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-␣/␤-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 virusinfected 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 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). Many, but not all, of the proteins encoded by these two groups of viruses carry out similar functions (2). 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) 2 -␣/␤-induced, ubiquitin-like protein (3). The bind-ing site for human ISG15 is located in the N-terminal 104 amino acids of NS1B (3). ISG15 is conjugated to more than 100 cellular proteins (4,5) through the sequential action of three conjugation enzymes that are also induced by IFN-␣/␤: the E1-activating enzyme (Ube1L) (3), the E2-conjugating enzyme (UbcH8) (6,7), and the E3 ligase (Herc5) (8,9). Human ISG15 consists of two ubiquitin-like domains connected by a short 5-amino acid hinge linker (10). The C-terminal ISG15 domain, which is recognized by Ube1L, contains the C-terminal LRLRGG motif that is conjugated to target proteins (11). The N-terminal domain (plus the hinge) is sufficient to bind NS1B.
ISG15 and/or its conjugation play an important role in the IFNinduced antiviral state against several viruses, including influenza A and B viruses (3,(12)(13)(14)(15)(16)(17)(18)(19). 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), 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 Ϫ/Ϫ ) and Ube1L Ϫ/Ϫ mice are more susceptible to influenza B virus infection, establishing that ISG15 conjugation inhibits influenza B virus replication (14,18). 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 ϩ/ϩ , Ube1L ϩ/ϩ ) mice.
Here we demonstrate that the NS1B protein exhibits speciesspecific 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.

EXPERIMENTAL PROCEDURES
Cells and Virus-All cell lines were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Influenza B/Yamanashi/1998 virus was generated using plasmid-based reverse genetics (20). HeLa cells were infected with this virus at a multiplicity of infection of five plaque-forming units/cell and were then incubated in Dulbecco's modified Eagle's medium in the absence of serum.
Transfection Assays-The cDNA encoding NS1B was cloned into a pCN vector, and the cDNA encoding the indicated ISG15 protein was fused downstream of GST in the pCN vector. A mutated 3Ј splice site was introduced into the NS1B open reading frame to block production of spliced NS2 mRNA. Canine and monkey ISG15 cDNAs were cloned from poly(I-C)-treated Madin-Darby canine kidney cells and Cos7 cells, respectively. A plasmid expressing mouse ISG15 cDNA was provided by Jon Huibregtse (University of Texas at Austin). A plasmid express-ing human ISG15 cDNA was described previously (3). All point mutants and chimeric ISG15 cDNAs were generated by PCR. Cells were co-transfected with the plasmids as described in the legends for Figs. 1 and 3 and were harvested at 36 h after transfection in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 2.5 mM MgCl 2 , 1% Nonidet P-40, 10% glycerol) supplemented with a protease inhibitor. Cell extracts were processed as described in the legends for Figs. 1 and 3.
Immunofluorescence-NS1B cDNA was cloned downstream of GFP in the pEGFP-C1 plasmid, and ISG15 cDNAs were cloned downstream of the 3xFLAG tag in the p3xFLAG-CMV-10 plasmid. HeLa cells were co-transfected with the plasmids indicated in the legend for Fig. 2A. Alternatively, HeLa cells were transfected with a plasmid encoding the 3xFLAGtagged ISG15 protein indicated in the legend for Fig. 2B, and 24 h later, the cells were infected with influenza B virus for 5 h. Cells from both protocols were fixed and permeabilized as described previously (21). The primary antibody was either mouse anti-FLAG M2 antibody or serum from ferrets infected with influenza B/Memphis/12/97 virus (provided by J. McCullers) (22). The cells were incubated with the primary antibody at a dilution of 1:200 for 1 h at room temperature and were then incubated in the dark for 30 min at room temperature with the secondary antibody (rhodamine-conjugated goat anti-mouse IgG or fluorescein-conjugated goat anti-ferret IgG) at a dilution of 1:200. Cells were visualized using the Leica SP2 AOBS confocal microscope.

Influenza B Virus NS1 Protein Binds to Specific Mammalian
ISG15 Proteins-The protein sequence of ISG15 is poorly conserved across different mammalian species. For example, canine and mouse ISG15 proteins are only 69 and 65% identical to human ISG15, respectively. In contrast, monkey ISG15 exhibits 92% identity to human ISG15. We hypothesized that only those ISG15 proteins that exhibit high homology with human ISG15 would bind to the NS1B protein. To test this hypothesis, human HEK 293T cells were co-transfected with plasmids encoding NS1B and either GST alone or GST fused to the N terminus of human, mouse, canine, or monkey ISG15, and the cell extracts were subjected to GST selection (Fig. 1). Immunoblotting of the GST-selected extracts showed that NS1B binds not only human ISG15 but also monkey ISG15 (top panel, lanes 2 and 5). In contrast, binding of NS1B to either mouse or canine ISG15 was not detected (top panel, lanes 3 and 4). Immunoblots with anti-GST antibody showed that comparable amounts of the different GST-ISG15 proteins were selected (middle panel). Immunoblotting of transfected cell extracts with anti-NS1B antibody confirmed that the expression of the NS1B was comparable in all the samples (bottom panel). These results demonstrate that NS1B binds ISG15 in a highly species-specific manner. The same species-specific binding was observed when this transfection assay was carried out in mouse NIH3T3 cells (lanes 6 -10) and in canine Madin-Darby canine kidney cells (lanes 11-15), demonstrating that the binding of NS1B to ISG15 is independent of species-specific host factors.

Influenza B Virus Infection Causes Intracellular Relocalization of Human and Monkey ISG15 Proteins-During
transfection and at early times after infection, the NS1B protein localizes to the nucleus and accumulates in intranuclear compartments known as splicing or SC35 speckles (23). We determined whether NS1B specifically relocalizes only human and monkey ISG15 to speckles during transfection and virus infection. When HeLa cells were co-transfected with plasmids encoding GFP-NS1B and either 3xFLAG-tagged human ISG15 or 3xFLAG-tagged mouse ISG15, GFP-NS1B accumulated in nuclear speckles ( Fig. 2A, GFP fluorescence). Only human ISG15, but not mouse ISG15, was relocalized from the cytoplasm to nuclear speckles (FLAG fluorescence), as verified in the Merge panels. The same species specificity was seen in influenza B virus-infected cells. HeLa cells were transfected with a plasmid encoding a 3xFLAG ISG15 protein 24 h prior to infection with influenza B virus. Immunofluorescence of infected cells with anti-FLAG antibody showed that human and monkey ISG15, but not mouse or canine ISG15, were relocalized to nuclear speckles in infected cells (Fig. 2B, middle and right columns). To verify that these cells were infected, immunofluorescence was carried out using anti-influenza B virus antibody, which detects all viral proteins, including the NS1B protein that is localized in nuclear speckles (right column). These results show that the NS1B protein in infected cells specifically binds and alters the localization of only human and monkey ISG15.
The Critical Role of the Hinge of Human ISG15 in Binding the NS1B Protein-We took advantage of the species specificity of ISG15 binding by the NS1B protein to identify regions of the human ISG15 molecule that are required for NS1B binding. We confirmed that the N-terminal domain (plus the hinge) of  1, 6, and 11), GST-human ISG15 (lanes 2, 7, and 12), GST-mouse ISG15 (lanes 3, 8, and 13), GST-canine ISG15 (lanes 4, 9, and 14), or GST-monkey ISG15 (lanes 5, 10, and 15). Cell extracts were subjected to glutathione-Sepharose selection, and the eluents were analyzed by immunoblots (WB) using anti-NS1B (top panel) or anti-GST antibody (middle panel). Aliquots of the cell extracts containing equal protein amounts were analyzed by an immunoblot using anti-NS1B antibody (bottom panel). MDCK, Madin-Darby canine kidney cells.
human ISG15 is sufficient to bind the NS1B protein (11) and showed that this was also the case for monkey ISG15 (supplemental Fig. S1). Remarkably, canine ISG15 acquired strong NS1B binding simply by replacing its hinge with the human hinge (Can/Hum-H) (Fig. 3A, lanes 1-3), demonstrating the critical role of the human hinge in NS1B binding. The results with mouse ISG15 were different. Substitution of the human hinge in mouse ISG15 (Mou/Hum-H) led to little or no increase in ISG15 binding (lanes 4 -6). We can conclude that region(s) in the N-terminal domain of ISG15 are also recognized by NS1B and that the N-terminal domain of mouse ISG15 lacks the appropriate binding sequence(s), in contrast to human, nonhuman primate, and canine ISG15 (see under "Discussion").
Substituting the mouse ISG15 hinge for the human hinge in human ISG15 resulted in a complete loss of NS1B binding (Fig. 3B, lane 2), confirming the critical role of the human hinge in NS1B binding. To determine the role of individual amino acids in the human hinge, we made the indicated human-to-mouse amino acid substitutions (D76Q, K77N, and D79S). Each of these substitutions resulted in essentially a total loss of NS1B binding (lanes 3-5). Consequently, only those mammalian ISG15 molecules that contain the human ISG15 hinge sequence would be able to bind the NS1B protein. The hinge sequence of the ISG15 proteins of non-human primates is identical to that of humans, except that Lys-77 is replaced by Arg in the African green monkey hinge (Fig. 3C). This Lys-to-Arg replacement does not affect NS1B binding because African green monkey ISG15 binds NS1B (Fig. 1). In contrast, the hinge sequences of the other mammalian ISG15 proteins show substantial deviation from the human/non-human primate ISG15 hinge sequence. Consequently, these other mammalian ISG15 proteins would not be expected to bind NS1B, as already documented for the canine and mouse ISG15 proteins.

DISCUSSION
Influenza B virus is predominately, if not totally, restricted to humans (1), 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 (14,18). 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 humanspecific 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). 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. 3 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.