Inhibition of Lymphocytic Choriomeningitis Virus Multiplication

by 2-(a-Hydroxybenzyl)benzimidazole

It has been reported that Z-(w-hydrosyberl~y-1).belizimidasole (TTBB) selectively inhibits only the picornaviruses. However, WC have found that three stains of LCM vinx-Traub, WCP, and C:Sl371-when grown in IIeLa cells, are inhibited by HBB. In all cases HBB, at, 55 MN, inhibited virus production by 70-90$;. HBB at 220 PAI did llot affect the multiplication rate of noninfected cells, bllt did noticeably inhibit multiplication 2 days after infectiotl. Furthermore, 220 ml IIBB did not change t,he in vitro inaclivatioll ra1.e of the virus, nor its adsorption to cells. It would appear then that inhibitiou of virus multiplication by IIBB is not entirely restricted to defined picornaviruses. IIowever, unlike other IIBB-seltsilivc viruses, the multiplication of LCM was not inhibited by 700 roll gllanidine hydrochloride.

INTRODUCTIOiX

The exist.ing information on basic charac- teristics of Iymphocgtie ehoriomeningit,is is not \-et) sufficient for a sound taxonomic class&:~tion of this virus. Its presumed RX.4 content and ils lahilit’y in et#her and high concentrations of divalent cations have let, to the suggestion t,hat t,he virus could belong t.o either the myxo- or arbovirus groups (I’fau et ab., 1965). Holvever, the report that 2-(aclh~droxybcnz;l)bcrleimida- zole (HBB) inhlbks LC1I strain SY621 (l<‘urus:l\\-a et al., 1964) is not in accord with the above conclusion. It has ‘been established (I<ggcrs and Tamm, l%(i) that HBB selec- tively inhibits many of the picornaviruscs, and 11:~s no effect on major groups of viruses such as arbo-, myxo-, reo-, herpes-, and pox- viruses. It, has been shon-n in several labora- tories that LCM is ct)her sensit)ive (An- dren-es and Horstmann, 1949; Pfau, 1965). E’rom this it \\-ould seem clear that LC1\1 does not ‘belong to the picorna virus group, but t)his is t.he only group reported to bc in- hibited by HBB.

The present report confirms the HBB sensitivit,y of strain SYGfLl, but sho1v-s that this is not a Iymphocytic choriomeningitis

virus. However, synt,hesis of classic LCM strains is indeed sensit#ive to this compound, and represents a primary exception to the narrolv virus-inhihit)ory spectrum of HBB.

MATE1~ISLS AND METIIODS

YGuses. The origin and passage history of the CA1371, WC]‘, and Traub strains of I/231 have been previously described (Camyre and I’fau, 196s).

T\\-o samples of the tissue culture-adaptled NY621 J/Z11 strain lv-ere obtained from Dr. E. F’uruxnn-a. The first had been passed 58 t’imes in KB cells before shipment and tlvice in HeT,a cells in our laboratory. The second sample, passed 65 times in KB cells, KM used direct)ly in our experiments. Both virus stocks, for the experiment#s reported here, exhi’bited the same degree of sensi- tivity t)o HBB. Polio type 1 \vas from Dr. S. Penman, and the WR strain of vaccinia uxs from Dr. W. Joltlik.

ConzpountIs. HBB (lot Ko. 579559-O-7) was a generous gift from ?\lerck, Sharp, and Dohme Research Laboratory, Rahx-ay, Nelv Jersey. It \vns suspended in Eagle’s basal medium (minus glutamine and bicarbonate) at, a conccnt~rntion of 54.5 ,ug/ml. The HBB

375

376 PFAU AND CAMYRE

was dissolved by shaking in a mechanical shaker for 1 hour at 37”. The solut’ion was stored at -20” until needed, and then made up as complete BME plus 5% calf serum. The final HBB concentration was 49 pg/ml (219 PM). Guanidine hydrochloride (ultra pure) was purchased from RIann Research Laboratories, New York. It was suspended and stored as above. The final guanidine HCI concentration was 67 pg/ml (700 PM).

CPE INHIBITION BY HBB

-NONE; ----219 PM

Cell cultwes. HeLa cells n-ere obtained from Dr. J. Hotchin (St,ate of New York, Department of Healt,h, Albany) and were propagated (at 37”) on 32-ounce prescrip- tion bott’les with Eagle’s basal medium plus 5% calf serum. For experimental use these cells were trypsinized, as previously de- scribed (Pfau et al., 196.5), and dispensed (4 to 6 X lo5 cells/s ml unit) in small plastic tissue culture flasks (Falcon Ko. 3012); 48-72 hours later they were used as described below.

Measurement of infective virus. The -50 % end point procedure (TCID,,) was used for titrating polio, vaccinia, and the NY621 LCM strain. The HeLa cultures used for this are described above. The end points of the titrations were observed on day 3 for polio and LCM NY621 ; and on day 6 for the vaccinia (in the latter titration the medium was changed on the t#hird day). The three other LCM strains, which nor- mally induce no CPE in tissue culture, were assayed in mice (Camyre and Pfau, 1968). lOOF

Determination of vi,rus inhibitory activity of HBB and guanicline HCZ. Cell cultures, as described above, were inoculated wit’h 100 TCIDSo of the CPE-inducing viruses and incubated in the presence or absence of HBB. Cultures receiving no virus were also incubated with or without HBB, and served as further internal controls. Groups of 3 or 4 cultures were used per variable. Incuba- tion was continued at 37”, and the cultures were observed by phase contrast microscopy every day. The same person estimated the amount of CPE in all experiments. For the

75 -

VACCINIA WR

I 2 3 4 5 6

DAYS polio and NY621 virus, this was based on the percentage of cells in the originally

FIG. 1. Specificity of HBB on virus replication. c on uent fl monolayers of HeLa cells, with or

confluent monolayer that had disintegrated, without HBB in the growth medium, were infected plus those that showed cellular retraction. with 100 TCIDW units of each of the viruses. The For vaccinia, this was based on the per- amount of CPE was recorded at daily intervals.

centage disintegrated plus those w&h ab-

INIIIBITIOS OF LCM VIRUS BY IIBB 377

normal morphology. The amount of CPE could be determined with an accuracy of ZtlO’X’.

The growth of LCRI virus strains inducing no CPE was monitored essentially as above except that at 1.5 hours before exposure to virus the medium was decanted and re- placed \vit,h the same volume (5 ml) of BRIE containing a specified amount of HBB Or

guanidine. At the time of infection, the medium was decanted and replaced with 1 ml of LCN (in BME minus HBB or guani- dine with a log,,, LDBO of 6.0/0.03 ml). Aft,er 1 hour at 37” the virus n-as poured off and the monolayers were washed three times n-it)h prewarmed BME. The medium was then replaced to the original volume, with or without HBB-the same as that used prior to the virus adsorption period. Sam- ples, 0.1 ml, were withdrawn from the flasks each day and assayed in mice. In t,his case growth curves of t’he HBB-treated cultures were expressed as percentage values of the maximum titers (usually with log,, LD50’s of .>.0/0.03 ml) achieved in nontreatcd cultures.

RESULTS

@feet of HBB on Virus Synthesis in HeLa Cells.

Figure 1 represents our data on the effects of HBB using previously investigated viruses. The curves for polio and vaccinia closely follow those already published (Eggers and Tamm, 1961). It is clear then that the delay in appearance of CPE in- duced in cells infected with t’he tissue cul- ture-adapted NY621 virus, is related to a specific effect of HBB. Under the same con- dit,ions the non-CI’E inducing strains of LCJ1 \vere also inhibited (Fig. 2). It should he noted t,hough that there is a basic differ- ence in t)he type of inhibition observed, With the Cl’E-inducing viruses t’here was no permanent’ inhibition but only a delay of about 1 day. With the ot,her viruses, however, t,here was an absolute inhibition of virus synt’hesis.

Even at, concentrations as low as 55 ~‘11 HBB there was significantly less virus pro- duced than in t,he controls. The highest concentration of drug used (219 PM) had no

INHIBITION OF LCM MULTIPLICATION BY HBB

- NONE . . . . . . . . . . . . . 5SpM .-.-.-. llOCIM ---- --21gpM

DAYS

FIG. 2. EKecl of IIBB 011 classic LCM slrains. Collfll~ellt monolayers of TIcLa cells, wilh or witholLt> variolls concentjratiotrs of IIBB in the growth medillm, were itlfect,ed with 107.G L&o units of each of the LCM strains. Noue of the cultures exhibited CPE, bllt they wcrc sampled and assayed ilr mice.

378 PFAU AN11 CAMYRE

effect on the mult,iplication of the non- infected HeLa cells, but’ it did not,iceably slow down the division rate of LCRI-in- fect.ed cells between 24 and 4S hours after infection. Under normal conditions the multiplication rate of the LCl\I-infected cells, in our laboratory, is slightly higher than in noninfected controls. It, should be noted here that not every strain of HeLa cells is suitable for the above t,ypes of ex- periments. Three HeLa lines (two from Grand Island Biological Company, one from Flow Laboratories, Inc.) gave no indication of supporting LC:\I replication. However, Trauh LCJl would grow on a strain of HeLa cells used by one of us (C. J. I’.) several years ago at Stabens Serum- instituh, Copenhagen, Denmark.

Effect of HBB on the Atlsolption of the TT*aub Strain of LCM Virus to HeLa Cells

Two confluent monolayers (grown in plastic flasks as outlined in the section on cell culture) were exposed to fresh medium, wit,h or without HBB, 1.5 hours before in- fection. The medium was then decanted and each flask received 1 ml of virus with an initial LD,, of 4.1 X l@/ml. After 1 hour at 37” the medium from each flask was de- canted, pooled wit’h those from the three succeeding washings, and then assayed. The amount, of virus t,hat could be recovered (nonadsorbed and/or reversibly adsorbed) from t’he HBB-treated cells represented 29 % of the input. Recoverable virus from the controls represented 35% of t,he input.

Inactivation of the Traub and WCP Strains

Both st,rains (with initial LDcO’s of 6.0,’ 0.03 ml) were diluted IO-fold into Eagle’s BRIE plus 5 % calf serum, wit’h or without 219 PM HBB. The infectivity of the prepara- tions was determined. The tubes were stoppered tightly, incubated at 37”, and at 6- or 12-hour intervals were sampled and assayed immediately. The duration of the experiment was 24 hours. As expected with virus in t’he presence of serum, there was less than one log unit drop in infectivity/O.03 ml (Pfau and Camyre, 1967) at the end of this period. All differences in LDSO’s with

or \I-ithout HBB, were lvithin the st,andard error of the titration technique (ho.3 log/ 0.03 ml).

E,ffect oJ Guamdine HCl on Vims Synthesis it2 HeLa Cells

Confluent monolayers of cells, \vith or without 700 PM guanidine in t.he growt,h medium, mere infected with 100 TCIDSo units of polio. At 24 hours after infection the untreated monolayers exhibited 30% CPE which increased to 55% after an addit,ional 8 hours of incubation. At both time intervals the guanidine-treated cultures appeared identical (no CI’E) to the noninfected con- trols. When confluent monolayers were in- fected with 107.5 LDso units of the CA1371 strain of LCJI, with or without 700 & guanidine in the growth medium, there were identical increases in t,iter (1.3 log,, LDsa units/O.03 ml) within the 24-hour postin- fection test period.

ChamcteGdcs of the Tissue Culture-Adapted LCM NY62?1 Strain

The highest concent,rat’ion of virus (lo5 TCIDsO) used in t,he following experiments was not pathogenic for mice. Since this was in striking contrast to the results obt.ained with other LC:\I strains, we wished to see whether bhis virus could protect mice against future challenge with classic LCM strains.

This was found not to be the case. If mice were injected (i.e.) with lo2 or lo5 TCID,, units of the NY621 virus 21 days prior to challenge 1vit.h the Traub strain (normally causing death 7-10 days after infection), the same LDS, was obtained as with mice originally injected with saline. Other proper- ties were then examined. The NY621 strain could not induce a carrier state when in- jected into newborn mice, and also would not produce a foot pad response (Hotchin, 1962.)

Furthermore, in our laboratory, the NY621 strain was found to be ether re- sistant. All other LCM strains that we have examined are ether sensitive (Camyre and Pfau, 1968). The NY621 strain was then sent to t#he Virus Reference Unit, National Communicable Disease Center, Atlanta. It was typed as a picornavirus.

INHIBITION OF LCM VIRUS BY lTBB 379

UISCESSION

Our initial interest in t#he NV621 strain was because of it,s ur~usual ability to produce CPE in HeLa cells (IJurusawa et al., 1964). Of the many reports in the literature of LC-\ I -induced cell damage (Ackermann, 1960; Benson and Hotchin, 1960; Chastel, 1963; Eagle et al., 1956; I’arikh, 1961; Stul- berg et al., 1956), the NY621-HeLa system was the only one \ve have found to give consistently cyt’opathic effects. Since use of an LCi\I strain t,hat would produce CPE in tissue culture would ‘bc of great practical advantage, \ve wished to examine it ext.en- sively. None of the tests with this virus, described above, indicated any similarities to the \vell documentJed properties of IX11 viruses.

Aside from the above findings, one report in t,he literat,ure still led us to check the HBB sensitivity of the more classic strains of LCJI. Yamazi and his co-workers (1963) found t,hat very high amounts of HBB (196- 347 pm) ret,arded the appearance of cyto- pat,hic changes in measles virus-infected cells, but the extent and reproducibility of the action was not too high. Our result,s (Fig. 2) clearly indicated that the three strains of LCR[ used, which produce no CPE, were inhibited by a rather low concen- tration (55 PM) of HBB. This compares favorably with the concentration range of the drug used by Tamm and Eggers (1962). They considered a virus to be inhibited if, in the presence of 219 ~!1/ HBB, the extent of virus-induced ccl1 damage was less than 25% of that in the unt’reated controls. The effect w-as observed with most, but not all, suscept,ible viruses in the presence of 98 pflf HBB, and for a few viruses with as little as 70 pM.

Although the mode of action of the drug is not8 known (Eggers and Tamm, 1966), n-e have explored most of the possible mecha- nisms previously considered. Our results mimic those of Eggers and Tamm (1962) in that (1) at virus-inhibitory concentrations the compound had no effect on normal host cell mult8iplication, and in our case evc)11 with virus-infected cells (since LCM induces no CPE) within the first 24 hours p.i.; (2) the compound had no effect on the direct in- activation of the virus; and (3) the com-

pound did not affect adsorption of the virus t’o cells. Because of limit,ed ‘biochemical knowledge of the synthesis of LCM virus, we were not a’blc to undertake experiments t,o show, as did Baltimore et al., (1963) \\-ith poliovirus, that, HBB inhibits the appear- ance of a virus-directed RNA polymerase. Thus, up to t,his point, \ve have 110 evidence to suggest a dissimilarity between t#he inhib- itory mechanism of HBB with respect t’o the LCnZ- and polio-systems.

IGnally, it should ‘be mentioned that Tamm and Eggers (1962) found that picornaviruses, on the basis of their scnsi- t’ivity to HBB and guanidine, could be grouped as follows: (1) HBB and guanidine susceptible; (2) HBB insuscept’ible, guani- dine susceptible; and (3) HBB and guanidinc insusceptible. LCM, an ether-sensitive RNA virus, does not fall into any of these groups in that it is HBB susceptible and guanidine insusceptible (700 PM guanidine, a concen- tration howl to inhibit all guanidine-sus- ceptible picornaviruses, had 110 detectable effect OII t’he synthesis of LCAI).

At t’his time then, the conclusion to be drawn from t,he result,s is t,hat HBB does inhibit, LCM virus multiplication and is a valid except#ion to the findings, thus far ob- -tained, t)hat’ only certain picornaviruses are affected by the drug.

This study was support,ed by t,he IJ. S. Pltblic Health Service Grant, AI-OG735 from the National Institute of Allergy and Infectia LIS Diseases and Research Career Program Award (5-K3-GM-8494) from the National Institllte of General Medical Sciences. The able technical assistance of Mrs. Judith Madsen is acknowledged.

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