Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
J. gen. Virol. (1977), 3 6 , ~ 459-469 459 Printed in Great Britain R N A Tumour Virus Phosphoproteins: Evidence for Virus Specificity of Phosphorylation By E D W A R D G. H A Y M A N , B I J A Y K. P A L AND P R A D I P R O Y - B U R M A N Departments of Pathology and Biochemistry, University of Southern California School of Medicine, Los Angeles, California 9o033, U.S.A. (Accepted 19 April I977) SUMMARY The purified I2OOO dalton (pI2) phosphoprotein of Rauscher (R) and wild mouse (WM) strains of murine leukaemia virus (MuLV) was analysed for the distribution patterns of its variously charged molecular species by urea-polyacrylamide gradient gel electrophoresis. The distribution patterns of the pI 2 of two different field isolates of WM viruses, 292 and I5O4, and the mouse-tropic and amphotropic clonal sub-populations of 15o4 field isolate were very similar but different from that of MuLV-R. A unique characteristic of the pI2 of the WM isolates is the presence of two major apparently non-phosphorylated species in approximately constant proportions relative to the phosphorylated species. Similar studies on the pI2 of the same virus (MuLV-R or WM viruses) grown in different host cells showed that the patterns of phosphorylated and non-phosphorylated species are virus-specific and independent of the cell lines of propagation. These analyses and their comparison with urea-gel patterns of the phosphoproteins of other mammalian type C viruses indicated that the number and relative proportion of the variously phosphorylated and non-phosphorylated species are predetermined for a virus. Therefore, the virus must have the genetic information for the phosphoprotein as well as other necessary genetic information which functions, perhaps in conjunction with appropriate cellular factors, in regulating the specific proportions of these multiple species. Possible biological significance of the variously charged molecular species in the phosphoprotein of RNA tumour viruses is discussed. INTRODUCTION Type C RNA tumour viruses contain phosphoproteins as their structural components (Pal et al. 1975; Pal & Roy-Burman, 1975). The proteins that are phosphorylated in vivo during virus synthesis belong to the major structural polypeptides. These phosphorylated proteins have been identified in various rodent type C viruses, in the exogenous and endogenous classes of feline type C viruses, and in type C viruses isolated from primates, including simian sarcoma associated virus, gibbon ape lymphosarcoma virus and baboon endogenous virus (Pal et al. 1975; Pal & Roy-Burman, 1975). In general, viruses originated from the lower mammalian species contain a major phosphoprotein of 12000 daltons (pI2) and the endogenous viruses of primate origin (baboon endogenous virus and the RD-II4 virus) contain a major phosphoprotein of 15ooo daltons (pI5). Besides the pI2 phosphoprotein, a second major phosphoprotein of IOOOO daltons (plo) is present only in viruses Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 09:53:23 ~1m46o E . G . H A Y M A N , B. K. P A L A N D P. R O Y - B U R M A N genetically related to the rat species. Recently it was shown that the major phosphorylated protein of avian type C viruses is the pl 9 protein (Lai, 1976). The phosphoprotein p15, pt2, or plo is present in the virion in several different phosphorylated states (Pal et al. 1975). These phosphorylated states and their relative proportions in the pI2 of Rauscher murine leukaemia virus (MuLV-R), murine leukaemia virus field isolates from wild mice (15o4, 292; Officer et al. 1973; Pal et al. 1973; Gardner et al. 1976), and the cloned mouse-tr0pic and amphotropic components (Bryant & Klement, 1976; Hartley & Rowe, 1976; Rasheed, Gardner & Chan, 1976) of the 15o4 isolate have been investigated for comparison after propagation of the viruses in different mouse and human cell lines. These results suggest that the phosphorylation patterns of the phosphoprotein are virus-specific and independent of the host cell line of propagation. In addition, these experiments reveal a unique characteristic of having two major non-phosphorylated species of pi2 in all uncloned or cloned isolates of wild mouse type C viruses tested. METHODS Reagents, media, and sera. Reagent or analytical grade chemicals were used in all experiments. All radiochemicals were obtained from New England Nuclear Corp., Boston, Massachusetts. Ultrapure urea and sucrose were purchased from Schwarz/Mann, Orangeburg, New York; agarose, acrylamide, and bisacrylamide from Bio-Rad Lab., Richmond, California; guanidine hydrochloride and Triton X-IOO from Sigma Chemical Co., St Louis, Missouri; pancreatic ribonuclease from Worthington Biochemical Corp., Freehold, New Jersey; Eagle's minimum essential medium (MEM) and foetal bovine serum from Flow Labs, Inglewood, California; gentamicin from Microbiological Associates, Bethesda, Maryland; and polybrene from Aldrich Chemical Co., San Leandro, California. Sodium dodecyl sulphate (SDS) obtained from Matheson, Los Angeles, California, was recrystallized before use. Cells and viruses. The Rauscher strain of MuLV was grown in NIH Swiss mouse embryo cells and in human rhabdomyosarcoma (RD) cells (McAllister et al. 1969). For infection of the cell lines, cells were pre-incubated for 18 to 24 h with 2/zg of polybrene per ml before exposure to the virus. Wild mouse (Mus musculus) virus field isolates, i5o4 and 292, were grown in NIH Swiss and cloned wild mouse embryo cell line (SC-I ; Hartley & Rowe, 1975). The mouse-tropic and amphotropic components of the t 5o4 virus were also grown separately in SC-I cells. All virus-infected cell lines were cultured in MEM supplemented with lO % foetal bovine serum, z mM-glutamine, and 8o #g of gentamicin per ml. Radiolabelling of virus and fractionation of virion phosphoproteins. Virus-producing cells were grown in 5o ~o phosphate-free MEM containing IO ~o dialysed foetal bovine serum in the presence of 3H-labelled L-amino acid mixture (2/zCi/ml) and carrier-free 32P-phosphate (8o #Ci/ml). Two changes of the medium were made at 24 h intervals, and the virus was purified from the pooled culture fluids as described (Pal et al. I973). The pelleted virus was then disrupted in 2o mM-tris-HC1 (pH 7"4) containing o.I °jo Nonidet P-4o at o °Cfor I h, treated with pancreatic ribonuclease (5oo #g/ml) at 37 °C for z h, and adjusted to 8 Mguanidine hydrochloride and o'3 % fl-mercaptoethanol. After heating at 56 °C for 45 min, the solution was applied on a Bio-Gel A-5m column for fractionation of virion proteins (Nowinski et aL 1972). The fractions containing the phosphoprotein were pooled, dialysed, and lyophilized (Pal et al. I975; Pal & Roy-Burman, I975). SDS-polyacrylamide gel electrophoresis. The protein peaks containing both ZH and 32p labels, isolated by the guanidine-agarose chromatography, were analysed by Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 09:53:23 R N A tumour virus phosphoproteins I I 46I ~ / I i i || ! ] 6 I |1 II X #4 " -- r~! I J × ,~ 2~ 2 m -i 10 30 50 Fraction number Fig. I. Urea-polyacrylamide gel electrophoresis of ~H-amino acid (0--(3) and 3~P-phosphate ( • - - - • ) labelled p 12 of Rauscher MuLV grown in NIH Swiss cells. Migration_wasfrom cathode (left) to anode (right). It should be noted that 8H was counted in a narrow channel to avoid 3~p spillover, and the values were not corrected for counting efficiency.The background values in the narrow 3H channel (8 ct/min) and 32p channel (20 ct/min) were deducted from the data shown. SDS-polyacrylamide gel electrophoresis (Pal et al. I975; Pal & Roy-Burman, 2975) for determination of the homogeneity of the labelled polypeptides. Urea-polyacrylamide gradient gel electrophoresis. Further separation of the variously charged species of polypeptides of similar size class was accomplished by electrophoresis in 4 to IO % gradient polyacrylamide gels with 3 % stacking gel in the presence of denaturing 5"25 M-urea. The procedure used was essentially the same as described previously (Pal et al. 1975). The electrophoresis was continued until the bromophenol blue marker reached the end of the gel. High voltage paper electrophoresis of virus phosphoprotein hydrolysates. The phosphoprotein (pI2) of WM-29z virus labelled in vivo with 32P-phosphate was hydrolysed in 6 N-HCI at I IO °C for 5 h and the hydrolysate was subjected to high voltage paper electrophoresis as described (Pal et al. 1975) in presence of o-phosphoserine and o-phosphothreonine markers. RESULTS Urea-gel electrophoresis patterns of the p 12 of MuL V-R The 32P-phosphate and 3H-amino acid labelled phosphoprotein of MuLV-R grown in N I H Swiss cells isolated by the guanidine agarose chromatography technique (Pal & RoyBurman, 1975) was analysed by SDS- and urea-gel electrophoresis. While a single homogeneous radioactive peak was obtained in guanidine-agarose gel filtration and in SDS-gels (Pal et al. I973; Pal & Roy-Burman, I975), resolution into 3 to 4 major components was accomplished in gradient gel electrophoresis in the presence of denaturing urea (Fig. I). As previously observed with the phosphoproteins of the RD-I I4, gibbon ape lymphosarcoma and feline leukaemia viruses (Pal et aL 1975), these multiple components apparently represent varying degrees of phosphorylation of the pI2 protein of MuLV-R. It was evident that all Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 09:53:23 462 E . G . H A Y M A N ~ B. J. P A L A N D P. R O Y - B U R M A N | I ! I I ! (a) X I I 40 50 (b) 4 3 2 1 i0 20 30 Fraction number Fig. 2. Comparison of the urea-polyacrylamide gel electrophoresis patterns of 3~P-phosphate labelled p i z of Rauscher MuLV grown in h u m a n rhabdomyosarcoma (RD) cells (a) and in N I H Swiss embryo cells (b). Migration was from left to right. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 09:53:23 RNA tumour virus phosphoproteins (a) I I I I 463 gilD I q II It II li It II f 1 115 I ,;,--, × 2-0 (b) 1.5 0 ll i - 10 30 Fraction number 50 Fig. 3. Comparison of the urea-polyacrylamide gel electrophoresis patterns of 3H-amino acid ((3--(3) and zzP-phosphate ( O - - - Q ) labelled pi2 of a wild mouse virus field isolate, strain 292, propagated in NIH Swiss cells (a) and in wild mouse cloned (SC-x) embryo cells (b). Migration was from left to right. major components were phosphorylated since all of them contained superimposable *~P and ZH counts. To determine whether the phosphorylation patterns exhibited in the urea-gel electrophoresis are virus- or host cell-specific, MuLV-R was propagated in a heterologous human cell line (RD) for isolation and analysis of the pI2. I f phosphorylation characteristics are virus-specific, then the urea-gel patterns of the virus p I z should be independent of the cell line in which it was propagated. The results (Fig. z) show that the phosphorylation patterns o f the p I z of MuLV-R grown in N I H Swiss cells (Fig. zb) were very similar to that o f M u L V - R grown in R D cells (Fig. 2a). Three to four major peaks were obtained in both cases and the relative proportion of these peaks appeared to be almost identical. These Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 09:53:23 Ifm464 E. G. H A Y M A N , B. K. P A L A N D 2"1.501 - T - ' l ' - ' l ' ( a ) P. 'l~ ~, T ROY-BURMAN T ] 0-5 (b) I X II ._= 4 I I I I I I i: i "t (c) i+ -] 10 30 50 Fraction number Fig, 4. Urea polyacrylamide gel electrophoresis patterns of 8H-amino acid ( 0 - - 0 ) and z2P-phosphate (O---O),~a~oefled pI2 protein of the (a) cloned mouse-tropic component of wild mouse virus, strain 15o4, grown in SC-I cells; (b) cloned amphotropic component of the I5O4virus grown in SC-I cells; and (c) uncloned I5o4 field isolate grown in NIH Swiss cells. Migration was from left to right. experiments were repeated and identical patterns were derived, suggesting that the number and amount of the various phosphorylated species are intrinsic characteristics of the virus. Urea-gel electrophoresis patterns of the pI 2 of wild mouse type C viruses Virus 292 field isolate was propagated in two different mouse cell lines, namely NIH Swiss embryo cells and SC-I cells. The pI2 phosphoprotein was isolated from these two separate virus preparations and was analysed by urea-gel electrophoresis. The data (Fig. 3) show that peak distribution patterns obtained in the two runs were very similar. Three phosphorylated species were recognized in each case. However, in contrast to the MuLV-R pI2 patterns, 292 pI 2 showed the presence of two major species which were not detectably phosphorylated. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 09:53:23 465 ul~ RNA tumour virus phosphoproteins T T l l 10 20 30 40 (a~ i X .=_ C~j 50 Fraction number Fig. 5- Urea polyacrylamide gel electrophoresis patterns of ~H-amino acid labelled p3o (a), p15 (b), and pxo (e) of W M q 5 o 4 virus grown in N I H Swiss cells. Migration was from left to right. 30 VIR 36 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 09:53:23 i(J466 E.G. HAYMAN, B. K. PAL AND P. ROY-BURMAN F p12 WM-1504 in WM embryo cells 1 10 p12 WM-1504 Mouse-tropic cloned tn SC-1 a cells 7 x f p12 FeLV 10 ~ i 2 I 2.0 ,, .e"J I p! 2 WM-292 in SC-I cells ! I 5 , L, f p12 1.2 0.4 ...___I~___.___L. / p12 MuLV-R 1-(~ p15 RD 114 0.5 10 30 50 I0 Fraction number 30 50 Fig. 6. Diagrammatic representation of the variously charged populations of molecules in phosphoproteins of mammalian type C RNA tumour viruses. Separation was accomplished by electrophoresis in urea-polyacrylamide gels. Migration was from left to right. Proteins were labelled with ~H-amino acids and ~zP-phosphateand the heights of the peaks represent 3H-radioactivity. Species of molecules also containing 3~P-radioactivity were shaded black (J) for distinguishing from those not detectably phosphorylated ([]). The data for the pI5 of RD-Ir4 al~d pI2 of GaLV were previously reported by Pal et al. 0975). Similar experiments were done with another wild mouse virus isolate, t5o 4. The field isolate of I5o4 was grown in N I H Swiss cells, and its cloned mouse-tropic and amphotropic components were grown separately in SC-r cells. The isolated p I z proteins were run in urea-gels for comparison (Fig. 4). Two major non-phosphorylated species were present in each case and the distribution of the phosphorylated species, although not well resolved, showed good similarity to each other. Again, these experiments were repeated and the Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 09:53:23 R N A tumour virus phosphoproteins 467 patterns were found to be reproducible. These data supported the observation with MuLV-R that the phosphorylation patterns are virus-specific and are not grossly altered by propagation of the virus in different host cells. In addition, the data revealed a unique characteristic of the pi2 of wild mouse derived viruses, whether uncloned or cloned, in having two major non-phosphorylated species whose relative concentration appeared to be constant. It should be noted that the charge heterogeneity detected in polyacrylamide gel electrophoresis under urea denaturing conditions was restricted to the virion phosphoproteins. The other major structural proteins showed primarily single species distribution under analogous conditions. This is illustrated in Fig. 5 showing the electrophoretic pattern of p3o, p I 5 and p I o proteins of the 15o4 virus in gradient gels in the presence of urea. Proteins p3o and plo of feline leukaemia virus (Pal et al. 1975) and p3o of mouse sarcoma virus (Kirsten) and avian sarcoma viruses also showed similar charge homogeneity in urea-gels (data not shown). Phosphoamino acids of the phosphoprotein of wiM mouse type C virus For identification of major phosphoamino acids, the ~2P-labelled p12 of WM-292 virus was hydrolysed in acid and subjected to high voltage paper electrophoresis with o-phosphoserine and o-phosphothreonine as markers (Pal et al. I975). Consistent with the previous observation on mouse type C viruses (Pal et al. 1975; Pal & Roy-Burman, I975), pI2 of 292 virus contained phosphoserine as the major phosphoamino acid. A small amount of phosphothreonine was also detected. DISCUSSION Consistent with our previous observation (Pal et al. i975) on the presence of several different phosphorylated species within the phosphoproteins of feline leukaemia virus (FeLV), gibbon ape lymphosarcoma virus (GaLV), and the endogenous cat type C virus (RD-II4), we report here the occurrence of multiple phosphorylated species in the pI2 phosphoproteins of the Rauscher (R) and wild mouse (WM) strains of murine leukaemia virus (MuLV). The number and relative proportion of the variously phosphorylated species are found to be a characteristic of the origin of the virus. This is diagrammatically illustrated in Fig. 6. The profiles of variously charged species reflect individual characteristics of the phosphoproteins of FeLV, GaLV, RD-114, MuLV-R, and WM viruses. A unique characteristic of the phosphoprotein of the wild mouse virus isolates, whether uncloned or cloned, for the mouse-tropic and amphotropic components is the occurrence of two distinct non-phosphorylated species, apparently in constant amounts relative to the phosphorylated species. These two non-phosphorylated species as well as the phosphorylated ones should be considered as pI2 because they all co-migrate as a single peak in both guanidine-agarose gel filtration and SDS-gel electrophoresis. A preparation of 3H-amino acid labelled pI2 of WM-292 virus isolated from the guanidine-agarose column was iodinated in vitro by 125I and run in SDS-gels. Again, a single major 12~I-labclledprotein peak ( > 95 % homogeneous) was obtained, corresponding to a molecular size of approx. 12ooo daltons (data not shown). Thus, all available information suggests that the non-phosphorylated species belong to the same size class as the phosphorylated species of the wild mouse viruses. They probably do not represent cellular proteins as their occurrence and proportion relative to the phosphorylated species do not change when the virus is propagated in widely different host cells like mouse and human. The possibility that the non-phosphorylated species may represent degraded larger virion proteins was also considered. However, their presence in constant proportion in the p12 protein fractions of all wild mouse isolates, cloned or uncloned, suggests that it is not very likely. Further, the method used for the isolation of the pI2 rules 30-2 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 09:53:23 468 E.G. HAYMAN, B. K. PAL AND P. ROY-BURMAN out the presence of at least some precursor degradation products like p~5E or pI2E (Buchhagen, Stutman & Fleissner, 1975; Naso et al. I976) which elute in the void volume of the guanidine-agarose column. The presence or absence of homology between the nonphosphorylated pI2 species and the phosphorylated pI2 species of the wild mouse type C viruses remains unknown. If the different species were to show overall similarity in peptide patterns, protein modifications other than phosphorylation have to be invoked to explain the charge differences between the two non-phosphorylated species. This possibility should be considered as proteins are known to be modified in nature by acetylation, sulphuration, carboxylation, and various other covalently linked charged substitutions. When the overall distribution patterns of the variously charged molecular species of the pI2 of wild mouse type C viruses are compared with that of MuLV-R, it appears that the p~2 of MuLV-R has lost the two non-phosphorylated species that are present on the pI2 of wild mouse viruses. The phosphorylated species of these wild mouse and laboratory MuLV strains, however, still maintain a good degree of similarity. These findings raise the following interesting questions. Is the absence of the major non-phosphorylated species in MuLV-R p12 a function of genetic selection during long term laboratory propagation? Are the additional non-phosphorylated pI2 species related to the neurotropic property (Gardner et al. I973, 1976; Officer et al. 1973) of the wild mouse virus isolates? Our results on the studies of the phosphoproteins of the same virus (MuLV-R or WM isolates) grown in different cell lines suggest that the patterns of phosphorylated and nonphosphorylated species are virus-specific and apparently independent of the cell lines of propagation, although it is recognized that the cell offers the setting for phosphorylation, dephosphorylation or other protein modifications. These results indirectly suggest that the information for the phosphoprotein resides in the virus genome, a concept that has already been established by analysis of the translation products of the gag gene of mammalian type C RNA tumour virus (Barbacid, Stephenson & Aaronson, 1976, and the references cited therein). Although the structural and biological significance of the occurrence of multiple species of phosphorylated polypeptides within RNA tumour virions is largely unknown, some recent studies strongly suggest that these phosphorylated polypeptides have a regulatory role in vivo. It has been shown that the pIz phosphoprotein of mammalian type C viruses binds specifically to the homologous virus RNA (Sen, Sherr & Todaro, 2976; Sen & Todaro, 1976) and the avian P~9 phosphoprotein to its RNA (Sen & Todaro, 1977). These binding studies also indicate that there are at least two functionally distinct populations of molecules in the pie protein preparations and one of these may be responsible for specific interaction with the virus RNA (Sen & Todaro, ~976). It would appear very likely that this population represents one or more of the several different species of p~z as described earlier (Pal et al. I975) and presented in this report. Recent data on the binding studies, in fact, show that, of the various phosphorylated forms of pIz protein of the Rauscher MuLV, only a species containing relatively little phosphate can bind in vitro to purified homologous virus RNA (A. Sen, C. J. Sherr & G. J. Todaro, personal communication). We thankV. Klement and M. Bryant for supplying mouse-tropic and amphotropic clones of WM 15o4 virus, M. B. Gardner for reading the manuscript, M. Akhavi for expert technical assistance, and A. Dawson for her assistance in preparing the manuscript. This investigation was supported by contract number NoI CP 535oo with the National Cancer Institute. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 09:53:23 RNA tumour virus phosphoproteins 469 REFERENCES BARBACID, M., STEPHENSON, J.R. & AARONSON, S. A. (1976)- Gag gene o f m a m m a l i a n type-C R N A t u m o r viruses. Nature, London 262, 554-559. BRYANT, M. L. & KLEMENT, V. (1976). Clonal heterogeneity o f wild m o u s e leukemia viruses: h o s t range a n d antigenicity. Virology 73, 532-536. BUCHHAGEN, D. L., STUTMAN, O. & FLEISSNER, E. (I975). C h r o m a t o g r a p h i c separation a n d antigeoic analysis o f proteins o f the oncornaviruses. Journal of Virology I5, I I 4 8 - ~ I 5 7 . GARDNER, M. B., HENDERSON, B. E.~ ESTES, J. D., RONGEY, R. W., CASAGRANDE~J., PIKE, M. & HUEBNER~ R.J. (I976). T h e epidemiology a n d virology o f C-type virus-associated hematological cancers a n d related diseases in wild mice. Cancer Research 36, 574-581. GARDNER, M.B., HENDERSON, B.E., OFFICER~ J.E., RONGEY~ R. W., PARKER~ J. C., OLIVER, C., ESTES, J.D. & nUEBNER, R.J. (1973). A s p o n t a n e o u s lower m o t o r n e u r o n disease apparently caused by indigenous type-C R N A virus in wild mice. Journal of the National Cancer Institute Sx, 1243-I 254. HARTLEY, J. W. & ROWE, W. ~. (1975). Clonal cell lines f r o m a feral m o u s e e m b r y o which lack h o s t r a n g e restriction for m u r i n e l e u k e m i a virus. Virology 65, 128-I34. HARTLEY, J. W. a ROWE, W. P. (I976). N a t u r a l l y occurring m u r i n e leukemia viruses in wild mice: characterization o f a new ' a m p h o t r o p i c ' class. Journal of Virology I9, I9-25. LAI, M. M. C. 0976). P h o s p h o p r o t e i n s of R o u s s a r c o m a viruses. Virology 74, 2 8 7 - 3 o L McALLISTER, R. M., MELNYK, J., FINKLESTEIN, J. Z., ADAMS, E. C. & GARDNER, M. B. (I969). Cultivation in vitro o f cells derived f r o m a h u m a n r h a b d o m y o s a r c o m a . Cancer 24, 520-526. NASO, R. B., ARCEMENT, L. S., KARSHIN, W. L., JAMJOOM, 13. A. & ARLINGHAUS, R. B. (1976). A fucose-deficient glycoprotein precursor to R a u s c h e r leukemia virus gp 69/7I. Proceedings of the National Academy of Sciences of the United States of America 73, 2326-233o. NOWINSKI, R. C., FLEISSNER, E., SARKAR, N. H. & AOKI, T. (1972). C h r o m a t o g r a p h i c separation a n d antigenic analysis o f proteins o f the oncornaviruses. I. M a m m a l i a n l e u k e m i a - s a r c o m a viruses. Journal of Virology 9, 359-366. OFFICER, J. E., TECSON, N., ESTES, J. D., FONTANILLA, E., RONGEY, R. W. & GARDNER, M. B. (I973). Isolation of neurotropic type C virus. Science 18I, 945-947. PAL, B. K., McALLISTER, R. M., GARDNER, M. B. & ROY-BURMAN,P. (I975). C o m p a r a t i v e studies o n the structural p h o s p h o p r o t e i n s o f m a m m a l i a n type C viruses. Journal of Virology i6, I23-131 . PAL, B. K. & ROY-BURMAN, P. (I975). P h o s p h o p r o t e i n s : structural c o m p o n e n t s o f oncornaviruses. Journal of Virology xS, 540-549. PAL, B. K., WRIGHT, M., OFFICER, J. E., GARDNER, M.B. & ROY-BURMAN, P. (t973)- Subviral c o m p o n e n t s o f a wild m o u s e embryo-derived type-C oncornavirus. Virology 56, 189-I97. RASHEED, S., GARDNER, M. B. & CHAN, E. (I976). A m p h o t r o p i c host range o f naturally occurring wild m o u s e leukemia viruses. Journal of Virology I9, I 3 - I 8 . SEN, A., SHERR, C. J. & TODARO, G. J. (I976). A specific binding o f the type C viral core protein p12 with purified viral R N A . Cell 7, 21-32. SEN, g. & TODARO, G. J, (1976). Specificity of in vitro binding of primate type C viral R N A a n d the h o m o l o g o u s viral p i 2 core protein. Science I93 , 326-328. SEN, A. & TODARO, G. J. (1977). T h e genome-associated, specific R N A binding proteins of avian a n d m a m m a l i a n type C viruses. Cell io, 91-99. (Received 8 February 1977) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 09:53:23