- Email: [email protected]
The inactivation of simian virus 40 infectivity and antigen-inducing capacity by ultraviolet light
DISCUSSION
AND
PRELIMINARY
RIF. As expected, inoculation either intravenously or by the wing web route with RII? alone induced no tumors. Pretreatment with heat-inactivated RIF also did not stimulate t,umor production. RSV-SR-Ql when injected by wing web inocuIation into specific-pathogen-free chickens induced 100 % tumors. Rubin has postulated that the SchmidtRuppin strain of RSV may be partially defective or defective in a quantitative sense (10). Our results cannot be fully evaluated at this time, but it would appear that they do support the a.bove hypothesis and that prior infect’ion of quail with various leukosis viruses serves to increase bhe quail’s susceptibility to infection with the Schmidt-Ruppin strain of RSV. The presence of infectious virus in the RSV-SR-&I material is demonstrated by the production of tumors in chickens and also by the observation that a low number of foci are produced by this material in CEF. The role played by the leukosis viruses in stimulating tumor production, however, remains yet to be elucidated. Tissue culture experiments with QEF have shown that even though foci are produced upon primary inoculation of RSV-SR, the cell-free tissue culture fluids from plat’es heavily transformed do not contain virus which will produce foci upon subsequent inoculation of sensitive QEF. Virus can be demonstrated, nevert’heless, in this tissue culture fluid by the production of foci in chick embryo fibroblasts. These foci are characteristic of those induced by RSV-SR. Studies are currently being conducted to determine whether the RSV-SR transformed quail cells cztn be induced by the addition of appropriate viruses of the avian leukosis complex to produce mature virus progeny capable of inducing transformation in a &SF system. ACKNOWLEDGMENT The Nowlin
techni’cal assistance of Mr. is gratefully acknowledged.
Elbert
L.
REFERENCES 1. SCHMIDT-RUPPIN,
Strahlentherapie
K. H., Sonderbtinde 41, 26-27 (1959).
zur
REPORTS
639
C. G., Natl. Cancer Inst. Monograph 17, 299-319 (1964). HARRIS, R. J. c., Nail. Cancer Inst. Jfono. graph 17, 321-335 (1964). JENSEN, F. C., GIRARDI, A. J., GILDEN, 1~. v., and KOPROWSKI, H., Proc. L\Tatl. AC&. f&. U.S. 52, 53-59 (1964). RA~SCHER, F. J., REYXIERS, J. A., and SACKSTEDER, M. R., J. Bacleriol. 84, 11341139 (1962). RAIJSCHER, F. J., REYNIERS, J. A., and
2. AHLSTR~M, 9. 4.
5.
6.
M. R.., Natl.
SACKSTEDER, 7. 8. 9. 10.
11. 12.
Cancer
Inst.
A!!on~ograph 17, 211-229 (1964). RUBIN, H., Virology 10, 2949 (1960). RUBIN, H., Proc. Xatl. Acad. Sci. CT.8. 46, 1105-1119 (1960). MOLONEY, J. B., J. Xatl. Cancer Inst. 16, 877-888 (1956). RUBIN, H., J. Celllllar Comp. Physiol. 64, Suppl. 1, 173-179 (1964). VOW, I’. K., Virology 25,237-247 (1965). HANAFUSA, H., vi’irology 25, 248-255 (1965). CHARLES SHIPMAX, JR. ALVIN
S. LEVINE
Department of Microbiology Indiana Ulziversity School of Medicine Indianapolis, Indiana &‘207 Accepted October 11, 1965
The
inactivation
of
Simian
and Antigen-Inducing Ultraviolet
Virus
40
Capacity
Infectivity by
Light
Infection of a wide variety of cell types with simian virus 40 (SV40) leads to the production of a nonvirion specific antigen1 that’ can be detected by a standard complement fixation test or by the immunofluorescent technique (1-S). All attempts t)o demonstrate STi40 ICFA reactivit,y in whole or degraded virus have failed (4). SV40 ICFA was first demonstrated in extracts of tumors induced by SV40 but has since been demonst,rat#ed in the lytic cycle of the virus in primary African green monkey kidney cells (GMK) (2). In the latter case, ICFA wa.s detectable prior t’o virus DNA synthesis (4-r). With the use of cycloheximide (Actidione, Upjohn) (8) to inhibit protein synthe1 Referred ment Fixing
to here as SV40 Induced Antigen (SV40 ICFA) (4).
Corn+-
640
I~)ISCIWSlON ANI) PRELIMINARY
sis and .i-fluolodcosvuridirlc to inhibit virus DS:\ ~vnthcsk (9), “it ~1s found t.hat ICP‘A synthek began IO-12 hours \)ostinfcction ivhereas virus DKA synthesis began 22-21 hours I)ostinfectiorl (Gilden and Carl), in prq)aration). The source of the informat,ion that codes for the ST40 ICFA is &ill uncertain. The available evidence suggests that, the antigen is coded for by the incoming virus genome,
I’LEPOIITti
infectious virus to yield a multiplicity of infection of 3, and bvhen untrcat,ed SV40 ~1s added, under the conditions out,lined, 80100 “/I of the cells %v’cre positive for SV40 ICl
linear relationship
between infectious
virus
not, by the host cell. This evidence includes the fact, that the antigen is induced in a wide variety of caells including chick embryo fibroblasts (10). I’urthermore, there exists a virus strain suqected of being a ‘(hybrid” of SV40 and adenovirus 7. This strain produces SV40 ICZ;&\ and tumors charact,eristic of SV40, but neither infected cells nor tumor cells ljroduce
infectious SV40 or virus antigen (10-12). This suggests that only a portion of t)hc SV40 virus DNA is required for ICFA l”roduc%ion. The follokng experiments present additional evidence in support of the hypothesis that the incoming virus DiYA codes for ICPA.
nIonolayer
cultures oi primary trypsinized
GRIT< cells were ljrepared by adding to 60-mm petri dishes, cells suspended in t3 1111 of Earle’s salt solution containing the mix-
ture of amino acids and vitamins recom mended by Eagle and 10%. calf serum. The lletri dishes mere incuba,ted at mosphere of 4L% COz in air. monolayers jvere formed after incubation. Immunofluorescent carried out using cells planted
37” in an atContinuous 6-8 days of studies were on coverslips
(11 X 22 mm). A pool of SV40 prepared
in primary
GRIT< cells yielded 107.j plaque-forming units (PFU)/ml lvhen titrated in primary GhIK. This virus pool was diluted equally
with Hanks Basic Salt Solution (BSS) and placed in petri dishes locat,ed 17.5 cm from an ultraviolet light source. At indicated intervals, samples mere removed and aliyuots were titrated for SV40 by the lllaque technique in primary an undiluted aliquot %vas added to primary GhII< cultures maintained on coverslips. After 50 hours t,he coverslips lvere removed and l,roccssed for SV40 ICFh by fluorescent staining
am (IS).Also,
(4). Untreated
aliquots cont
FIG. 1. Effect of ultraviolet irradiation of ICFA synthesis and virus infectivit,y. A pool of S\-40 was exposed to U\T irradiation for the times indicated and then analyzed for virus infectivity and for 8V40 lCl?A-producing capacity. The U\source was a We&inghouse Sterilamp@ (;37T(iI, emitting more than 845~;of its energy at 253(i A and 2652 A and less t,han 0.251;~ of its energy at 2809 A and 2894 A. The points on the graph represent the results (immunofluorescent~ positive cells ant1 virus titer) obtained with UT--t,reated samples expressed as a percentage of the results obtained with an untreated sample. Five experiments were performed, and a typical experiment is recorded in this figure. The inactivation rates (ICFA: 1.3::; per second; virus: 4.9’jA per second) obtained in all experiments did not differ significantly from each other.
DISCUSSION
AND
PRELIMINARY
input and the number of fluorescent positive cells, as has been shown previously (10). The results of a typical UV inact’ivation experiment are shown in Fig. 1. These results indicate that the ability of a virus pool to induce SV40 ICFA decreases wit,h the time of exposure t,o UV. The rate of inactivat’ion followed first-order kinetics through the doses studied. Virus infectivity was also inact’ivstcd following first-order kinet’ics for the first, 3-4 minutes but at a faster rate (approximat,ely 3- to 4-fold) than t,he inactivation of ICFA producing capacit’y. To ensure that multiplicity reactivatjion (14) was not a factor in the results obtained in the SV40 ICFA determinations, the samples obtained after UT: exposure were diluted lo-fold and the proportion of positive cells was determilled. Rates of inactivation of SV40 ICFA--inducing potential were virtually identical when the undiluted and the 1:lO dilutions of t,he UV-exposed samples were used. UV inactivation rates of infectivity and ICFA production obt.ained in the dark paralleled the rates obtained in white light. These results indicate that photoreactivation did not affect the inactivation rates. The fact t*hat’ t’he ability of SV40 to induce antigen production was reduced by exposure of the virus t,o UV, strongly indicat,es that. SV40 ICFA is coded for by the genonie of the incoming virus. Despite this evidence, and t.he factors discussed above, there remains a remote possibility that the cell corltains repressed information for ICFA production and that virus DSA derepresses this function. A final resolution of t,his question may be obtained by the demonstration of ICFA production in a cell-free system dependent on either cell DKA or SV40 DNA for protein synthesis (for example, see reference 15). The hypothesis that SV4O ICE’A is coded by the virus gcnome implies that at least part of the genome persists in some way in the transformed cells, since these cells con t,ain SV40 ICFA without any evidence of infectious virus (4). The faster r&e of inactivation of infectivity, compared to SV40 ICFA product’ion, is probably rela.ted to the greater target size of the infectivity. Presumably, synthesis of
641
REPORTS
several proteins is required to obtain an in fectious product whereas SV40 ICFA is probably a single protein. If the relative inactivation rates are assumed t,o be an indication of functional genome size, t,hen the port,ion of the SV40 genome coding for ICI;A should not be greater than one-third t’o onefourth of the total essential DNA. REFERENCES f. BLACK, I’. H., ROIVE, W. P., TURNER, H. C., and HUEBNER, I<. J. Proc. ;Vatl. Acnd. Sci. U.S. 50, 1148 (1963). 2. POPE, J. H., and RowE, W. P., J. Expll. Med. 120, 121 (1964). 3. RAPP, F., BLYTEL, J. S., and %IELNICK, J. L., Proc. Sot. Exptl. Biol. Med. 116, 131 (1964). 4. GILDEN, 12. V., CARP, R. I., TAWCHI, F., and DEFENDI, V., Proc. Sail. Acad. Sci. U.S. 53, 684 (1965). 5. RAPP, F., KITAHARA, T., BUTEL, J. S., and MELNICK, J. L., Proc. Natl. Acad. Sci. U.S. 52, 1138 (1964). 6. HOGGAN, R/I. D., ROWE, W. P., BLACK, P. H., and HIZEBNER, R. J., Proc. ,Vatl. Acnd. Sci. U.S. 53, 12 (1965). 7. SABIN, A. B., and KOCH, M. A., Proc. h;atl. Acad. Sci. L’.S. 32, 1131 (1964). 8. ENNIS, H. L., and LVBIX, M., Science 146, 1474 (1964). 9. COHEX, S. S., FLAKS, J. G., BARNER, H. I)., LOEB, &!I. Ii., and LICHTESSTEIN, J., Proc. ~V’atl. ricad. Sci. U.S. 44, 1004 (1958). 10. ROWE, W. P., md BAUM, s. G., Proc. Satl. hod. Sci. b’.S. 32, 1340 (1965). 11. HUEBNER, R. J., CEIANOCK, It. M., RUBIE, B. A., C~seu, RI. J., Proc. Satl. ~lcacl. Sci. U.S. 32, 1333 (1965). 12. RAPP, F., RIELNI~K, J. L.: BLTBL, J. S., and KITAHARA, T., Proc. -Vatl. :lcad. Sci. GT.S. 52, 1348 (1965). 13. CARP, R. I., CHI:DNO\V, I. I., KOPROWSKI, H., and KRITCHEVSKY, D., Proc. Sot. Bxptl. Biol. Med. 113, 5ti9 (1963). 14. ABEL, I’., Viwlogy 17, 511 (1962). 16. BOSNER, J., HVANG, 1:. C., alld GILDES, 11. I’., Proc. Null. .2cad. Sci. U.S. SO, 893 (1963). 1:. I. CARP 11. v. &LDEX* The W’istar Institute of Anatomy and Biology Philadelphia, Pennsylvania Accepted October 6, 1965 * Present address: ville, Maryland.
Flow
Laboratories,
Itock-
AND
PRELIMINARY
RIF. As expected, inoculation either intravenously or by the wing web route with RII? alone induced no tumors. Pretreatment with heat-inactivated RIF also did not stimulate t,umor production. RSV-SR-Ql when injected by wing web inocuIation into specific-pathogen-free chickens induced 100 % tumors. Rubin has postulated that the SchmidtRuppin strain of RSV may be partially defective or defective in a quantitative sense (10). Our results cannot be fully evaluated at this time, but it would appear that they do support the a.bove hypothesis and that prior infect’ion of quail with various leukosis viruses serves to increase bhe quail’s susceptibility to infection with the Schmidt-Ruppin strain of RSV. The presence of infectious virus in the RSV-SR-&I material is demonstrated by the production of tumors in chickens and also by the observation that a low number of foci are produced by this material in CEF. The role played by the leukosis viruses in stimulating tumor production, however, remains yet to be elucidated. Tissue culture experiments with QEF have shown that even though foci are produced upon primary inoculation of RSV-SR, the cell-free tissue culture fluids from plat’es heavily transformed do not contain virus which will produce foci upon subsequent inoculation of sensitive QEF. Virus can be demonstrated, nevert’heless, in this tissue culture fluid by the production of foci in chick embryo fibroblasts. These foci are characteristic of those induced by RSV-SR. Studies are currently being conducted to determine whether the RSV-SR transformed quail cells cztn be induced by the addition of appropriate viruses of the avian leukosis complex to produce mature virus progeny capable of inducing transformation in a &SF system. ACKNOWLEDGMENT The Nowlin
techni’cal assistance of Mr. is gratefully acknowledged.
Elbert
L.
REFERENCES 1. SCHMIDT-RUPPIN,
Strahlentherapie
K. H., Sonderbtinde 41, 26-27 (1959).
zur
REPORTS
639
C. G., Natl. Cancer Inst. Monograph 17, 299-319 (1964). HARRIS, R. J. c., Nail. Cancer Inst. Jfono. graph 17, 321-335 (1964). JENSEN, F. C., GIRARDI, A. J., GILDEN, 1~. v., and KOPROWSKI, H., Proc. L\Tatl. AC&. f&. U.S. 52, 53-59 (1964). RA~SCHER, F. J., REYXIERS, J. A., and SACKSTEDER, M. R., J. Bacleriol. 84, 11341139 (1962). RAIJSCHER, F. J., REYNIERS, J. A., and
2. AHLSTR~M, 9. 4.
5.
6.
M. R.., Natl.
SACKSTEDER, 7. 8. 9. 10.
11. 12.
Cancer
Inst.
A!!on~ograph 17, 211-229 (1964). RUBIN, H., Virology 10, 2949 (1960). RUBIN, H., Proc. Xatl. Acad. Sci. CT.8. 46, 1105-1119 (1960). MOLONEY, J. B., J. Xatl. Cancer Inst. 16, 877-888 (1956). RUBIN, H., J. Celllllar Comp. Physiol. 64, Suppl. 1, 173-179 (1964). VOW, I’. K., Virology 25,237-247 (1965). HANAFUSA, H., vi’irology 25, 248-255 (1965). CHARLES SHIPMAX, JR. ALVIN
S. LEVINE
Department of Microbiology Indiana Ulziversity School of Medicine Indianapolis, Indiana &‘207 Accepted October 11, 1965
The
inactivation
of
Simian
and Antigen-Inducing Ultraviolet
Virus
40
Capacity
Infectivity by
Light
Infection of a wide variety of cell types with simian virus 40 (SV40) leads to the production of a nonvirion specific antigen1 that’ can be detected by a standard complement fixation test or by the immunofluorescent technique (1-S). All attempts t)o demonstrate STi40 ICFA reactivit,y in whole or degraded virus have failed (4). SV40 ICFA was first demonstrated in extracts of tumors induced by SV40 but has since been demonst,rat#ed in the lytic cycle of the virus in primary African green monkey kidney cells (GMK) (2). In the latter case, ICFA wa.s detectable prior t’o virus DNA synthesis (4-r). With the use of cycloheximide (Actidione, Upjohn) (8) to inhibit protein synthe1 Referred ment Fixing
to here as SV40 Induced Antigen (SV40 ICFA) (4).
Corn+-
640
I~)ISCIWSlON ANI) PRELIMINARY
sis and .i-fluolodcosvuridirlc to inhibit virus DS:\ ~vnthcsk (9), “it ~1s found t.hat ICP‘A synthek began IO-12 hours \)ostinfcction ivhereas virus DKA synthesis began 22-21 hours I)ostinfectiorl (Gilden and Carl), in prq)aration). The source of the informat,ion that codes for the ST40 ICFA is &ill uncertain. The available evidence suggests that, the antigen is coded for by the incoming virus genome,
I’LEPOIITti
infectious virus to yield a multiplicity of infection of 3, and bvhen untrcat,ed SV40 ~1s added, under the conditions out,lined, 80100 “/I of the cells %v’cre positive for SV40 ICl
linear relationship
between infectious
virus
not, by the host cell. This evidence includes the fact, that the antigen is induced in a wide variety of caells including chick embryo fibroblasts (10). I’urthermore, there exists a virus strain suqected of being a ‘(hybrid” of SV40 and adenovirus 7. This strain produces SV40 ICZ;&\ and tumors charact,eristic of SV40, but neither infected cells nor tumor cells ljroduce
infectious SV40 or virus antigen (10-12). This suggests that only a portion of t)hc SV40 virus DNA is required for ICFA l”roduc%ion. The follokng experiments present additional evidence in support of the hypothesis that the incoming virus DiYA codes for ICPA.
nIonolayer
cultures oi primary trypsinized
GRIT< cells were ljrepared by adding to 60-mm petri dishes, cells suspended in t3 1111 of Earle’s salt solution containing the mix-
ture of amino acids and vitamins recom mended by Eagle and 10%. calf serum. The lletri dishes mere incuba,ted at mosphere of 4L% COz in air. monolayers jvere formed after incubation. Immunofluorescent carried out using cells planted
37” in an atContinuous 6-8 days of studies were on coverslips
(11 X 22 mm). A pool of SV40 prepared
in primary
GRIT< cells yielded 107.j plaque-forming units (PFU)/ml lvhen titrated in primary GhIK. This virus pool was diluted equally
with Hanks Basic Salt Solution (BSS) and placed in petri dishes locat,ed 17.5 cm from an ultraviolet light source. At indicated intervals, samples mere removed and aliyuots were titrated for SV40 by the lllaque technique in primary an undiluted aliquot %vas added to primary GhII< cultures maintained on coverslips. After 50 hours t,he coverslips lvere removed and l,roccssed for SV40 ICFh by fluorescent staining
am (IS).Also,
(4). Untreated
aliquots cont
FIG. 1. Effect of ultraviolet irradiation of ICFA synthesis and virus infectivit,y. A pool of S\-40 was exposed to U\T irradiation for the times indicated and then analyzed for virus infectivity and for 8V40 lCl?A-producing capacity. The U\source was a We&inghouse Sterilamp@ (;37T(iI, emitting more than 845~;of its energy at 253(i A and 2652 A and less t,han 0.251;~ of its energy at 2809 A and 2894 A. The points on the graph represent the results (immunofluorescent~ positive cells ant1 virus titer) obtained with UT--t,reated samples expressed as a percentage of the results obtained with an untreated sample. Five experiments were performed, and a typical experiment is recorded in this figure. The inactivation rates (ICFA: 1.3::; per second; virus: 4.9’jA per second) obtained in all experiments did not differ significantly from each other.
DISCUSSION
AND
PRELIMINARY
input and the number of fluorescent positive cells, as has been shown previously (10). The results of a typical UV inact’ivation experiment are shown in Fig. 1. These results indicate that the ability of a virus pool to induce SV40 ICFA decreases wit,h the time of exposure t,o UV. The rate of inactivat’ion followed first-order kinetics through the doses studied. Virus infectivity was also inact’ivstcd following first-order kinet’ics for the first, 3-4 minutes but at a faster rate (approximat,ely 3- to 4-fold) than t,he inactivation of ICFA producing capacit’y. To ensure that multiplicity reactivatjion (14) was not a factor in the results obtained in the SV40 ICFA determinations, the samples obtained after UT: exposure were diluted lo-fold and the proportion of positive cells was determilled. Rates of inactivation of SV40 ICFA--inducing potential were virtually identical when the undiluted and the 1:lO dilutions of t,he UV-exposed samples were used. UV inactivation rates of infectivity and ICFA production obt.ained in the dark paralleled the rates obtained in white light. These results indicate that photoreactivation did not affect the inactivation rates. The fact t*hat’ t’he ability of SV40 to induce antigen production was reduced by exposure of the virus t,o UV, strongly indicat,es that. SV40 ICFA is coded for by the genonie of the incoming virus. Despite this evidence, and t.he factors discussed above, there remains a remote possibility that the cell corltains repressed information for ICFA production and that virus DSA derepresses this function. A final resolution of t,his question may be obtained by the demonstration of ICFA production in a cell-free system dependent on either cell DKA or SV40 DNA for protein synthesis (for example, see reference 15). The hypothesis that SV4O ICE’A is coded by the virus gcnome implies that at least part of the genome persists in some way in the transformed cells, since these cells con t,ain SV40 ICFA without any evidence of infectious virus (4). The faster r&e of inactivation of infectivity, compared to SV40 ICFA product’ion, is probably rela.ted to the greater target size of the infectivity. Presumably, synthesis of
641
REPORTS
several proteins is required to obtain an in fectious product whereas SV40 ICFA is probably a single protein. If the relative inactivation rates are assumed t,o be an indication of functional genome size, t,hen the port,ion of the SV40 genome coding for ICI;A should not be greater than one-third t’o onefourth of the total essential DNA. REFERENCES f. BLACK, I’. H., ROIVE, W. P., TURNER, H. C., and HUEBNER, I<. J. Proc. ;Vatl. Acnd. Sci. U.S. 50, 1148 (1963). 2. POPE, J. H., and RowE, W. P., J. Expll. Med. 120, 121 (1964). 3. RAPP, F., BLYTEL, J. S., and %IELNICK, J. L., Proc. Sot. Exptl. Biol. Med. 116, 131 (1964). 4. GILDEN, 12. V., CARP, R. I., TAWCHI, F., and DEFENDI, V., Proc. Sail. Acad. Sci. U.S. 53, 684 (1965). 5. RAPP, F., KITAHARA, T., BUTEL, J. S., and MELNICK, J. L., Proc. Natl. Acad. Sci. U.S. 52, 1138 (1964). 6. HOGGAN, R/I. D., ROWE, W. P., BLACK, P. H., and HIZEBNER, R. J., Proc. ,Vatl. Acnd. Sci. U.S. 53, 12 (1965). 7. SABIN, A. B., and KOCH, M. A., Proc. h;atl. Acad. Sci. L’.S. 32, 1131 (1964). 8. ENNIS, H. L., and LVBIX, M., Science 146, 1474 (1964). 9. COHEX, S. S., FLAKS, J. G., BARNER, H. I)., LOEB, &!I. Ii., and LICHTESSTEIN, J., Proc. ~V’atl. ricad. Sci. U.S. 44, 1004 (1958). 10. ROWE, W. P., md BAUM, s. G., Proc. Satl. hod. Sci. b’.S. 32, 1340 (1965). 11. HUEBNER, R. J., CEIANOCK, It. M., RUBIE, B. A., C~seu, RI. J., Proc. Satl. ~lcacl. Sci. U.S. 32, 1333 (1965). 12. RAPP, F., RIELNI~K, J. L.: BLTBL, J. S., and KITAHARA, T., Proc. -Vatl. :lcad. Sci. GT.S. 52, 1348 (1965). 13. CARP, R. I., CHI:DNO\V, I. I., KOPROWSKI, H., and KRITCHEVSKY, D., Proc. Sot. Bxptl. Biol. Med. 113, 5ti9 (1963). 14. ABEL, I’., Viwlogy 17, 511 (1962). 16. BOSNER, J., HVANG, 1:. C., alld GILDES, 11. I’., Proc. Null. .2cad. Sci. U.S. SO, 893 (1963). 1:. I. CARP 11. v. &LDEX* The W’istar Institute of Anatomy and Biology Philadelphia, Pennsylvania Accepted October 6, 1965 * Present address: ville, Maryland.
Flow
Laboratories,
Itock-