch. Virol.60:249-255.
    OpenUrlCrossRefPubMed
  26. 25.↵
    Ryman, K. D., K. C. Meier, E. M. Nangle, S. L. Ragsdale, N. L. Korneeva, R. E. Rhoads, M. R. MacDonald, and W. B. Klimstra. 2005. Sindbis virus translation is inhibited by a PKR/RNase L-independent effector induced by alpha/beta interferon priming of dendritic cells. J. Virol.79:1487-1499.
    OpenUrlAbstract/FREE Full Text
  27. 26.↵
    Samuel, C. E. 2001. Antiviral actions of interferons. Clin. Microbiol. Rev.14:778-809.
    OpenUrlAbstract/FREE Full Text
  28. 27.↵
    Stanwick, T. L., and J. V. Hallum. 1974. Role of interferon in six cell lines persistently infected with rubella virus. Infect. Immun.10:810-815.
    OpenUrlAbstract/FREE Full Text
  29. 28.↵
    Strauss, J. H., and E. G. Strauss. 1994. The alphaviruses: gene expression, replication, and evolution. Microbiol. Rev.58:491-562.
    OpenUrlAbstract/FREE Full Text
  30. 29.↵
    Taniguchi, T., and A. Takaoka. 2001. A weak signal for strong responses: interferon-alpha/beta revisited. Nat Rev. Mol. Cell. Biol.2:378-386.
    OpenUrlCrossRefPubMedWeb of Science
  31. 30.↵
    Truant, A. L., and J. V. Hallum. 1977. A persistent infection of baby hamster kidney-21 cells with mumps virus and the role of temperature-sensitive variants. J. Med. Virol.1:49-67.
    OpenUrlPubMed
  32. 31.↵
    Zhang, Y., C. W. Burke, K. D. Ryman, and W. B. Klimstra. 2007. Identification and characterization of interferon-induced proteins that inhibit alphavirus replication. J. Virol.81:11246-11255.
    OpenUrlAbstract/FREE Full Text
  33. 32.↵
    Zhao, C., C. Denison, J. M. Huibregtse, S. Gygi, and R. M. Krug. 2005. Human ISG15 conjugation targets both IFN-induced and constitutively expressed proteins functioning in diverse cellular pathways. Proc. Natl. Acad. Sci. USA102:10200-10205.
    OpenUrlAbstract/FREE Full Text
View Abstract
PreviousNext
Back to top
Download PDF
Citation Tools
The Zinc Finger Antiviral Protein Acts Synergistically with an Interferon-Induced Factor for Maximal Activity against Alphaviruses
Margaret R. MacDonald, Erica S. Machlin, Owen R. Albin, David E. Levy
Journal of Virology Nov 2007, 81 (24) 13509-13518; DOI: 10.1128/JVI.00402-07

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Journal of Virology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
The Zinc Finger Antiviral Protein Acts Synergistically with an Interferon-Induced Factor for Maximal Activity against Alphaviruses
(Your Name) has forwarded a page to you from Journal of Virology
(Your Name) thought you would be interested in this article in Journal of Virology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
The Zinc Finger Antiviral Protein Acts Synergistically with an Interferon-Induced Factor for Maximal Activity against Alphaviruses
Margaret R. MacDonald, Erica S. Machlin, Owen R. Albin, David E. Levy
Journal of Virology Nov 2007, 81 (24) 13509-13518; DOI: 10.1128/JVI.00402-07
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

Carrier Proteins
Interferon-alpha
Proteins
Sindbis virus

Related Articles

Cited By...

About

  • About JVI
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #Jvirology

@ASMicrobiology

       

 

JVI in collaboration with

American Society for Virology

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0022-538X; Online ISSN: 1098-5514