VIRUS GENES 6:2, 131-141, 1992 © Kluwer Academic Publishers, Manufactured in The Netherlands
Computer Prediction of Antigenic and Topogenic Domains in HSV-1 and HSV-2 Glycoprotein B (gB) YECHIEL BECKER
Department of Molecular Virology, Faculty of Medicine, Hebrew UniversiO, of Jerusalem, Jerusalem, Israel Received March 21, 1991 Accepted June 24, 1991 Requests for reprints should be addressed to Yechiel Becker, Department of Molecular Virology, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel. Key words: antigenic domains, topogenic domains, computer prediction, HSV-1/HSV-2, glycoprotein B (gB)
Abstract
The envelope glycoprotein B (gB) coded for by the herpes simplex virus type 1 (HSV-1) Ut27 gene is similar to the amino acid (aa) sequence of the gB coded by a homologous gene in HSV-2 DNA. The putative antigenic domains in HSV-1 and HSV-2 gB glycoproteins were analyzed on a comparative basis by suitable computer programs, which allowed the prediction of putative antigenic and topogenic domains. The computer-derived domains were compared to experimentally reported antigenic domains in HSV-1 gB glycoprotein. The computer-predicted antigenic domains in the HSV-1 gB glycoprotein matched well with the reported experimentally derived antigenic domains. The aa sequence of antigenic domain 1 was noted to resemble the amino acid sequence in ApoE that is involved in the attachment of this protein to LDL receptors. The clusters of hydrophobic aa domains are conserved in the two viral glycoproteins and are signals for transfer of the viral proteins through the cellular membrane. Introduction
The envelope glycoprotein B (gB) of herpes simplex virus type 1 (HSV-I) is coded by a gene (UL27) in the viral genome (1). This protein is involved in virus entry into cells and induces fusion of the virion envelope with the host cell membrane. The amino acid (aa) sequence of HSV-1 gB is a long polypeptide chain with hydrophobic domains (2,3). The HSV-1 gB revealed a close similarity to the aa sequence of the gB coded by HSV-2 (4). The HSV-1 gB was also found to have homology with the protein product of the Epstein-Barr virus (EBV) BAL4
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BECKER
open reading flame (ORF) (5) and the varicella-zoster virus (VZV) gene 31 protein product (6). Further studies by Pereira and associates (7-10) revealed that HSV-I gB polypeptides formed dimers on the cell membrane, and neutralizable antigenic epitopes were found to cluster in two domains: one in the amino-terminal 190 aa residues (seven continuous epitopes, five of which are HSV-1 specific). Antibodies reacted with synthetic peptides from the N terminus of HSV-1 gB glycoprotein mapped with epitopes between residues 1 and 47 in the mature polypeptide from which the 30 aa signal peptide was removed (8,9). The second domain is composed of discontinuous epitopes, including domains between aa residues 273 and 298. Mester et al. (11) searched for antigenic domains in HSV-1 gB glycoprotein by synthesizing three overlapping peptides (two 20-mers and one 18-mer) spanning aa 63-1t0 of the immature polypeptide chain (including the 30 aa of the signal peptide). These authors used the Garnier algorithm to predict the secondary structure of the above polypeptide sequence and reported that a monoclonal antibody reacted with the two peptides spanning aa 63-95 (with an overlap at aa 78-81) in HSV-1 gB. The antigenic domain is present in a random conformation of the polypeptide between a 13-turn and an a-helix. Antigenic epitopes of HSV-I gB were characterized by Chapsal and Pereira (7). Monoclonal antibodies that recognized native proteins reacted with discontinuous epitopes of gB dimers, whereas monoclonal antibodies reactive with denatured gB polypeptide recognized continuous epitopes on monomeric or dimeric proteins. Since computer programs that provide information on the physical properties of aa in polypeptide chains can be used to predict putative antigenic domains (continuous epitopes) in viral glycoproteins (12-14), it was of interest to determine if the experimentally proven antigenic domains in gB glycoproteins of HSV-1 and HSV-2 predicted by the computer analyses agree with the experimentally proven antigenic epitopes. Since the computer programs (15-25) provide information on the hydrophobicity and hydrophilicity of the aa, it is possible to use these physical properties for the definition of topogenic functional domains in the viral protein. Such domains may be involved in the interactions of HSV-1 virions with receptors on cell membranes, and in the interaction of the viral protein with the signal recognition protein (SRP) complex (26), which is involved in the intracytoplasmic transfer of the glycoprotein through the cell membrane during the genesis of the viral membrane. The aim of the present study is to define, with the help of computer programs, topogenic domains in HSV-1 and HSV-2 gB gtycoproteins and to assess their functional and biological properties in comparison with experimentally defined domains.
Methods Primary amino acid sequence
The aa sequences of gB glycoproteins of HSV-I(F), HSV-I(Synl7), HSVI(KOS), and HSV-2(333), as well as VZV, and EBV-related proteins and apolipo
HSV-I AND HSV-2 gB
133
proteins E and B, were obtained through the NBRF Protein Sequence Data Bank from the N B R F Protein Identification Resource (15). This program is available from the University of Wisconsin Genetics Computer Group (GCG) software (16).
Computer programs The compilation of seven algorithms in one program by Wolf et al. (17) provides information for analyzing the properties of aa in a polypeptide chain by using the primary aa sequence of the peptide. The following properties of aa were studied: a) hydrophilicity according to Hopp and Woods (18,19) or Kyte and Doolittle (20), b) surface probability according to Emini et al. (21), c) chain flexibility according to Kai~lus and Schulz (22), d) secondary structure according to Chou and Fasman (23) and Garnier-Osguthorpe-Robson (24), and e) the antigenicity index according to Wolf et al. (17). All of these provide quantitative estimates for hydrophilicity, surface probability, chain flexibility, and the antigenicity index. The GAP program (16) was used to compare the sequences of HSV-1 gB to HSV-2 gB. The properties of the two gB aa sequences were determined by the programs compiled by Jameson and Wolf (25) and Wolf et al. (17). In the present analysis an antigenic domain was selected on the basis of the highest values of hydrophilicity, surface probability, and flexibility of consecutive aa in the gB. The predicted secondary structure of the polypeptide was also taken into account, since domains in polypeptides with a 13-turn conformation were found to have a better antigenicity value than aa sequences with a [3-sheet conformation. To obtain a numerical value for the physical property of each aa in the polypeptide that constitutes a putative antigenic domain, the normalized value was determined by summing up the specific values (such as hydrophilicity, etc.) of all aa in the domain and dividing them by the number of aa in that domain. This normalized value per aa was used for the comparison of different putative antigenic domains.
Results
Putative antigenic domains in HSV-1 and HSV-2 gB glycoproteins Computer analysis of the HSV-1 gB polypeptide (Table 1) revealed eight aa domains that have properties compatible with antigenic domains (e.g., a relative high hydrophilicity, surface probability, and antigenic index, and a 13-turn or a-helix conformation). Six putative antigenic domains are in the outer portion of the glycoprotein, which is inserted into the cellular membrane, and two are in the intracytoplasmic aa sequence. It was noted that the aa sequence aa 62-81 (putative antigen domain 1) has the most pronounced computer-derived physical properties of an antigenic domain in the polypeptide chain. When compared with the seven other putative antigenic domains, the hydrophilicity value of the aa sequence for domain I is the highest (the normalized value per aa is 2.31). A high
2.31 2.1 2.28 1.48 1.92 1.55 1.9 2.13
No. aa
62-81 a 127-141 223-230 250-257 463-481 700-712 855-867 879-888
Domain no,
1 2 3 4 5 6 7 8
1.8 2,44 2,64 2.29 3,19 2,05 2,98 3.9
Surface probability per aa 1.8 1.06 1.02 1.03 1.04 1 1.04 1.02
Flexibility prediction per aa 1.4 1.3 1.03 1.15 1.02 0.88 0.9 1.13
Antigenic index per aa
aThe amino acid number is the same as in the immature polypeptide (containing the 30 aa of the signal peptide).
(20) (15) (8) (8) (19) (13) (13) (10)
Hydrophilicity per aa
Table 1. Probable antigenic domains in HSV-1 (F) glycoprotein B
T T T+H T H+T h H h+t+B
CF prediction
T T H T H+T T H H+T
GOR p~diction
¢'3 7~
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HSV-I A N D HSV-2 gB
Table 2. Putative antigenic d o m a i n s in HSV-2 (333) glycoprotein B
Domain no.
Domain
1 2 3 4 5 6 7 8
60-79 ~ 122-138 219-238 245-253 459-474 700-707 957-867 882-889
No. aa
Surface probability per aa
Hydrophilicity per aa
20 17 10 9 16 8 1t 8
2.43 2.92 2.06 1.46 2.15 2.06 2.23 2.02
5.47 2.28 2.44 2.24 3.99 1.37 3.33 3.69
Flexibility per aa
Antigenic index per aa
1.07 1.05 1.02 1.03 1.02 1.03 1.04 1.03
1.2 1.24 0.99 1.19 1 1.2 0.94 1.33
Conformation ................. CF GOR h+t T T+H T H+t h H t
T T H T H+T T H T
aThe a m i n o acid n u m b e r is the s a m e as in the immature polypeptide (containing the 27 aa of the signal peptide),
surface probability value (the normalized value per aa is 5.47), with the highest values of flexibility and antigenicity noted in domain 1 of HSV-2 gB (Table 2). The secondary structure of aa domain 1 is a [3-turn, as determined by two computer programs (Table 1). Comparison of the aa sequence of this predicted antigenic domain with the putative antigenic domain 1 in HSV-2 gB (Table 2) revealed a marked homology with the aa of the two putative antigenic domains:
HSV-l(F)(66)
T K P K K N K K P K N P P P R P
t t f
I
I
HSV-2(333)(59)T K A R K R K Y K K -
If
(81)
It
P P K R P
(73)
Seven additional putative antigenic domains were detected by computer analysis, but only three (domains 1, 2, and 4) have the predicted 13-turn conformation, while the others had a combination of an a-helix and a combined [3-turn with an a-helix (Table 1). A similar situation can be noted with the putative antigenic domains of HSV-2 gB glycoprotein (Table 2). Since the GAP analysis of HSV-I gB glycoprotein to HSV-2 gB glycoprotein yielded 92.522% similarity and 87.723% identity, all putative antigenic domains in both glycoproteins resembled each other in the primary aa sequence. The predicted antigenic domains 3-6 are present in the outer portion of the gB, while domains 7 and 8 are located in the intracytoplasmic portion of the gB. The latter putative antigenic domains seem, on the basis of the physical properties of the aa in the antigenic domain, to be slightly better antigenically than the putative antigenic domains 5 and 6. Comparison of the aa sequences in the predicted antigenic domains 5, 6, and 7 of HSV-1 gB to the related sequences in HSV-2 gB, revealed a marked identity in the primary aa sequence.
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BECKER
Comparison of the computer-predicted putative antigenic domains with experimentally proven antigenic domains On the basis of computer analysis, putative antigenic domain 1 in HSV-I and HSV-2 gB glycoproteins is the dominant antigenic domain of the viral glycoprotein. This observation agrees with the findings of Mester et al. (11), who used the Garnier algorithm to predict the secondary structure of the major antigenic domain of HSV-1 gB glycoprotein. These authors produced antibodies to synthetic peptides, made according to the sequence of aa 63-t 10, and showed that a monoclonal antibody reacted with the synthetic peptide with the sequence aa 63-82. The computer prediction of the putative antigenic domains of HSV-I gB were compared with the experimental results reported by Periera et al. (8). Most of the experimentally proven antigenic domains in the extracytoplasmic portion of HSV-1 gB were also predicted by computer analysis.
Hydrophobic domains along the HSV-1 and HSV-2 gB glycoprotein serve as transfer signals and a stop-transfer signal is present at the membrane anchor hydrophobic domain In the aa sequence of HSV-1 or HSV-2 gB glycoproteins clusters of hydrophobic aa can be noted (Tables 3 and 4). Altogether 19 hydrophobic domains, with a total of 115 hydrophobic aa, ranging from 5-30 aa per domain, are present in the gB glycoproteins of both viruses. The signal peptide is the N terminus of the glycoproteins (aa 1-30 in HSV-1 and aa 1-27 in HSV-2), and the three hydrophobic domains 15-17 contain 13, 25, and 27 hydrophobic aa, respectively. These hydrophobic domains are considered to be the membrane-anchoring sequences of HSV-1 gB, and the polypeptide spans the membrane three times (6,10). It is suggested that the multiple hydrophobic domains in the viral polypeptide are involved in the process of transfer of the viral gB glycoprotein through the cellular membrane by the signal recognition complex (SRP) [recently reviewed by Saier et al. (26)]. It was reported (26) that the signal recognition protein (53 kD) interacts with the signal peptide of the viral glycoprotein and later interacts with the protein-RNA complex that is bound to the docking protein that is inserted into the inner side of the cellular membrane. The RNA-protein-polypeptide complex transfers the signal peptide of the glycoprotein through a hole in the membrane and translocates the polypeptide through the membrane by pushing the polypeptide through this hole. When the hydrophobic aa sequences that anchor the viral gB polypeptide chain are inserted into the membrane the transfer of the polypeptide comes to a hault when the SRP complex encounters a signal to stop transfer (14). Our studies (Y. Becker et al., to be published) revealed the presence of the expression of several SRP complex genes in cells infected by HSV-1. Thus the insertion of the viral glycoproteins into the cell membrane and the transfer of the viral polypeptide through the membrane is most probably
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HSV-1 A N D HSV-2 gB
Table 3. H y d r o p h o b i c a m i n o acid d o m a i n s in HSV-I(F) gB glycoprotein Conformation Function
Position
No. aa
1 Signal peptide 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M e m b e r a n c h o r 16 M e m b e r a n c h o r 17 M e m b e r anchor 18 19
1-30 a 37-43 88-93 120-124 144-151 267-274 285-297 444-453 456-462 521-528 549-553 563-580 642-647 663-675 727-739 745-769 771-797 847-854 868-874
[30] [7} [6] [5] [8] [8] [13] [10] [7] 18] [5] [18] 16] [13] [13] [25] [27] 18] [7]
Stop transfer
M R L Q(801)
CF
GOR
H+T+B+H T+H h+ H B B B B B+T+B t+b b h B T B H h+ B H B L
T+B -H B H B B+T+B T+B -H B B T B H B H H H
aHydrophobic d o m a i n s (//-5 aa).
Table 4. H y d r o p h o b i c a m i n o acid domain in HSV-2 (333) gB glycoprotein
Function
Position
No. aa
1 Signal peptide 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M e m b r a n e anchor 16 M e m b r a n e a n c h o r 17 M e m b r a n e a n c h o r 18 19
aa
[27] [10] [7] [5] [51 [8] [8] [13] [tl] [7] [8] [13] [7] [13] [13] [25] [27] [8] [6]
1-27 ~ 34-43 83-89 107-111 116-120 140-147 263-270 281-293 439-449 452-458 519-526 561-573 639-645 661-673 725-737 743-767 769-795 848-855 869-874
aHydrophobic d o m a i n s (/>5 aa).
Stop transfer
L Q L Q (799)
Secondary structure .... CF GOR T+ B+ H h+H h B B B B B+T+B b b b+h h+B B+T B H B h+ B B h
T+B+H H+T+B H B B H T+B T+B H H H B B+T B+H H B H H B
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BECKER
Table 5. Comparisons of the matjor antigenic domain in HSV-I and HSV-2 glycoprotein B and the amino acid sequence in ApoE that th binds to the LDL receptors HSV-I (17) HSV-1 (KOS) HSV-t (F) HSV-2 (333) Apo E Apo B
[67] [66] [661 [59] [167] [3386]
P K P K K NR K P K P P K P P R P P K P K K N K K P K N P T P P R P
T KP KKNKKP KNP P P P R P T K A R K R K T K K P P K R P
L R KL RKRL L R DADDL R L T R K R G L K L A
[83] [82] [82] [72] [173] [3396]
carried out by the SPR complex using the hydrophobic domains in the polypeptide chain. Further studies in this area are in progress. The stop-transfer signal in HSV-I gB glycoprotein is (798)MRLQ(801) (Table 3), and in HSV-2 gB glycoprotein it is (796)LQLQ(799) (Table 4). These stoptransfer sequences differ from those in the HSV-1 gD glycoprotein (RRHT) or the HSV-2 gD glycoprotein (RRRR) (14). The reason for the difference in the two types stop-transfer signals and its significance are not known.
Possible function o f the putative antigenic domains 1 in HSV-I and HSV-2 glycoproteins Antibodies to the major antigenic domain at the N terminus of HSV-1 gB were reported to neutralize virus infectivity (8,9,11). Quadri et al. (10) indicated that gB glycoprotein is required for the penetration of virions into the infected cells. Since HSV-1 and HSV-2 have a conserved antigenic domain 1 (Table 5), it was of interest to determine if there is sequence homology to other proteins that are known to interact with specific cellular receptors. Recently, Dyer and Curtiss (27) identified the aa sequence in the human plasma apolipoprotein E [Apo E (28)] that binds to the low-density lipid (LDL) receptor [ L D L R (29)] binding domains (Table 5) Analysis of the aa sequence of apo E and apo B (30) revealed a homology with the R K R sequence of apoE amino acid, which is involved in binding to the L D L R binding domain. Comparison of these sequences to the aa sequence o f HSV-2 putative antigenic domain 1 (Table 5) revealed that the viral gB glycoprotein contains a sequence that mimicks part of the L D L R binding sequence of apo E and apo B. HSV-2 gB putative domain 1 also resembles the apo E binding sequence. The significance of this observation and the question of the involvement of L D L R in the entry of HSV-1 and HSV-2 into cells are currently under investigation.
Discussion The c o m p u t e r programs compiled by Wolf et al. (10) and Jameson and Wolf (18) that are available in the U W G C G software allow the analysis o f the properties
HSV-I AND HSV-2 gB
139
of the aa in a polypeptide, providing a basis for defining antigenic domains in polypeptides, as well as topogenic domains. The definition of a putative antigenic domain is based on the computed values for individual aa in the polypeptide chain, using algorithms that calculate the hydrophilicity, surface probability, flexibility, antigenic index, and conformational organization of the peptide. A distinct antigenic domain is identified by its high hydrophilicity value, a high value for surface probability, and the conformation of a 13-turn or an a-helix. To compare the properties of different putative antigenic domains in a polypeptide, the computed values for a predicted antigenic domain are normalized to obtain the average value per aa in the putative antigenic domain. The normalized value for each of the physical properties is used in the comparison of different predicted antigenic domains in the same polypeptides and in polypeptides coded by related viruses of the same virus family. The usefulness of the computer predictions has also been studied in analyses of HIV-1 envelope proteins (12-14). In this study the computer programs were able to detect antigenic domains in the envelope protein that had been reported to be antigenic on the basis of experimental studies in which synthetic peptides were used. The present study deals with the prediction of antigenic and topogenic domains in HSV-! gB, and in HSV-2 gB glycoproteins, which are thought to be involved in the penetration of the herpes simplex virions into cells (10). The sequence of the HSV-1 UL27 gene and the aa sequence of the glycoprotein were reported (1-6). The antigenic properties of the viral glycoprotein were studied with the help of monoclonal antibodies. Periera et al. (8-10) experimentally mapped the antigenic domains in HSV-1 gB glycoproteins. It was therefore of interest to use computer programs for the prediction of putative antigenic domains and to compare them with the experimentally reported domains. The Garnier algorithm was applied by Mester et al. (11) to the analysis of the major antigenic domain 1 of HSV-I gB glycoprotein, and the antigenicity of this domain was determined by producing antibodies to synthetic peptides modelled according to the aa sequence of the antigenic domain. The present computer prediction agrees with the results of Mester et al. (11) and Periera et al. (8) in suggesting that the strongest antigenic domain in HSV-1 and HSV-2 gB glycoprotein resides in aa 62-81 (Table 1) and aa 60-79 (Table 2), respectively. The additional computer-derived putative antigenic domains in the extraceilular sequence of gB are in agreement with the antigenic domains reported by Pereira and collaborators (8-10). Studies by Chapsal and Pereira (7) showed that the antigenic epitopes of four potent neutralizing antibodies map within the strongly hydrophitic peak of HSV-1 gB: "Twenty antibodies recognized both denatured and native forms of gB, thus reacting with a continuous set of amino acids present on monomers and dimers: nine reactive with surface domains had neutralizing activity; of these, five were HSV-1 specific and one was HSV-2 specific. Seven failed to recognize surface domains and five antibodies failed to precipitate gB made in Vero cells." The present computer analyses of putative antigenic domains provide six antigenic domains in the extracellular portion of gB. This does not rule out the possibility
140
BECKER
that additional aa sequences in HSV-1 gB are antigenic. The discontinous antigenic epitopes cannot be predicted by computer analysis. Putative antigenic domain I is located in HSV-1 or HSV-2 gB polypeptide very close to the N terminus from which the signal peptide is removed after transfer of the polypeptide through the cellular membrane. The aa in this antigenic domain are conserved in HSV-I and HSV-2 gB glycoproteins, suggesting a possible function for the aa domains. Since the fibroblast growth-factor receptor (FGFR) was suggested by Kaner et al. (31) as the cellular receptor and a portal of cellular entry for Herpes simplex 1, the aa sequence of fibroblast growth factor (FGF) was compared to the aa sequence of the gB major antigenic domain. No aa homology was found. In contrast, homology was found with the aa sequence of apoE reported by Dyer and Curtiss (27) to interact with its receptor (LDLR). Since LDL receptors are present on the outer membranes of all cell types (32) and are active in endocytosis of the receptor-bound LDL, the resemblance of apoB, and the apoE aa sequence involved in the binding to the LDLR, were compared to the aa sequence of the major antigenic domain in HSV-1 and HSV-2. The results (Table 5) suggest that both types o f a a sequences resemble each other. This analysis might suggest that HSV-1 and HSV-2 entry into the infected cell after their initial interaction with heparan sulfate present on the cell membranes (33) might be by internalization due to the interaction with LDLR. Preliminary experiments on the role of L D L R in the entry of HSV-I into cells suggest that infection by HSV-1 requires the presence of LDLR on cells (Y. Becker et al., to be published). The computer analysis of HSV-t and HSV-2 glycoprotein gB also provided information on the hydrophobic aa domains along the polypeptide chain. This information might be of value when the mechanisms of transfer of the gB polypeptide through the cellular membrane are elucidated. The polypeptide transfer process by the signal recognition protein (SRP) complex (26) has yet to be investigated. Computer analysis of virus-coded polypeptides may provide valuable information on the properties of antigenic and topogenic domains in such proteins, with the potential of helping in the design of experimental approaches to the study of viral glycoproteins.
Acknowledgments This study was supported by a grant from the Foundation for the Study of Molecular Virology and Cell Biology, Phoenix, Arizona.
References 1. McGeoch D.J., Dalrymple M.A., Davison A.J, Dolan A., Frame M.C., NcNab D., Perry L.J., Scott J.E., and Taylor P., J Gen Virol 69, 1531-1574, 1987.
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2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.
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Pellet P.E., Kousoulas K.G., Pereira L., and Roizman B., J Viro153, 243-253, 1985. Bzik DA., Fox B.A., DeLuca N.A., and Person S., Virology 133, 301-314, 1984. Bzik D.J., Debroy C., Fox B., Pederson N.E., and Person S., Virology 155, 322-333, 1986. Pellet P.E., Biggen M.D., Barrell B , and Roizman B.J., Virology 56, 807-813, 1985. Pellet P.E., Kousoulas K.G., Pereira L., and Roizman B., J Virol 53, 243-253, 1985. Chapsal J.M. and Pereira L., Virology 164,427-434, 1988. Pereira L., Ali M., Kousoulas K., Huo B., and Banks T., Virology 172, 11-24, 1989. Kousoulas K., Arsenakis M., and Pereira L., J Gen Virol 70, 135-152, 1991. Quadri I., Gimeno C., Navarro D., and Pereira L., Virology 180, 135-152, 1991. Mester J.C., Highlander S.L., Osmand A.P., Glorioso J.C., and Rouse B.T. J Viro164, 5277-5283, 1990. Becker Y., Virus Genes 3,323-341, 1990. Becket Y., Virus Genes 3,267-282, 1990. Becker Y., Virus Genes 5, 287-312, 1991. George D.G., Barker W.C., and Hunt L . T , Nucleic Acids Res 14, 11-16, 1986. Devereux J., Haberlin P., and Smithies O., Nucleic Acid Res 12, 387-395, 1984. WolfH., Modrow S., Motz M., Jameson B.A., Hermann G., and F6rtsch B., CABIOS4, 187-191, 1988. Hopp T.P. and Woods K.R., Proc Natl Acad Sci USA 78, 3824-3828, 1981. Hopp T.P. and Woods K.R., Molec lmmunol 20, 483-489, 1983. Kyte J. and Doolittle R.F., J Mol Biol 157, 105-132, 1982. Emini E.A., Hughes J.V., Perlow D.S., and Boger J., J Virol 55, 836-839, 1985. Karplus P.A. and Schulz G.E., Naturwissenschaften 72, 212-213, 1985. Chou P.Y, and Fasman G.D., Ann Rev Biochem 47, 251-276, 1978. Garnier J., Osguthorpe D.J., and Robson B., J Mol Biol 120, 97-120, 1978. Jameson B.A. and Wolf H., CABIOS 4, 181-186, 1988. Saier M.H. Jr., Werner P.K., and Muller M., Microbiol Rev 53, 333-366, 1989. Dyer C.A. and Curtiss L.K. (Page Award Abstract) Circulation 82 (Suppl 3), III-0, 1990. Zannis V . I , McPherson J., Goldberger G., Karathanasis S.K., and Breslow J.L., J Biot Chem 259, 5495-5499, 1984. Yamamoto Y , Davis C.G., Brown M.S., Schneider W.J., Casey M.L., Goldstein M.L., and Russell D.W., Cell 39, 27-38, t984. Cladaras C., Hadzopoulou-Cladaras M., Nolte R.T., Atkinson D., and Zannis V.I. Embo J 5, 3495-3507, 1986. Kaner R.J., Baird A., Mansukhani A., Basilico C., Summers B.D., Florkiewicz R.Z., and Hajjar D.P., Science 248, 1410-1413, 1990. Goldstein J.L., and Brown M.S., Ann Rev Biochem 46, 897-930, 1977. WuDunn D. and Spear P.G., J Virol 63, 52-58, 1989.
Computer Prediction of Antigenic and Topogenic Domains in HSV-1 and HSV-2 Glycoprotein B (gB) YECHIEL BECKER
Department of Molecular Virology, Faculty of Medicine, Hebrew UniversiO, of Jerusalem, Jerusalem, Israel Received March 21, 1991 Accepted June 24, 1991 Requests for reprints should be addressed to Yechiel Becker, Department of Molecular Virology, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel. Key words: antigenic domains, topogenic domains, computer prediction, HSV-1/HSV-2, glycoprotein B (gB)
Abstract
The envelope glycoprotein B (gB) coded for by the herpes simplex virus type 1 (HSV-1) Ut27 gene is similar to the amino acid (aa) sequence of the gB coded by a homologous gene in HSV-2 DNA. The putative antigenic domains in HSV-1 and HSV-2 gB glycoproteins were analyzed on a comparative basis by suitable computer programs, which allowed the prediction of putative antigenic and topogenic domains. The computer-derived domains were compared to experimentally reported antigenic domains in HSV-1 gB glycoprotein. The computer-predicted antigenic domains in the HSV-1 gB glycoprotein matched well with the reported experimentally derived antigenic domains. The aa sequence of antigenic domain 1 was noted to resemble the amino acid sequence in ApoE that is involved in the attachment of this protein to LDL receptors. The clusters of hydrophobic aa domains are conserved in the two viral glycoproteins and are signals for transfer of the viral proteins through the cellular membrane. Introduction
The envelope glycoprotein B (gB) of herpes simplex virus type 1 (HSV-I) is coded by a gene (UL27) in the viral genome (1). This protein is involved in virus entry into cells and induces fusion of the virion envelope with the host cell membrane. The amino acid (aa) sequence of HSV-1 gB is a long polypeptide chain with hydrophobic domains (2,3). The HSV-1 gB revealed a close similarity to the aa sequence of the gB coded by HSV-2 (4). The HSV-1 gB was also found to have homology with the protein product of the Epstein-Barr virus (EBV) BAL4
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BECKER
open reading flame (ORF) (5) and the varicella-zoster virus (VZV) gene 31 protein product (6). Further studies by Pereira and associates (7-10) revealed that HSV-I gB polypeptides formed dimers on the cell membrane, and neutralizable antigenic epitopes were found to cluster in two domains: one in the amino-terminal 190 aa residues (seven continuous epitopes, five of which are HSV-1 specific). Antibodies reacted with synthetic peptides from the N terminus of HSV-1 gB glycoprotein mapped with epitopes between residues 1 and 47 in the mature polypeptide from which the 30 aa signal peptide was removed (8,9). The second domain is composed of discontinuous epitopes, including domains between aa residues 273 and 298. Mester et al. (11) searched for antigenic domains in HSV-1 gB glycoprotein by synthesizing three overlapping peptides (two 20-mers and one 18-mer) spanning aa 63-1t0 of the immature polypeptide chain (including the 30 aa of the signal peptide). These authors used the Garnier algorithm to predict the secondary structure of the above polypeptide sequence and reported that a monoclonal antibody reacted with the two peptides spanning aa 63-95 (with an overlap at aa 78-81) in HSV-1 gB. The antigenic domain is present in a random conformation of the polypeptide between a 13-turn and an a-helix. Antigenic epitopes of HSV-I gB were characterized by Chapsal and Pereira (7). Monoclonal antibodies that recognized native proteins reacted with discontinuous epitopes of gB dimers, whereas monoclonal antibodies reactive with denatured gB polypeptide recognized continuous epitopes on monomeric or dimeric proteins. Since computer programs that provide information on the physical properties of aa in polypeptide chains can be used to predict putative antigenic domains (continuous epitopes) in viral glycoproteins (12-14), it was of interest to determine if the experimentally proven antigenic domains in gB glycoproteins of HSV-1 and HSV-2 predicted by the computer analyses agree with the experimentally proven antigenic epitopes. Since the computer programs (15-25) provide information on the hydrophobicity and hydrophilicity of the aa, it is possible to use these physical properties for the definition of topogenic functional domains in the viral protein. Such domains may be involved in the interactions of HSV-1 virions with receptors on cell membranes, and in the interaction of the viral protein with the signal recognition protein (SRP) complex (26), which is involved in the intracytoplasmic transfer of the glycoprotein through the cell membrane during the genesis of the viral membrane. The aim of the present study is to define, with the help of computer programs, topogenic domains in HSV-1 and HSV-2 gB gtycoproteins and to assess their functional and biological properties in comparison with experimentally defined domains.
Methods Primary amino acid sequence
The aa sequences of gB glycoproteins of HSV-I(F), HSV-I(Synl7), HSVI(KOS), and HSV-2(333), as well as VZV, and EBV-related proteins and apolipo
HSV-I AND HSV-2 gB
133
proteins E and B, were obtained through the NBRF Protein Sequence Data Bank from the N B R F Protein Identification Resource (15). This program is available from the University of Wisconsin Genetics Computer Group (GCG) software (16).
Computer programs The compilation of seven algorithms in one program by Wolf et al. (17) provides information for analyzing the properties of aa in a polypeptide chain by using the primary aa sequence of the peptide. The following properties of aa were studied: a) hydrophilicity according to Hopp and Woods (18,19) or Kyte and Doolittle (20), b) surface probability according to Emini et al. (21), c) chain flexibility according to Kai~lus and Schulz (22), d) secondary structure according to Chou and Fasman (23) and Garnier-Osguthorpe-Robson (24), and e) the antigenicity index according to Wolf et al. (17). All of these provide quantitative estimates for hydrophilicity, surface probability, chain flexibility, and the antigenicity index. The GAP program (16) was used to compare the sequences of HSV-1 gB to HSV-2 gB. The properties of the two gB aa sequences were determined by the programs compiled by Jameson and Wolf (25) and Wolf et al. (17). In the present analysis an antigenic domain was selected on the basis of the highest values of hydrophilicity, surface probability, and flexibility of consecutive aa in the gB. The predicted secondary structure of the polypeptide was also taken into account, since domains in polypeptides with a 13-turn conformation were found to have a better antigenicity value than aa sequences with a [3-sheet conformation. To obtain a numerical value for the physical property of each aa in the polypeptide that constitutes a putative antigenic domain, the normalized value was determined by summing up the specific values (such as hydrophilicity, etc.) of all aa in the domain and dividing them by the number of aa in that domain. This normalized value per aa was used for the comparison of different putative antigenic domains.
Results
Putative antigenic domains in HSV-1 and HSV-2 gB glycoproteins Computer analysis of the HSV-1 gB polypeptide (Table 1) revealed eight aa domains that have properties compatible with antigenic domains (e.g., a relative high hydrophilicity, surface probability, and antigenic index, and a 13-turn or a-helix conformation). Six putative antigenic domains are in the outer portion of the glycoprotein, which is inserted into the cellular membrane, and two are in the intracytoplasmic aa sequence. It was noted that the aa sequence aa 62-81 (putative antigen domain 1) has the most pronounced computer-derived physical properties of an antigenic domain in the polypeptide chain. When compared with the seven other putative antigenic domains, the hydrophilicity value of the aa sequence for domain I is the highest (the normalized value per aa is 2.31). A high
2.31 2.1 2.28 1.48 1.92 1.55 1.9 2.13
No. aa
62-81 a 127-141 223-230 250-257 463-481 700-712 855-867 879-888
Domain no,
1 2 3 4 5 6 7 8
1.8 2,44 2,64 2.29 3,19 2,05 2,98 3.9
Surface probability per aa 1.8 1.06 1.02 1.03 1.04 1 1.04 1.02
Flexibility prediction per aa 1.4 1.3 1.03 1.15 1.02 0.88 0.9 1.13
Antigenic index per aa
aThe amino acid number is the same as in the immature polypeptide (containing the 30 aa of the signal peptide).
(20) (15) (8) (8) (19) (13) (13) (10)
Hydrophilicity per aa
Table 1. Probable antigenic domains in HSV-1 (F) glycoprotein B
T T T+H T H+T h H h+t+B
CF prediction
T T H T H+T T H H+T
GOR p~diction
¢'3 7~
135
HSV-I A N D HSV-2 gB
Table 2. Putative antigenic d o m a i n s in HSV-2 (333) glycoprotein B
Domain no.
Domain
1 2 3 4 5 6 7 8
60-79 ~ 122-138 219-238 245-253 459-474 700-707 957-867 882-889
No. aa
Surface probability per aa
Hydrophilicity per aa
20 17 10 9 16 8 1t 8
2.43 2.92 2.06 1.46 2.15 2.06 2.23 2.02
5.47 2.28 2.44 2.24 3.99 1.37 3.33 3.69
Flexibility per aa
Antigenic index per aa
1.07 1.05 1.02 1.03 1.02 1.03 1.04 1.03
1.2 1.24 0.99 1.19 1 1.2 0.94 1.33
Conformation ................. CF GOR h+t T T+H T H+t h H t
T T H T H+T T H T
aThe a m i n o acid n u m b e r is the s a m e as in the immature polypeptide (containing the 27 aa of the signal peptide),
surface probability value (the normalized value per aa is 5.47), with the highest values of flexibility and antigenicity noted in domain 1 of HSV-2 gB (Table 2). The secondary structure of aa domain 1 is a [3-turn, as determined by two computer programs (Table 1). Comparison of the aa sequence of this predicted antigenic domain with the putative antigenic domain 1 in HSV-2 gB (Table 2) revealed a marked homology with the aa of the two putative antigenic domains:
HSV-l(F)(66)
T K P K K N K K P K N P P P R P
t t f
I
I
HSV-2(333)(59)T K A R K R K Y K K -
If
(81)
It
P P K R P
(73)
Seven additional putative antigenic domains were detected by computer analysis, but only three (domains 1, 2, and 4) have the predicted 13-turn conformation, while the others had a combination of an a-helix and a combined [3-turn with an a-helix (Table 1). A similar situation can be noted with the putative antigenic domains of HSV-2 gB glycoprotein (Table 2). Since the GAP analysis of HSV-I gB glycoprotein to HSV-2 gB glycoprotein yielded 92.522% similarity and 87.723% identity, all putative antigenic domains in both glycoproteins resembled each other in the primary aa sequence. The predicted antigenic domains 3-6 are present in the outer portion of the gB, while domains 7 and 8 are located in the intracytoplasmic portion of the gB. The latter putative antigenic domains seem, on the basis of the physical properties of the aa in the antigenic domain, to be slightly better antigenically than the putative antigenic domains 5 and 6. Comparison of the aa sequences in the predicted antigenic domains 5, 6, and 7 of HSV-1 gB to the related sequences in HSV-2 gB, revealed a marked identity in the primary aa sequence.
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BECKER
Comparison of the computer-predicted putative antigenic domains with experimentally proven antigenic domains On the basis of computer analysis, putative antigenic domain 1 in HSV-I and HSV-2 gB glycoproteins is the dominant antigenic domain of the viral glycoprotein. This observation agrees with the findings of Mester et al. (11), who used the Garnier algorithm to predict the secondary structure of the major antigenic domain of HSV-1 gB glycoprotein. These authors produced antibodies to synthetic peptides, made according to the sequence of aa 63-t 10, and showed that a monoclonal antibody reacted with the synthetic peptide with the sequence aa 63-82. The computer prediction of the putative antigenic domains of HSV-I gB were compared with the experimental results reported by Periera et al. (8). Most of the experimentally proven antigenic domains in the extracytoplasmic portion of HSV-1 gB were also predicted by computer analysis.
Hydrophobic domains along the HSV-1 and HSV-2 gB glycoprotein serve as transfer signals and a stop-transfer signal is present at the membrane anchor hydrophobic domain In the aa sequence of HSV-1 or HSV-2 gB glycoproteins clusters of hydrophobic aa can be noted (Tables 3 and 4). Altogether 19 hydrophobic domains, with a total of 115 hydrophobic aa, ranging from 5-30 aa per domain, are present in the gB glycoproteins of both viruses. The signal peptide is the N terminus of the glycoproteins (aa 1-30 in HSV-1 and aa 1-27 in HSV-2), and the three hydrophobic domains 15-17 contain 13, 25, and 27 hydrophobic aa, respectively. These hydrophobic domains are considered to be the membrane-anchoring sequences of HSV-1 gB, and the polypeptide spans the membrane three times (6,10). It is suggested that the multiple hydrophobic domains in the viral polypeptide are involved in the process of transfer of the viral gB glycoprotein through the cellular membrane by the signal recognition complex (SRP) [recently reviewed by Saier et al. (26)]. It was reported (26) that the signal recognition protein (53 kD) interacts with the signal peptide of the viral glycoprotein and later interacts with the protein-RNA complex that is bound to the docking protein that is inserted into the inner side of the cellular membrane. The RNA-protein-polypeptide complex transfers the signal peptide of the glycoprotein through a hole in the membrane and translocates the polypeptide through the membrane by pushing the polypeptide through this hole. When the hydrophobic aa sequences that anchor the viral gB polypeptide chain are inserted into the membrane the transfer of the polypeptide comes to a hault when the SRP complex encounters a signal to stop transfer (14). Our studies (Y. Becker et al., to be published) revealed the presence of the expression of several SRP complex genes in cells infected by HSV-1. Thus the insertion of the viral glycoproteins into the cell membrane and the transfer of the viral polypeptide through the membrane is most probably
137
HSV-1 A N D HSV-2 gB
Table 3. H y d r o p h o b i c a m i n o acid d o m a i n s in HSV-I(F) gB glycoprotein Conformation Function
Position
No. aa
1 Signal peptide 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M e m b e r a n c h o r 16 M e m b e r a n c h o r 17 M e m b e r anchor 18 19
1-30 a 37-43 88-93 120-124 144-151 267-274 285-297 444-453 456-462 521-528 549-553 563-580 642-647 663-675 727-739 745-769 771-797 847-854 868-874
[30] [7} [6] [5] [8] [8] [13] [10] [7] 18] [5] [18] 16] [13] [13] [25] [27] 18] [7]
Stop transfer
M R L Q(801)
CF
GOR
H+T+B+H T+H h+ H B B B B B+T+B t+b b h B T B H h+ B H B L
T+B -H B H B B+T+B T+B -H B B T B H B H H H
aHydrophobic d o m a i n s (//-5 aa).
Table 4. H y d r o p h o b i c a m i n o acid domain in HSV-2 (333) gB glycoprotein
Function
Position
No. aa
1 Signal peptide 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M e m b r a n e anchor 16 M e m b r a n e a n c h o r 17 M e m b r a n e a n c h o r 18 19
aa
[27] [10] [7] [5] [51 [8] [8] [13] [tl] [7] [8] [13] [7] [13] [13] [25] [27] [8] [6]
1-27 ~ 34-43 83-89 107-111 116-120 140-147 263-270 281-293 439-449 452-458 519-526 561-573 639-645 661-673 725-737 743-767 769-795 848-855 869-874
aHydrophobic d o m a i n s (/>5 aa).
Stop transfer
L Q L Q (799)
Secondary structure .... CF GOR T+ B+ H h+H h B B B B B+T+B b b b+h h+B B+T B H B h+ B B h
T+B+H H+T+B H B B H T+B T+B H H H B B+T B+H H B H H B
138
BECKER
Table 5. Comparisons of the matjor antigenic domain in HSV-I and HSV-2 glycoprotein B and the amino acid sequence in ApoE that th binds to the LDL receptors HSV-I (17) HSV-1 (KOS) HSV-t (F) HSV-2 (333) Apo E Apo B
[67] [66] [661 [59] [167] [3386]
P K P K K NR K P K P P K P P R P P K P K K N K K P K N P T P P R P
T KP KKNKKP KNP P P P R P T K A R K R K T K K P P K R P
L R KL RKRL L R DADDL R L T R K R G L K L A
[83] [82] [82] [72] [173] [3396]
carried out by the SPR complex using the hydrophobic domains in the polypeptide chain. Further studies in this area are in progress. The stop-transfer signal in HSV-I gB glycoprotein is (798)MRLQ(801) (Table 3), and in HSV-2 gB glycoprotein it is (796)LQLQ(799) (Table 4). These stoptransfer sequences differ from those in the HSV-1 gD glycoprotein (RRHT) or the HSV-2 gD glycoprotein (RRRR) (14). The reason for the difference in the two types stop-transfer signals and its significance are not known.
Possible function o f the putative antigenic domains 1 in HSV-I and HSV-2 glycoproteins Antibodies to the major antigenic domain at the N terminus of HSV-1 gB were reported to neutralize virus infectivity (8,9,11). Quadri et al. (10) indicated that gB glycoprotein is required for the penetration of virions into the infected cells. Since HSV-1 and HSV-2 have a conserved antigenic domain 1 (Table 5), it was of interest to determine if there is sequence homology to other proteins that are known to interact with specific cellular receptors. Recently, Dyer and Curtiss (27) identified the aa sequence in the human plasma apolipoprotein E [Apo E (28)] that binds to the low-density lipid (LDL) receptor [ L D L R (29)] binding domains (Table 5) Analysis of the aa sequence of apo E and apo B (30) revealed a homology with the R K R sequence of apoE amino acid, which is involved in binding to the L D L R binding domain. Comparison of these sequences to the aa sequence o f HSV-2 putative antigenic domain 1 (Table 5) revealed that the viral gB glycoprotein contains a sequence that mimicks part of the L D L R binding sequence of apo E and apo B. HSV-2 gB putative domain 1 also resembles the apo E binding sequence. The significance of this observation and the question of the involvement of L D L R in the entry of HSV-1 and HSV-2 into cells are currently under investigation.
Discussion The c o m p u t e r programs compiled by Wolf et al. (10) and Jameson and Wolf (18) that are available in the U W G C G software allow the analysis o f the properties
HSV-I AND HSV-2 gB
139
of the aa in a polypeptide, providing a basis for defining antigenic domains in polypeptides, as well as topogenic domains. The definition of a putative antigenic domain is based on the computed values for individual aa in the polypeptide chain, using algorithms that calculate the hydrophilicity, surface probability, flexibility, antigenic index, and conformational organization of the peptide. A distinct antigenic domain is identified by its high hydrophilicity value, a high value for surface probability, and the conformation of a 13-turn or an a-helix. To compare the properties of different putative antigenic domains in a polypeptide, the computed values for a predicted antigenic domain are normalized to obtain the average value per aa in the putative antigenic domain. The normalized value for each of the physical properties is used in the comparison of different predicted antigenic domains in the same polypeptides and in polypeptides coded by related viruses of the same virus family. The usefulness of the computer predictions has also been studied in analyses of HIV-1 envelope proteins (12-14). In this study the computer programs were able to detect antigenic domains in the envelope protein that had been reported to be antigenic on the basis of experimental studies in which synthetic peptides were used. The present study deals with the prediction of antigenic and topogenic domains in HSV-! gB, and in HSV-2 gB glycoproteins, which are thought to be involved in the penetration of the herpes simplex virions into cells (10). The sequence of the HSV-1 UL27 gene and the aa sequence of the glycoprotein were reported (1-6). The antigenic properties of the viral glycoprotein were studied with the help of monoclonal antibodies. Periera et al. (8-10) experimentally mapped the antigenic domains in HSV-1 gB glycoproteins. It was therefore of interest to use computer programs for the prediction of putative antigenic domains and to compare them with the experimentally reported domains. The Garnier algorithm was applied by Mester et al. (11) to the analysis of the major antigenic domain 1 of HSV-I gB glycoprotein, and the antigenicity of this domain was determined by producing antibodies to synthetic peptides modelled according to the aa sequence of the antigenic domain. The present computer prediction agrees with the results of Mester et al. (11) and Periera et al. (8) in suggesting that the strongest antigenic domain in HSV-1 and HSV-2 gB glycoprotein resides in aa 62-81 (Table 1) and aa 60-79 (Table 2), respectively. The additional computer-derived putative antigenic domains in the extraceilular sequence of gB are in agreement with the antigenic domains reported by Pereira and collaborators (8-10). Studies by Chapsal and Pereira (7) showed that the antigenic epitopes of four potent neutralizing antibodies map within the strongly hydrophitic peak of HSV-1 gB: "Twenty antibodies recognized both denatured and native forms of gB, thus reacting with a continuous set of amino acids present on monomers and dimers: nine reactive with surface domains had neutralizing activity; of these, five were HSV-1 specific and one was HSV-2 specific. Seven failed to recognize surface domains and five antibodies failed to precipitate gB made in Vero cells." The present computer analyses of putative antigenic domains provide six antigenic domains in the extracellular portion of gB. This does not rule out the possibility
140
BECKER
that additional aa sequences in HSV-1 gB are antigenic. The discontinous antigenic epitopes cannot be predicted by computer analysis. Putative antigenic domain I is located in HSV-1 or HSV-2 gB polypeptide very close to the N terminus from which the signal peptide is removed after transfer of the polypeptide through the cellular membrane. The aa in this antigenic domain are conserved in HSV-I and HSV-2 gB glycoproteins, suggesting a possible function for the aa domains. Since the fibroblast growth-factor receptor (FGFR) was suggested by Kaner et al. (31) as the cellular receptor and a portal of cellular entry for Herpes simplex 1, the aa sequence of fibroblast growth factor (FGF) was compared to the aa sequence of the gB major antigenic domain. No aa homology was found. In contrast, homology was found with the aa sequence of apoE reported by Dyer and Curtiss (27) to interact with its receptor (LDLR). Since LDL receptors are present on the outer membranes of all cell types (32) and are active in endocytosis of the receptor-bound LDL, the resemblance of apoB, and the apoE aa sequence involved in the binding to the LDLR, were compared to the aa sequence of the major antigenic domain in HSV-1 and HSV-2. The results (Table 5) suggest that both types o f a a sequences resemble each other. This analysis might suggest that HSV-1 and HSV-2 entry into the infected cell after their initial interaction with heparan sulfate present on the cell membranes (33) might be by internalization due to the interaction with LDLR. Preliminary experiments on the role of L D L R in the entry of HSV-I into cells suggest that infection by HSV-1 requires the presence of LDLR on cells (Y. Becker et al., to be published). The computer analysis of HSV-t and HSV-2 glycoprotein gB also provided information on the hydrophobic aa domains along the polypeptide chain. This information might be of value when the mechanisms of transfer of the gB polypeptide through the cellular membrane are elucidated. The polypeptide transfer process by the signal recognition protein (SRP) complex (26) has yet to be investigated. Computer analysis of virus-coded polypeptides may provide valuable information on the properties of antigenic and topogenic domains in such proteins, with the potential of helping in the design of experimental approaches to the study of viral glycoproteins.
Acknowledgments This study was supported by a grant from the Foundation for the Study of Molecular Virology and Cell Biology, Phoenix, Arizona.
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