Polyacrylamide Gel Electrophoresis of Visna Virus Polypeptides Isolated by Agarose Gel Chromatography
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1 22-538X/78/25-27$2./ JOURNAL OF VIROLOGY, Jan. 1978, p Copyright 1978 American Society for Microbiology Vol. 25, No. 1 Printed in U.S.A. Polyacrylamide Gel Electrophoresis of Visna Virus Polypeptides Isolated by Agarose Gel Chromatography FU HAI LIN New York State Institute for Basic Research in Mental Retardation, Staten Island, New York 1314 Received for publioation 5 August 1977 The proteins of visna are separated into nine major peaks by agarose gel chromatography in 6 M guanidine hydrochloride (GuHCl). The polypeptides in eack peak were isolated by acid precipitation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The patterns of SDS- PAGE show that the excluded material from the GuHCl column contains an aggregate of 1 non-glycosylated polypeptides. It is shown that this aggregate represents virus substructures that are not completely solubilized by GuHCl. Two glycoproteins, gp175 and gpll5, were isolated from the column eluate. The major glycoprotein gpll5 was coeluted with P9, P68, and P61 in GuHCl 4. Each of the four major peaks (GuHCl 5 to 8) contains more than one nonglycosylated polypeptide. However, a small polypeptide, P12, can be isolated in a homogeneous form in the last peak, GuHCl 9. Analysis of the virus proteins (1 ug) by SDS-PAGE shows that 2 radioactive bands can be recognized. During fractionation of the protein on agarose gel columns followed by analysis with SDS-PAGE, a number of minor polypeptides that were not detected before became clearly recognizable. Thus, the combined use of column chromatography and SDS-PAGE shows that visna virus is composed of 25 proteins. Visna virus shares a number of important biochemical properties with oncornaviruses (12), such as the presence of 6 to 78 RNA (7, 14, 16) and an RNA-dependent DNA polymerase in the virions (15, 23). Although the proteins and virion DNA polymerase of oncornaviruses have been extensively studied (4), very little is known about the counterparts of visna virus. The slow progress in this area of investigation is probably due to the low yield of visna virus, as compared with that of oncornaviruses. Since visna virus is a prototype of slow infection, it is of interest to learn more about the biological properties of the virus polypeptides. We initiated the study of visna virus protein (17) by using agarose gel column chromatography in 6 M guanidine hydrochloride (GuHCl) because it has been demonstrated by a number of workers that proteins of oncornaviruses can be resolved into homogeneous components and their antigenicity can be restored after removal of the denaturing agent by dialysis (5, 9-11, 21). The radioactively labeled protein of visna virus can be resolved into 1 peaks by GuHCl (17). However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the virus protein showed that visna virus contains 14 to 15 polypeptides (13, 2). These results suggest that some of the GuHCl peaks may contain more than one component. Thus, it is necesary to examine the homogeneity of the pooled fractions of each GuHCl peak before studying the biological properties of isolated polypeptides. This paper reports the results of analyses of polypeptide content in GuHCl peaks by SDS- PAGE. MATERIALS AND METHODS Reagents and chemicals. [35S]methionine, [14C]glucosamine, and D-[3H]glucosamine were purchased from New England Nuclear Corp., Boston, Mass., and from Schwarz/Mann, Orangeburg, N.Y. Other reagents were obtained from the same sources as described previously (17). Standard buffers. TNE buffer (ph 7.4) contained 1 mm Tris-hydrochloride,.1 M NaCl, and 1. mm EDTA. Buffer A contained.5 M Tris-hydrochloride (ph 8.5), 8 M GuHCl, 2% 2-mercaptoethanol, and 1. mm EDTA. Buffer B (ph 6.5) contained 2 mm sodium phosphate, 6 M GuHCl, and 1 mm dithiothreitol Buffer C was composed of 62 mm Tris-hydrochloride (ph 6.8), 2% SDS, 1% sucrose, 5% 2-mercaptoethanol, and.1% bromophenol blue. Buffer D (ph 8.3) contained 25 mm Tris, 192 mm glycine, and.1% SDS. Cell cultures and media. The preparation and propagation of sheep choroid plexus cells have been described previously (17). Preparation of radioactive virus. The sheep choroid plexus cells were grown in a 25-ml Falcon flask and inoculated with visna virus K796 with a 27
2 28 LIN multiplicity of about 1 infectious dose per cell. The culture was incubated in 4 ml of maintenance medium (16) containing 1,uCi of [3S]methionine (487 Ci/mmol) per ml at 37C. Three to four days after the inoculation, when the cytopathic effect was widely spread on the monolayer, the infectious fluid was collected. The virus was usually purified immediately or stored at 4 C for no more than 1 week. Similar procedures were followed for labeling [14C]glucosamine and D-[3H]glucosamine. Purification of virus. Visna virus was purified by three successive cycles of equilibrium centrifugation on 1 to 5% potassium tartrate gradient as described previously (17). The centrifugation was performed at 4Cfor24h. Gel filtration. The gel filtration procedure has been described previously (17). Samples (.1 ml) were taken from each fraction and mixed with 1 ml of Aquasol-2 (New England Nuclear). The radioactivity was measured in a Packard Tricarb model 338 scintillation counter. PAGE. A 5 to 2% gradient of polyacrylamide gel containing.1% SDS was prepared essentially by the method of Baum et al. (2). The pooled fractions of GuHCl eluate were dialyzed against deionized distilled water for 24 h with two to three changes of water. The polypeptides in each pool were precipitated with trichloroacetic acid, washed twice with cold acetone, and dissolved in 4 pl of buffer C by boiling for 3 min. Each sample contained about 1 to 2 jig of protein. Electrophoresis was performed at room temperature at 6 V (constant voltage) for 6 to 8 h by using buffer D as the electrode solution. After electrophoresis, the gel was stained with Coomassie brilliant blue and destained with 7.5% glacial acetic acid containing 25% methanol as previously described (19). Autoradiography. The gel was immersed in 1% dimethyl sulfoxide with three changes at 1-h intervals, followed by treatment with 16% 2,5-diphenyloxazole in dimethyl sulfoxide, also with three changes, and dried overnight at 82C. DuPont Cronex 2c X-ray film was exposed to the dried gel for 1 to 2 weeks. Protein determination. The protein was measured by the method of Lowry et al. (18) with bovine serum albumin as the standard protein. RESULTS SDS-PAGE analysis of polypeptide composition in GuHCI fractions. Visna virus was purified through three rounds of gradient centrifugation on potassium tartrate gradient. It banded sharply at a peak with a density of 1.13 to 1.14 g per ml. No other radioactivity peak was detected in the second and third gradients. For convenient reference, a representative elution profile of [3S]methionine-labeled polypeptides from an agarose gel column is illustrated in Fig. 1. The radioactivity in the excluded volume accounted for 4% of the total eluate. The bars indicate the fractions pooled for SDS- PAGE analysis, and the numbers correlate each rp -2 - x z U I. *~~~~~~~~~ 9.5~~~~~~~~~i FRACTION NUMBER J. VIROL FIG. 1. Agarose gel column chromatography ofthe proteins of visna virus. [rs]methionine-labeled virus was dissociated with 8 M GuHCI, loaded onto a Bio- Gel (A-5M, Bio-Rad) column (1.5 by 85 cm), and eluted with 6 M GuHCI (buffer B). Fractions (1 ml) were collected, and.1-mi samples of each fraction were mixed with 1 ml of Aquasol-2. The radioactivity was measured in a Packard Tricarb liquid scintillation counter. Fractions were pooled as indicated by bars on the top of the graph. The numbers above bars identify the pools and correspond to the numbers on the top ofpolyacrylamide gels. pool to the corresponding slot of the polyacrylamide gel. Figure 2 shows SDS-PAGE patterns of polypeptides in pools 1 through 15. Pool 2 consisted of peak fractions in the excluded volume. The predominant polypeptides in this pool were P25 (the nomenclature for proteins of oncornaviruses is adopted for visna proteins [1]), P14, P68, and P9. They were excluded from the column along with the virus RNA (unpublished data). In addition, P51 and P53, which are minor polypeptides of visna virus, were found in large quantities in the void volume. P61, P46, P38-4, and two unidentified high-molecular-weight polypeptides were also present. It was also seen that the excluded volume contained some radioactive material that did not enter the gel. The major visna glycopolypeptide gpll5 was not found in the polyacrylamide gel. The small polypeptide P12 was barely detectable. A small peak located between fractions 5 and 6 (Fig. 1) represents a minor component of the virus with an estimated molecular weight of 11,. Analysis of this material by SDS-PAGE showed a faint band with a molecular weight of 175,. This protein was found to associate with glucosine (Fig. 6 and 7). The radioactive peak located between fractions 76 and 86 of Fig. 1 was divided into two
3 VOL. 25, 1978 ELECTROPHORESIS OF VISNA VIRUS POLYPEPTIDES Stds P p94-- p9-7 p51 l g pl 75- P.-'- -Phos. A -BSA *b -Ovalb. p32-pi34.: p 8/p25- Q d -Chy. -Cyt.c FIG. 2. SDS-PAGE of visna polypeptides isolated from pools 2 to 15 of Fig. 1. A 5 to 2% gradient of slab gel was used in this and subsequent experiments. Polypeptides in each pool were precipitiated with trichloroacetic acid and subjected to electrophoresis. The gel was dried, and autoradiography was done by exposing a DuPont X-ray film to the gel. The numbers on the top of the figure correlate to those of the GuHCI pools as indicated in Fig. 1. Standard proteins (Stds.) run in the same gel were phosphorylase A (Phos. A, 94,); bovine serum albumin (BSA, 68,); ovalbumin (Ovalb., 45,); chymotrypsinogen A (Chy., 25,), and cytochrome C (Cyt. C, 12,). pools (pool 1 and 11). This peak was previously designated as GuHCl 3 and 4 (17), with molecular weights of 75, and 7,, respectively. The 75,-dalton component was also shown to contain glucosamine. Pool 1 (GuHCl 3) contains P68, P9, P94, and P115 (Fig. 2). In pool 11 (GuHCl 4), P61, P68, P9, and P115 were present as major components. A diffused band with an estimated molecular weight of 73, was also visible. As will be shown later, P115 is a major glycoprotein of visna virus. P9 and P115 were eluted first, followed by P68 and P61, which were crossed over to the next peak. In pools 13 and 14 (Fig. 2), which cover the area of GuHCl 5 (Fig. 1), P46 was the major component. Polypeptides P51 and P53 were found in this peak. In pools 14 and 15 (Fig. 2), polypeptides P32, P33, and P34 were located. Pool 15 consisted of fractions located between peaks GuHCl 5 and 6. P46 and P25 were found in this pool. Figure 3 shows SDS-PAGE patterns of pools 16 through 2. Pool 16 consisted of peak fractions of GuHCl 6 (Fig. 1). The principal constituents of this pool were P25, P28, and P32. The presence of minor polypeptides, such as P16, P18, P2, and P23, in this peak is evident. The heterogeneity of pool 17 is shown by the presence of P14, P16, P18, P23, and P25. Pool 17 corresponds to GuHCl 7 as reported previously (17). Pool 18 is also heterogeneous as evidenced by the presence of P12, P14, P16, and P18. Based on SDS-PAGE patterns of pools 17 and 18, it can be concluded that P14, P16, and P18 are the resident components of these two pools, whereas P25 and P12 represent contaminants from the peaks before and behind GuHCl 7 and 8. The SDS-PAGE pattern showed that pools
4 21 LIN V Stds. J. VIROL. gpl7o - gp115-i p94 F* p 9 p 68 p73 p6l-~p 53 p5i--- P48 p36--- p32- p34'i4 p28 p25 b a 4* - Phos. A - BSA - OvaIb. - Chy. p1 4 pl Cyt. C FIG. 3. SDS-PAGE of visna polypeptides isolated from pools 16 to 2 and of visna virus. The procedures were the same as those described in the legend to Fig. 1. About 1 pg of visna virus (V) protein (44, cpm) was applied to the gel. See legend to Fig. 2 for abbreviations. 19 and 2 contain the same single polypeptide P12, indicating the homogeneity of the last peak of the column. To serve as reference, about 1,ug of virus protein with a radioactivity of 44, cpm was run in the gel (Fig. 3). Autoradiography of the gel showed that 2 bands were recognizable (Fig. 3). All the polypeptides loaded onto the column were recovered. Minor polypeptides, particularly P16, P18, and P28, were not detected in the unfractionated virus protein preparation, but became reco ble after fractionation on the GuHCl column. Chromatographic and SDS-PAGE patterns of visna polypeptides released from virions by treatment with NP-4. In our previous report (17), we found that virus substructures can be separated into two major fractions by gradient centrifugation of Nonidet P- 4 (NP-4)-treated virions. One fraction (light) was located on the top of the gradient (p = 1.1 g/ml), accounting for about 9% of the initial material, and the other (heavy fraction) banded at a density of 1.24 g/ml. To further analyze the polypeptide composition of the light fraction, [3S]methionine-labeled virions were incubated with.1% NP-4 for 1 min at room temperature, and the mixture was fractionated on a potassium tartrate gradient as described previously (17). The light fraction was chromatographed on an agarose gel in GuHCl, and eluate fractions were pooled and analyzed by SDS- PAGE as described above. Figures 4 and 5 illustrate the results of these experiments. In contrast to Fig. 1, the radioactivity in the void volume represented in Fig. 4 accounted for about 3%. More than 9% of the radioactivity was found in the three major peaks GuHCl 6 through 9. SDS-PAGE analysis (Fig. 5) of the excluded material showed that no detectable radioactive band was observed in the polyacrylamide gel, in marked contrast with the SDS-PAGE pattern shown in Fig. 2. Electrophoresis of the acidprecipitable material isolated from othier peaks (Fig. 4) showed polypeptide patterns (Fig. 5) similar to those isolated from the corresponding peaks of Fig. 1 (see Fig. 2 and 3), with the notable exception that GuHCl 5 and its major constituent, P46, were almost absent. GuHCl elution pattem of the heavy fraction (p 1.24 g/ml) showed = a similar profile as
5 VoLS- 25, 1978 ELECTROPHORESIS OF VISNA VIRUS POLYPEPTIDES 211 previously reported (17). SDS-PAGE analysis of the major peak (GuHCl 6) indicated that P25 is the major constituent. The heavy fraction also contained P46 and P14, which were eluted in l) 3. x Z- 2 - z I I.-I 7, 2 j.: 2''-..1 In) IT~~~~ FRACTION NUMBER FIG. 4. Agarose gel column chromatography of visna polypeptides in light fiaction. (us]methioninelabeled visna virus was treated with.1% NP-4. The mixture was cenrifuged on a 1 to 5% potassiwn tartrate gradient for 24 h, the light material that banded near the top (p = 1.1 g/ml) was couected and chromatographed on a Bio-gel column, and the GuHCI fractions were pooled as described ut the legend to Fig. 1. GuHCl 5 and 7, respectively. Analysis of visna virus glycoproteins by SDS-PAGE. Visna virus was labeled with ["4C]glucosamine or D-[3H]glucosamine and chromatographed on agarose gel column as described previously (17). Figure 6 depicts a profile of GuHCl elution. The D-[3H]glucosamine label was eluted in two major peaks, one in the excluded volume and the other around fraction 7, which was previously designated as GuHCl 3 (17). Fractions showing radioactivity were pooled (Fig. 6) and subjected to electrophoresis as described above. Figure 7 shows an SDS-PAGE pattern of the isolated glycopolypeptides. The [3H]glucosamine label eluted in the void volume (GuHCl 1) did not enter the gel (pool 1; Fig. 7). The small radioactive peak consistng of fractions 5 through 54 (Fig. 6; GuHCl 2) was found to contain a glycopolypeptide with an estimated molecular weight of 175, in SDS-PAGE. The SDS-PAGE pattern of GuHCl 3 (Fig. 6), covering fractions 69 through 75, showed that a single radioactive band was detected at a position with a molecular weight of 115,. Electrophoresis of pooled preparations from 81 through 86 (Fig. 4; GuHCl 5) did not show any glucosamine band in the polyacrylamide gel, although proteins with molecular weights of 68, and 73, were heavily stained by Coomassie brilliant blue. When visna Origin, * " g pi75-- gp115-- e3 Pp6 p6dl--- p53 psl p46- p34 p33-,- p323-- p p23-- p12-- Ms 4 f -BSA -Ovalb. -Chy. -Cyt.C FIG. 5. SDS-PAGE of visna polypeptides isolated from pool 1 through 12 of Fig. 4. See legend to Fig. 2 for a description of the procedures used and for abbreviations.
6 2 12 LIN J. VIROL. -3 x z 2 z D C-2 C-) -J ~1 DISCUSSION The primary objective of the present work was to determine the feasibility of using agarose- GuHCl chromatography in preparing homogeneous polypeptides of visna virus for biological study. Because of the low yield of the virus, it is of special interest to find a system that confers a power of high resolution and is capable of recovering most of the initial proteins. The -b,, 2 oeb- 5 GuHCl chromatographic procedures seem to fulfill this purpose in that the proteins of visna virus can be resolved into five distinct major 3 7'O 8 9 IOC *i2 ' peaks. Purification of polypeptides in FRACTION NUMBER each peak can, therefore, be simplified. Thus, FIIG. 6. Agarose gel chromatography of visna pro- most of the major proteins can be isolated in tein labeled with D-IH]glucosamine. Visna virus one virus preparation. In the case of P12, a pure was ilabeled with D-[H]glucosamine and chromato- form can be obtained in quantity in a grap single Aed on a Bio-Gel column as described step in the legenid to Fig. 1. Fractions were pooled as indicated of foran. operation. for StDS-PAGE analysis. The material excluded from the agarose column (Fig. 1) consists of almost all the major proteins of visna virus (Fig. 2). Their exclusion from the column is most likely due to incomplete solubilization of the virus substructures by 8 M O r' 9g n GuHCl. This conclusion is substantiated by the experiments represented in Fig. 4 and 5. When virions were pretreated with NP-4 followed by preliminary fractionation on potassium tartrate gradient centrifugation to separate the virus core (heavy fraction) from other substructures including the envelope (light fraction), chroma- 175 S * tography of both fractions showed that little material was excluded from the column (Fig. 4). Analysis of the excluded material by SDS- PAGE (Fig. 5) showed that none of the polypeptides depicted in Fig. 2 (pool 2) were present. 115 These results indicate that pretreatment of virions with NP-4 facilitates solubilization of the virus proteins in GuHCI. Fleissner (9) reported that electrophoresis of the material excluded from an agarose gel column resulted in detecting a glycoprotein with an estimated molecular weight of 32,. In contrast, the glucosamine-containingmaterialeluted in the void volume in the present study was never resolved into a molecule small enough to enter the polyacrylamide gel system, despite repeated attempts to dissociate the aggregate. + Two glycoproteins of visna virus were eluted in FI G. 7. SDS-PAGE of glycopolypeptides isolated GuHCl 2 and GuHCl 3 to 4 to molecular weights from pools 1 to 5 of Fig. 6. The procedures were the of 11, and 75,, respectively (17; Fig. 6 same as those described in the legend to Fig. 2. No and 7). However, the molecular weights of these detec ltable radioactive band was observed on the slot two glycoproteins were estimated to be 175, load vir alyz ed with pool 5. and 115,, respectively, by the SDS-PAGE method. The weight values of glycoproteins obs was labeled with ["4C]glucosamine and an- tained from the GuHCl method are probably more reliable than that of the SDS-PAGE be- cause glycoproteins behave abnormally in SDS- ed by the same methods, no different pattem was seen.
7 VOL. 25, 1978 PAGE and their molecular weights vary with the concentration of the gel (22, 24). It is of interest that the major glycoprotein of avian oncornaviruses was found to have a molecular weight of about 1, by Duesberg et al. (8) and Bolognesi et al. (6) as measured by SDS- PAGE, whereas the corresponding molecule was estimated to be 7, daltons in GuHCl (9). It appears, therefore, that the major glycoprotein (gpll5) of visna virus is similar to that of avian oncornaviruses. The molecular weights of non-glycosylated proteins measured in GuHCl (17) are about the same as those estimated by SDS-PAGE. Since most of the GuHCl peaks are heterogeneous, the weight values estimated by the SDS-PAGE method will be adopted for the polypeptides of visna virus, with a reservation for the glycoproteins whose molecular weights may be overestimated by SDS-PAGE. Based on the elution property in GuHCl, the proteins of visna virus can be grouped into five classes, and each of these classes can be represented by a major protein. The first class comprises P61, P68, P9, and gpll5 and is represented by P68. This conclusion is drawn from data reported previously (17) that this group of proteins was eluted in a GuHCl position corresponding to a molecular weight of about 7, to 75,. As discussed above, gpll5 probably has a true molecular weight of about 7,. P9 is probably the same protein that was identified to be a glycoprotein by Haase and Baringer (13) and Haase (12). Therefore, the molecular weight of this protein could also be overestimated. In light of this argument, it can be concluded that the true sizes of these proteins are within close range. The second class of visna protein consists of P46, P51, and P53. These three proteins as a group can be readily separated from other groups by the GuHCl column (Fig. 1, 2, 4, and 5). It has been found that P46 is located at the center of core, whereas P51 and P53 are detected in an envelope fraction (Lin and Brown, submitted for publication). Thus, this internal protein can be obtained free of P51 and P53 from isolated cores. The third class is composed primarily of P25, P28, and P32. This group is dominated by P25, which is an active antigen against antibody prepared from rabbits hyperimmunized with visna virus (19). The fourth group is represented by P14, accompanied by P16 and P18. These three proteins were eluted in a broad peak, which was designated as GuHCl 7 and 8 (17). Judging by the relative intensity of radioactive bands (Fig. 3; pools 17 and 18), it can be concluded that P16 was eluted primarily in ELECTROPHORESIS OF VISNA VIRUS POLYPEPTIDES 213 GuHCl 7, whereas both P18 and P14 were found in a proportionally higher concentration in GuHCl 8. The parallel proportionality in intensity of the latter two polypeptides in GuHCl 7 and 8 seems to suggest that P18 and P14 are eluted in GuHCl as one component. Thus, the elution and SDS-PAGE patterns of visna P18 and P14 are the reverse of those of P1 and P15 of avian leukosis virus (9). The last class of visna protein consists of a single homogeneous polypeptide P12. This small protein can be isolated in pure form in one step of purification. It is possible to avoid contamination of P14 in P12 by preliminary separation of the visna envelope from the virus core by gradient centrifugation of nonionic, detergent-treated virions because P12 locates in the envelope, whereas P14 resides in the core (Lin and Brown, submitted for publication). The number of visna virus proteins has increased to 25, with a total mass of about 1.3 x 16 daltons. These results can be attributed to the high resolution power of SDS-PAGE for small polypeptides and to the concentration of the minor components by the GuHCl column. This conclusion is supported by the detection of P16, P18, P23, and P28 (Fig. 3) after gel filtration. If the genomic complexity of 3.5 x 16 for visna virus (3) is accepted, it is capable of coding about 25% of the proteins. It can be argued that the virus preparation was contaminated with cellular material. However, measures were taken to purify the virus exhaustively to exclude cellular contaminants. The purification procedures were very similar to those of Haase and Baringer (13), who showed that almost all cellular material was separated from the virus band in their reconstruction experiments. It is unlikely, therefore, that a massive cellular contamination of our virus preparation had occurred. Nevertheless, inclusion of proteins not coded by the virus genome is not unequivocally ruled out. The origin ofsome visna virus proteins is under study. ACKNOWLEDGMENTS I than]k Halldor Thormar for his support, Michael C. Papini for his technical assitance, Robert J. McCreary for providing tissue culture, and Edith M. Riedel for preparing the manuscript. LITERATURE CITED 1. August, J. T., D. P. Bolognesi, E. Fleissner, R. V. Gilden, and R. C. Nowinski A proposed nomenclature for the virion proteins of oncogenic RNA virses. Virology 6: Baum, S. G., M. S. Horwitz, and J. V. Maizel, Jr Studies of the mechanism of enhancement of human adenovirus infection in monkey cells by simian virus 4. J. Virol. 1:
8 214 LIN 3. Beemon, K. L, A. J. Faras, A. T. Haase, P. H. Duesberg, and J. E. Maisel Genomic complexities of murine leukemia and sarcoma, reticuloendotheliosis, and visna viruses. J. Virol. 17: Bolognesi, D. P Structural components of RNA tumor viruses. Adv. Virus Res. 19: Bolognesi, D. P., and H. Bauer Polypeptides of avian RNA tumor vimses. I. Isolation and physical and chemical analysis. Virology 42: Bolognesi, D. P., H. Bauer, H. Gelderblom, and G. Huper Polypeptides of avian RNA tumor viruses. IV. Components of the viral envelope. Virology 47: Brahic, M*, J. Tamalet, and C. Chippaux-Hyppolite Visna virus: isolation of an RNA of high molecular weight. C. R. Acad. Sci. 272: Duesberg, P. H., G. S. Martin, and P. K. Vogt Glycoprotein components of avian and murine RNA tumor viruses. Virology 41: Fleissner, E Chromatographic separation and antigenic analysis of proteins of the oncornaviruses. I. Avian leukemia-sarcoma virues. Virology 8: Green, W., and D. P. Bolognesi Isolation of proteins by gel Sltration in 6 M guanidine chloride: application to RNA tumor viruses. Anal. Biochem. 57: Green, W., D. P. Bolognesi, W. Schafer, L Pister, G. Hunsmn, and F. DeNoronha Polypeptides of mammalian oncornaviruses. I. Isolation and serological analysis of polypeptides from murine and feline C- type viruses. Virology 56: Haase, A. T The slow infection caused by visna virus. Curr. Top. Microbiol. Immunol. 72: Haase, A. T., and J. Baringer The structural polypeptides ofrna slow viruses. Virology 57: J. VIROL. 14. Harter, D. H., J. Schlom, and S. Spiegelman Characterization of visna virus nucleic acid. Biochim. Biophys. Acta 24: Ln, F. H., and H. Thormar Ribonucleic aciddependent deoxyribonucleic acid polymerase in visna virus. J. Virol. 6: Uin, F. H., and H. Thormar Characterization of ribonucleic acid from visna virus. J. Virol. 7: Uin, F. H., and H. Thormar Substructures and polypeptides of visna virus. J. Virol. 14: Lowry,. H., N. J. Rosebrough, A. L Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Mehta, P. D., F. H. Lin, and H. Thormar Antigenic analysis of isolated polypeptides from visna virus. Infect. Immun. 13: Mountcastle, W. E., D. H. Harter, and P. W. Choppin The proteins of visna virus. Virology 47: Nowinski, R. C., E. Fleissner, N. H. Sarkar, and T. Aoki Chromatographic separation and antigenic analysis of proteins of the oncornaviruses. II. Mammalian leukemia-sarcoma viruses. J. Virol. 9: Russ, G., and K. Polakova The molecular weight detennination of proteins and glycoproteins of RNA enveloped viruses by polyacrylamide gel electrophoresis in SDS. Biochem. Biophys. Res. Commun. 55: Schlom, J., D. H. Harter, A. Burny, and S. Spiegelman DNA polymerase activities in virions of visna virus. A causative agent of a "slow" neurological disease. Proc. Natl. Acad. Sci. U.S.A. 68: Tung, J. S., and C. A. Knight The coat protein subunits of cucumber viruses 3 and 4 and a comparison of methods for determining their molecular weights. Virology 48:
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