Herc5 Attenuates Influenza A Virus by Catalyzing ISGylation of Viral NS1 Protein

YuJie Tang, Gongxun Zhong, Lianhui Zhu, Xing Liu, Yufei Shan, Huapeng Feng, ZhiGao Bu, Hualan Chen and Chen Wang
J Immunol May 15, 2010, 184 (10) 5777-5790; DOI: https://doi.org/10.4049/jimmunol.0903588

Abstract

Ubiquitin-like protein ISG15, which is robustly induced by IFN or virus, is implicated to inhibit influenza A virus (IAV) in vivo. But the underlying mechanism still remains largely unknown. In this study, we report that Herc5 could catalyze conjugation of ISG15 onto IAV-NS1 protein, the critical virulence factor of IAV. This modification produces two more species, respectively mapped to IAV-NS1 at lysine 20, 41, 217, 219, and 108, 110, and 126. The ISGylated IAV-NS1 fails to form homodimers and inhibits relevant antiviral processes. Knockdown of Herc5 or ISG15 could partially alleviate IFN-β–induced antiviral activities against IAV, whereas ectopic expression of the Herc5-mediated ISGylation system could distinctly potentiate IFN-β–induced antiviral effects against IAV. Notably, IAV-NS1s of H5N1 avian IAVs display less ISGylation species than that of IAV-PR8/34 (human H1N1). Consistently, IAV-PR8/34 mutants deprived of IAV-NS1’s ISGylation exhibit augmented viral propagation and virulence in both cultured cells and mice. Our study reports the first microbial target of ISGylation and uncovers the direct antiviral function and mechanism of this novel modification.

Influenza A viruses (IAVs) are respiratory pathogens responsible for both seasonal flu outbreaks and periodic worldwide pandemics. They are negative-sense ssRNA viruses (orthomyxoviruses) encoding 11 proteins. NS1 protein (nonstructural) of IAV (IAV-NS1) is ∼230 aa and expressed abundantly in permissive cells (1). IAV-NS1 has been well characterized as a critical virulence factor of IAV, which contributes significantly to the disease pathogenesis by subverting host antiviral and physiological processes (2–4). For example, IAV-NS1 could inhibit the induction of type I IFNs upon virus infection (5), block the activation of two IFN-inducible antiviral proteins, protein kinase R (PKR) and oligoadenylate synthetase (6, 7), activate PI3K–Akt signaling pathway (8), selectively enhance viral mRNA translation (9), and interfere with cellular mRNA processing (10, 11).

To achieve these effects, IAV-NS1 depends on its versatile ability to participate in both protein-protein and protein–RNA interactions. The N-terminal of IAV-NS1 harbors an RNA–binding domain (RBD; 1–73 aa) that binds to several RNA species (12), whereas its C-terminal effector domain (ED; 74–230 aa) is predominantly responsible for binding to cellular proteins, such as PKR, p85β, Cleavage and polyadenylation specificity factor subunit 30 (CPSF30), and PABII (8, 10, 13, 14). Both domains contribute to the formation of IAV-NS1 homodimers, a prerequisite for IAV-NS1 to interact with its RNA targets and maintain its function (15–17). Interestingly, phosphorylation of IAV-NS1 has been reported to be functionally important for some IAV strains, and T215 is identified to be one of the potential target sites (18–20). However, modulation of IAV-NS1’s action by host innate immune response is still poorly understood. So it is intriguing to identify host factors that regulate IAV-NS1’s function during IAV infection.

The type I IFN system is a key component of innate immunity and represents the first line of defense against viral infection. Type I IFNs exert their effects through interaction with their cognate receptors that consequently activate the JAK/STAT and other signaling pathways. As a result, hundreds of IFN-stimulated genes (ISGs) are induced to perform a myriad of antiviral and immunoregulatory functions. Some of the ISG proteins have been well characterized to impair virus replication and spread in host cells, including PKR, RnaseL, and Mx (21). However, the potential functions for the rest of the ISG proteins remain to be identified.

ISG15 (15 kD) was reported to be induced rapidly and robustly by IFNs or viruses three decades ago. Recent years have witnessed some progress concerning the function and underlying mechanism of ISG15 as an ubiquitin homolog (21, 22). It is made up of two ubiquitin-like domains, in which the C-terminal LRLRGG motif is responsible for its conjugating (ISGylation) onto target proteins (23, 24). Formation of this isopeptide bond is catalyzed consecutively by a series of inducible enzymes: E1 activating enzyme Ube1L (25), E2 conjugating enzyme UbcH8 (26, 27), E3 ligase Herc5 or EFP (28–30), and deconjugating enzyme UBP43 (31). Unlike ubiquitination, ISGylation typically does not promote degradation of the target proteins. A couple of proteomics studies have identified >100 cellular proteins as potential targets of ISGylation (32–35). These proteins cover a wide spectrum of biological processes, including transcriptional regulation, signal transduction, inflammation, and control of cell growth. Notably, ISG15 and its conjugation system (E1, E2, and E3) were overexpressed in these proteomics studies, which made the observations possibly artificial. Until now, only a few of them have been validated as authentic substrates of ISGylation in vivo, including 4EHP and filamin B (36, 37). Interestingly, RIG-I, JAK1, STAT1, MxA, PKR, and RNaseL, which were either IFN-induced antiviral proteins or components of innate immune signaling pathways, were also suggested as potential targets for this modification (33–35). Hence, it remains to address whether ISGylation will influence the corresponding antiviral actions of these cellular proteins.

Herc5, initially described as cyclin E-binding protein 1, belongs to the Herc protein family, which is characterized by the presence of a HECT domain and one or more RCC1-like domains. The expression of Herc5 is especially high in testis and fetal brain but low in most tissues (38). Interestingly, Herc5 is induced by the proinflammatory cytokines TNF-α and the β form of pro-IL-1 (39). Herc5 is also markedly induced by type I IFNs or viruses and functions as an ISG15 E3 ligase to promote overall ISGylation in host cells (29, 30). But the authentic substrates and physiological function of Herc5-mediated ISGylation in response to IFN stimulation or virus infection remains largely unknown. Therefore, it is interesting to address whether and how Herc5, as an ISG15 E3 ligase, plays a role in host antiviral response.

Several recent studies have suggested a role of ISG15 in attenuating virus infection (21, 22). It is reported that mice deficient in ISG15 have increased susceptibility to influenza A and B, Sindbis virus, HSV-1, murine γ-herpes virus, and vaccinia virus (40, 41). Ectopic expression of ISG15 could protect IFN-α/βR−/− and ISG15−/− mice against lethality following Sindbis virus infection (41, 42). ISG15 could also interfere with HIV-1 release or Ebola virus VP40 virus-like particle budding, possibly by inhibiting ubiquitination of its target proteins (43–45). Interestingly, the increased lethality of IFN-α/βR−/− and ISG15−/− mice upon Sindbis virus infection can only be rescued by a recombinant virus expressing wild-type (WT) ISG15, but not ISG15 (AA) mutant, which cannot form conjugates (41, 42). Mice lacking Ube1L, the ISG15 E1 enzyme, show increased susceptibility to both Sindbis virus and influenza B virus infection (46, 47). In addition, influenza B virus NS1 binds ISG15 at its N terminus and blocks cellular ISGylation (25). Several viral-encoded proteases have been found to mediate de-ISGylation (48, 49). Together, these results have defined ISG15 as a critical IFN-induced antiviral molecule. However, the mechanism of ISG15’s antiviral action still remains largely unclear.

Although considerable progress has been made in understanding ISGylation of cellular target proteins, no investigation has yet explored the possibility of this modification concerning any viral proteins. In this study, we report on IAV-NS1 as the first microbial target of ISGylation and uncover Herc5 as a novel and critical host antiviral protein against IAV by catalyzing the ISGylation of IAV-NS1.

Materials and Methods

Plasmids and recombinant proteins

Full-length IAV-NS1 cDNAs of the different IAVs (PR8: A/PR8/34; GX27: A/Duck/Guangxi/27; GX35: A/Duck/Guangxi/35; HN12: A/Chicken/Henan/12; and JS7: A/Chicken/Jiangsu/7) and all the IAV-NS1 truncation mutants were derived by PCR from the corresponding pGEM-T-NS plasmids and subcloned into the indicated vectors to make IAV-NS1 expression constructs. The pGEM-T-NS (PR8, HN12, and JS7) plasmids are gifts from Prof. Ze Chen (Wuhan Institute of Virology, Chinese Academy of Sciences [CAS], Shanghai, China). The pGEM-T-NS (GX27 and GX35) plasmids were generated as described previously (50). Human ISG15, Ube1L, UbcH8, Herc5, EFP, PKR-NT, p85β-inSH2, and poly(A)-binding protein 1 (PABP1)-CT cDNAs were constructed by PCR from the human thymus plasmid cDNA library (BD Clontech, Palo Alto, CA) and subsequently cloned into the indicated expression vectors. All point mutations were introduced into the indicated expression constructs by using a QuickChange XL site-directed mutagenesis method (Stratagene, La Jolla, CA). All constructs were confirmed by sequencing. rGST-fusion proteins were purified from Escherichia coli (BL21) using glutathione-Sepharose 4B resin (GE Healthcare, Piscataway, NJ).

Cells and reagents

HEK293T, HEK293, A549, and MDCK cells were all cultured using DMEM (Invitrogen, Carlsbad, CA) plus 10% FBS (Hyclone, Thermo Fisher Scientific, Waltham, MA), supplemented with 1% penicillin-streptomycin (Invitrogen). Transient transfections were performed with the calcium phosphate method or Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Anti-Flag M2 affinity Gel and N-ethylmaleimide were purchased from Sigma-Aldrich (St. Louis, MO). Recombinant human IFN-β was purchased from PBL InterferonSource (Piscataway, NJ).

Purification of IAV-NS1 binding proteins and mass spectrometry

Twenty-four hours posttreatment of IFN-β (1000 U/ml), A549 cells were collected and lysed with TBS buffer (50 mM Tris-Cl [pH 7.4], 150 mM NaCl) supplemented with 1% Triton-X 100, 1 mM PMSF, and a complete protease inhibitor mixture (Roche, Basel, Switzerland). Postcentrifuged supernatants were then incubated at 4°C for 4 h with GST resin (GE Healthcare), which had already been loaded with rGST or GST-PR8-NS1 proteins. Postincubation, GST beads were washed extensively with lysis buffer, and proteins bound to GST beads were separated on 7.5% SDS-PAGE gels. After silver staining (Sigma-Aldrich), specific protein bands were excised and analyzed by mass spectrometry.

Immunoprecipitation analysis and immunoblot analysis

For immunoprecipitation (IP) analysis, cells were lysed in TBS buffer (50 mM Tris-Cl [pH 7.4], 150 mM NaCl) supplemented with 1% Triton-X 100, 1 mM PMSF, and a protease inhibitor mixture (Roche). After preclearing for 1 h, cell extracts were incubated with the indicated Ab for 4 h to overnight at 4°C. Two hours after adding protein A/G-agarose, the immunoprecipitates were extensively washed with lysis buffer and eluted with SDS loading buffer by boiling for 3–5 min. The anti-Flag M2 affinity gel was used to immunoprecipitate Flag-tagged protein when needed.

For immunoblot analysis, the samples were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The following primary Abs were used: anti-Flag (1:5,000; Sigma-Aldrich), anti–β-actin (1:10,000; Sigma-Aldrich), anti-hemagglutinin (HA) (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Herc5 (1:1,000; Abmart, Shanghai, China), anti-ISG15 (1:2,000; Abmart), anti-Myc (1:2,000; Santa Cruz Biotechnology), anti-PKR (1:2,000; Santa Cruz Biotechnology), and anti-GST (1:2,000; Santa Cruz Biotechnology); anti-NS1 and anti-NP primary Abs were kindly provided by Prof. Bing Sun (Shanghai Pasteur Institute, CAS). The proteins were visualized by using an NBT/BCIP Western blotting system (Promega, Madison, WI) or a SuperSignal West Pico chemiluminescence ECL kit (Pierce, Rockford, IL).

Real-time RT-PCR

RNA extractions and reverse transcriptions of purified RNA were performed as described previously (51). The quantifications of gene transcripts were performed by real-time PCR using Power SYBR GREEN PCR MASTER MIX (Applied Biosystems, Foster City, CA). All values were normalized to the level of β-actin mRNA. The primers were listed as follows: β-actin: sense (5′-AAAGACCTGTACGCCAACAC-3′) and antisense (5′-GTCATACTCCTGCTTGCTGAT-3′); IFN-β: sense (5′-ATTGCCTCAAGGACAGGATG-3′) and antisense (5′-GGCCTTCAGGTAATGCAGAA-3′); ISG56: sense (5′-GCCATTTTCTTTGCTTCCCCTA-3′) and antisense (5′-TGCCCTTTTGTAGCCTCCTTG-3′); and RANTES: sense (5′-TACACCAGTGGCAAGTGCTC-3′) and antisense (5′-ACACACTTGGCGGTTCTTTC-3′).

ISG15 and Herc5 siRNAs

The sequences of small interfering (si)RNAs used in this study were as follows: NC: 5′-UUCUCCGAAGGUGUCACGU-3′; siISG15: 5′-UGAGCACCGUGUUCAUGAA-3′; siISG15m: 5′-UGAGGUCCUGGUUCACAAA-3′; siHerc5: 5′- GGACUAGACAAUCAGAAAG-3′; and siHerc5m: 5′- GGAC-UAGACGCUCAGAGCC-3′.

All siRNA duplexes were chemically synthesized by Gene-Pharma (Shanghai, China) and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Ni-NTA pulldown analysis

For Ni-nitrilotriacetic acid resin (NTA) pulldown analysis, cells were lysed in His-Lysis Buffer (50 mM Tris-Cl [pH 7.4], 300 mM NaCl, 1% Triton-X 100, 10 mM imidazole, 10 mM 2-ME) supplemented with 5 mM N-ethylmaleimide, 1 mM PMSF, and a protease inhibitor mixture (Roche). Ni-NTA agarose (Qiagen, Valencia, CA) was then added into the postcentrifuged cell extracts and incubated for 4 h to overnight at 4°C. After extensively washing with His-Lysis Buffer containing 20 mM imidazole, the precipitates were subjected to SDS-PAGE followed by immunoblot analysis or eluted with TBS containing 300 mM imidazole and subsequently subjected to GST-pulldown or RNA-pulldown analysis.

GST-pulldown and RNA-pulldown analysis

For GST-pulldown analysis, purified rGST-fusion proteins were loaded onto GST resin (GE Healthcare) by mixing for 1 h followed by extensive washing. Cells were harvested and lysed with TBS buffer (50 mM Tris-Cl [pH 7.4], 150 mM NaCl) supplemented with 1% Triton-X 100, 1 mM PMSF, and a protease inhibitor mixture (Roche). Postcentrifugation, cell extracts were incubated with preloaded GST resin and incubated for 2–4 h at 4°C. Precipitates were extensively washed and subjected to SDS-PAGE followed by immunoblot analysis.

For RNA-pulldown analysis, biotin-labeled RNA was prepared by performing in vitro transcription reaction with the Biotin RNA Labeling Mix (Roche) and T7/SP6 RNA polymerase (Ambion, Austin, TX). Biotin-labeled dsRNA was made by annealing two complementary ssRNAs. Cell extracts were made with RP buffer (50 mM Tris-Cl [pH 7.4], 150 mM NaCl, 2 mM MgCl2, 2 mM DTT, 1 mM EDTA, 2.5% glycerol, 0.5% Triton-X 100, 0.2 mg/ml heparin, 0.1 mg/ml tRNA, 200 U/ml RNasin) supplemented with 1 mM PMSF, and a protease inhibitor mixture (Roche), and then mixed with biotin-labeled RNA for 2 h at 4°C before Streptavidin Magnetic Particles (Roche) were added. After 2 h incubation, precipitates were extensively washed and subjected to SDS-PAGE followed by immunoblot analysis.

Generation of IAV-PR8/34 mutants and virus infection

Human H1N1 IAV strain IAV-PR8/34 was kindly provided by Prof. Ze Chen (Wuhan Institute of Virology, CAS). All the IAV-PR8/34 mutants were generated by reverse genetics and propagated in 11-d-old chicken eggs as described previously (50). Viral infection was performed when 80–90% cell confluence was reached. After 1 h of incubation, the inoculums were removed, and the cells were washed twice with PBS and then overlaid with DMEM with or without N-acetylated trypsin. At the indicated time point, the media was collected and titrated for virus infectivity in eggs as described previously (50). Alternatively, cell extracts were made and subjected to immunoblot analysis with the indicated Abs. Where indicated, the cells were pretreated with rIFN-β before virus infection.

Animal experiments

For the mouse study, groups of 3–6-wk-old female BALB/c mice (Beijing Experimental Animal Center, Beijing, China) were lightly anesthetized with CO2 and inoculated intranasally with 104 50% egg infectious dose (EID50) of the IAV in a volume of 50 μl. The mice in each group were euthanized on days 2 and 3 postinoculation. Lungs were collected and titrated for virus infectivity in eggs as described previously (50). The 50% mouse lethal dose was determined by inoculating groups of five mice with 10-fold serial dilutions containing 101–106 EID50 of the IAV in a 50-μl volume and calculated by using the method of Reed and Muench (52). The mice groups were monitored for 14 d for weight change and mortality. Weight changes of mice groups were determined by measuring the average weight change of each group of mice instead of weight change of each single mouse in every group. These mice challenging experiments were repeated three times, and the data of each time exhibited the same trend. Therefore, only one representative group of data was presented. The mouse studies have been approved by the Review Board of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.

Statistics

Student t test was used for the statistical analysis of two independent treatments. For all tests, a p value of <0.05 was considered statistically significant.

Results

Herc5 interacts with NS1 during IAV infection

To explore whether there was any host factor regulating IAV-NS1 during the innate immune response, A549 cells were first treated with or without 1000 U/ml IFN-β for 24 h. Then the cell extracts were subjected to GST-pulldown analysis with rGST or GST-NS1 proteins. The precipitates were resolved and silver stained. Interestingly, a protein band (∼120 kD) was specifically pulled down by GST-NS1 in IFN-β–treated cell extract, but not in mock-treated cell extract (Fig. 1A). Mass spectrometry analysis exclusively identified it as Herc5/cyclin E-binding protein 1. Consistently, the band could be detected by anti-Herc5 Ab (Fig. 1A). Alternatively, ectopically expressed Flag-Herc5 could also be specifically pulled down by GST-NS1 (Fig. 1B).

FIGURE 1.
FIGURE 1.

Herc5 interacts with IAV-NS1 during IAV infection. A, A549 cells were treated with or without 1000 U/ml IFN-β for 24 h. The cell extracts were then subjected to GST-PD analysis with rGST or GST-NS1 proteins. The precipitates were resolved in SDS-PAGE followed by silver staining (left panel). The specific band noted by the asterisk (*) was excised for mass spectroscopy identification. The same samples were immunoblotted with the indicated Abs (right panel). B, HEK293T cells were transfected with Flag-Herc5 or empty vector for 48 h. The cell extracts were then subjected to GST-PD analysis with GST or GST-NS1 proteins, followed by immunoblot analysis with the indicated Abs. C, HEK293T cells were transfected with Flag-Herc5 and HA-NS1 as indicated for 48 h. The cell extracts were then immunoprecipitated and immunoblotted with the indicated Abs. D, A549 cells were infected with IAV-PR8/34 or Sendai virus for 24 h. Equal amounts of the cell extracts were then immunoprecipitated and immunoblotted with the indicated Abs or control IgG. E, HEK293T cells were transfected with Flag-Herc5 for 48 h. The cell extracts were then subjected to GST-PD analysis with the indicated GST fusion proteins, followed by immunoblot analysis with anti-Flag Ab or CBB staining. F, HEK293T cells were transfected with Myc-Herc5 (C994A) for 48 h. The cell extracts were then subjected to GST-PD analysis with GST or GST-NS1 proteins, followed by immunoblot analysis with anti-Myc Ab or CBB staining. CBB, Coomassie brilliant blue; GST-PD, GST pulldown.

To further confirm the interaction between IAV-NS1 and Herc5, HA-NS1 and Flag-Herc5 were transfected individually or together into HEK293T cells. The cell extracts were immunoprecipitated with anti-HA or anti-Flag Abs. As expected, HA-NS1 and Flag-Herc5 could coimmunoprecipitate with each other (Fig. 1C). Likewise, the interaction between endogenous IAV-NS1 and Herc5 was confirmed by immunoprecipitation (IP) analysis of IAV-PR8/34–infected A549 cell extracts using anti-NS1 Ab or control IgG (Fig. 1D). To determine which domain of NS1 was responsible for binding to Herc5, ectopically expressed Flag-Herc5 was subjected to GST-pulldown analysis with GST-NS1-RBD or GST-NS1-ED. Neither of them could interact with Flag-Herc5, suggesting that both RBD and ED of IAV-NS1 were essential for its interaction with Herc5 (Fig. 1E). Additionally, Herc5 (C994A), the enzymatically deficient mutant of Herc5 carrying a point mutation in its HECT domain, could also be pulled down by GST-NS1 (Fig. 1F).

IAV-NS1 is a novel target of Herc5-mediated ISGylation

Because Herc5 is markedly induced by type I IFNs or viruses and functions as an ISG15 E3 ligase to promote overall protein ISGylation in host cells, we wondered if IAV-NS1 was a target of Herc5-mediated ISGylation. Therefore, IAV-NS1 was coexpressed with different combinations of the His-ISGylation system components, including His-ISG15, Myc-Ube1L, and Myc-UbcH8. Ni-NTA pulldown analysis demonstrated that IAV-NS1 was marginally modified by ISG15 without any E3 ligase, and two ISGylated bands of IAV-NS1 were observed (Fig. 2A, bottom band I and top band II). Notably, this modification could be dramatically enhanced by Herc5, but not by EFP (Fig. 2A). In contrast, Herc5 (C994A), the enzymatically deficient mutant of Herc5, could barely catalyze the ISGylation of IAV-NS1 (Fig. 2B). Similarly, the ISGylation of IAV-NS1 was markedly attenuated when other components of the His-ISGylation system were individually mutated (Fig. 2B). Alternatively, IP analysis of HA-NS1 also revealed that IAV-NS1 was ISGylated as two bands dependent on the ISGylation system (Fig. 2C). To probe the ISGylation of IAV-NS1 in IAV-infected cells, HEK293T cells were transfected with the His-ISGylation system, and then infected with IAV-PR8/34. Consistently, Ni-NTA pulldown analysis revealed two ISGylated bands of IAV-NS1, which were markedly enhanced by Herc5 (Fig. 2D).

FIGURE 2.
FIGURE 2.

Herc5 catalyzes ISGylation of IAV-NS1. A, HEK293T cells were transfected with pcDNA3-IAV-NS1, His-ISG15, Myc-Ube1L, Myc-UbcH8, Flag-EFP, and Flag-Herc5 plasmids as indicated for 48 h. The cell extracts were then subjected to Ni-NTA PD analysis, followed by immunoblot analysis with anti-NS1 Ab. The two ISGylated bands of IAV-NS1 were indicated: the bottom band I and the top band II. B, HEK293T cells were transfected with the indicated plasmids (WT or mutant) for 48 h. The cell extracts were then analyzed as above. C, HEK293T cells were transfected with the indicated plasmids for 48 h. The cell extracts were then immunoprecipitated with anti-HA Ab, followed by immunoblot analysis with the indicated Abs. D, HEK293T cells were transfected with the indicated plasmids and infected with or without IAV-PR8/34 for 24 h. The cell extracts were then subjected to Ni-NTA PD analysis, followed by immunoblot analysis with anti-NS1 Ab. Ni-NTA PD, Ni-NTA pulldown.

Furthermore, we used a knockdown approach to investigate the modification of IAV-NS1 by endogenous Herc5-mediated ISGylation in response to IFN treatment or IAV infection. Effective siRNAs against Herc5 or ISG15 were screened out and could markedly diminish IFN-β–induced protein ISGylation (Fig. 3A). HEK293 cells were transfected with HA-NS1 and treated with 1000 U/ml IFN-β for 48 h. IP analysis of HA-NS1 clearly indicated that IAV-NS1 was ISGylated as two bands (Fig. 3B). Notably, this modification was severely attenuated when the specific siRNAs against Herc5 or ISG15 were employed (Fig. 3B). Additionally, when the extracts of IAV-PR8/34–infected A549 cells or IFN-β–pretreated HEK293 cells were subjected to IP analysis with anti-NS1 Ab, IAV-NS1 was also found to be ISGylated with identical pattern as above observations (Fig. 3C, 3D). Taken together, these data indicated that IAV-NS1 could be modified by ISG15 in response to IFN treatment or IAV infection, which was catalyzed by Herc5.

FIGURE 3.
FIGURE 3.

Herc5 is essential for the ISGylation of IAV-NS1 upon IFN stimulation or IAV infection. A, The effectiveness of siRNAs against Herc5 or ISG15. Cells were transfected with or without siRNAs and then treated with 1000 U/ml IFN-β as indicated. The cell extracts were subjected to immunoblot analysis with the indicated Abs. B, HEK293 cells were transfected with HA-NS1 and siRNAs as indicated and then treated with 1000 U/ml IFN-β for 48 h. The cell extracts were immunoprecipitated with anti-HA Ab, followed by immunoblot analysis with the indicated Abs. C, HEK293 cells were transfected with siRNAs and pretreated with 1000 U/ml IFN-β as indicated. The cells were then infected with an equal amount of IAV-PR8/34 or not. The cell extracts were immunoprecipitated with anti-NS1 Ab, followed by immunoblot analysis with the indicated Abs. D, A549 cells were transfected with siRNAs as indicated. The cells were then infected with an equal amount of IAV-PR8/34 or not. The cell extracts were analyzed as above.

Herc5-mediated ISGylation targets multiple lysines of IAV-NS1

Next, we carried out a systematic lysine (K) to arginine (R) mutation scanning to identify the potential ISGylation target sites on IAV-NS1. The IAV-NS1 constructs harboring different combinations of K to R point mutations were expressed along with His-ISGylation system, followed by Ni-NTA pulldown and IP analysis. We found that when the four lysines (K20/41/217/219) at the N- or C-terminal of IAV-NS1 were all mutated to arginines, the bottom ISGylated band I disappeared, suggesting that K20/41/217/219 were clearly linked to the bottom ISGylated band I (Fig. 4A). Similarly, three other lysines (K108/110/126) in the middle part of IAV-NS1 were unequivocally linked to the top ISGylated band II (Fig. 4B). We further generated a IAV-NS1 (7M) construct carrying all the seven lysines (K20/41/108/110/126/217/219) mutated to arginines. It was found that ISGylation (both band I and II) of this IAV-NS1 mutant was completely abolished, indicating that these seven lysines were necessary for Herc5-mediated ISGylation of IAV-NS1 (Fig. 4C).

FIGURE 4.
FIGURE 4.

Herc5-mediated ISGylation targets multiple lysines of IAV-NS1. A–D, HEK293T cells were transfected with the His-ISGylation system (His-ISG15 /E1/E2/E3) together with empty vector or pcDNA3-IAV-NS1 (WT and mutant) as indicated. The cell extracts were subjected to Ni-NTA PD analysis, followed by immunoblot analysis with anti-NS1 Ab. The cell extracts were also immunoprecipitated with anti-NS1 Ab and immunoblotted with the indicated Abs. 7M: K20/41/108/110/126/217/219R. E, Schematic diagram of IAV-NS1’s ISGylation pattern. K20/41/108/110/126/217/219 residues of IAV-NS1 are the target sites of Herc5-mediated ISGylation. They can be divided into two modification groups, in which K20/41/217/219 contributes to the bottom band I and K108/110/126 to the top band II.

On the background of the IAV-NS1 (KO) mutant, of which all 12 lysines were mutated to arginines, we generated five other IAV-NS1 mutants for which, for a given mutant, only the indicated one or two adjacent lysines were kept intact, whereas the rest were all mutated to arginines. We tested the ISGylation status of these IAV-NS1 mutants and found that they all retained the ability to be modified by Herc5-mediated ISGylation (Fig. 4D). Interestingly, the bottom ISGylated band I reappeared when K20, K41, or K217/219 were reintroduced into IAV-NS1 individually (Fig. 4D). Likewise, the top ISGylated band II reappeared when K108/110 or K126 were restored (Fig. 4D). Collectively, these data established that the seven lysines (K20/41/108/110/126/217/219) of IAV-NS1 were potential target sites of Herc5-mdiated ISGylation. These lysines could be divided into two modification groups, in which K20/41/217/219 contributed to the bottom band I and K108/110/126 to the top band II (Fig. 4E).

The action of IAV NS1 is attenuated by Herc5-mediated ISGylation

To determine the function of IAV-NS1’s ISGylation, ISGylated IAV-NS1 was generated and purified to test its interaction with the protein or RNA targets in comparison with unmodified IAV-NS1, according to the experimental design in Fig. 5A. To purify ISGylated IAV-NS1, HA-NS1 was coexpressed along with His-ISGylation system (E1/E2/E3/His-ISG15) in HEK293T cells. The cell extracts were purified by Ni-NTA agarose, and the elute fraction (∼300 mM imidazole) was subjected to GST-pulldown or RNA-pulldown analysis. Interestingly, comparable amounts of both ISGylated and unmodified HA-NS1 were present in the elute fraction, as shown by immunoblot analysis with anti-HA Ab (Fig. 5B, 5C, INPUT lane). The unmodified HA-NS1 was found to be pulled down by nonspecific interaction with the Ni-NTA agarose, which could serve as an internal positive control, as was exploited previously in the case of 4EHP (37). Expectedly, the unmodified HA-NS1 could be pulled down by all the indicated GST-fusion proteins (GST-NS1-RBD/GST-p85β-inSH2/GST-PKR-NT/GST-PABP1-CT) but not by GST (Fig. 5B). In contrast, the ISGylated HA-NS1 could not be pulled down by GST-PKR-NT or GST-NS1-RBD, indicating that ISGylation of IAV-NS1 could disrupt its interaction with PKR and the dimerization of its RBDs (Fig. 5B). Notably, the ISGylated HA-NS1 could still be pulled down by GST-p85β-inSH2 and GST-PABP1-CT, suggesting that ISGylation of IAV-NS1 selectively impaired its interaction with a subgroup of the binding partners (Fig. 5B).

FIGURE 5.
FIGURE 5.

The action of IAV-NS1 is attenuated by Herc5-mediated ISGylation. A, The flow charts of indirect and direct GST-PD/RNA-PD analysis. HEK293T cells were transfected with HA-NS1 together with or without His-ISGylation system (His-ISG15/E1/E2/E3). When HA-NS1 was coexpressed with His-ISGylation system, the cell extracts were firstly purified by Ni-NTA agarose, and then the elute fractions (∼300 mM imidazole) were subjected to GST-PD or RNA-PD analysis (left panel); When HA-NS1 was expressed alone, the cell extracts were made and subjected directly to GST-PD or RNA-PD analysis (right panel). B and C, HEK293T cells were transfected with HA-NS1 together with or without His-ISGylation system. The cell extracts were analyzed by indirect or direct GST-PD analysis (B) or RNA-PD analysis (C) as indicated according to the experimental design described in A. The ISGylated and unmodified HA-NS1 are indicated on the right side. D, HEK293T cells were transfected with the indicated plasmids, then infected with equal amount of Sendai virus for 12 h. The inductions of IFN-β, ISG56, and RANTES mRNAs were measured by real-time RT-PCR. The results are presented as means ± SD from at least three independent experiments. **p < 0.01. CT, C-terminal; NT, N-terminal.

The RBD dimer of IAV-NS1 has been demonstrated to be responsible for its interaction with RNA targets, including U6snRNA, dsRNA, poly-A RNA, and viral RNA (16, 17). So we next carried out the RNA-pulldown analysis with U6snRNA and dsRNA, which were in vitro transcribed and labeled with biotin. Consistently, the unmodified HA-NS1 could interact with both RNA targets, whereas the ISGylated HA-NS1 could not, indicating that ISGylation of IAV-NS1 could also impair its interaction with RNA targets (Fig. 5C).

PR8-NS1 is reported to be able to block the activation of IFN regulatory factor 3 (IRF-3), AP-1, and NF-κB signaling pathways, possibly by preventing intracellular sensors from accessing to viral dsRNA, thus inhibiting the induction of type I IFNs and other antiviral genes upon virus infection (5, 53, 54). Therefore, we further examined if ISGylation of IAV-NS1 could interfere with its inhibitory effect on the induction of antiviral genes upon virus infection. It is known that Sendai virus could induce robust expression of such antiviral genes as IFN-β, ISG56, and RANTES, which could be dramatically attenuated by ectopic expression of IAV-NS1 in cells (Fig. 5D). Significantly, the inhibitory effect of IAV-NS1 was partially alleviated when Herc5-mediated ISGylation system was coexpressed (Fig. 5D). This deinhibition failed when the ISGylation system was incomplete or mutated (Fig. 5D). In addition, it was found that coexpression of the Herc5-mediated ISGylation system apparently did not affect the inhibitory effect of IAV-NS1 (7M) mutant, which could not be ISGylated (Fig. 5D). Taken together, these data demonstrated that the action of NS1 was crippled when it was modified by Herc5-mediated ISGylation.

Herc5 displays antiviral activities against IAV in cultured cells

Given that IFN-β markedly induces the expression of ISG15, Herc5, and protein ISGylation in HEK293 cells, we took knockdown and overexpression approaches to explore if Herc5 was important for IFN-β–induced antiviral effects against IAV. Initially, we confirmed the antiviral effect of IFN-β against IAV by showing that pretreatment of cells with IFN-β could distinctly reduce the expression levels of IAV-NS1 and IAV-NP in IAV-PR8/34–infected HEK293 cells (Fig. 6A). Consistently, this effect was dependent on the concentration and duration of the IFN-β applied (Fig. 6A). Next, the cells were transfected with siRNAs and treated with IFN-β as indicated, followed by infection with equal amount of IAV-PR8/34. The expression levels of IAV-NS1 and IAV-NP were detected by immunoblot analysis to monitor the propagation status of the virus. Interestingly, the expressions of both IAV-NS1 and IAV-NP were partially rescued when either Herc5 or ISG15 was knocked down in the IFN-β–pretreated cells, suggesting that both Herc5 and ISG15 were essential for IFN-β–induced antiviral effects against IAV (Fig. 6B). To further substantiate this observation, Herc5-mediated ISGylation system was overexpressed in HEK293 cells, and the cells were treated with IFN-β followed by IAV-PR8/34 infection. Consistently, the expression levels of IAV-NS1 and IAV-NP were distinctly further downregulated when Herc5-mediated ISGylation system was ectopically expressed, whereas no obvious downregulation was observed when ISG15 was expressed alone. The results revealed that Herc5-mediated ISGylation could potentiate IFN-β–induced inhibitory effects against IAV (Fig. 6C).

FIGURE 6.
FIGURE 6.

Herc5 displays antiviral activities against IAV in cultured cells. The experimental design is shown at the top of each panel. A, HEK293 cells were pretreated with IFN-β as indicated, followed by infection with equal amount of IAV-PR8/34 for 8 h. The cell extracts were made and subjected to immunoblot analysis with the indicated Abs. B, HEK293 cells were transfected with the indicated siRNAs for 24 h, then treated with or without 1000 U/ml IFN-β for another 24 h, followed by infection with equal amount of IAV-PR8/34. Eight hours postinfection, the cell extracts were made and subjected to immunoblot analysis with the indicated Abs. C, HEK293 cells were transfected with the indicated plasmids for 24 h, then treated with or without 100 U/ml IFN-β for another 12 h, followed by infection with equal amount of IAV-PR8/34. Eight hours postinfection, the cell extracts were made and subjected to immunoblot analysis with the indicated Abs. D, A549 cells were transfected with the indicated siRNAs for 24 h, followed by infection with equal amount of IAV-PR8/34. Forty-eight hours postinfection, the culture media were collected and measured for virus infectivity in eggs. The results are presented as means ± SD. *p < 0.05; **p < 0.01.

In A549 cells, IAV-PR8/34 infection per se could induce robust expression of Herc5, ISG15, and protein ISGylation, which are comparable to IFN-β treatment. So we chose A549 cells to explore Herc5-mediated antiviral effects against IAV without IFN-β pretreatment. The cells were transfected with the indicated siRNAs and infected with an equal amount of IAV-PR8/34. The propagation status of the virus was examined by measuring the virus infectivity of the cell culture media. It was found that IAV-PR8/34 exhibited augmented viral propagation in either Herc5 or ISG15 knockdown cells, indicating that Herc5 and ISG15 both displayed antiviral activities against IAV in A549 cells (Fig. 6D).

IAV-NS1s of H5N1 avian IAVs display less ISGylation species than that of IAV-PR8/34 (human H1N1)

H5N1 highly pathogenic avian IAVs, which were originally identified as catastrophic pathogens for poultry, have now become a severe threat to human health and may cause a future fatal worldwide influenza pandemic. Therefore, we examined if IAV-NS1 of H5N1 avian IAV could be modified by Herc5-mediated ISGylation. HA-GX27-NS1 and HA-GX35-NS1 of H5N1 avian IAVs were coexpressed with the Herc5-mediated ISGylation system in cells. IP analysis with anti-HA Ab revealed that both H5N1-NS1s could be ISGylated (Fig. 7A). Surprisingly, only one ISGylated band was detected (Fig. 7A). To further determine the ISGylation pattern of H5N1-NS1, Flag-HN12-NS1 of H5N1 avian IAV and Flag-JS7-NS1 of H9N2 avian IAV were examined by IP analysis with anti-Flag M2 affinity Gel and still displayed only one ISGylated band (Fig. 7A). The ISGylated band of these H5N1-NS1s corresponded to the bottom band I of PR8-NS1 mapped above in terms of the migration position in gel. Indeed, the ISGylated band disappeared when the corresponding lysines of band I (K20/41/217/219/221) were all mutated to arginines (Fig. 7B). Furthermore, we aligned and compared the protein sequences of these IAV-NS1s to explore the molecular basis of the different ISGylation patterns between H5N1-NS1 and PR8-NS1. Intriguingly, all of the lysines corresponding to the top ISGylated band II (K108/110/126) of PR8-NS1 remained intact in the H5N1-NS1s, indicating that the loss of ISGylation species did not result from the absence of lysines at the corresponding sites (Fig. 7C). In addition, GST-pulldown analysis with rGST-NS1 protein revealed that the affinity of Herc5 to PR8-NS1 was apparently higher than to HN12-NS1, suggesting that the difference of ISGylation pattern may in some degree result from the different affinity between IAV-NS1 and Herc5 (Fig. 7D).

FIGURE 7.
FIGURE 7.

IAV-NS1s of H5N1 avian IAVs display less ISGylation species than that of IAV-PR8/34. A, HEK293T cells were transfected with empty vector or the indicated IAV-NS1 plasmids (PR8: human H1N1; GX27, GX35 and HN12: avian H5N1; JS7: avian H9N2) together with His-ISGylation system for 48 h. The cell extracts were immunoprecipitated and immunoblotted with the indicated Abs. B, HEK293T cells were transfected with the indicated IAV-NS1 plasmids (WT and mutant) together with His-ISGylation system for 48 h. The cell extracts were immunoprecipitated and immunoblotted with the indicated Abs. 5M: K20/41/217/219/221R; 4M: K20/41/214/216R. C, Schematic diagram of ISGylation patterns of the indicated IAV-NS1s. The ISGylated sites of each IAV-NS1 are indicated and aligned. D, HEK293T cells were transfected with Flag-Herc5 for 48 h. The cell extracts were then subjected to GST-PD analysis as indicated.

K126 and K217 of IAV-NS1 are critical target sites of Herc5-mediated ISGylation in vivo

To explore the functional significance of IAV-NS1 ISGylation, we systematically generated IAV-PR8/34 mutant viruses, each individually carrying K to R mutation on one or two potential target sites of IAV-NS1 ISGylation. The viral propagations of these mutant viruses were then tested in MDCK and A549 cells in comparison with WT IAV-PR8/34. Notably, only PR8-K126R and PR8-K217E exhibited distinctly augmented viral propagation in both cell lines, whereas the other mutant viruses behaved similar to WT IAV-PR8/34, indicating that K126 and K217 were critical target sites of IAV-NS1 ISGylation in cultured cells (Fig. 8A, 8B). Consistently, these two mutant viruses were less sensitive to knocking down either Herc5 or ISG15 in A549 cells than WT IAV-PR8/34, indicating that they were more resistant to the inhibitory effects due to IAV-NS1 ISGylation (Fig. 8G). Furthermore, we assessed the viral propagation and virulence of PR8-K126R and PR8-K217E in mice and found that these two mutant viruses also exhibited augmented viral propagation and virulence in mice in comparison with WT IAV-PR8/34 (Fig. 8C–F). Additionally, a PR8-K126R/K217E mutant virus was generated, and it exhibited stronger viral propagation and virulence than PR8-K126R or PR8-K217E in both cultured cells and mice (Fig. 8A–G). Taken together, these results revealed that Herc5 catalyzed ISGylation of IAV-NS1 on its two lysines K126 and K217, and this was critical for the host antiviral response against IAV in vivo.

FIGURE 8.
FIGURE 8.

K126 and K217 of IAV-NS1 are critical target sites of Herc5-mediated ISGylation in vivo. A, Viral replication of the indicated IAVs (WT and mutants) in MDCK cells determined on 48 h postinfection by measuring virus infectivity in eggs. The results are presented as means ± SD. B, Viral replication of the indicated IAVs (WT and mutants) in A549 cells determined on 48 h postinfection by measuring virus infectivity in eggs. The results are presented as means ± SD. C, Viral replication of the indicated IAVs (WT and mutants) in lungs of infected mice determined on days 2 and 3 postinfection by measuring virus infectivity in eggs. The results are presented as means ± SD (n = 3). D, Kaplan-Meier survival curves of mice (n = 5) infected with 104 EID50 (top panel) or 103 EID50 (bottom panel) of the indicated IAVs (WT and mutants). E, Comparison of weight changes in mice (n = 5) infected with 104 EID50 (top panel) or 103 EID50 (bottom panel) of the indicated IAVs (WT and mutants). F, The 50% mouse lethal dose of the indicated IAVs (WT and mutants). G, A549 cells were transfected with the indicated siRNAs for 24 h, followed by infection with indicated IAVs (WT or mutants). Forty-eight hours postinfection, the culture media were collected and measured for virus infectivity in eggs. The results are presented as means ± SD. *p < 0.05; **p < 0.01.

Discussion

Hosts and viruses coevolve to maintain a dynamic balance of competition and antagonism. The ultimate outcome of infection depends on the timing and efficiency of both sides to achieve supremacy. The type I IFNs are instrumental to inhibit viral infection and propagation via inducing antiviral proteins. Accordingly, many viruses encode specific viral proteins to antagonize IFN-induced host antiviral activities (55, 56). For IAVs, IAV-NS1 serves as the major IFN antagonist and a critical virulence factor to antagonize host antiviral innate immune response. It has been demonstrated that IAV-NS1 not only inhibits the induction of IFNs upon IAV infection, but also directly impairs the antiviral functions of IFN-induced host proteins (2–4). However, it remains poorly understood whether and how host cell specifically cripples the action of IAV-NS1 to dampen IAV.

In this study, we identified Herc5 to interact with IAV-NS1 upon IAV infection. We did not observe any distinct regulatory effect of IAV-NS1 on the ISG15 E3 activity of Herc5. Instead, we found that Herc5 could catalyze ISGylation of IAV-NS1 during IAV infection. This novel posttranslational modification contributed significantly to host innate immune antiviral response against IAV by limiting IAV-NS1’s action. Several lines of evidence in this study strongly support this finding:

  1. His-pulldown assay and IP assay unequivocally established that IAV-NS1 could be modified by ISG15 at seven lysines (K20/41/108/110/126/217/219). Importantly, this modification could be specifically catalyzed by Herc5 via its HECT domain.

  2. ISGylation of IAV-NS1 resulted in its inability to interact with PKR and form homodimers, which impaired its interaction with its RNA targets.

  3. IAV-NS1 could markedly inhibit the expression of antiviral genes (IFN-β, ISG56, and RANTES) induced by Sendai virus infection. Interestingly, this inhibition could be partially alleviated by ectopic expression of the Herc5-mediated ISGylation system.

  4. Pretreatment of HEK293 cells with IFN-β–induced antiviral activities against IAV infection. Knockdown of either Herc5 or ISG15 could partially attenuate these antiviral activities, indicating that Herc5 is an important host antiviral protein.

  5. Similarly, IAV exhibited augmented viral propagation in either Herc5 or ISG15 knockdown A549 cells.

  6. Alternatively, ectopic expression of the Herc5-mediated ISGylation system but not ISG15 alone in HEK293 cells could distinctly potentiate IFN-β–induced antiviral activities against IAV, suggesting that Herc5 exerted its antiviral function by catalyzing ISGylation.

  7. Notably, IAV-NS1s of H5N1 avian IAVs displayed less ISGylation species than that of IAV-PR8/34 (human H1N1), which is in agreement with the virulence of the corresponding IAV toward human and mice.

  8. K126 and K217 of IAV-NS1 were critical target sites of Herc5-mediated ISGylation in vivo.

Taken together, our study uncovers Herc5 as a novel and critical host antiviral protein, which inhibits IAV by catalyzing ISGylation of IAV-NS1.

Induction of ISG15 and its conjugation (ISGylation) has been well established as a rapid host response to IFN stimulation or virus infection (21, 22). To explore the potential function of ISG15, all of the previous studies focused mainly on identification of host proteins that might be modified and regulated by ISG15. For example, 4EHP and filamin B have been reported to be modified by ISG15, and their modifications may play important roles in IFN-induced innate immune response (36, 37). So far, >100 cellular proteins had been suggested as potential targets of ISGylation via a proteomics approach, in which RIG-I, JAK1, STAT1, MxA, PKR, and RNaseL were either IFN-induced antiviral proteins or components of innate immune signaling pathways (32–35). However, it is hard to figure out why host cells need one more layer of regulation for these proteins when they could perform their antiviral functions directly. Therefore, it remains to be determined that whether these proteins are modified by ISG15 during virus infection and how ISGylation affects their functions.

ISG15 has been reported to inhibit IAVs both in mice and human cultured cells (41, 57), but it is still unclear how ISG15 achieves this effect. To our knowledge, there has been no investigation to explore if any microbial protein is authentic target of ISGylation. In this study, we characterized IAV-NS1 as the first microbial target of ISGylation and demonstrated the importance of this novel modification in host antiviral process. Arguably, this insight will change our perspective on the functions of ISGylation and direct our attention to microbial proteins as regulatory targets. Besides IAV, several other viruses are also reported to be attenuated by ISG15 in vivo, including influenza B virus, Sindbis virus, HSV-1, murine γ-herpes virus, and vaccinia virus (40, 41). In light of our current study, it will be intriguing to explore whether other viral proteins could also be modified and regulated by ISGylation.

Although the seven lysines (K20/41/108/110/126/217/219) of IAV-NS1 were identified as potential target sites of Herc5-mediated ISGylation in vitro, K126 and K217 were found to be critical in vivo. One possible explanation for this observation is that not all of the seven potential target sites of IAV-NS1 ISGylation are exposed during IAV infection in vivo, because viral or host factors will bind IAV-NS1 and mask some of the lysines. Interestingly, we noticed that although K126 remains intact in IAV-NS1s of H5N1 highly pathogenic avian IAVs, it could not be modified by ISGylation. We speculated that H5N1-NS1 might have a different protein conformation from PR8-NS1, which prevented K126 from being ISGylated. This assumption is tentatively supported by the observation that Herc5 interacts much more weakly with HN12-NS1 than PR8-NS1. Hopefully, structural studies will shed important light on this functional difference, revealing the molecular basis of the extremely high pathogenicity and virulence of H5N1 avian IAVs in mammalian hosts. Additionally, we also found that K217 was most frequently mutated in a systematical analysis of IAV-NS1 protein sequences of H1N1 and H5N1 IAVs at the IAV database of the National Center for Biotechnology Information’s Influenza Virus Resource (58), especially on IAV-NS1s of H5N1 highly pathogenic avian IAVs and new outbreak 2009-H1N1 IAVs. These observations further substantiated the critical roles of K126 and K217 in IAV-NS1 ISGylation, highlighting the functional significance of their ISGylation for host antiviral response against IAV in vivo.

Although type I IFNs could induce the components of ISG15 modification system, it has been recently reported that inflammatory cytokines and type II IFNs could also induce them. In addition, bacteria and virus infection could directly induce the ISG15 modification system, possibly via IRF3/IRF7 transcription factors (39, 59, 60). Because IAV-NS1 is a strong inhibitor of type I IFN induction during IAV infection, it would be difficult to reveal the functional difference of ISG15 modification toward IAV in IFNR−/− and WT mice, because both types of mice would induce ISG15 modification system to almost the same extent during IAV infection (61–63). In our in vitro experiment, the cells were firstly pretreated with type I IFN to induce the ISG15 modification system (knocking down or not Herc5 or ISG15) and then infected with IAV. In this way, it is easy to reveal the function of ISG15 modification in inhibiting IAV. For in vivo experiments, although IAV-NS1 will block the induction of type I IFNs, IAV infection could induce ISG15 modification via other signaling pathways, which will alleviate the inhibitory effects mediated by IAV-NS1. Given the complication of IFN-α/β signaling, it will be important in future investigations to explore the dynamics of ISGylation system during in vivo IAV infection.

In summary, our study characterizes Herc5 as a novel and critical host antiviral protein. Herc5 catalyzes ISGylation of IAV-NS1 and cripples its virulence function in mammalian hosts. Arguably, this study uncovers the first microbial target of ISGylation and opens a new perspective to explore the potential function and mechanism of ISGylation in innate immunity.

Acknowledgments

We thank Drs. Dong-Yan Jin and K. Y. Yuen (University of Hong Kong, Hong Kong, China), Bing-Sun (Shanghai Pasteur Institute, CAS), and Zhe Chen (Wuhan Institute of Virology, CAS) for providing reagents in this study.

Disclosures The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by grants from the Ministry of Science and Technology of Shanghai (09XD1404800), the Ministry of Science and Technology of China (2007CB914504, 2009ZX10004-105, 2005CB523005), and the Chinese Academy of Sciences (KSCX1-YW-R-06).

  • Abbreviations used in this paper:

    CAS
    Chinese Academy of Sciences
    CBB
    Coomassie brilliant blue
    CT
    C-terminal
    ED
    effector domain
    EID50
    50% egg infectious dose
    GST-PD
    GST pulldown
    HA
    hemagglutinin
    IAV
    influenza A virus
    IP
    immunoprecipitation
    IRF3
    IFN regulatory factor 3
    ISG
    IFN-stimulated gene
    Ni-NTA PD
    Ni-NTA pulldown
    NT
    N-terminal
    NTA
    nitrilotriacetic acid
    PABP1
    poly(A)-binding protein 1
    PD
    pulldown
    PKR
    protein kinase R
    RBD
    RNA-binding domain
    si
    small interfering
    WT
    wild-type.

  • Received November 5, 2009.
  • Accepted March 9, 2010.

References

  1. ↵
    1. Palese P.,
    2. M. L. Shaw
    . 2007. Orthomyxoviridae: the viruses and their replication. In Fields Virology, 5th ed, Knipe D. M., P. M. Howley, eds. Lippincott Williams & Wilkins, Philadelphia, p. 1647–1689.
  2. ↵
    1. Hale B. G.,
    2. R. E. Randall,
    3. J. Ortín,
    4. D. Jackson
    . 2008. The multifunctional NS1 protein of influenza A viruses. J. Gen. Virol. 89: 2359–2376.
    OpenUrlCrossRefPubMed
    1. Noah D. L.,
    2. R. M. Krug
    . 2005. Influenza virus virulence and its molecular determinants. Adv. Virus Res. 65: 121–145.
    OpenUrlCrossRefPubMed
  4. ↵
    1. García-Sastre A.
    2001. Inhibition of interferon-mediated antiviral responses by influenza A viruses and other negative-strand RNA viruses. Virology 279: 375–384.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Wang X.,
    2. M. Li,
    3. H. Zheng,
    4. T. Muster,
    5. P. Palese,
    6. A. A. Beg,
    7. A. García-Sastre
    . 2000. Influenza A virus NS1 protein prevents activation of NF-kappaB and induction of alpha/beta interferon. J. Virol. 74: 11566–11573.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Min J. Y.,
    2. R. M. Krug
    . 2006. The primary function of RNA binding by the influenza A virus NS1 protein in infected cells: Inhibiting the 2′-5′ oligo (A) synthetase/RNase L pathway. Proc. Natl. Acad. Sci. USA 103: 7100–7105.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Bergmann M.,
    2. A. Garcia-Sastre,
    3. E. Carnero,
    4. H. Pehamberger,
    5. K. Wolff,
    6. P. Palese,
    7. T. Muster
    . 2000. Influenza virus NS1 protein counteracts PKR-mediated inhibition of replication. J. Virol. 74: 6203–6206.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Hale B. G.,
    2. D. Jackson,
    3. Y. H. Chen,
    4. R. A. Lamb,
    5. R. E. Randall
    . 2006. Influenza A virus NS1 protein binds p85beta and activates phosphatidylinositol-3-kinase signaling. Proc. Natl. Acad. Sci. USA 103: 14194–14199.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. de la Luna S.,
    2. P. Fortes,
    3. A. Beloso,
    4. J. Ortín
    . 1995. Influenza virus NS1 protein enhances the rate of translation initiation of viral mRNAs. J. Virol. 69: 2427–2433.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Nemeroff M. E.,
    2. S. M. Barabino,
    3. Y. Li,
    4. W. Keller,
    5. R. M. Krug
    . 1998. Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3’end formation of cellular pre-mRNAs. Mol. Cell 1: 991–1000.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Fortes P.,
    2. A. Beloso,
    3. J. Ortín
    . 1994. Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport. EMBO J. 13: 704–712.
    OpenUrlPubMed
  12. ↵
    1. Qian X. Y.,
    2. C. Y. Chien,
    3. Y. Lu,
    4. G. T. Montelione,
    5. R. M. Krug
    . 1995. An amino-terminal polypeptide fragment of the influenza virus NS1 protein possesses specific RNA-binding activity and largely helical backbone structure. RNA 1: 948–956.
    OpenUrlAbstract
  13. ↵
    1. Chen Z.,
    2. Y. Li,
    3. R. M. Krug
    . 1999. Influenza A virus NS1 protein targets poly(A)-binding protein II of the cellular 3′-end processing machinery. EMBO J. 18: 2273–2283.
    OpenUrlAbstract
  14. ↵
    1. Min J. Y.,
    2. S. Li,
    3. G. C. Sen,
    4. R. M. Krug
    . 2007. A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis. Virology 363: 236–243.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Wang X.,
    2. C. F. Basler,
    3. B. R. Williams,
    4. R. H. Silverman,
    5. P. Palese,
    6. A. García-Sastre
    . 2002. Functional replacement of the carboxy-terminal two-thirds of the influenza A virus NS1 protein with short heterologous dimerization domains. J. Virol. 76: 12951–12962.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Wang W.,
    2. K. Riedel,
    3. P. Lynch,
    4. C. Y. Chien,
    5. G. T. Montelione,
    6. R. M. Krug
    . 1999. RNA binding by the novel helical domain of the influenza virus NS1 protein requires its dimer structure and a small number of specific basic amino acids. RNA 5: 195–205.
    OpenUrlAbstract
  17. ↵
    1. Chien C. Y.,
    2. Y. Xu,
    3. R. Xiao,
    4. J. M. Aramini,
    5. P. V. Sahasrabudhe,
    6. R. M. Krug,
    7. G. T. Montelione
    . 2004. Biophysical characterization of the complex between double-stranded RNA and the N-terminal domain of the NS1 protein from influenza A virus: evidence for a novel RNA-binding mode. Biochemistry 43: 1950–1962.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Mahmoudian S.,
    2. S. Auerochs,
    3. M. Gröne,
    4. M. Marschall
    . 2009. Influenza A virus proteins PB1 and NS1 are subject to functionally important phosphorylation by protein kinase C. J. Gen. Virol. 90: 1392–1397.
    OpenUrlCrossRefPubMed
    1. Hale B. G.,
    2. A. Knebel,
    3. C. H. Botting,
    4. C. S. Galloway,
    5. B. L. Precious,
    6. D. Jackson,
    7. R. M. Elliott,
    8. R. E. Randall
    . 2009. CDK/ERK-mediated phosphorylation of the human influenza A virus NS1 protein at threonine-215. Virology 383: 6–11.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Petri T.,
    2. S. Patterson,
    3. N. J. Dimmock
    . 1982. Polymorphism of the NS1 proteins of type A influenza virus. J. Gen. Virol. 61: 217–231.
    OpenUrlPubMed
  21. ↵
    1. Sadler A. J.,
    2. B. R. Williams
    . 2008. Interferon-inducible antiviral effectors. Nat. Rev. Immunol. 8: 559–568.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Pitha-Rowe I. F.,
    2. P. M. Pitha
    . 2007. Viral defense, carcinogenesis and ISG15: novel roles for an old ISG. Cytokine Growth Factor Rev. 18: 409–417.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Narasimhan J.,
    2. M. Wang,
    3. Z. Fu,
    4. J. M. Klein,
    5. A. L. Haas,
    6. J. J. Kim
    . 2005. Crystal structure of the interferon-induced ubiquitin-like protein ISG15. J. Biol. Chem. 280: 27356–27365.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Potter J. L.,
    2. J. Narasimhan,
    3. L. Mende-Mueller,
    4. A. L. Haas
    . 1999. Precursor processing of pro-ISG15/UCRP, an interferon-beta-induced ubiquitin-like protein. J. Biol. Chem. 274: 25061–25068.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Yuan W.,
    2. R. M. Krug
    . 2001. Influenza B virus NS1 protein inhibits conjugation of the interferon (IFN)-induced ubiquitin-like ISG15 protein. EMBO J. 20: 362–371.
    OpenUrlAbstract
  26. ↵
    1. Zhao C.,
    2. S. L. Beaudenon,
    3. M. L. Kelley,
    4. M. B. Waddell,
    5. W. Yuan,
    6. B. A. Schulman,
    7. J. M. Huibregtse,
    8. R. M. Krug
    . 2004. The UbcH8 ubiquitin E2 enzyme is also the E2 enzyme for ISG15, an IFN-alpha/beta-induced ubiquitin-like protein. Proc. Natl. Acad. Sci. USA 101: 7578–7582.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Kim K. I.,
    2. N. V. Giannakopoulos,
    3. H. W. Virgin,
    4. D. E. Zhang
    . 2004. Interferon-inducible ubiquitin E2, Ubc8, is a conjugating enzyme for protein ISGylation. Mol. Cell. Biol. 24: 9592–9600.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Zou W.,
    2. D. E. Zhang
    . 2006. The interferon-inducible ubiquitin-protein isopeptide ligase (E3) EFP also functions as an ISG15 E3 ligase. J. Biol. Chem. 281: 3989–3994.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Wong J. J.,
    2. Y. F. Pung,
    3. N. S. Sze,
    4. K. C. Chin
    . 2006. HERC5 is an IFN-induced HECT-type E3 protein ligase that mediates type I IFN-induced ISGylation of protein targets. Proc. Natl. Acad. Sci. USA 103: 10735–10740.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Dastur A.,
    2. S. Beaudenon,
    3. M. Kelley,
    4. R. M. Krug,
    5. J. M. Huibregtse
    . 2006. Herc5, an interferon-induced HECT E3 enzyme, is required for conjugation of ISG15 in human cells. J. Biol. Chem. 281: 4334–4338.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Malakhov M. P.,
    2. O. A. Malakhova,
    3. K. I. Kim,
    4. K. J. Ritchie,
    5. D. E. Zhang
    . 2002. UBP43 (USP18) specifically removes ISG15 from conjugated proteins. J. Biol. Chem. 277: 9976–9981.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Takeuchi T.,
    2. S. Inoue,
    3. H. Yokosawa
    . 2006. Identification and Herc5-mediated ISGylation of novel target proteins. Biochem. Biophys. Res. Commun. 348: 473–477.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Zhao C.,
    2. C. Denison,
    3. J. M. Huibregtse,
    4. S. Gygi,
    5. R. M. Krug
    . 2005. Human ISG15 conjugation targets both IFN-induced and constitutively expressed proteins functioning in diverse cellular pathways. Proc. Natl. Acad. Sci. USA 102: 10200–10205.
    OpenUrlAbstract/FREE Full Text
    1. Giannakopoulos N. V.,
    2. J. K. Luo,
    3. V. Papov,
    4. W. Zou,
    5. D. J. Lenschow,
    6. B. S. Jacobs,
    7. E. C. Borden,
    8. J. Li,
    9. H. W. Virgin,
    10. D. E. Zhang
    . 2005. Proteomic identification of proteins conjugated to ISG15 in mouse and human cells. Biochem. Biophys. Res. Commun. 336: 496–506.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Malakhov M. P.,
    2. K. I. Kim,
    3. O. A. Malakhova,
    4. B. S. Jacobs,
    5. E. C. Borden,
    6. D. E. Zhang
    . 2003. High-throughput immunoblotting. Ubiquitiin-like protein ISG15 modifies key regulators of signal transduction. J. Biol. Chem. 278: 16608–16613.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Jeon Y. J.,
    2. J. S. Choi,
    3. J. Y. Lee,
    4. K. R. Yu,
    5. S. M. Kim,
    6. S. H. Ka,
    7. K. H. Oh,
    8. K. I. Kim,
    9. D. E. Zhang,
    10. O. S. Bang,
    11. C. H. Chung
    . 2009. ISG15 modification of filamin B negatively regulates the type I interferon-induced JNK signalling pathway. EMBO Rep. 10: 374–380.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Okumura F.,
    2. W. Zou,
    3. D. E. Zhang
    . 2007. ISG15 modification of the eIF4E cognate 4EHP enhances cap structure-binding activity of 4EHP. Genes Dev. 21: 255–260.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Mitsui K.,
    2. M. Nakanishi,
    3. S. Ohtsuka,
    4. T. H. Norwood,
    5. K. Okabayashi,
    6. C. Miyamoto,
    7. K. Tanaka,
    8. A. Yoshimura,
    9. M. Ohtsubo
    . 1999. A novel human gene encoding HECT domain and RCC1-like repeats interacts with cyclins and is potentially regulated by the tumor suppressor proteins. Biochem. Biophys. Res. Commun. 266: 115–122.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Kroismayr R.,
    2. U. Baranyi,
    3. C. Stehlik,
    4. A. Dorfleutner,
    5. B. R. Binder,
    6. J. Lipp
    . 2004. HERC5, a HECT E3 ubiquitin ligase tightly regulated in LPS activated endothelial cells. J. Cell Sci. 117: 4749–4756.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Guerra S.,
    2. A. Cáceres,
    3. K. P. Knobeloch,
    4. I. Horak,
    5. M. Esteban
    . 2008. Vaccinia virus E3 protein prevents the antiviral action of ISG15. PLoS Pathog. 4: e1000096.
    OpenUrlCrossRefPubMed
  41. ↵
    1. Lenschow D. J.,
    2. C. Lai,
    3. N. Frias-Staheli,
    4. N. V. Giannakopoulos,
    5. A. Lutz,
    6. T. Wolff,
    7. A. Osiak,
    8. B. Levine,
    9. R. E. Schmidt,
    10. A. García-Sastre,
    11. et al
    . 2007. IFN-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and Sindbis viruses. Proc. Natl. Acad. Sci. USA 104: 1371–1376.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Lenschow D. J.,
    2. N. V. Giannakopoulos,
    3. L. J. Gunn,
    4. C. Johnston,
    5. A. K. O’Guin,
    6. R. E. Schmidt,
    7. B. Levine,
    8. H. W. Virgin 4th.
    . 2005. Identification of interferon-stimulated gene 15 as an antiviral molecule during Sindbis virus infection in vivo. J. Virol. 79: 13974–13983.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    1. Okumura A.,
    2. P. M. Pitha,
    3. R. N. Harty
    . 2008. ISG15 inhibits Ebola VP40 VLP budding in an L-domain-dependent manner by blocking Nedd4 ligase activity. Proc. Natl. Acad. Sci. USA 105: 3974–3979.
    OpenUrlAbstract/FREE Full Text
    1. Malakhova O. A.,
    2. D. E. Zhang
    . 2008. ISG15 inhibits Nedd4 ubiquitin E3 activity and enhances the innate antiviral response. J. Biol. Chem. 283: 8783–8787.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    1. Okumura A.,
    2. G. Lu,
    3. I. Pitha-Rowe,
    4. P. M. Pitha
    . 2006. Innate antiviral response targets HIV-1 release by the induction of ubiquitin-like protein ISG15. Proc. Natl. Acad. Sci. USA 103: 1440–1445.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    1. Lai C.,
    2. J. J. Struckhoff,
    3. J. Schneider,
    4. L. Martinez-Sobrido,
    5. T. Wolff,
    6. A. García-Sastre,
    7. D. E. Zhang,
    8. D. J. Lenschow
    . 2009. Mice lacking the ISG15 E1 enzyme UbE1L demonstrate increased susceptibility to both mouse-adapted and non-mouse-adapted influenza B virus infection. J. Virol. 83: 1147–1151.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. Giannakopoulos N. V.,
    2. E. Arutyunova,
    3. C. Lai,
    4. D. J. Lenschow,
    5. A. L. Haas,
    6. H. W. Virgin
    . 2009. ISG15 Arg151 and the ISG15-conjugating enzyme UbE1L are important for innate immune control of Sindbis virus. J. Virol. 83: 1602–1610.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    1. Lindner H. A.,
    2. V. Lytvyn,
    3. H. Qi,
    4. P. Lachance,
    5. E. Ziomek,
    6. R. Ménard
    . 2007. Selectivity in ISG15 and ubiquitin recognition by the SARS coronavirus papain-like protease. Arch. Biochem. Biophys. 466: 8–14.
    OpenUrlCrossRefPubMed
  49. ↵
    1. Frias-Staheli N.,
    2. N. V. Giannakopoulos,
    3. M. Kikkert,
    4. S. L. Taylor,
    5. A. Bridgen,
    6. J. Paragas,
    7. J. A. Richt,
    8. R. R. Rowland,
    9. C. S. Schmaljohn,
    10. D. J. Lenschow,
    11. et al
    . 2007. Ovarian tumor domain-containing viral proteases evade ubiquitin- and ISG15-dependent innate immune responses. Cell Host Microbe 2: 404–416.
    OpenUrlCrossRefPubMed
  50. ↵
    1. Jiao P.,
    2. G. Tian,
    3. Y. Li,
    4. G. Deng,
    5. Y. Jiang,
    6. C. Liu,
    7. W. Liu,
    8. Z. Bu,
    9. Y. Kawaoka,
    10. H. Chen
    . 2008. A single-amino-acid substitution in the NS1 protein changes the pathogenicity of H5N1 avian influenza viruses in mice. J. Virol. 82: 1146–1154.
    OpenUrlAbstract/FREE Full Text
  51. ↵
    1. Sun S.,
    2. Y. Tang,
    3. X. Lou,
    4. L. Zhu,
    5. K. Yang,
    6. B. Zhang,
    7. H. Shi,
    8. C. Wang
    . 2007. UXT is a novel and essential cofactor in the NF-kappaB transcriptional enhanceosome. J. Cell Biol. 178: 231–244.
    OpenUrlAbstract/FREE Full Text
  52. ↵
    1. Reed L. J.,
    2. H. Muench
    . 1938. A simple method of estimating fifty percent endpoints. Am. J. Epidemiol. 27: 493–497.
    OpenUrlFREE Full Text
  53. ↵
    1. Ludwig S.,
    2. X. Wang,
    3. C. Ehrhardt,
    4. H. Zheng,
    5. N. Donelan,
    6. O. Planz,
    7. S. Pleschka,
    8. A. García-Sastre,
    9. G. Heins,
    10. T. Wolff
    . 2002. The influenza A virus NS1 protein inhibits activation of Jun N-terminal kinase and AP-1 transcription factors. J. Virol. 76: 11166–11171.
    OpenUrlAbstract/FREE Full Text
  54. ↵
    1. Talon J.,
    2. C. M. Horvath,
    3. R. Polley,
    4. C. F. Basler,
    5. T. Muster,
    6. P. Palese,
    7. A. García-Sastre
    . 2000. Activation of interferon regulatory factor 3 is inhibited by the influenza A virus NS1 protein. J. Virol. 74: 7989–7996.
    OpenUrlAbstract/FREE Full Text
  55. ↵
    1. García-Sastre A.,
    2. C. A. Biron
    . 2006. Type 1 interferons and the virus-host relationship: a lesson in détente. Science 312: 879–882.
    OpenUrlAbstract/FREE Full Text
  56. ↵
    1. Katze M. G.,
    2. Y. He,
    3. M. Gale Jr..
    . 2002. Viruses and interferon: a fight for supremacy. Nat. Rev. Immunol. 2: 675–687.
    OpenUrlCrossRefPubMed
  57. ↵
    1. Hsiang T. Y.,
    2. C. Zhao,
    3. R. M. Krug
    . 2009. Interferon-induced ISG15 conjugation inhibits influenza A virus gene expression and replication in human cells. J. Virol. 83: 5971–5977.
    OpenUrlAbstract/FREE Full Text
  58. ↵
    1. Bao Y.,
    2. P. Bolotov,
    3. D. Dernovoy,
    4. B. Kiryutin,
    5. L. Zaslavsky,
    6. T. Tatusova,
    7. J. Ostell,
    8. D. Lipman
    . 2008. The influenza virus resource at the National Center for Biotechnology Information. J. Virol. 82: 596–601.
    OpenUrlFREE Full Text
  59. ↵
    1. Harty R. N.,
    2. P. M. Pitha,
    3. A. Okumura
    . 2009. Antiviral activity of innate immune protein ISG15. J. Innate Immun. 1: 397–404.
    OpenUrlCrossRefPubMed
  60. ↵
    1. Malakhova O.,
    2. M. Malakhov,
    3. C. Hetherington,
    4. D. E. Zhang
    . 2002. Lipopolysaccharide activates the expression of ISG15-specific protease UBP43 via interferon regulatory factor 3. J. Biol. Chem. 277: 14703–14711.
    OpenUrlAbstract/FREE Full Text
  61. ↵
    1. Coro E. S.,
    2. W. L. Chang,
    3. N. Baumgarth
    . 2006. Type I IFN receptor signals directly stimulate local B cells early following influenza virus infection. J. Immunol. 176: 4343–4351.
    OpenUrlAbstract/FREE Full Text
    1. García-Sastre A.,
    2. R. K. Durbin,
    3. H. Zheng,
    4. P. Palese,
    5. R. Gertner,
    6. D. E. Levy,
    7. J. E. Durbin
    . 1998. The role of interferon in influenza virus tissue tropism. J. Virol. 72: 8550–8558.
    OpenUrlAbstract/FREE Full Text
  63. ↵
    1. Price G. E.,
    2. A. Gaszewska-Mastarlarz,
    3. D. Moskophidis
    . 2000. The role of alpha/beta and gamma interferons in development of immunity to influenza A virus in mice. J. Virol. 74: 3996–4003.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section