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Similar Structural Models of the Transmembrane Proteins of Ebola and Avian Sarcoma Viruses
Cell, Vol. 85, 477–478, May 17, 1996, Copyright 1996 by Cell Press
Letter to the Editor
Similar Structural Models of the Transmembrane Proteins of Ebola and Avian Sarcoma Viruses Epidemics of African hemorrhagic fever caused by viruses of the Filoviridae, Ebola and Marburg, have recently appeared with increasing frequency (Centers for Disease Control and Prevention, 1995). These agents have enormous potential for human morbidity and mortality. This virus family has a genomic structure and replication strategy similar to that of the Paramyxoviridae, but very little direct homology to this or other virus families at the RNA or protein levels (Sanchez et al., 1993). In 1992, Volchkov et al. noted that the carboxyterminal region of the Ebola glycoprotein (GP) bore some overall resemblance to the transmembrane (TM) protein of oncogenic Retroviruses, particularly over a 26 amino acid region that defines a conserved immunosuppressive peptide (Cianciolo et al., 1985). Subsequent discussion has been limited to speculation as to the significance of this region in Ebola infection. We have shown that Retroviruses generally dissimilar in sequence can have limited sequence identities and similar structural propensities that can generate an overall model for homologous proteins that may otherwise not be apparent (Gallaher et al., 1989). The model of the gp41 TM protein of human immunodeficiency virus (HIV) thus generated has since been supported in a number of laboratories (Decroly et al., 1993; Wild et al., 1995; Blacklow et al., 1995). Applying similar methods to a comparison of Ebola and avian Retroviruses, it has been found that models for the carboxy-terminal 181 amino acids of Ebola GP and Rous sarcoma virus (RSV) TM are very similar (Figure 1). A description of the similarities between the two proteins is given below, beginning from the N-terminal side of the models in Figure 1. At 152-155 amino acids prior to membrane insertion, there is a polybasic region in both viruses that in RSV serves as the site of precursor cleavage yielding a new amino-terminus (Schwartz et al., 1983). Since cleavage of Ebola GP has not been reported, the functional significance of this similarity is unknown. Within 8-9 amino acids, there is in both viruses a 37-46 amino acid region bounded by cysteines, which could form a disulfidedefined loop structure. This region has at its center a sequence of 13-16 uncharged and hydrophobic amino acids in the same relative position as the fusion peptides critical for viral entry and cell fusion in the Retroviridae, most highly detailed for HIV (Freed et al., 1990). Marburg contains within this region a canonical fusion tripeptide FFG, conservatively substituted with YFG in Ebola, and with FLG in HIV (Richardson et al., 1980; Gallaher, 1987). Further, this hydrophobic region exhibits the highest degree of identity within this loop between Ebola and Marburg (Sanchez et al., 1993).
Next, proceeding for 42 amino acids, there is in Ebola and RSV a region with high propensity to form an amphipathic helix similar to other Retroviridae (Gallaher et al., 1989). Mutations in this region of HIV gp41 abolish infectivity and fusion (Cao et al., 1993). Homology of Ebola with Retroviral TM proteins is further reinforced by the finding of 18 of 42 amino acids identical between Ebola and RSV distributed throughout this region, including a site for N-linked glycosylation 7 amino acids after this second cysteine. Within 7-8 amino acids beyond this helical region, there is a set of cysteines separated by 6 amino acids that define a conserved motif (CX6CC) in Retroviral glycoproteins. In HIV this defines an antigenic site (Gnann et al., 1987). Overlying the latter third of the amphipathic helix and CX6CC is the immunosuppressive peptide similarity previously identified, 50% identical between Ebola and RSV. Within 2-9 residues beyond CX 6CC there is a second N-linked glycosylation site that is conserved in the Retrovirus family and shared by Ebola and Marburg. This lies near or within a second region with high charge density and potential to form an amphipathic helix. Over the 41 amino acids between CX6 CC and membrane insertion of Ebola GP, the identical amino acids are centered in the region of highest helical potential, with 9 of 24 positions identical between Ebola and RSV, and 10 of 24 between Ebola and Marburg. Beyond this region toward membrane insertion, the sequence diverges between Ebola and RSV. Even among the Retroviridae there is variation after the CX 6CC motif, such that diversity between Ebola and RSV in this region does not detract from the overall similarity observed. Identification of regions with particular structural propensities align the highly charged helical region of Ebola GP with a region in HIV gp41 that is similar in character, if only 10% identical. A peptide analogue centered on this charged region in HIV-1 (Wild et al., 1993), and based on our prior identification of this region (Gallaher et al., 1989), has been found to inhibit HIV infection and fusion. Peptide analogues centered about these helical regions of Ebola and Marburg may have significant antiviral activity. Similar roles for the proposed helices of Filoviruses and Retroviruses are supported by the finding that both of these glycoproteins form trimers (Hunter and Swanstrom, 1990; Feldmann et al., 1991). In HIV-1 and SIV these helices play a role as coiled-coils in trimer formation (Gallaher et al., 1989; Wild et al., 1995; Blacklow et al., 1995), as shown previously for HA2 of influenza virus (Wilson et al., 1981). The noted similarities of Filovirus and Retrovirus transmembrane proteins thus immediately suggest investigations into Ebola and Marburg that
Cell 478
Figure 1. Models of the Carboxy-Terminal Transmembrane Region of Ebola and Rous Sarcoma Virus Glycoproteins Proposed helices are indicated in helical net projection with sequential amino acids connected by solid lines. Proposed disulfide linkages are indicated by double dotted lines. Hydrophobic amino acids are grouped with a solid background; neutral amino acids with a heavily outlined circle; hydrophilic amino acids with a light circle. Double circles indicate identical amino acids between Ebola and Rous viruses. Asterisk indicates an additional amino acid needed to align the identities. Sequences are of Ebola, Zaire (amino acid residues 497-650, Genbank U31033) and Rous, Prague C (amino acid residues 402-554, Genbank J02342).
could aid our understanding of molecular pathogenesis and medical intervention. A typical TM structure, including a fusion peptide motif, indicates that this region of the Filovirus GP is likely to play the same role in virus entry and cytopathogenicity as does the homologous TM protein superfamily in the Orthomyxoviridae, Paramyxoviridae, and Retroviridae. Once Filovirus GP genes are available in mammalian expression vectors, finely targeted site-directed mutagenesis should rapidly and safely elucidate the role of these peptide regions in Filovirus membrane biology and infectivity. William R. Gallaher Department of Microbiology, Immunology, and Parasitology, and Stanley S. Scott Cancer Center Louisiana State University Medical Center New Orleans, Louisiana 70112–1393 References Blacklow, S.C., Lu, M., and Kim, P.S. (1995). Biochemistry 34, 14955– 14962. Cao, J., Bergeron, L., Helseth, E., Thali,M., Repke, H., and Sodroski, J. (1993). J. Virol. 67, 2747–2755. Centers for Disease Control and Prevention (1995). Morb. Mort. Weekly Rpt. 44, 381–382; 399. Cianciolo, G.J., Copeland, T.D., Oroszlan, S., and Snyderman, R. (1985). Science 230, 453–455. Decroly, E., Cornet, B., Martin, I., Ruysschaert, J.-M., and Vandenbranden, M. (1993). J. Virol. 67, 3552–3560. Feldmann, H, Will, C., Schikore, M., Slenczka, W., and Klenk, H.-D. (1991). Virology 182, 353–356. Freed, E., Myers, D., and Risser, R. (1990). Proc. Natl. Acad. Sci. USA 87, 4650–4654.
Gallaher, W.R. (1987). Cell 50, 327–328. Gallaher, W.R., Ball, J.M., Garry, R.F., Griffin, M. C., and Montelaro, R.C. (1989). AIDS Res. Human Retroviruses 5, 431–440. Gnann, J.W.Jr., Nelson, J.A., and Oldstone, M.A.B. (1987). J. Virol. 61, 2639–2641. Hunter E., and Swanstrom R. (1990). Curr. Top. Microbiol. Immunol. 157, 187–253. Richardson, C., Scheid, A., and Choppin, P.W. (1980). Virology 105, 205–222. Sanchez, A., Kiley, M.P., Holloway, B.P., and Auperin, D.D. (1993). Virus Res. 29, 215–240. Schwartz, D.E., Tizard, R., and Gilbert, W. (1983). Cell 32, 853–869. Volchkov, V.E., Blinov, V.M., and Netesov, S.V. (1992). FEBS Lett. 305, 181–184. Wild, C., Dubay, J.W., Greenwell, T., Baird T.Jr., Oas, T.G., McDanal, C., Hunter, E., and Matthews, T. (1995). Proc. Natl. Acad. Sci. USA 91, 12676–12680. Wild, C., Greenwell, T., and Matthews, T. (1993). AIDS Res. Human Retroviruses 9, 1051–1053. Wilson, I.A., Skehel, J.J., and Wiley, D.C. (1981). Nature 289, 366–373.
Letter to the Editor
Similar Structural Models of the Transmembrane Proteins of Ebola and Avian Sarcoma Viruses Epidemics of African hemorrhagic fever caused by viruses of the Filoviridae, Ebola and Marburg, have recently appeared with increasing frequency (Centers for Disease Control and Prevention, 1995). These agents have enormous potential for human morbidity and mortality. This virus family has a genomic structure and replication strategy similar to that of the Paramyxoviridae, but very little direct homology to this or other virus families at the RNA or protein levels (Sanchez et al., 1993). In 1992, Volchkov et al. noted that the carboxyterminal region of the Ebola glycoprotein (GP) bore some overall resemblance to the transmembrane (TM) protein of oncogenic Retroviruses, particularly over a 26 amino acid region that defines a conserved immunosuppressive peptide (Cianciolo et al., 1985). Subsequent discussion has been limited to speculation as to the significance of this region in Ebola infection. We have shown that Retroviruses generally dissimilar in sequence can have limited sequence identities and similar structural propensities that can generate an overall model for homologous proteins that may otherwise not be apparent (Gallaher et al., 1989). The model of the gp41 TM protein of human immunodeficiency virus (HIV) thus generated has since been supported in a number of laboratories (Decroly et al., 1993; Wild et al., 1995; Blacklow et al., 1995). Applying similar methods to a comparison of Ebola and avian Retroviruses, it has been found that models for the carboxy-terminal 181 amino acids of Ebola GP and Rous sarcoma virus (RSV) TM are very similar (Figure 1). A description of the similarities between the two proteins is given below, beginning from the N-terminal side of the models in Figure 1. At 152-155 amino acids prior to membrane insertion, there is a polybasic region in both viruses that in RSV serves as the site of precursor cleavage yielding a new amino-terminus (Schwartz et al., 1983). Since cleavage of Ebola GP has not been reported, the functional significance of this similarity is unknown. Within 8-9 amino acids, there is in both viruses a 37-46 amino acid region bounded by cysteines, which could form a disulfidedefined loop structure. This region has at its center a sequence of 13-16 uncharged and hydrophobic amino acids in the same relative position as the fusion peptides critical for viral entry and cell fusion in the Retroviridae, most highly detailed for HIV (Freed et al., 1990). Marburg contains within this region a canonical fusion tripeptide FFG, conservatively substituted with YFG in Ebola, and with FLG in HIV (Richardson et al., 1980; Gallaher, 1987). Further, this hydrophobic region exhibits the highest degree of identity within this loop between Ebola and Marburg (Sanchez et al., 1993).
Next, proceeding for 42 amino acids, there is in Ebola and RSV a region with high propensity to form an amphipathic helix similar to other Retroviridae (Gallaher et al., 1989). Mutations in this region of HIV gp41 abolish infectivity and fusion (Cao et al., 1993). Homology of Ebola with Retroviral TM proteins is further reinforced by the finding of 18 of 42 amino acids identical between Ebola and RSV distributed throughout this region, including a site for N-linked glycosylation 7 amino acids after this second cysteine. Within 7-8 amino acids beyond this helical region, there is a set of cysteines separated by 6 amino acids that define a conserved motif (CX6CC) in Retroviral glycoproteins. In HIV this defines an antigenic site (Gnann et al., 1987). Overlying the latter third of the amphipathic helix and CX6CC is the immunosuppressive peptide similarity previously identified, 50% identical between Ebola and RSV. Within 2-9 residues beyond CX 6CC there is a second N-linked glycosylation site that is conserved in the Retrovirus family and shared by Ebola and Marburg. This lies near or within a second region with high charge density and potential to form an amphipathic helix. Over the 41 amino acids between CX6 CC and membrane insertion of Ebola GP, the identical amino acids are centered in the region of highest helical potential, with 9 of 24 positions identical between Ebola and RSV, and 10 of 24 between Ebola and Marburg. Beyond this region toward membrane insertion, the sequence diverges between Ebola and RSV. Even among the Retroviridae there is variation after the CX 6CC motif, such that diversity between Ebola and RSV in this region does not detract from the overall similarity observed. Identification of regions with particular structural propensities align the highly charged helical region of Ebola GP with a region in HIV gp41 that is similar in character, if only 10% identical. A peptide analogue centered on this charged region in HIV-1 (Wild et al., 1993), and based on our prior identification of this region (Gallaher et al., 1989), has been found to inhibit HIV infection and fusion. Peptide analogues centered about these helical regions of Ebola and Marburg may have significant antiviral activity. Similar roles for the proposed helices of Filoviruses and Retroviruses are supported by the finding that both of these glycoproteins form trimers (Hunter and Swanstrom, 1990; Feldmann et al., 1991). In HIV-1 and SIV these helices play a role as coiled-coils in trimer formation (Gallaher et al., 1989; Wild et al., 1995; Blacklow et al., 1995), as shown previously for HA2 of influenza virus (Wilson et al., 1981). The noted similarities of Filovirus and Retrovirus transmembrane proteins thus immediately suggest investigations into Ebola and Marburg that
Cell 478
Figure 1. Models of the Carboxy-Terminal Transmembrane Region of Ebola and Rous Sarcoma Virus Glycoproteins Proposed helices are indicated in helical net projection with sequential amino acids connected by solid lines. Proposed disulfide linkages are indicated by double dotted lines. Hydrophobic amino acids are grouped with a solid background; neutral amino acids with a heavily outlined circle; hydrophilic amino acids with a light circle. Double circles indicate identical amino acids between Ebola and Rous viruses. Asterisk indicates an additional amino acid needed to align the identities. Sequences are of Ebola, Zaire (amino acid residues 497-650, Genbank U31033) and Rous, Prague C (amino acid residues 402-554, Genbank J02342).
could aid our understanding of molecular pathogenesis and medical intervention. A typical TM structure, including a fusion peptide motif, indicates that this region of the Filovirus GP is likely to play the same role in virus entry and cytopathogenicity as does the homologous TM protein superfamily in the Orthomyxoviridae, Paramyxoviridae, and Retroviridae. Once Filovirus GP genes are available in mammalian expression vectors, finely targeted site-directed mutagenesis should rapidly and safely elucidate the role of these peptide regions in Filovirus membrane biology and infectivity. William R. Gallaher Department of Microbiology, Immunology, and Parasitology, and Stanley S. Scott Cancer Center Louisiana State University Medical Center New Orleans, Louisiana 70112–1393 References Blacklow, S.C., Lu, M., and Kim, P.S. (1995). Biochemistry 34, 14955– 14962. Cao, J., Bergeron, L., Helseth, E., Thali,M., Repke, H., and Sodroski, J. (1993). J. Virol. 67, 2747–2755. Centers for Disease Control and Prevention (1995). Morb. Mort. Weekly Rpt. 44, 381–382; 399. Cianciolo, G.J., Copeland, T.D., Oroszlan, S., and Snyderman, R. (1985). Science 230, 453–455. Decroly, E., Cornet, B., Martin, I., Ruysschaert, J.-M., and Vandenbranden, M. (1993). J. Virol. 67, 3552–3560. Feldmann, H, Will, C., Schikore, M., Slenczka, W., and Klenk, H.-D. (1991). Virology 182, 353–356. Freed, E., Myers, D., and Risser, R. (1990). Proc. Natl. Acad. Sci. USA 87, 4650–4654.
Gallaher, W.R. (1987). Cell 50, 327–328. Gallaher, W.R., Ball, J.M., Garry, R.F., Griffin, M. C., and Montelaro, R.C. (1989). AIDS Res. Human Retroviruses 5, 431–440. Gnann, J.W.Jr., Nelson, J.A., and Oldstone, M.A.B. (1987). J. Virol. 61, 2639–2641. Hunter E., and Swanstrom R. (1990). Curr. Top. Microbiol. Immunol. 157, 187–253. Richardson, C., Scheid, A., and Choppin, P.W. (1980). Virology 105, 205–222. Sanchez, A., Kiley, M.P., Holloway, B.P., and Auperin, D.D. (1993). Virus Res. 29, 215–240. Schwartz, D.E., Tizard, R., and Gilbert, W. (1983). Cell 32, 853–869. Volchkov, V.E., Blinov, V.M., and Netesov, S.V. (1992). FEBS Lett. 305, 181–184. Wild, C., Dubay, J.W., Greenwell, T., Baird T.Jr., Oas, T.G., McDanal, C., Hunter, E., and Matthews, T. (1995). Proc. Natl. Acad. Sci. USA 91, 12676–12680. Wild, C., Greenwell, T., and Matthews, T. (1993). AIDS Res. Human Retroviruses 9, 1051–1053. Wilson, I.A., Skehel, J.J., and Wiley, D.C. (1981). Nature 289, 366–373.