- Email: [email protected]
Synthesis and processing of a 22–26K murine cytomegalovirus glycoprotein recognized by a neutralizing monoclonal antibody
VIROLOGY
169,474-478
Synthesis
(1989)
and Processing
of a 22-26K Murine Cytomegalovirus Glycoprotein by a Neutralizing Monoclonal Antibody LAMBERT
Department
of Microbiology,
C. LOH’
University of Saskatchewan,
ReceivedAugust
Recognized
Saskatoon,
25, 1988; accepted December
Saskatchewan,
Canada S7N OWO
22, 1988
A 22-26K glycoprotein (gp24) of the murine cytomegalovirus (MCMV) virion was immunoprecipitated by a monoclonal antibody (MAb) 6A1.21A that neutralized MCMV infectivity only in the presence of complement. Pulse-chase experiments demonstrated that gp24, which contained only N-linked, complex-type oligosaccharides, was processed from an 18.4K high-mannose precursor (gp18.4). Analyses by two-dimensional (nonreducing/reducing) gel electrophoresis have shown that both gp18.4 and gp24 are present as disulfide-linked complexes, and rapid oligomerization of the 18.4K precursor is an early step in the processing pathway of gp24. Finally, we demonstrated that gp24 belongs to the “late” class of MCMV proteins. o 1989 Academic press. ~nc.
During herpesvirus infections, induction of neutralizing antibodies against viral glycoproteins is an important part of the host’s humoral immune response. My laboratory has initiated a series of studies aimed at identifying and characterizing viral proteins that are targets of the immune response during MCMV infections, and have previously reported the characterization of two groups of MCMV glycoproteins recognized by the neutralizing monoclonal antibodies 8Dl. 1 1A (1) and 8G5.12A (2). Here we describe the characterization of a third group of MCMV glycoproteins recognized by a monoclonal antibody 6A1.21A which neutralizes MCMV infectivity only in the presence of complement. As shown in Table 1, the neutralizing activity of MAb 6A1.2 1A is specifically directed against MCMV, since it cannot neutralize HSV-1 or Ad5 even at the lowest dilution of 1: 10. The neutralizing titer and the extent of neutralization varied slightly with different preparations of ascitic fluid. Typically, 80 to 90% of the infectious virus is neutralized by 6A1.21A (dilution 1: 10) whereas close to 100% of MCMV infectivity is neutralized by immune sera. This slight difference may reflect the fact that the MAb only blocks one of several possible routes of virus entry. To identify viral proteins recognized by the MAb 6A1.21A, [35S]methionine-labeled MCMV virion proteins and lysates of MCMV-infected 3T3-Ll cells were immunoprecipitated with either 6A1.21A or normal mouse serum (NMS) and analyzed by 9% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). As shown in Fig. 1A, a 22-26K virion protein (Fig. 1A, lane 3) and an additional 18.4K protein that was present
only in MCMV-infected cells (Fig. 1A, lane 4) were specifically immunoprecipitated by the MAb 6A1.2 1A. Although other less prominent radiolabeled bands were seen, they were considered nonspecific, as they could also be precipitated by NMS (Fig. lA, lanes 1 and 2). Both proteins contained N-linked carbohydrates, as they were radiolabeled by [‘4C]glucosamine only in the absence of tunicamycin (Fig. 1 B). Deglycosylation of the two glycoproteins with /V-glycanase which removed all N-linked oligosaccharide chains produced a 14.6K polypeptide (Fig. 1C, lane 1) which was also the only [35S]methionine-labeled polypeptide immunoprecipitated from lysates of MCMV-infected cells grown in the presence of tunicamycin (Fig. 1C, lane 3) demonstrating that this 14.6K protein was the unglycosylated form of the 22-26K (gp24) and 18.4K (gp18.4) glycoproteins. To further define the type of oligosaccharide chains on gp24 and gp18.4, [35S]methionine-labeled, 6A1.21A-immunoprecipitated proteins were digested with different combinations of glycosidases. As shown in Fig. 2A, the mobilities of gp24 and gp18.4 on SDSpolyacrylamide gels were not affected by treatment with neuraminidase, which removes terminal sialic acid residues, or 0-glycanase, which hydrolyzes Olinked gal-/3-(1,3)galNAc (gal, galactose; galNAc, Nacetylgalactosamine) core disaccharides (3), demonstrating the lack of such carbohydrate structures on these glycoproteins. However, the conversion of gp18.4 to its 14.6K unglycosylated form after endoglycosidase H (endo H) digestion indicated that it contained only high-mannose type N-linked oligosaccharides (Fig. 2A). In contrast, gp24 contained only complex type oligosaccharide chains, as evidenced by its susceptibility to endo F (New England Nuclear) diges-
’ To whom requests for reprints should be addressed. 0042-6822189
$3.00
Copyright 0 1989 by Academic Press. Inc. All rights of reproduction I” any form resewed.
474
475
SHORT COMMUNICATIONS
present only in infected cell extracts (Fig. 1A, lane 4) suggest that gp18.4 is the precursor to gp24. To test this hypothesis, MCMV-infected 3T3-Ll cells were pulse-labeled for 10 min with [35S]methionine (500 &i/ ml) and chased for 1, 2, and 5 hr in the presence of 50 times the normal amount of unlabeled methionine. Lysates of the pulse-chase samples were then immunoprecipitated with either NMS or 6A1.21A and analyzed by 12% SDS-PAGE. The results are shown in Fig. 3A. Only gp18.4 was immunoprecipitated by 6A1.21A during the pulse-labeling period (Fig. 3A, lane 2) and the amount of precipitated gp18.4 continually decreased during the 5-hr chase period. On the other hand, the gp24 band was barely visible after a I-hr chase period and increased in intensity throughout the chase period (Fig. 3A, lanes 4, 6, and 8). Thus the kinetics of the synthesis of these two glycoproteins are consistent with our hypothesis that gp18.4 is the precursor to gp24. Furthermore, the unglycosylated 14.6K polypeptide was never seen even in a shorter 5-min pulselabeling period, and gp18.4 was sensitive to endo H throughout the experiment (data not shown), thus we concluded that N-linked high-mannose type chains on gp18.4 were cotranslationally added to its polypeptide backbone. The formation of disulfide-linked glycoprotein complexes is quite common among herpesviruses including both human (5-9) and mouse cytomegaloviruses (1, 2). To investigate whether gp24 and its precursor also form such complexes, lysates of [35S]methioninelabeled, MCMV-infected 3T3-Ll cells from a pulse-
TABLE 1 NEUTRALIZATION ASSAYS WITH MCMV-SPECIFIC MONOCLONAL ANTIBODIES
Neutralization of MCMV* (titer) Antibody (subclass) 6A1,21A(lgM) 6A1.21A 6A1.21A Immune mouse serum Normal mouse serum
Source”
With complement
Without complement
Virus
-
AF” AF AF
f(400) -
N.D.d N.D.
MCMV HSV-le Ad5’
Mouse
+(410)
+
MCMV
Mouse
-
-
MCMV
a All assays were done with 1: 10 dilutions of ascitic fluid or serum unless otherwise indicated. ‘Antibodies showing 50% or more in plaque reduction were considered positive. ’ Ascitic fluid. d Not determined. e Herpes simplex virus type 1 (McIntyre). ‘Adenovirus 5, from Dr. P. Branton, McMaster University, Hamilton, Ontario.
tion at pH 6 where the enzyme was known to cleave both high-mannose and complex type chains (4). This was further confirmed when gp24 was immunoprecipitated from MCMV virion proteins free of gp18.4 and digested with endo H or endo F (Fig. 2B). Data from our analysis of the oligosaccharide chains on the two glycoproteins and the fact that gp18.4 is B
Tunicamycin
C
+ -
‘_“lI :’ _,
1234
‘2” s4,’ r. i.’ :;,
8P24 ] 22-28 k
18dk 14.8k
-18dk NMS
6A1.21A
NM8
6A1.21A
FIG. 1. (A) The MAb 6A1.21A immmunoprecipitated 18.4K and 22 - 26K proteins. [%]methionine-labeled proteins immunoprecipitated by normal mouse serum (NMS) or MAb 6A1.21A from purified MCMV virions (lanes 1 and 3) and lysates of MCMV-infected 3T3-Ll cells (lanes 2 and 4) were analyzed by 9% SDS-PAGE and visualized by autoradiography. (B) The MAb 6A1.21A immunoprecipitated 18.4K and 22-26K glycoproteins. [‘4C]glucosamine-labeled glycoproteins were immunoprecipitated with normal mouse serum (NMS) or MAb 6A1.21Afrom lysates of MCMV-infected 3T3-Ll cells grown in the presence or absence of tunicamycin (5 pg/ml), analyzed by 9% SDS-PAGE, and visualized by autoradiography. (C) A 14.6K protein is the unglycosylated form of gp24. Lysates of [%]methionine-labeled, MCMV-infected 3T3-Lt cells grown in the presence (lane 3) or absence (lanes 1, 2, and 4) of 5 pg/ml of tunicamycin were immunoprecipitated with MAb 6A1.21A. The immunoprecipitates were either left untreated (lanes 2. 3, and 4) or digested with N-glycanase (lane l), and analyzed by 12% SDS-PAGE.
476
SHORT COMMUNICATIONS A E NF z i-k+ :NOO
Y N F
FIG. 2. (A) Glycoproteins immunoprecipitated by MAb 6A1.21A contained N-linked oligosaccharide chains. [%]methionine-labeled proteins immunoprecipitated by MAb 6A1.21Afrom lysates of MCMV-infected 3T3-Ll cells were either left untreated (control), digested with neuraminidase (N), digested sequentially with neuraminidase and 0-glycanase (N + O), digested sequentially with neuraminidase, endo F, and O-glycanase (N + F + 0), or digested sequentially with neuraminidase and endo F (N + F) as indicated above each lane. Alternatively, the immunoprecipitated gtycoproteins were incubated in the presence or absence of endo H. Afterward, the digestion products were analyzed by 12% SDSPAGE and visualized by autoradiography. (6) gp24 contained N-linked, complex type oligosacchande chains. [35S]methionine-labeled proteins immunoprecipitated by MAb 6A1.21A from purified MCMV virions were either left untreated, or digested with endo H or endo F as indicated above each lane. The digestion products were then analyzed by 12% SDS-PAGE, and visualized by autoradiography.
B
A 0) w
+ Chase
-
3
2
1
0.
---II ,”
i
5
Hours
C Chase
(D Chase J!,----
2-l
I3125
0’
1 2
-BME 5
t
Hours
(’,i” ‘I q’,f’ ,i, ,’
I 9P24 -18.4 k
1
2
3
4
5
6
7
8
--
I
NMS
fllfl
6A1.21A
FIG. 3. (A) The 18.4K glycoprotein is the precursor to gp24. MCMV-infected 3T3-Lt cells were pulse-labeled 20 hr postinfection with [%]methionine (500 MCi/ml) for 10 min (lanes 1 and 2) and chased for 1 hr (lanes 3 and 4) 2 hr (lanes 5 and 6) and 5 hr (lanes 7 and 8). Lysates of the pulse-chase samples were immunoprecipitated with normal mouse serum (lanes 1, 3, 5. and 7) or MAb 6A1.21A (lanes 2, 4, 6, and 8) and analyzed by 1296 SDS-PAGE. (E?)Analysis of pulse-labeled proteins immunoprecipitated with MAb 6A1.21A by 7.5% SDS-PAGE under nonreducing conditions. MCMV-infected 3T3-Ll cells were pulse-labeled 20 hr postinfection with [%]methionine (500 &i/ml) for 10 min and chased for 1, 2, and 5 hr. Lysates of the pulse-chase samples were immunoprecipitated with normal mouse serum (NMS) or MAb 6A1.21A, and the immunoprecipitates were analyzed by 7.5% SDS-PAGE under nonreducing conditions. Numbers on the left side represent sizes of molecular weight markers used. (C) Analysis of the pulse-labeled 18.4K glycoprotein by two-dimensional (nonreducing/reducing) gel electrophoresis. Lysates from MCMV-infected 3T3-Ll cells pulse-labeled with [%]methionine as described in (B) were immunoprecipitated with MAb 6A1.21A and analyzed by two-dimensional gel electrophoresis (a 7.5% nonreducing tube gel in the first dimension, and a 10% reducing slab gel in the second dimension). Protein bands of the first dimension gel were assumed to be identical to those shown in (B) and are displayed at the bottom of the two-dimensional gel for comparison with the dots in the autoradiogram. Arrows indicate proteins specifically immunoprecipitated by MAb 6A1.21A.
SHORT COMMUNICATIONS
477
A Time
post-infection in
:
hours
?I .?
N
d
(D
co0ol*(0co
0
,,
.:
1,
18.4k
FIG. 4. (A) The 18.4K precursor was first synthesized between 12 and 14 hr postinfection. Lysates from MCMV-infected 3T3-Ll cells labeled with [?S]methionine for consecutive 2-hr intervals between 0 and 20 hr postinfection were immunoprecipitated with MAb 6A1.21A and analyzed by 12% SDS-PAGE. (5) Synthesis of the 18.4K precursor in the presence of viral DNA synthesis inhibitors. MCMV-infected 3T3-Ll cells were either treated normally (control), or incubated with viral DNA synthesis inhibitors phosphonoacetic acid (PAA) at 200 @i/ml or cytosine arabinoside (Ara C) at 50 pg/ml for 18 hr post-infection and then labeled with [35S]methionine (50 pCi/ml) for 45 min. Lysates of the infected cells were immunoprecipitated with MAb 6A1.21A and analyzed by 12% SDS-PAGE.
chase experiment were immunoprecipitated with MAb 6A1.21A or NMS and analyzed by 7.5% SDS-PAGE under nonreducing conditions. The results are shown in Fig. 3B. During the chase periods, the monomeric forms of gp18.4 and gp24 were not seen, suggesting that they were present as disulfide-linked complexes migrating near the top of each lane. However, during the pulse-labeling period, multiple bands with apparent molecular weights ranging from 28K to over 300K were immunoprecipitated by the MAb 6A1.21A (Fig. 3B) and not by NMS. Since only a single prominent glycoprotein (gp18.4) was precipitated by 6A1.21A under reducing conditions during the same period (Fig. 3A), these extra bands probably consisted of oligomers of gp18.4. To confirm this, the pulse-labeled polypeptides immunoprecipitated by MAb 6A1.21A were analyzed by twodimensional (nonreducing/reducing) gel electrophoresis as described in the legend to Fig. 3C. The radiolabeled bands specifically precipitated by MAb 6A1.2 1A (marked by arrows in Fig. 3C) dissociated into gp18.4 monomers under reducing conditions, and were visible on the autoradiogram as a line of dots migrating close to the bottom of the second-dimension slab gel. The multiple oligomers of gp18.4 disappeared after a 1-hr chase period when the endo H-resistant gp24 appeared (compare Fig. 3A, lane 4 and Fig. 3B), indicating that the oligomerization process was completed within 1 hr. Therefore the data presented here suggested that oligomerization of gp18.4 through disulfide bonding is an early step in the processing pathway and preceded the appearance of the endo H-resistant gp24. Thus the oligomerization process probably took place before or
during the transport of gp18.4 from the endoplasmic reticulum (ER) to the Golgi complex, or at least to the medial compartment of the Golgi complex where endo H resistance was acquired (10). It has been proposed that formation of correctly folded quaternary structures constitute a key event regulating the transport of influenza hemagglutinin trimers out of the ER (11, 12). Perhaps the oligomerization process assured the correct folding of gp18.4 for transport to the Golgi complex or between Golgi compartments for further processing. In addition, the presence of multimeric forms of gp18.4 implied that gp24 was present in the MCMV virion as disulfide-linked multimers, and is consistent with our analysis of gp24 complexes immunoprecipitated from virion proteins (data not shown). To determine which kinetic class gp24 belongs to, 3T3-Ll cells were infected with MCMV at a multiplicity of infection of 10 and labeled with [35S]methionine for consecutive 2-hr periods between 0 and 20 hr postinfection. The proteins immunoprecipitated by MAb 6A1.2 1A from lysates of these infected cells were then analyzed by 12% SDS-PAGE. As shown in Fig. 4A, a radiolabeled protein band corresponding to gp18.4 first became visible during the 12th-to 14th-hr labeling period. Since MCMV DNA replication begins around 12 hr postinfection in asynchronous 3T3 cells (13) it appeared that gp24 belonged to the “late” class of MCMV proteins according to its time of synthesis. To confirm that synthesis of gp18.4 did not commence until viral DNA replication has begun, 3T3-Ll cells infected with MCMV were incubated with viral DNA synthesis inhibitors cytosine arabinoside (Ara C) at 50 pgl
478
SHORT COMMUNICATIONS
ml (14) or phosphonoacetic acid (PAA) at 200 pg/ml (15). The synthesis of gp18.4 was then monitored by immunoprecipitation with the MAb 6A1.2 1A followed by analysis on 12% SDS-PAGE. As shown in Fig. 4B, the 18.4K precursor was only synthesized when Ara C or PAA were absent, clearly demonstrating that gp24 expression requires viral DNA synthesis. In summary, we have characterized a MCMV virion glycoprotein gp24 that was the target of a complement-dependent neutralizing monoclonal antibody, suggesting that it may play a role in virus attachment or penetration. The apparent molecular weight of gp24 is much lower than that of the smallest glycoprotein GP6 (38K) described by Kim et a/. (16) and may represent a previously unreported MCMV virion glycoprotein. In addition, our data indicated that gp24 was present in the virion as high molecular weight disulfide-linked multimers (Fig. 3B), and the oligomerization process occurred prior to conversion of its 18.4K precursor to an endo H-resistant form (Fig. 3). Experiments are in progress to investigate the possible biological functions of gp24. ACKNOWLEDGMENTS I am grateful to Vicki Keeler for her excellent technical assistance and Dr. P. E. Branton for his gifts of Ad5 and 293 cells. This study
was supported 8196.
by Medical
Research Council of Canada Grant MA-
REFERENCES
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
LOH, L. C.. and QUALTIERE,L. F., virology 162,498-502 (1988). LOH, L. C., BALACHANDRAN, N., and QUALTIERE. L. F., Virology 166,206-216(1988). UMEMOTO, J., BHAVANANDAN, V. P., and DAVIDSON, E. A., J. Biol. Chem. 252,8609-86 14 (1977). PLUMMER, T. H., JR., ELDER,J. H., ALEXANDER,S., PHELAN, A. W., and TARENTINO, A. L., J. Biol. Chem. 259, 10,700-l 0,704 (1984). BRITT, W. J., virology 135, 369-378 (1984). LAW, K. M., WILTON-SMITH, P., and FARRAR, G. H., 1. Med. l&o/. 17,255-266(1985). FARRAR, G. H., and GREENAWAY,P. J., J. Gen. viral. 67, 14691473 (1986). KARI, B., LUSSENHOP,N., GOERTZ,R., WABUKE-BUNOTI,M., RADEKE, R., and GEHRZ, R. J. Viral. 60, 345-352 (1986). GRETCH, D. R., SARI, B., RASMUSSEN,L., GEHRZ, R. C., and STINSKI, M. F., J. Viral. 62, 875-881 (1988). DUNPHY, W. G., and ROTHMAN, J. E.. Cell42, 13-21 (1985). COPELAND, C. S., DOMS, R. W., BOLZAU, E. M., WEBSTER, R. G., and HELENIUS,A., J. Ce//. Biol. 103, 1179-l 191 (1986). COPELAND,C. S., ZIMMER, K., WAGNER, K. R., HEALEY,G. A., MELLMAN, I., and HELENIUS,A., Cell 53, 197-209 (1988). MULLER, M. T., and HUDSON, J. B., J. l&o/. 22, 267-272 (1977). HONESS, R. W., and ROIZMAN, B., J. viral. 14, 8-l 9 (1974). HUANG. E., HUANG. C., HUONG, S., and SELGRADE,M., Yale 1. of Biol. Med. 49, 93-98 (1976). KIM, K. S.. SAPIENZA.V. J., CARP, R. I., and MOON, H. M., 1. Viral. 17,906-915(1976).
169,474-478
Synthesis
(1989)
and Processing
of a 22-26K Murine Cytomegalovirus Glycoprotein by a Neutralizing Monoclonal Antibody LAMBERT
Department
of Microbiology,
C. LOH’
University of Saskatchewan,
ReceivedAugust
Recognized
Saskatoon,
25, 1988; accepted December
Saskatchewan,
Canada S7N OWO
22, 1988
A 22-26K glycoprotein (gp24) of the murine cytomegalovirus (MCMV) virion was immunoprecipitated by a monoclonal antibody (MAb) 6A1.21A that neutralized MCMV infectivity only in the presence of complement. Pulse-chase experiments demonstrated that gp24, which contained only N-linked, complex-type oligosaccharides, was processed from an 18.4K high-mannose precursor (gp18.4). Analyses by two-dimensional (nonreducing/reducing) gel electrophoresis have shown that both gp18.4 and gp24 are present as disulfide-linked complexes, and rapid oligomerization of the 18.4K precursor is an early step in the processing pathway of gp24. Finally, we demonstrated that gp24 belongs to the “late” class of MCMV proteins. o 1989 Academic press. ~nc.
During herpesvirus infections, induction of neutralizing antibodies against viral glycoproteins is an important part of the host’s humoral immune response. My laboratory has initiated a series of studies aimed at identifying and characterizing viral proteins that are targets of the immune response during MCMV infections, and have previously reported the characterization of two groups of MCMV glycoproteins recognized by the neutralizing monoclonal antibodies 8Dl. 1 1A (1) and 8G5.12A (2). Here we describe the characterization of a third group of MCMV glycoproteins recognized by a monoclonal antibody 6A1.21A which neutralizes MCMV infectivity only in the presence of complement. As shown in Table 1, the neutralizing activity of MAb 6A1.2 1A is specifically directed against MCMV, since it cannot neutralize HSV-1 or Ad5 even at the lowest dilution of 1: 10. The neutralizing titer and the extent of neutralization varied slightly with different preparations of ascitic fluid. Typically, 80 to 90% of the infectious virus is neutralized by 6A1.21A (dilution 1: 10) whereas close to 100% of MCMV infectivity is neutralized by immune sera. This slight difference may reflect the fact that the MAb only blocks one of several possible routes of virus entry. To identify viral proteins recognized by the MAb 6A1.21A, [35S]methionine-labeled MCMV virion proteins and lysates of MCMV-infected 3T3-Ll cells were immunoprecipitated with either 6A1.21A or normal mouse serum (NMS) and analyzed by 9% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). As shown in Fig. 1A, a 22-26K virion protein (Fig. 1A, lane 3) and an additional 18.4K protein that was present
only in MCMV-infected cells (Fig. 1A, lane 4) were specifically immunoprecipitated by the MAb 6A1.2 1A. Although other less prominent radiolabeled bands were seen, they were considered nonspecific, as they could also be precipitated by NMS (Fig. lA, lanes 1 and 2). Both proteins contained N-linked carbohydrates, as they were radiolabeled by [‘4C]glucosamine only in the absence of tunicamycin (Fig. 1 B). Deglycosylation of the two glycoproteins with /V-glycanase which removed all N-linked oligosaccharide chains produced a 14.6K polypeptide (Fig. 1C, lane 1) which was also the only [35S]methionine-labeled polypeptide immunoprecipitated from lysates of MCMV-infected cells grown in the presence of tunicamycin (Fig. 1C, lane 3) demonstrating that this 14.6K protein was the unglycosylated form of the 22-26K (gp24) and 18.4K (gp18.4) glycoproteins. To further define the type of oligosaccharide chains on gp24 and gp18.4, [35S]methionine-labeled, 6A1.21A-immunoprecipitated proteins were digested with different combinations of glycosidases. As shown in Fig. 2A, the mobilities of gp24 and gp18.4 on SDSpolyacrylamide gels were not affected by treatment with neuraminidase, which removes terminal sialic acid residues, or 0-glycanase, which hydrolyzes Olinked gal-/3-(1,3)galNAc (gal, galactose; galNAc, Nacetylgalactosamine) core disaccharides (3), demonstrating the lack of such carbohydrate structures on these glycoproteins. However, the conversion of gp18.4 to its 14.6K unglycosylated form after endoglycosidase H (endo H) digestion indicated that it contained only high-mannose type N-linked oligosaccharides (Fig. 2A). In contrast, gp24 contained only complex type oligosaccharide chains, as evidenced by its susceptibility to endo F (New England Nuclear) diges-
’ To whom requests for reprints should be addressed. 0042-6822189
$3.00
Copyright 0 1989 by Academic Press. Inc. All rights of reproduction I” any form resewed.
474
475
SHORT COMMUNICATIONS
present only in infected cell extracts (Fig. 1A, lane 4) suggest that gp18.4 is the precursor to gp24. To test this hypothesis, MCMV-infected 3T3-Ll cells were pulse-labeled for 10 min with [35S]methionine (500 &i/ ml) and chased for 1, 2, and 5 hr in the presence of 50 times the normal amount of unlabeled methionine. Lysates of the pulse-chase samples were then immunoprecipitated with either NMS or 6A1.21A and analyzed by 12% SDS-PAGE. The results are shown in Fig. 3A. Only gp18.4 was immunoprecipitated by 6A1.21A during the pulse-labeling period (Fig. 3A, lane 2) and the amount of precipitated gp18.4 continually decreased during the 5-hr chase period. On the other hand, the gp24 band was barely visible after a I-hr chase period and increased in intensity throughout the chase period (Fig. 3A, lanes 4, 6, and 8). Thus the kinetics of the synthesis of these two glycoproteins are consistent with our hypothesis that gp18.4 is the precursor to gp24. Furthermore, the unglycosylated 14.6K polypeptide was never seen even in a shorter 5-min pulselabeling period, and gp18.4 was sensitive to endo H throughout the experiment (data not shown), thus we concluded that N-linked high-mannose type chains on gp18.4 were cotranslationally added to its polypeptide backbone. The formation of disulfide-linked glycoprotein complexes is quite common among herpesviruses including both human (5-9) and mouse cytomegaloviruses (1, 2). To investigate whether gp24 and its precursor also form such complexes, lysates of [35S]methioninelabeled, MCMV-infected 3T3-Ll cells from a pulse-
TABLE 1 NEUTRALIZATION ASSAYS WITH MCMV-SPECIFIC MONOCLONAL ANTIBODIES
Neutralization of MCMV* (titer) Antibody (subclass) 6A1,21A(lgM) 6A1.21A 6A1.21A Immune mouse serum Normal mouse serum
Source”
With complement
Without complement
Virus
-
AF” AF AF
f(400) -
N.D.d N.D.
MCMV HSV-le Ad5’
Mouse
+(410)
+
MCMV
Mouse
-
-
MCMV
a All assays were done with 1: 10 dilutions of ascitic fluid or serum unless otherwise indicated. ‘Antibodies showing 50% or more in plaque reduction were considered positive. ’ Ascitic fluid. d Not determined. e Herpes simplex virus type 1 (McIntyre). ‘Adenovirus 5, from Dr. P. Branton, McMaster University, Hamilton, Ontario.
tion at pH 6 where the enzyme was known to cleave both high-mannose and complex type chains (4). This was further confirmed when gp24 was immunoprecipitated from MCMV virion proteins free of gp18.4 and digested with endo H or endo F (Fig. 2B). Data from our analysis of the oligosaccharide chains on the two glycoproteins and the fact that gp18.4 is B
Tunicamycin
C
+ -
‘_“lI :’ _,
1234
‘2” s4,’ r. i.’ :;,
8P24 ] 22-28 k
18dk 14.8k
-18dk NMS
6A1.21A
NM8
6A1.21A
FIG. 1. (A) The MAb 6A1.21A immmunoprecipitated 18.4K and 22 - 26K proteins. [%]methionine-labeled proteins immunoprecipitated by normal mouse serum (NMS) or MAb 6A1.21A from purified MCMV virions (lanes 1 and 3) and lysates of MCMV-infected 3T3-Ll cells (lanes 2 and 4) were analyzed by 9% SDS-PAGE and visualized by autoradiography. (B) The MAb 6A1.21A immunoprecipitated 18.4K and 22-26K glycoproteins. [‘4C]glucosamine-labeled glycoproteins were immunoprecipitated with normal mouse serum (NMS) or MAb 6A1.21Afrom lysates of MCMV-infected 3T3-Ll cells grown in the presence or absence of tunicamycin (5 pg/ml), analyzed by 9% SDS-PAGE, and visualized by autoradiography. (C) A 14.6K protein is the unglycosylated form of gp24. Lysates of [%]methionine-labeled, MCMV-infected 3T3-Lt cells grown in the presence (lane 3) or absence (lanes 1, 2, and 4) of 5 pg/ml of tunicamycin were immunoprecipitated with MAb 6A1.21A. The immunoprecipitates were either left untreated (lanes 2. 3, and 4) or digested with N-glycanase (lane l), and analyzed by 12% SDS-PAGE.
476
SHORT COMMUNICATIONS A E NF z i-k+ :NOO
Y N F
FIG. 2. (A) Glycoproteins immunoprecipitated by MAb 6A1.21A contained N-linked oligosaccharide chains. [%]methionine-labeled proteins immunoprecipitated by MAb 6A1.21Afrom lysates of MCMV-infected 3T3-Ll cells were either left untreated (control), digested with neuraminidase (N), digested sequentially with neuraminidase and 0-glycanase (N + O), digested sequentially with neuraminidase, endo F, and O-glycanase (N + F + 0), or digested sequentially with neuraminidase and endo F (N + F) as indicated above each lane. Alternatively, the immunoprecipitated gtycoproteins were incubated in the presence or absence of endo H. Afterward, the digestion products were analyzed by 12% SDSPAGE and visualized by autoradiography. (6) gp24 contained N-linked, complex type oligosacchande chains. [35S]methionine-labeled proteins immunoprecipitated by MAb 6A1.21A from purified MCMV virions were either left untreated, or digested with endo H or endo F as indicated above each lane. The digestion products were then analyzed by 12% SDS-PAGE, and visualized by autoradiography.
B
A 0) w
+ Chase
-
3
2
1
0.
---II ,”
i
5
Hours
C Chase
(D Chase J!,----
2-l
I3125
0’
1 2
-BME 5
t
Hours
(’,i” ‘I q’,f’ ,i, ,’
I 9P24 -18.4 k
1
2
3
4
5
6
7
8
--
I
NMS
fllfl
6A1.21A
FIG. 3. (A) The 18.4K glycoprotein is the precursor to gp24. MCMV-infected 3T3-Lt cells were pulse-labeled 20 hr postinfection with [%]methionine (500 MCi/ml) for 10 min (lanes 1 and 2) and chased for 1 hr (lanes 3 and 4) 2 hr (lanes 5 and 6) and 5 hr (lanes 7 and 8). Lysates of the pulse-chase samples were immunoprecipitated with normal mouse serum (lanes 1, 3, 5. and 7) or MAb 6A1.21A (lanes 2, 4, 6, and 8) and analyzed by 1296 SDS-PAGE. (E?)Analysis of pulse-labeled proteins immunoprecipitated with MAb 6A1.21A by 7.5% SDS-PAGE under nonreducing conditions. MCMV-infected 3T3-Ll cells were pulse-labeled 20 hr postinfection with [%]methionine (500 &i/ml) for 10 min and chased for 1, 2, and 5 hr. Lysates of the pulse-chase samples were immunoprecipitated with normal mouse serum (NMS) or MAb 6A1.21A, and the immunoprecipitates were analyzed by 7.5% SDS-PAGE under nonreducing conditions. Numbers on the left side represent sizes of molecular weight markers used. (C) Analysis of the pulse-labeled 18.4K glycoprotein by two-dimensional (nonreducing/reducing) gel electrophoresis. Lysates from MCMV-infected 3T3-Ll cells pulse-labeled with [%]methionine as described in (B) were immunoprecipitated with MAb 6A1.21A and analyzed by two-dimensional gel electrophoresis (a 7.5% nonreducing tube gel in the first dimension, and a 10% reducing slab gel in the second dimension). Protein bands of the first dimension gel were assumed to be identical to those shown in (B) and are displayed at the bottom of the two-dimensional gel for comparison with the dots in the autoradiogram. Arrows indicate proteins specifically immunoprecipitated by MAb 6A1.21A.
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477
A Time
post-infection in
:
hours
?I .?
N
d
(D
co0ol*(0co
0
,,
.:
1,
18.4k
FIG. 4. (A) The 18.4K precursor was first synthesized between 12 and 14 hr postinfection. Lysates from MCMV-infected 3T3-Ll cells labeled with [?S]methionine for consecutive 2-hr intervals between 0 and 20 hr postinfection were immunoprecipitated with MAb 6A1.21A and analyzed by 12% SDS-PAGE. (5) Synthesis of the 18.4K precursor in the presence of viral DNA synthesis inhibitors. MCMV-infected 3T3-Ll cells were either treated normally (control), or incubated with viral DNA synthesis inhibitors phosphonoacetic acid (PAA) at 200 @i/ml or cytosine arabinoside (Ara C) at 50 pg/ml for 18 hr post-infection and then labeled with [35S]methionine (50 pCi/ml) for 45 min. Lysates of the infected cells were immunoprecipitated with MAb 6A1.21A and analyzed by 12% SDS-PAGE.
chase experiment were immunoprecipitated with MAb 6A1.21A or NMS and analyzed by 7.5% SDS-PAGE under nonreducing conditions. The results are shown in Fig. 3B. During the chase periods, the monomeric forms of gp18.4 and gp24 were not seen, suggesting that they were present as disulfide-linked complexes migrating near the top of each lane. However, during the pulse-labeling period, multiple bands with apparent molecular weights ranging from 28K to over 300K were immunoprecipitated by the MAb 6A1.21A (Fig. 3B) and not by NMS. Since only a single prominent glycoprotein (gp18.4) was precipitated by 6A1.21A under reducing conditions during the same period (Fig. 3A), these extra bands probably consisted of oligomers of gp18.4. To confirm this, the pulse-labeled polypeptides immunoprecipitated by MAb 6A1.21A were analyzed by twodimensional (nonreducing/reducing) gel electrophoresis as described in the legend to Fig. 3C. The radiolabeled bands specifically precipitated by MAb 6A1.2 1A (marked by arrows in Fig. 3C) dissociated into gp18.4 monomers under reducing conditions, and were visible on the autoradiogram as a line of dots migrating close to the bottom of the second-dimension slab gel. The multiple oligomers of gp18.4 disappeared after a 1-hr chase period when the endo H-resistant gp24 appeared (compare Fig. 3A, lane 4 and Fig. 3B), indicating that the oligomerization process was completed within 1 hr. Therefore the data presented here suggested that oligomerization of gp18.4 through disulfide bonding is an early step in the processing pathway and preceded the appearance of the endo H-resistant gp24. Thus the oligomerization process probably took place before or
during the transport of gp18.4 from the endoplasmic reticulum (ER) to the Golgi complex, or at least to the medial compartment of the Golgi complex where endo H resistance was acquired (10). It has been proposed that formation of correctly folded quaternary structures constitute a key event regulating the transport of influenza hemagglutinin trimers out of the ER (11, 12). Perhaps the oligomerization process assured the correct folding of gp18.4 for transport to the Golgi complex or between Golgi compartments for further processing. In addition, the presence of multimeric forms of gp18.4 implied that gp24 was present in the MCMV virion as disulfide-linked multimers, and is consistent with our analysis of gp24 complexes immunoprecipitated from virion proteins (data not shown). To determine which kinetic class gp24 belongs to, 3T3-Ll cells were infected with MCMV at a multiplicity of infection of 10 and labeled with [35S]methionine for consecutive 2-hr periods between 0 and 20 hr postinfection. The proteins immunoprecipitated by MAb 6A1.2 1A from lysates of these infected cells were then analyzed by 12% SDS-PAGE. As shown in Fig. 4A, a radiolabeled protein band corresponding to gp18.4 first became visible during the 12th-to 14th-hr labeling period. Since MCMV DNA replication begins around 12 hr postinfection in asynchronous 3T3 cells (13) it appeared that gp24 belonged to the “late” class of MCMV proteins according to its time of synthesis. To confirm that synthesis of gp18.4 did not commence until viral DNA replication has begun, 3T3-Ll cells infected with MCMV were incubated with viral DNA synthesis inhibitors cytosine arabinoside (Ara C) at 50 pgl
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ml (14) or phosphonoacetic acid (PAA) at 200 pg/ml (15). The synthesis of gp18.4 was then monitored by immunoprecipitation with the MAb 6A1.2 1A followed by analysis on 12% SDS-PAGE. As shown in Fig. 4B, the 18.4K precursor was only synthesized when Ara C or PAA were absent, clearly demonstrating that gp24 expression requires viral DNA synthesis. In summary, we have characterized a MCMV virion glycoprotein gp24 that was the target of a complement-dependent neutralizing monoclonal antibody, suggesting that it may play a role in virus attachment or penetration. The apparent molecular weight of gp24 is much lower than that of the smallest glycoprotein GP6 (38K) described by Kim et a/. (16) and may represent a previously unreported MCMV virion glycoprotein. In addition, our data indicated that gp24 was present in the virion as high molecular weight disulfide-linked multimers (Fig. 3B), and the oligomerization process occurred prior to conversion of its 18.4K precursor to an endo H-resistant form (Fig. 3). Experiments are in progress to investigate the possible biological functions of gp24. ACKNOWLEDGMENTS I am grateful to Vicki Keeler for her excellent technical assistance and Dr. P. E. Branton for his gifts of Ad5 and 293 cells. This study
was supported 8196.
by Medical
Research Council of Canada Grant MA-
REFERENCES
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