Tumorigenicity of the antigen-forming defective virions of simian virus 40

Short Tumorigenicity Defective

Communications plaque method with primary or secondary G;\IK cells, and t,he final reading was made on day 14 (6). The T or V antigen-forming ability was determined by the fluorescent antibody technique, and expressed as T or V antigen-forming units (TZ:U or VFU; comparable units with PFU), as described in detail in the preceding paper (6). In G;\IK cultures, the number of stained cells with anti-T or anti-V fluorescent ant’ibody at 72 hours after infection is proportiona t,o the reciprocal of dilution of inoculum in the range of low multiplicities of infection (ant,iSV40 serum was added to t,he maintenance medium to prevent, secondary infection). The ratio (k) of the plaque-forming titer of inoculum to the number of stained cells was determined with a reference (i.e., dilute passage) preparation. Defining 1 PE’IT of the reference material as being equal to 1 TlcC or 1 VFU, the antigen-forming titer of a preparation containing defective virions is determined from the number of stained cells (N) and the dilution of inoculum (l/D) (TFU or VFU = IcND). In this case, TFU or VFU represents total titer of plaque formers and defective antigen farmers in the sample. For the assay of tumorigenicit,y, groups of newborn Syrian hamsters (within 3 days after birth) were inoculated subcutaneously with lo-fold dilubions of each virus preparation. The animals were observed for 10 months. The dose of virus producing tumors in 50 % of inoculat8cd animals (TDw) was calculat,ed by the method of KBrber (879). A comparison of tumorigenicity between dilute and undiluted passage fluids (supernatant at 3000 rpm for 20 minutes) was made in preliminary studies. The ratio of PFU to TD,, was found to be 1Ofi,3 and 106.” for two dilute passage materials, and 104,6 and 104.1 for materials of the second and the third undiluted passage, respectively. The results support the suggestion that

of the Antigen-Forming Virions

of Simian

Virus

40

The radiation target of the transforming ability of polyoma virus has been reported to be considerably smaller t,han that of the plaque-forming ability (1-3). These st,udies indicate that the complete integrity of the viral genome is not required for the transformation by polyoma virus. However, the oncogenicity in hamster of DI\;A tumor viruses (polyoma, SV40, and LLE 46-a hybrid of adenovirus 7 and SV40) has recently been reported to increase after ultraviolet or gamma radiation (4). Successive undiluted passages of SV40 (small plaque) in African green monkey kidney (GMK) cells result in the production of het,erogeneous defect.ive virions with lower densities, accompanied by decreased production of infectious virus (5, 6). The light virions contain shorter circular DNA molecules of various lengths depending on their dens&y (7). The presence of two classes of antigen-forming defective particles (T and V part,icles) is demonstrated in the light virion population. T part,icles produce only T antigen, and V part’icles are functional up to the formation of V (capsid) antigen in nuclei of GMK cells (6). The present report deals wit,h the results of study on the tumorigenicity of the defective virions, which st’rongly suggest that the defective antigen formers are as tumorigenic as plaque formers in the hamster. Strain 777 was employed unless otherwise stated. A twice-plaque-purified clone ‘Sa” from the strain was also used in an experiment,. The infect’ivity of both viruses decreases markedly during serial undilut,ed passages, but the production of the defective antigen formers in “Sa” is lower than that in the parental strain. The method of passage and the procedures for purification of the virus have been reported (5, 6). Infectivity (PFU) of 6he virus was assayed by the 166

SHORT

defective viruses present in undiluted passage fluids may participate in the tumor formation in hamsters. The tumorigenicity of the defective virion preparations, which MWC characterized wit,h respect to the buoyant density and biological activitk, was compared lvith that of the plaque former preparation. The samples wrc selected from appropriate fractions obtained by CsCl density gradient ccntrifugation of the purified vu~ons from the third dilute and undilut,ed passage yields. The cent,rifugation has been described in detail elsewhere (6). The specific biological activities and the buoyant, densities of the materials used in the expcriment~ are listed in Table 1. Sample FlS is regarded as a homogeneous population of plaque formcrs. :Uthough the virions of dilute passage seemed to consist of fxirl) homogeneous particles as indicated by an approximatel\Gaussian distribution of optical density at 238 mp, a small amount, of T particles was found in the fractions at densities lower than that at the peak of infectivity (Fig. 3a and Table 3 in reference 6). Therefore, the fraction (FlS) at, a slightly higher density than the peak xw selected as :I sample practically free of defective antigen formers. Samples RH22, RAI26, and RI,28 represent defective virion preparations. The distribut8ion profile of the virions of undiluted passage in a CsCl density gradient n-as broader than that of plaque formers and sl;el\.ed considerably toward lower densities TABT,E SPECIFIC

PaSSage

IXlllte Undiluted

a Fract,ions of the third b A slightly c Optical fractions of d ISstimated E \‘E’U = ’ TFU =

BIOLOGICIL Fraction

Ac~rrv~~r~~:s No.

167

COMMUNICATIOM

(E’ig. 3) and Tahlc 3 in reference 6). The heavy, middle, and light’ fractions in the first, centrifugation \\-we recenkifuged separat,cly, and the fractions (I~H22, R;\126, RL28) at the peak of each virion distribution were selected as samples each containing various numbers of defcct’ive p&iclcs and differing in t’hc buo>-ant density. The results of tlhe experiment’ with the purified virions and the crude samples of the clone ‘%a” t)ested simultaneously are summarized in Table 2. Ratios of PIPIJ, VPI,?, and TI:I! to TDbc, are also she\\-n in the table to stud!- t,he possible correlation bet,\\-een tumorigenicity in hamsters and biological activit)ies determined in GAIT< cells. III the “plaque former” and “dilute passage” preparations (MS and “Sa” dilute), t,he amount of plaque formers causing tumors in 50% of Dhe inoculkted animals was about 10” I’E’T:, as expect cd from preliminary studies. In t’hc case of undiluted passage materials, t,he ratio of I’IX to TD,, varied from OIW preparat,ion to another. Tumorigenicity (TD50) parallclcd most closely wit,h T antigen-forming activity (TFU). TI:I’/ TD5” uxs fourld to be about 10” in all preparations tested, irrespective of the types of passage, t)he specific activity of t’he sample, and the density of virions. The presence of SV40 T antigen was confirmed in all tumors found in the experiment by t,he complement fixation test. The most likely explanation of the results 1

OF FILNXION.LTI~:D

Estimated buoyant density (g/cm:‘)

F18h RFI22C

1.3

Ii1126c

1.33

RL28”

1 .a2

1.34

VIILION

PFU/particles” x 104

PRIGPAILY~IONS” VFCe/particles x 104

TFUf/particles x 10”

100 l.i

100 23

0.13

2

2-i

0.17

1

15

100 I .i

obtained by CsCl density gradient centrifugation of virions purified from infected cells dilute or undiluted passage. heavier fraction than the peak (Fly) of infectivity distribution. density maximl~m of fractions obtained by recentjrifugation of the heavy, middle, and light the first, centrifllgation. from optical densit,y (I 01) -;7,3 c,,P unit ‘=. 6.4 X lW* virions). Y antigen-forming rurit. T antigen-forming lmit.

168

SHORT

COMMUNICATIONS TABLE

TUMORIGICNICITY Preparation”

‘;y{

100

F18 RH22 RM26 RL28 “Sa” “Sa”

108.O 107-1 107.0 dilutec undilutedc

Virus

107.8 107 .9 107 .9

6/6” 8/B

6/6 7/7

f3/6 6/6

2

OF PURIFIED inoculated

VIRION

PREPARATIONS

(0.2 ml/hamster)

Virionse/ TDi,’

10-l

10-z

10-a

10-4

T/7

l/4 S/8

O/4

l/6

O/6 l/8

108.1 108.4 108.8 109.’ --y

-

F/8 3/6 7/7 4/6

W6

l/4 O/4 4/6

O/7 o/7

O/7 o/4 O/4 o/5

2/6

O/6

o/a

PFU/ TDao 106 1 105.6 10” .9 104.” lo”.’

105.7

VFW/ TDjo 106.’ 105.6 105.1 105.1 10~~’

105.7

$FDy”:( : 106.1 105 8 106 2 lO6.8 loo.’

10” .I

(1 Specific activities are shown in Table 1. Samples were diluted to give approximately equal T antigen-forming activity (TFU). b TFU = T antigen-forming unit. c Culture fhlid of the dilute or the third undiluted passage of the plaque-purified clone “Sa” of strain 777. d Number of hamsters with tumors/total number of hamsters bearing tumors and surviving without tumors. Observation period: about 300 days. e Estimated from optical density (1 OD 208 ,r,a unit ‘F 6.4 X 1Ol2 virions). J TIY = 50% tumor-forming dose calcldated by the method of KLrber. Q Not determined. h VFU = V antigen-forming unit.

is that all types of T antigen former (plaque formers, V and T particles; TFlJ represents total titer of these particles in the sample) are equally t’umorigenic in hamsters, regardless of the lengt’h of circular DNA molecule contained therein. The explanat’ion is based 011 the assumption that the plaque former does not multiply significant,ly in hamsters. The assumption appears to be reasonable, since the multiplicaCon of SV40 in hamsters is not demonstrated during pretumor period (10) and t’umor induction in newborn hamst,ers by polyoma virus takes place rapidly and directly, and the viral replicat,ion, if any, does not seem to play any significant role in t’umorigenesis (II). The explanation is consistent with the data indicating t,hat orlly a part of viral genome is required for the transformat,ion of target cells (1-S). It may be compatible xvith the report by Defend1 and Jensen (4), since irradiation of the virus with ultraviolet light or y-rays may induce some chemical alterations in the genetic material which may increase the efficiency of tumor formation. The other possible explanation may be that the t,umorigenic ability of the plaque formers present in the light fractions is about, one hundred times as great as that, of

the reference plaque formers. These two explanations may not be mutually exclusive. A possibility that the multiplicity reactivation of defective virions is involved in the tumor formation also may not be ruled out complet’ely from the present studies. Further investigation to elucidate t,hese questions is in progress. It has been reported that infectious SV40 can be recovered from “virus-free” tumor or transformed cells by the method of “contact hvith susceptible cells (GMK) culture” (12, 13). However, this has not been successful in all instances (14, 15). It) is quite conceivable that no infectious virus would be recovered from t,he cells transformed by the defective viral genome even by the application of the improved cell fusion technique (16, 17). Attempts are non- being made to determine the efficiencies of the recovery of infectious virus from mouse 3T3 cells transformed by “plaque former” and “defective virion” preparat’ions. REFERENCES

1. BENJAMIN,

T. L., Proc. Natl. 54, 121-124 (1965). S. BASILICO, C., and DI ?ulauo~ca, ilcad. Sci. Lr.S. 54, 125-127

Acad. Sci. U.S. Cr., Proc. (1965).

AV~tZ.

SHORT 3. L.\T.~RJI;T, 1~ ., CII.\MI-R, II., and L., r~~imzogy 33, 104-111 (1967). \:., atlld JENSI~N, F., 4. I)ISFI:NDI, 703-iO5 (1967).

5.

7. 8.

MONTAGNIEIL,

Science

Gi,

s., and KATZ, At., (1966). UCHII).\, 8., I-OSHIII ad FUI~UXO, A., Virology 34, l-8 (1968). Y0SHIIIil:, Ii., ~‘iT’O~Ogy in pIE%S. K.%I~I~~.II, G., .t~~h. Ezptl. Palhol. I’hurtnukol. 162, f8OkJ83 (1931). UCHII1.1,

s..

~~.\T.zN.%III~:,

l’iroZog~/

6.

COMVlUNICSTIONS

28, 1X-141

9. 10. BIAU<,

1’. II.,

Cancer

11.

kHk:L,

K.,

12,

12.

(;I:RUI:R,

13.

BL.I~‘I~,

:llld

P.,

J.

Sail.

It.

J.,

vi?dOgy

11., J.

28,

Xall.

501-509

Cancer

(1966).

Inst.

37, 487-

(1966).

J. L.,

MLLNICI~,

23,

1, \NI).\IJ,

B.

:illd

Uiol.

17.

w. (1964).

sILVI~2RHkXLG,

1.irdogy

I’.,

I’.

h.,

16.

Rowe:, 253%26.?

(1960).

l’indogl/

15.

32,

46%-ii6

493

14.

md

Inst.

J.,

K. S., ad

KHI~I~.~,

X1OGi32 L.\RSON,

~~IIrLII\1.\N.

RAPP,

F.,

(t964). LT. hI., Al.

It.,

L>IsWRS,

P,‘OC.

SOC.

ct. E.f:@.

.llctl.

122, 1174-1182 (1966). II.. JP:NSISN, F. C., and Rs~‘~:~~L1.x sm, Z., I’tw. :\:afl. .1ccctl. Sci. U.S. 58, 127-133 (1967). R.\~arss, J. s., :md I~uLI,I~:c~Y~, It., Proc. ?Vctll. .tcarZ. Sci. I:.S. 38, 1396.-1403 (1967). KOPRI)VSI~I,

Effect the

of Magnesium Infectivity Mosaic

s.

UcIIII~.\

s.

WATANAl3I~:

Trisilicate

on

of Tobacco Virus

Hecht-I’oinar and Yarwood (2) reported that’ magnesium trisilicate increased t,he infectivit’p of some crude-juice viral inocula, but that* it’ had only a slight effect on tobacco mosaic virus (TMV) in tobacco extract or in purified preparations. In our screening t,est,s for chemicals influencing viral infections, magnesium t’risilicate increased t’he infectivity of purified preparations of TMV when assayed on Scotia beans. This finding prompt,ed us to investigate the mechanism of magnesium trisilicate action in increasing

16’3

the infect,ivity of viral inoculum. This paper gives result,s lvhich, in part,, confirm the report, by Hecht-l’oinnr and Yarrvood and, in addition, suggest,that, the chemical acts primarily as an abrasive. Virus used was a Rockefeller Institute subcult,ure of tobacco mosaic virus, Marrrror tabaci Holmes, maint,ained in living t,obacco plants. Partially purified virus (2.X mg/ml) was prcparcd .b~.t,hrec differen&d sedimentat,ions from infect’ious crude juice of Turkish t,obacco, Nicotiarra tabaculrhI,. Magnesium t8risilic:tte (Mallinckrodt~, U.S. 1’. powdcr for prescription compounding) was easily suspendedin wat,er. Its solubility in dist,illcd wat#er was 14.9% as determined by the dry weight of tbc supernatant liquid (1000 CJ10 minutes) of a 1% suspensiondried to a constant, weight at 60” and 60 mm of mercury pressure. Aqueous suspensionswere pH 93. Virus infectivit,v was assayed by inoculnting the upper s&ace of primary leaves of a single-plant selection of Scot,ia beans, PAaseolus vulywis I,. (3). Treatment and cont,rol inocula were applied to opposite leaves 14 days after planting the seed.l’rior to inoculation all inocula were adjust,ed to pH 7.0 lvith 1 N HCl or KOH unless stated othcrmisc. Infcct~ion index, as used herein, is the ratio of the number of lesions from the treaiment to that of t,he control. To ascertain whether the infect,ionincreasing cnpacit,y of magnesium trisilicat’e is associated with the insoluble par-ticulates or the soluble fract,ion, a 3.5 % aqueous suspensionwas filtered t,hrough a membrane (0.1 p pore size Bacto-T-Flex membrane) at GOmm of mercury pressure. The residue was resuspendedin waier (original volume). The liquids were mixed with virus, adjust,ed to pH 7.0, and assayed for infect)ivity. The residue suspension(Table 1) increased infectivit,y 5 times more t,han did the filtrate. The increase evoked by each of t,hese two liquids separately, however, was ahout half t,he increase from recombined residue and filtrat,c, or the unfihered suspension. Thus t,he infect8ion-increasing factor(s) was not appreciably affected during filtration. To assesst)he relation of t)he amount of