Glycoprotein Synthesis by D-Glucosamine Hydrochloride

Size: px

Start display at page:

Download "Glycoprotein Synthesis by D-Glucosamine Hydrochloride"

Jane Johnson
5 years ago
Views:

JOURNAL OF VIROLOGY, Apr. 1974, p. 775-779 Copyright 0 1974 American Society for Microbiology Vol. 13, No. 4 Printed in U.S.A. Selective Inhibition of Newcastle Disease Virus-Induced Glycoprotein Synthesis by D-Glucosamine Hydrochloride A.

1 JOURNAL OF VIROLOGY, Apr. 1974, p Copyright American Society for Microbiology Vol. 13, No. 4 Printed in U.S.A. Selective Inhibition of Newcastle Disease Virus-Induced Glycoprotein Synthesis by D-Glucosamine Hydrochloride A. C. R. SAMSON' AND C. FRED FOX Department of Bacteriology and the Molecular Biology Institute, University of California, Los Angeles, California Received for publication 26 September 1974 D-Glucosamine selectively affected the synthesis of virus-induced glycoproteins in Newcastle disease virus-infected chick fibroblasts, but had no effect upon post-translational cleavage of the glycoprotein precursor for one of the two major glycoproteins of the mature virion. In response to glucosamine, at least one of the two virus-induced glycoproteins is formed in reduced quantity and the second is either not formed or has an electrophoretic mobility identical to that of another virus-induced protein. Purified Newcastle disease virions contain three major and at least three minor polypeptides as revealed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) (7). In addition to the three major virion polypeptides, a fourth major polypeptide which has a precursor role can be detected in Newcastle disease virus (NDV)-infected chicken embryo fibr6blasts (9). The major virion polypeptide having the highest molecular weight (I, 78,400) is a glycoprotein. Both the neuraminidase and hemagglutinin (HA) activities of the virion are associated with this glycoprotein (10). The nonvirion precursor polypeptide (PII, 64,800 daltons) and its cleavage product (53,500 daltons) are also glycoproteins (our results here and those of Kaplan and Bratt, Abstracts of the annual meeting of the American Society for Microbiology, p. 242, 1973). The second major virion protein (II, 53,500 daltons) has the same electrophoretic mobility in SDS-PAGE as the PII cleavage product (9). This protein is non-glycosylated and is the nucleocapsid protein (6). The third major protein (III, 37,600 daltons) thought to be a membrane protein of the enveloped virion (10) is also non-glycosylated. The identity of electrophoretic mobility in SDS-PAGE of both nucleocapsid protein and the PII cleavage product has led to ambiguity in interpretation of pulse-chase experimental data (9). Since both PII and its cleavage product are glycosylated proteins, whereas nucleocapsid protein is not, we have also studied the effects of glucosamine upon the synthesis of NDV- 1Present address: Department of Genetics, University of Newcastle Upon Tyne, Newcastle Upon Tyne, England. induced proteins. In the fowl plague virus system, the addition of high concentrations of glucosamine selectively inhibited the formation of functional hemagglutinin (3, 4). Moreover, glucosamine appears to inhibit a post-translational cleavage step in the maturation of the HA (4), an observation of possible relevance to the NDV system. MATERIALS AND METHODS The experimental design, conditions of growth, and methods of SDS-PAGE analysis used were as previously described (9). Chicken embryo fibroblast cultures were grown to confluency (1.8 x 107 cells per 60-mm diameter plastic dish) in medium 199IC supplemented with 2 jig of L-isoleucine per ml (9). Confluent monolayers of cells were either used uninfected or infected with 120 PFU per cell of the L. Kansas strain of NDV, and 14C-isoleucine (30,Ci per dish) was added to provide the SDS-PAGE polypeptide synthesis pattern of uninfected cells. Four parallel NDV-infected cell monolayers were incubated in medium 1991C plus 2 gg of isoleucine per ml for 5.5 h prior to a 15-min pulse incubation with 3Hisoleucine (70 uci per dish). To test the effects of glucosamine on NDV-induced glycoprotein synthesis, D-glucosamine hydrochloride was added at 5 mg/ml to one of the infected cultures 30 min prior to the addition of 3H-isoleucine. This culture and a control pulse-labeled culture (not treated with D- glucosamine-hydrochloride), were boiled in a solution which contained 2% SDS and 2% mercaptoethanol in the gel sample buffer (5) for 2 min directly ifter the 15-min incubation with 3H-isoleucine. The remaining two pulse-labeled, NDV-infected cultures were subjected to a 30-min chase incubation in medium 199IC containing 200 Mg of unlabeled isoleucine per ml after the pulse incubation with 3H-isoleucine. To test the influence of glucosamine on precursor cleavage, D-glucosamine was added at 5 mg/ml to one of these cultures at the beginning of the

2 776 SAMSON AND FOX J. VIROL. c) 0~ X LUJ z Cr) 0 ZD -J L--- PO I~ FIG. 1. Acrylamide gel electrophoretic analysis of an extract of 3H-glucosamine-labeled NDV-infected chick cells showing the portion of the gel containing the four major NDV-induced polypeptides. A, An SDS extract of NDV-infected cells labeled for 15 min with 100 MCi of 3H-glucosamine. B, An SDS extract chase incubation. Both cultures were extracted with boiling 2% SDS-2% mercaptoethanol as above. The four 3H-labeled SDS extracts of NDV-infected cells were mixed separately with a portion of the "4C-labeled SDS extract of uninfected cells, and the mixtures were subjected to SDS-PAGE analysis as previously described (9). To study the gel electrophoretic pattern of viral proteins labeled with glucosamine, cells grown in medium 199IC containing 2 ug of isoleucine per ml were labeled with 100 MCi of carrier-free 3H-glucosamine per dish (New England Nuclear Corp., 7.5 Ci/mmol). Procedures for growth and infection with NDV L. Kansas were identical to those used in experiments for amino acid labeling. For chase incubations, the medium containing the labeled glucosamine was removed, and fresh medium containing 1 mg of unlabeled glucosamine per ml was added. RESULTS Our first concern was to establish whether the precursor protein (PH) is or is not a glycoprotein. We therefore studied the labeling patterns of NDV-infected cultures incubated with 3Hglucosamine. The SDS-PAGE patterns of radioactivity derived from labeled glucosamine are shown in Fig. 1. Figure 1A describes an experiment in which NDV-infected cells were labeled with glucosamine during a 15-min pulse incubation prior to solubilization with SDS. Two major peaks of radioactivity are observed in the area of the gel where the major viral polypeptides migrate. These correspond to polypeptide regions I and PH. No distinct peaks of radioactivity were detected in the regions designated II and III. Glucosamine labeling of a control (uninfected) culture revealed no distinct peaks of radioactivity in the regions of the gel designated I, PH, II, or III (now shown). Figure 1B describes an extension of the experiment in Fig. 1A. After the 15-min pulse incubation with labeled glucosamine, unlabeled glucosamine was added to the culture. The cells were then subjected to a 30-min chase incubation prior to solubilization of the proteins with detergent. During the chase incubation there was an increase in radioactivity in region I, indicating that labeled glucosamine leaves the cellular pool slowly compared with the behavior of labeled amino acids during similar chase incubations (9). There is also a decrease in the height of the peak of radioactivity that stands out above background in region PII, and a distinct peak of radioactivity can be detected in of chick cells labeled as in A and then incubated for a 30-min chase incubation (Materials and Methods) with unlabeled glucosamine. Arrows I, II, and III are the mobilities for the major polypeptides of NDV(relative to the tracker dye) obtained in a separate gel.

VOL. 13, 1974 + 20- INHIBITION OF NDV GLYCOPROTEIN SYNTHESIS I region II of the gel.

3 VOL. 13, INHIBITION OF NDV GLYCOPROTEIN SYNTHESIS I region II of the gel. These experiments show that viral polypeptides electrophoresing in regionsi, PII, and II are glycoproteins, and confirm our earlier findings thatp1ii is a precur- II ofthegels. We have also done the experiment described in A ' sor ofaproteinmigrating inregion Fig. 1 by using the procedure of Mountcastle et +15 al. (6) to separate envelope and nucleocapsid P11 m fractions prior to electrophoretic separation and II analysis of radioactivity. The results of these experiments indicate that the radioactivity derived from glucosamine is associated with the +10_ envelope fraction, and not with the nucleocapsid Ṫo study the effects of high levels of glucosamine on the formation of NDV-induced glyco- +5 proteins and on the cleavage of the precursor glycoprotein, we extended our studies using amino acid labels. The difference profiles in Fig. o 18 2 and 3 represent NDV-induced polypeptide I \ \ I W synthesis. Difference profiles were obtained by > O )0 - - _ subtracting the "4C counts per minute from the 0V 3H counts per minute after normalizing the14c counts per minute to 3H counts per minute in (f) areas of the gel pattern by NDV infection (9). known not to be affected Figure 2 shows the gel electrophoresis pattern of NDV-induced polypeptides synthesized dur- LU ing a 15-min pulse incubation with 3H-isoleu- ( cine (A) in the absence and (B) in the presence x Li -10- _ of glucosamine. The effects of glucosamine are exerted primarily upon the contents of regions I +l _0 - ll l and PII of the gels. Table 1 summarizes the 1T effects of glucosamine on NDV-induced poly- peptide synthesis and shows that relative to 1m radioactivity in peak III, glucosamine caused decreases in radioactivity in peaks I and PII of approximately 90 and 60%, respectively. The radioactivity in peak II of the sample from + 5 P11 glucosamine-treated cells is increased nearly 30% by the same basis of comparison. This latter effect could be caused by a relative increase in the radioactivity of polypeptides comprising peak II relative to radioactivity in peak III, or by an effect of glucosamine on host-directed protein synthesis in NDV-infected cells. The data in Fig. 3 and Table 2 indicate that - 5 _ high medium glucosamine concentration had no I I mmin with 3H-isoleucine with no glucosamine added, or B, an SDS extract of NDV-infected cells incubated with 5 mg of glucosamine per ml 30 min prior to and also during the 15-min incubation with 'H-isoleucine. FIG. 2. Difference profiles (3H minus 14C counts Chick cells infected with NDV (120 PFU per cell) per minute) derived from SDS-PAGE patterns of an were subjected to the 15-min incubation with 'H- SDS extract of uninfected chick cells labeled for 6 h isoleucine 5.5 h postinfection. Arrows I, II, and III with "4C-isoleucine electrophoresed with either: A, are the predicted positions for the three major polyan SDS extract of NDV-infected cells labeled for 15 peptides in purified NDV virions I 777

4 778 SAMSON AND FOX J. VIROL. r(9 a- x Lli + FIG. 3. Difference profiles (3H minus 14C counts per minute) derived from SDS-PAGE patterns of an SDS extract of uninfected chick cells labeled for 6 h with i4c_isoleucine electrophoresed with an SDS ex- TABLE 1. Effect of glucosamine upon NDV-induced polypeptide synthesis Without glucosamine With glucosamine Polvpeptide Excess 'H Molar Excess 3H Molar counts in rat.io countsin rat io peak' ( ) peaka ( r ) I i PII II III a Comprising the three fractions having the highest counts per minute in the peak area. b Polypeptide peak III expressed as unity. TABLE 2. Effect of glucosamine upon cleavage of polypeptide PII Without glucosamine With glucosamine Polvpeptide Excess 3H Excess 3H counts in b ratio' peak' C(') counts in peak' ('7) ratiob I PIl II III a Peak comprised of the three fractions having the highest counts per minute in the peak area. h Polypeptide III expressed as unity. effect on the post-translational cleavage of the presumably fully glycosylated precursor protein. With reference to studies with the fowl plague virus system (4), however, a carbohydrate-deficient precursor protein might not be cleaved. DISCUSSION Choppin and his colleagues have identified at least six proteins in mature NDV virions (6, 7, 10). Two of the six are glycoproteins, and both are located in the viral envelope. One of these glycoproteins has both neuraminidase and HA activity (10) (protein 1, designation of Mountcastle et al.; I, ours). The second virion glycoprotein (protein 2, designation of Mountcastle et al.; a region II protein, ours) may play an essential role in viral infiltration through the tract of NDV-infected cells incubated 15 min with 3Hisoleucine in the absence of glucosamine, and subjected to a 30-min chase incubation in the presence of 200 gg of unlabeled isoleucine per ml. A, No glucosamine present; B, 5 mg of glucosamine per ml present in the chase media. NDV infection and time of 31H incubation is the same as in Fig. 1. Arrows I, II, and III are the predicted positions for the three major polypeptides in purified NDV virions.

VOL. 13, 1974 INHIBITION OF NDV GLYCOPROTEIN SYNTHESIS 779 cell surface membrane and in membrane fusion (P. W. Choppin, personal communication). The data in Fig.

5 VOL. 13, 1974 INHIBITION OF NDV GLYCOPROTEIN SYNTHESIS 779 cell surface membrane and in membrane fusion (P. W. Choppin, personal communication). The data in Fig. 1 indicate that the protein migrating in the Pll region is also a glycoprotein, and these and our previously published data indicate that the protein in region Pll is the precursor of an envelope glycoprotein (2, designation of Mountcastle et al.) that has an electrophoretic mobility close to that of nucleocapsid protein (9). We were unable to do effective pulse-chase experiments with radioactive glucosamine. Our experience with this label was much like that of Plagemann and Erbe, who observed that labeled glucosamine both enters and leaves the cellular pool slowly (8). Addition of glucosamine to the medium at 5 mg/ml appeared to cause a selective inhibition of viral glycoprotein synthesis. Virtually no radioactivity was detected in the HA region of the gel (I, Fig. 2A) in the glucosamine-treated culture, and radioactivity migrating in the PII region was reduced disproportionately compared to that in regions II or III. High medium concentrations of glucosamine have a number of effects. Plagemann and Erbe reported that 10 mm glucosamine inhibited protein synthesis in rat hepatoma cells by approximately 85% (8). Glucosamine also interferes with the glycosylation and further maturation of viral glycoproteins (2-4, 11). In contrast to the properties of NDV-infected chick cells, glucosamine appeared to have no selective effect on the inhibition of synthesis of the non-glycosylated precursors of the glycoproteins of fowl plague virus (3). In the NDV system the non-glycosylated precursors of polypeptides I and Pll may be synthesized, but they are synthesized in decreased quantity relative to the non-glycosylated proteins (Fig. 2 and Table 1). Since glucosamine apparently inhibits protein glycosylation, it is not possible to predict the gel positions of the carbohydrate-deficient polypeptides that would be found in regions I and PII if they had their full complements of carbohydrate. Bretscher has observed that carbohydrate can increase the molecular weights of proteins determined by SDS-PAGE (1). (A more thorough treatment of molecular weight artifacts in SDS-PAGE appears in reference 12.) From the appearance of Fig. 2B it is obvious that glucosamine either completely inhibited the synthesis of protein in region I, or that this protein appeared either in region PII, or in regions II or III. We cannot predict, however, the identity of the protein in region Pll of Fig. 2B. Whatever the identity of this protein, a high medium glucosamine concentration definitely affected a selective decrease in the synthesis of either the protein with both HA and neuraminidase function (I), the precursor glycoprotein (Pll), or both. ACKNOWLEDGMENTS This work was supported by Public Health Service grants GM and AI from the National Institute of General Medical Sciences and the National Institute of Allergy and Infectious Diseases, respectively, by grant DRG-1153 from the Damon Runyon Memorial Fund, and by grant BC79 from the American Cancer Society. A. C. R. S. was supported by Damon Runyon Memorial Fund for Cancer Research Postdoctoral Fellowship DRF-640. C. F. F. is the recipient of Public Health Service Research Career Development Award GM from the National Institute of General Medical Sciences. LITERATURE CITED 1. Brestcher, M. S Major human erythrocvte glycoprotein spans the cell membrane. Nature N. Biol. 231: Gandhi, S. S., P. Stanley,,J. M. Taylor, and D. 0. White Inhibition of influenza viral glvcoprotein svnthesis by sugars. Microbios 5: Kaluza, G., C. Scholtissek. and R. Rott Inhibition of the multiplication of enveloped RNA-viruses by glucosamine and 2-deoxy-D-glucose. J. Gen. Virol. 14: Klenk, H.-D., C. Scholtissek, and R. Rott Inhibition of glycoprotein biosynthesis of influenza virus by D-glucosamine and 2-deoxy-D-glucose. Vriology 49: Laemmli. U. K Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: Mountcastle, W. E., R. W. Compans, L. A. Caliguiri, and P. W. Choppin Nucleocapsid protein subunits of simian virus 5, Newcastle disease virus, and Sendai virus. J. Virol. 6: Mountcastle, W. E., R. W. Compans, and P. W. Choppin Proteins and glycoproteins of paramvxoviruses: a comparison of simian virus 5, Newcastle disease virus, and Sendai virus. J. Virol. 7: Plagemann, P. G. W., and J. Erbe Transport and metabolism of glucosamine by cultured Novikoff rat hepatoma cells and effects on nucleotide pools. Cancer Res. 33: Samson, A. C. R., and C. F. Fox A precursor protein for Newcastle disease virus. J. Virol. 12: Scheid, A., and P. W. Choppin Isolation and purification of the envelope proteins of Newcastle disease virus. J. Virol. 11: Stanley, P., S. S. Gandhi, and D. 0. White The polypeptides of influenza virus VII. Synthesis of the hemagglutinin. Virology 53: Steck, T. L., and C. F. Fox Membrane proteins, p In C. F. Fox and A. D. Keith (ed.), Membrane molecular biology, Sinauer Associates, Stamford, Conn.

Formation of Influenza Virus Proteins

Formation of Influenza Virus Proteins JOURNAL OF VROLOGY, June 1973, p. 823-831 Copyright 6 1973 American Society for Microbiology Vol. 11, No. 6 Printed in U.SA. Formation of nfluenza Virus Proteins HANS-DETER KLENK AND RUDOLF ROT nstitut