JOURNAL OF VIROLOGY,0022-538X/99/$04.0010

Sept. 1999, p. 7193–7198 Vol. 73, No. 9

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Picornavirus Receptor Down-Regulation by PlasminogenActivator Inhibitor Type 2

D. R. SHAFREN,1* J. GARDNER,2 V. H. MANN,3 T. M. ANTALIS,3 AND A. SUHRBIER2

Picornaviral Research Unit, Discipline of Immunology and Microbiology, Faculty of Medicine and Health Sciences,University of Newcastle, Newcastle, New South Wales 2300,1 and Cellular Oncology Laboratory3 and

Australian Centre for International & Tropical Health & Nutrition,2 Queensland Institute ofMedical Research and University of Queensland, Brisbane, Queensland 4029, Australia

Received 4 February 1999/Accepted 21 May 1999

Therapeutic interference with virus-cell surface receptor interactions represents a viable antiviral strategy.Here we demonstrate that cytoplasmic expression of the serine protease inhibitor (serpin), plasminogenactivator inhibitor type 2 (PAI-2), affords a high level of protection from lytic infection by multiple humanpicornaviruses. The antiviral action of PAI-2 was mediated primarily through transcriptional down-regulationof the following virus receptors: intercellular adhesion molecule 1 (ICAM-1, a cellular receptor for the majorgroup of rhinoviruses), decay-accelerating factor (a cellular receptor for echoviruses and coxsackieviruses),and to a lesser extent the coxsackie-adenovirus receptor protein (a cellular receptor for group B coxsackievi-ruses and group C adenoviruses). Expression of related cell surface receptors, including membrane cofactorprotein and the poliovirus receptor, remained unaffected. These findings suggest that PAI-2 and/or relatedserpins may form the basis of novel antiviral strategies against picornavirus infections and also therapeuticinterventions against ICAM-1-mediated respiratory inflammation.

Picornaviruses are small, spherical, naked viruses (20 to 25nm) containing positive-sense genomic RNA and comprise agenus of the family Picornaviridae. The two largest subgroupsare rhinoviruses and enteroviruses. There are ;100 rhinovirusserotypes, which collectively constitute the most importantcause of mild upper respiratory illnesses in adults. The humanenteroviruses consist of polioviruses (3 serotypes), echoviruses(34 serotypes), group A coxsackieviruses (24 serotypes), andgroup B coxsackieviruses (6 serotypes). The human enterovi-ruses are ubiquitous; transmitted primarily by the oral-fecalroute, they are capable of producing a wide range of clinicalsyndromes varying in severity. They are mainly found in thegastrointestinal tracts of infected individuals but also have thepotential to infect the meninges, central nervous system, myo-cardium, pericardium, striated muscles, respiratory tract, andskin (23).

Identification and molecular characterization of virus-spe-cific receptors and receptor-mediated processes are importantnot only because receptors determine host range, susceptibilityto infection, and pathogenesis but also because virus receptorsconstitute potential targets for antiviral therapy. Three majorgroups of cell surface molecules have been shown to be in-volved with distinct stages of picornaviral cell attachment andmembrane penetration: the immunoglobulin-like supergenefamily, complement regulatory proteins, and integrins. Thepoliovirus receptor (PVR) (24), intercellular adhesion mole-cule 1 (ICAM-1) (16), and coxsackievirus-adenovirus receptor(CAR) (8, 36) are all members of the immunoglobulin-likesupergene family (Fig. 1) and are able to facilitate productivecell infection by polioviruses (24), a major group of rhinovi-ruses (16), and some group A (31) and group B (8, 33, 36)

coxsackieviruses, respectively. In contrast, the complementregulatory protein, decay-accelerating factor (DAF) (28) (Fig.1), is employed as an attachment or sequestering receptor bymany enteroviruses (6, 7, 17, 30, 32, 41) but cannot by itselfmediate entry of virus into the cell (7, 30) unless cross-linkedby specific antibody (34, 34a). Additionally, a number of inte-grins have been shown to bind human picornaviruses, but theirrole in viral cell entry is uncertain (1, 5, 29). Recently, anumber of picornaviruses have been shown to employ com-plexes of the above-mentioned receptors to enter cells, thesereceptor complexes consisting of distinct attachment and cellinternalizing components (32, 33).

The cellular expression of many picornavirus receptors isstrongly influenced by the action of inflammatory cytokines(10, 26, 35, 37). Human rhinoviruses (HRVs) enhance theirown dissemination by up-regulating the cell surface expressionof ICAM-1 on neighboring cells through cytokine induction.ICAM-1 is highly susceptible to up-regulation via the inflam-matory cytokines tumor necrosis factor alpha (TNF-a) andinterleukin-1b (IL-1b) (37), and during initial stages of cellularlytic infection by HRVs, cytokines such as TNF-a and IL-1bare released by infected cells (35). Furthermore, the expressionof CAR on the surface of colon carcinoma cells is markedlyincreased by the action of TNF-a (1a).

The expression of DAF, a major picornavirus attachmentreceptor, has been shown to be significantly up-regulated bythe action of TNF-a (10, 26). In contrast, another member ofthe complement regulatory protein family, membrane cofactorprotein (MCP; CD46), which is also a cellular receptor formeasles viruses, is resistant to TNF-a-induced up-regulation(10, 26, 38). Thus, the ability to negate TNF-a-induced up-regulation of cellular virus receptors might be regarded a po-tential antiviral strategy for controlling human picornavirusinfections.

TNF-a, in addition to up-regulating numerous cellular pro-teins, is also able to promote apoptosis in many cells (40).Recently, cells expressing plasminogen activator inhibitor type2 (PAI-2) were shown to be protected from TNF-a-induced

* Corresponding author. Mailing address: Picornaviral ResearchUnit, Discipline of Immunology and Microbiology, Faculty of Medi-cine and Health Sciences, University of Newcastle, Level 3, DavidMaddison Clinical Sciences Building, Royal Newcastle Hospital, New-castle, New South Wales 2300, Australia. Phone: 61 2 4923 6158. Fax:61 2 4923 6814. E-mail: dshafren@mail.newcastle.edu.au.

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apoptosis (13, 21). PAI-2 is a serine proteinase inhibitor (ser-pin), which is a major product of macrophages in response toendotoxin and inflammatory cytokines (20). Under physiolog-ical conditions, PAI-2 expression is limited to a select numberof cell types, which include differentiated keratinocytes, acti-vated monocytes and macrophages, placental trophoblasts, andsome tumor cell lines (20). PAI-2 is well characterized as aninhibitor of the extracellular proteinase urokinase-type plas-minogen activator (20, 39). Recently, an additional, distinct,intracellular function for PAI-2 as a regulator of the intracel-lular signal transduction pathway(s) has been postulated (3,13). Intracellular PAI-2 expression in HeLa cells was associ-ated with resistance to TNF-a-mediated apoptosis (13) andwith increased alpha/beta interferon (IFN-a/b) activity (3).PAI-2 is a member of the ovalbumin group of serpins (ov-serpins), which lack hydrophobic signal sequences. This resultsin inefficient secretion and translocation and thus a primarilycytoplasmic location of these proteins. In the case of HeLacells expressing PAI-2, no secreted PAI-2 is detectable. Apartfrom PAI-2, a number of other ov-serpins have now beenshown to have cytoplasmic functions; these include CrmA (15),protease inhibitor 9 (9), and protease inhibitor 6 (27).

Here we show that expression of PAI-2 in HeLa cells signif-icantly reduced picornavirus infection through transcriptionaldown-regulation of the expression of TNF-a-inducible virusreceptors. These findings may find application in the develop-ment of a novel serpin-based antiviral strategies for control ofenteric picornavirus infections, and also for the therapeuticreduction of ICAM-1-mediated respiratory inflammations.

MATERIALS AND METHODS

Cells and viruses. Echovirus type 7 (EH7; Wallace), poliovirus type 1 (PV1;Sabin), coxsackievirus A21 (CVA21; KuyKendall), coxsackievirus B3 (CVB3;Nancy), and HeLa B cells were obtained from Margery Kennett, Enterorespi-ratory Laboratory, Fairfield Hospital, Melbourne, Victoria, Australia. HRV type14 (HRV14) was obtained from the American Type Culture Collection. Allviruses were grown in HeLa B cells.

A panel of individual cloned HeLa cell lines expressing sense (S1a and S1b)and antisense (A2/7) PAI-2 cDNA was generated as previously described (13) byinserting a DNA fragment containing the entire PAI-2 coding sequence and the39 untranslated region in both orientations into the expression vector pRcCMVunder control of the constitutive cytomegalovirus V promoter. Stable transfec-tants containing these constructs were selected by resistance to G418 and char-

acterized by Northern blot, immunoblot, and immunofluorescence analyses (13,14). HeLa and A2/7 cells express no detectable PAI-2 mRNA or protein. S1a andS1b cell lines each express cytoplasmic PAI-2 at levels similar to or less thanthose detected in activated monocytic cells (13).

Antibodies. The anti-ICAM-1 monoclonal antibody (MAb) WEHI (11) wasobtained from Andrew Boyd, Queensland Institute of Medical Research, Bris-bane, Australia. The anti-DAF MAbs IA10, VIIIA7, and IIH6 (18) were gener-ous gifts from Taroh Kinoshita, Department of Immunoregulation, Osaka Uni-versity, Osaka, Japan; MAb IH4 and anti-MCP MAb E4.3 (12) were from BruceLoveland, Austin Research Institute, Heidelberg, Victoria, Australia. The anti-PVR MAb 280 (25) was supplied by Philip Minor, National Institute for Bio-logical Standards and Control, Potters Bar, United Kingdom, and the anti-CARMAb (8) was from Jeffery Bergelson, Dana-Farber Cancer Institute, Boston,Mass.

Virus infectivity assay. Cell monolayers in 96-well culture plates were chal-lenged with 100 ml of 10-fold serial dilutions of the above-mentioned picornavi-ruses and then incubated at 37°C for 48 h. To quantitate cell survival, monolayerswere stained with a crystal violet-methanol solution and washed with distilledwater, and the plates were read at a wavelength of 540 nm. Results are expressedas mean percentages of cell lysis relative to the uninfected control cell mono-layers of triplicate wells 1 standard deviation.

Radiolabeled virus binding assays. Viruses were radiolabeled in Dulbecco’smodified essential medium containing [35S]methionine and purified by velocitycentrifugation in 5 to 30% sucrose gradients as previously described (32). Mono-layers of PAI-2-expressing S1a and S1b cells and parental HeLa cells wereincubated with radiolabeled viruses (2 3 104 to 5 3 104 cpm) in serum-freeDulbecco’s modified essential medium for 1 h at 37°C. After three washes, thecell monolayers were dissolved in 200 ml of 0.2 M NaOH–1.0% sodium dodecylsulfate (SDS). The amount of labeled virus bound was measured by liquidscintillation counting.

Flow cytometry. Cells (5 3 105) were incubated with the appropriate MAbs(5.0 mg/ml) diluted in phosphate-buffered saline (PBS) containing 1% bovineserum albumin (PBS-BSA) at 0°C for 30 min, after which the cells were washedwith 5.0 ml of PBS-BSA. The cells were then pelleted at 1,000 3 g for 5 min andresuspended in 100 ml of phycoerythrin-conjugated goat anti-mouse immuno-globulin G (heavy plus light chains) (Silenus, Melbourne, Victoria, Australia)diluted in PBS-BSA. After incubation at 0°C for 30 min, the cells were washedand pelleted as described above, resuspended in PBS-BSA, and analyzed with aFACStar analyzer (Becton Dickinson, Sydney, New South Wales, Australia).

Surface biotinylation and immunoprecipitation. Cells (5 3 106) were de-tached from the surface of plastic tissue culture flasks by using an EDTA solutionand washed once in PBS. Washed cells were resuspended into 3 ml of biotiny-lation buffer (10 nM sodium borate [pH 8.8], 150 nM NaCl). Biotin-amidocap-roate N-hydroxysuccinimide ester was added to 50 mg/ml, and the sample stoodat room temperature for 15 min. The reaction was terminated by the addition ofNH4Cl to 10 mM. Cells were washed twice in PBS and resuspended into bufferA (138 mM NaCl, 2.9 mM KCl, 0.5 mM MgCl2, 12 mM NaHCO3, 0.3 mMNaH2PO4, 5.5 mM glucose, 10 mM HEPES [pH 7.4]). Cell lysates were placedon ice for 60 min and then centrifuged at 15,000 3 g for 15 min to removedetergent-insoluble material. Soluble lysates were precleared with rabbit anti-mouse immunoglobulin G coupled to Sepharose 4B. Immunocomplexes werewashed three times with lysis buffer and resolved (reduced) by SDS-polyacryl-amide gel electrophoresis. Resolved proteins were electrophoretically trans-ferred to nitrocellulose membranes. Streptavidin-biotin-horseradish peroxidasecomplexes were used to probe the membrane, and labeled proteins were visu-alized by using enhanced chemiluminescence (Amersham Life Sciences, LittleChalfont, United Kingdom).

Northern blot analysis. Total RNA isolated from S1a, S1b, A2/7, and parentalHeLa cells was separated by denaturing gel electrophoresis and transferred toHybond-N nylon membranes as described elsewhere (2). The blot was hybridizedwith [32P]dCTP-labeled purified DNA fragments encoding cDNAs for ICAM-1(30), DAF (30), CAR (8), and PVR (24). Blots were hybridized at 65°C andwashed to a final stringency of 0.13 SSC (13 SSC is 0.15 M NaCl plus 0.015 Msodium citrate)–0.1% SDS at 65°C. The level of 18S rRNA was used as ameasure of RNA loading in each lane.

RESULTS

PAI-2 expression inhibits HeLa cell lytic infection by manypicornaviruses. Monolayers of S1a, S1b, A2/7, and parentalHeLa cells were challenged (105 PFU/well) with PV1, HRV14,E7, CAV21, and CVB3 and examined for cytopathic effectfollowing a 48-h incubation period. The cell lines not express-ing PAI-2 (HeLa and A2/7) were susceptible to lytic infectionby all of these picornaviruses (Fig. 2). In contrast, PAI-2-expressing S1a cells were refractory to lytic infection byHRV14, E7, CAV21, and CVB3 but not PV1 (Fig. 2).

To determine whether infection of PAI-2-expressing cellswas noncytopathic but still productive, monolayers of HeLa

FIG. 1. Schematic representation of structures of the virus receptorsICAM-1, PVR, CAR, MCP, and DAF. ICAM-1, PVR, and CAR belong to theimmunoglobulin supergene family, and MCP and DAF are complement regula-tory proteins. ICAM-1 is an internalization receptor for rhinoviruses and somegroup A coxsackieviruses. CAR is an internalization receptor for group B cox-sackieviruses. DAF is a sequestration receptor for coxsackieviruses, echoviruses,and enteroviruses. C2, constant region 2; V, variable region; SCR, short consen-sus repeat.

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and S1a cells in six-well plates were infected with inocula of 105

and 103 PFU of CVB3. Following incubation for 24 h, cell lysiswas evident only in the inoculated HeLa cells. Analysis ofinfectious virus production by plaque assay revealed thatS1a cells yielded titers attributable to that of the input virus,whereas HeLa cell lysates yielded increases of 103- and105-fold, respectively, over the input inoculum (data notshown).

To further quantitate the antiviral effect of PAI-2 expres-sion, monolayers of HeLa, A2/7, and S1a cells were infectedwith 10-fold serial dilutions of PV1, HRV14, E7, CAV21, and

CVB3. HeLa and A2/7 cells showed higher levels of cell lysisthan S1a cells for all dilutions of HRV14, E7, CAV21, andCVB3 (Fig. 3). S1b cells behaved similarly to S1a cells in theseassays (data not shown). At low dilutions (or high multiplicityof infection [MOI]) of PV1, no difference between cytopathiceffect of S1a cells and control cells was observed; however, athigh dilutions of virus (low MOI), S1a cells showed increasedsurvival (low percent lysis) relative to control lines (Fig. 3).

The lower cell lysis values for E7 and HRV infection ofcontrol cell lines at high virus dilutions than for CAV21 andCVB3 infection reflect a lower replication competence of E7

FIG. 2. Picornavirus-induced lytic infection of PAI-2-expressing S1a cells and control A2/7 and parental HeLa cells. Monolayers of S1a, A2/7, and HeLa cells in96-well culture plates were inoculated (approximately 105 PFU/well) with PV1, HRV14, E7, CAV21, and CVB3. Following incubation for 48 h at 37°C, the cellmonolayers were inspected for signs of cell lysis and then photographed at an original magnification of 320.

FIG. 3. Dose-dependent picornavirus-induced lytic infection of S1a, A2/7, and HeLa cells, using a range of input multiplicities of PV1, HRV14, E7, CAV21, andCVB3.

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and HRV in HeLa cells and thus less lysis within the 48-h assayperiod.

Thus, HeLa and A2/7 cells were substantially more suscep-tible than PAI-2-expressing cells to the cytopathic effects ofHRV14, E7, CAV21, and CVB3 infection. Picornaviral rep-lication appeared not to be modified by PAI-2 cytoplasmicexpression, as HeLa and PAI-2-expressing S1a cells yieldedcomparable levels of infectious CAV21 and CVB3 whentransfected with CAV21 and CVB3 viral RNAs, respectively(data not shown).

Reduced binding of picornaviruses to PAI-2-expressingcells. To determine whether inhibition of picornavirus lyticinfection in S1a and S1b cells was due to interference withcellular attachment, preparations of gradient-purified picorna-viruses were radiolabeled (Fig. 4A) and incubated with S1a,S1b, and HeLa cells to monitor relative levels of virus-cellsurface binding. Cellular attachment of E7, HRV14, CAV21,and CVB3 was reduced by between 80 and 95% in S1a and S1bcells compared to HeLa controls (Fig. 4B). PV1 bound equallyto S1a, S1b, and HeLa cell lines, albeit with a slight increase inbinding to S1b cells compared with HeLa cells (Fig. 4B).

The overall pattern of reduced virus binding to the panel ofcells was similar to the pattern observed for inhibition of virus-induced lytic infection shown in Fig. 2 and 3, suggesting thatthe presence of PAI-2 reduced the surface expression of pi-cornavirus cellular receptors.

PAI-2 reduces surface expression of picornavirus receptors.To determine whether PAI-2-transfected cells expressed lowerlevels of virus receptors, which would result in less virus bind-ing (Fig. 4) and less cell infection (Fig. 2 and 3), the levels ofthe picornavirus receptor molecules PVR, CAR, DAF, andICAM-1 were assessed by flow cytometry. The cellular recep-tor of the unrelated measles virus, MCP, was included as acontrol. Measurement of the mean peak fluorescence intensi-ties showed that expression of CAR, DAF, and ICAM-1 on thesurface of S1a and S1b cells compared to A2/7 cells was re-duced by 50 to 90% (Fig. 5A). The surface expression of PVRand MCP was similar among all cell lines, indicating that thesereceptors were unaffected by PAI-2 expression. (Results for

the parent HeLa cell line were similar to those for A2/7 cells inthese assays [data not shown]). The slight increase in PV1binding to S1b cells observed in Fig. 4B was mirrored by aslight increase in the level of PVR on S1b cells (Fig. 5A). Thedecreased picornavirus receptor expression on the surface ofPAI-2-expressing HeLa cells was not due to an accident of thetransfection and selection process, as the antisense PAI-2clone A2/7 (Fig. 5) and a chloramphenicol acetyltransferase-expressing HeLa clone generated by the same protocol as usedfor the S1a and S1b cells showed no signs of reduced surfaceICAM-1 expression (35a).

To confirm that PAI-2 selectively reduced surface expressionof CAR, DAF, and ICAM-1, S1a cells and A2/7 cells weresurface biotinylated and immunoprecipitated with anti-DAF,anti-CAR, and anti-ICAM-1 MAbs. The level of surface ex-pression of biotinylated DAF and CAR was significantly lesson the surface of S1a cells than on the surface of A2/7 cells.ICAM-1 expression differed dramatically between the two celllines, being undetectable on the surface of S1a cells (Fig. 5B).The reduction in CAR, DAF, and ICAM-1 expression on thesurface of PAI-2-expressing S1a cells (Fig. 5B) mirrored thatdetected by flow cytometry (Fig. 5A).

These data clearly show a reduction in the expression of thepicornavirus receptors CAR, DAF, and ICAM-1 in PAI-2 ex-pressing cells, while PVR and MCP were unaffected.

PAI-2-mediated picornavirus receptor down-regulation oc-curs at the transcriptional level. To ascertain whether PAI-2-induced picornavirus receptor down-regulation was mediatedby transcriptional or posttranscriptional activity, mRNA levelsfor ICAM-1, DAF, and PVR were examined in PAI-2-express-ing and control cell lines. Significant levels of PVR mRNAwere detected in each cell line (S1a, S1b, A2/7, and HeLa)independent of expression of PAI-2 (Fig. 6). ICAM-1 andDAF mRNAs were present in HeLa and A2/7 cells, but noICAM-1 or DAF mRNA was detected in PAI-2-expressing S1aand S1b cells (Fig. 6). A marked reduction in the CAR mRNAlevel was observed in the S1a cells but not in S1b cells (data notshown). This may be due to the finding that S1a cells expresssignificantly higher levels of intracellular PAI-2 than S1b cells

FIG. 4. Picornavirus binding to S1a, A2/7, and parental HeLa cells. (A) 35S-labeled preparations of PV1, HRV14, E7, CAV21, and CVB3 were separated on a 15%slab SDS-polyacrylamide gel, and individual proteins identified by autoradiography. (B) Monolayers of PAI-2-expressing S1a and S1b cells and parental HeLa cells wereincubated with radiolabeled viruses and washed, and the amount of labeled virus bound was measured by liquid scintillation counting. Results are expressed as the meanof triplicate wells 1 standard deviation.

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(3a), which raises the possibility that the intracellular thresholdPAI-2 level required for CAR down-regulation is higher thanthat needed for DAF and ICAM-1 down-regulation. The levelsof the loading control, 18S and 28S rRNAs, were comparablein all the samples. These findings demonstrated that the re-duction in the expression of picornavirus receptors in PAI-2-expressing cells was due to a reduction in their gene transcrip-tion.

DISCUSSION

This study demonstrated that expression of PAI-2 protectedthe highly permissive HeLa cells against lytic infection by se-lected human picornaviruses (Fig. 2 and 3). The primary modeof action was a PAI-2-dependent transcriptional down-regula-tion of the surface expression of three picornavirus cellularreceptors, DAF, CAR, and ICAM-1 (Fig. 6). The surface ex-pression of other virus receptors, namely, PVR and MCP,although closely related to CAR/ICAM-1 and DAF (Fig. 1),respectively, were unaffected by cytoplasmic expression ofPAI-2 (Fig. 5).

Cytoplasmic expression of PAI-2 has been shown to protectcells against TNF-a-mediated apoptosis (13, 21), and here weshow that PAI-2 expression significantly reduced the surfaceexpression of the virus receptor molecules DAF, CAR, andICAM-1, which are all known to be TNF-a inducible (10, 26,37). In contrast, PVR and MCP were not inducible by TNF-a,and their expression was largely unaffected by PAI-2 expres-

sion. The structural relatedness of PVR, CAR, and ICAM-1and of DAF and MCP (Fig. 1) appeared to have no bearing onthe PAI-2-mediated effects. Thus, PAI-2 appeared to selec-tively down-regulate TNF-a-inducible receptors, although theexpression of the TNF-a-inducible HLA class I was not af-fected by PAI-2 expression (data not shown). These experi-ments suggest that PAI-2 has activity as a specific intracellularregulator of gene expression, consistent with previous reportsof a PAI-2-mediated influence on the signal transduction path-way(s) (3, 13, 14).

Antalis et al. (3) recently described another phenotype ofPAI-2-expressing HeLa cells whereby the cells were protectedfrom the rapid cytopathic effects of alphavirus infection via aPAI-2-mediated induction of constitutive IFN-a/b production(3). The replication of many picornaviruses has been shown tobe inhibited by the action of IFN-a/b (19, 22). The antiviralaction of IFN-a/b may provide an explanation for the protec-tion against poliovirus lytic infection seen in PAI-2-expressingcells at low MOI (Fig. 3), in the absence of receptor down-regulation or reduction in viral binding (Fig. 4 and 5). Theantipicornaviral activity of PAI-2 may thus be twofold: (i) themajor component via receptor down-regulation and reductionof viral binding and (ii) a minor component via inhibition ofviral replication through the action of autocrine IFN-a/b.

Picornavirus infections are a major cause of morbidity in ourcommunity, with symptoms ranging from the common cold topericarditis. For many of these viruses, the large number ofserotypes makes vaccine development difficult. In recent years,much research effort has been directed at characterizing thecellular receptors used by viruses and the molecular basis ofvirus-receptor interactions, with the ultimate aim of developinggeneric antiviral strategies. Here we show that a specific serpincan transcriptionally down-regulate specific viral receptors, il-lustrating the potential for novel serpin based antiviral strate-gies for a large proportion of picornavirus infections.

ICAM-1 plays an important role in the pathogenesis of notonly rhinovirus infection but also Plasmodium falciparum in-fection (4) and the exacerbations of asthma, chronic bronchitis,and cystic fibrosis (42). ICAM-1 was the most susceptible tothe action of cytoplasmic PAI-2 expression, indicating thatPAI-2-based therapy may also find application in the reductionof ICAM-1-mediated respiratory inflammations.

FIG. 5. Analysis of picornavirus receptor expression on the surface of S1a,S1b, and HeLa cells. (A) Flow cytometric analysis of receptor surface expression.Suspensions of S1a, S1b, A2/7, and HeLa cells were incubated with MAbs againstthe virus receptors PVR, ICAM-1, DAF, CAR, and MCP. (B) Immunoprecipi-tation of biotinylated virus receptors. S1a and A2/7 cells were surface biotinyl-ated and then immunoprecipitated with anti-DAF, anti-ICAM-1, and anti-CARMAbs, as indicated. Sizes are indicated in kilodaltons.

FIG. 6. Northern blot analysis of virus receptor expression in PAI-2-express-ing S1a and S1b cells and control A2/7 and HeLa cells. Total RNA was isolatedfrom each cell line, separated by denaturing gel electrophoresis, and Northernblotted, and the membranes were probed with radiolabeled cDNA fragmentsencoding each receptor. Levels of 18S and 28S rRNAs are shown as a measureof RNA loading in each lane.

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ACKNOWLEDGMENTS

We thank Margery Kennett for the stock picornaviral preparations,and we thank Rebecca Ingham and Simone Cross for excellent tech-nical assistance.

This research was supported by grants from the National Health andMedical Research Council of Australia, Hunter Medical ResearchFoundation, Australian Center for International & Tropical Health &Nutrition, and the Australian Commonwealth AIDS Research GrantsProgram.

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