VIROLOGY
185, 843-846
(199 1)
Fate of the 6K Membrane
Protein
of Semliki
Forest
Virus during Virus Assembly
SARI LUSA, HENRIK GAROFF, AND PETER LILIESTR~~M’ Department
of Molecular Received
Biology,
Karolinska
Institute,
July 8, 199 1; accepted
S- 14 1 57 Huddinge,
September
Sweden
4, 199 1
The Semliki Forest virus directs the synthesis of three virus-specific transmembrane proteins p62,6K, all are made in equimolar amounts from a polyprotein precursor molecule. The p62 and El spike proteins dimeric complexes in the endoplasmic reticulum before being transported to the cell surface where occurs. In this study we show that the 6K protein becomes associated to the p62El complex in the reticulum and transported with the complex to the cell surface. During virus budding, El and p62 (which into the E2 protein) are incorporated into new virions whereas the 6K is mostly excluded. Virus particles infected BHK cells contain only about 3% of 6K in their membrane as compared to the spike protein relevance of these findings for the mechanism of SFV assembly is discussed. o 1991 Academic PWSS. I~C.
The budding of Semliki Forest virus (SFV), an enveloped positive-strand RNA virus, is dependent on the interactions between cytoplasmically formed nucleocapsids and heterodimeric spike complexes at the plasma membrane (PM). All the structural proteins of SFV are synthesized as a polyprotein precursor in the order C-p62-6K-El (76) from which each individual protein is cotranslationally released by a proteolytic processing event. The capsid protein (C) is formed by an autoproteolytic cleavage (74), while the other three proteins are formed by signal peptidase cleavages during their translocation and insertion into the endoplasmic reticulum (ER) membrane (7, 9). In ER, the p62 and El proteins form heterodimers (78, 79) which subsequently are transported to PM. The p62 precursor protein is cleaved to E2 and E3 at a very late stage during transport from the transGolgi compartment to the PM (3). The p62 and El proteins are known to have important functions in virus assembly and disassembly (7 I73, 75, 77, 78), but the role of the 6K protein in virus replication remains unsolved. During synthesis of the viral membrane proteins at ER the C-terminal portion of 6K functions as a signal peptide for El. However, we have shown that removal of 6K from the genome allows normal synthesis and intracellular transport of the p62El heterodimeric complex to the cell surface, but does reduce virus release to about 2% of wild-type frequency (9, 70). Interestingly, the produced virus had similar infectivity as compared to wild-type virus (70). A portion of the SFV and of the closely related Sindbis virus 6K protein becomes palmitylated in infected ’ To whom
reprint
requests
should
and El, which form heterovirus budding endoplasmic has matured released from content. The
chicken embryo fibroblast (CEF) cells, and substitution mutations which cause underacylation of the 6K protein lead to a reduction in virus release and to the formation of multicored virions at the PM (4, 6). Only small amounts (up to 10%) of 6K are found in mature virions released from infected CEF cells (5). Together these results suggest that 6K plays an important role in virus maturation. To follow the fate of 6K after synthesis BHK cells infected with wild-type SFV were pulse-labeled for 10 min and chased for various times. After a short 10 min chase, all three membrane proteins ~62, El, and 6K were readily detected (Fig. lA, lane 1). With longer chase times (30-180 min), the p62 and El proteins were transported out of ER, as visualized by the postGolgi cleavage of p62 to E2 and E3 (3) and by the slight increase in apparent molecular weight of El due to its sialylation in the trans-Golgi compartment (8) (Fig. 1A, lanes 2-4). During this time a substantial portion of the synthesized p62 and El proteins were incorporated into budding virions (Fig. 1A, lanes 5-8). However, the 6K protein was not readily detectable in released virus particles. Since the structural proteins are synthesized as a common precursor protein, there should be equimolar amounts of ~62, El, and 6K proteins in the cell lysates. Quantitations of the gels showed that the ratio of p62/ El heterodimers to 6K immediately after synthesis (10 min chase) was 1.32, i.e., 76% of the 6K proteins synthesized were detected (Table 1). After prolonged chase periods (30, 90, and 180 min) the ratio of p62/El to 6K was gradually reduced, although the amount of 6K detected in the total cell lysates remained the same (Table 1). This can be explained by the fact that while 6K remained inside the cell, a substantial portion of the
be addressed. 843
0042-6822191 Copyright All rights
$3.00
0 1991 by Academic Press. Inc. of reproduction ID any form reserved.
SHORT COMMUNICATIONS
844
A
1234
B
5678
12
3
4
567
8
107kDap62 3
E3 -
C
12
p62 -
D
~62
6K
FIG. 1. (A) Proteins from SFV-infected BHK-cell lysates and growth medium run on a 1 O-20% gradient polyacrylamide gel. Cells were labeled with [36S]methionine for 10 min and then chased for 10, 30, 90, or 180 min, after which cells were iysed in 1% NP40-buffer. Before lysis, the growth medium was collected, cleared by low speed centrifugation to remove residual cells, and remaining virus particles in the medium were collected by ultracentrifugation. Lanes l-4, lysates after 10, 30, 90, and 180 min chase period; lanes 5-8, collected growth media of corresponding lysates. The positions of the ~62, E2, El, C, and 6K proteins are indicated. (B) lmmunoprecipitations of SFV proteins from the lysates shown in (A), using anti-El and anti-E2 monoclonal antibodies (7). Lanes l-4, immunoprecipitations with anti-El after 10, 30, 90, and 180 min chase period; lanes 5-8, immunoprecipitations with anti-E2 after 10, 30, 90, and 180 min chase period. (C) Analysis of surface-labeled SFV membrane proteins. W/-infected BHK-cells were labeled with [YSlmethionine for 2 hr before biotinylation. Lane 1, lysate of the biotinylated cells. Lane 2, streptavidine precipitation of corresponding lysate. Note that four times more material was loaded onto lane 2, to compensate for the fact that only a portion of the membrane proteins are present on the cell surface. (D) Association of 6K with ~62. Cells were transfected with a El deletion mutant and 12 hr after transfection pulse-labeled for 15 min and chased for 15 min, after which cells were lysed in 1% NP40-buffer, Lane 1, total cell lysate, lane 2, immunoprecipitation with anti-E2. Note that in (A) and (B) the lower part of the gel covering the area of E3 and 6K has been exposed for a longer time period to better show these small and less labeled protein species.
E2/El heterodimers was chased out into new virus particles. Indeed, when the amounts of p62(E2)/El heterodimers present in the virus particles were added to the corresponding amounts in the cell lysates, the spike/6K ratio remained rather constant during the
whole chase period (Table 1). In a previous report an underacylated form of 6K, termed 4K, was found in infected CEF cells (5). Despite extensive effort, we have not been able to detect a 4K protein species in our assays, so the situation might be somewhat differ-
SHORT COMMUNICATIONS TABLE 1 QUANTITATION OF [35S]-M~~~~~~~~-~~~~~~ SFV MEMBRANE PROTEINS FROM INFECTED AND PULSE-LABELED BHK CELLSB
Chase timeb 10 30 90 180
min min min min
p62(E2) + El16Kd
a-El*
c~-E2*
6K (cpm)
Lys
Lys + Sup
E1/6K
p62(E2)/6K
5526 5285 6254 5643
1.32 1.21 0.83 0.54
1.32 1.36 1.05 1.32
2.50 2.14 1.71 1.08
2.49 2.08 0.91 0.94
’ Radioactivity was determined by excising bands from dried SDS gels. The gel slices were solubilized in Protosol (DuPont) according to the instructions of the manufacturer before scintillation counting. b Ceils were pulsed for 10 min followed by various chase times as indicated. ‘Total amounts of 6K label detected. d Molar ratios between the SFV glycoproteins and 6K. Values are calculated by equalizing methionine content of the p62 (E2) + El proteins with the 6K protein. ’ Molar ratios between the SFV glycoproteins and 6K after immunoprecipitations with either El or E2 monoclonal antibodies.
ent in BHK cells. Since our detection level (76%) for 6K was the same as that of 6K+4K reported earlier (5) we feel confident that 6K species analyzed in this work represents the bulk portion relevant for virus assembly. These initial tests indicated that no significant amounts of the 6K protein was incorporated into new virions. By extensive overexposure of the gels some 6K protein could be seen (data not shown). Subsequent quantitation showed that 6K was present in virus particles to only 3% of the amount of the spike proteins p62 and El. The result confirms earlier obtained data which showed that Sindbis virus released from CEF cells incorporates up to 10% of 6K protein into new virions (5). This raised the question whether the bulk of 6K is transported out of ER at all and, if so, whether or not it is associated with the other two membrane proteins p62 and El. To test this we employed monoclonal antibodies, which are known to coimmunoprecipitate the p62 (E2) and El proteins as heterodimers (18). Indeed, already after a 10 min chase 6K coprecipitated with the heterodimer, indicating that it became associated with the spike protein complex soon after synthesis (Fig. 1 B, lanes 1 and 5). The same result was obtained after longer chase times (30-l 80 min), when the p62 and El proteins had been transported out of ER and through the trans-Golgi compartment and p62 cleaved to E2 and E3 (Fig. 1 B, lanes 2-4 and 6-8). This suggested that 6K was also transported out from the ER. The analysis was also performed using infected CEF cells, with exactly the same result (data not shown). We find it most likely that the association of 6K with E2
845
and/or El is specific, since monoclonal antibodies against either E2 or El coprecipitated the 6K protein (Fig. 1 B), whereas an anti-capsid monoclonal antibody did not (data not shown). To measure the actual amounts of 6K associated with the complex, the coimmunoprecipitation gel analysis was quantitated (Table 1). After a short chase, the ratio of p62/El to 6K was rather high (2.50) indicating that only a portion of 6K was coprecipitating. However, by longer chase times, during which the p62 and El proteins were transported out of ER, the ratio approached one. We explain the initial high spike to 6K ratio to be due to instability of the complex soon after synthesis, and that this complex becomes more resistant to antibody precipitation as it matures. Most importantly, since the ratio of E2/El to 6K after a long chase period was one, we conclude, that every spike protein heterodimer is in complex with 6K at least as far as a post-Golgi compartment, where p62 is cleaved to E2 and E3. This being the case, it was highly probable that 6K is transported all the way to the cell surface. To study this, we performed cell surface labeling with biotin (2). The biotin reagent NHS-LC-biotin is membrane impermeable and reacts with primary amines. Since none of the luminal amino acid residues of 6K has such an amino group, 6K cannot be biotinylated at the cell surface. However, if 6K is in complex with the E2/El heterodimer, it might coprecipitate when these biotinylated cell surface proteins are precipitated with streptavidine agarose. SFV-infected BHK-cells were pulselabeled for 2 hr and the cell surface proteins biotinylated at 0°C. Subsequent precipitation by streptavidine showed that both 6K and the spike proteins were efficiently precipitated with streptavidine agarose, whereas no C protein was precipitated (Fig. 1C). The ratio pf p62 to E2/El differed significantly in the lysate and in the biotinylated sample. Most importantly, the ratio of 6K to spike proteins appeared to be the same as in immunoprecipitates from total cell lysates, indicating that 6K indeed had been transported all the way to the cell surface. To analyze the association of 6K to the E2/El heterodimeric complex in more detail, we utilized an El deletion derivative of the SFV cDNA clone (IO), kindly provided by Antti Salminen in our laboratory. The construct carries two amber stop codons immediately after the 6K coding unit and therefore does not produce any El protein. In vitro-made RNA was transfected into BHK cells (70) and the cells were pulse-labeled for 15 min and chased for 15 min. Immunoprecipitation with the monoclonal antibody directed against E2 showed that 6K still coprecipitated with p62 (Fig. 1 D), and subsequent quantitation revealed that the ra-
846
SHORT
COMMUNICATIONS
tio of p62 to 6K was 2.26, i.e., quite similar to the wild type situation soon after synthesis (see Table 1). We conclude that 6K binds at least to ~62, which of course does not exclude the possibility that it also might bind to El. In conclusion, it seems that all three transmembrane proteins ~62, 6K, and El immediately after synthesis form a stable complex in ER and remain so during their transport to the cell surface. Since 6K is not incorporated into virus particles to any great extent, it must dissociate from the complex before or during the budding process. The fact that removal of 6K still allows production of virus particles (albeit at low frequency) with normal infectivity (10) suggests that 6K plays no major role as part of the virus particle. It seems, therefore, that 6K modulates virus assembly and/or release, exerting its main function intracellularly during the maturation of the virion. ACKNOWLEDGMENTS We acknowledge Antti Salminen for providing the El deletion derivative of the SFV cDNA clone and W. A. M. Boere forthe generous gift of antibodies. This work was supported by The Swedish Medical Research Council (Grant B88-12X-0872.Ol A), The Swedish National Board for Technical Development (Grant 87-0275OP), and The Swedish Natural Science Research Council (Grant B-BU 9353-301).
REFERENCES 1. BOERE, W. A. M., HARMSEN, T., VINJE, J.. BENAISSA-TROUW, B. J., KRAAIJEEVELD, C. A., and SNIPPE, H., J. Viral. 52, 575-582 (1984).
2. BFI&NDLI, A., PARTON, R. G., and SIMONS, K., J. Cell Biol. 111, 2909-292 1 (1990). 3. DE CURTIS, I., and SIMONS, K., Proc. Nat/. Acad. Sci. USA 85, 8052-8056 (1988). 4. GAEDIGK-NITSCHKO, K., DING, M., LEVY, M. A., and SCHLESINGER, M., Virology 175, 282-291 (1990). K., and SCHLESINGER, M., virology 175,2745. GAEDIGK-NITSCHKO, 281 (1990). 6. GAEDIGK-NITSCHKO, K., and SCHLESINGER, M. J., Virology 183, 206-214 (1991). 7. GAROFF, H., HUYLEBROECK, D., ROBINSON, A., TILLMAN, U., and LILIESTR~M, P., J. Cell Biol. 111, 867-876 (1990). 8. GREEN, J., GRIFFITHS, G., LOUVARD, D., QUINN, P., and WARREN, G., 1. Mol. Biol. 152, 663-698 (1981). 9. LIUESTRBM, P., and GAROFF. H., J. Viral. 65, 147-154 (1991). 10. LIUESTR~M, P., LUSA, S., HUYLEBROECK, D.. and GAROFF, H., /. Viral. 65, 4107-41 13 (1991). (1990). 11. LOEIGS, M., and GAROFF, H., /. viral. 64, 1233-1240 12. LOBIGS, M., WAHLBERG, J. M., and GAROFF, H., J. Viral. 64,52145218 (1990). 13. LOBIGS, M., ZHAO. H., and GAROFF, H., J. Viral. 64, 4346-4355 (1990). P., and GAROFF, H., J. Wol. 61, 1301-1309 (1987). 14. MELANCON, 15. SALMINEN, A., WAHLBERG. J. M., LOBIGS, M., LILJESTR~M, P., and GAROFF, H.. /. Cell Biol., in press (1991). 16. SCHLESINGER, S. S., and SCHLESINGER, M. J., IN “THE TOGAVIRIDAE AND FLAVIVIRIDAE” (S. S. SCHLESINGER AND M. J. SCHLESINGER, EDS.), PP. 121-l 48. PLENUM PRESS, NEW YORK, 1986. 17. WAHLBERG, J., and GAROFF. H., J. Cell Biol., in press (1991). 18. WAHLBERG, J. M., BOERE, W. A., and GAROFF, H., J. Viral. 63, 4991-4997 (1989). 19. ZIEMIECKI, A., GAROFF, H., and SIMONS. K., J. Gem Viral. 50, 1 1 l123 (1980).
185, 843-846
(199 1)
Fate of the 6K Membrane
Protein
of Semliki
Forest
Virus during Virus Assembly
SARI LUSA, HENRIK GAROFF, AND PETER LILIESTR~~M’ Department
of Molecular Received
Biology,
Karolinska
Institute,
July 8, 199 1; accepted
S- 14 1 57 Huddinge,
September
Sweden
4, 199 1
The Semliki Forest virus directs the synthesis of three virus-specific transmembrane proteins p62,6K, all are made in equimolar amounts from a polyprotein precursor molecule. The p62 and El spike proteins dimeric complexes in the endoplasmic reticulum before being transported to the cell surface where occurs. In this study we show that the 6K protein becomes associated to the p62El complex in the reticulum and transported with the complex to the cell surface. During virus budding, El and p62 (which into the E2 protein) are incorporated into new virions whereas the 6K is mostly excluded. Virus particles infected BHK cells contain only about 3% of 6K in their membrane as compared to the spike protein relevance of these findings for the mechanism of SFV assembly is discussed. o 1991 Academic PWSS. I~C.
The budding of Semliki Forest virus (SFV), an enveloped positive-strand RNA virus, is dependent on the interactions between cytoplasmically formed nucleocapsids and heterodimeric spike complexes at the plasma membrane (PM). All the structural proteins of SFV are synthesized as a polyprotein precursor in the order C-p62-6K-El (76) from which each individual protein is cotranslationally released by a proteolytic processing event. The capsid protein (C) is formed by an autoproteolytic cleavage (74), while the other three proteins are formed by signal peptidase cleavages during their translocation and insertion into the endoplasmic reticulum (ER) membrane (7, 9). In ER, the p62 and El proteins form heterodimers (78, 79) which subsequently are transported to PM. The p62 precursor protein is cleaved to E2 and E3 at a very late stage during transport from the transGolgi compartment to the PM (3). The p62 and El proteins are known to have important functions in virus assembly and disassembly (7 I73, 75, 77, 78), but the role of the 6K protein in virus replication remains unsolved. During synthesis of the viral membrane proteins at ER the C-terminal portion of 6K functions as a signal peptide for El. However, we have shown that removal of 6K from the genome allows normal synthesis and intracellular transport of the p62El heterodimeric complex to the cell surface, but does reduce virus release to about 2% of wild-type frequency (9, 70). Interestingly, the produced virus had similar infectivity as compared to wild-type virus (70). A portion of the SFV and of the closely related Sindbis virus 6K protein becomes palmitylated in infected ’ To whom
reprint
requests
should
and El, which form heterovirus budding endoplasmic has matured released from content. The
chicken embryo fibroblast (CEF) cells, and substitution mutations which cause underacylation of the 6K protein lead to a reduction in virus release and to the formation of multicored virions at the PM (4, 6). Only small amounts (up to 10%) of 6K are found in mature virions released from infected CEF cells (5). Together these results suggest that 6K plays an important role in virus maturation. To follow the fate of 6K after synthesis BHK cells infected with wild-type SFV were pulse-labeled for 10 min and chased for various times. After a short 10 min chase, all three membrane proteins ~62, El, and 6K were readily detected (Fig. lA, lane 1). With longer chase times (30-180 min), the p62 and El proteins were transported out of ER, as visualized by the postGolgi cleavage of p62 to E2 and E3 (3) and by the slight increase in apparent molecular weight of El due to its sialylation in the trans-Golgi compartment (8) (Fig. 1A, lanes 2-4). During this time a substantial portion of the synthesized p62 and El proteins were incorporated into budding virions (Fig. 1A, lanes 5-8). However, the 6K protein was not readily detectable in released virus particles. Since the structural proteins are synthesized as a common precursor protein, there should be equimolar amounts of ~62, El, and 6K proteins in the cell lysates. Quantitations of the gels showed that the ratio of p62/ El heterodimers to 6K immediately after synthesis (10 min chase) was 1.32, i.e., 76% of the 6K proteins synthesized were detected (Table 1). After prolonged chase periods (30, 90, and 180 min) the ratio of p62/El to 6K was gradually reduced, although the amount of 6K detected in the total cell lysates remained the same (Table 1). This can be explained by the fact that while 6K remained inside the cell, a substantial portion of the
be addressed. 843
0042-6822191 Copyright All rights
$3.00
0 1991 by Academic Press. Inc. of reproduction ID any form reserved.
SHORT COMMUNICATIONS
844
A
1234
B
5678
12
3
4
567
8
107kDap62 3
E3 -
C
12
p62 -
D
~62
6K
FIG. 1. (A) Proteins from SFV-infected BHK-cell lysates and growth medium run on a 1 O-20% gradient polyacrylamide gel. Cells were labeled with [36S]methionine for 10 min and then chased for 10, 30, 90, or 180 min, after which cells were iysed in 1% NP40-buffer. Before lysis, the growth medium was collected, cleared by low speed centrifugation to remove residual cells, and remaining virus particles in the medium were collected by ultracentrifugation. Lanes l-4, lysates after 10, 30, 90, and 180 min chase period; lanes 5-8, collected growth media of corresponding lysates. The positions of the ~62, E2, El, C, and 6K proteins are indicated. (B) lmmunoprecipitations of SFV proteins from the lysates shown in (A), using anti-El and anti-E2 monoclonal antibodies (7). Lanes l-4, immunoprecipitations with anti-El after 10, 30, 90, and 180 min chase period; lanes 5-8, immunoprecipitations with anti-E2 after 10, 30, 90, and 180 min chase period. (C) Analysis of surface-labeled SFV membrane proteins. W/-infected BHK-cells were labeled with [YSlmethionine for 2 hr before biotinylation. Lane 1, lysate of the biotinylated cells. Lane 2, streptavidine precipitation of corresponding lysate. Note that four times more material was loaded onto lane 2, to compensate for the fact that only a portion of the membrane proteins are present on the cell surface. (D) Association of 6K with ~62. Cells were transfected with a El deletion mutant and 12 hr after transfection pulse-labeled for 15 min and chased for 15 min, after which cells were lysed in 1% NP40-buffer, Lane 1, total cell lysate, lane 2, immunoprecipitation with anti-E2. Note that in (A) and (B) the lower part of the gel covering the area of E3 and 6K has been exposed for a longer time period to better show these small and less labeled protein species.
E2/El heterodimers was chased out into new virus particles. Indeed, when the amounts of p62(E2)/El heterodimers present in the virus particles were added to the corresponding amounts in the cell lysates, the spike/6K ratio remained rather constant during the
whole chase period (Table 1). In a previous report an underacylated form of 6K, termed 4K, was found in infected CEF cells (5). Despite extensive effort, we have not been able to detect a 4K protein species in our assays, so the situation might be somewhat differ-
SHORT COMMUNICATIONS TABLE 1 QUANTITATION OF [35S]-M~~~~~~~~-~~~~~~ SFV MEMBRANE PROTEINS FROM INFECTED AND PULSE-LABELED BHK CELLSB
Chase timeb 10 30 90 180
min min min min
p62(E2) + El16Kd
a-El*
c~-E2*
6K (cpm)
Lys
Lys + Sup
E1/6K
p62(E2)/6K
5526 5285 6254 5643
1.32 1.21 0.83 0.54
1.32 1.36 1.05 1.32
2.50 2.14 1.71 1.08
2.49 2.08 0.91 0.94
’ Radioactivity was determined by excising bands from dried SDS gels. The gel slices were solubilized in Protosol (DuPont) according to the instructions of the manufacturer before scintillation counting. b Ceils were pulsed for 10 min followed by various chase times as indicated. ‘Total amounts of 6K label detected. d Molar ratios between the SFV glycoproteins and 6K. Values are calculated by equalizing methionine content of the p62 (E2) + El proteins with the 6K protein. ’ Molar ratios between the SFV glycoproteins and 6K after immunoprecipitations with either El or E2 monoclonal antibodies.
ent in BHK cells. Since our detection level (76%) for 6K was the same as that of 6K+4K reported earlier (5) we feel confident that 6K species analyzed in this work represents the bulk portion relevant for virus assembly. These initial tests indicated that no significant amounts of the 6K protein was incorporated into new virions. By extensive overexposure of the gels some 6K protein could be seen (data not shown). Subsequent quantitation showed that 6K was present in virus particles to only 3% of the amount of the spike proteins p62 and El. The result confirms earlier obtained data which showed that Sindbis virus released from CEF cells incorporates up to 10% of 6K protein into new virions (5). This raised the question whether the bulk of 6K is transported out of ER at all and, if so, whether or not it is associated with the other two membrane proteins p62 and El. To test this we employed monoclonal antibodies, which are known to coimmunoprecipitate the p62 (E2) and El proteins as heterodimers (18). Indeed, already after a 10 min chase 6K coprecipitated with the heterodimer, indicating that it became associated with the spike protein complex soon after synthesis (Fig. 1 B, lanes 1 and 5). The same result was obtained after longer chase times (30-l 80 min), when the p62 and El proteins had been transported out of ER and through the trans-Golgi compartment and p62 cleaved to E2 and E3 (Fig. 1 B, lanes 2-4 and 6-8). This suggested that 6K was also transported out from the ER. The analysis was also performed using infected CEF cells, with exactly the same result (data not shown). We find it most likely that the association of 6K with E2
845
and/or El is specific, since monoclonal antibodies against either E2 or El coprecipitated the 6K protein (Fig. 1 B), whereas an anti-capsid monoclonal antibody did not (data not shown). To measure the actual amounts of 6K associated with the complex, the coimmunoprecipitation gel analysis was quantitated (Table 1). After a short chase, the ratio of p62/El to 6K was rather high (2.50) indicating that only a portion of 6K was coprecipitating. However, by longer chase times, during which the p62 and El proteins were transported out of ER, the ratio approached one. We explain the initial high spike to 6K ratio to be due to instability of the complex soon after synthesis, and that this complex becomes more resistant to antibody precipitation as it matures. Most importantly, since the ratio of E2/El to 6K after a long chase period was one, we conclude, that every spike protein heterodimer is in complex with 6K at least as far as a post-Golgi compartment, where p62 is cleaved to E2 and E3. This being the case, it was highly probable that 6K is transported all the way to the cell surface. To study this, we performed cell surface labeling with biotin (2). The biotin reagent NHS-LC-biotin is membrane impermeable and reacts with primary amines. Since none of the luminal amino acid residues of 6K has such an amino group, 6K cannot be biotinylated at the cell surface. However, if 6K is in complex with the E2/El heterodimer, it might coprecipitate when these biotinylated cell surface proteins are precipitated with streptavidine agarose. SFV-infected BHK-cells were pulselabeled for 2 hr and the cell surface proteins biotinylated at 0°C. Subsequent precipitation by streptavidine showed that both 6K and the spike proteins were efficiently precipitated with streptavidine agarose, whereas no C protein was precipitated (Fig. 1C). The ratio pf p62 to E2/El differed significantly in the lysate and in the biotinylated sample. Most importantly, the ratio of 6K to spike proteins appeared to be the same as in immunoprecipitates from total cell lysates, indicating that 6K indeed had been transported all the way to the cell surface. To analyze the association of 6K to the E2/El heterodimeric complex in more detail, we utilized an El deletion derivative of the SFV cDNA clone (IO), kindly provided by Antti Salminen in our laboratory. The construct carries two amber stop codons immediately after the 6K coding unit and therefore does not produce any El protein. In vitro-made RNA was transfected into BHK cells (70) and the cells were pulse-labeled for 15 min and chased for 15 min. Immunoprecipitation with the monoclonal antibody directed against E2 showed that 6K still coprecipitated with p62 (Fig. 1 D), and subsequent quantitation revealed that the ra-
846
SHORT
COMMUNICATIONS
tio of p62 to 6K was 2.26, i.e., quite similar to the wild type situation soon after synthesis (see Table 1). We conclude that 6K binds at least to ~62, which of course does not exclude the possibility that it also might bind to El. In conclusion, it seems that all three transmembrane proteins ~62, 6K, and El immediately after synthesis form a stable complex in ER and remain so during their transport to the cell surface. Since 6K is not incorporated into virus particles to any great extent, it must dissociate from the complex before or during the budding process. The fact that removal of 6K still allows production of virus particles (albeit at low frequency) with normal infectivity (10) suggests that 6K plays no major role as part of the virus particle. It seems, therefore, that 6K modulates virus assembly and/or release, exerting its main function intracellularly during the maturation of the virion. ACKNOWLEDGMENTS We acknowledge Antti Salminen for providing the El deletion derivative of the SFV cDNA clone and W. A. M. Boere forthe generous gift of antibodies. This work was supported by The Swedish Medical Research Council (Grant B88-12X-0872.Ol A), The Swedish National Board for Technical Development (Grant 87-0275OP), and The Swedish Natural Science Research Council (Grant B-BU 9353-301).
REFERENCES 1. BOERE, W. A. M., HARMSEN, T., VINJE, J.. BENAISSA-TROUW, B. J., KRAAIJEEVELD, C. A., and SNIPPE, H., J. Viral. 52, 575-582 (1984).
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