teins and RNA sequences. This paper will present brief descriptions of these viruses. in most of the studies were obtained from fertile eggs (SPA-

Transcription

1 Proc. Nat. cad. Sci. US Vol. 73, No., pp , pril 1976 Microbiology Pheasant virus: New class of ribodeoxyvirus [avian oncornavirus/viral proteins/dn.rn hybridization/rn-dependent DN polymerase (nucleotidyltransferase)/helper virus] T. HNFUS*, H. HNFUS*, C.. MTROK*, W. S. HYWRD*, C. W. RTTNMR*, R. C.; SWYR*, R. M. DOUGHRTYt, ND H. S. DSTFNOt * The Rockefeller University, New York, N.Y ; and t State University of New York, Upstate Medical Center, Syracuse, N.Y Communicated by James. Darnell, Jr., January 26,1976 BSTRCT Cocultivation of cells derived from embryos of golden pheasants or mherst pheasants with chicken embryo cells infected with Bryan strain of Rous sarcoma virus resulted in the detection of viruses which appear to be endogenous in these pheasant cells. The pheasant viruses (PV) were similar to avian leukosis-sarcoma viruses (LSV) in their gross morphology, in the size of their RN, in the presence of a virion-associated RN-dependent DN polymerase (DN nucleotidyltransferase; deoxynucleoside triphosphate: DN deoxynucleotidyltransferase; C ), and in their growth characteristics. PV also serves as a helper for the glycoprotein-defective Rous sarcoma virus. However, PV was shown to be different from both LSV and reticuloendotheliosis virus in the following properties: (i) PV does not have LSV group specific antigens; (ii) the protein composition of PV is different from those of the other two groups of viruses; (iii) PV fails to complement the defective polymerase of alpha type Rous sarcoma virus; and (iv) PV RN shows no detectable homology with nucleic acids of the other two groups of viruses. Thus, PV appears to be a new class of RN viruses which contain RN-dependent DN polymerase. The presence of viral genetic information of RN tumor virus in normal uninfected chicken cells is well known (1-5). Genetic information of the "endogenous" viral genes can often be recovered in progeny viruses after infecting these cells with avian leukosis-sarcoma viruses (LSV). Rous-associated virus 60 (RV-60) isolated from chicken cells in this manner (6) has been shown to contain nucleic acid sequences of both the endogenous virus genes and the exogenously infecting viruses (7). Likewise, endogenous viral genes were shown to exist in ring-necked pheasant and golden pheasant cells, and the new viruses isolated from these cells were called RV-61 and golden pheasant virus (GPV) respectively (8, 9). The main criteria for the recovery of new viral genetic information in these isolates were the properties of viruses specified by the viral envelope, such as host range, sensitivity to interference, and antigenicity, which are the basis for the subgroup classification of LSV. n the previous studies, we found no clear correlation between the expression of group specific antigens of LSV in pheasant embryo cultures and the ease with which RV-61 was recovered from the particular cultures (8). Further studies on the correlation of these two phenomena in other pheasant cells led us to the discovery that the GPV and another similar isolate from mherst pheasants are different from the members of known LSV in their structural pro- bbreviations: chf, chicken helper factor; LSV, avian leukosis-sarcoma virus; RV, Rous associated virus (avian leukosis virus); B- RSV, Bryan strain of Rous sarcoma virus; B-RSV(-), B-RSV produced from chf negative chicken cells; B-RSV(chf), B-RSV produced from chf positive chicken cells; RV, reticuloendotheliosis virus, TDSNV, Trager duck spleen necrosis virus; PV; pheasant virus; GPV, golden pheasant virus; PV, mherst pheasant virus; cdn, DN complementary to virus RN; NaDodSO, sodium dodecyl sulfate; NP0, Nonidet P0; FFU, focus-forming units teins and RN sequences. This paper will present brief descriptions of these viruses. MTRLS ND MTHODS Cells and Viruses. Chick embryo cells of C/ type used in most of the studies were obtained from fertile eggs (SP- FS nc., Norwich, Conn.) (10). Chicken eggs of line 15 (11) were kindly supplied by Dr. L. B. Crittenden, Regional Poultry Research Laboratory, U.S. Department of griculture, ast Lansing, Mich. Chicken cells used were free from the expression of chicken helper factor (chf). ggs of golden pheasants (Chrysolophus pictus) and mherst pheasants (Chrysolophus amherstiae) were supplied by Mr. R.. Reynolds and Mr. Carol Ganoung, Richard. Reynolds New York State Game Farm, thaca, N.Y. The group specific antigens of LSV were determined by the complement fixation test (10) and by the radioimmunoassay (12, 13). Chicken cells transformed by Bryan Rous sarcoma virus (B- RSV) were prepared by the method described before (10, 38). LSVs utilized for the comparison of their envelope properties were those described before (6, 7, 10). GPV and B-RSV(GPV) generously supplied from Drs. P. K. Vogt and Y. Chen, University of Southern California, were also used as reference viruses. Trager duck spleen necrosis virus (TDSNV) isolated by Trager (1) was kindly supplied by Dr. H. Temin, University of Wisconsin, and grown in chick embryo cells. Gel lectrophoresis of Viral Proteins. Cultures infected with various viruses were incubated with medium containing 25 gci/ml of L-[,5-3H]leucine (6 Ci/mmol), 5,qCi/ml of. L-['C]leucine (320 mci/mmol), or 10,uCi/ml of D-[6-3H]glucosamine hydrochloride (10 Ci/mmol) for successive 12 hr intervals. The labeled virus was purified and subjected to protein analysis as described before (15). The samples were analyzed by sodium dodecyl sulfate (NaDodSO) polyacrylamide gel electrophoresis, which was performed either in a 7% cylindrical gel (15) or in a % gradient slab gel (16), both containing NaDodSO at 0.1%. Polymerase ssay. Virus-associated RN-dependent DN polymerase (DN nucleotidyltransferase, deoxynucleosidetriphosphate:dn deoxynucleotidyltransferase; C ) activity was determined as previously described (3), using (rc)n.(dg)12-s as a template-primer complex. xtraction of Labeled Viral RN. Virus-infected cells were incubated with medium containing 200,uCi of [3H]uridine per ml. Culture fluids were collected at 12 hr intervals. Virus was pelleted and RN was isolated after treatment with Pronase-NaDodSO as described by Bishop et al. (17). Viral RN was centrifuged in a 5-20% sucrose gradient in a buffer containing 0.01 M Tris-HCl (ph 7.), M DT, and 0.1 M NaC, before or after heating RN at 800 for 3 min.

2 133 Microbiology: Hanafusa et al. Homologies among Viral Nucleic cids. DN complementary to viral RN was synthesized in the presence of actinomycin D from detergent-treated virions as described before (13, 18). 3H-Labeled complementary DN (cdn) (approximately 2.0 X 107 cpm/gg) was hybridized with RN extracted from virus-infected cells in a reaction mixture containing 0.5 M NaCl, 0.05 M sodium citrate (ph 7.), 5 mm DT, 0.1% NaDodSO, and 50% formamide (13). The reaction mixture was incubated at 500. Hybrid formation was detected by treatment with the single-strandspecific S-1 nuclease (18). Radioimmunoassay of LSV Proteins. The techniques were described previously (12, 13). Viral proteins p27, p19, and p15 of RV-2, and rat antisera for these purified proteins were prepared by Dr. J. H. Chen (13). lectron Microscopy. Cell cultures were fixed in situ for 1 hr with an osmium preparation described previously (35), consisting of 1% OS0 in equal parts of 7.3% polyvinylpyrrolidinone and 0.25 M sucrose (ph adjusted to 7.2 with 0.1 M NaOH). The cells were subsequently washed with 0.85% NaCi, dehydrated, and embedded in Maraglas. Sections were stained with uranyl acetate followed by basic lead citrate and examined with a Norelco M300 electron microscope. RSULTS solation of Virus from Pheasant Cells. Culture fluids of cells from three embryos each of golden pheasant and mherst pheasant were shown to be free from LSV by the absence of virus particles detectable by DN polymerase or of helper activity for formation of pseudotypes of defective B- RSV. Helper viruses were isolated from both species of pheasant cells by essentially the same manner as that utilized before (8, 9). Pheasant cells were cocultured together with approximately the same number of chicken cells which had been transformed by B-RSV(-). The culture fluid was assayed on chicken cells for focus-forming virus. Since these chickentransformed cells alone produce only noninfectious RSV(-), detection of focus-forming virus by assay in C/ type chicken cell cultures would indicate the formation of a phenotypically new virus as a result of interaction with the pheasant cells. The focus-forming virus became detectable shortly after coculture: in 3 days focus-forming units (FFU)/ml of RSV were found in culture fluids by assaying on chicken cells, and the titer increased to greater than 105 FFU/ml in 1 week. The presence of chicken cells seemed to facilitate isolation because the rescued virus grew well in chicken cells. However, transforming virus with the same properties could also be isolated directly from pheasant cells within 2 weeks following infection by B-RSV(chf). By both methods, transforming viruses were obtained from all embryos of the two species of pheasants. The infectivity for various avian cells (chicken, quail, duck, ringnecked pheasant, mherst pheasant, and golden pheasant) of the transforming virus isolated from both golden and mherst pheasants was similar to that of GPV previously isolated by Fujita et al. (9) which had been classified as subgroup G. By repeated passages of these viruses at terminal dilutions, nontransforming helper viruses were isolated from both preparations of transforming viruses. Since our helper virus isolates were indistinguishable from the previously isolated GPV (9) in host range and virus-induced interference, we will designate the virus isolated from our golden pheasants as GPV-ny. The virus from mherst pheasants will be ; b 2 x 0 U Proc. Nat. cad. Sci. US 73 (1976) B B %.., : C? 2 o x o 1O Fraction Number FG. 1. lectrophoresis of the proteins of GPV-ny, PV, RV-2, and TDSNV in NaDodSO (0.1%)-polyacrylamide (7%) gel. The polypeptides migrated from left to right. () RV-2 labeled with ['C]leucine (0) was subjected to coelectrophoresis with GPV-ny labeled with [3H]leucine (0). GPV-ny labeled with [3H]glucosamine () was analyzed in a parallel gel and the data are superimposed. (B) TDSNV labeled with ['C]leucine (0) and GPV-ny with [3H]leucine (0) were coelectrophoresed. TDSNV labeled with [3H]glucosamine () in a parallel run was superimposed. designated as mherst pheasant virus (PV). GPV or PV pseudotypes of B-RSV were obtained by adding GPV or PV to cells transformed by B-RSV. Both GPV-ny and PV produce interference with RSV(GPV), RSV(GPV-ny), or RSV(PV) but no interference with other LSVs which belong to subgroup to F, nor with the pseudotype of a new isolate, RV-62 from Hungarian partridge embryos, which may be assigned as subgroup H (T. Hanafusa, unpublished results). nfectivity of PV and GPV-ny was enhanced by D-dextran, as is that of most members of LSV. Viral Proteins of GPV and PV. During the course of isolation, we recognized that chicken cell cultures fully infected with GPV-ny and PV, as well as GPV, were negative for group specific antigens of LSV by the complement fixation test, even though the cultures produced virions detectable by both infectivity (about 106 infectious units/ml) and polymerase activity. The absence of proteins immunologically reactive with antibodies against LSV proteins was confirmed by the more sensitive radioimmunoassay. The amounts of three major LSV proteins, p27, p19, and p15, in GPV-ny-infected chicken cells were comparable to those found in uninfected (chf-negative) chicken cells. Since the above results strongly suggested that the proteins of GPV and PV are quite different from those of regular LSV, we examined the composition of viral proteins of GPV-ny and PV by gel electrophoresis. Cultures infected with the pheasant viruses were labeled with [3H]leucine; and the 3H-labeled virus was purified. RV-2 labeled with [1C]leucine was prepared in the same manner. The two purified viruses were mixed, heated in the presence of NaDod- S0, and subjected to cylindrical gel electrophoresis. 1C- Labeled viruses were also separately prepared for GPV, RV-2, -7, -60, and -61, and proteins were analyzed on slab gels. s shown in Figs. 1 and 2, the profiles of proteins from 3 2

3 Microbiology: Hanafusa et al. Proc. Nat. cad. Sci. US 73 (1976) 1335 B C D,.V1., GP85 11 P12 P27 P19 P15 FG. 2. utoradiogram of [1C]leucine-labeled proteins from purified GPV-ny and LSVs after gel electrophoresis % gradient polyacrylamide slab gel, 1.25 mm thick, was used with electrophoresis buffer consisting of M Tris (ph 8.9), 0.2 M glycine, and 0.1% NaDodSO. The migration was from left to right. Following electrophoresis at 10 m., the gel was fixed and stained with Coomassie blue and dried. For autoradiography the gel was exposed against Dupont Cronex 2DC safety film. () GPV-ny; (B) RV-61 (subgroup F); (C) RV-60 (subgroup ); (D) RV-7 (subgroup C); () RV-2 (subgroup B). ll viruses were grown in chick embryo cells with the exception of RV-60, which was grown in Japanese quail cells. GPV and PV were quite different from that of LSV. ll strains of LSV thus far tested are known to have the same composition of structural proteins, of which only p19 is antigenically distinguishable for different strains (19). This was confirmed in the radioautogram shown in Fig. 2, in which differences in mobility were demonstrated only for p19 among LSV tested. None of the proteins of GPV or PV comigrated with those of RV-2. Two slow-moving pheasant virus proteins were labeled with glucosamine, as shown in Fig.. The protein composition of pheasant viruses was also compared (Fig. B) with that of TDSNV, which is one of the members of reticuloendotheliosis viruses (RV). RV is known to be similar to avian leukosis virus in its structure and life cycle, but their protein compositions are different. The electropherograms of TDSNV proteins were essentially the same as those described by Halpern et al. (20) and Mosser et al. (21). gain none of the proteins had the same mobility as those of PV. The protein composition of the original isolate of GPV was also the same as those of GPV-ny and PV. Table 1 summarizes the comparison of proteins of Table 1. Polypeptide composition of LSV, TDSNV, and PV RV-2 TDSNV PV gp85 p80 gp65 gp75 gp5 gp32 p50 p3 p27 p30 p29 p19 gp22 p12 p1 p15 plo (plo)* The results of analyses of polypeptides of viral proteins of RV-2, TDSNV, and PV by NaDodSO-polyacrylamide gels are summarized. The polypeptides are shown by their apparent molecular weight (X10-3), and glycoproteins are indicated by gp. The apparent molecular weight of each polypeptide was calculated from its mobility relative to the following proteins: phosphorylase (9,000 daltons), bovine serum albumin (68,000 daltons), ovalbumin (3,000 daltons), chymotrypsinogen (25,500 daltons), and cytochrome c (12,500 daltons). * LSV plo was not seen in either cylindrical or slab gels. 20 C W 20 / a.: /, -o_-o2so+ 60 B X,^ //to 10 o10' 12 1o3 o Ct (mol sec ifter-1) FG. 3. Hybridization of viral RN in infected cells with viral [3H]cDN. Cr is initial RN concentration in moles of nucleotide/ liter; t is time in seconds. () GPV-ny [3H]cDN was hybridized with RN of cells infected with GPV-ny (0), RV-0 (0), B-RSV (), or TDSMV (X). (B) RV-0 [3H]cDN (---- ) or RV-60 [3H]cDN (-) was hybridized with RN of cells infected with RV-60 (+), RV-0 (o), B-RSV (), or GPV-ny (0, 0). Line 15 chicken cells were used for infection with RV-0, and Japanese quail cells for RV-60. SPFS chicken cells were used for all other viruses. these three classes of viruses. The molecular weights of pheasant virus proteins were estimated from their mobility in the gel electrophoresis. Lack of Homologies among Nucleic cids of Pheasant Viruses, LSV, and RV. 3H-Labeled complementary DN (cdn) was synthesized from purified GPV-ny in the presence of Nonidet P0 (NP0), Mg2+, and actinomycin D, using reaction conditions which have been utilized for DN synthesis with LSV (7). The [3H]cDN product hybridized more than 90% with RN isolated from GPV-ny-infected chicken cells (Fig. 3). No hybridization (<2%) was observed with RN from uninfected chicken cells, or with RN from cells infected with TDSNV, RV-O, or B-RSV. By contrast, RV-0 and RV-60 [3H]cDNs hybridized 85-95% with the RN from LSV-infected cells, but did not hybridize with the GPV-ny-infected cell RN. These data, and those described in the previous section, clearly indicate that these pheasant viruses are substantially different from both LSV and RV in their genome RN and their structural proteins. To distinguish these viruses from known avian RN tumor viruses, the GPV and PV may be called pheasant virus (PV) collectively. Other Biological and Biochemical Properties. Despite these differences, PV appears to be distantly related to LSV, since the glycoproteins of the former virus can be effectively utilized by RSV deficient in the synthesis of the glycoproteins. RV was shown to be incapable of acting as a helper for the defective RSV (20). Further, in this study cultures fully infected with TDSNV did not produce any interference with B-RSV(RV-2), B-RSV(GPV-ny), or B- RSV(PV), indicating a difference between the envelope glycoproteins of TDSNV and PV.

4 1336 Microbiology: Hanafusa et al. 1L~~~~ i0~~~~~~~~~~~~~~~ L Final Mg* or Mn* concentration (rmm) FG.. ffect of divalent cation concentration on exogenous RN-dependent DN polymerase reaction. Virus was concentrated from equal volumes of culture medium by centrifugation into a pellet and assayed for polymerase activity in the presence of 7. g of (rc)n-(dg)1si1 (7:3). The reaction mixture in 0.1 ml contained 0.1% NP0, 0.05 M Tris-HCl (ph 8.3), 0.06 M NaCl, 0.02 M dithiothreitol, 2 nmol of [3H]dGTP (about 500 cpm/pmol), and the indicated final concentration of either Mg acetate or Mn acetate. The mixture was incubated for 1 hr at 370 and trichloroacetic acidinsoluble radioactivity was determined. () ffect of Mg2+ concentration; (B) ffect of Mn2+ concentration. RV-2 (0), GPV-ny (o), TDSNV (0). Unlike the enzyme of RV, the DN polymerase of PV worked well with both exogenous and endogenous templates, and cdn product of the endogenous reaction hybridized nearly 100% with the PV RN. The PV polymerase more closely resembles the LSV enzyme than the RV enzyme by its strong preference for Mg2+ to Mn2+ over a broad range of concentrations using the template-primer poly(rc).oligo(dg) (Fig. ). Unlike RV polymerase, PV polymerase gives only low levels of incorporation using Mn2+ as compared to the incorporation at optimal Mg2+ concentrations. However, another characteristic which distinguished PV from LSV was the failure of the former to act as a helper for B-RSVa which is known to be deficient in both genes coding for RN dependent DN polymerase and envelope glycoproteins. Cells transformed by B-RSVa produce noninfectious particles which lack both the polymerase and the glycoproteins (38). nfection of these transformed cells with nondefective LV results in the production of infectious RSV containing polymerase and glycoproteins supplied by the superinfecting virus. Superinfection of the B-RSVatransformed cells with PV or GPV failed to form infectious RSV, suggesting that some molecular differences exist between the DN polymerases of PV and of LSV. The density of PV in sucrose gradients was greater than that of LSV: i 0.05 g/cm3 for the former and g/cm3 for the latter. The size and subunit structure of PV RN seem to be similar to those of LSV. The viral RN of PV sediments as a 70-75S complex which is converted to a form sedimenting at about 35 S by heating at 800 for.. : 66_ a '[3 He he a,{ +'..,. '. ' -i,.e :_ >'R:s x S s + * ;Xf * Proc. Nat. cad. Sci. US 73 (1976) lf, TC, k 2ML NM, 9W OF i ft-%.".w L,- a FG. 5 Comparative morphology of typical GPV and RV-1 particles a b and c are three stages in the maturation of GPV (budding form, free immature, and mature particles, respectively); di e, and f are comparable stages in the maturation of RV-1. Compare space between core membrane and envelope of immature particles (b versus e) and space between nucleoid and core membrane of mature particles (c versus f). X 151,000 3 min. No further characterization of viral RN was made in this study. Morphologically PV has most characteristics of type-c virus, but in detail it differs from LSV. n fixed preparations of PV, the core membranes were located close to the outer envelope so that a larger space was visible between nucleoid and core membranes. n immature particles the core of PV was consistently more electron lucent than that of LSV (Fig. 5). DSCUSSON The virions of pheasant viruses described here have a morphology similar to C-type RN tumor viruses or RV. They contain a high-molecular weight RN which can be dissociated to a smaller subunit(s) by heating. The viruses also have a polymerase which synthesizes polydeoxyribonucleotides using either endogenous viral RN or synthetic poly(rc).oligo(dg) as templates and primers. They establish persistent infection in chicken or pheasant cells. Based on all of these characteristics, PV should certainly be considered a member of the large virus group called ribodeoxyviruses (36) or leukoviruses (37), which include oncornaviruses, RV, visna virus; and foamy viruses. Because GPV acts similarly to avian leukosis virus in complementing the defective strain of RSV and in causing virusinduced resistance, GPV was previously classified as a r 0 - N P. l W"

5 Microbiology: Hanafusa et al. subgroup of LSV (9). The present study clearly shows that structural proteins of both GPV and PV are substantially different in composition from any of those of LSV (15, 22), mammalian oncornaviruses (23), RV (20, 21), visna virus (2, 25), rhabdovirus (26), myxovirus (27, 28), or paramyxovirus (29). The PV proteins were also shown to be immunologically unrelated to three major LSV proteins. Further, there was no detectable homology between PV RN and RN of members of LSV or RV by nucleic acid hybridization, which has been shown to be specific; high levels of homology are found among all of the members of the LSV class, but not with members of other classes of virus such as murine leukemia virus or RV (30, 31). Finally the DN polymerase of PV does not appear to be utilized by an RSV mutant which is defective for this enzyme. Therefore, PV seems to be sufficiently different from LSV to be classified separately. t should be mentioned however, that the term PV used in this paper does not mean to imply the presence of this type of virus in all pheasant species, or that they will be isolated only from pheasants. GPV and PV were isolated from pheasants which belong to the same genus, Chrysolophus. RV-61 (8), which was isolated from ringnecked pheasants (Phasianus colchilus), appears to contain structural proteins essentially indistinguishable from other members of LSV (Fig. 2). t is conceivable, however, that RV-61 is a recombinant between the RSV genome and virus genes endogenous to the pheasant, in which the gene for internal structural proteins may have originated from RSV. Further investigations are required to better establish the classification of these viruses and their distribution among different species of pheasants. The lack of homology between RNs of PV and LSV raises an interesting question about the mechanism of formation of GPV or PV. Thus far, we have been unable to observe the spontaneous production of viruses from golden or mherst pheasant cells. f, as in the case of formation of RV-60 or RV-61, genetic recombination between endogenous and exogenous virus genes plays a role in the formation of PV, the isolates should contain some sequences homologous to the exogenous virus, B-RSV in this case. More information is needed to clarify the mechanism for the formation of PV. The only known biological interaction between PV and LSV is the helper activity of PV for defective B-RSV. Since RV lacks this activity, one might argue that PV is closer to LSV than RV. However, envelope glycoprotein is known to be exchangeable between LSV and the unrelated vesicular stomatitis virus (32, 33, 39). We are grateful to Ms. Kathryn Brier for excellent technical assistance, and to Mr. R.. Reynolds and Mr. Carol Ganoung for generously providing us with pheasant eggs. The work was supported by Grants C 1018, C 1935, C 1936, and C from the National Cancer nstitute. R.C.S. is a fellow of The Jane Coffin Childs Memorial Fund for Medical Research. 1. Weiss, R.. (1969) J. Gen. Virol. 5, Hanafusa, H., Miyamoto, T. & Hanafusa, T. (1970) Proc. Nat. cad. Sci. US 66, Proc. Nat. cad. Sci. US 73 (1976) Rosenthal, P. N., Robinson, H. L., Robinson, W. S., Hanafusa, T. & Hanafusa, H. (1971) Proc. Nat. cad. Sci. US 68, Varmus, H.., Weiss, R.., Friis, R. R., Levinson, W. & Bishop, J. M. (1972) Proc. Nat. cad. Sci. US 69, Baluda, M.. (1972) Proc. Nat. cad. Sci. US 69, Hanafusa, T., Hanafusa, H. & Miyamoto, T. (1970) Proc. Nat. cad. Sci. US 67, Hayward, W. S. & Hanafusa, H. (1975) J. Virol. 15, Hanafusa, T. & Hanafusa, H. (1973) Virology 51, Fujita, D. J., Chen, Y. C., Friis, R. R. & Vogt, P. K. (197) Virology 60, Hanafusa, T., Hanafusa, H., Miyamoto, T. & Fleissner,. 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