Species Specificity of the NS1 Protein of Influenza B Virus

Influenza B viruses, which cause a highly contagious respiratory disease every year, are restricted to humans, but the basis for this restriction had not been determined. Here we provide one explanation for this restriction: the species specificity exhibited by the NS1 protein of influenza B virus (NS1B protein). This viral protein combats a major host antiviral response by binding the interferon-α/β-induced, ubiquitin-like ISG15 protein and inhibiting its conjugation to an array of proteins. We demonstrate that the NS1B protein exhibits species-specific binding; it binds human and non-human primate ISG15 but not mouse or canine ISG15. In both transfection assays and virus-infected cells, the NS1B protein binds and relocalizes only human and non-human primate ISG15 from the cytoplasm to nuclear speckles. Human and non-human primate ISG15 proteins consist of two ubiquitin-like domains separated by a short hinge linker of five amino acids. Remarkably, this short hinge plays a large role in the species-specific binding by the NS1B protein. The hinge of human and non-human primate ISG15, which has a sequence that differs from that of other mammalian ISG15 proteins, including mouse and canine ISG15, is absolutely required for binding the NS1B protein. Consequently, the ISG15 proteins of humans and non-human primates are the only mammalian ISG15 proteins that would bind NS1B. Influenza B viruses, which cause a highly contagious respiratory disease every year, are restricted to humans, but the basis for this restriction had not been determined. Here we provide one explanation for this restriction: the species specificity exhibited by the NS1 protein of influenza B virus (NS1B protein). This viral protein combats a major host antiviral response by binding the interferon-α/β-induced, ubiquitin-like ISG15 protein and inhibiting its conjugation to an array of proteins. We demonstrate that the NS1B protein exhibits species-specific binding; it binds human and non-human primate ISG15 but not mouse or canine ISG15. In both transfection assays and virus-infected cells, the NS1B protein binds and relocalizes only human and non-human primate ISG15 from the cytoplasm to nuclear speckles. Human and non-human primate ISG15 proteins consist of two ubiquitin-like domains separated by a short hinge linker of five amino acids. Remarkably, this short hinge plays a large role in the species-specific binding by the NS1B protein. The hinge of human and non-human primate ISG15, which has a sequence that differs from that of other mammalian ISG15 proteins, including mouse and canine ISG15, is absolutely required for binding the NS1B protein. Consequently, the ISG15 proteins of humans and non-human primates are the only mammalian ISG15 proteins that would bind NS1B. IntroductionInfluenza A and B viruses cause a highly contagious respiratory disease in humans. Influenza A viruses infect a wide variety of species, whereas influenza B viruses infect only humans (1.Wright P.F. Webster R.G. Knipe D.M. Howley P.M. Fields Virology. 4th Ed. Lippincott Williams & Wilkins, Philadelphia2001: 1533-1579Google Scholar). Many, but not all, of the proteins encoded by these two groups of viruses carry out similar functions (2.Lamb R.A. Krug R.M. Knipe D.M. Howley P.M. Fields Virology. 4th Ed. Lippincott Williams & Wilkins, Philadelphia2001: 1487-1531Google Scholar). Here we focus on a function of the NS1 protein of influenza B virus (NS1B protein) that is not shared by the NS1 protein of influenza A virus (NS1A protein). NS1B, but not NS1A, binds human ISG15, an interferon (IFN) 2The abbreviations used are: IFNinterferonGSTglutathione S-transferaseGFPgreen fluorescent protein. -α/β-induced, ubiquitin-like protein (3.Yuan W. Krug R.M. EMBO J. 2001; 20: 362-371Crossref PubMed Scopus (415) Google Scholar). The binding site for human ISG15 is located in the N-terminal 104 amino acids of NS1B (3.Yuan W. Krug R.M. EMBO J. 2001; 20: 362-371Crossref PubMed Scopus (415) Google Scholar). ISG15 is conjugated to more than 100 cellular proteins (4.Zhao C. Denison C. Huibregtse J.M. Gygi S. Krug R.M. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 10200-10205Crossref PubMed Scopus (363) Google Scholar, 5.Giannakopoulos N.V. Luo J.K. Papov V. Zou W. Lenschow D.J. Jacobs B.S. Borden E.C. Li J. Virgin H.W. Zhang D.E. Biochem. Biophys. Res. Commun. 2005; 336: 496-506Crossref PubMed Scopus (178) Google Scholar) through the sequential action of three conjugation enzymes that are also induced by IFN-α/β: the E1-activating enzyme (Ube1L) (3.Yuan W. Krug R.M. EMBO J. 2001; 20: 362-371Crossref PubMed Scopus (415) Google Scholar), the E2-conjugating enzyme (UbcH8) (6.Zhao C. Beaudenon S.L. Kelley M.L. Waddell M.B. Yuan W. Schulman B.A. Huibregtse J.M. Krug R.M. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 7578-7582Crossref PubMed Scopus (243) Google Scholar, 7.Kim K.I. Giannakopoulos N.V. Virgin H.W. Zhang D.E. Mol. Cell. Biol. 2004; 24: 9592-9600Crossref PubMed Scopus (181) Google Scholar), and the E3 ligase (Herc5) (8.Dastur A. Beaudenon S. Kelley M. Krug R.M. Huibregtse J.M. J. Biol. Chem. 2006; 281: 4334-4338Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 9.Wong J.J. Pung Y.F. Sze N.S. Chin K.C. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10735-10740Crossref PubMed Scopus (184) Google Scholar). Human ISG15 consists of two ubiquitin-like domains connected by a short 5-amino acid hinge linker (10.Narasimhan J. Wang M. Fu Z. Klein J.M. Haas A.L. Kim J.J. J. Biol. Chem. 2005; 280: 27356-27365Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The C-terminal ISG15 domain, which is recognized by Ube1L, contains the C-terminal LRLRGG motif that is conjugated to target proteins (11.Chang Y.G. Yan X.Z. Xie Y.Y. Gao X.C. Song A.X. Zhang D.E. Hu H.Y. J. Biol. Chem. 2008; 283: 13370-13377Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The N-terminal domain (plus the hinge) is sufficient to bind NS1B.ISG15 and/or its conjugation play an important role in the IFN-induced antiviral state against several viruses, including influenza A and B viruses (3.Yuan W. Krug R.M. EMBO J. 2001; 20: 362-371Crossref PubMed Scopus (415) Google Scholar, 12.Lenschow D.J. Giannakopoulos N.V. Gunn L.J. Johnston C. O'Guin A.K. Schmidt R.E. Levine B. Virgin 4th, H.W. J. Virol. 2005; 79: 13974-13983Crossref PubMed Scopus (222) Google Scholar, 13.Okumura A. Lu G. Pitha-Rowe I. Pitha P.M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 1440-1445Crossref PubMed Scopus (282) Google Scholar, 14.Lenschow D.J. Lai C. Frias-Staheli N. Giannakopoulos N.V. Lutz A. Wolff T. Osiak A. Levine B. Schmidt R.E. García-Sastre A. Leib D.A. Pekosz A. Knobeloch K.P. Horak I. Virgin 4th, H.W. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1371-1376Crossref PubMed Scopus (408) Google Scholar, 15.Malakhova O.A. Zhang D.E. J. Biol. Chem. 2008; 283: 8783-8787Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 16.Okumura A. Pitha P.M. Harty R.N. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 3974-3979Crossref PubMed Scopus (216) Google Scholar, 17.Guerra S. Cáceres A. Knobeloch K.P. Horak I. Esteban M. PLoS Pathog. 2008; 4: e1000096Crossref PubMed Scopus (112) Google Scholar, 18.Lai C. Struckhoff J.J. Schneider J. Martinez-Sobrido L. Wolff T. García-Sastre A. Zhang D.E. Lenschow D.J. J. Virol. 2009; 83: 1147-1151Crossref PubMed Scopus (102) Google Scholar, 19.Hsiang T.Y. Zhao C. Krug R.M. J. Virology. 2009; 83: 5971-5977Crossref PubMed Scopus (91) Google Scholar). The first evidence for the antiviral role of ISG15 conjugation was the finding that the NS1B protein of influenza B virus binds ISG15 and blocks its conjugation (3.Yuan W. Krug R.M. EMBO J. 2001; 20: 362-371Crossref PubMed Scopus (415) Google Scholar), suggesting that ISG15 and/or its conjugation are inhibitory to the replication of influenza B virus. Subsequently, it was shown that both ISG15 knock-out (ISG15−/−) and Ube1L−/− mice are more susceptible to influenza B virus infection, establishing that ISG15 conjugation inhibits influenza B virus replication (14.Lenschow D.J. Lai C. Frias-Staheli N. Giannakopoulos N.V. Lutz A. Wolff T. Osiak A. Levine B. Schmidt R.E. García-Sastre A. Leib D.A. Pekosz A. Knobeloch K.P. Horak I. Virgin 4th, H.W. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1371-1376Crossref PubMed Scopus (408) Google Scholar, 18.Lai C. Struckhoff J.J. Schneider J. Martinez-Sobrido L. Wolff T. García-Sastre A. Zhang D.E. Lenschow D.J. J. Virol. 2009; 83: 1147-1151Crossref PubMed Scopus (102) Google Scholar). However, it was not clear why NS1B did not protect influenza B virus from the antiviral effects of ISG15 and its conjugation system in wild-type (ISG15+/+, Ube1L+/+) mice.Here we demonstrate that the NS1B protein exhibits species-specific binding. It binds human and non-human primate ISG15 but not mouse or canine ISG15. These results explain why influenza B virus replication is inhibited in wild-type mice that express mouse ISG15 and Ube1L. Surprisingly, we show that the 5-amino acid hinge of human and non-human primate ISG15, which has a different sequence than the ISG15 hinges of other mammalian species, is absolutely required for binding the NS1B protein. This study provides the first example of an influenza B virus protein that exhibits human (and non-human primate)-specific properties and thus provides one explanation for the restriction of influenza B virus to humans.DISCUSSIONInfluenza B virus is predominately, if not totally, restricted to humans (1.Wright P.F. Webster R.G. Knipe D.M. Howley P.M. Fields Virology. 4th Ed. Lippincott Williams & Wilkins, Philadelphia2001: 1533-1579Google Scholar), but the basis for this restriction had not been determined. Here we provide one explanation for this restriction, namely that the influenza B virus NS1B protein binds only human and non-human primate ISG15. Consequently, the NS1B protein would only be able to protect influenza B virus from the antiviral effects of ISG15 and ISG15 conjugation in humans, and presumably in non-human primates. No protection would be possible in other mammalian species, as already documented by the finding that ISG15 conjugation inhibits influenza B virus replication in mice (14.Lenschow D.J. Lai C. Frias-Staheli N. Giannakopoulos N.V. Lutz A. Wolff T. Osiak A. Levine B. Schmidt R.E. García-Sastre A. Leib D.A. Pekosz A. Knobeloch K.P. Horak I. Virgin 4th, H.W. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1371-1376Crossref PubMed Scopus (408) Google Scholar, 18.Lai C. Struckhoff J.J. Schneider J. Martinez-Sobrido L. Wolff T. García-Sastre A. Zhang D.E. Lenschow D.J. J. Virol. 2009; 83: 1147-1151Crossref PubMed Scopus (102) Google Scholar). Because influenza B virus would not be protected from this IFN-induced antiviral system, it would not be able to be maintained in these other mammalian species. However, influenza B virus might also be found in non-human primates, a possibility that has not yet been explored. Future experiments will determine whether other influenza B virus proteins also exhibit human-specific properties.Surprisingly, the small 5-amino acid hinge between the two ubiquitin-like domains of ISG15 is crucial for NS1B binding. Only the human and primate hinges are suitable for binding. Replacement of individual amino acids in the human hinge sequence with the corresponding mouse amino acid was sufficient to eliminate NS1B binding. In addition, substitution of the human hinge sequence for the canine hinge sequence in the canine ISG15 molecule was sufficient to result in optimum binding of the NS1B protein. It has been proposed that the hinge of human ISG15 is flexible and might adopt a different orientation upon binding other proteins (10.Narasimhan J. Wang M. Fu Z. Klein J.M. Haas A.L. Kim J.J. J. Biol. Chem. 2005; 280: 27356-27365Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Perhaps only the human hinge sequence allows an orientation suitable for the NS1B protein to bind to a region in the N-terminal domain. Alternatively, human hinge amino acids may interact directly with the NS1B protein.The N-terminal domain of human ISG15 contains a binding site for the NS1B protein, as shown by the results with mouse ISG15. Substitution of the human hinge in mouse ISG15 resulted in little or no NS1B binding, indicating that the N-terminal mouse domain contains sequences that are inhibitory to NS1B binding. Such inhibitory sequences likely include some, or all, of the 8 amino acids in the N-terminal domain of mouse ISG15 that differ from the corresponding amino acids in both human and canine ISG15. To definitively identify the binding site in the N-terminal domain of human ISG15, we and our collaborators are determining the x-ray crystal structure of human ISG15 in complex with an N-terminal fragment of the NS1B protein. 3R. Guan, H. Sridharan, C. Zhao, R. M. Krug, and G. T. Montelione, unpublished results. This structure should also identify the NS1B amino acids that directly interact with human ISG15 and hence would enable us to generate a recombinant influenza B virus encoding a NS1B protein that does not bind human ISG15. Such a virus would be expected to be susceptible to inhibition by ISG15 conjugation in human cells, thereby enabling us to identify the mechanism by which IFN-induced ISG15 conjugation inhibits influenza B virus replication in human cells. IntroductionInfluenza A and B viruses cause a highly contagious respiratory disease in humans. Influenza A viruses infect a wide variety of species, whereas influenza B viruses infect only humans (1.Wright P.F. Webster R.G. Knipe D.M. Howley P.M. Fields Virology. 4th Ed. Lippincott Williams & Wilkins, Philadelphia2001: 1533-1579Google Scholar). Many, but not all, of the proteins encoded by these two groups of viruses carry out similar functions (2.Lamb R.A. Krug R.M. Knipe D.M. Howley P.M. Fields Virology. 4th Ed. Lippincott Williams & Wilkins, Philadelphia2001: 1487-1531Google Scholar). Here we focus on a function of the NS1 protein of influenza B virus (NS1B protein) that is not shared by the NS1 protein of influenza A virus (NS1A protein). NS1B, but not NS1A, binds human ISG15, an interferon (IFN) 2The abbreviations used are: IFNinterferonGSTglutathione S-transferaseGFPgreen fluorescent protein. -α/β-induced, ubiquitin-like protein (3.Yuan W. Krug R.M. EMBO J. 2001; 20: 362-371Crossref PubMed Scopus (415) Google Scholar). The binding site for human ISG15 is located in the N-terminal 104 amino acids of NS1B (3.Yuan W. Krug R.M. EMBO J. 2001; 20: 362-371Crossref PubMed Scopus (415) Google Scholar). ISG15 is conjugated to more than 100 cellular proteins (4.Zhao C. Denison C. Huibregtse J.M. Gygi S. Krug R.M. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 10200-10205Crossref PubMed Scopus (363) Google Scholar, 5.Giannakopoulos N.V. Luo J.K. Papov V. Zou W. Lenschow D.J. Jacobs B.S. Borden E.C. Li J. Virgin H.W. Zhang D.E. Biochem. Biophys. Res. Commun. 2005; 336: 496-506Crossref PubMed Scopus (178) Google Scholar) through the sequential action of three conjugation enzymes that are also induced by IFN-α/β: the E1-activating enzyme (Ube1L) (3.Yuan W. Krug R.M. EMBO J. 2001; 20: 362-371Crossref PubMed Scopus (415) Google Scholar), the E2-conjugating enzyme (UbcH8) (6.Zhao C. Beaudenon S.L. Kelley M.L. Waddell M.B. Yuan W. Schulman B.A. Huibregtse J.M. Krug R.M. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 7578-7582Crossref PubMed Scopus (243) Google Scholar, 7.Kim K.I. Giannakopoulos N.V. Virgin H.W. Zhang D.E. Mol. Cell. Biol. 2004; 24: 9592-9600Crossref PubMed Scopus (181) Google Scholar), and the E3 ligase (Herc5) (8.Dastur A. Beaudenon S. Kelley M. Krug R.M. Huibregtse J.M. J. Biol. Chem. 2006; 281: 4334-4338Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 9.Wong J.J. Pung Y.F. Sze N.S. Chin K.C. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10735-10740Crossref PubMed Scopus (184) Google Scholar). Human ISG15 consists of two ubiquitin-like domains connected by a short 5-amino acid hinge linker (10.Narasimhan J. Wang M. Fu Z. Klein J.M. Haas A.L. Kim J.J. J. Biol. Chem. 2005; 280: 27356-27365Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The C-terminal ISG15 domain, which is recognized by Ube1L, contains the C-terminal LRLRGG motif that is conjugated to target proteins (11.Chang Y.G. Yan X.Z. Xie Y.Y. Gao X.C. Song A.X. Zhang D.E. Hu H.Y. J. Biol. Chem. 2008; 283: 13370-13377Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The N-terminal domain (plus the hinge) is sufficient to bind NS1B.ISG15 and/or its conjugation play an important role in the IFN-induced antiviral state against several viruses, including influenza A and B viruses (3.Yuan W. Krug R.M. EMBO J. 2001; 20: 362-371Crossref PubMed Scopus (415) Google Scholar, 12.Lenschow D.J. Giannakopoulos N.V. Gunn L.J. Johnston C. O'Guin A.K. Schmidt R.E. Levine B. Virgin 4th, H.W. J. Virol. 2005; 79: 13974-13983Crossref PubMed Scopus (222) Google Scholar, 13.Okumura A. Lu G. Pitha-Rowe I. Pitha P.M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 1440-1445Crossref PubMed Scopus (282) Google Scholar, 14.Lenschow D.J. Lai C. Frias-Staheli N. Giannakopoulos N.V. Lutz A. Wolff T. Osiak A. Levine B. Schmidt R.E. García-Sastre A. Leib D.A. Pekosz A. Knobeloch K.P. Horak I. Virgin 4th, H.W. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1371-1376Crossref PubMed Scopus (408) Google Scholar, 15.Malakhova O.A. Zhang D.E. J. Biol. Chem. 2008; 283: 8783-8787Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 16.Okumura A. Pitha P.M. Harty R.N. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 3974-3979Crossref PubMed Scopus (216) Google Scholar, 17.Guerra S. Cáceres A. Knobeloch K.P. Horak I. Esteban M. PLoS Pathog. 2008; 4: e1000096Crossref PubMed Scopus (112) Google Scholar, 18.Lai C. Struckhoff J.J. Schneider J. Martinez-Sobrido L. Wolff T. García-Sastre A. Zhang D.E. Lenschow D.J. J. Virol. 2009; 83: 1147-1151Crossref PubMed Scopus (102) Google Scholar, 19.Hsiang T.Y. Zhao C. Krug R.M. J. Virology. 2009; 83: 5971-5977Crossref PubMed Scopus (91) Google Scholar). The first evidence for the antiviral role of ISG15 conjugation was the finding that the NS1B protein of influenza B virus binds ISG15 and blocks its conjugation (3.Yuan W. Krug R.M. EMBO J. 2001; 20: 362-371Crossref PubMed Scopus (415) Google Scholar), suggesting that ISG15 and/or its conjugation are inhibitory to the replication of influenza B virus. Subsequently, it was shown that both ISG15 knock-out (ISG15−/−) and Ube1L−/− mice are more susceptible to influenza B virus infection, establishing that ISG15 conjugation inhibits influenza B virus replication (14.Lenschow D.J. Lai C. Frias-Staheli N. Giannakopoulos N.V. Lutz A. Wolff T. Osiak A. Levine B. Schmidt R.E. García-Sastre A. Leib D.A. Pekosz A. Knobeloch K.P. Horak I. Virgin 4th, H.W. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1371-1376Crossref PubMed Scopus (408) Google Scholar, 18.Lai C. Struckhoff J.J. Schneider J. Martinez-Sobrido L. Wolff T. García-Sastre A. Zhang D.E. Lenschow D.J. J. Virol. 2009; 83: 1147-1151Crossref PubMed Scopus (102) Google Scholar). However, it was not clear why NS1B did not protect influenza B virus from the antiviral effects of ISG15 and its conjugation system in wild-type (ISG15+/+, Ube1L+/+) mice.Here we demonstrate that the NS1B protein exhibits species-specific binding. It binds human and non-human primate ISG15 but not mouse or canine ISG15. These results explain why influenza B virus replication is inhibited in wild-type mice that express mouse ISG15 and Ube1L. Surprisingly, we show that the 5-amino acid hinge of human and non-human primate ISG15, which has a different sequence than the ISG15 hinges of other mammalian species, is absolutely required for binding the NS1B protein. This study provides the first example of an influenza B virus protein that exhibits human (and non-human primate)-specific properties and thus provides one explanation for the restriction of influenza B virus to humans.