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Apical expression of herpes simplex virus type 2 glycoproteins in human neuroblastoma cells
185,908-910
VIROLOGY
(1991)
Apical Expression
of Herpes
Simplex
Virus Type 2 Glycoproteins
in Human
Neuroblastoma
Cells
LARS N. NIELSEN, RICHARD J. WHITLEY, AND SUBHENDRA CHAITERJEE’ University
of Alabama
at Birmingham, Received
June
Department
of Pediatrics,
14, 199 1; accepted
August
Birmingham,
Alabama
35294
30, 199 1
The expression of herpes simplex virus type 2 (HSV-2) glycoproteins on the surface of human neuroblastoma cells has been investigated using Millipore Millicell culture plate inserts. Utilizing a modified radioimmunoassay, we learned that glycoproteins B, C, D, E, and I were expressed predominantly on the apical membrane domain of the infected neuroblastoma cells. The unidirectional transport of HSV-2 glycoproteins was substantiated by the analysis of extracellular glycoproteins released from neuroblastoma cells. The results suggest that the evaluated HSV-2 glycoproteins o is% Academic were transported primarily to the apical plasma membrane domain of human neuroblastoma cells.
used neuroblastoma cells because of the association between HSV and infection of neuronal cells, a target for establishment of latency (2 1,22). The unidirectional transport of HSV-2 glycoproteins was confirmed by the analysis of extracellular glycoproteins released from neuroblastoma cells. To determine the expression of HSV-2 glycoproteins on apical and basolateral surfaces, the human neuroblastoma cells, grown on Millicell-HA culture plate inserts (Millipore Corp., Bedford, MA), were infected with strain G with a multiplicity of infection of five. The Millicell-HA insert, consisting of a 0.45-pm poresize microporous membrane (mixed esters of cellulose), was sealed to a cylindrical polystyrene holder. Forty-eight hours postinfection both sides of the membrane were blocked for 1 hr with phosphate-buffered saline containing 3% nonfat dry milk, 0.01% antifoam A, and 0.01% sodium azide (BLOTTO; 23, 24). Monoclonal antibodies against HSV glycoproteins B, C, D, E, and I (gB, gC, gD. gE, and gl) were added inside the culture plate inserts separately of one set (for the analysis of the apical surface) and to the lower surface of the membrane inserts separately of another set (for the analysis of the basolateral surface). Type-common monoclonal antibodies against HSV gB, gC, gD, gE, and gl were prepared in this laboratory (6, 25, 26). The cells were incubated with the antibodies at room temperature for 1 hr and washed twice with BLOTTO. Finally, the cells were reacted with ‘251-labeled protein A and incubated further for 1 hr at room temperature. The membranes of the inserts were removed and placed in a gamma counter for quantitation. The results of the above experiments are summarized in Table 1. The distribution ratio between the apical and the basolateral plasma membrane indicated that all five
Herpes simplex virus (HSV) glycoproteins are important mediators of the biological functions of this virus, including adsorption, penetration, neutralization, and cell-to-cell fusion. There are at least seven well-characterized HSV-specific glycoproteins, B, C, D, E, G, H, and I (7-6). These glycoproteins are, in general, synthesized, processed, and transported inside infected cells in a sequential manner. Although several investigators have studied the expression and intracellular transport pathways of the HSV glycoproteins in cells of different origin, including human neuroblastoma cells (7- 74), the details of the intracellular trafficking of these glycoproteins remain unclear. The intracellular expression and trafficking of several viral glycoproteins in polarized epithelial cells have been repot-ted (15-20). Polarized epithelial cells, such as the Madin-Darby canine kidney (MDCK) cell line, are characterized by the presence of distinct apical and basolateral membranes separated by tight junctions. Vesicular stomatitis virus (VSV) and Type C retroviruses assemble and bud from the basolateral surface (17), whereas influenza and Sendai viruses assemble and bud from the apical plasma membrane (15, 18, 19). Thus, polarized cells must have specialized mechanisms for sorting and targeting membrane proteins to different surfaces. This type of directional transport of viral glycoproteins in cells of neuronal origin has not been reported for HSV. Using a characterized human neuroblastoma cell line, we showed that HSV type 2 (HSV-2) glycoproteins B, C, D, E, and I were transported primarily to the apical plasma membrane. We
’ To whom reprint requests should Alabama at Birmingham, 988, Basic mingham, AL 35294. 0042.6822/91
$3.00
CopyrIght 0 1991 by Academic Press. Inc. All raghts of reproductnn in any form reserved
be addressed at University Health Science Building,
of Bir-
908
SHORT COMMUNICATIONS TABLE 1 APICAL AND BA.SOLATEWL EXPRESSION OF HUMAN NEUROUSTOMA
HSV-2 GLYCOPROTEINS
IN
CELLS’
cpmb Glycoproteins
Apical surface
Basolateral surface
Ratio upper:lower
B C D E I
445 447 2436 1339 885
57 10 437 398 179
7.8:1 44.7:1 5.6:1 3.4:1 4.9:1
a Human neuroblastoma cells, grown on Millicell inserts were infected with strain G of HSV-2, provided by Dr. B. Roizman, University of Chicago, Chicago, IL. The human neuroblastoma cells were provided by Dr. T. Howard, University of Alabama at Birmingham, Birmingham, AL. These cells exhibited rosette formation, had microtubule arrays and neurofilaments, and exhibited neuron-specific enolase activity. b cpm, after subtracting the counts obtained from the uninfected cells.
glycoproteins were expressed primarily on the apical plasma membrane of the neuroblastoma cells. The ratio of apical:basolateral distributions were 7.8: 1, 44.7:1, 5.6:1, 3.4:1, and 4.9:1 for glycoproteins B, C, D, E, and I, respectively. The above result was confirmed by the autoradiography of the membrane inserts as shown in Fig. 1. The autoradiography of the inserts clearly demonstrated the expression of gB, gC, gD, gE, and gl on the infected upper cell surface. Since HSV-2 glycoproteins were expressed on the apical surface of the neuroblastoma cells, it was of interest to determine the apical expression of glycoproteins in released extracellular supernatants. Supernatants from apical and basolateral sides of infected neuroblastoma cells, seeded onto membrane inserts, were collected at 46 hr postinfection after a media change at 42 hr. The supernatants were centrifuged at 35,000 rpm for 1 hr. The pellets were dissolved in lysis buffer (2% SDS, 5% @-mercaptoethanol, 0.05 mlMTris, and 3% sucrose). The samples were analyzed by SDS-polyacrylamide gel electrophoresis as described
gB
909
previously (24, 27). The resulting gel was processed for immunoblotting as previously described (24, 28). The quantities of the extracellular HSV-2 glycoproteins B, C, D, E, and I can be seen in Fig. 2. The expression of all the glycoproteins tested were detected primarily in the supernatants collected from the apical side of the HSV-2-infected cell membranes. Our knowledge regarding the intracellular trafficking and expression of HSV-2 glycoproteins on the cell surface is limited. Such knowledge is especially limited for the cells of neuronal origin, a site for establishment of latency. It is known that HSV-2, in some areas of the United States, is responsible for at least 75% of primary genital herpes (29). In addition, the frequency of recurrences is greater after HSV-2 infection of the genital tract than HSV-1 (30). The trafficking of viral components inside neuronal cells could provide useful data regarding transport of virions and their components from the site of infection to a target for the establishment of latency. Using a human neuroblastoma cell line, we investigated the transport and expression of five HSV-2 glycoproteins on the infected cell surface. In this report, utilizing Millicell culture plate inserts, we demonstrated that HSV-2 glycoproteins, B, C, D, E, and I primarily were transported to the apical surface of the human neuroblastoma cells. This finding was supported by the similar type of polarity observed after the analysis of extracellular glycoproteins released from neuroblastoma cells. At present it is unknown whether the signal for the sorting and transport of HSV-2 glycoproteins reside on the glycoprotein molecule or involve other viral components. Moreover, the participation of cellular components may also be necessary in the sorting and trafficking of viral glycoproteins. This type of unidirectional transport of viral glycoproteins has previously been observed in polarized epithelial cells, MDCK, which are characterized by the presence of distinct apical and basolateral membrane domains separated by tight junctions. It has been shown that VSV and C-type retroviruses assemble and bud from the basolateral membrane (7 7) whereas influenza and Sendai virus assemble at the apical plasma mem-
gc
FIG. 1. Expression of HSV2 glycoproteins after autoradiography of the infected upper and lower membrane inserts. After quantitation, the membrane inserts derived from the experiment described in Table 1 were exposed to Kodak X-OMAT film for autoradiography.
SHORT
910
12
34
5
6
COMMUNICATIONS
78
REFERENCES (1979). 1. BAUCKE, R. B., and SPEAR, P. G., 1. viral. 32, 779-789 R. J., J. Viral. 36, 665-675 (1980). 3. MARSDEN, H. S., BUCKMASTER, A., PALFREYMAN, J. W., HOPE, R. G., and MINSON, A. C., J. Viral. 50, 547-554 (1984). 4. ROIZMAN, B., NORRILD, B., CHAN, C., and PEREIRA. L., Virology 133,242-247 (1984). 5. GOMPELS, U., and MINSON, A.. virology 153, 230-247 (1986). 6. LONGNECKER, R., CHATTERJEE, S., WHITLEY, R. J., and ROIZMAN, B., Proc. Nat/. Acad. Sci. USA 84, 4303-4307 (1987). 7. JOHNSON, D. C., and SPEAR, P. G., J. Viral. 43, 1102-l 112 (1982). 8. NORRILD, B., and PEDERSEN, B., J. Vifol. 43, 395-402 (1982). 9. Su. H. K., EBERLE, R., and COURTNEY, R. J.. 1. Viral. 61, 17351737 (1987). 10. Su, H. K., and COURTNEY, R. J., J. VifoL 62, 3668-3674 (1988). 11. GLORIOSO. J., SZCZESIUL, M. S., MARLIN, S. D., and LEVINE, M.. Virology 126, l-18 (1983). 12. JOHNSON, D. C., and SPEAR, P. G.. Cell 32,987-997 (1983). 13. CHATTEFUEE. S.. and BURNS, P., J. Viral. 64, 5209-5213 (1990). S., and WHITLEY, R. J., Biochem. 14. CHATTERJEE, S., NISHIMURO, Biophys. Res. Commun. 167, 1139-l 145 (1990). E., and SABATINI, D. D., Proc. Nat/. Acad. 15. RODRIGUEZ-BOULAN, Sci. USA 75, 5071-5075 (1978). 16. BFIASITUS, T. A., TALL, A. R., SCHACHTER, D., and MOMOUNERS, T. G.. Gastroenterology 76, 1107 (1979). 17. RICHARDSON, J. C. W., and SIMMONS, N. L., FE&S Leff. 105,201204 (1979). 18. ROTH, M. G., SRINIVAS, R. V., and COMPANS. R. W., 1. Viral. 45, 1065-1073 (1983). 19. ROTH, M. G., COMPANS, R. W., GIUSTI, L., DAVIS, A. R., NAYAK, D. P.. GETHING, M.-J., and SAMBROOK. J., Cell 33, 435-443 (1983). 20. LISANTI, M. P., SARGIACOMO, M., GRAEVE, L., SALTIEL, A. R., and RODRIGUU-BOULAN, E., Proc. Natl. Acad. SC;. USA 85, 9557956 1 (1988). 21. BARINGER, J. R.. and SWOVELAND, P.. N. Engl. J. Med. 288, 648650 (1973). 22. BASTIAN, F. O., RABSON, A. S., YEE, C. L., and TRALKA, T. S., Science 178, 306-307 (1972). 23. JOHNSON, D. A., GAUTSCH, J. W., SPORTSMAN, 1. R.. and ELDER, J. H., Gene Anal. Techn. 1, 3-8 (1984). 24. CHA~ERJEE, S., HUNTER, E., and WHITLEY. R., J. Viral. 56, 419425 (1985). 25. KOGA. J., CHATTEFUEE, S., and WHITLEY, R. J., virology 151, 385389 (1986). 26. CHAT~ERJEE, S., KOGA, J., and WHITLEY, R. I., J. Gen. Viral. 70, 2157-2162 (1989). 27. LAEMMLI, U. K., Nature 227, 680-685 (1970). 28. TOWBIN, H., STAEHELIN, T.. and GORDON, J., Proc. Nat/. Acad. Sci. USA 76,4350-4354 (1979). 29. COREY, L., ADAMS, H.. BROWN, Z.. and HOLMES, K., Ann. Intn. Med. 98,958-972 (1983). 30. REEVES, W. C., COREY, L., ADAMS, H. G., VONTVER, L. A., and HOLMES, K. K.. N. Engl. J. Med. 305, 315-319 (1981). 31. SRINIVAS, R. V., BALACHANDRAN. N., ALONSO-CAPLEN, F. V., and COMPANS, R. W., J. Vifol. 58, 689-693 (1986). 32. DOTTI, C. G., and SIMONS, K., Cell 62, 63-72 (I 990).
2. EBERLE, R., and COURTNEY,
FIG. 2. lmmunoblot analysis of the released extracellular HSV-2 glycoproteins. Lanes 1, 3, 5, and 7, extracellular glycoproteins released from the apical surface. Lanes 2, 4, 6, and 8, extracellular glycoproteins released from the basolateral surface. Lanes 1 and 2, incubated with antibody to gB. Lanes 3 and 4, incubated with antibody to gC. Lanes 5 and 6, incubated with antibodies to gD and gE. Lanes 7 and 8, incubated with antibody to gl.
brane (15, 18, 19) of the cultured epithelial cells. In case of DNA viruses, a previous report indicated that HSV-2 glycoproteins B, C, D, E, and G are expressed on the basolateral surface of MDCK cells (31). The exact reason for the difference observed between the human neuroblastoma and MDCK cells is unclear, although the species, origin, and type of cells may be responsible for such differences, as surface polarities in neuronal cells and epithelia are morphologically different. Recently, Dotti and Simons (32) found that the same viral glycoproteins are sorted in a polarized fashion in both hippocampal neuronal and epithelial cells. This result suggested that the molecular mechanisms of protein sorting share common features in these two different cell types. It is evident that the neuroblastoma cells used in our study must have a mechanism for sorting and targeting these HSV-2 glycoproteins to the apical membrane domain. Further investigation with other neuronal cells and different strains of HSV will be necessary to understand the exact mechanism for sorting and intracellular transport of viral glycoproteins in infected cells. ACKNOWLEDGMENTS We thank P. Burns for her excellent technical assistance. This work was supported by the Public Health Service Grant AI-251 20 from the National Institute of Health. L. N. Nielsen was in part supported by the Danish Medical Research Council Grant 12-0462-l and by the Danish Cancer Society Grant 90-003.
VIROLOGY
(1991)
Apical Expression
of Herpes
Simplex
Virus Type 2 Glycoproteins
in Human
Neuroblastoma
Cells
LARS N. NIELSEN, RICHARD J. WHITLEY, AND SUBHENDRA CHAITERJEE’ University
of Alabama
at Birmingham, Received
June
Department
of Pediatrics,
14, 199 1; accepted
August
Birmingham,
Alabama
35294
30, 199 1
The expression of herpes simplex virus type 2 (HSV-2) glycoproteins on the surface of human neuroblastoma cells has been investigated using Millipore Millicell culture plate inserts. Utilizing a modified radioimmunoassay, we learned that glycoproteins B, C, D, E, and I were expressed predominantly on the apical membrane domain of the infected neuroblastoma cells. The unidirectional transport of HSV-2 glycoproteins was substantiated by the analysis of extracellular glycoproteins released from neuroblastoma cells. The results suggest that the evaluated HSV-2 glycoproteins o is% Academic were transported primarily to the apical plasma membrane domain of human neuroblastoma cells.
used neuroblastoma cells because of the association between HSV and infection of neuronal cells, a target for establishment of latency (2 1,22). The unidirectional transport of HSV-2 glycoproteins was confirmed by the analysis of extracellular glycoproteins released from neuroblastoma cells. To determine the expression of HSV-2 glycoproteins on apical and basolateral surfaces, the human neuroblastoma cells, grown on Millicell-HA culture plate inserts (Millipore Corp., Bedford, MA), were infected with strain G with a multiplicity of infection of five. The Millicell-HA insert, consisting of a 0.45-pm poresize microporous membrane (mixed esters of cellulose), was sealed to a cylindrical polystyrene holder. Forty-eight hours postinfection both sides of the membrane were blocked for 1 hr with phosphate-buffered saline containing 3% nonfat dry milk, 0.01% antifoam A, and 0.01% sodium azide (BLOTTO; 23, 24). Monoclonal antibodies against HSV glycoproteins B, C, D, E, and I (gB, gC, gD. gE, and gl) were added inside the culture plate inserts separately of one set (for the analysis of the apical surface) and to the lower surface of the membrane inserts separately of another set (for the analysis of the basolateral surface). Type-common monoclonal antibodies against HSV gB, gC, gD, gE, and gl were prepared in this laboratory (6, 25, 26). The cells were incubated with the antibodies at room temperature for 1 hr and washed twice with BLOTTO. Finally, the cells were reacted with ‘251-labeled protein A and incubated further for 1 hr at room temperature. The membranes of the inserts were removed and placed in a gamma counter for quantitation. The results of the above experiments are summarized in Table 1. The distribution ratio between the apical and the basolateral plasma membrane indicated that all five
Herpes simplex virus (HSV) glycoproteins are important mediators of the biological functions of this virus, including adsorption, penetration, neutralization, and cell-to-cell fusion. There are at least seven well-characterized HSV-specific glycoproteins, B, C, D, E, G, H, and I (7-6). These glycoproteins are, in general, synthesized, processed, and transported inside infected cells in a sequential manner. Although several investigators have studied the expression and intracellular transport pathways of the HSV glycoproteins in cells of different origin, including human neuroblastoma cells (7- 74), the details of the intracellular trafficking of these glycoproteins remain unclear. The intracellular expression and trafficking of several viral glycoproteins in polarized epithelial cells have been repot-ted (15-20). Polarized epithelial cells, such as the Madin-Darby canine kidney (MDCK) cell line, are characterized by the presence of distinct apical and basolateral membranes separated by tight junctions. Vesicular stomatitis virus (VSV) and Type C retroviruses assemble and bud from the basolateral surface (17), whereas influenza and Sendai viruses assemble and bud from the apical plasma membrane (15, 18, 19). Thus, polarized cells must have specialized mechanisms for sorting and targeting membrane proteins to different surfaces. This type of directional transport of viral glycoproteins in cells of neuronal origin has not been reported for HSV. Using a characterized human neuroblastoma cell line, we showed that HSV type 2 (HSV-2) glycoproteins B, C, D, E, and I were transported primarily to the apical plasma membrane. We
’ To whom reprint requests should Alabama at Birmingham, 988, Basic mingham, AL 35294. 0042.6822/91
$3.00
CopyrIght 0 1991 by Academic Press. Inc. All raghts of reproductnn in any form reserved
be addressed at University Health Science Building,
of Bir-
908
SHORT COMMUNICATIONS TABLE 1 APICAL AND BA.SOLATEWL EXPRESSION OF HUMAN NEUROUSTOMA
HSV-2 GLYCOPROTEINS
IN
CELLS’
cpmb Glycoproteins
Apical surface
Basolateral surface
Ratio upper:lower
B C D E I
445 447 2436 1339 885
57 10 437 398 179
7.8:1 44.7:1 5.6:1 3.4:1 4.9:1
a Human neuroblastoma cells, grown on Millicell inserts were infected with strain G of HSV-2, provided by Dr. B. Roizman, University of Chicago, Chicago, IL. The human neuroblastoma cells were provided by Dr. T. Howard, University of Alabama at Birmingham, Birmingham, AL. These cells exhibited rosette formation, had microtubule arrays and neurofilaments, and exhibited neuron-specific enolase activity. b cpm, after subtracting the counts obtained from the uninfected cells.
glycoproteins were expressed primarily on the apical plasma membrane of the neuroblastoma cells. The ratio of apical:basolateral distributions were 7.8: 1, 44.7:1, 5.6:1, 3.4:1, and 4.9:1 for glycoproteins B, C, D, E, and I, respectively. The above result was confirmed by the autoradiography of the membrane inserts as shown in Fig. 1. The autoradiography of the inserts clearly demonstrated the expression of gB, gC, gD, gE, and gl on the infected upper cell surface. Since HSV-2 glycoproteins were expressed on the apical surface of the neuroblastoma cells, it was of interest to determine the apical expression of glycoproteins in released extracellular supernatants. Supernatants from apical and basolateral sides of infected neuroblastoma cells, seeded onto membrane inserts, were collected at 46 hr postinfection after a media change at 42 hr. The supernatants were centrifuged at 35,000 rpm for 1 hr. The pellets were dissolved in lysis buffer (2% SDS, 5% @-mercaptoethanol, 0.05 mlMTris, and 3% sucrose). The samples were analyzed by SDS-polyacrylamide gel electrophoresis as described
gB
909
previously (24, 27). The resulting gel was processed for immunoblotting as previously described (24, 28). The quantities of the extracellular HSV-2 glycoproteins B, C, D, E, and I can be seen in Fig. 2. The expression of all the glycoproteins tested were detected primarily in the supernatants collected from the apical side of the HSV-2-infected cell membranes. Our knowledge regarding the intracellular trafficking and expression of HSV-2 glycoproteins on the cell surface is limited. Such knowledge is especially limited for the cells of neuronal origin, a site for establishment of latency. It is known that HSV-2, in some areas of the United States, is responsible for at least 75% of primary genital herpes (29). In addition, the frequency of recurrences is greater after HSV-2 infection of the genital tract than HSV-1 (30). The trafficking of viral components inside neuronal cells could provide useful data regarding transport of virions and their components from the site of infection to a target for the establishment of latency. Using a human neuroblastoma cell line, we investigated the transport and expression of five HSV-2 glycoproteins on the infected cell surface. In this report, utilizing Millicell culture plate inserts, we demonstrated that HSV-2 glycoproteins, B, C, D, E, and I primarily were transported to the apical surface of the human neuroblastoma cells. This finding was supported by the similar type of polarity observed after the analysis of extracellular glycoproteins released from neuroblastoma cells. At present it is unknown whether the signal for the sorting and transport of HSV-2 glycoproteins reside on the glycoprotein molecule or involve other viral components. Moreover, the participation of cellular components may also be necessary in the sorting and trafficking of viral glycoproteins. This type of unidirectional transport of viral glycoproteins has previously been observed in polarized epithelial cells, MDCK, which are characterized by the presence of distinct apical and basolateral membrane domains separated by tight junctions. It has been shown that VSV and C-type retroviruses assemble and bud from the basolateral membrane (7 7) whereas influenza and Sendai virus assemble at the apical plasma mem-
gc
FIG. 1. Expression of HSV2 glycoproteins after autoradiography of the infected upper and lower membrane inserts. After quantitation, the membrane inserts derived from the experiment described in Table 1 were exposed to Kodak X-OMAT film for autoradiography.
SHORT
910
12
34
5
6
COMMUNICATIONS
78
REFERENCES (1979). 1. BAUCKE, R. B., and SPEAR, P. G., 1. viral. 32, 779-789 R. J., J. Viral. 36, 665-675 (1980). 3. MARSDEN, H. S., BUCKMASTER, A., PALFREYMAN, J. W., HOPE, R. G., and MINSON, A. C., J. Viral. 50, 547-554 (1984). 4. ROIZMAN, B., NORRILD, B., CHAN, C., and PEREIRA. L., Virology 133,242-247 (1984). 5. GOMPELS, U., and MINSON, A.. virology 153, 230-247 (1986). 6. LONGNECKER, R., CHATTERJEE, S., WHITLEY, R. J., and ROIZMAN, B., Proc. Nat/. Acad. Sci. USA 84, 4303-4307 (1987). 7. JOHNSON, D. C., and SPEAR, P. G., J. Viral. 43, 1102-l 112 (1982). 8. NORRILD, B., and PEDERSEN, B., J. Vifol. 43, 395-402 (1982). 9. Su. H. K., EBERLE, R., and COURTNEY, R. J.. 1. Viral. 61, 17351737 (1987). 10. Su, H. K., and COURTNEY, R. J., J. VifoL 62, 3668-3674 (1988). 11. GLORIOSO. J., SZCZESIUL, M. S., MARLIN, S. D., and LEVINE, M.. Virology 126, l-18 (1983). 12. JOHNSON, D. C., and SPEAR, P. G.. Cell 32,987-997 (1983). 13. CHATTEFUEE. S.. and BURNS, P., J. Viral. 64, 5209-5213 (1990). S., and WHITLEY, R. J., Biochem. 14. CHATTERJEE, S., NISHIMURO, Biophys. Res. Commun. 167, 1139-l 145 (1990). E., and SABATINI, D. D., Proc. Nat/. Acad. 15. RODRIGUEZ-BOULAN, Sci. USA 75, 5071-5075 (1978). 16. BFIASITUS, T. A., TALL, A. R., SCHACHTER, D., and MOMOUNERS, T. G.. Gastroenterology 76, 1107 (1979). 17. RICHARDSON, J. C. W., and SIMMONS, N. L., FE&S Leff. 105,201204 (1979). 18. ROTH, M. G., SRINIVAS, R. V., and COMPANS. R. W., 1. Viral. 45, 1065-1073 (1983). 19. ROTH, M. G., COMPANS, R. W., GIUSTI, L., DAVIS, A. R., NAYAK, D. P.. GETHING, M.-J., and SAMBROOK. J., Cell 33, 435-443 (1983). 20. LISANTI, M. P., SARGIACOMO, M., GRAEVE, L., SALTIEL, A. R., and RODRIGUU-BOULAN, E., Proc. Natl. Acad. SC;. USA 85, 9557956 1 (1988). 21. BARINGER, J. R.. and SWOVELAND, P.. N. Engl. J. Med. 288, 648650 (1973). 22. BASTIAN, F. O., RABSON, A. S., YEE, C. L., and TRALKA, T. S., Science 178, 306-307 (1972). 23. JOHNSON, D. A., GAUTSCH, J. W., SPORTSMAN, 1. R.. and ELDER, J. H., Gene Anal. Techn. 1, 3-8 (1984). 24. CHA~ERJEE, S., HUNTER, E., and WHITLEY. R., J. Viral. 56, 419425 (1985). 25. KOGA. J., CHATTEFUEE, S., and WHITLEY, R. J., virology 151, 385389 (1986). 26. CHAT~ERJEE, S., KOGA, J., and WHITLEY, R. I., J. Gen. Viral. 70, 2157-2162 (1989). 27. LAEMMLI, U. K., Nature 227, 680-685 (1970). 28. TOWBIN, H., STAEHELIN, T.. and GORDON, J., Proc. Nat/. Acad. Sci. USA 76,4350-4354 (1979). 29. COREY, L., ADAMS, H.. BROWN, Z.. and HOLMES, K., Ann. Intn. Med. 98,958-972 (1983). 30. REEVES, W. C., COREY, L., ADAMS, H. G., VONTVER, L. A., and HOLMES, K. K.. N. Engl. J. Med. 305, 315-319 (1981). 31. SRINIVAS, R. V., BALACHANDRAN. N., ALONSO-CAPLEN, F. V., and COMPANS, R. W., J. Vifol. 58, 689-693 (1986). 32. DOTTI, C. G., and SIMONS, K., Cell 62, 63-72 (I 990).
2. EBERLE, R., and COURTNEY,
FIG. 2. lmmunoblot analysis of the released extracellular HSV-2 glycoproteins. Lanes 1, 3, 5, and 7, extracellular glycoproteins released from the apical surface. Lanes 2, 4, 6, and 8, extracellular glycoproteins released from the basolateral surface. Lanes 1 and 2, incubated with antibody to gB. Lanes 3 and 4, incubated with antibody to gC. Lanes 5 and 6, incubated with antibodies to gD and gE. Lanes 7 and 8, incubated with antibody to gl.
brane (15, 18, 19) of the cultured epithelial cells. In case of DNA viruses, a previous report indicated that HSV-2 glycoproteins B, C, D, E, and G are expressed on the basolateral surface of MDCK cells (31). The exact reason for the difference observed between the human neuroblastoma and MDCK cells is unclear, although the species, origin, and type of cells may be responsible for such differences, as surface polarities in neuronal cells and epithelia are morphologically different. Recently, Dotti and Simons (32) found that the same viral glycoproteins are sorted in a polarized fashion in both hippocampal neuronal and epithelial cells. This result suggested that the molecular mechanisms of protein sorting share common features in these two different cell types. It is evident that the neuroblastoma cells used in our study must have a mechanism for sorting and targeting these HSV-2 glycoproteins to the apical membrane domain. Further investigation with other neuronal cells and different strains of HSV will be necessary to understand the exact mechanism for sorting and intracellular transport of viral glycoproteins in infected cells. ACKNOWLEDGMENTS We thank P. Burns for her excellent technical assistance. This work was supported by the Public Health Service Grant AI-251 20 from the National Institute of Health. L. N. Nielsen was in part supported by the Danish Medical Research Council Grant 12-0462-l and by the Danish Cancer Society Grant 90-003.