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Prediction of tyrosine sulfation sites in animal viruses
BBRC Biochemical and Biophysical Research Communications 312 (2003) 1154–1158 www.elsevier.com/locate/ybbrc
Prediction of tyrosine sulfation sites in animal viruses Henry C. Lin,a Kevin Tsai,a Brian L. Chang,a Justin Liu,a Melinda Young,a Willy Hsu,a Samuel Louie,b Hugh B. Nicholas Jr.,c and Grace L. Rosenquista,* b
a Section of Neurobiology, Physiology and Behavior, University of California, Davis, CA 95616, USA Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of California, Davis, Sacramento, CA 95817, USA c Pittsburgh Supercomputing Center, Pittsburgh, PA 15213, USA
Received 22 October 2003
Abstract Post-translational modification of proteins by tyrosine sulfation enhances the affinity of extracellular ligand–receptor interactions important in the immune response and other biological processes in animals. For example, sulfated tyrosines in polyomavirus and varicella-zoster virus may help modulate host cell recognition and facilitate viral attachment and entry. Using a Position-SpecificScoring-Matrix with an accuracy of 96.43%, we analyzed the possibility of tyrosine sulfation in all 1517 animal viruses available in the Swiss-Prot database. From a total of 97,729 tyrosines, we predicted 5091 sulfated tyrosine sites from 1024 viruses. Our site predictions in hemagglutinin of influenza A, VP4 of rotavirus, and US28 of cytomegalovirus strongly suggest an important link between tyrosine sulfation and viral disease mechanisms. In each of these three viral proteins, we observed highly conserved amino acid sequences surrounding predicted sulfated tyrosine sites. Tyrosine sulfation appears to be much more common in animal viruses than is currently recognized. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Tyrosine; Sulfation; Post-translational modification; Virus; Influenza A; Rotavirus; Cytomegalovirus
Tyrosine sulfation occurs when tyrosylprotein sulfotransferases (TPSTs) catalyze the transfer of a negatively charged sulfate from 30 -phosphoadenosine 50 -phosphosulfate (PAPS) to the hydroxyl group of a tyrosine residue on a polypeptide [1]. Since the demonstration of its prevalence in diverse mammalian cells in 1982 [2], tyrosine sulfation has been considered to be the most common post-translational modification of tyrosines transported through the trans-Golgi network [3]. Approximately 1% of all tyrosines may be sulfated [4]. Tyrosine sulfation has been experimentally shown to be essential for extracellular protein–protein interactions in many important biological processes in animals. For instance, sulfated tyrosines play a critical role in the immune response, such as in leukocyte rolling [5] and complement cascade [6], and promote HIV infection of T-helper lymphocytes [7–9].
We postulate that tyrosine sulfation performs similar functions in viruses. Based on our current understanding, all viral proteins that undergo tyrosine sulfation, such as capsid protein VP1 in polyomavirus and envelope glycoprotein gpI in varicella-zoster virus, must be modified in the host cell trans-Golgi apparatus during replication [10,11]. The purpose of tyrosine sulfation in these examples remains to be determined, but it is believed that specific sulfated tyrosines enhance target cell recognition, attachment, and entry by viruses [10,11]. Tyrosine sulfation in viruses has not been studied extensively since these publications. Detailed analyses of this post-translational modification in viral proteins may provide insights into the mechanisms of viral infection and the design of new therapies.
Materials and methods *
Corresponding author. Fax: 1-530-752-5582. E-mail addresses: [email protected], [email protected] (G.L. Rosenquist). 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.11.047
Acquisition of animal viral tyrosine sites. We obtained all known proteins of 1517 available animal viruses with the Site Retrieval System (http://us.expasy.org/srs5/) based on Swiss-Prot Release 41.19 of
H.C. Lin et al. / Biochemical and Biophysical Research Communications 312 (2003) 1154–1158 August 4th, 2003, using the keyword “virus” on the Organism Classification (OC) line of the database entry. As many as 97,729 tyrosine sites, which consist of target tyrosines and up to 5 flanking amino acids on each side [3], were extracted from these viral proteins using the Pick Sites program available at the Pittsburgh Supercomputing Center (Pittsburgh, PA). Position-Specific-Scoring-Matrix. The training set used in this study contained all currently known 69 sulfated and 454 non-sulfated tyrosine sites. The sulfated tyrosine sites have been identified experimentally and the non-sulfated tyrosine sites, extracted from sulfated proteins, are the remaining tyrosines for which sulfation has not been demonstrated. The Position-Specific-Scoring-Matrix (PSSM) was used to compare the distributions of amino acids around the query tyrosine sites with those of known sulfated and non-sulfated tyrosine sites. Each query tyrosine site was assigned a score measuring the similarity of the amino acids around the tyrosine to those around either the known sulfated tyrosine sites or those around non-sulfated tyrosine sites. Tyrosine sites with a high score are taken to have a high probability of sulfation. A detailed description of the construction of the PSSM was published previously [3]. When the PSSM was used to predict the occurrence of tyrosine sulfation in the training set, the sulfated tyrosine sites received a mean score of 6.890 with a standard deviation of 2.911, while the non-sulfated tyrosine sites received a mean score of )3.526 with a standard deviation of 3.234. To evaluate the ability of the PSSM to recognize sulfated tyrosine sites not included in its training set, 69 jackknifed PSSMs, each containing 68 sulfated and 454 non-sulfated sites, were used to evaluate the excluded sulfated site in each training set. The Receiver Operating Characteristic (ROC) [12] was then used to evaluate the predictions of the jackknifed PSSMs. The resulting ROC score of 0.9643 indicates that the PSSM is expected to have an accuracy of 96.43% in the prediction of unknown tyrosine sulfation sites. In order to minimize both the numbers of false positives and false negatives, the threshold score for a predicted tyrosine sulfation site was set at 2.5.
Results and discussion The PSSM was applied to animal viral proteins from Swiss-Prot and 5091 sulfated tyrosine sites were predicted in 1024 viruses. We focused on our predictions in three viruses responsible for causing human infectious diseases. Potential new roles for sulfated tyrosines were hypothesized based on our predictions in hemagglutinin of influenza A, VP4 of rotavirus, and US28 of cytomegalovirus. Although prior investigators suggested that tyrosine sulfation might be required for specific host recognition and optimal viral attachment and entry [10,11], no further corroborating studies have since been published. Our numerous predictions of sulfated tyrosines in viral proteins and the high conservation of predicted sites were initially surprising but led us to hypothesize that tyrosine sulfation is necessary for viral infection of host cells. We discuss below the specific predictions in hemagglutinin of influenza A, VP4 of rotavirus, and US28 of cytomegalovirus and the implications in mechanisms of disease. Hemagglutinin in influenza A Influenza viruses, which consist of types A, B, and C, belong to the family Orthomyxoviridae [13]. Of the three types, influenza type A is most responsible for deadly
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human pandemics [14]. For attachment to and subsequent invasion of target cells, the virus relies on the protein hemagglutinin (HA), which binds to sialic acid on human epithelial cells [13]. Once the virus has been internalized by the host cell endosome, HA is cleaved by host proteases into HA1 and HA2. The cleavage allows for conformational changes in HA that facilitate the fusion of viral and host membranes and release of viral particles into the cytosol, initiating infection. Previous studies have attributed the unique virulence and adaptability of influenza A to its frequent mutation in HA [14]. Our PSSM predictions reveal a near-perfect conservation of amino acid sequences surrounding predicted tyrosine sulfation sites in HA across diverse influenza strains (Table 1). Most notably, the predicted tyrosine sulfation site in the Bangkok/1/79 strain is perfectly conserved in 23 other strains. While the predicted tyrosine sulfation sites are almost perfectly conserved, we also observe that the overall HA amino acid sequences are approximately 80% identical on average. Thus, the slightly higher degree of conservation within predicted tyrosine sulfation sites as compared to the entire protein may not be functionally significant. Yet, even in HA sequences with low overall conservation, such as HEMA_IAMAB, which is at most 66% identical to any of the other HA with predicted tyrosine sulfation sites, amino acids around predicted sulfated tyrosines still exhibit a near-perfect conservation (Table 1). The high degree of sequence conservation around predicted sulfated tyrosine sites may implicate the functional significance of sulfated tyrosines in HA. Known structures of HA in complex with sialic acid have presented strong evidence for the importance of our predicted tyrosine sulfation sites in viral binding to host cells. While the primary binding site for sialic acid is located at the surface of the HA molecule [15], there exists a secondary binding site located below the primary binding site in the 3D structure [16]. The predicted sulfated tyrosine site at position 105 (Table 1) in the X31 strain is one of the contact residues in the secondary binding site [16]. Although the biological significance of the secondary binding site is unknown, it may promote viral attachment as an auxiliary receptor and effect a conformational change necessary for the fusion of virus to host cell membranes [16]. This sequence conservation across diverse strains of HA may be key for successful viral propagation. VP4 in rotavirus Rotaviruses are members of the Reoviridae family and are the leading cause of severe gastroenteritis in young children and animals worldwide [17]. Unlike many porcine rotaviruses that are integrin-independent and cause asymptomatic infections, laboratory-adapted, disease-causing human, simian, and bovine rotaviruses
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Table 1 Conservation of hemagglutinin precursor protein in influenza A Strain
Swiss-Prot name
Tyrosine No.
Bangkok/1/79a Equine/Fontainebleau/76b Mallard/Astrakhan/263/82c Duck/Alberta/78/76 Memphis/102/72 Swine/Ukkel/1/84
HEMA_IABAN HEMA_IAHFO HEMA_IAMAB HEMA_IADA3 HEMA_IAME2 HEMA_IAZUK
105 120 121 121 121 121
Site
Score 3.522 4.142 3.466 6.921 3.424 4.491
% Conservation
Additional strains of influenza A with identical sites: a Aichi/2/68; Duck/Hokkaido (10/85, 33/80, 5/77, 7/82, 8/80, 9/85); Duck/Hong Kong (231/77, 64/76, 7/75); Duck/Memphis/928/74; England/321/ 77; Equine/Algiers/72; Equine/Miami/1/63; Goose/Hong Kong/10/76; Mallard/New York/6874/78; Memphis (1/71, 6/86); NT/60/68; Swine/Colorado/ 1/77; Udorn/307/72; Victoria/3/75; Strain X-31. b Equine/Kentucky (2/86, 1/87); Equine/New Market/76; Equine/Romania/80; Equine/Santiago/1/85; Equine/Suffolk/89; Equine/Tennessee/5/86; Equine/Uruguay/1/63; Duck/Ukraine/1/63. c Mallard/Astrakhan/244/82; Budgerigar/Hokkaido/1/77; Chicken/Alabama/1/75; Duck/Alberta/28/76; Duck/New Zealand/31/76; Seal/Massachusetts/133/82; Turkey/Minnesota/833/80.
Mutagenesis of D308 and G309 to alanine in VP4 has recently been shown to decrease viral binding and infection in simian cells dramatically, therefore confirming the importance of the a2b1 ligand motif’s interaction with integrin [17]. Interestingly, the mutation of D308 to alanine decreases the PSSM score from 3.033 to 0.913, which is below the cut-off for prediction of tyrosine sulfation. These observations provide further support that Y303 sulfation near D308 may be necessary within the a2b1 binding site for successful attachment of rotavirus to host cell membranes.
rely on integrin-dependent mechanisms for target cell entry and infection [17]. For the integrin-dependent viruses, the interaction between host integrin a2b1 and the viral surface spike protein VP4 is crucial in mediating initial viral attachment [17]. The a2b1 ligand motif, DGE, located at residues 308–310 of VP4, is responsible for the interaction with a2b1 [17]. In addition, VP4 has been implicated in host cell penetration, hemagglutination, antibody neutralization, and virulence [18,19]. The acidic a2b1 ligand motif in VP4 contains D308, which is just five residues away from the predicted tyrosine sulfation site at Y303. Sulfation of Y303 may enhance the viral binding affinity to host a2b1. Consistent with patterns of integrin dependence and virulence, we have predicted VP4 tyrosine sulfation sites in only human, simian, and bovine rotaviruses, but not in porcine strains (Table 2). As in HA in influenza A, we observed a high degree of conservation in amino acids surrounding the predicted tyrosine sulfation sites, which suggests that the preservation of function is regulated by tyrosine sulfation despite viral mutability.
US28 encoded by human cytomegalovirus The human cytomegalovirus (HCMV), a member of the family Herpesviridae, is the primary cause of congenital virus infections and may also initiate fatal infections in immunocompromised individuals [20,21]. HCMV encodes the exogenous chemokine receptor US28, which is expressed on host cells after infection [21]. Natural chemokine receptors are a member of Gprotein coupled, 7-transmembrane (7TM) receptors that
Table 2 Conservation of outer layer protein VP4 in rotavirus Strain
Swiss-Prot name
Tyrosine No.
Bovine 993/83 Human 116ea Human 1076b Bovine 61a Bovine uk Human 69mc Human 1845d Human au-1e
VP4_ROTB9 VP4_ROTHU VP4_ROTH1 VP4_ROTB6 VP4_ROTBU VP4_ROTH6 VP4_ROTHY VP4_ROTH3
299 302 302 303 303 303 303 303
Site
Additional strains of rotavirus with identical sites: a Bovine (a44, b223, kk3). b Human (ku, m37, p, st. thomas 3, va70, wa, mcn13, gottfried). c Bovine (a5, c486); Rhesus; Simian (sa11-fem, sa11-sem, sa11-both). d Canine (k9); Feline (frv-64). e Human (k8, mc35); Feline (frv-1).
Score 5.956 4.768 3.805 2.582 4.163 3.034 4.368 3.175
% Conservation
H.C. Lin et al. / Biochemical and Biophysical Research Communications 312 (2003) 1154–1158 Table 3 Predicted tyrosine sulfation sites in human cytomegalovirus Swiss-Prot name
Tyrosine No.
Site
Score
US28_HCMVA US28_HCMVA US28_HCMVA
16 177 179
ttefdYdedat qcmtdYdylev mtdydYlevsy
9.943 4.739 5.821
are important for the immune response system, especially in the development, differentiation, and distribution of leukocytes [21]. We predicted three tyrosine sulfation sites in US28 (Table 3), of which two, Y177 and Y179, are located in close proximity to one another. This is not surprising; clustering of tyrosine sulfation sites is a common phenomenon. It occurs because tyrosines may be more readily sulfated if they are positioned close to an existing sulfated tyrosine, which imparts a negative charge. A known example of this pattern occurs in the chemokine receptor, CCR5, which has experimentally confirmed tyrosine sulfation sites at residues 3, 10, 14, and 15 [8]. The structural positions of the predicted tyrosine sulfation sites in US28 are consistent with data from previously published literature and may reflect functional significance. Experimentally confirmed tyrosine sulfation sites in 7TM receptors are primarily localized to the N-terminal extracellular tail and the second extracellular loop, and so are the predicted cytomegalovirus sites: Y16 of US28 can be mapped to the N-terminal extracellular tail, and Y177 and Y179 to the second extracellular loop between transmembrane helices 4 and 5. These two domains have been implicated in the ligand binding of 7TM receptors [6]. Furthermore, all currently known tyrosine sulfation sites in chemokine receptors are located on the N-terminal extracellular tail and have been shown to be crucial for proper receptor functioning [22–25]. US28 may play a critical role in the survival of HCMV by allowing the virus to confuse and evade the host immune system. Previous studies have demonstrated that US28 is highly promiscuous and capable of binding with chemokines MIP-1a, MIP-1b, RANTES, MCP-1, MCP-3, and fractalkine [21]. Fibroblast cells expressing US28 after HCMV infection have been shown to deplete endogenous RANTES and MCP-1, thus preventing the activation of their corresponding chemokine receptors that would otherwise initiate a host immune response [26,27]. It has also been postulated that the binding of US28 to fractalkine may impart adhesive properties to US28, which can then facilitate cell–cell propagation of the virus [21]. The requirement for strong ligand-binding affinities in these instances may account for the preservation of tyrosine sulfation sites in animal viruses predicted by our PSSM. Our PSSM predictions for hemagglutinin of influenza A, VP4 of rotavirus, and US28 of cytomegalovirus
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suggest an unrecognized link between tyrosine sulfation and viral disease mechanisms. We observed a high degree of conservation in amino acid sequences surrounding predicted sulfated tyrosine sites in each of the above viral proteins. Future investigations of tyrosine sulfation through site-directed mutagenesis may confirm our predictions, and subsequent structural studies using X-ray crystallography and NMR may further our knowledge and understanding of the basic mechanisms of viral infection.
Acknowledgments We thank Michelle Louie and Kristine Yu for editorial assistance. The research was supported by the National Center for Research Resources (RR06009) and the National Human Genome Research Institute (HG00015).
References [1] K.L. Moore, The biology and enzymology of protein tyrosine Osulfation, J. Biol. Chem. 278 (2003) 24243–24246. [2] W.B. Huttner, Sulphation of tyrosine residues—a widespread modification of proteins, Nature 299 (1982) 273–276. [3] H.B. Nicholas Jr., S.S. Chan, G.L. Rosenquist, Reevaluation of the determinants of tyrosine sulfation, Endocrine 11 (1999) 285– 292. [4] W.B. Huttner, Tyrosine sulfation and the secretory pathway, Annu. Rev. Physiol. 50 (1988) 363–376. [5] M.P. Bernimoulin, X.L. Zeng, C. Abbal, S. Giraud, M. Martinez, O. Michielin, M. Schapira, O. Spertini, Molecular basis of leukocyte rolling on PSGL-1. Predominant role of core-2 Oglycans and of tyrosine sulfate residue 51, J. Biol. Chem. 278 (2003) 37–47. [6] J. Gao, H. Choe, D. Bota, P.L. Wright, C. Gerard, N.P. Gerard, Sulfation of tyrosine 174 in the human C3aR is essential for binding of C3a Anaphylatoxin3t, J. Biol. Chem. 278 (2003) 37902–37908. [7] J.W. Kehoe, C.R. Bertozzi, Tyrosine sulfation: a modulator of extracellular protein-protein interactions, Chem. Biol. 7 (2000) R57–R61. [8] M. Farzan, T. Mirzabekov, P. Kolchinsky, R. Wyatt, M. Cayabyab, N.P. Gerard, C. Gerard, J. Sodroski, H. Choe, Tyrosine sulfation of the amino terminus of CCR5 facilitates HIV-1 entry, Cell 96 (1999) 667–676. [9] H. Choe, W. Li, P.L. Wright, N. Vasilieva, M. Venturi, C.C. Huang, C. Grundner, T. Dorfman, M.B. Zwick, L. Wang, E.S. Rosenberg, P.D. Kwong, D.R. Burton, J.E. Robinson, J.G. Sodroski, M. Farzan, Tyrosine sulfation of human antibodies contributes to recognition of the CCR5 binding region of HIV-1 gp120, Cell 114 (2003) 161–170. [10] J.W. Ludlow, R.A. Consigli, Polyomavirus major capsid protein VP1 is modified by tyrosine sulfuration, J. Virol. 61 (1987) 1708– 1711. [11] C.M. Edson, Tyrosine sulfation of varicella-zoster virus envelope glycoprotein gpl, Virology 197 (1993) 159–165. [12] C.E. Metz, Basic principles of ROC analysis, Semin. Nucl. Med. 8 (1978) 283–298. [13] S.J. Baigent, J.W. McCauley, Influenza type A in humans, mammals and birds: determinants of virus virulence, host-range and interspecies transmission, Bioessays 25 (2003) 657–671.
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[14] R.G. Webster, The importance of animal influenza for human disease, Vaccine 20 (2002) S16–S20. [15] W. Weis, J.H. Brown, S. Cusack, J.C. Paulson, J.J. Skehel, D.C. Wiley, Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid, Nature 333 (1988) 426–431. [16] N.K. Sauter, J.E. Hanson, G.D. Glick, J.H. Brown, R.L. Crowther, S.J. Park, J.J. Skehel, D.C. Wiley, Binding of influenza virus hemagglutinin to analogs of its cell-surface receptor, sialic acid: analysis by proton nuclear magnetic resonance spectroscopy and X-ray crystallography, Biochemistry 31 (1992) 9609–9621. [17] K.L. Graham, P. Halasz, Y. Tan, M.J. Hewish, Y. Takada, E.R. Mackow, M.K. Robinson, B.S. Coulson, Integrin-using rotaviruses bind a2b1 integrin a2 I domain via VP4 DGE site and recognize aXb2 and aVb3 by using VP7 during cell entry, J. Virol. 77 (2003) 9969–9978. [18] V. Enouf, S. Chwetzoff, G. Trugnan, J. Cohen, Interactions of rotavirus VP4 spike protein with the endosomal protein Rab5 and the prenylated Rab acceptor PRA1, J. Virol. 77 (2003) 7041–7047. [19] F. Mota-Hernandez, J.J. Calva, C. Gutierrez-Camacho, S. VillaContreras, C.F. Arias, L. Padilla-Noriega, H. Guiscafre-Gallardo, M. de Lourdes Guerrero, S. Lopez, O. Munoz, J.F. Contreras, R. Cedillo, I. Herrera, F.I. Puerto, Rotavirus diarrhea severity is related to the VP4 type in Mexican children, J. Clin. Microbiol. 41 (2003) 3158–3162. [20] A.L. Bissinger, C. Sinzger, E. Kaiserling, G. Jahn, Human cytomegalovirus as a direct pathogen: correlation of multiorgan involvement and cell distribution with clinical and pathological findings in a case of congenital inclusion disease, J. Med. Virol. 67 (2002) 200–206. [21] P.M. Murphy, M. Baggiolini, I.F. Charo, C.A. Hebert, R. Horuk, K. Matsushima, L.H. Miller, J.J. Oppenheim, C.A. Power,
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International Union of Pharmacology. XXII. Nomenclature for chemokine receptors, Pharmacol. Rev. 52 (2000) 145–176. B.J. Doranz, M.J. Orsini, J.D. Turner, T.L. Hoffman, J.F. Berson, J.A. Hoxie, S.C. Peiper, L.F. Brass, R.W. Doms, Identification of CXCR4 domains that support co-receptor and chemokine receptor functions, J. Virol. 73 (1999) 2752–2761. F.S. Monteclaro, I.F. Charo, The amino-terminal extracellular domain of the MCP-1 receptor, but not the RANTES/MIP1alpha receptor, confers chemokine selectivity. Evidence for a two-step mechanism for MCP-1 receptor activation, J. Biol. Chem. 271 (1996) 19084–19092. C. Blanpain, B.J. Doranz, J. Vakili, J. Rucker, C. Govaerts, S.S. Baik, O. Lorthioir, I. Migeotte, F. Libert, F. Baleux, G. Vassart, R.W. Doms, M. Parmentier, Multiple charged and aromatic residues in CCR5 amino-terminal domain are involved in high affinity binding of both chemokines and HIV-1 Env protein, J. Biol. Chem. 274 (1999) 34719–34727. L.S. Mizoue, J.F. Bazan, E.C. Johnson, T.M. Handel, Solution structure and dynamics of the CX3C chemokine domain of fractalkine and its interaction with an N-terminal fragment of CX3CR1, Biochemistry 38 (1999) 1402–1414. B. Bodaghi, T.R. Jones, D. Zipeto, C. Vita, L. Sun, L. Laurent, F. Arenzana-Seisdedos, J.L. Virelizier, S. Michelson, Chemokine sequestration by viral chemoreceptors as a novel viral escape strategy: withdrawal of chemokines from the environment of cytomegalovirus-infected cells, J. Exp. Med. 188 (1998) 855–866. J. Vieira, T.J. Schall, L. Corey, A.P. Geballe, Functional analysis of the human cytomegalovirus US28 gene by insertion mutagenesis with the green fluorescent protein gene, J. Virol. 72 (1998) 8158–8165.
Prediction of tyrosine sulfation sites in animal viruses Henry C. Lin,a Kevin Tsai,a Brian L. Chang,a Justin Liu,a Melinda Young,a Willy Hsu,a Samuel Louie,b Hugh B. Nicholas Jr.,c and Grace L. Rosenquista,* b
a Section of Neurobiology, Physiology and Behavior, University of California, Davis, CA 95616, USA Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of California, Davis, Sacramento, CA 95817, USA c Pittsburgh Supercomputing Center, Pittsburgh, PA 15213, USA
Received 22 October 2003
Abstract Post-translational modification of proteins by tyrosine sulfation enhances the affinity of extracellular ligand–receptor interactions important in the immune response and other biological processes in animals. For example, sulfated tyrosines in polyomavirus and varicella-zoster virus may help modulate host cell recognition and facilitate viral attachment and entry. Using a Position-SpecificScoring-Matrix with an accuracy of 96.43%, we analyzed the possibility of tyrosine sulfation in all 1517 animal viruses available in the Swiss-Prot database. From a total of 97,729 tyrosines, we predicted 5091 sulfated tyrosine sites from 1024 viruses. Our site predictions in hemagglutinin of influenza A, VP4 of rotavirus, and US28 of cytomegalovirus strongly suggest an important link between tyrosine sulfation and viral disease mechanisms. In each of these three viral proteins, we observed highly conserved amino acid sequences surrounding predicted sulfated tyrosine sites. Tyrosine sulfation appears to be much more common in animal viruses than is currently recognized. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Tyrosine; Sulfation; Post-translational modification; Virus; Influenza A; Rotavirus; Cytomegalovirus
Tyrosine sulfation occurs when tyrosylprotein sulfotransferases (TPSTs) catalyze the transfer of a negatively charged sulfate from 30 -phosphoadenosine 50 -phosphosulfate (PAPS) to the hydroxyl group of a tyrosine residue on a polypeptide [1]. Since the demonstration of its prevalence in diverse mammalian cells in 1982 [2], tyrosine sulfation has been considered to be the most common post-translational modification of tyrosines transported through the trans-Golgi network [3]. Approximately 1% of all tyrosines may be sulfated [4]. Tyrosine sulfation has been experimentally shown to be essential for extracellular protein–protein interactions in many important biological processes in animals. For instance, sulfated tyrosines play a critical role in the immune response, such as in leukocyte rolling [5] and complement cascade [6], and promote HIV infection of T-helper lymphocytes [7–9].
We postulate that tyrosine sulfation performs similar functions in viruses. Based on our current understanding, all viral proteins that undergo tyrosine sulfation, such as capsid protein VP1 in polyomavirus and envelope glycoprotein gpI in varicella-zoster virus, must be modified in the host cell trans-Golgi apparatus during replication [10,11]. The purpose of tyrosine sulfation in these examples remains to be determined, but it is believed that specific sulfated tyrosines enhance target cell recognition, attachment, and entry by viruses [10,11]. Tyrosine sulfation in viruses has not been studied extensively since these publications. Detailed analyses of this post-translational modification in viral proteins may provide insights into the mechanisms of viral infection and the design of new therapies.
Materials and methods *
Corresponding author. Fax: 1-530-752-5582. E-mail addresses: [email protected], [email protected] (G.L. Rosenquist). 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.11.047
Acquisition of animal viral tyrosine sites. We obtained all known proteins of 1517 available animal viruses with the Site Retrieval System (http://us.expasy.org/srs5/) based on Swiss-Prot Release 41.19 of
H.C. Lin et al. / Biochemical and Biophysical Research Communications 312 (2003) 1154–1158 August 4th, 2003, using the keyword “virus” on the Organism Classification (OC) line of the database entry. As many as 97,729 tyrosine sites, which consist of target tyrosines and up to 5 flanking amino acids on each side [3], were extracted from these viral proteins using the Pick Sites program available at the Pittsburgh Supercomputing Center (Pittsburgh, PA). Position-Specific-Scoring-Matrix. The training set used in this study contained all currently known 69 sulfated and 454 non-sulfated tyrosine sites. The sulfated tyrosine sites have been identified experimentally and the non-sulfated tyrosine sites, extracted from sulfated proteins, are the remaining tyrosines for which sulfation has not been demonstrated. The Position-Specific-Scoring-Matrix (PSSM) was used to compare the distributions of amino acids around the query tyrosine sites with those of known sulfated and non-sulfated tyrosine sites. Each query tyrosine site was assigned a score measuring the similarity of the amino acids around the tyrosine to those around either the known sulfated tyrosine sites or those around non-sulfated tyrosine sites. Tyrosine sites with a high score are taken to have a high probability of sulfation. A detailed description of the construction of the PSSM was published previously [3]. When the PSSM was used to predict the occurrence of tyrosine sulfation in the training set, the sulfated tyrosine sites received a mean score of 6.890 with a standard deviation of 2.911, while the non-sulfated tyrosine sites received a mean score of )3.526 with a standard deviation of 3.234. To evaluate the ability of the PSSM to recognize sulfated tyrosine sites not included in its training set, 69 jackknifed PSSMs, each containing 68 sulfated and 454 non-sulfated sites, were used to evaluate the excluded sulfated site in each training set. The Receiver Operating Characteristic (ROC) [12] was then used to evaluate the predictions of the jackknifed PSSMs. The resulting ROC score of 0.9643 indicates that the PSSM is expected to have an accuracy of 96.43% in the prediction of unknown tyrosine sulfation sites. In order to minimize both the numbers of false positives and false negatives, the threshold score for a predicted tyrosine sulfation site was set at 2.5.
Results and discussion The PSSM was applied to animal viral proteins from Swiss-Prot and 5091 sulfated tyrosine sites were predicted in 1024 viruses. We focused on our predictions in three viruses responsible for causing human infectious diseases. Potential new roles for sulfated tyrosines were hypothesized based on our predictions in hemagglutinin of influenza A, VP4 of rotavirus, and US28 of cytomegalovirus. Although prior investigators suggested that tyrosine sulfation might be required for specific host recognition and optimal viral attachment and entry [10,11], no further corroborating studies have since been published. Our numerous predictions of sulfated tyrosines in viral proteins and the high conservation of predicted sites were initially surprising but led us to hypothesize that tyrosine sulfation is necessary for viral infection of host cells. We discuss below the specific predictions in hemagglutinin of influenza A, VP4 of rotavirus, and US28 of cytomegalovirus and the implications in mechanisms of disease. Hemagglutinin in influenza A Influenza viruses, which consist of types A, B, and C, belong to the family Orthomyxoviridae [13]. Of the three types, influenza type A is most responsible for deadly
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human pandemics [14]. For attachment to and subsequent invasion of target cells, the virus relies on the protein hemagglutinin (HA), which binds to sialic acid on human epithelial cells [13]. Once the virus has been internalized by the host cell endosome, HA is cleaved by host proteases into HA1 and HA2. The cleavage allows for conformational changes in HA that facilitate the fusion of viral and host membranes and release of viral particles into the cytosol, initiating infection. Previous studies have attributed the unique virulence and adaptability of influenza A to its frequent mutation in HA [14]. Our PSSM predictions reveal a near-perfect conservation of amino acid sequences surrounding predicted tyrosine sulfation sites in HA across diverse influenza strains (Table 1). Most notably, the predicted tyrosine sulfation site in the Bangkok/1/79 strain is perfectly conserved in 23 other strains. While the predicted tyrosine sulfation sites are almost perfectly conserved, we also observe that the overall HA amino acid sequences are approximately 80% identical on average. Thus, the slightly higher degree of conservation within predicted tyrosine sulfation sites as compared to the entire protein may not be functionally significant. Yet, even in HA sequences with low overall conservation, such as HEMA_IAMAB, which is at most 66% identical to any of the other HA with predicted tyrosine sulfation sites, amino acids around predicted sulfated tyrosines still exhibit a near-perfect conservation (Table 1). The high degree of sequence conservation around predicted sulfated tyrosine sites may implicate the functional significance of sulfated tyrosines in HA. Known structures of HA in complex with sialic acid have presented strong evidence for the importance of our predicted tyrosine sulfation sites in viral binding to host cells. While the primary binding site for sialic acid is located at the surface of the HA molecule [15], there exists a secondary binding site located below the primary binding site in the 3D structure [16]. The predicted sulfated tyrosine site at position 105 (Table 1) in the X31 strain is one of the contact residues in the secondary binding site [16]. Although the biological significance of the secondary binding site is unknown, it may promote viral attachment as an auxiliary receptor and effect a conformational change necessary for the fusion of virus to host cell membranes [16]. This sequence conservation across diverse strains of HA may be key for successful viral propagation. VP4 in rotavirus Rotaviruses are members of the Reoviridae family and are the leading cause of severe gastroenteritis in young children and animals worldwide [17]. Unlike many porcine rotaviruses that are integrin-independent and cause asymptomatic infections, laboratory-adapted, disease-causing human, simian, and bovine rotaviruses
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Table 1 Conservation of hemagglutinin precursor protein in influenza A Strain
Swiss-Prot name
Tyrosine No.
Bangkok/1/79a Equine/Fontainebleau/76b Mallard/Astrakhan/263/82c Duck/Alberta/78/76 Memphis/102/72 Swine/Ukkel/1/84
HEMA_IABAN HEMA_IAHFO HEMA_IAMAB HEMA_IADA3 HEMA_IAME2 HEMA_IAZUK
105 120 121 121 121 121
Site
Score 3.522 4.142 3.466 6.921 3.424 4.491
% Conservation
Additional strains of influenza A with identical sites: a Aichi/2/68; Duck/Hokkaido (10/85, 33/80, 5/77, 7/82, 8/80, 9/85); Duck/Hong Kong (231/77, 64/76, 7/75); Duck/Memphis/928/74; England/321/ 77; Equine/Algiers/72; Equine/Miami/1/63; Goose/Hong Kong/10/76; Mallard/New York/6874/78; Memphis (1/71, 6/86); NT/60/68; Swine/Colorado/ 1/77; Udorn/307/72; Victoria/3/75; Strain X-31. b Equine/Kentucky (2/86, 1/87); Equine/New Market/76; Equine/Romania/80; Equine/Santiago/1/85; Equine/Suffolk/89; Equine/Tennessee/5/86; Equine/Uruguay/1/63; Duck/Ukraine/1/63. c Mallard/Astrakhan/244/82; Budgerigar/Hokkaido/1/77; Chicken/Alabama/1/75; Duck/Alberta/28/76; Duck/New Zealand/31/76; Seal/Massachusetts/133/82; Turkey/Minnesota/833/80.
Mutagenesis of D308 and G309 to alanine in VP4 has recently been shown to decrease viral binding and infection in simian cells dramatically, therefore confirming the importance of the a2b1 ligand motif’s interaction with integrin [17]. Interestingly, the mutation of D308 to alanine decreases the PSSM score from 3.033 to 0.913, which is below the cut-off for prediction of tyrosine sulfation. These observations provide further support that Y303 sulfation near D308 may be necessary within the a2b1 binding site for successful attachment of rotavirus to host cell membranes.
rely on integrin-dependent mechanisms for target cell entry and infection [17]. For the integrin-dependent viruses, the interaction between host integrin a2b1 and the viral surface spike protein VP4 is crucial in mediating initial viral attachment [17]. The a2b1 ligand motif, DGE, located at residues 308–310 of VP4, is responsible for the interaction with a2b1 [17]. In addition, VP4 has been implicated in host cell penetration, hemagglutination, antibody neutralization, and virulence [18,19]. The acidic a2b1 ligand motif in VP4 contains D308, which is just five residues away from the predicted tyrosine sulfation site at Y303. Sulfation of Y303 may enhance the viral binding affinity to host a2b1. Consistent with patterns of integrin dependence and virulence, we have predicted VP4 tyrosine sulfation sites in only human, simian, and bovine rotaviruses, but not in porcine strains (Table 2). As in HA in influenza A, we observed a high degree of conservation in amino acids surrounding the predicted tyrosine sulfation sites, which suggests that the preservation of function is regulated by tyrosine sulfation despite viral mutability.
US28 encoded by human cytomegalovirus The human cytomegalovirus (HCMV), a member of the family Herpesviridae, is the primary cause of congenital virus infections and may also initiate fatal infections in immunocompromised individuals [20,21]. HCMV encodes the exogenous chemokine receptor US28, which is expressed on host cells after infection [21]. Natural chemokine receptors are a member of Gprotein coupled, 7-transmembrane (7TM) receptors that
Table 2 Conservation of outer layer protein VP4 in rotavirus Strain
Swiss-Prot name
Tyrosine No.
Bovine 993/83 Human 116ea Human 1076b Bovine 61a Bovine uk Human 69mc Human 1845d Human au-1e
VP4_ROTB9 VP4_ROTHU VP4_ROTH1 VP4_ROTB6 VP4_ROTBU VP4_ROTH6 VP4_ROTHY VP4_ROTH3
299 302 302 303 303 303 303 303
Site
Additional strains of rotavirus with identical sites: a Bovine (a44, b223, kk3). b Human (ku, m37, p, st. thomas 3, va70, wa, mcn13, gottfried). c Bovine (a5, c486); Rhesus; Simian (sa11-fem, sa11-sem, sa11-both). d Canine (k9); Feline (frv-64). e Human (k8, mc35); Feline (frv-1).
Score 5.956 4.768 3.805 2.582 4.163 3.034 4.368 3.175
% Conservation
H.C. Lin et al. / Biochemical and Biophysical Research Communications 312 (2003) 1154–1158 Table 3 Predicted tyrosine sulfation sites in human cytomegalovirus Swiss-Prot name
Tyrosine No.
Site
Score
US28_HCMVA US28_HCMVA US28_HCMVA
16 177 179
ttefdYdedat qcmtdYdylev mtdydYlevsy
9.943 4.739 5.821
are important for the immune response system, especially in the development, differentiation, and distribution of leukocytes [21]. We predicted three tyrosine sulfation sites in US28 (Table 3), of which two, Y177 and Y179, are located in close proximity to one another. This is not surprising; clustering of tyrosine sulfation sites is a common phenomenon. It occurs because tyrosines may be more readily sulfated if they are positioned close to an existing sulfated tyrosine, which imparts a negative charge. A known example of this pattern occurs in the chemokine receptor, CCR5, which has experimentally confirmed tyrosine sulfation sites at residues 3, 10, 14, and 15 [8]. The structural positions of the predicted tyrosine sulfation sites in US28 are consistent with data from previously published literature and may reflect functional significance. Experimentally confirmed tyrosine sulfation sites in 7TM receptors are primarily localized to the N-terminal extracellular tail and the second extracellular loop, and so are the predicted cytomegalovirus sites: Y16 of US28 can be mapped to the N-terminal extracellular tail, and Y177 and Y179 to the second extracellular loop between transmembrane helices 4 and 5. These two domains have been implicated in the ligand binding of 7TM receptors [6]. Furthermore, all currently known tyrosine sulfation sites in chemokine receptors are located on the N-terminal extracellular tail and have been shown to be crucial for proper receptor functioning [22–25]. US28 may play a critical role in the survival of HCMV by allowing the virus to confuse and evade the host immune system. Previous studies have demonstrated that US28 is highly promiscuous and capable of binding with chemokines MIP-1a, MIP-1b, RANTES, MCP-1, MCP-3, and fractalkine [21]. Fibroblast cells expressing US28 after HCMV infection have been shown to deplete endogenous RANTES and MCP-1, thus preventing the activation of their corresponding chemokine receptors that would otherwise initiate a host immune response [26,27]. It has also been postulated that the binding of US28 to fractalkine may impart adhesive properties to US28, which can then facilitate cell–cell propagation of the virus [21]. The requirement for strong ligand-binding affinities in these instances may account for the preservation of tyrosine sulfation sites in animal viruses predicted by our PSSM. Our PSSM predictions for hemagglutinin of influenza A, VP4 of rotavirus, and US28 of cytomegalovirus
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suggest an unrecognized link between tyrosine sulfation and viral disease mechanisms. We observed a high degree of conservation in amino acid sequences surrounding predicted sulfated tyrosine sites in each of the above viral proteins. Future investigations of tyrosine sulfation through site-directed mutagenesis may confirm our predictions, and subsequent structural studies using X-ray crystallography and NMR may further our knowledge and understanding of the basic mechanisms of viral infection.
Acknowledgments We thank Michelle Louie and Kristine Yu for editorial assistance. The research was supported by the National Center for Research Resources (RR06009) and the National Human Genome Research Institute (HG00015).
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