JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 15:27-30 (1995)Mary Ann Liebert, Inc., Publishers

2',5'-01igoadenylate Synthetase in Interferon- - andAcyclovir-Treated Herpes Simplex Virus-Infected Cells

J.L. TAYLOR, J P.L. WITT,13 A. IRIZARRY,3 P. TOM,1 and W.J. O'BRIEN 1,2

ABSTRACT

The 2',5'-oligoadenylate (2-5A) synthetase pathway, induced by interferon- (IFN- ), has been shown to beresponsible for the antiviral action of IFN- against some viruses. Studies were done to determine the role ofthis pathway in the anti-herpes simplex virus (HSV) action of IFN- alone or in combination with acyclovir(ACV), a combination that leads to synergistic anti-HSV activity. Treatment of human corneal cells or Verocells with 100 IU/ml of IFN- induced expression of 2-5A synthetase mRNA and a 10-fold increase in 2-5Asynthetase production compared with untreated cells. HSV infection alone did not induce 2-5A synthetaseproduction, but when IFN-a-treated cells were infected with HSV, enzyme level was significantly increased(p < 0.05) compared with that in IFN-a-treated, uninfected cells. HSV infection actually decreased the level of2-5A synthetase mRNA in IFN-a-treated cells. Although IFN- treatment induced high levels of 2-5A syn¬thetase with or without HSV infection, no activation of the latent endonuclease was detected by specificcleavage of ribosomal RNA. Treatment of infected cells with 5 µ ACV alone or combined with IFN- did notincrease 2-5A synthetase or endonuclease activities above those detected in cells not treated with ACV. Thedata indicate that the 2-5A synthetase pathway was inducible in corneal cells and Vero cells but did not appearto contribute to the anti-HSV activity of IFN- alone or the synergistic activity of IFN- combined with ACV.

INTRODUCTION

INTERFERON-a (IFN-a) treatment of human cornea stromalcells or the continuous monkey kidney cell line, Vero cells,

can reduce the replication of herpes simplex virus 1 (HSV) to a

limited extent, but when IFN- is combined with the nucleosideanalog acyclovir (ACV), synergistic inhibition of virus replica¬tion occurs."'2' The mechanism(s) by which the IFN-a+ ACV combination exerts its synergistic anti-HSV activityhas not been determined, although modification of nucleosidemetabolism in HSV-infected cells may contribute to the syner¬gistic anti-HSV activity.<3)

IFNs induce the transcription of several cellular genes whoseproducts, including the gene for 2',5'-oligoadenylate (2-5A)synthetase, participate in the development of antiviral activ¬ity.(4) Synthesis of this enzyme has been shown to initiate a

pathway that can lead to the establishment of an antiviral statein some cells. The enzyme is synthesized in an inactive formand is activated by double-stranded RNA (dsRNA) to synthe¬size oligomers of adenylate-linked 2'-5'. These oligomers can

activate a latent endonuclease that then cleaves single-stranded

RNA, including mRNA and rRNA. Activation of this pathwayhas been shown to be responsible for the antiviral action ofIFN- against the picornaviruses, encephalomyocarditis virus(EMCV)(5) and mengovirus.(6> The purpose of the studies re¬

ported here was to determine whether the 2-5A synthetase path¬way is induced by HSV infection and to determine whether theanti-HSV activity of IFN- , alone or in combination withACV, is mediated by this pathway.

MATERIALS AND METHODS

Treatment of cells

Human cornea stromal cell cultures, established as previ¬ously described,'" or Vero cells, a continuous monkey kidneycell line, were pretreated with 100 IU/ml ofhuman recombinantIFN-a2a (Hoffman-La Roche, Nutley, NJ) or mock treated withDulbecco's modified Eagle's medium containing 2% fetal bo¬vine serum. After 24 h, medium was removed and cells wereinfected with the McKrae strain of HSV-1 at a multiplicity of

Departments of 'Microbiology and 2Ophthalmology and 3The Cancer Center, Medical College of Wisconsin, Milwaukee, WI 53226.

27

28 TAYLOR ET AL.

infection (MOI) of 5 plaque-forming units (PFU)/cell. Follow¬ing 1 h incubation for virus adsorption, medium was replacedwith medium with or without 5 µ ACV (Burroughs-Well-come, Co., Research Triangle Park, NC). At 24 h after infec¬tion, cells were harvested by scraping, pelleted, and storedfrozen at

—

80CC,'until assay of enzyme activity.

Measurement of 2-5A synthetase activityPelleted cells were lysed by Nonidet P-40 (NP-40) treatment,

the enzyme bound to agarose beads coated with the syntheticdsRNA, poly(I)/poly(C), and 2-5A synthetase activity wasmeasured by incorporation of 3H-ATP as described previ¬ously.(7) Binding of the enzyme to poly(I)/poly(C)-coatedbeads partially purifies the enzyme and also activates it; thus theactivity of total enzyme in a cell extract is measured. Enzymeactivity was determined as pmol ATP incorporated/h/105 cells.All samples were assayed in duplicate. Data from three experi¬ments were averaged, and the amount of enzyme present fol¬lowing treatments of cells was compared with untreated valuesby Student's t test.

Northern blotsCells were lysed with NP-40, and total cytoplasmic RNA

was phenol-chloroform extracted'8' at 24 h after IFN- treat¬ment and at 6 h after HSV infection. RNA was electrophoresedon formaldehyde agarose gels,'8·9' blotted to nitrocellulose, andcross-linked to the membrane by ultraviolet light. The probe(supplied by Dhan Kalvakolanu, Medical College of Wiscon¬sin, Milwaukee) used to detect 2-5A synthetase mRNA was the1491 bp 9-2 cloned cDNA for murine 2-5A synthetase.'10'Sequences within this clone show high levels of homology withthe human 2-5A synthetase gene encoding the small (40^16kD) forms of synthetase. It detects human 1.6 and 1.8 kbmRNAs resulting from the differential splicing of the humangene. The probe was 32P labeled by nick translation and hybrid¬ized to northern blots as previously described." " Hybridizationwas detected by autoradiography.

Endonuclease cleavage of ribosomal RNA

At 24 h after infection with HSV, total cellular RNA was

extracted,'8' electrophoresed on formaldehyde gel, stained withethidium bromide, and photographed. As a positive control foractivation of the endonuclease, cells were treated with 100IU/ml of IFN- and after 24 h infected with EMCV, a virus forwhich antiviral activity of the IFN has been shown to correlatewith activation of the 2-5A synthetase pathway and endonu¬clease activity.'512'

RESULTS

Treatment of uninfected human cornea stromal cells withIFN- resulted in the induction of 2-5A synthetase mRNAproduction. At 24 and 30 h after treatment, a single 1.6 kbmRNA species was detected (Fig. 1, lanes 2 and 4). Enzymelevels were significantly (p < 0.05) increased by IFN treatment(Fig. 2A), from 4.8 ± 2.5 units (n = 6) in untreated stromal

2-SA

FIG. 1. Induction of 2-5A synthetase mRNA expression inhuman corneal stromal cells. Confluent cultures were treatedwith 100 IU/ml of IFN- or not treated. After 24 h, cytoplasmicRNA was extracted from some cultures (not treated, lane 1 orIFN- treated, lane 2). IFN-a-containing medium was removedfrom the remaining cultures, and cells were infected with HSVat MOI = 5 PFU/cell or mock infected. After 1 h adsorption,medium was replaced with medium containing 5 µ ACV orno additive. After 6 h, cytoplasmic RNA was extracted (lanes3-8) and RNA analyzed by northern blots with a 2-5A syn-thetase-specific probe. (Lane 3, uninfected, untreated; lane 4,uninfected, IFN treated; lane 5, HSV infected; lane 6, IFN-atreated, HSV infected; lane 7, HSV infected, ACV treated; lane8, IFN- treated, HSV infected, ACV treated). Size corre¬

sponding to 2-5A 1.6 kb mRNAs indicated at left.

cells to 50.9 ± 8.0 units (n = 6) in cells treated with IFN- for48 h. HSV infection alone did not induce activity above thatdetected in untreated cells (Fig. 2A), nor did it induce detect¬able mRNA synthesis (Fig. 1, lane 5). HSV infection of cornea

stromal cells that were pretreated with IFN- reduced the 1.6 kb2-5A synthetase mRNA below detectable levels (Fig. 1, lane6). Although 2-5A synthetase mRNA was no longer detectable,enzyme activity was significantly higher (p < 0.05) in IFN-a-treated, HSV-infected corneal cells (116.3 ± 18.0 units, = 6) than in cells treated with IFN- alone (50.9 ±8.0 units, = 6). ACV treatment of HSV-infected cells with or withoutIFN- pretreatment did not significantly alter the levels of en¬

zyme present in cells 24 h postinfection. The induction ofenzyme in Vero cells followed the same pattern as in cornea

stromal cells (Fig. 2B).Because the assay'7' used for measurement of 2-5A syn¬

thetase did not discriminate between active and inactive en¬

zyme, it was not known whether the enzyme detected in IFN-a-treated cells led to the production of 2-5A oligomers that werefunctional in activation of the latent endonuclease. As shown inFig. 3, EMCV infection of IFN-treated corneal cells resulted inthe specific cleavage"3' of cellular ribosomal RNA as has beenreported in other cells."4' In cornea stromal cells infected withHSV with or without IFN- pretreatment, there was no detect¬able cleavage of rRNA. Similar results were obtained in Verocells (data not shown). These results indicate that, although theproduction of 2-5A synthetase was induced in cells treated with

2-5A SYNTHETASE IN HSV-INFECTED CELLS 29

g »OIoC 200

< 150

100

50

0HSV IFNtHSV HSV*ACV IFN+HSV+ACV

Treatment

FIG. 2. Induction of 2-5A synthetase in human cornea stro¬mal cells (A) or Vero cells (B). Cells were treated with or

without 100 IU/ml of IFN- , incubated for 24 h, infected withHSV at MOI = 5 or not infected, and after 1 h overlain withmedium with or without 5 µ ACV to yield six groups: unin¬fected, untreated (untreated); uninfected, IFN- treated(IFN); infected, untreated (HSV); IFN- treated, infected(IFN + HSV); infected, ACV treated (HSV + ACV); and in¬fected, treated with both IFN- and ACV (IFN + HSV +ACV). After 24 h, cells were harvested and the amount of 2-5Asynthetase present in cell extracts determined. Values for stro¬mal cells represent a mean of six replicates from three differentcell preparations. Error bars indicate standard deviation. Valuesfor Vero cells represent mean of duplicate samples.

IFN- and further increased by HSV infection of IFN-a-treatedcells, this increase in enzyme did not lead to the activation ofendonuclease.

EndonucleaseCleavage Products

V V V

IFN + EMC

IFN + HSV + ACV

IFN + HSV

HSV

28S 18S

FIG. 3. Endonuclease activation in Vero cells. Cells weretreated with or without 100 IU/ml of IFN- , incubated for 24 h,infected with HSV or EMC virus at MOI = 5 or mock infected,and after 1 h overlain with medium with or without 5 µ ACV.After 24 h, total cell RNA was extracted, electrophoresed on

agarose gel, and stained with ethidium bromide. Arrowheadsindicate the characteristic cleavage products of ribosomal RNAby 2-5A-dependent endonuclease.

ACV blocks HSV replication by inhibiting viral DNA syn¬thesis."5' As a result, virus replication remains stalled in theearly stages, with continued expression of some early genes.Because HSV encodes genes on both strands of its double-stranded genome and RNA transcripts with complementary re¬

gions are produced,"6' it was possible that sufficient levels ofdsRNA might accumulate in the presence of ACV to activate2-5A synthetase and lead to activation of the endonuclease.However, ACV treatment of HSV-infected cells did not resultin induction of the endonuclease, even when high levels ofsynthetase were present in cells that were also treated withIFN-a.

DISCUSSION

These studies indicate that, like many other cell types, hu¬man cornea stromal cells as well as Vero cells respond to IFN-atreatment by increased expression of 2-5A synthetase mRNA,leading to increased production of 2-5A synthetase. HSV infec¬tion of IFN-a-treated cells induced a higher level of synthetase,although HSV infection of untreated cells did not induce en¬

zyme synthesis.This enzyme has been detected in several forms (40, 44-46,

69, and 100 kD) in cells, apparently representing the productsof differentially spliced mRNAs from at least two genes."718'A single 1.6 kb transcript was detected in corneal cells at 24 hafter IFN- treatment. The elevation of activity in IFN-a-treated, HSV-infected cells compared with IFN-a-treated cellsalone did not result from synthesis of differentially spliced 2-5Asynthetase mRNA species (i.e., the 1.8 kb mRNA), and in fact,detectable mRNA was lost by 6 h after HSV infection in IFN-a-treated cells. It is possible that the genes encoding the larger (69and 100 kD) 2-5A synthetase forms may be the source of theincrease in enzyme. Despite elevated levels of enzyme, no

activation of the 2-5A-dependent endonuclease occurred in ei¬ther corneal cells or Vero cells following HSV infection.

In an effort to correlate the anti-HSV action of IFN with 2-5Asynthetase production, Rysiecki et al.'5' introduced a 2-5A syn¬thetase cDNA-containing plasmid into human glioblastomacells. Although constitutive expression of the 1.6 kb transcriptof 2-5A synthetase led to resistance to EMCV, cells were notresistant to HSV infection. When Fujihara et al."9' treatedHSV-infected baby hamster kidney fibroblasts with oligomersof 2-5A, the product of activated 2-5A synthetase, replicationof virus was inhibited. These results suggest that in HSV-infected cells that have been induced by IFN- treatment to

produce the synthetase, the enzyme does not become activatedto produce oligomers capable of activating the endonuclease. Insupport of this idea, Cayley et al.'20' found that 2-5A oligomerswere synthesized in IFN-treated, HSV-infected human Changcells; however, these oligomers were poor activators of the2-5A-dependent endonuclease and, in fact, inhibited its activa¬tion by authentic 2-5A oligomers. Our results in IFN-a-treated,HSV-infected human corneal cells and Vero cells are consistentwith these earlier reports that the 2-5A synthetase pathway doesnot contribute to the anti-HSV activity of IFN- and demon¬strate that HSV infection turns off the production of the 1.6 kb2-5A synthetase mRNA. Addition of ACV to the IFN- treat-

30 TAYLOR ET AL.

ment of cells led to synergistic antiviral action,'1' but that actionappeared to be caused by some mechanism other than the acti¬vation of the 2-5A synthetase pathway because production of2-5A synthetase mRNA and enzyme activity were not increasedand endonuclease was not activated following ACV treatmentof IFN-a-treated cells.

ACKNOWLEDGMENTS

This work was supported in part by Grant Nos. R01-EY-06990 and P30-EY-01931 from the National Eye Institute, an

unrestricted grant from Research to Prevent Blindness, and theCancer Center of the Medical College of Wisconsin. Corneasfor the production of cell cultures were provided by the Wiscon¬sin Lions Eye Bank, Milwaukee, WI.

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Address reprint requests to:Dr. Jerry Lynn Taylor

Department of MicrobiologyMedical College of Wisconsin8701 Watertown Plank Road

Milwaukee, WI 53226

Received 20 May 1994/Accepted 9 August 1994