The mechanism of genetic resistance of chick embryo cells to infection by Rous sarcoma virus-Bryan strain (BS-RSV)

The Mechanism Infection

of Genetic

Resistance

by Rous Sarcoma

of Chick

Virus-Bryan

Embryo

Strain

Cells

to

(BS-RSV)

The early steps of Rous sarcoma virus-Bryan strain (BS-IMY infection of genel.itally resistant and susceptible chick embryo cells were investigated. The methods used were t)he uptake of virus bv cells grown in monolayer, and the ability of infected cells to form foci of transformed cells after plating on resistnut and susceptible assay plates. It was found I hat the initial attachment of virtls to either genetic type of cells did not differ appreciably. Attachment 011susceptible cells was immediately followed bl penetjration, which TVBS70% complete nft,er 1 hour. Attachment on resistant cells u-as not followed by penetration of the ~11 membraue. It was concllxled that t,he genetic block of resistance 1o infecl ion cjf chick embryo cells by BS-RSI- occlux at t hr step of viral peuet rat iou of the cell membrane. INTIZOIIUCTIOn’

At least two distinct subgroups of avian tumor viruses (A and B) have been recognized so far on the basis of host range property (Vogt and Ishjzaki, 1965) and antigenic similarities (Ishizaki and J’ogt’, 1966). (Anetically determined ccllulnr resistance to Rous sarcoma virus (RST’) appears to act selectively toward these viral subgroups (Vogt and Ishizaki, 1965; Payne and Biggs, 1966). The susceptibility of cells to infection by RSY of subgroups &I, (Raters and Burmester, 1961; Payne and Biggs, 1964b) and B (Payne and Biggs, 1966; Rubin, 1965) appears to be governed by nutosomal genes, located at independent loci’ with susceptibilit’y dominant over resistance. Certain of the RSV &rains are defective in the sense that msture virus particles are not formed in the absence of a concurrent infection with an avian leukosis virus or so-called “helper virus” (Hanafusa rt al., 1963). The “helper virus” donates it,s outer prot’ein coat to RSV particles, and therefore determines several important phenotypic properties. 1 Personal communication from l>r. L. Crit,tellden, Regional Poultry I,aboratory, U.S.D.h., I’:ast Lansing, ?rIichigan.

These include host range and virus interfcrencc patterns (Hanafusa, 1965) as well as thcl immunogenic properties associated with the protein ma,terial of the outer coat (Ishizaki and Vogt’, 1966). Since the phenotypic properties of RSV are determined by the outer coat only, Rubin (1965) and others (Vogt and Tshizaki, 1965) have suggested that the cellular genes which control resist,ance patterns to RSV could regulate Ihe synthesis of wceptors involved in the early steps of infection, namely virus adsorption or penetration. The present, studies are primarily concerned wit,h the early strps of infection of BS-RSV in chick embryo cells derived from a highly inbred strain of chickens (line 7) resistant, to BS-RSV (Waters and Burrnester, 1961). The basis of this rcsist,ancc has been demonstr&d t,o be exclusively genetic in origin (Vogt and Ishizaki, 196.5; Waters and Burmcster, 1961; Crittenden ct.al., 1963). Shortly after infection with US-RSV, genetically resistant and susceptible chick embryo cells were examined for the ability to form foci of transformed cells under :I vnri+of cxperimcntal conditions, when plntrd on monolayers of one cell tyJw or t,he 700

GEXETIC

RESISTANCE

other. Both types of cells in monolayer cultures were examined for the ability to remove BS-RSV from a fluid suspension of viral particles. The results pf these studies are presented in this report. RfAT&RISI,S

ANL)

METHOIXS

1. Chicl
TO RSV

‘701

@SV has been characterized as a subgroup h type virus (Vogt and Ishizalti, 1965). R. Prepalption oj” assay plates. Secondary cultures of chick fibroblast cells were prepared from embryos 9-11 days old; the general procedures used wcrc described bg~ Rubin (1960) and T&n and Rubin (195s) The assay plates consisted of 1.5 X 10F cells incubated overnight in ;i ml of secontlar:, chick cell growth medium. The medium contained 11 199,2 O.SO; calf strum, 0.10; tryptose phosphatcl broth, 0.10; supplemented with penicillin G and streptomycin. X11 cells were grown in I”:ilcon plastic cow taincrs (60 X 15 mm), and all incub:lt,ion was done at 37” in a carbon dioside-rich humidified xtmosphcrc. The plates were used for the assav of free virus as w-e11as for t,hc assay of infeclcd cells for focus formation. In dither case the fluid was decanted and cells or virus allowed to attach to the monolayer for 1 hour bc>fore adding hard agar overlap medium, which consisted of serondary growth medium plus 0.007 p:lrt Difco Purifkd Agar.” 3. BS-RSTY. BS-RSV was obtainctl from Dr. Ra!- Bryan of the Sat~ional Cancer IrAit’ute as lot, number CT 916. A pool of CT 916 was prepared from supcrnat~nnt fluids of infectctl ~11s after one passage in cell cultures of MYt8mcr H 3 embryos. The titer of the pool was 5 X 10” 1WU per millit,cr of fluid. 4. Assay of BS-RSI’. The assay of BSRSV was based on the property of this virus t,o induct, foci of trannformc~d chick fibroblast’ ~11s using t)he proccdurc described by Temin and 1tubin (195s). B8-RSlT ~1s adsorbed on duplicate assay plates for 40 minutes at 37”. The cells mcrc then overlaid with hard agnr nut’rient medium :md reincubnkl for *j--7 da\-s in a hurnidifictl chamber gassed with k-5 ‘; carbon dioxide. The foci wcrc counted at, about’ 30 X magnificntion using a Zeiss Inverted microscope, ant1 the average number of foci detcrminctl.

regist,ctr as focus formers after plating on cell monolayers prepared from susceptible or resist,ant embryos was done generally according to the technique described by Rubin (1960). Duplicate assay plates were infecOed with virus at a multiplicity of infection equal t,o about 1.0 (m.o.i. = 1.0). Virus was adsorbed for 1 hour at, 37”, the cells were washed thrice with 3-5 ml of Tris saline (pH 7.4) to remove unadsorbed virus and were reincubated for an additional 5-24 hours in fresh growth medium. The appropriate cells of each embryo were then pooled, packed by low speed centrifugation, and resuspended in fluid medium. When appropriate, the cells were disrupted by three cycles of freezing and thawing in a dry iceacetone bath mixture, and aliquots of whole or disrupted cells were then added to assay plates in volumes of 0.1 ml per plate. The plates were then incubated for 1 hour at 37” to allow attachment of the cells to the monolayer, and tested for focus formation as previously described. Control studies on each step of the infected cell assay indicated that only virus attached to either whole or disrupted cells served as a source of focus-forming activity. 6. Sttachment of BS-RSV to chick fibroblast cells. Chick fibroblast cell monolayers prepared from pools of cells derived from e&her resistant, line 7 or sensitive Kramer embryos were inoculated with 0.1 ml of undiluted BS-RSV and incubated at 37” for various periods of time. Kne-tenths milliter of M 199 was then added to the monolayer to harvest unadsorbed virus. The virus was st,ored at, -62” until assayed for residual focus-forming activity on susceptible cell assay plates. 7. Treatment of infected cells with BS-RSV antibody prior to plating th,e cells for focus formatio~l 011 assay plates. BS-RSV hyperimmune turkey serum was obtained from Dr. Ray Bryan of the National Cancer InstiMe as lot number AR 7. A 1: 100 serum dilution neutralized 5 X lo5 FFU of virus. Assay plates were inoculated with 0.2 ml of undiluted virus, and adsorption was allowed to occur for 10 minutes at 37”. All plates were then washed 3 times wit’h 5 ml of Tris saline and 2.0 ml of a 110: dilution of t#urkeO

AR 7 serum was added per plate O-60 minutes after adsorption. The plates were then incubated for a total time period of 5 hours after adsorption with turkey serum in place. The AR 7 t’urkey serum was then removed from all plates by three cycles of washing as before, and the cells were then tested for focus formation as previously described. RESULTS 1. Plating of Whole ancl Disrupted Cells for Ability to Form Foci of BS-RSV Transformed Cells 5-6 Hours after Infection Resistant and susceptible cell monolayers prepared from individual embryos were inoculated with BS-RSV and incubated at 37” for 5-6 hours. Whole and disrupted cells were then tested for focus formation on resistant and susceptible cell assay plates as described earlier. The results of two experiments with 3 susceptible and 7 resistant embryos are given in Table 1. The fractions of plated whole cells derived from 3 susceptible embryos which formed foci on susceptible assay plates ranged from 0.0561 to 0.0969. The fractions obtained are about the proportions expected for susceptible cells plated shortly after infection (Rubin 1960). Comparable fractions of disrupted cells ranged from 6- to Sl-fold less. The fractions of whole cells derived from line 7 resistant embryos which formed foci on susceptible assay plates ranged from 0.0089 to 0.0456. Comparable fractions of disrupted resistant cells were only 2- to 4- fold less. These results suggested that virus adsorption occurred on both resistant and susceptible cells. Probably, virus adsorbed on SUHceptible cells penetrated and %ncoated,” but virus adsorbed on resistant cells remained at the cell surface. Such resistant cells, when plated on susceptible assay plates, could be expected to score as focus formers almost as efficiently for disrupted cells as for whole cells. This would not bc the case for susceptible cells. The results of the platings on line 7 assay plates strengthens this suggestion. In this case, foci which form on line 7 assay plat’es could only occur by growth of transformed cells, since BS-RSV cannot multiply in re-

GENETIC

RESISTBNCE

703

TO RSV

TABLE 1 FK.KTIONS OF CELLS DERIVED FROM BS-RSV SUSCEPTIBLE AND RESISTANT EMBRYOS WHICH INDUCED Focus FORMATION ON Ass.*u PLATES WHEN PLATED $6 HOURS AFTER INFECTION Fractions obtained with cells plated on Number of cells plated

Source of cells

Susceptible embryos wa #I Kc xl K ~2 Resistant embryos L7b #l L7 n2 L7 13 L7 #4 I,7 ~5 L7 #6 L7 #7

Susceptible assay plates Whole cells

Disrupted

cell

6,000 7,130 12,100

0 0561 0.0819 0.0969

0.0093 0.0019 0.0012

(6)” (43) (81)

md 0.0239 (3) 0.0336 (3)

ND 0.0000 0.0001

4,800 7,100 7,300 9,300 6,600 8,900 7,300

0.0089 0.0193 0.0278 0.0234 0.0463 0.0422 0.0456

0.0042 0.0112 0.0145 0.0140 0.0175 0.0112 0.0105

(2) (2) (2) (2) (3) (4) (4.l

Nl) o.oooo 0.0000 0.0000 0.0000 0.0000 o.oooo

ND 0.0000 0.0000 0.0000 0.0000 0.0000 0.0040

a Witmer chicken embryos. b Line 7 chicken embryos. r Kramer chicken embryos. li Not done. e Figures in parentheses indicate magnitude on susceptible assay plates (column 3 divided

F.,

and

Disrupted cells

whole cells plated

2.1

-0

IO

20 TIME

(MIN.)

FIG. 1. Attachment susceptible cells.

.

SS-RSV

0

RESISTANT

a

SUSCEPTIBLE

30 AFTER

40

CONTROL CELLS CELLS

50

60

INOCULATION

of BS-RSV

t,o resistant

and

on Susceptible and

The resuks of the previous experiments suggested that virus at.tachment occurred on 4 Piraino, data, 1965.

m’hole cells

of decrease compared to homologous by the indicated column).

&ant cells4 (Payne and Biggs, 1964a). Whole cells derived from susceptible embryos formed foci at a rate of about 3-fold less than that obtained when these cells were plated on susceptible assay plates. Whole or disrupted cells derived from resistant embryos failed to form a single focus. These results conclusively showed that foci which developed on susceptible assay plates after plat’ing with cells derived from resistant embryos were not BS-RSV transformed line 7 cells. It should be noted here that disrupted cells of Kramer No. 2 embryo, when plated on line 7 assay plates, formed foci at a rate of about 1 per 10,000 cells plated. From this finding it was estimated that the freeze and thaw procedure used in this study killed about 99 5%of t’he viable cell population. 2. Attachment of Virus Resistant Cells

Line 7 assay plates

Solomon,

J.,

unpublished

resistant as well as susceptible cells. An attempt was made to determine whether attachment on resistant cells differed in any way from attachment on susceptible cells. Virus was inoculated on resistant and susceptible cell monolayers; at various

n n

SUSCEPTIBLE

0 RESISTANT

Generally, no differences in the at,tacbmerit of virus to susceptible and resistant cells were observed. It appears that virus attachment on resistant cells is at leas: as efficient’ as attachment on susceptible cells.

CELLS CELLS

3. Fate of BS-RSV Attached to ResistaTlt Cells

OL 0

6

TIME E‘IG.

2.

susceptil>le infection.

12

(HRSI

xnmber

disrupted

AFTER

0

0

18

24

INOCULATION

of foci indl~ced cells plated

by

resistant

G-24 hours

and

after

periods of time the virus fluid was harvested and teskd for residual virus activity as described. The result’s of this experiment, are given in Fig. 1. About half of the virus activity was rcmoved by both susceptible and resistant cells within 1 minute aft,er inoculation. The init,inl rapid rate of attachment of BS-RSV on chick cells was reminiscent of the adsorption of certain myxoviruses on red blood cells (Burnet, 1952). The flat arm of the experimental curve probably represent,s, in large part, the t’hermal inactivation of unadsorbed virus, since the slope of this portion of the curve is similar to the survival curve of BS-RSV inoculated in the same manner but in the absence of cells (BS-RSV control). The maximum amounts of attached virus (the plateau region of the curve) appear to be similar for both types of cells.

a. Plating of disrupted cells on susceptible assay plates &‘&J hours after infectiotr . Resistant and susceptible cell monolaycrs were inoculated with BS-RSV. After incubation from 6 to 24 hours, the cells were disrupted, and about 2 to 3 X lo4 cells were plated on susceptible assay plates for focus formation. The reklk obtained with cells derived from two Kramer and two line 7 embryos are given in Fig. 2. The focus-forming activity of cells (FE‘A) derived from suscept,ible embryos decreased about one log during the 6-12 hour interval. Between 12 and 24 hours a burst in I;Fh was observed. The burst in FFA probably was related to the formation of infectious particles after one cycle of growth. The FFA of resistant cells decreased logarithmically to a complete loss of 1VA by 24 hours. The loss of FFA by resistant cells is best explained as thermal”iIlnctivatiorl rjf att’ached virus, since the loss observed hercl is well within the range of the half-life of BS-RST: (Vogt, 1965). Of p:uticul:Lr interest is the similar rate of loss of F’FA by both resist~ant~ and susceptible cells during the first 12 hours after inoculation. In t)licxcase of suscept,ible cells, this loss cannot be due t,o virus penetration of the cell membrane CJr ‘Luncoating” of the virus particle, since pcnc~tration of susceptible chick ~11s by HS-IiS\is complete within SO minutes at 37" (&cl< and Rubin, 1966). It is more likely that this loss represents attached virus which 11:~s failed to penetrate the cell and snbsquent~ly remains at, the cell surface until destroyed 1~~. heat, in a manner similar t,o virus nttarhed WI resistant cells. b. Treatmcttt oj” i@xted resistant cd1.s with BS-RSV antibody. From the forcgoitlg studies, it appeared that BS-RST’ attached t,o resist~ant,~11s rcmaincd at, or near t,hc ~11 surface until inact’iv:ated by physical factors. To :answcr th(, clue&on \vhct8hcr at’tachcd virus remained at or near the cell surfaccx or whcthcr it, n-as able to pcnrtrxto ~11 n1cn1-

Source of embryos Kramer cells

.intibody treatment’”

I min)

SOlIt: 10 80 GO

Espt. so. 1 CVhole Cells

----~---I--Expt.

Expt. No. 2 Disrupted

Cells

LVhole Cells

Emb. 1

Emh. 2

Emh. 1

Emb. 2

Emb. 1

Xi0 260 NIY

1620 280 n-1)

4io 0 1

2800 0 I

,520 0 ND

GGO

0

0

970

‘,‘I? 7 ce1’s No. 1

0

~_~_ Expt. No. 2

Disrupted

Emh. 2

Cells

Emb. 1

l’mb. 2

(I ND

1GM 0 1

1xx0 0 0

0

2

1

Ml

~1All plates were inoculated with 0.2 ml of undiluted BS-RSV. Virus was adsorbed for 10 minutes at 3T” and then replaced with 2 ml of a 1:lO dilution of IX-RSV hyperimmune tlwkey serum added at 10, 30, and 60 minut,es after adsorption. The turkey serum remained in place until the cells were plated for FFA on susceptible assay plates. The data are given as FFU’s obtained per 0.1 ml of cell illoculum plated. Each 0.1 ml plated for FFA contained about 200.000 cells. /) Sot done.

bmnes, penetration of BS-RSV in susceptible and resistant cells was studied by plating infected cells for PFA after treatment with BS-RSV antibody. Monolayers prepared from individual resistant and susceptible embryos were inoculated with BS-RSV. Virus was adsorbed for 10 minutes at 37”, and at 10, 30, and 60 minutes later BS-RSV hyperimmune turkey serum was added and left in place until plated for FFA 5 hours after addition of BS-RSV. The results of two experiments, in which either whole or disrupted cells were plated for FFA are given in Table 2. In each experiment cells derived from two resistant and two susceptible embryos were tested. So evidence was obt,ained that BS-RSV penetrates resistant cells after virus attachment. The FFA of both whole and disrupted line 7 cells was sensitive to antibody treatment even when t,he antibody was added as late as 60 minutes after virus attachment. The results obtained using Kramer cells indicat,ed that. 22 c/dof the FFA of whole cells was insensitive t,o antibody added as early as 10 minutes after virus attachment, and 70 % was insensitive after 60 minutes. These data indicut’e that penet’ration of BS-RSV into susceptible cells occurs as early as 10 minutes after adsorpCon, and is 70% complete after 60 minutes. Steck and Rubin (1966) obtained similar results using a more direct method. They found that penct,ration of RSV (RAV

1) into suscept’ible cells occurred as early as 5 minutes after adsorption and was practically complete after SOminutes. Of particular interest in the present studies was that the FFA of Kramer disrupted cells remained sensitive to antibody treatment throughout, the experimental period. DISCUSSION i b

Foci of BS-RSV transformed cells which developed after plating line 7-infected cells were induced by virus attached to the cell surface, which subsequently infected susceptible cells of the assay plate. This conclusion was supported by the findings that: one, foci induced by infected line 7 cells developed on susceptible assay plates (Table 1) but, not resistant assay plates, and, two, prior treatment of infected whole cells with BS-RSV antibody abolished the focus-forming activity of line i cells, but not susceptible cells (Table 2). These results suggested the presence of an efficient process of virus attachment on line 7 cells. Subsequent experiments (Fig. 1) indicated that the attachment of virus to resistant cells was about as efficient as that of attachment to susceptible cells. Virus attached to resistant cells apparently remained at the site of virus adsorption until inactivated by heat or other physical factors. No evidence was obtained that attachment n-as followed by viral pene-

VIRAL CELL

GENOME MEMBRANE

)

ADSORPTION-PENETRATION-UNCOATING ( BUDDING-VIRAL RELEASE

FIG. 3. Mechanism

for BS-RSV

pelletrat,ioll.

tration of the cell membrane. These conclusions are supported by the findings that (1) the loss of focus-forming activity of line 7-infected cells was correlated roughly with the half-life of BS-RSV at 37°C (Fig. 2) and (2) at no time during the experimental period of 1 hour was the FFA of line 7 cells insensitive to antibody treatment (Table 2). Apparently, a substantial amount of virus attached to susceptible cells is at least much delayed in penetration of the cell membrane. This suggestion is prompted by the finding that as late as 12 hours after infection many disrupted susceptible cells were able to initiate focus formation (Fig. 2). It is difficult to explain this finding at present, since it was found here and elsewhere (Steck and Rubin, 1966) that RSV penet)ration is practically complete after 60 minutes. Overall, these results strongly suggest that the mechanism of line 7 genetic cellular resistance to BS-RSV involves a block at the step of viral penetration, and in this respect, has a basis similar t,o that observed for an avian leukosis virus-induced cellular resistance to another RSV strain, RSV (RAV-1) (Steck and Rubin, 1966). Of special interest in these studies was the apparent failure to detect infective virus in disrupted susceptible cells during the 60minute period when penetration of the virus was most active (Table 2). It appears possibIe that infective virus may have been bound internally within the cell matrix in such a way as to prevent its detection by plating cells for focus formation.

Cell bound antibody may also be invoked as an explanation for the lack of FFA in diarupted susceptible cells. It is possible t,hat HS-RSV antibody bound at t,he cell surface was released during the disruption process and inactivated virus locat’ed internally. It appears unlikely, though, that, cell bound antibody which cannot bc removed by I’(‘peated washing will subsequently become dislodged after cell disruption. However, it appears more likely t,hat the absence of infectivity was related to “UIIcoating” of the virus envelope after penet,ration. This would suggest that the processes of penetration and ‘Luncoating” are closely relat,ed in t,ime, and in fact, t,he two ma.y be identical-that is, two propert.ies of the same process. A relatively simple mechanism for such a unified process is given in Fig. 3. The mechanism of viral penetration given in this figure could be closely related t,o the budding process observed in the release of mature RSV viral particles (Vogt, 1965) except that “budding” related to penetration would occur in the reverse direction. Besides accounting for the simultaneous physiological processes of bot,h loss of vim1 infectivity and virus penetration of the ccl1 membrane, this mechanism predicts that the cell membrane would play an important role in viral oncology as well as in virus-associated aut,oimmune disease. REFEREXCES BURPU‘ET, F. M.

(1952). Hemagglutination in relation to host cell-virlls interaction. ,lnn. lfw. Microbial. 6, 229-246. CRITTEXDEN, I,. B., OKAZAKI, W., and GLEAMER, I(. (1963). Genetic resistance to Rous sarcoma virus in embryo cell cultures and embryos. Virology 20, 541-515. HASAFUSA, H. (1965). Analysis of t.he defect,iveness of Rous sarcoma virus. III. Determining influence of a new helper virlls on the host range and susceptibility to interference of IiSV. Virology 25, W-255. HAKAFUSA, H., HANAFUSA, T., and RUBIN, H. (1963). The defectiveness of 110~s sarcoma virtw. Proc. Xntl. ilcad. Sci. U.S. 49, 572-580. ISHIZAKI, R., and VOGT, P. K. (1966). Immnnological relationships among envelope antigens of avian tumor viruses. ViroZog?/ 30, 375-387. PAYNE, L. N., and BIGOS, P. hZ. (1964a). A difference in suscept,ibi!ity to lymphoid leukosis

GENETIC

RESISTANCE

virus and Rous sarcoma virus between cells from two inbred lines of domestic fowl. Sature 203, 1306. PAYNE, L. N., and BIGGS, P. M. (1961b). Differences between highly inbred lrnes of chickens in the response to Rous sarcoma virus of the chorioallantoic membrane and of embryonic cells in tissue culture. Virology 24, 610-616. PAYNE, L. N., and BIGGS, P. &I. (1966). Genetic basis of cellular susceptibility to the SchmidtRuppin and Harris strains of Rous sarcoma virus. Virology 29, 190-198. PRINCE, A. M. (1958). Quantitative studies on ROW sarcoma virus. II. Mechanism of resistance of chick embryos to chorio-allantoic inoculation of Rous sarcoma virus. J. IValZ. Cancer 1nnst. 20, 843450. RUBIN, H. (1960). An analysis of the assay of Rous center sarcoma cells in ~ilro by the infective techllique. Viiirology 10,29-49. RUBIN, H. (1961). The nature of a virus-induced cellular resistance to Rous sarcoma virus. Virology 13, 200-206.

TO RSV

707

RUBIN, H. (1965). Genetic control of cellular suscept,ibility to pseudotypes of Rous sarroma virus. Virology 26, 27@276. STECR, F. T., and RUBIN, H. (1966). The mechanism of interference between an avian leukosis and Rous sarcoma virus. II. Early steps of infection by RSV of cells under condit,ions of interference. Virology 29, (i-22-653. TEMIN, H., and RUBIK, H. (1958). Characteristics of an assay for Rous sarcoma vinls aud RO~IS sarcoma cells in tissue crrlt,nre. Virology 6, 669688. VOGT, P. K. (1965). Avian tumor virtlses. ;l&an. VirusRes. 11,293-38.5. VOGT, P. K., and ISHIZABI, R. I. (1965). Reciprocal patterns of genetic resistance to avian tumor viruses in t,wo lines of chickens. Vi/dog!/ 26, 664672. WATERS, N. F., and BURMESTER, B. R. (1961). Mode of inheritance of resistance to Rous sarcoma virus in chickens. J. Sail. Cancer Inst. 27, 655M61.