ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1974, p. 153-160Copyright O 1974 American Society for Microbiology

Vol. 5, No. 2Printed in U.S.A.

Anti-Herpesvirus Action of IsoprinosinePAUL GORDON', BRUCE RONSEN, AND ERIC R. BROWN

Departments of Pharmacology and Microbiology, Chicago Medical School, University of Health Sciences,Chicago, Illinois 60612

Received for publication 13 August 1973

Anti-herpesvirus effects of Isoprinosine were found to be condition-dependentin vitro and in vivo. A herpes type 1 strain, grown in primary rabbit kidney cells,was not susceptible to Isoprinosine when cytopathic effect was evaluated, butwas modestly inhibited when plaque-forming methocel overlay was used in thesame cell line. However, the growth of a herpes type 2 strain was inhibited byIsoprinosine in this system when cytopathic effect was evaluated. Both virusesproduced an encephalitis in hamsters after application by corneal abrasion. Theencephalitis produced by a 1.5 mean lethal dose of herpesvirus was strikinglysuppressed by large doses of Isoprinosine given ad libitum in the drinking water;this protection was not seen, however, at 15 mean lethal doses. Anti-herpeseffects of Isoprinosine were suppressed by additional treatment with cortisone,both in tissue culture and in vivo. Isoprinosine, in the absence of virus infection,produced significant effects on the metabolism, structure, and function of brainribosomes. In vivo, RNA components were labeled more rapidly and gave uptheir radioactivity more slowly after treatment with Isoprinosine. This enhancedcytoplasmic radioactivity was associated with a loss of incorporated radioactivitylocated in the nucleus and an increase in radioactivity located in a ribonucleo-protein fraction >4S and

154 GORDON, RONSEN, AND BROWN

Two herpesviruses were used in the tissue culturestudies. One was herpes simplex virus 35, type 1,passage 6 (obtained from Fred Rapp of Penn StateUniversity College of Medicine). The other was herpessimplex virus 27, type 2, passage 2 (obtained fromTe-Wen Chang of Tufts University School of Medi-cine).

Animal virology. For hamster studies, bothherpesviruses were passed once into 100-g femalehamsters by corneal abrasion, and were harvestedfrom the brains of moribund animals as a supernatantfrom brain homogenate. For herpes type 2, twodilutions of the supematant (approximate virulenceof 1.5 and 15 mean lethal dose [LD50]) were adminis-tered by unilateral corneal abrasion. Drug was admin-istered ad libitum in the drinking water, beginning 24h after infection, in solution concentrations of 0.33,0.50, 0.75, and 1.0%. Mortality was evaluated at 12days.These same procedures were used in a study of

herpes type 1 at 1.5 LD50 where treatment was givenin the drinking water at the 1.0% concentration.

Extracted cytoplasmic RNA. Three hours priorto sacrifice, 48 2-month-old female Sprague Dawleyrats were divided into six groups of eight animals eachand treated intraperitoneally with normal saline (twogro zps) or with Isoprinosine at 125 mg/kg (one group),250 mg/kg (one group) or 500 mg/kg (two groups). Onecontrol group and one group treated with 500 mg ofIsoprinosine per kg were injected intraperitoneallywith 500 ACi of [5-3H]orotic acid per kg 20 min beforesacrifice. The remaining groups were injected withthis dose of isotope 45 min before sacrifice.

Animals were sacrificed by guillotine, brains werequickly excised and chilled in buffer A (0.05 Mtris(hydroxymethyl)aminomethane [Tris 1-hydrochlo-ride, 0.01 M MgCl2, 0.025 M KCl at pH 7.4) contain-ing 0.25 M sucrose. Excess buffer was removed andtissues were weighed and homogenized in buffer A, 1ml/g, in a glass-Teflon Potter-Elvehjem system. Thehomogenates were centrifuged for 20 min at 10,000rpm in a Sorvall RC2-B centrifuge by using an SM-34rotor. The resulting postmitochondrial supernatantswere extracted for RNA by the method of DiGiro-lamo et al. (5). Weighed portions of this RNA fractionwere examined by orcinol (14) and Lowry (16) metho-dologies for RNA and protein content, respectively,and were counted for radioactivity by using a Packardmodel no. 3310 liquid scintillation spectrometer.

Distribution of radioactivity between nuclearand ribosome-bound cytoplasmic RNA. Forty-eight5-month-old female Sprague Dawley rats were di-vided into two groups; one group was pretreated for 3days with 1% Isoprinosine ad libitum in the drinkingwater. On day 3, all animals received an intracisternalinjection of 42 uCi of [6-"4C]orotic acid in 0.02 ml ofbuffered saline. Eight animals from each group weresacrificed at 24, 72, and 120 h. Drug was continuedthroughout the experiment. Brains were homogenizedby the method described above. Homogenates werefiltered through four layers of cheesecloth, and fil-trates were centrifuged for 10 min at 2,400 rpm in aSorvall RC2-B, SM-34 rotor.The crude nuclear pellets were processed according

to a modified method of Blobel and Potter (2),

wherein the pellets were resuspended in buffer B(0.05 M Tris-hydrochloride, 0.04 M CaCl2, 0.025 MKCI, 0.2% Triton X-100) containing 1.0 M sucrose, 1ml per g of tissue, and layered over buffer B contain-ing 1.5 M sucrose to form a discontinuous sucrosegradient in a volume ratio of 1: 2. Centrifugation wascarried out in a Beckman L2 centrifuge by using aSpinco 50.1 rotor at 40,000 rpm for 90 min. Nucleiwere stained and examined under a light microscopeand were found to be, on the average, 75% intact.The supernatants generated by the 2,400 rpm

centrifugation were recentrifuged at 10,000 rpm for 20min in a Sorvall SM-34 rotor. Portions of the resultingsupernatants were extracted for total cytoplasmicRNA as previously described (5). The remainingsupernatants were centrifuged according to themethod of Campagnoni and Mahler (3), resulting inpellets rich in free ribosomes. These pellets wererehomogenized in buffer A, and portions were made to0.01 M Na2 ethylenediaminetetraacetic acid (EDTA)and 0.25 M KCl, and were allowed to incubate for 1 hat 0 to 4 C according to the method of Wilson andHoaglund (23). A 1.0-ml portion from each group waslayered over a 14-ml linear sucrose density gradient, 5to 25% (wt/vol), and centrifuged for 3.5 h at 27,000rpm in a Beckman L2, SW-27 rotor equipped withsmall buckets. The developed gradients were eluted,continuously monitored at 254 nm, and fractionatedinto 0.25-ml fractions. These fractions were countedfor radioactivity. The remaining EDTA incubantswere diluted in buffer A and filtered through anAmicon Diaflo no. XM-100A filter, 43 mm diameter.Equal fractions of the filtrates were then assayed forradioactivity.

Protein synthesis in cell-free brain polyribo-somes. In two identical studies, 32 rats (8 each of 2, 6,11, and 32 months of age) were employed per study,with the animals from each age category divided intoa control and a treatment group. Treated animalsreceived 1% Isoprinosine in their drinking water for 3days. On day 3, all animals received intracisternalinjections of [5-3H]orotic acid, 100 ,uCi/kg of bodyweight, 45 min prior to sacrifice. Polyribosomes wereisolated from brain tissue according to the method ofZomzely (24). The purified ribosomes were resus-pended in buffer A, and a 1-ml portion was layered ona 15 to 40% (wt/vol) linear sucrose density gradient.Centrifugation for 155 min at 27,000 rpm in the SW-27rotor was followed by elution with continuous moni-toring at 254 nm. The separated fractions from thesucrose density gradients were examined for opticaldensity at 254 and 350 nm. These fractions wereindividually hydrolyzed by a procedure similar to thatof Schlessinger (21).

Polyribosome samples from control and treated ratswere assayed in an in vitro protein synthesizingsystem designed to examine the incorporation of[14C ]phenylalanine into nascent protein. The proce-dures generally followed were those of Zomzely (24).The synthesizing mixtures (0.5 ml each) were run

in triplicate, incubated for 20 min at 37 C. At the endof the incubation period, 0.1 ml of each reactionmixture was assayed for protein by Lowry procedures(16). The remaining mixtures were assayed for radio-activity incorporation by elution on a Whatman no. 2

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filter paper wick (1 by 3 cm), washed with 2 ml ofdistilled water. The filters were solubilized in hya-mine hydroxide and subjected to liquid scintillationcounting.The active synthesizing system was tested for

specific amino acid incorporation into polypeptidesby deleting critical components of the system. Thedeletions were as follows: polysomes, pH 5 fraction,yeast transfer RNA, adenosine 5'-triphosphate-generating system, or guanosine triphosphate. Baseline values for in vitro incorporation were alwaystaken as that control which lacked the added poly-somes.

RESULTSTissue culture. In primary rabbit kidney

cells, the growth of herpes simplex virus 35, type1, was not affected by Isoprinosine in theabsence of, but was modestly suppressed in thepresence of, a methocel overlay. Isoprinosinereduced plaque formation by 60% by day 8; thisprotection was absent by day 10. Cortisoneadded to the tissue culture medium reversedthis modest protective effect (Table 1).

In the absence of methocel in a systememploying either primary rabbit kidney cells orhuman embryo WI-38 cells of low passage andherpes simplex virus 27, type 2, Isoprinosinesuppressed virus infectivity by 1.5 logs at 10yg/ml and by 1.0 log at 1 ug/ml.Animal virology. Isoprinosine, when given in

the drinking water, significantly suppressedmortality in hamsters infected with eitherherpes virus at an LD50 of 1.5 (Table 2). Duringthe first 4 days, the average water consumptionof animals drinking the 1.0% solution was 12.6ml/24 h (1,260 mg per kg per 24 h). The averagewater consumption of the other treated groupsin the herpes 2 study was 11.8 ml/24 h. Theconsumption of drug during this time wasinversely related to final mortality. As thedisease progressed, average water consumptionwas reduced and no attempt was made todrug-dose ill or moribund animals. All deathsoccurred between day 6 and day 12.

TABLE 1. Effect of Isoprinosine on plaque formationproduced by herpes simplex virus 35p7a with

methocel overlayNo. of plaques

Day Isoprinosineay Control Isoprinosine (20+ g/ml)Cnrl (20 Ag/ml) cortisone(5 ug/ml)

7 58 22 398 > 150 58 150

a At 100 TCID5O in primary rabbit kidney cells.

TABLE 2. Effect of oral Isoprinosine on herpessimplex mortality in hamstersa

Isoidrnking Herpes sim- Herpes sim-water plex 35, plex 27,type 1 type 2

Mor- % Mor-Concn mg/kg tality Mor- tality Mor-tality tlty

Control 11/15 73 18/25 720.33% 390 13/25 520.50% 590 9/25 360.75% 885 6/25 241.00% 1,260 2/15 13 3/25 12

1% + 5 mg of cor- 19/25 76tisone per kgaVirus dose = 1.5 LD50.

The anti-herpes action of Isoprinosine in vivowas reversed by the daily injection of 5 mg ofcortisone per kg (Table 2). This additionaltreatment did not alter the amount of drugingested by the animals in their drinking water,and had no significant effect on the controlinfection.When herpes type 2 was used at a dose of 15

LD5,, Isoprinosine prolonged the survival ofparalyzed animals, delaying death by an aver-age of 3 days, so that animals died between day10 and day 14. At this level of viral infectivity,Isoprinosine did not decrease the ultimate mor-tality frequency of 23/25, or 92%.The disease produced by these viruses, which

had been adapted to the brain, was not accom-panied by keratitis, nor did this conditiondevelop in survivors up to 30 days.Extracted cytoplasmic RNA. Isoprinosine

increased the specific activity of extracted ratbrain rapidly labeled cytoplasmic RNA as afunction of drug dose. Specific activity in-creased by approximately 40% over controls atthe highest dose used during both labelingperiods (Table 3). This effect was not accom-panied by change in cytoplasmic RNA content,control animals averaging 2.6 mg per g of tissue(wet weight), and treated animals averaging 2.4mg/g.Distribution of radioactivity between nu-

clear and ribosome-bound cytoplasmic RNA.At 24, 72, and 120 h after administration of[6-"C]orotic acid, the incorporated counts thatlocate in the nucleus are reduced by drugtreatment, whereas the specific activity of poly-ribosomal RNA is increased (Tables 4 and 5).This effect was greatest at 120 h, and occurredin the absence of a significant effect on nuclearor cytoplasmic RNA content.

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156 GORDON, RONSEN, AND BROWN

TABLE 3. Isoprinosine effect on incorporation ofradioactive precursor into cytoplasmic RNA

20-Min labeling 45-Min labelingperiod period

Drug dosage(mg/kg) Extracted % Extracted %

cytoplas- Above cytoplas- Abovemic RNAa control mic RNAa control

0 561 4 23 1,917 81125 2,176 71 +13250 2,362 72 +23500 783 26 +40 2,886 78 +41

aAll treated entries are significantly greater thanrespective controls, P < 0.05 to 0.01. Measured indisintegrations per minute per milligram.

TABLE 4. Isoprinosine effect on distribution ofradioactivity incorporated into RNA

Time RNA sp actapost- Ribosomal-bound cyto-

isotope Nuclear plasmiccinjec-

tion (h) Control Treated Control Treated

24 9.5 0.50 7.2 0.40d 1.6 0.07 3.8 0.19'72 5.1 0.26 4.9 0.26 2.8 0.15 3.2 0.16'120 4.9 i 0.25 2.5 0.14d 0.12 0.01 1.1 0.06'

aAll figures were multiplied by 10'."Disintegrations per minute per milligram of trichloro-

acetic acid-washed nuclear pellet.cDisintegrations per minute per milligram of RNA.dSignificantly less than respective control, P < 0.05 to

0.01."Significantly greater than respective control, P < 0.05

to 0.01.

TABLE 5. RNA specific activity ratio reflectingtransport

Time post-isotope Nuclear: cytoplasmicinjection (h) Control Treated

24 5.93 0.30 1.89 0.14a72 1.82 0.12 1.53 0.11a120 40.80 0.26 2.27 0.15a

aSignificantly less than respective control, P