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Expression of type C virus p30 in mouse cells infected with herpes simplex virus
VIROLOGY
83, 438-443 (1977)
Expression
of Type C Virus p30 in Mouse Cells Infected Simplex Virus
with Herpes
BERGE HAMPAR,‘* JOHN R. STEPHENSON,? ANN BOYD,S JEFFREY G. DERGE,S ASHLEY BROWN,* AND STEPHEN OROSZLANS * Laboratory of DNA Tumor Viruses, National Cancer Institute, National Znstitutes of Health, Bethesda, Maryland 20014, t Laboratory of RNA Tumor Viruses, National Cancer Institute, National Znstitutes of Health, Bethesda, Maryland 20014, and $ Viral Oncology Program, NCZ Frederick Cancer Research Center, P.O. Box B, Frederick, Maryland 21701 Accepted August 3,1977 Expression of type C virus p30 in BALB/c and NIH Swiss mouse cells infected with live (productive infection) or uv-irradiated herpes simplex virus (uv-HSV) types 1 and 2 was studied by use of either immunofluorescent (FA) or radioimmunoassay (RIA) procedures. No evidence for enhanced expression of p30 was obtained with either of these procedures although activation of type C virus in uv-HSV-infected BALB/c cells was readily demonstrated at low frequencies (-10m4) by infectious center assay. While FA staining with rabbit anti-murine leukemia virus (MuLV) p30 serum was observed in mouse cells productively infected with HSV or infected with uv-HSV, this was shown to be nonspecific. It was concluded that activation of type C virus in uv-HSVinfected BALB/c cells does not occur in significantly more cells than detected by infectious center assay.
Activation of endogenous xenotropic type C virus in transformed (1) and nontransformed (2) BALB/c mouse cells following infection with uv-irradiated herpes simplex virus (uv-HSV) has been reported. Type C virus activation in transformed cells was demonstrated by an infectious center assay, while activation in nontransformed cells was measured indirectly by cocultivation with permissive sarcoma virus-transformed feline (FSl) cells. The present studies were undertaken to determine whether type C virus activation in uv-HSV-infected BALB/c cells could be detected by procedures which did not require synthesis of infectious virions. The methods employed included immunofluorescent (FA) - and radioimmunoassay (RIA) procedures for detection of the major nonglycosylated murine leukemia virus (MuLV) structural protein, ~30. During the course of these studies, a report appeared (3) suggesting that productive in1 Author addressed.
to whom reprint
requests should be
Copyright 8 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
fection of mouse cells with HSV results in the induced synthesis of p30 as measured by FA. In view of this report, our studies were extended to include testing of BALB/ c and NIH Swiss mouse cells productively infected with HSV. The llc cell line, a clonal isolate of BALB/c embryo cells derived in this laboratory, and a thymidine kinase-deficient line of NIH Swiss cells (N cl A cl 10) obtained from Dr. R. Goldberg (NCI) were grown in Eagle’s minimal essential medium (EMEM) supplemented with nonessential amino acids, vitamins, 10% heatinactivated fetal calf serum (FCS), penicillin, and streptomycin. A feline cell line (F81) nonproductively transformed with the Moloney strain of murine sarcoma virus (4) was grown in McCoy’s medium 5A supplemented with 15% heat-inactivated FCS and antibiotics. Vero cells were grown in EMEM supplemented with 10% heat-inactivated FCS and antibiotics. The type 1 Miyama and the type 2 333 strains of HSV were plaque purified and 438 ISSN
0042-6622
SHORT COMMUNICATIONS
propagated in Vero cells (2). Hyperimmune rabbit sera to HSV types 1 and 2 were .prepared as described (5). Rabbit antiserum was prepared against AKR MuLV p30 purified by isoelectric focusing (6). Two weekly subcutaneous injections of 200 pg of protein with complete Freund’s adjuvant were followed by a third injection (100 pg of protein) given intravenously (iv) without adjuvant. A booster injection of 100 Fg of protein was given iv after 2.5 months. Serum lot NC1 E71-2097 was a pool from three bleedings obtained at lo- to 14-day intervals following the last injection. Sera were absorbed with either control llc cells, HSV type l-infected llc cells harvested 24 hr postinfection, or llc cells chronically infected with the Rauscher strain of MuLV (F&V). Frozen-thawed cell pellets (2 x 10’ cells) were resuspended in 1 ml of serum previously diluted fivefold with phosphate-buffered saline (PBS). The mixtures were shaken for 1 hr at 25” and then for 18 hr at 4”. Following centrifugation at 2500 g for 30 min, the sera were subjected to a second absorption cycle prior to testing. Staining by indirect FA was carried out following fixation in acetone for 3 min as described (3). The fured cells were reacted for 30 min at 25” with diluted serum, washed in PBS, reacted for 30 min at 25” with fluorescein isothiocyanate-conjugated goat anti-rabbit 7 S globulin (Meloy Laboratories, Inc.), washed in PBS, counterstained with Evans’ blue, and mounted on slides with Elvanol for microscopic examination. FA titers were expressed as the highest dilution at which an intensity of fluorescence of +2 based on a scale of 0 to +4 was observed. Cell lines and virus pools were routinely tested by R. Del Guidice (FCRC) and were found free of mycoplasma contamination. An infectious center assay for type C virus activation in mouse cells infected with uv-HSV (1) was developed using F81 indicator cells which are permissive for xenotropic but not ecotropic virus ( 7). Replicate cultures of llc or N cl A cl 10 cells were infected with uv-HSV at the desired multiplicity of infection (m.o.i.1 based on titrations of unirradiated virus in Vero
439
cells. The cultures were incubated for 2 hr, washed, and trypsinized, and the cells were reseeded and incubated for 20-24 hr. Mitomycin (25 kg ml-‘) was added and the cells were incubated for 1 hr, washed, refed, incubated for 2 hr, and trypsinized, and 5 x 105cells were added in duplicate to 100~mmpetri dishes seeded 24 hr previously with 1.5 x lo6 F81 cells in medium containing Polybrene (2 pg ml-‘). Cultures were incubated for 4 days, 10 ml of fresh medium was added, and the cells were incubated for an additional 3 days. The cells were refed, incubated for 24 hr, fmed with methanol, and stained with Giemsa for quantitation of foci. Preliminary studies indicated that both llc and N cl A cl 10 cells could support the replication of HSV types 1 and 2. Vero, llc, and N cl A cl 10 cells were infected with HSV at an m.o.i. of 3 to 5, and the cells were fixed after 7 to 8 hr for testing by indirect FA. At the time of fixation, the infected cells showed typical HSV cytopathic effects, and 2 75% showed positive FA staining with various sera. The results with llc and N cl A cl 10 cells were essentially the same, and only those observed with llc cells are presented. The pertinent findings (Table 1) which led us to conclude that significant nonspecific FA staining occurs with rabbit sera in llc and N cl A cl 10 cells productively infected with HSV may be summarized as follows. First, only the anti-MuLV p30 serum reacted with RLV infected cells, and this reactivity was effectively reduced by absorption with RLV-infected cells but not by absorption with control or HSVinfected cells. Second, the anti-HSV, antiMuLV ~30, and normal rabbit sera reacted to high titers with HSV type l-infected llc cells, and the titers were not reduced by absorption with control, HSV-infected, or RLV-infected llc cells. Staining by the anti-HSV serum was observed in the nucleus and cytoplasm at dilutions up to 160 to 320, and only in the cytoplasm at higher dilutions. Staining by the anti-MuLV p30 and normal rabbit sera was limited to the cytoplasm at all dilutions (Fig. 1). In contrast to the results with llc cells, only the anti-HSV serum reacted to high titers
440
SHORT
COMMUNICATIONS TABLE
IYYUNOFLUOREBCENT
Rabbit serum’
OF RABBIT
SERA
llc cells
Control
HSV-l-infected 1280 1280 1280
<20
<20
NDd ND
ND ND
ND
ND
Anti-HSV type lc Absorbed llc cells Absorbed HSV-l-infected cells Absorbed RLV-infected cells
<20
<20
ND ND
ND ND
ND
ND
Anti-MuLV p30 Absorbed llc cells Absorbed HSV-l-infected cells Absorbed RLV-infected cells
<20
160 160 160
ND ND
Vera cells
RLV-infected
Controlb Absorbed llc cells Absorbed HSV-l-infected cells Absorbed RLV-infected cells
B Control and anti-MuLV anti-HSV serum neutralized absorption with control or infected llc cells. * A second control serum ’ Anti-HSV type 2 serum d ND, not tested.
1
TITERS
ND
-
2560 1280 1280
HSV-P-infected
Control
HEW-l-infected
HSV-2-h fected
80 80 40
<20
ND ND
40 20 20
20 <20 20
80
ND
20
<20
640 640 80
(20 ND
ND
640 640 40
160 160 20
640
ND
640
160
<20
40 20 40
20 <20 20
40
20
80 80
2560 1280 1280
80
ND ND
1280
80
ND
p30 sera showed no neutralizing activity (~20) against HSV type 1 or 2. The HSV types 1 and 2 at a titer of 640-1280. The titers were not reduced by RLV-infected llc cells, but were reduced to <20 by absorption with HSVwas tested with essentially showed similar results
with HSV type l-infected Vero cells, and this reactivity was effectively reduced by absorption with HSV-infected cells. Finally, the anti-HSV serum reacted to high titers with llc cells infected with HSV type 2, and this reactivity was reduced by absorption with HSV-infected cells. In contrast, the anti-MuLV p30 and normal rabbit sera reacted to low but significant titers with HSV type 2-infected llc cells, and the titers were not reduced by absorption with control, HSV-infected, or RLVinfected cells. If the intense staining observed with the anti-MuLV p30 serum in 375% of HSV-infected mouse cells (Fig. 1) was due to induced p30 synthesis, this might be expected to be reflected in elevated levels of p30 measured by RIA. To test this possibility, llc and N cl A cl 10 cells were infected with HSV types 1 and 2 at an m.o.i. of 5 and harvested at 8 hr for testing
similar
results.
by RIA using previously described procedures (8). Parallel cultures tested by FA with anti-HSV, anti-MuLV ~30, and normal rabbit sera showed the typical staining pattern described above (Table 1 and Fig. 1). The results by RIA were similar for llc (Fig. 2) and N cl A cl 10 cells, and indicated no enhanced levels of p30 above the previously establshed levels in mouse cells (9). As a positive control, llc and N cl A cl 10 cells chronically infected with RLV were included, and these were shown to express high levels of ~30. An additional experiment was carried out with cells productively infected at an m.o.i of 5 with HSV type 1 for 22 hr or infected with 6-min uv-HSV under conditions previously determined optimal for detecting type C virus activation by infectious center assay. Type C virus activation in llc cells was observed at a frequency of 9 x 1O-5 (control cells show an activation
SHORT
FIG. 1. Appearance
of FA staining
COMMUNICATIONS
by anti-MuLV
Cellular
Protein
Concentration
(tog,gtiglml)
FIG. 2. RIA for MuLV p30 antigen in llc cells infected with HSV types 1 or 2 at an m.o.i. of 5. Cells were harvested at 8 hr for testing. Control uninfected cells (A); HSV type l-infected cells (0); HSV type a-infected cells (0); RLV-infected cells (0).
p30 serum in HSV type l-infected
441
llc cells at 8 hr.
frequency of G 10m6).No enhancement of p30 expression was evident by RIA at 22 hr in cells productively infected with HSV or infected with uv-HSV. Further, FA staining of uv-HSV-infected llc cells with anit-p30 serum was not correlated with type C virus activation by infectious center assay, and the staining observed was considered nonspecific based on the same criteria used with cells productively infected with HSV (Table 1). Finally, no evidence was obtained for activation of type C virus by uv-HSV in NIH Swiss derived N cl A cl 10 cells. The findings of the present study argue against induced synthesis of p30 in BALB/ c or in NIH Swiss mouse cells productively infected with HSV types 1 or 2. While FA staining of HSV-infected mouse cells was observed with anit-MuLV ~30 serum, this was shown to be nonspecific. Nonspecific
442
SHORT COMMUNICATIONS
staining by various rabbit sera was most pronounced in mouse cells infected with HSV type 1 (Miyama strain), while HSV type 2 (strain 333)infected cells showed low but still significant levels of nonspecific staining. The inability to induce MuLV p30 expression by productive infection of mouse cells with HSV was also indicated by the absence of enhanced p30 levels measured by RIA. The nonspecific staining observed with various rabbit sera against mouse cells productively infected with HSV was also evident in cells infected with uv-HSV. In this regard, the RIA results with uv-HSV-infected llc cells were relevant since they indicated no increase in p30 levels under conditions where type C virus activation was readily detectable by infectious center assay. Our findings, employing rabbit sera comparable to. those used by Reed and Rapp (3), indicated significant nonspecific FA staining against mouse cells infected with live or uv-irradiated HSV. Nonspecific staining is also seen in cells infected with cytomegalovirus (10, 111, another member of the herpesvirus group. We are of the opinion that the discrepancy between our findings and those reported by Reed and Rapp (3) can be attributed to nonspecific staining encountered with rabbit sera in HSV-infected mouse cells. At present, therefore, we conclude that the only evidence for activation of endogenous type C virus of mouse cells by uv-HSV is derived from studies in which infectious type C virus has been isolated and characterized (I, 2, and the present study). Our inability, thus far, to demonstrate type C virus activation by uv-HSV in NIH Swissderived fibroblasts is consistent with other reports (9, 12) indicating that cell lines derived from mice of this strain lack chemically inducible endogenous type C virus. The possibility was considered previously (I) that type C virus activation by UV-HSV may occur in many more cells than detected in the infectious center assay which requires synthesis of infectious virions. If one accepts p30 as a valid indicator of type C virus genome expression at the translational level, our findings by FA and RIA suggest that uv-HSV infec-
tion of BALB/c cells (11~) did not result in type C virus activation at levels significantly greater than measured in the infectious center assay. This does not exclude the possibility that uv-HSV may induce expression of the type C virus genome limited to transcriptional products. The results reported here agree with previous findings (I, 2) that sensitive biologic assays must be employed for detection of type C virus activated by uv-HSV in BALB/c cells. The inability to detect enhanced p30 expression by RIA in uvHSV-infected BALB/c cells under conditions where type C virus activation was detected by infectious center assay was consistent with the fact that the normal levels of p30 expression in mouse cells are between 0.1 and 1% of those found in cells productively infected with type C virus (12). Thus, a frequency of activation of at least 1O-3 would be required for a significant increase in p30 expression, and this is approximately lo-fold higher than levels we have observed in uv-HSV-infected BALB/c cells. ACKNOWLEDGMENTS The authors wish to thank Mrinal Chakrabarty and Donald Simms for excellent technical assistance. This work was supported in part by Contract NOl-CO-25423 with the National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014. REFERENCES 1. HAMPAR. B., AARONSON, S. A., DERGE. J. G., CHAKRABARY, M., SHOWALTER, S. D., and DUNN, C. Y., Proc. Nat. Acad. Sci. USA 73, 646-650 (1976). 2. HAMPAR, B., HATANAKA, M., AULAKH, G., DERGE. J. G., LEE, L., and SHOWALTER, S. D., Virology 76, 876-881 (1977). 3. REED, C. L., and RAPP. F., J. Viral. 19, 102% 1033 (1976). 4. FIBCHINGER. P. J., PEEBLES. P. T., NOMURA, S., and HAAPALA. D. K., J. Viral. 11, 976-985 (1973). 5. HAMPAR. B., STEVENS, D. A., MARTOS. L. M., ABLASHI, D. V., BURROUGHS. M. A. K., and WELLB.G. A., J. Immunol. 162,397-403 (1969). 6. OROSZLAN. S., FISHER. C. L., STANLEY, T. B., and GILDEN. R. V., J. Gen. Viral. 8, l-10 (1970). 7. FIBCHINGER. P. J., and NOIUURA. S., Virology
SHORT COMMUNICATIONS 65, 304-307 (1975). J. R., REYNOLDS, R. K., TRONICK. S. R., and AARONSON. S. A., Virology 67, 404-414 (1975). 9. STEPHENSON. J. R., and AARONSON, S. A., PFOC. Nat. Acad. Sci. USA 71, 4925-4929 (1974). 10. JACK, I., and WARK, M. C., In “Oncogenesis 8.
STEPHENSON,
443
and Herpesviruses,” pp. 339-340. 1972. B., and MARTOS, L. M. In “The Herpesviruses” (A. S. Kaplan, ed.), pp. 221-259. Academic Press, New York, 1973. 12. STEPHENSON, J. R., TRONICK. S. R., REYNOLDS, R. K., and AARONSON. S. A., J. Exp. Med. 139, 427-438, (1974).
Il.
HAMPAR,
83, 438-443 (1977)
Expression
of Type C Virus p30 in Mouse Cells Infected Simplex Virus
with Herpes
BERGE HAMPAR,‘* JOHN R. STEPHENSON,? ANN BOYD,S JEFFREY G. DERGE,S ASHLEY BROWN,* AND STEPHEN OROSZLANS * Laboratory of DNA Tumor Viruses, National Cancer Institute, National Znstitutes of Health, Bethesda, Maryland 20014, t Laboratory of RNA Tumor Viruses, National Cancer Institute, National Znstitutes of Health, Bethesda, Maryland 20014, and $ Viral Oncology Program, NCZ Frederick Cancer Research Center, P.O. Box B, Frederick, Maryland 21701 Accepted August 3,1977 Expression of type C virus p30 in BALB/c and NIH Swiss mouse cells infected with live (productive infection) or uv-irradiated herpes simplex virus (uv-HSV) types 1 and 2 was studied by use of either immunofluorescent (FA) or radioimmunoassay (RIA) procedures. No evidence for enhanced expression of p30 was obtained with either of these procedures although activation of type C virus in uv-HSV-infected BALB/c cells was readily demonstrated at low frequencies (-10m4) by infectious center assay. While FA staining with rabbit anti-murine leukemia virus (MuLV) p30 serum was observed in mouse cells productively infected with HSV or infected with uv-HSV, this was shown to be nonspecific. It was concluded that activation of type C virus in uv-HSVinfected BALB/c cells does not occur in significantly more cells than detected by infectious center assay.
Activation of endogenous xenotropic type C virus in transformed (1) and nontransformed (2) BALB/c mouse cells following infection with uv-irradiated herpes simplex virus (uv-HSV) has been reported. Type C virus activation in transformed cells was demonstrated by an infectious center assay, while activation in nontransformed cells was measured indirectly by cocultivation with permissive sarcoma virus-transformed feline (FSl) cells. The present studies were undertaken to determine whether type C virus activation in uv-HSV-infected BALB/c cells could be detected by procedures which did not require synthesis of infectious virions. The methods employed included immunofluorescent (FA) - and radioimmunoassay (RIA) procedures for detection of the major nonglycosylated murine leukemia virus (MuLV) structural protein, ~30. During the course of these studies, a report appeared (3) suggesting that productive in1 Author addressed.
to whom reprint
requests should be
Copyright 8 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
fection of mouse cells with HSV results in the induced synthesis of p30 as measured by FA. In view of this report, our studies were extended to include testing of BALB/ c and NIH Swiss mouse cells productively infected with HSV. The llc cell line, a clonal isolate of BALB/c embryo cells derived in this laboratory, and a thymidine kinase-deficient line of NIH Swiss cells (N cl A cl 10) obtained from Dr. R. Goldberg (NCI) were grown in Eagle’s minimal essential medium (EMEM) supplemented with nonessential amino acids, vitamins, 10% heatinactivated fetal calf serum (FCS), penicillin, and streptomycin. A feline cell line (F81) nonproductively transformed with the Moloney strain of murine sarcoma virus (4) was grown in McCoy’s medium 5A supplemented with 15% heat-inactivated FCS and antibiotics. Vero cells were grown in EMEM supplemented with 10% heat-inactivated FCS and antibiotics. The type 1 Miyama and the type 2 333 strains of HSV were plaque purified and 438 ISSN
0042-6622
SHORT COMMUNICATIONS
propagated in Vero cells (2). Hyperimmune rabbit sera to HSV types 1 and 2 were .prepared as described (5). Rabbit antiserum was prepared against AKR MuLV p30 purified by isoelectric focusing (6). Two weekly subcutaneous injections of 200 pg of protein with complete Freund’s adjuvant were followed by a third injection (100 pg of protein) given intravenously (iv) without adjuvant. A booster injection of 100 Fg of protein was given iv after 2.5 months. Serum lot NC1 E71-2097 was a pool from three bleedings obtained at lo- to 14-day intervals following the last injection. Sera were absorbed with either control llc cells, HSV type l-infected llc cells harvested 24 hr postinfection, or llc cells chronically infected with the Rauscher strain of MuLV (F&V). Frozen-thawed cell pellets (2 x 10’ cells) were resuspended in 1 ml of serum previously diluted fivefold with phosphate-buffered saline (PBS). The mixtures were shaken for 1 hr at 25” and then for 18 hr at 4”. Following centrifugation at 2500 g for 30 min, the sera were subjected to a second absorption cycle prior to testing. Staining by indirect FA was carried out following fixation in acetone for 3 min as described (3). The fured cells were reacted for 30 min at 25” with diluted serum, washed in PBS, reacted for 30 min at 25” with fluorescein isothiocyanate-conjugated goat anti-rabbit 7 S globulin (Meloy Laboratories, Inc.), washed in PBS, counterstained with Evans’ blue, and mounted on slides with Elvanol for microscopic examination. FA titers were expressed as the highest dilution at which an intensity of fluorescence of +2 based on a scale of 0 to +4 was observed. Cell lines and virus pools were routinely tested by R. Del Guidice (FCRC) and were found free of mycoplasma contamination. An infectious center assay for type C virus activation in mouse cells infected with uv-HSV (1) was developed using F81 indicator cells which are permissive for xenotropic but not ecotropic virus ( 7). Replicate cultures of llc or N cl A cl 10 cells were infected with uv-HSV at the desired multiplicity of infection (m.o.i.1 based on titrations of unirradiated virus in Vero
439
cells. The cultures were incubated for 2 hr, washed, and trypsinized, and the cells were reseeded and incubated for 20-24 hr. Mitomycin (25 kg ml-‘) was added and the cells were incubated for 1 hr, washed, refed, incubated for 2 hr, and trypsinized, and 5 x 105cells were added in duplicate to 100~mmpetri dishes seeded 24 hr previously with 1.5 x lo6 F81 cells in medium containing Polybrene (2 pg ml-‘). Cultures were incubated for 4 days, 10 ml of fresh medium was added, and the cells were incubated for an additional 3 days. The cells were refed, incubated for 24 hr, fmed with methanol, and stained with Giemsa for quantitation of foci. Preliminary studies indicated that both llc and N cl A cl 10 cells could support the replication of HSV types 1 and 2. Vero, llc, and N cl A cl 10 cells were infected with HSV at an m.o.i. of 3 to 5, and the cells were fixed after 7 to 8 hr for testing by indirect FA. At the time of fixation, the infected cells showed typical HSV cytopathic effects, and 2 75% showed positive FA staining with various sera. The results with llc and N cl A cl 10 cells were essentially the same, and only those observed with llc cells are presented. The pertinent findings (Table 1) which led us to conclude that significant nonspecific FA staining occurs with rabbit sera in llc and N cl A cl 10 cells productively infected with HSV may be summarized as follows. First, only the anti-MuLV p30 serum reacted with RLV infected cells, and this reactivity was effectively reduced by absorption with RLV-infected cells but not by absorption with control or HSVinfected cells. Second, the anti-HSV, antiMuLV ~30, and normal rabbit sera reacted to high titers with HSV type l-infected llc cells, and the titers were not reduced by absorption with control, HSV-infected, or RLV-infected llc cells. Staining by the anti-HSV serum was observed in the nucleus and cytoplasm at dilutions up to 160 to 320, and only in the cytoplasm at higher dilutions. Staining by the anti-MuLV p30 and normal rabbit sera was limited to the cytoplasm at all dilutions (Fig. 1). In contrast to the results with llc cells, only the anti-HSV serum reacted to high titers
440
SHORT
COMMUNICATIONS TABLE
IYYUNOFLUOREBCENT
Rabbit serum’
OF RABBIT
SERA
llc cells
Control
HSV-l-infected 1280 1280 1280
<20
<20
NDd ND
ND ND
ND
ND
Anti-HSV type lc Absorbed llc cells Absorbed HSV-l-infected cells Absorbed RLV-infected cells
<20
<20
ND ND
ND ND
ND
ND
Anti-MuLV p30 Absorbed llc cells Absorbed HSV-l-infected cells Absorbed RLV-infected cells
<20
160 160 160
ND ND
Vera cells
RLV-infected
Controlb Absorbed llc cells Absorbed HSV-l-infected cells Absorbed RLV-infected cells
B Control and anti-MuLV anti-HSV serum neutralized absorption with control or infected llc cells. * A second control serum ’ Anti-HSV type 2 serum d ND, not tested.
1
TITERS
ND
-
2560 1280 1280
HSV-P-infected
Control
HEW-l-infected
HSV-2-h fected
80 80 40
<20
ND ND
40 20 20
20 <20 20
80
ND
20
<20
640 640 80
(20 ND
ND
640 640 40
160 160 20
640
ND
640
160
<20
40 20 40
20 <20 20
40
20
80 80
2560 1280 1280
80
ND ND
1280
80
ND
p30 sera showed no neutralizing activity (~20) against HSV type 1 or 2. The HSV types 1 and 2 at a titer of 640-1280. The titers were not reduced by RLV-infected llc cells, but were reduced to <20 by absorption with HSVwas tested with essentially showed similar results
with HSV type l-infected Vero cells, and this reactivity was effectively reduced by absorption with HSV-infected cells. Finally, the anti-HSV serum reacted to high titers with llc cells infected with HSV type 2, and this reactivity was reduced by absorption with HSV-infected cells. In contrast, the anti-MuLV p30 and normal rabbit sera reacted to low but significant titers with HSV type 2-infected llc cells, and the titers were not reduced by absorption with control, HSV-infected, or RLVinfected cells. If the intense staining observed with the anti-MuLV p30 serum in 375% of HSV-infected mouse cells (Fig. 1) was due to induced p30 synthesis, this might be expected to be reflected in elevated levels of p30 measured by RIA. To test this possibility, llc and N cl A cl 10 cells were infected with HSV types 1 and 2 at an m.o.i. of 5 and harvested at 8 hr for testing
similar
results.
by RIA using previously described procedures (8). Parallel cultures tested by FA with anti-HSV, anti-MuLV ~30, and normal rabbit sera showed the typical staining pattern described above (Table 1 and Fig. 1). The results by RIA were similar for llc (Fig. 2) and N cl A cl 10 cells, and indicated no enhanced levels of p30 above the previously establshed levels in mouse cells (9). As a positive control, llc and N cl A cl 10 cells chronically infected with RLV were included, and these were shown to express high levels of ~30. An additional experiment was carried out with cells productively infected at an m.o.i of 5 with HSV type 1 for 22 hr or infected with 6-min uv-HSV under conditions previously determined optimal for detecting type C virus activation by infectious center assay. Type C virus activation in llc cells was observed at a frequency of 9 x 1O-5 (control cells show an activation
SHORT
FIG. 1. Appearance
of FA staining
COMMUNICATIONS
by anti-MuLV
Cellular
Protein
Concentration
(tog,gtiglml)
FIG. 2. RIA for MuLV p30 antigen in llc cells infected with HSV types 1 or 2 at an m.o.i. of 5. Cells were harvested at 8 hr for testing. Control uninfected cells (A); HSV type l-infected cells (0); HSV type a-infected cells (0); RLV-infected cells (0).
p30 serum in HSV type l-infected
441
llc cells at 8 hr.
frequency of G 10m6).No enhancement of p30 expression was evident by RIA at 22 hr in cells productively infected with HSV or infected with uv-HSV. Further, FA staining of uv-HSV-infected llc cells with anit-p30 serum was not correlated with type C virus activation by infectious center assay, and the staining observed was considered nonspecific based on the same criteria used with cells productively infected with HSV (Table 1). Finally, no evidence was obtained for activation of type C virus by uv-HSV in NIH Swiss derived N cl A cl 10 cells. The findings of the present study argue against induced synthesis of p30 in BALB/ c or in NIH Swiss mouse cells productively infected with HSV types 1 or 2. While FA staining of HSV-infected mouse cells was observed with anit-MuLV ~30 serum, this was shown to be nonspecific. Nonspecific
442
SHORT COMMUNICATIONS
staining by various rabbit sera was most pronounced in mouse cells infected with HSV type 1 (Miyama strain), while HSV type 2 (strain 333)infected cells showed low but still significant levels of nonspecific staining. The inability to induce MuLV p30 expression by productive infection of mouse cells with HSV was also indicated by the absence of enhanced p30 levels measured by RIA. The nonspecific staining observed with various rabbit sera against mouse cells productively infected with HSV was also evident in cells infected with uv-HSV. In this regard, the RIA results with uv-HSV-infected llc cells were relevant since they indicated no increase in p30 levels under conditions where type C virus activation was readily detectable by infectious center assay. Our findings, employing rabbit sera comparable to. those used by Reed and Rapp (3), indicated significant nonspecific FA staining against mouse cells infected with live or uv-irradiated HSV. Nonspecific staining is also seen in cells infected with cytomegalovirus (10, 111, another member of the herpesvirus group. We are of the opinion that the discrepancy between our findings and those reported by Reed and Rapp (3) can be attributed to nonspecific staining encountered with rabbit sera in HSV-infected mouse cells. At present, therefore, we conclude that the only evidence for activation of endogenous type C virus of mouse cells by uv-HSV is derived from studies in which infectious type C virus has been isolated and characterized (I, 2, and the present study). Our inability, thus far, to demonstrate type C virus activation by uv-HSV in NIH Swissderived fibroblasts is consistent with other reports (9, 12) indicating that cell lines derived from mice of this strain lack chemically inducible endogenous type C virus. The possibility was considered previously (I) that type C virus activation by UV-HSV may occur in many more cells than detected in the infectious center assay which requires synthesis of infectious virions. If one accepts p30 as a valid indicator of type C virus genome expression at the translational level, our findings by FA and RIA suggest that uv-HSV infec-
tion of BALB/c cells (11~) did not result in type C virus activation at levels significantly greater than measured in the infectious center assay. This does not exclude the possibility that uv-HSV may induce expression of the type C virus genome limited to transcriptional products. The results reported here agree with previous findings (I, 2) that sensitive biologic assays must be employed for detection of type C virus activated by uv-HSV in BALB/c cells. The inability to detect enhanced p30 expression by RIA in uvHSV-infected BALB/c cells under conditions where type C virus activation was detected by infectious center assay was consistent with the fact that the normal levels of p30 expression in mouse cells are between 0.1 and 1% of those found in cells productively infected with type C virus (12). Thus, a frequency of activation of at least 1O-3 would be required for a significant increase in p30 expression, and this is approximately lo-fold higher than levels we have observed in uv-HSV-infected BALB/c cells. ACKNOWLEDGMENTS The authors wish to thank Mrinal Chakrabarty and Donald Simms for excellent technical assistance. This work was supported in part by Contract NOl-CO-25423 with the National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014. REFERENCES 1. HAMPAR. B., AARONSON, S. A., DERGE. J. G., CHAKRABARY, M., SHOWALTER, S. D., and DUNN, C. Y., Proc. Nat. Acad. Sci. USA 73, 646-650 (1976). 2. HAMPAR, B., HATANAKA, M., AULAKH, G., DERGE. J. G., LEE, L., and SHOWALTER, S. D., Virology 76, 876-881 (1977). 3. REED, C. L., and RAPP. F., J. Viral. 19, 102% 1033 (1976). 4. FIBCHINGER. P. J., PEEBLES. P. T., NOMURA, S., and HAAPALA. D. K., J. Viral. 11, 976-985 (1973). 5. HAMPAR. B., STEVENS, D. A., MARTOS. L. M., ABLASHI, D. V., BURROUGHS. M. A. K., and WELLB.G. A., J. Immunol. 162,397-403 (1969). 6. OROSZLAN. S., FISHER. C. L., STANLEY, T. B., and GILDEN. R. V., J. Gen. Viral. 8, l-10 (1970). 7. FIBCHINGER. P. J., and NOIUURA. S., Virology
SHORT COMMUNICATIONS 65, 304-307 (1975). J. R., REYNOLDS, R. K., TRONICK. S. R., and AARONSON. S. A., Virology 67, 404-414 (1975). 9. STEPHENSON. J. R., and AARONSON, S. A., PFOC. Nat. Acad. Sci. USA 71, 4925-4929 (1974). 10. JACK, I., and WARK, M. C., In “Oncogenesis 8.
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