Envelope Antigen Relationships among Three Hamster-specific Sarcoma Viruses and a Hamster-specific Helper Virus

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1 J. gen. Virol. (197o), 9, I9-26 I9 Printed in Great Britain Envelope Antigen Relationships among Three Hamster-specific Sarcoma Viruses and a Hamster-specific Helper Virus By G. KELLOFF AND R. J. HUEBNER Viral Carcinogenesis Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 2ooi 4 AND NAM HO CHANG, YONG KI LEE AYD R. V. GILDEN Flow Laboratories, Inc., Rockville, Maryland (Accepted 2I May 197o) SUMMARY The immunological relationships among three hamster-specific sarcoma viruses and a non-sarcomagenic virus were determined by neutralization and interference tests. These viruses were all derived from hamster tumours induced originally by murine sarcoma viruses but were no longer capable of growth on mouse cells. The four viruses shared at least one envelope antigen responsible for neutralization reactions. Specific interference for the sarcoma viruses was obtained in cultures infected with the non-sarcomagenic virus. In both assays these viruses were clearly distinguishable from murine C-type viruses. INTRODUCTION Several workers have isolated hamster-specific sarcoma viruses from hamster tumours induced by murine sarcoma viruses (Bassin et al. I968; Kelloff et al. I97oa; Klement et al. ~969; Sarma, Log & Gilden, ~97o; Perk et al. 1969). Such viruses are no longer active in mice or mouse tissue cultures. By in vitro neutralization tests these viruses are antigenically distinct from the original turnout-inducing murine viruses (Klement et al. ~969; Kelloff et al. ~97oa). In addition to sarcoma viruses, we have also isolated a non-sarcomagenic hamster specific virus from one of these isolates, i.e. that induced by the Gross pseudotype of the MOLONEY strain of murine sarcoma virus, M-MSV(GLV) (Kelloff et al. ~97oa). The nonfocus-forming hamster virus is loosely termed a leukaemia virus to indicate failure to induce transformation in vitro in hamster cells which support virus growth and also failure to induce sarcoma in vivo; however, this virus has not yet induced leukaemias in the hamster. The sarcoma virus preparation was designated M-MSV(GLV)(O-H), following the nomenclature of Klement et al. 0969~. This report and the accompanying report describe further studies establishing immunological cross-reactivity among these viruses and two other hamster specific sarcoma viruses derived from hamster tumours induced by murine sarcoma viruses M-MSV(O-H) (R. J. Huebner & W. T. Lane, unpublished data) and Ki-MSV(O-H) (Klement et al. ~969), and finally establishes the immunological identity of these hamster specific viruses with an indigenous C-type virus of the hamster. On this basis we suggest that MSV(GLV)(O-H) andits' helper' virus would be more appropriately termed M-MSV(HaLV)- hamster leukaemia virus pseudotype of the MOLONEY strain of murine sarcoma virus, and HaLV-hamster leukaemia virus, respectively. For consistency, other (O-H) designations will be changed to (HaLV). This terminology is explained in the footnote to Table ~.

2 20 G. KELLOFF AND OTHERS METHODS Viruses. M-MSV(HaLV)G (Table 2) was obtained from a cell-free passage line established in hamsters by injection of M-MSV(GLV), the Gross virus pseudotype of MOLOYEY sarcoma virus. Virus concentrates (Moloney, ~96o) from this tumour line were used as a virus source. A cell line derived from hamster embryo fibroblasts that had been inoculated with a dilution of M-MSV(HaLV)G tenfold beyond its focus end-point provided the source of HaLV (Kelloff et al. ~97oa). The source of the Ki-MSV(HaLV) and M-MSV(HaLV) viruses were virus concentrates (Moloney, I96o) oftumour transplant lines that had originally induced in hamsters by their respective murine 'precursor' viruses Ki-MSV (Klement et al. ~969) and M-MSV (R. J. Huebner & W. T. Lane, unpublished data). The Gross pseudotype Of MOLONEY sarcoma virus, M-MSV(GLV), and the MOLONEY sarcoma virus, M-MSV, were obtained from passage in mouse embryo fibroblasts. The Rauscher pseudotype of MOLONEY sarcoma virus, M-MSV(RLV), adapted to grow in rat ceils, was a generous gift of Dr Robert C. Ting (Ting & Bader, I969). Table I. Suggested new nomenclature for hamster specific sarcoma viruses based on immunological data Original tumour-inducing Original abbreviation of Suggested new murine virus hamster specific virus abbreviation M-MSV(GLV)* M-MSV(GLV)(O-H) M-MSV(HaLV)G'~ M-MSV M-MSV(O-H) M-MSV(HaLV) Ki-MSV Ki-MSV(O-H) Ki-MSV(HaLV) * As described in detail by Huebner et al. 0969), the initial letter symbol indicates strain (M = MOI.ONEV, Ki = KIRSrEN) of sarcoma virus, the second sequence indicates the species origin of the sarcoma genes (MSV = murine sarcoma virus), andthe symbols in parentheses indicate the envelope type of the helper virus which was used to rescue the sarcoma genes from non-virus-producing tumour cells (GLV = Gross leukaemia virus, HaLV-hamster leukaemia virus). Such rescued viruses have been readily produced in vitro (Huebner et al. I966) and we have assumed a similar situation occurred in vivo to produce M-MSV(HaLV). t ' G' is used to indicate the origin of the hamster tumour from the Gross pseudotype of MSV. There is at present no indication that GLV has contributed any genetic information to M-MSV(HaLV)G, but some means of distinguishing this virus from that isolated from hamster tumours induced by M-MSV is necessary. Tissue culture and focus assay techniques were described by Kelloff et al. (I97o a), Hartley et al. 0965) and Hartley & Rowe (~966). Animals. Syrian hamsters were obtained from the National Institutes of Health animal production colony. The LSH, LHC and MHA inbred strains of Syrian hamsters were obtained from the Lakeview Hamster Colony, New Field, New Jersey. Interference test. Hamster embryo fibroblasts were inoculated with dilutions of the virus to be tested for interfering activity. Infected tissue cultures and uninfected controls were then transferred twice weekly at a split ratio of I : 3 (approximately 3 x io 5 cells/5o mm. Petri dish) and challenged at 21 days with 5o to 7o focus-forming units (f.f.u.) of virus. Cultures were scored for foci 5 to 6 days after challenge. Antisera and neutralization test. Antisera were obtained from weanling hamsters bearing transplants of a tumour line originally induced by M-MSV(HaLV)G and from guinea-pigs hyperimmunized with M-MSV(HaLV)G purified by rate zonal centrifugation in sucrose density gradients. Neutralization tests were performed as described by Kelloff et al. (197o a). [3H]Uridine labelling of virus particles. Various tissue culture preparations were tested for virus synthesis by following the incorporation of [ah]uridine into particles having a buoyant density of ~ 1.16 g./c.c, in sucrose gradients (Duesberg & Robinson, 1966). Cultures were

3 Hamster-tropic C-type viruses incubated with zo #c [ah]uridine (2oc/mmole)/ml. for 24 hr. Supernatant fluids were then collected, clarified by centrifugation at Io,ooo rev./min, for IO rain. and layered on I5 to 60 % sucrose gradients prepared in o.or M-tris + HC1 buffer, ph 7.2. Tubes were centrifuged for 9o rain. in the Spinco SW4I rotor at 4o,ooo rev./min. After centrifugation, o.2 ml. fractions were collected dropwise from the bottom of punctured tubes and radioactivity was then determined in a liquid scintillation counter. The presence of significant radioactivity in the appropriate density range (~'r5 to r'i7 g./c.c.) showing a symmetrical distribution was considered evidence of virus synthesis, since control cultures never yielded radioactivity in this region of the gradient. Electron microscopy. Electron microscopic observations were made by Mr J. Walker, Flow Laboratories, using the thin section technique on material which had been fixed in glutaraldehyde and osmium tetroxide, embedded in a mixture of Epon 812 and Araldite and double-stained with uranyl acetate and lead citrate. Sections were examined using an Hitachi HUr re electron microscope at a scanning magnification of 25,0o0 diameters. A culture was considered positive for virus if typical budding or extracellular C-type particles were clearly seen. Complement-fixation test. Complement-fixation tests were made in the Microtitre System using I'8 to 2.0 exact units of complement and overnight fixation at 5 (Huebner et al. r963). Studies on HaL V RESULTS Kelloff et al. (197o a) established that supernatant fluids and suspensions of freeze-thawed cells from a tissue culture line shedding this virus did not yield focus forming activity. However, an infectivity assay was required to prove that this virus was indeed a non-focusforming virus and not merely M-MSV(HaLV)G of insufficient titre to produce loci. Hamster embryo fibroblasts were infected with dilutions of HaLV, and at 7 days the infected cultures were found to be positive for virus by the [3H]uridine labelling technique and by electron microscope examination. Both techniques gave titres of IO infectious units/o. 4 ml., yet no foci were present in plates receiving undiluted inoculum (Table 2). Several pools of HaLV have now been tested and titres of Io 4 to IO 5 infectious units/o. 4 ml. were repeatedly obtained with no evidence of cell transformation. Since we have found repeatedly (see below) that HaLV is present in only IO- to ioo-fold excess of the focusforming activity in the M-MSV(HaLV)G stocks, we are sure that HaLV is an entity distinct from the focus-forming virus in the M-MSV(HaLV)G stocks. Duplicate cultures receiving dilutions of HaLV were also challenged with 5o to 7o f.f.u. of M-MSV(HaLV)G at 7, I4, 2i and 28 days and also with Ki-MSV(HaLV) and M-MSV(HaLV) at 2 t days after infection. Foci were counted 5 to 6 days after challenge. Interfering activity rose steeply from 7 to 2I days after infection, reaching a maximum at this time (Fig. I). Tissue cultures harvested at 2I days were also positive in complement-fixation tests with antisera prepared against the purified group-specific antigen of M-MSV(HaLV)G (Kelloff et al. I97ob; Oroszlan et al. 197o), thus establishing an assay which has been useful with other C-type viruses (Hartley et al. I965; Sarma, Turner & Huebner, i964) (Table 2). Challenge with Ki-MSV(HaLV) and M-MSV(HaLV) showed that HaLV interfered with these viruses to about the same extent as with M-MSV(HaLV) (Table 3)- To establish that the interference observed was not mediated by interferon, cells infected with dilutions of the helper virus and uninoculated controls were serially transferred and at 21 days challenged with I oo p.f.u, of vesicular stomatitis virus. No interference with vesicular stomatitis virus plaque-forming ability was observed in the HaLV-infected cells, as 2I

4 G. KELLOFF AND OTHERS compared to uninoculated controls (Table 3). In preliminary experiments, HaLV-infected cells were found to he susceptible to transformation by a strain of murine sarcoma virus, M-MSV (RLV), passaged in rat cells and able to replicate in hamster cells (Ting & Bader, I969). This provided a further distinction from the murine C-type viruses exhibiting reciprocal interference among all strains tested (Sarma et al I967) and emphasized the specificity of the interference by HaLV for hamster C-type viruses J t J I Days Fig. i. Development of interference in hamster embryo fibroblasts infected with HaLV. Table 2. Titration of hamster specific non-focus-inducing virus (HaLV) by several methods* Interference with Dilution of Electron [~H]Uridine- Complement M-MSV(HaLV)G HaLV microscopyt labelling:~ fixation (f.f.u./culture)ll IO -1 l- + -~- 0 - I0 -~ I I IO s Uninfected hamster cells * Hamster embryo fibroblasts (LSH strain) in 50 mm. Petri dishes tested 2t days after infection. y + = presence of C-type particles. $ + = clear presence of radioactive peak at H6 g/c.c, in sucrose gradients of culture fluid after 24 hr labelling with io mc/ml, of [ah]uridine. + = positive fixation of 2o % cell suspensions using guinea-pig antiserum against hamster gs antigen (Kelloff et al. 197ob ). II Approximately 5o focus-forming units added at 2I days. Foci counted 5 to 6 days later. M-MSV(GLV) stocks were tested for the presence of HaLV by infecting hamster embryo fibroblasts with IO TCD 5o (as assayed on mouse embryo cells), and at 2I days testing these cells in complement-fixation tests with antisera made against the purified gs antigen of the hamster C-type virus. No HaLV antigen was detectable in these cells.

5 Hamster-tropic C-type viruses 2 3 Titre of helper virus, interference patterns and host range of M-MS V(HaL V)G, Ki-MS V(HaL V) and M-MS V(HaL V) Hamster embryo fibroblasts were inoculated with serial tenfold dilutions of the three virus stocks. The focus-forming and infectious virus titres (as measured by the [ah]uridine labelling technique) were obtained at 7 days, and the infected cells were subsequently transferred twice a week and challenged with the three viruses at 2I days. The infectious virus titres of the M-MSV(HaLV)G stocks were consistently found to be tenfold higher than the focus-forming titres, whereas the infectious virus titres of the Ki-MSV(HaLV) and M-MSV Table 3. Interference with three hamster-specific sarcoma viruses by HaL V Challenge virus* Dilution of ~- HaLV M-MSV(HaLV)G Ki-MSV(HaLV) M-MSV(HaLV) I0-1 0"~ O ~ I0 -a I a 4 IO 8 IO-~ Uninfected 56 6o 5 i HEF Vesicular stomatitis virus (p.f.u./culture) * Focus-forming virus added to hamster embryo cells 2r days after infection; foci counted 5 to 6 days after challenge. Number of transformed cell foci per culture. Table 4- Attempts to demonstrate interfering virus in excess of focus-forming virus in three hamster-specific sarcoma virus preparations Challenge viruses* No challenge M-MSV(HaLV)G M-MSV(HaLV) Ki-MSV(HaLV) Uninoculated HEF control o 6o'~ M-MSV(HaLV)G: IO ~ TNTC~ O O o M-MSV(HaLV): IO a TNTC I o -4 o l Ici-MSV(HaLV) : IO -3 TNTC IO * Challenge virus added 21 days after infection, foci counted 5 to 6 days after challenge. ~- Number of transformed cell foci per culture. $ Unchallenged cultures which had received focus-forming virus in the initial inoculum contained many transformed cells and confluent foci which could not be counted accurately; these cultures were not challenged (HaLV) stocks were never in excess of their respective focus-forming titres, These results were consistent with the interference results shown in Table 4- The cells infected with M-MSV (HaLV)G which had received a dilution of virus stock tenfold greater than the focus-forming end-point dilution were completely resistant to challenge with all three sarcoma viruses, whereas no interference was observed in cells receiving a dilution of Ki-MSV(HaLV) and

6 2 4 G. KELLOFF AND OTHERS M-MSV(HaLV) higher than their respective focus-forming titres. Studies are in progress to establish whether or not a helper virus is present in these stocks which is not in excess of the focus-forming viruses. Cells that were originally infected with focus-forming virus, but not subsequently challenged, yielded tissue culture lines with many transformed cells and non-discrete foci (Table 4). Studies of interfering ability of one focus-forming virus for the other two could therefore not be accurately made; but the envelope relationships of these viruses could be satisfactorily established by immunological methods (see below). Table 5. Relative susceptibility of embryo fibroblasts from four strains of hamster to three hamster sarcoma viruses Hamster strain Virus strain LSH MHA LHC NIH M-MSV(HaLV)G M-MSV(HaLV) 1 ~ o o-o 0.0 0" Ki-MSV(HaLV) I 0"22 0'0 0"28 Table 6. Cross neutralization of three hamster-specific sarcoma viruses by antisera to one of them* Dilution of anti- Test virus serum against M-MSV(HaLV)G c ~' M-MSV(HaLV)G M-MSV(HaLV) Ki-MSV(HaLV) I/40 o t o o I[80 O O I 1/16o /320 I3 II 5 t/64o Normal hamster I/IO * Heat inactivated serum (56, 30 rain.) incubated with virus at 37, 30 rain. before inoculation on hamster embryo cells. ec Number of transformed cell foci per culture. Embryo fibroblasts from the three inbred hamster strains, LSH, LHC and MHA, obtained from Lakeview Farms, and the NIH strain from the National Institutes of Health, were tested for their relative susceptibility to the three sarcoma viruses in attempts to find distinguishing host range differences. Serial dilutions of the three sarcoma viruses were titrated on the four hamster embryo strains. Virus titres on LSH were given a relative value of one. The virus titres on the other three strains were expressed as proportions of the titre on LSH cells. A difference in susceptibility to the three viruses was found for each strain, but each strain appeared to be equally susceptible to the three viruses (Table 5). LSH was the most susceptible, while LHC cells yielded no foci with irlocula containing as much as ~ o 5 f.f.u. ph]uridine-labelling and electron microscopic examination of LHC cultures revealed no C-type particles, so the resistance of these cells is assumed to be genetic in origin. Neutralization studies Neutralizing antisera to M-MSV(HaLV)G were obtained from hamsters bearing a tumour transplant line originally induced by M-MSV(HaLV)G and from guinea-pigs hyperimmunized with purified intact M-MSV(HaLV)G. In addition to the homologous virus, these antisera also consistently neutralized Ki-MSV(HaLV) and M-MSV(HaLV). No differences in serum

7 Hamster-tropic C-type viruses 25 end-points were obtained with equivalent concentrations of the three sarcoma viruses (Table 6), indicating that these viruses shared at least one critical envelope antigen; however, more rigorous studies are required to establish complete identity of their envelopes. As previously reported, these antisera consistently failed to neutralize equivalent doses of M-MSV(GLV) and M-MSV. Conversely, antisera known to neutralize these murine viruses failed to neutralize the three hamster sarcoma viruses. Neutralization of HaLV was studied by inhibition of interference induction. Four neutralizing units of the hamster virus specific antisera were used in tests with IO ~ interfering units of helper virus. The interfering activity of HaLV was completely neutralized by such antiserum (Table 7). Table 7. Neutralization of HaLV-intetfering activity by antiserum to M-MS V(HaLV)G from tumour-bearing hamsters Dilution of HaLV Normal hamster 1/I o io 0 o ~ I0 2 0 IO 3 0 lo Anti M-MSV(HaLV) t/4o 52 * Number of transformed cell foci per culture. DISCUSSION This report confirms the presence of a non-focus-forming C-type virus in the stocks of M-MSV(HaLV)G (Kelloff et al. I97oa). Inability to detect this virus in our stocks of M-MSV (GLV) suggests that the source of this virus is the hamster and that it represents the indigenous C-type virus of the hamster; further evidence for this is provided in the accompanying report (Kelloff et al. I97ob). The development of interference, independent of interferon production, in cultures infected with HaLV is consistent with previous studies on the C-type viruses (Rubin & Vogt, I962; Sarma et al. 1967). The ability of this virus to interfere with superinfection by M-MSV(HaLV)G, Ki-MSV(HaLV) and M-MSV(HaLV) is strong evidence that these four viruses share a common envelope antigen. The ability of antisera against one of these viruses to neutralize all four hamster specific viruses provides further proof of these conclusions and serves to justify the nomenclature which appears best to describe these viruses. The question of the origin of the sarcomagenic genome in M-MSV(HaLV)G has been discussed by Kelloff et al. 097oa). They assumed that this sarcomagenic genome was the same as that in the murine virus inoculum MSV(GLV), and not an activation of an indigenous sarcoma virus of the hamster, although proof of this cannot be obtained in the absence of genetic markers for the sarcoma genome. It is important to note here that the morphology of transformed cells observed with Ki-MSV(HaLV) is consistently distinguishable from that observed with M-MSV(HaLV)G and M-MSV(HaLV), suggesting that these sarcomagenic genomes are distinct and do not represent activation of an indigenous sarcoma virus of the hamster. This work was supported by Contracts PH43-67-I 396 and NIH69-97 from the National Cancer Institute of the National Institutes of Health, Bethesda, Maryland. We thank Mr R. Toni for excellent technical assistance and Mrs Nancy C. Tucker for preparation of the manuscript.

8 26 G, KELLOFF AND OTHERS REFERENCES BASSIN, R. H., SIMONS, P. S., CHESTERMAN, F. C. & HARVEY, J. J. (I968). Murine sarcoma virus (HARVEY). Characteristics of focus formation in mouse embryo cell cultures and virus production by hamster tumor cells. International Journal of Cancer 3, 265. DUESBERG, P.H. & ROBINSON, W. S, (I966). Nucleic acid and proteins isolated from the Rauscher mouse leukemia virus (MLV). Proceedings of the National Academy of Sciences of the United States of America 55, 219. HARTLEY, J. W. & ROWE, W. P. (I966). Production of altered cell loci in tissue culture by defective MOLONEY sarcoma virus particles. Proceedings of the National Academy of Sciences of the United States of America 55, 780. HARTLEY, J. W., ROWE, W. P., CAPPS, W. I. & HUEBNER, R. J. (1965). Complement-fixation and tissue culture assays for mouse leukemia viruses. Proceedings of the National Academy of Sciences of the United States of America 53, 931. HUEBNER, R. J., HARTLEY, J. W., ROWE, W. V., LANE, W. 7. & CAPPS, W. I. (1966). Rescue of the defective genome of MOLONEY sarcoma virus from a noninfectious hamster tumor and the production of pseudotype sarcoma viruses with various routine leukemia viruses. Proceedings of the National Academy of Sciences of the United States of America 56, HUEBNER, R.J., ROWE, W. e., TURNER, H. C. & LANE, W.T. (1963). Specific adenovirus complement-fixing antigens in virus-free hamster and rat tumors. Proceedings of the National Academy of Sciences of the United States' qf America 50, 379- HUEBNER, R.J., TODARO, G.J., SARMA, P., HARTLEY~ J.W., FREEMAN, A.E., PETERS, R.L.~ WHITMIRE, C.E., MEIER, H. & GILDEN, R. V. (I969). 'Switched Off' vertically transmitted C-type RNA tumor viruses as determinants of spontaneous and induced cancer: A new hypothesis of viral carcinogenesis. Second International Symposium on Tumor Viruses, Paris'. KELLOFF, G., HUEBNER, R. J., LEE, Y. K., TONI, R. & GIEDEN, R. V. (I97oa). Hamster-tropic sarcomagenic and nonsarcomagenic virus(es) derived from hamster tumors induced by the Gross pseudotype of MOLONEY sarcoma virus. Proceedings of the National Academy of Sciences of the United States of America 65, 3IO. KELLOFF, G., HUEBNER, R. J., OROSZLAN, S., TONI, R. & GILDEN, R. V. (I97ob). Immunological identitity of the group-specific antigen of hamster-specific C-type viruses and an indigenous hamster virus. Journal of General Virology 9, 27. KLEMENT, V., HARTLEY, J. W., ROWE, W. P. & HUEBNER, R. J. (1969). Recovery of a hamster-specific focus forming and sarcomagenic virus from a 'noninfectious' hamster tumor induced by the KIRSTEN mouse sarcoma virus. Journal of the National Cancer Institute 43, 925. MOLONEY, J. B. (1960). Biological studies on a lymphoid-leukemia virus extract from Sarcoma 37. I. Origin and introductory investigations. Journal of the National Cancer Institute :a4, 933- OROSZLAN, S., FISHER, C. L., KELLOFF, G. & GILDEN, R. V. (1970). Purification and properties of the groupspecific antigen of hamster C-type viruses. (In preparation.) PERK, K., VIOLA, M. V., SMITH, K. L., W1VEL, N. A. & MOLONEY, J. B. 0969)- Biologic studies on hamster tumors induced by the murine sarcoma virus (MOLONEY). Cancer Research 29, Io89. RUBIN, H. & roar, P. (I962). An avian leukosis virus associated with stocks of Rous sarcoma virus. Virology 17, t84. SARMA, P., CHEONG, M. P., HARTLEY, J. W. & HUEBNER, R. J. (I967). A viral interference test for mouse leukemia viruses. Virology 33, 18o. SARMA, V., LO6, T. & 6ILDEN, R. V. (1970). Studies of hamster-specific virus derived from hamster tumors induced by ~IRSTEN murine sarcoma virus. Proceedings of the Society for Experimental Biology and Medicine I33, 718. SARMA, V. S., TURNER, H.. & Ht:EBNER, R. J. (I964). An avian leukosis group-specific complement-fixation reaction. Application for the detection and assay of non-cytopathogenic leukosis viruses. Virology 23, 313. TING, R. C. & BADER, A. V. (I969). A quantitative study of transformation of hamster embryo cells in vitro by murine sarcoma viruses (HARVEY & MOLONEY). Virology 39, 194. (Received I7 March I97O)