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The Extracellular Domain of La Crosse Virus G1 Forms Oligomers and Undergoes pH-Dependent Conformational Changes
VIROLOGY
225, 243–247 (1996) 0596
ARTICLE NO.
SHORT COMMUNICATION The Extracellular Domain of La Crosse Virus G1 Forms Oligomers and Undergoes pH-Dependent Conformational Changes ANDREW PEKOSZ*,†,‡ and FRANCISCO GONZA´LEZ-SCARANO†,‡,1 *Cell and Molecular Biology Graduate Group, and Departments of †Neurology and ‡Microbiology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania Received July 3, 1996; accepted August 27, 1996 The La Crosse virus G1 glycoprotein plays a critical role in virus binding to susceptible cells and in the subsequent fusion of viral and cellular membranes. A soluble form of the G1 glycoprotein (sG1) prepared in a recombinant baculovirus system mimics the cell-binding pattern of La Crosse virus and inhibits La Crosse virus infection (A. Pekosz et al., Virology 214, 339–348, 1995), presumably by competing for a cellular receptor, a finding that implies that sG1 can perform some functions absent G2, the smaller of the two bunyavirus glycoproteins. We have performed experiments to determine whether sG1 is present as an oligomer and whether it undergoes the conformational changes associated with fusion (F. Gonzalez-Scarano, Virology 140, 209–216, 1985). Our results indicate that both sG1 and native G1 undergo similar changes in conformation after exposure to an acidic environment, as detected by reactivity with monoclonal antibodies. Furthermore, using chemical cross-linking, both proteins were detected as oligomers (most likely dimers). Sucrose density gradient analysis of sG1 verified that it was present in monomeric and oligomeric forms. These results demonstrate that the isolated G1 glycoprotein can undergo a pH-dependent change in conformation in the absence of its transmembrane and cytoplasmic tail domains and that the extracellular portion of the glycoprotein can oligomerize. q 1996 Academic Press, Inc.
La Crosse virus is a member of the California encephalitis serogroup of bunyaviruses, a group of viruses associated with pediatric encephalitis in areas of North America and Europe where the mosquitoes that vector the virus are endemic (1). La Crosse virus binding, pHdependent fusion and entry have been studied in some detail (2–4) and evidence indicates that viral entry is dependent on the acidic pH environment found in endosomes (5). Like all Bunyaviridae, La Crosse virus has two glycoproteins termed G1 and G2. The larger, G1, plays an important role in entry, first as the virus attachment protein, and then by undergoing a pH-dependent conformational change in concert with the activation of virus– cell or cell-to-cell fusion (6). Using La Crosse virus glycoproteins expressed in recombinant vaccinia, it has been determined that in order for cell-to-cell fusion to take place both of the glycoproteins must be present, presumably because G2 contains the critical fusion sequences (3, 7). We have previously described the construction and expression of a recombinant La Crosse virus G1 protein in a baculovirus expression system (8). Soluble G1 (sG1) consists of the entire extracellular domain of La Crosse virus G1 (amino acids 474–1387 of the La Crosse virus
M segment open reading frame) and lacks the transmembrane and cytoplasmic domains of the native G1. The sG1 glycoprotein (110 kDa) is similar to the native G1 (125 kDa) because (i) it reacts with conformation sensitive anti-G1 monoclonal antibodies (MAb), (ii) it elicits a protective humoral response in mice, (iii) it binds to cells with specificity similar to that of La Crosse virions, and (iv) it inhibits infection of cultured cells by La Crosse and other California serogroup bunyaviruses (4, 8). This last finding—which was the most surprising—implies that not only is sG1 conformationally accurate, but that it can interfere with the normal functions of the G1 protein during the entry process. We undertook these studies to determine whether or not the native G1 protein forms oligomers, and if so, whether sG1 would assume a similar quaternary structure. Given the results indicating that both G1 and G2 must be expressed in concert before cell-to-cell fusion will take place, we also wanted to ascertain if an association between these two glycoproteins is important in inducing the conformational changes in G1 that are associated with virus entry and virus–cell fusion. Previous studies (2, 6, 9) have shown that the binding of specific anti-G1 MAbs is markedly reduced after exposure of intact virions to acid (pH 5.8 or lower). These data together with changes in the pattern of digestion obtained with trypsin were taken as evidence of a confor-
1 To whom correspondence and reprint requests should be addressed. Fax: (215) 573-2029. E-mail: [email protected].
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mational change in the protein. To determine whether the isolated sG1 undergoes a similar conformational alteration, an aliquot of the 35S-labeled protein was acidified to pH 5.6 for 30–60 sec, neutralized with 50 ml of 1 M Tris–HCl, and then immunoprecipitated with either individual anti-G1 MAbs (3, 4, 8) or with polyclonal mouse sera raised against La Crosse virus. As demonstrated in Fig. 1A, either the anti-La Crosse sera or MAb 807-15 recognized both the native and the acidified forms of sG1. In contrast, the signals obtained with either MAb 807-22 or MAb 813-48 were drastically reduced or absent, consistent with the known sensitivity of these epitopes to acidification. A comparable pattern was seen when 35S-labeled La Crosse virions which had been partially purified by ultracentrifugation over a 20% sucrose cushion were used in the assay (Fig. 1B). These results
indicated that the pH-dependent structural changes which take place in G1 during La Crosse virus acidification also occur in sG1, and thus do not require the presence of any other viral structural proteins, viral or cellular membranes, or the transmembrane and cytoplasmic domains of G1. Another important property of many viral glycoproteins is their ability to form either homo- or hetero-oligomers (10). To determine the oligomeric state of sG1, we exposed it to chemical crosslinking reagents and the crosslinked proteins were then immunoprecipitated with MAb 807-15. Using various concentrations of the chemical crosslinker bis(sulfosuccinimidyl) suberate (BS3 , spacer arm length of 11.4 Angstroms), several high molecular weight bands were detected (Fig. 2A). First, a doublet signal is at a position approximately corresponding to that expected of sG1 homodimers (labeled as 21 sG1). There was another band near the top of the gel but, due to its position outside the regions of linear resolution, a molecular weight could not be assigned. Labeled as 41 sG1 in Fig. 2A, this high molecular weight band could be a trimer instead of a tetramer. When the samples were acidified, neutralized, and subsequently crosslinked, we saw a similar pattern, indicating that these higher order oligomers are not dissociated by the pH-dependent conformational changes that take place in sG1. As expected, in both cases a corresponding decrease in the amount of sG1 was seen with increasing concentrations of the crosslinker (Fig. 2A). Native G1 (35S-labeled La Crosse virions) gave a similar pattern (Fig. 2B): a doublet at the approximate molecular weight of dimeric G1 (21 G1) and a much higher molecular weight species (41 G1). There was no change in the cross-linking pattern when the virions were briefly acidified. A second cross linking agent, ethylene glycolbis(sulfosuccinimidylsuccinate) (EGS, spacer arm length of 16.1 Angstroms), was also used in this assay, with comparable results (data not shown) and varying the time of crosslinking (30–120 min) or incubation temperature (47 or room temperature) did not produce more efficient crosslinking. The appearance of a doublet when either sG1 or native G1 was crosslinked could be due to the inability of some portion of the crosslinked protein to completely denature before SDS–PAGE analysis, thus giving it a slightly aberrant molecular weight. In the experiments described above, none of the bands corresponded to any of a number of potential crosslinked G1/G2 heterodimers. Experiments where the crosslinked La Crosse proteins were immunoprecipitated with anti-G2 MAbs were also negative for such a combination (data not shown). Nevertheless, G1 and G2 coprecipitate when MAbs against either glycoproteins are used under conditions that eliminate background precipitation of the other two structural proteins N and L (Fig. 3). This result is evidence that the two glycoproteins have a strong noncovalent interaction, particularly considering the
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FIG. 1. Acid treatment alters MAb binding to sG1 and native G1. 35Slabeled (A) sG1 or (B) native G1 (La Crosse virions) were either untreated or acidified by dropwise addition of H2SO4 to a pH of 5.6 for 30–60 sec and then neutralized with 50 ml of 1 M Tris–HCl, pH 7.4, or 1 M phosphate buffer, pH 7.1. The samples were immunoprecipitated with 15 ml of each of the indicated antibodies and protein G agarose as described (3), washed with 0.05 M Tris–HCl, pH 7.5, 0.15 M NaCl, 0.1% SDS, 1% Triton X-100, and 1% deoxycholate, followed by 0.01 M Tris–HCl, pH 8.0, 0.4 M LiCl, and 2 M urea, and then resuspended in sample buffer and heated at 377 for 10 min before resolution in SDS– 10% PAGE (8). MAbs 807-22 and 813-48 showed reduced or absent signal when immunoprecipitating either the native or truncated, acidified protein, indicating loss of these epitopes. MAb 807-15 and polyclonal mouse sera served as controls, since they immunoprecipitated both the native and the truncated forms of the protein equally well, regardless of acidification. Abbreviations: anti-LAC, polyclonal anti-La Crosse virus mouse sera; NMS, normal mouse sera; DpH, samples that have been acidified and then neutralized.
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FIG. 2. Cross-linking of 35S-labeled sG1 and native G1. The nonreducible chemical crosslinker BS3 was added to either untreated or acidified and neutralized 100-ml samples of (A) sG1 or (B) native G1 (La Crosse virions) at the indicated concentrations, and the samples were then incubated for 2 hr at 47. After quenching with 50 ml of 1 M Tris–HCl, pH 7.4, they were immunoprecipitated with MAb 807-15 and analyzed on SDS–5% PAGE. The positions of monomeric sG1 and La Crosse virus associated G1 as well as the possible dimeric (21, which appears as a doublet) and trimeric/ tetrameric (41) species are indicated. The samples that were acidified before crosslinking showed no change in the profile, indicating the oligomeric state is not effected by acidification.
stringency of the immunoprecipitation (see Fig. 1 legend). In support of this interpretation, analysis of the intracellular localization of La Crosse virus glycoproteins with re-
FIG. 3. La Crosse virus G1 and G2 glycoproteins are associated noncovalently. Sucrose density gradient purified La Crosse virions were loaded directly on to an SDS–12.5% PAGE (lane, 0) or immunoprecipitated with a mixture of the anti-G1 MAbs 807-31, 807-33, and 80725 (lane, aG1) or the anti-G2 MAb 9B7.5 (lane, aG2) before loading. Immunoprecipitation for either glycoprotein coimmunoprecipitated the other, while no L or N proteins were detectable, implying an association between G1 and G2. Immunoprecipitation with mouse sera raised to a thymidine kinase negative vaccinia virus (arVVtk-) and with sera from mice which had survived a La Crosse virus challenge (8) are shown as negative and positive immunoprecipitation controls, respectively.
combinant vaccinia viruses has demonstrated that G1 colocalizes with Golgi markers only when it is expressed in concert with G2, indicating at least a transient association responsible for transport (7). However, this interaction may simply not be detectable with chemical crosslinking. Conceivably, this could be due to the absence of lysines in the appropriate domains, since free amino groups are the substrates for these reagents (11). Alternatively, the G1–G2 interaction domain may involve the transmembrane and/or cytoplasmic tails of the proteins, two regions that cannot be crosslinked with BS3 . To further analyze the oligomeric state of sG1, we performed sucrose density gradient ultracentrifugation analysis on the 35S-labeled sG1. Samples of sG1 that were either untreated (Fig. 4A) or acidified and neutralized (Fig. 4B) were layered over 5–20% linear sucrose gradients and ultracentrifuged at 40,000 RPM for 18 hr in an SW41 rotor. The gradients were then fractionated from the bottom, and every other fraction was immunoprecipitated and analyzed on SDS–PAGE. sG1 sedimented in two peaks, one in fractions 9–11, and a second in fractions 19–23 (from a total of 34 fractions collected) (Fig. 4A). Acidification of sG1 prior to sucrose gradient analysis yielded a very similar profile (Fig. 4B). To control for the efficiency of the gradient, a parallel experiment was performed on a sample of rat brain extract, which was fractionated and analyzed for acetylcholinesterase (AChE) activity as described by Llanes et al. (12). The peak activities of the tetrameric (41 AChE, approximate molecular weight 272 kDa) and the dimeric (21 AChE, approximate
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FIG. 4. Sucrose density sedimentation of 35S-labeled sG1 shows the presence of higher order oligomers. Linear sucrose density gradients (5– 20% in phosphate-buffered saline with 0.1% Triton X-100, total volume of 13 ml) were prepared on a Biocomp Radian Gradient Maker. Approximately 500 ml of 35S-labeled sG1 with 0.1% Triton X-100 which were either (A) untreated or (B) acidified and neutralized were layered on top of the gradient and the samples were spun in an SW41 rotor at 40,000 RPM for 18 hr at 157. The samples were then fractionated in 34 aliquots beginning at the bottom of the gradient. Every other fraction was immunoprecipitated with 15 ml of mouse polyclonal antisera and analyzed on 10% SDS–PAGE. Both untreated and acidified sG1 showed a similar bimodal peak, indicating the presence of monomeric and oligomeric (most likely dimeric) forms of sG1. A parallel gradient containing rat brain extract was analyzed for acetylcholinesterase (AChE, monomer Å 68 kDa) activity as described (12) and the peak positions of the tetrameric (41) and dimeric (21) forms of the enzyme are indicated. A similar pattern of sG1 sedimentation is seen when Triton X-100 is left out of the sucrose gradient (data not shown).
molecular weight 136 kDa) forms of the enzyme are indicated as a rough gauge of molecular weight (12). The data indicate that sG1 is present in monomeric and dimeric (and possibly higher order) forms. Although a comparison experiment with native G1 would have been informative, we were unable to separate the glycoproteins from the virus ribonucleoprotein complexes with either Triton X-100 (0.5–1.0%) or Nonidet P-40 (0.1–0.5%). (Note the stringent washes used for the immunoprecipitations in Figs. 1 and 3.) Our data indicate that a deletion mutant of the La Crosse virus G1 glycoprotein lacking its transmembrane and cytoplasmic domains can undergo pH-dependent conformational changes that appear to be identical to those of the native G1. This observation proves that the disappearance of MAb epitopes following acid treatment is the result of structural changes in the G1 extracellular domain, and not just due to masking of a G1 epitope by structural changes in the G2 glycoprotein, an interpretation which could not be ruled out in studies using La Crosse virions (6). Furthermore, the loss of MAb epitopes after acidification is not a result of simple dissociation of a G1 oligomer, because pH changes seem to have little or no effect on the amount of oligomeric sG1 present. Since the sG1 glycoprotein can oligomerize, the transmembrane and cytoplasmic tails of G1 probably do not play a major role in this function, although they may help stabilize the oligomer once it has formed (13, 14).
Data regarding the quaternary structure of bunyavirus glycoproteins is limited. Studies on their intracellular distribution have mapped the Golgi targeting function to only one of the two surface glycoproteins, which brings along its other by hetero-oligomeric association (7, 15–17). Using the vaccinia virus T7 polymerase expression system, Chen and Compans, 1991, demonstrated the formation of Punta Toro virus G1/G2 heterodimers and G2 homodimers (18). Uukuniemi virus has stable G1 homodimers that do not dissociate with acidification (19). Because the nomenclature of bunyavirus glycoproteins is based on electrophoretic mobility in SDS–PAGE and not glycoprotein function, these results may seem contradictory. However, the bunyavirus glycoproteins, which are encoded in the M RNA segment, are translated as a single open reading frame that is cotranslationally processed; their order in this open reading frame is a better index of their function than their molecular mobility. The G1 proteins of Uukuniemi and La Crosse and the G2 protein of Punta Toro are all encoded as the downstream protein in the M segment and may have similar functions. That they can be found as homodimers is thus quite consistent among members of the family. Truncated proteins composed of the extracellular domains of spike glycoproteins are a powerful tool for experiments elucidating viral entry. For example, a soluble form of the influenza virus hemagglutinin (HA) can be generated by bromelain digestion of purified virions (20)
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or by expression of a mutated cloned cDNA (13). The protein expressed from the mutated cDNA is secreted both as a monomer and trimer (13), although HAs derived from different virus isolates showed varied ability to form oligomers. While the virion-associated HA trimers were stable, regardless of acidification, the soluble ectodomains dissociated into monomers and dimers after exposure to acidic conditions, suggesting that the transmembrane and cytoplasmic domains help stabilize the oligomeric structure (21). We suggest that a G1–G2 association may be similarly mediated. As in our system, a truncated form of the rabies virus G glycoprotein can also be secreted from cells as oligomers (22). X-ray crystallography and time-resolved cryoelectron microscopy has given new insight into the physical changes in the alphavirus surface proteins that are associated with entry (23, 24). In combination with previous work using soluble forms of the E1 and E2 glycoproteins (25, 26) these studies have produced an in depth analysis of the complicated changes in conformation and oligomeric state that are required for alphavirus fusion with susceptible cells. The sG1 glycoprotein will be a tool to identify the receptor binding, oligomerization, and conformational domains of the G1 glycoprotein and set the stage for a comparable structural analysis of the protein. Due to the relatively low amount of glycosylation of G1 (4), sG1 may also be a suitable candidate molecule for crystallographic studies, which could further elucidate the entry pathway mechanism for bunyaviruses. ACKNOWLEDGMENTS This work was supported by PHS Grants NS-20904 and NS-30606. We thank D. Kolson, Department of Neurology, University of Pennsylvania for performing AChE assays and all the members of the GonzalezScarano laboratory for helpful discussions.
REFERENCES 1. Gonzalez-Scarano, F., and Nathanson, N., in ‘‘Virology’’ (Fields, B. N., Knipe, D. M., and Howley, P. M. Eds.), 3rd ed., pp. 1473– 1504. Lippincott-Raven, New York, NY, 1996.
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2. Gonzalez-Scarano, F., Janssen, R. S., Najjar, J. A., Pobjecky, N., and Nathanson, N., J. Virol. 54, 757–763 (1985). 3. Jacoby, D. R., Cooke, C., Prabakaran, I., Boland, J., Nathanson, N., and Gonzalez-Scarano, F., Virology 193, 993–996 (1993). 4. Pekosz, A., Griot, C., Nathanson, N., and Gonzalez-Scarano, F., Virology 214, 339–348 (1995). 5. Pobjecky, N., Smith, J., and Gonzalez-Scarano, F., Microb. Pathog. 1, 491–501 (1986). 6. Gonzalez-Scarano, F., Virology 140, 209–216 (1985). 7. Bupp, K., Stillmock, K., and Gonzalez-Scarano, F., Virology 220, 485–490 (1996). 8. Pekosz, A., Griot, C., Stillmock, K., Nathanson, N., and GonzalezScarano, F., J. Virol. 69, 3475–3481 (1995). 9. Gonzalez-Scarano, F., Shope, R. E., Calisher, C. E., and Nathanson, N., Virology 120, 42–53 (1982). 10. Doms, R. W., Lamb, R. A., Rose, J. K., and Helenius, A., Virology 193, 545–562 (1993). 11. Fazakerley, J. K., Gonzalez-Scarano, F., Strickler, J., Dietzschold, B., Karush, F., and Nathanson, N., Virology 167, 422–432 (1988). 12. Llanes, C., Collman, R. G., Hrin, R., and Kolson, D. L., J. Neurosci. Res. 42, 791–802 (1995). 13. Singh, A., Doms, R. W., Wagner, K. R., and Helenius, A., EMBO J. 9, 631–639 (1990). 14. Gilbert, J. M., Hernandez, L. D., Chernov-Rogan, T., and White, J. M., J. Virol. 67, 6889–6892 (1993). 15. Chen, S.-Y., Matsuoka, Y., and Compans, R. W., Virology 183, 351– 365 (1991). 16. Lappin, D. F., Nakitare, G. W., Palfreyman, J. W., and Elliot, R. M., J. Gen. Virol. 75, 3441–3451 (1994). 17. Ruusala, A., Persson, R., Schmaljohn, C., and Petterson, R. F., Virology 186, 53–64 (1992). 18. Chen, S.-Y., and Compans, R. W., J. Virol. 65, 5902–5909 (1991). 19. Ronka, H., Hilden, P., Von Bonsdorff, C.-H., and Kuismanen, E., Virology 211, 241–250 (1995). 20. Skehel, J. J., Bayley, P. M., Brown, E. B., Martin, S. R., Waterfield, M. D., White, J. M., Wilson, I. A., and Wiley, D. C., Proc. Natl. Acad. Sci. USA 79, 968–972 (1982). 21. Doms, R. W., and Helenius, A., J. Virol. 60, 833–839 (1986). 22. Wojczyk, B., Shakin-Eshleman, S. H., Doms, R. W., Xiang, Z.-Q., Ertl, H. C. J., Wunner, W. H., and Spitalnick, S. L., Biochemistry 34, 2599–2609 (1995). 23. Fuller, S. D., Berriman, J. A., Butcher, S. J., and Gowen, B. E., Cell 81, 715–725 (1995). 24. Cheng, R. H., Kuhn, R. J., Olson, N. H., Rossman, M. G., Choi, H.-K., Smith, T. J., and Baker, T. S., Cell 80, 621–630 (1995). 25. Justman, J., Klimjack, M. R., and Kielian, M., J. Virol. 67, 7597–9607 (1993). 26. Klimjack, M. R., Jeffrey, S., and Kielian, M., J. Virol. 68, 6940–6946 (1994).
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SHORT COMMUNICATION The Extracellular Domain of La Crosse Virus G1 Forms Oligomers and Undergoes pH-Dependent Conformational Changes ANDREW PEKOSZ*,†,‡ and FRANCISCO GONZA´LEZ-SCARANO†,‡,1 *Cell and Molecular Biology Graduate Group, and Departments of †Neurology and ‡Microbiology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania Received July 3, 1996; accepted August 27, 1996 The La Crosse virus G1 glycoprotein plays a critical role in virus binding to susceptible cells and in the subsequent fusion of viral and cellular membranes. A soluble form of the G1 glycoprotein (sG1) prepared in a recombinant baculovirus system mimics the cell-binding pattern of La Crosse virus and inhibits La Crosse virus infection (A. Pekosz et al., Virology 214, 339–348, 1995), presumably by competing for a cellular receptor, a finding that implies that sG1 can perform some functions absent G2, the smaller of the two bunyavirus glycoproteins. We have performed experiments to determine whether sG1 is present as an oligomer and whether it undergoes the conformational changes associated with fusion (F. Gonzalez-Scarano, Virology 140, 209–216, 1985). Our results indicate that both sG1 and native G1 undergo similar changes in conformation after exposure to an acidic environment, as detected by reactivity with monoclonal antibodies. Furthermore, using chemical cross-linking, both proteins were detected as oligomers (most likely dimers). Sucrose density gradient analysis of sG1 verified that it was present in monomeric and oligomeric forms. These results demonstrate that the isolated G1 glycoprotein can undergo a pH-dependent change in conformation in the absence of its transmembrane and cytoplasmic tail domains and that the extracellular portion of the glycoprotein can oligomerize. q 1996 Academic Press, Inc.
La Crosse virus is a member of the California encephalitis serogroup of bunyaviruses, a group of viruses associated with pediatric encephalitis in areas of North America and Europe where the mosquitoes that vector the virus are endemic (1). La Crosse virus binding, pHdependent fusion and entry have been studied in some detail (2–4) and evidence indicates that viral entry is dependent on the acidic pH environment found in endosomes (5). Like all Bunyaviridae, La Crosse virus has two glycoproteins termed G1 and G2. The larger, G1, plays an important role in entry, first as the virus attachment protein, and then by undergoing a pH-dependent conformational change in concert with the activation of virus– cell or cell-to-cell fusion (6). Using La Crosse virus glycoproteins expressed in recombinant vaccinia, it has been determined that in order for cell-to-cell fusion to take place both of the glycoproteins must be present, presumably because G2 contains the critical fusion sequences (3, 7). We have previously described the construction and expression of a recombinant La Crosse virus G1 protein in a baculovirus expression system (8). Soluble G1 (sG1) consists of the entire extracellular domain of La Crosse virus G1 (amino acids 474–1387 of the La Crosse virus
M segment open reading frame) and lacks the transmembrane and cytoplasmic domains of the native G1. The sG1 glycoprotein (110 kDa) is similar to the native G1 (125 kDa) because (i) it reacts with conformation sensitive anti-G1 monoclonal antibodies (MAb), (ii) it elicits a protective humoral response in mice, (iii) it binds to cells with specificity similar to that of La Crosse virions, and (iv) it inhibits infection of cultured cells by La Crosse and other California serogroup bunyaviruses (4, 8). This last finding—which was the most surprising—implies that not only is sG1 conformationally accurate, but that it can interfere with the normal functions of the G1 protein during the entry process. We undertook these studies to determine whether or not the native G1 protein forms oligomers, and if so, whether sG1 would assume a similar quaternary structure. Given the results indicating that both G1 and G2 must be expressed in concert before cell-to-cell fusion will take place, we also wanted to ascertain if an association between these two glycoproteins is important in inducing the conformational changes in G1 that are associated with virus entry and virus–cell fusion. Previous studies (2, 6, 9) have shown that the binding of specific anti-G1 MAbs is markedly reduced after exposure of intact virions to acid (pH 5.8 or lower). These data together with changes in the pattern of digestion obtained with trypsin were taken as evidence of a confor-
1 To whom correspondence and reprint requests should be addressed. Fax: (215) 573-2029. E-mail: [email protected].
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mational change in the protein. To determine whether the isolated sG1 undergoes a similar conformational alteration, an aliquot of the 35S-labeled protein was acidified to pH 5.6 for 30–60 sec, neutralized with 50 ml of 1 M Tris–HCl, and then immunoprecipitated with either individual anti-G1 MAbs (3, 4, 8) or with polyclonal mouse sera raised against La Crosse virus. As demonstrated in Fig. 1A, either the anti-La Crosse sera or MAb 807-15 recognized both the native and the acidified forms of sG1. In contrast, the signals obtained with either MAb 807-22 or MAb 813-48 were drastically reduced or absent, consistent with the known sensitivity of these epitopes to acidification. A comparable pattern was seen when 35S-labeled La Crosse virions which had been partially purified by ultracentrifugation over a 20% sucrose cushion were used in the assay (Fig. 1B). These results
indicated that the pH-dependent structural changes which take place in G1 during La Crosse virus acidification also occur in sG1, and thus do not require the presence of any other viral structural proteins, viral or cellular membranes, or the transmembrane and cytoplasmic domains of G1. Another important property of many viral glycoproteins is their ability to form either homo- or hetero-oligomers (10). To determine the oligomeric state of sG1, we exposed it to chemical crosslinking reagents and the crosslinked proteins were then immunoprecipitated with MAb 807-15. Using various concentrations of the chemical crosslinker bis(sulfosuccinimidyl) suberate (BS3 , spacer arm length of 11.4 Angstroms), several high molecular weight bands were detected (Fig. 2A). First, a doublet signal is at a position approximately corresponding to that expected of sG1 homodimers (labeled as 21 sG1). There was another band near the top of the gel but, due to its position outside the regions of linear resolution, a molecular weight could not be assigned. Labeled as 41 sG1 in Fig. 2A, this high molecular weight band could be a trimer instead of a tetramer. When the samples were acidified, neutralized, and subsequently crosslinked, we saw a similar pattern, indicating that these higher order oligomers are not dissociated by the pH-dependent conformational changes that take place in sG1. As expected, in both cases a corresponding decrease in the amount of sG1 was seen with increasing concentrations of the crosslinker (Fig. 2A). Native G1 (35S-labeled La Crosse virions) gave a similar pattern (Fig. 2B): a doublet at the approximate molecular weight of dimeric G1 (21 G1) and a much higher molecular weight species (41 G1). There was no change in the cross-linking pattern when the virions were briefly acidified. A second cross linking agent, ethylene glycolbis(sulfosuccinimidylsuccinate) (EGS, spacer arm length of 16.1 Angstroms), was also used in this assay, with comparable results (data not shown) and varying the time of crosslinking (30–120 min) or incubation temperature (47 or room temperature) did not produce more efficient crosslinking. The appearance of a doublet when either sG1 or native G1 was crosslinked could be due to the inability of some portion of the crosslinked protein to completely denature before SDS–PAGE analysis, thus giving it a slightly aberrant molecular weight. In the experiments described above, none of the bands corresponded to any of a number of potential crosslinked G1/G2 heterodimers. Experiments where the crosslinked La Crosse proteins were immunoprecipitated with anti-G2 MAbs were also negative for such a combination (data not shown). Nevertheless, G1 and G2 coprecipitate when MAbs against either glycoproteins are used under conditions that eliminate background precipitation of the other two structural proteins N and L (Fig. 3). This result is evidence that the two glycoproteins have a strong noncovalent interaction, particularly considering the
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FIG. 1. Acid treatment alters MAb binding to sG1 and native G1. 35Slabeled (A) sG1 or (B) native G1 (La Crosse virions) were either untreated or acidified by dropwise addition of H2SO4 to a pH of 5.6 for 30–60 sec and then neutralized with 50 ml of 1 M Tris–HCl, pH 7.4, or 1 M phosphate buffer, pH 7.1. The samples were immunoprecipitated with 15 ml of each of the indicated antibodies and protein G agarose as described (3), washed with 0.05 M Tris–HCl, pH 7.5, 0.15 M NaCl, 0.1% SDS, 1% Triton X-100, and 1% deoxycholate, followed by 0.01 M Tris–HCl, pH 8.0, 0.4 M LiCl, and 2 M urea, and then resuspended in sample buffer and heated at 377 for 10 min before resolution in SDS– 10% PAGE (8). MAbs 807-22 and 813-48 showed reduced or absent signal when immunoprecipitating either the native or truncated, acidified protein, indicating loss of these epitopes. MAb 807-15 and polyclonal mouse sera served as controls, since they immunoprecipitated both the native and the truncated forms of the protein equally well, regardless of acidification. Abbreviations: anti-LAC, polyclonal anti-La Crosse virus mouse sera; NMS, normal mouse sera; DpH, samples that have been acidified and then neutralized.
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FIG. 2. Cross-linking of 35S-labeled sG1 and native G1. The nonreducible chemical crosslinker BS3 was added to either untreated or acidified and neutralized 100-ml samples of (A) sG1 or (B) native G1 (La Crosse virions) at the indicated concentrations, and the samples were then incubated for 2 hr at 47. After quenching with 50 ml of 1 M Tris–HCl, pH 7.4, they were immunoprecipitated with MAb 807-15 and analyzed on SDS–5% PAGE. The positions of monomeric sG1 and La Crosse virus associated G1 as well as the possible dimeric (21, which appears as a doublet) and trimeric/ tetrameric (41) species are indicated. The samples that were acidified before crosslinking showed no change in the profile, indicating the oligomeric state is not effected by acidification.
stringency of the immunoprecipitation (see Fig. 1 legend). In support of this interpretation, analysis of the intracellular localization of La Crosse virus glycoproteins with re-
FIG. 3. La Crosse virus G1 and G2 glycoproteins are associated noncovalently. Sucrose density gradient purified La Crosse virions were loaded directly on to an SDS–12.5% PAGE (lane, 0) or immunoprecipitated with a mixture of the anti-G1 MAbs 807-31, 807-33, and 80725 (lane, aG1) or the anti-G2 MAb 9B7.5 (lane, aG2) before loading. Immunoprecipitation for either glycoprotein coimmunoprecipitated the other, while no L or N proteins were detectable, implying an association between G1 and G2. Immunoprecipitation with mouse sera raised to a thymidine kinase negative vaccinia virus (arVVtk-) and with sera from mice which had survived a La Crosse virus challenge (8) are shown as negative and positive immunoprecipitation controls, respectively.
combinant vaccinia viruses has demonstrated that G1 colocalizes with Golgi markers only when it is expressed in concert with G2, indicating at least a transient association responsible for transport (7). However, this interaction may simply not be detectable with chemical crosslinking. Conceivably, this could be due to the absence of lysines in the appropriate domains, since free amino groups are the substrates for these reagents (11). Alternatively, the G1–G2 interaction domain may involve the transmembrane and/or cytoplasmic tails of the proteins, two regions that cannot be crosslinked with BS3 . To further analyze the oligomeric state of sG1, we performed sucrose density gradient ultracentrifugation analysis on the 35S-labeled sG1. Samples of sG1 that were either untreated (Fig. 4A) or acidified and neutralized (Fig. 4B) were layered over 5–20% linear sucrose gradients and ultracentrifuged at 40,000 RPM for 18 hr in an SW41 rotor. The gradients were then fractionated from the bottom, and every other fraction was immunoprecipitated and analyzed on SDS–PAGE. sG1 sedimented in two peaks, one in fractions 9–11, and a second in fractions 19–23 (from a total of 34 fractions collected) (Fig. 4A). Acidification of sG1 prior to sucrose gradient analysis yielded a very similar profile (Fig. 4B). To control for the efficiency of the gradient, a parallel experiment was performed on a sample of rat brain extract, which was fractionated and analyzed for acetylcholinesterase (AChE) activity as described by Llanes et al. (12). The peak activities of the tetrameric (41 AChE, approximate molecular weight 272 kDa) and the dimeric (21 AChE, approximate
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FIG. 4. Sucrose density sedimentation of 35S-labeled sG1 shows the presence of higher order oligomers. Linear sucrose density gradients (5– 20% in phosphate-buffered saline with 0.1% Triton X-100, total volume of 13 ml) were prepared on a Biocomp Radian Gradient Maker. Approximately 500 ml of 35S-labeled sG1 with 0.1% Triton X-100 which were either (A) untreated or (B) acidified and neutralized were layered on top of the gradient and the samples were spun in an SW41 rotor at 40,000 RPM for 18 hr at 157. The samples were then fractionated in 34 aliquots beginning at the bottom of the gradient. Every other fraction was immunoprecipitated with 15 ml of mouse polyclonal antisera and analyzed on 10% SDS–PAGE. Both untreated and acidified sG1 showed a similar bimodal peak, indicating the presence of monomeric and oligomeric (most likely dimeric) forms of sG1. A parallel gradient containing rat brain extract was analyzed for acetylcholinesterase (AChE, monomer Å 68 kDa) activity as described (12) and the peak positions of the tetrameric (41) and dimeric (21) forms of the enzyme are indicated. A similar pattern of sG1 sedimentation is seen when Triton X-100 is left out of the sucrose gradient (data not shown).
molecular weight 136 kDa) forms of the enzyme are indicated as a rough gauge of molecular weight (12). The data indicate that sG1 is present in monomeric and dimeric (and possibly higher order) forms. Although a comparison experiment with native G1 would have been informative, we were unable to separate the glycoproteins from the virus ribonucleoprotein complexes with either Triton X-100 (0.5–1.0%) or Nonidet P-40 (0.1–0.5%). (Note the stringent washes used for the immunoprecipitations in Figs. 1 and 3.) Our data indicate that a deletion mutant of the La Crosse virus G1 glycoprotein lacking its transmembrane and cytoplasmic domains can undergo pH-dependent conformational changes that appear to be identical to those of the native G1. This observation proves that the disappearance of MAb epitopes following acid treatment is the result of structural changes in the G1 extracellular domain, and not just due to masking of a G1 epitope by structural changes in the G2 glycoprotein, an interpretation which could not be ruled out in studies using La Crosse virions (6). Furthermore, the loss of MAb epitopes after acidification is not a result of simple dissociation of a G1 oligomer, because pH changes seem to have little or no effect on the amount of oligomeric sG1 present. Since the sG1 glycoprotein can oligomerize, the transmembrane and cytoplasmic tails of G1 probably do not play a major role in this function, although they may help stabilize the oligomer once it has formed (13, 14).
Data regarding the quaternary structure of bunyavirus glycoproteins is limited. Studies on their intracellular distribution have mapped the Golgi targeting function to only one of the two surface glycoproteins, which brings along its other by hetero-oligomeric association (7, 15–17). Using the vaccinia virus T7 polymerase expression system, Chen and Compans, 1991, demonstrated the formation of Punta Toro virus G1/G2 heterodimers and G2 homodimers (18). Uukuniemi virus has stable G1 homodimers that do not dissociate with acidification (19). Because the nomenclature of bunyavirus glycoproteins is based on electrophoretic mobility in SDS–PAGE and not glycoprotein function, these results may seem contradictory. However, the bunyavirus glycoproteins, which are encoded in the M RNA segment, are translated as a single open reading frame that is cotranslationally processed; their order in this open reading frame is a better index of their function than their molecular mobility. The G1 proteins of Uukuniemi and La Crosse and the G2 protein of Punta Toro are all encoded as the downstream protein in the M segment and may have similar functions. That they can be found as homodimers is thus quite consistent among members of the family. Truncated proteins composed of the extracellular domains of spike glycoproteins are a powerful tool for experiments elucidating viral entry. For example, a soluble form of the influenza virus hemagglutinin (HA) can be generated by bromelain digestion of purified virions (20)
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or by expression of a mutated cloned cDNA (13). The protein expressed from the mutated cDNA is secreted both as a monomer and trimer (13), although HAs derived from different virus isolates showed varied ability to form oligomers. While the virion-associated HA trimers were stable, regardless of acidification, the soluble ectodomains dissociated into monomers and dimers after exposure to acidic conditions, suggesting that the transmembrane and cytoplasmic domains help stabilize the oligomeric structure (21). We suggest that a G1–G2 association may be similarly mediated. As in our system, a truncated form of the rabies virus G glycoprotein can also be secreted from cells as oligomers (22). X-ray crystallography and time-resolved cryoelectron microscopy has given new insight into the physical changes in the alphavirus surface proteins that are associated with entry (23, 24). In combination with previous work using soluble forms of the E1 and E2 glycoproteins (25, 26) these studies have produced an in depth analysis of the complicated changes in conformation and oligomeric state that are required for alphavirus fusion with susceptible cells. The sG1 glycoprotein will be a tool to identify the receptor binding, oligomerization, and conformational domains of the G1 glycoprotein and set the stage for a comparable structural analysis of the protein. Due to the relatively low amount of glycosylation of G1 (4), sG1 may also be a suitable candidate molecule for crystallographic studies, which could further elucidate the entry pathway mechanism for bunyaviruses. ACKNOWLEDGMENTS This work was supported by PHS Grants NS-20904 and NS-30606. We thank D. Kolson, Department of Neurology, University of Pennsylvania for performing AChE assays and all the members of the GonzalezScarano laboratory for helpful discussions.
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2. Gonzalez-Scarano, F., Janssen, R. S., Najjar, J. A., Pobjecky, N., and Nathanson, N., J. Virol. 54, 757–763 (1985). 3. Jacoby, D. R., Cooke, C., Prabakaran, I., Boland, J., Nathanson, N., and Gonzalez-Scarano, F., Virology 193, 993–996 (1993). 4. Pekosz, A., Griot, C., Nathanson, N., and Gonzalez-Scarano, F., Virology 214, 339–348 (1995). 5. Pobjecky, N., Smith, J., and Gonzalez-Scarano, F., Microb. Pathog. 1, 491–501 (1986). 6. Gonzalez-Scarano, F., Virology 140, 209–216 (1985). 7. Bupp, K., Stillmock, K., and Gonzalez-Scarano, F., Virology 220, 485–490 (1996). 8. Pekosz, A., Griot, C., Stillmock, K., Nathanson, N., and GonzalezScarano, F., J. Virol. 69, 3475–3481 (1995). 9. Gonzalez-Scarano, F., Shope, R. E., Calisher, C. E., and Nathanson, N., Virology 120, 42–53 (1982). 10. Doms, R. W., Lamb, R. A., Rose, J. K., and Helenius, A., Virology 193, 545–562 (1993). 11. Fazakerley, J. K., Gonzalez-Scarano, F., Strickler, J., Dietzschold, B., Karush, F., and Nathanson, N., Virology 167, 422–432 (1988). 12. Llanes, C., Collman, R. G., Hrin, R., and Kolson, D. L., J. Neurosci. Res. 42, 791–802 (1995). 13. Singh, A., Doms, R. W., Wagner, K. R., and Helenius, A., EMBO J. 9, 631–639 (1990). 14. Gilbert, J. M., Hernandez, L. D., Chernov-Rogan, T., and White, J. M., J. Virol. 67, 6889–6892 (1993). 15. Chen, S.-Y., Matsuoka, Y., and Compans, R. W., Virology 183, 351– 365 (1991). 16. Lappin, D. F., Nakitare, G. W., Palfreyman, J. W., and Elliot, R. M., J. Gen. Virol. 75, 3441–3451 (1994). 17. Ruusala, A., Persson, R., Schmaljohn, C., and Petterson, R. F., Virology 186, 53–64 (1992). 18. Chen, S.-Y., and Compans, R. W., J. Virol. 65, 5902–5909 (1991). 19. Ronka, H., Hilden, P., Von Bonsdorff, C.-H., and Kuismanen, E., Virology 211, 241–250 (1995). 20. Skehel, J. J., Bayley, P. M., Brown, E. B., Martin, S. R., Waterfield, M. D., White, J. M., Wilson, I. A., and Wiley, D. C., Proc. Natl. Acad. Sci. USA 79, 968–972 (1982). 21. Doms, R. W., and Helenius, A., J. Virol. 60, 833–839 (1986). 22. Wojczyk, B., Shakin-Eshleman, S. H., Doms, R. W., Xiang, Z.-Q., Ertl, H. C. J., Wunner, W. H., and Spitalnick, S. L., Biochemistry 34, 2599–2609 (1995). 23. Fuller, S. D., Berriman, J. A., Butcher, S. J., and Gowen, B. E., Cell 81, 715–725 (1995). 24. Cheng, R. H., Kuhn, R. J., Olson, N. H., Rossman, M. G., Choi, H.-K., Smith, T. J., and Baker, T. S., Cell 80, 621–630 (1995). 25. Justman, J., Klimjack, M. R., and Kielian, M., J. Virol. 67, 7597–9607 (1993). 26. Klimjack, M. R., Jeffrey, S., and Kielian, M., J. Virol. 68, 6940–6946 (1994).
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