ABSTRACT
Canine influenza viruses (CIVs) are the causative agents of canine influenza, a contagious respiratory disease in dogs, and include the equine-origin H3N8 and the avian-origin H3N2 viruses. Influenza A virus (IAV) nonstructural protein 1 (NS1) is a virulence factor essential for counteracting the innate immune response. Here, we evaluated the ability of H3N8 CIV NS1 to inhibit host innate immune responses. We found that H3N8 CIV NS1 was able to efficiently counteract interferon (IFN) responses but was unable to block general gene expression in human or canine cells. Such ability was restored by a single amino acid substitution in position 186 (K186E) that resulted in NS1 binding to the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a cellular protein involved in pre-mRNA processing. We also examined the frequency distribution of K186 and E186 among H3N8 CIVs and equine influenza viruses (EIVs), the ancestors of H3N8 CIV, and experimentally determined the impact of amino acid 186 in the ability of different CIV and EIV NS1s to inhibit general gene expression. In all cases, the presence of E186 was responsible for the control of host gene expression. In contrast, the NS1 protein of H3N2 CIV harbors E186 and blocks general gene expression in canine cells. Altogether, our results confirm previous studies on the strain-dependent ability of NS1 to block general gene expression. Moreover, the observed polymorphism on amino acid 186 between H3N8 and H3N2 CIVs might be the result of adaptive changes acquired during long-term circulation of avian-origin IAVs in mammals.
IMPORTANCE Canine influenza is a respiratory disease of dogs caused by two CIV subtypes, the H3N8 and H3N2 viruses, of equine and avian origins, respectively. Influenza NS1 is the main viral factor responsible for the control of host innate immune responses, and changes in NS1 can play an important role in host adaptation. Here we assessed the ability of H3N8 CIV NS1 to inhibit host innate immune responses and gene expression. The H3N8 CIV NS1 did not block host gene expression, but this activity was restored by a single amino acid substitution (K186E), which was responsible for NS1 binding to the host factor CPSF30. In contrast, the H3N2 CIV NS1, which contains E186, blocks general gene expression. Our results suggest that the ability to block host gene expression is not required for influenza virus replication in mammals but might be important in the long-term adaptation of avian-origin influenza viruses to mammals.
INTRODUCTION
Influenza A viruses (IAVs) are single-stranded, negative-sense, segmented RNA viruses that belong to the Orthomyxoviridae family (1, 2). They are classified on the basis of the two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) (1, 2). While IAVs circulate in wild aquatic birds mostly (3–6), some have become established as novel lineages in various avian and mammalian hosts (7, 8). In addition, many IAVs cause sporadic spillover infections in humans and other mammals (4, 8, 9).
Two canine influenza viruses (CIVs) have emerged in the last decade. The first one is of the H3N8 subtype and was first reported during an outbreak of respiratory disease in Florida, USA, in 2004 (10–12). H3N8 CIV originated from equine influenza virus (EIV), an avian-origin IAV that has been circulating in horses since the 1960s (10, 13). Another recently emerged CIV is the avian-origin H3N2 subtype, originated in China, which has been circulating in Asia since ∼2006 (14) and was introduced to North America in 2015 (15). Since CIVs are the result of successful cross-species virus transfers, it is of interest to understand how they have succeeded in their new host and to establish whether they constitute potential threats to the global canine population (7, 8, 16) and perhaps to other species, including humans. Understanding the determinants of pathogenesis and host range of CIVs is essential to shed light on the mechanisms of disease in dogs and to provide insights into the general mechanisms of the IAV host range in other species.
The host innate immune responses constitute an essential part of the host defenses that restrict viral infections. These are mediated mainly by the induction of an interferon (IFN) response that results in the expression of a broad range of IFN-stimulated genes (ISGs), some of which possess antiviral activity (17). IAVs encode the nonstructural protein 1 (NS1), a multifunctional protein that is a molecular determinant of IAV virulence and is the main viral antagonist of the IFN response (18–20). NS1 acts at multiple levels of the IFN signaling pathway: it has been shown to impair the expression of ISGs (18) and to directly inhibit specific ISGs, such as protein kinase R (PKR) (18) and 2′-5′ oligoadenylate synthetase and RNase L (RNase L) (21). NS1 can also inhibit the production of IFN at the pretranscriptional level by sequestering double-stranded RNA (dsRNA) and subsequently decreasing the activation of retinoic acid-inducible gene 1 (RIG-I) (22–24) or by inhibiting the tripartite motif family 25 (TRIM-25)-mediated RIG-I ubiquitination (22, 24). NS1 can also impair the IFN response by blocking general host gene expression by binding the 30-kDa subunit of the cleavage and polyadenylation specificity factor 30 (CPSF30). The ability of NS1 to bind CPSF30 and block host gene expression is not conserved in all IAVs. Indeed, it has been shown that naturally occurring IAVs that infect humans, such as the swine origin 2009 pandemic H1N1 (pH1N1) virus (25), the avian-origin H7N9 (26) and H5N1 (27) viruses, and the laboratory strain A/Puerto Rico/8/34 (PR8) (28), do not bind CPSF30. The amino acid(s) involved in the interaction with CPSF30 have been mapped to different NS1 residues (25, 26, 28–34).
Here we examined the ability of H3N8 CIV NS1 to counteract the IFN response and found that this protein blocks the induction of IFN and ISGs without blocking general host gene expression. However, a single amino acid substitution (K186E) results in the recovery of the ability to block general host transcription. These results confirm the notion that the specific functions of NS1 protein are strain dependent and likely vary with the level of virus adaptation to the host.
RESULTS
H3N8 CIV NY09 NS1 protein does not block host gene expression in human or canine cells.To evaluate whether H3N8 CIV NS1 protein could block host gene expression, human HEK293T or Madin-Darby canine kidney (MDCK) cells were cotransfected with plasmids encoding green fluorescent protein (GFP) or Gaussia luciferase (Gluc) together with individual plasmids encoding the NS1 genes of either A/canine/NY/dog23/2009 H3N8 (CIV NY09), A/Puerto Rico/8/34 H1N1 (PR8), or A/Brevig Mission/1/18 H1N1 (BM/18) (Fig. 1). Cells transfected with empty plasmid were also used as an internal control. We have previously described that PR8 NS1 is not able to block host gene expression. However, such ability can be restored by introducing amino acid substitutions S103F and I106M in PR8 NS1 (28). In contrast, the NS1 protein from BM/18 is a strong inhibitor of host gene expression (28, 35). At 24 h posttransfection (hpt), GFP expression was evaluated using fluorescence microscopy (Fig. 1A and D) and Gluc expression levels were quantified in a luminometer (Fig. 1B and E). As expected, PR8 NS1 protein did not block expression of GFP and/or Gluc whereas BM/18 NS1 inhibited protein expression of both reporter genes (28). CIV NY09 NS1 protein did not inhibit either GFP or Gluc expression. The NS1 proteins from PR8 or CIV NY09 were detected by Western blotting (Fig. 1C and F), but this was not the case for the NS1 protein of BM/18, because this NS1 inhibits its own synthesis (28). Our results obtained with both human (Fig. 1A to C) and canine (Fig. 1D to F) cells were similar, suggesting that the inability of CIV NY09 NS1 protein to block host gene expression does not depend on the origin of the cells.
H3N8 CIV NS1 protein does not inhibit general gene expression in human HEK293T or canine MDCK cells. HEK293T (A to C) and MDCK (D to F) cells (12-well plate format, 3 × 105 cells/well, triplicates) were transiently cotransfected with 50 ng of pCAGGS expression plasmids encoding GFP and Gluc together with 2 μg of pCAGGS plasmids encoding the indicated NS1 proteins fused to an HA tag or an empty (E) plasmid as a control. At 24 hpt, cells were analyzed for GFP expression using a fluorescence microscope (A and D) and Gluc activity from TCS (B and E). NS1 protein expression levels (C and F) from total cell lysates were determined by Western blotting using an anti-HA polyclonal antibody (HA-NS1). Actin was included as a loading control. Representative images of three independent transfections are shown. Bar, 100 μm. Results represent the means and standard deviations of triplicate values. *, P < 0.005 using Student's t test. PR8, influenza A/Puerto Rico/8/34 H1N1; BM/18, A/Brevig Mission/01/1918 H1N1; CIV NY09, A/canine/NY/dog23/2009 H3N8.
A single substitution (K186E) in the NS1 protein of H3N8 CIV NY09 restores its ability to block host gene expression.To identify the amino acid(s) in H3N8 CIV NY09 NS1 protein responsible for the lack of inhibition of host gene expression, we aligned the NS1 protein sequences of the viruses used in our reporter-based assay (Fig. 1) and also included two additional viruses: A/Texas/36/91 H1N1 (TX/91, which binds CPSF30) and the pandemic A/California/04/09 H1N1 (pH1N1, which does not bind CPSF30) (Fig. 2A). We identified eight amino acids (P77, D96, T112, E139, F185, K186, K193, and I194) in the H3N8 CIV NY09 NS1 protein that could have a potential role in the lack of inhibition of host gene expression (Fig. 2A, gray). To test the contribution of these residues to the inhibition of host gene expression, we introduced mutations P77L, D96E, T112A, E139D, F185L, K186E, K193R, and I194V into the CIV NY09 NS1 protein and tested the mutants' ability to block general gene expression in our reporter-based assay in MDCK cells (Fig. 2B and C). A single substitution (K186E) introduced the ability of H3N8 CIV NY09 NS1 protein to inhibit general host gene expression as measured by GFP (Fig. 2B) and Gluc (Fig. 2C) expression levels. Moreover, the K186E mutant construct was the only one that was not detected by Western blotting, suggesting that it was inhibiting its own expression (Fig. 2D). We next evaluated if the ability of H3N8 CIV NY09 NS1 K186E protein to inhibit host gene expression was dose dependent (Fig. 3A and B). MDCK cells were cotransfected with the pCAGGS plasmid expressing Gluc together with different amounts of the plasmids encoding wild-type (WT) CIV NY09 NS or the K186E mutant. Clearly, CIV NY09 NS1 K186E protein showed a dose-dependent inhibitory effect on Gluc expression (Fig. 3A) and inhibition of its own expression (Fig. 3B). H3N8 CIV NY09 NS1 WT protein did not have an effect on reporter gene expression (Fig. 3A) and was detected by Western blotting only when 2, 1, or 0.5 μg of plasmid was transfected, most likely because of the limit of detection of the assay (Fig. 3B).
A single amino acid substitution (K186E) restores H3N8 CIV NS1's ability to block host gene expression. (A) Amino acid sequence alignment of IAV NS1 proteins: residues 70 to 194 from NS1 proteins that do not inhibit (PR8 and pH1N1) or inhibit (BM/18 and TX/91) host gene expression were aligned to H3N8 CIV NY09 NS1 protein (bottom). Gray shadow highlights amino acid changes in the H3N8 CIV NY09 NS1 effector domain, which could have a role in the lack of inhibition of host gene expression (P77, D96, T112, E139, F185, K186, K193, and I194). Abbreviations are as described for Fig. 1. pH1N1, A/California/04/09 H1N1; TX/91, A/Texas/36/91 H1N1. (B to D) Inhibition of host gene expression. MDCK cells (12-well plate format, 3 × 105 cells/well, triplicates) were transiently cotransfected with pCAGGS plasmids encoding GFP and Gluc (50 ng) together with plasmids encoding the indicated HA-tagged H3N8 CIV WT or single amino acid mutant (2 μg) NS1s or an empty (E) plasmid as a control. At 24 hpt, cells were analyzed for GFP (B) and Gluc (C) expression; NS1 protein expression levels from total cell lysates were determined by Western blotting using an anti-HA polyclonal antibody (HA-NS1) (D). Actin was included as a loading control. Representative images of three independent transfections are shown. Bar, 100 μm. Gluc expression levels in cells transfected with H3N8 CIV NS1 WT protein were considered to be 100% for comparison to expression levels from H3N8 CIV NS1 single-amino-acid individual mutant-transfected cells. Results represent the means and standard deviations of triplicate values. *, P < 0.05 using Student's t test.
Characterization of H3N8 CIV NS1 K186E mutation. (A and B) Inhibition of host gene expression. MDCK cells (12-well plate format, 3 × 105 cells/well, triplicates) were transiently cotransfected as described for Fig. 1, using different amounts of plasmids encoding N-terminal HA-tagged H3N8 CIV NS1 WT or K186E proteins. The total amount of transfected plasmid DNA was maintained constant with empty plasmid. At 24 hpt, cells were analyzed for Gluc activity from TCS (A). NS1 protein expression levels were determined by Western blotting using an anti-HA polyclonal antibody (HA-NS1) and actin as a loading control (B). (C to E) H3N8 CIV NS1 K186E does not inhibit T7-driven RNA polymerase expression. Rodent BSRT7 cells (12-well plate format, 3 × 105 cells/well, triplicates) constitutively expressing the T7 RNA polymerase were transiently cotransfected with the indicated NS1 expression plasmids or an empty (E) plasmid as a control (2 μg), together with GFP and FFluc T7-driven reporter pCITE-T7 plasmids (0.5 μg). At 24 hpt, GFP expression was evaluated under a fluorescence microscope (C) and Gluc activity was assessed from TCS (D). NS1 protein expression levels from total cell lysates were determined by Western blotting using an anti-HA polyclonal antibody (HA-NS1) (E). Actin was included as a loading control. Representative images from three independent transfections are shown. Bar, 100 μm. Results represent the means and standard deviations of triplicate values. *, P < 0.05 using Student's t test. Abbreviations are as described for Fig. 1.
To test if CIV NY09 NS1 K186E protein inhibition of host gene expression was due to inhibiting transcripts produced by the cellular RNA polymerase II, we used a variation of the assay whereby reporter gene expression (GFP and firefly luciferase [FFluc]) was under the control of a bacteriophage T7 polymerase promoter (36). BSR-T7 cells constitutively expressing T7 RNA polymerase (37) were cotransfected with reporter GFP and FFluc plasmids under the control of the T7 polymerase, together with plasmids encoding the H3N8 CIV NY09 NS1 WT or K186E protein or the NS1 proteins from PR8 or BM/18 or with an empty plasmid as an internal control. At 24 hpt, GFP and FFluc expressions were analyzed using fluorescence microscopy (Fig. 3C) and a luminometer (Fig. 3D), respectively. In this assay, none of the NS1 constructs showed any effect on reporter gene expression, demonstrating that only nuclear transcripts produced by cellular RNA polymerase II were targets of the inhibitory effect of NS1 WT or mutant constructs. As expected, when NS1 protein expression was analyzed by Western blotting, H3N8 CIV NY09 K186E and BM/18 NS1 proteins were not detected (Fig. 3E).
K186E substitution introduces the ability of H3N8 CIV NY09 NS1 protein to bind to CPSF30.It has been shown that NS1 proteins of some IAV strains interact with CPSF30, blocking host gene expression (25, 26, 28–34). The interaction of NS1 and CPSF30 is mediated by the effector domain of the former and the F2/F3 region of the latter (25, 26, 28–34). Importantly, the F2/F3 region of CPSF30 is conserved between species, including the ones relevant for this study (human, dogs, and horses) (data not shown). Given the ability of the H3N8 CIV NY09 NS1 K186E protein to inhibit host gene expression, we hypothesized that this phenotype was due to a restored ability to bind CPSF30. We evaluated the interaction between H3N8 CIV NY09 NS1 WT or K186E proteins and CPSF30 by coimmunoprecipitation (Fig. 4A). Cell extracts from human HEK293T cells transfected with a plasmid encoding an N-terminal FLAG-tagged F2/F3 region of CPSF30 (34) were incubated with in vitro-transcribed and -translated H3N8 CIV NY09 NS1 WT and K186E proteins or with PR8 NS1 protein as a control and agarose beads conjugated with an anti-FLAG antibody. As expected, PR8 NS1 and H3N8 CIV NY09 NS1 WT proteins did not coimmunoprecipitate with CPSF30 (28); however, CIV NY09 NS1 K186E protein did (Fig. 4A). The three-dimensional structure of influenza A/Udorn/72 H3N2 NS1 bound to the F2/F3 domain of CPSF30, which shows that NS1 residue 186 is closely located to the CPSF30 protein (Fig. 4B), providing a likely explanation for the effect of this amino acid change in the binding to CPSF30 (29). Altogether, our results indicate that the K186E substitution restores the ability of H3N8 CIV NY09 NS1 protein to bind CPSF30.
K186E substitution in H3N8 CIV NS1 protein restores binding to CPSF30. (A) Analysis of NS1-CPSF30 interaction by coimmunoprecipitation. A FLAG-tagged F2/F3 region of CPSF30 was expressed in HEK293T cells, mixed with in vitro-synthesized PR8 or H3N8 CIV (WT or K186E) NS1 proteins, and immunoprecipitated using an anti-FLAG resin. Following SDS-PAGE, input (top) and immunoprecipitated (IP, bottom) proteins were detected by Western blotting using antibodies specific for the FLAG (region F2/F3 of CPSF30) or the HA (NS) tags. (B) Tridimensional structure of NS1 effector domain in complex with the F2/F3 region of CPSF30. Influenza NS1 A/Udorn/72 H3N2 monomers conforming the dimer are shown in magenta or blue, and the monomers of the F2/F3 region of CPSF30 are in green or brown. Amino acid residue K186 in influenza NS1 is indicated in yellow. The structure was generated with Cn3D and is based on the NS1 of influenza A/Udorn/72 H3N2 (PDB entry 2RHK ) (29).
Effect of H3N8 CIV NY09 NS1 protein on inhibition of IFN responses.To analyze the effect of H3N8 CIV NY09 NS1 WT and K186E proteins to counteract IFN responses, human HEK293T cells were cotransfected with pCAGGS plasmids expressing PR8, BM/18, and CIV NY09 WT and K186E NS1 proteins, together with plasmids expressing FFluc and GFP under the control of the IFN-β promoter (Fig. 5A to C) or FFluc and RFP under the control of an IFN-stimulated response element (ISRE) promoter (Fig. 5D to F). At 18 hpt, cells were mock infected or infected with strain Cantell of Sendai virus (SeV) for 18 h to induce activation of both promoters. Then, IFN-β and ISRE promoter activation was determined by measuring the expression levels of GFP and RFP (Fig. 5A and D) and FFluc (Fig. 5B and E). As expected, SeV infection induced robust activation of the promoters IFN-β (Fig. 5A and B) and ISRE (Fig. 5D and E) in cells transfected with empty plasmid. On the other hand, and consistent with previous results (18), SeV-infected cells transfected with the different NS1-expressing plasmids showed less activation for both promoters than did cells transfected with empty plasmid. Moreover, when NS1 protein expression levels were evaluated by Western blotting, only PR8 and H3N8 CIV NY09 WT NS1 proteins were detected (Fig. 5C and F). These results indicate that the K186E substitution did not affect the ability of H3N8 CIV NY09 NS1 protein to inhibit SeV-mediated activation of IFN-β or ISRE promoters. In addition, these results suggest that H3N8 CIV NY09 NS1 protein has properties antagonistic to IFN despite not being able to block general host gene expression in either human or canine cells (Fig. 1).
H3N8 CIV NS1 protein is an IFN antagonist. HEK293T cells (12-well plate format, 3 × 105 cells/well, triplicates) were transiently cotransfected, using CaPO4, with reporter plasmids encoding GFP and FFluc under the control of the IFN-β promoter (pIFN-β-GFP/CAT and pIFN-β-FFluc, respectively) (0.5 μg) (A to C) or expressing RFP and FFluc under the control of an ISRE promoter (pISRE-RFP/CAT and pISRE-FFluc, respectively) (D to F), and the indicated N-terminal HA-tagged NS1 protein expressing plasmids, or empty (E) plasmid as control (1 μg). At 18 hpt, cells were infected (MOI, 3) with SeV strain Cantell, and at 18 hpi, cells were analyzed for IFN-β (A and B) or ISRE (D and E) promoter activation by GFP (A) and RFP (D) expression under a fluorescence microscope and for FFluc activity (B and E). NS1 protein expression levels from total cell lysates were determined by Western blotting using an anti-HA polyclonal antibody (HA-NS1) (C and F). Actin was included as a loading control. Representative images from three independent transfections are shown. Bar, 100 μm. FFluc activity is represented as fold induction normalized to empty plasmid-transfected, mock-infected cells. Results represent the means and standard deviations of triplicate values. *, P < 0.05 using Student's t test. Abbreviations are as described for Fig. 1.
Generation and characterization of WT and K186E H3N8 NY09 CIVs.To analyze whether the H3N8 CIV NY09 NS1 K186E mutation had an effect on virus replication, we generated recombinant H3N8 NY09 CIVs containing the WT NS1 or the NS1 with the K186E substitution (13). We first confirmed the identity of the recombinant viruses by reverse transcription (RT)-PCR and restriction analysis of the NS segment. The K186E substitution removed a SwaI restriction site in NS1, which was used as a genetic marker to distinguish H3N8 NY09 WT and K186E recombinant CIVs (Fig. 6A). In addition, the NS viral RNA (vRNA) was sequenced to ensure the absence of additional mutations (data not shown). The amino acid substitution K186E in H3N8 NY09 NS1 protein did not affect the amino acid sequence of the viral nuclear export protein (NEP), which is produced using an alternative splicing mechanism from the same NS vRNA (18, 19). Next, we compared the two viruses' ability to spread between neighboring cells by plaque assay in MDCK cells (Fig. 6B), as well as their multicycle and single-cycle growth kinetics in MDCK (Fig. 6C and D, respectively) and in A72 (Fig. 6E and F, respectively) canine cells. Canine A72 cells are a well-established tissue culture system to study viruses infecting dogs, including parvovirus and CIV (13, 38). CIV NY09 NS1 K186E displayed a plaque phenotype in MDCK cells similar to that of the parental virus (Fig. 6B). Interestingly, multicycle replication experiments using MDCK (Fig. 6C) or A72 (Fig. 6E) cells infected at a low multiplicity of infection (MOI) (0.001) revealed that the recombinant H3N8 NY09 WT and K186E CIVs grew with similar kinetics. However, when cells were infected at a high MOI (3), the WT virus grew at lower titers than the K186E mutant in both cell lines (Fig. 6D and F). These data indicate that the defect in efficient CPSF30 binding and inhibition of host protein expression by CIV NY09 NS1 protein only slightly affects virus replication in vitro, as previously described for other IAVs (18, 25, 28, 30, 33).
Characterization of H3N8 NS1 WT and K186E CIVs. (A) Phenotypic characterization. MDCK cells (6-well plate format, 1 × 106 cells/well) were infected (MOI of 3) with H3N8 NS1 WT and K186E CIVs. At 18 hpi, RNA was collected and the NS viral segments were amplified by RT-PCR and undigested (−) or digested (+) with SwaI restriction endonuclease. Expected DNA molecular sizes (in nucleotides) are indicated on the left. (B) Plaque assay. The plaque sizes of H3N8 NS1 WT and K186E CIVs in MDCK cells (6-well plate format, 1 × 106 cells/well) were evaluated at 3 dpi by immunostaining using an anti-NP monoclonal antibody (HB-65). (C to F) Growth kinetics. MDCK (C and D) or A72 (E and F) cells (12-well plate format, 5 × 105 cells/well, triplicates) were infected with low (C and E) or high (D and F) MOI (0.001 or 3, respectively), and TCS were collected at the indicated hours postinfection. Viral titers were determined by immunofocus assay (in focus-forming units per milliliter). Data represent the means of the results determined for triplicate wells. *, P < 0.05 using Student's t test.
To further analyze the ability of the recombinant NY09 H3N8 NS1 WT and K186E mutant CIVs to inhibit ISG IFN-induced protein with tetratricopeptide repeats 2 (IFIT2) (Fig. 7A and B) or IFN-β (Fig. 7C and D) responses, MDCK cells were infected (MOI, 3), and at 12 (Fig. 7A and C) or 24 (Fig. 7A and C) hours postinfection (hpi), expression levels of IFIT2 and IFN-β were measured at the mRNA level by quantitative RT-PCR (qRT-PCR). Interestingly, the levels of IFIT2 expression were higher in NY09 H3N8 NS1 WT-infected MDCK cells than in mutant K186E-infected cells at both times postinfection (Fig. 7A and B). These data indicate that the amino acid change K186E increases the ability of NS1 to inhibit ISGs during viral infection. On the other hand, the two viruses were able to inhibit the expression of IFN-β at similar levels (Fig. 7C and D).
Induction of innate immune responses by H3N8 NS1 WT and K186E mutant CIVs. MDCK cells (12-well plate format, 5 × 105 cells/well, triplicates) were infected (MOI of 3) with H3N8 NS1 WT and mutant K186E CIVs. At 12 (A and C) or 24 (B and D) hpi, total RNA was collected and mRNA expression levels of the ISG IFIT2 (A and B) and IFN-β (C and D) were quantified by qRT-PCR analysis. Expression fold changes were calculated relative to mock-infected cells Data represent the averages and standard deviations of triplicate values. *, P < 0.05 using Student's t test.
The ability of H3N8 CIV NS1 protein to block host gene expression was lost during virus evolution.We next investigated if the inability to block host gene expression by H3N8 CIV NS1 protein was conserved along its evolution by comparing the NS1 amino acid sequences of H3N8 viruses phylogenetically related to CIV NY09, including A/canine/Florida/2004 H3N8 (CIV FL04) and the equine influenza viruses (EIVs) A/equine/Pennsylvania/1/2007 H3N8 (EIV Pen07) and the early A/equine/Miami/1963 H3N8 (EIV Mi63) (Fig. 8). K186 was mostly conserved, as EIV Mi63 is the only virus that displays E186 (Fig. 8A and 9). We evaluated the ability of the aforementioned NS1 proteins to block host gene expression in MDCK cells, as previously described (Fig. 1). As expected, only the NS1 protein of EIV Mi63 blocked expression of GFP and/or Gluc (Fig. 8B and C). Similar results were obtained when NS1 protein expression levels were evaluated by Western blotting (Fig. 8D). We confirmed that amino acid substitution E186K was responsible for the lack of inhibition of host gene expression when we introduced E186 in CIV FL04 and EIV Pen07 NS1 proteins and K186 in EIV Mi63 NS1 (Fig. 8B to D).
The ability to block host gene expression was lost during the evolution of H3N8 EIV. (A) Amino acid sequence alignment of H3N8 CIV and EIV NS1 proteins. Amino acid residues 161 to 230 from CIV NY09, A/canine/Florida/2004 (CIV FL04), A/equine/Pennsylvania/1/2007 (EIV Pen07), and A/equine/Miami/1963 (EIV Mi63) H3N8 NS1 proteins are shown. Gray shadow highlights amino acid 186. (B to D) Amino acid 186 is responsible for the lack of inhibition of host gene expression in H3N8 CIV and EIV NS1 proteins. MDCK cells (12-well plate format, 3 × 105 cells/well, triplicates) were transiently cotransfected with expression plasmids encoding GFP and Gluc (50 ng), together with plasmids encoding the indicated H3N8 CIV or EIV WT and mutant NS1 proteins or empty (E) plasmid as a control (2 μg). At 24 hpt, cells were evaluated for GFP expression (B) or Gluc activity (C). NS1 protein expression levels from total cell lysates were determined by Western blotting using an anti-HA polyclonal antibody (HA-NS1) (D). Actin was included as a loading control. Bar, 100 μm. Results represent the means and standard deviations of triplicate values. *, P < 0.05 using Student's t test.
Frequency of identified NS1 186 mutation in H3N8 EIV and CIV isolates over time. Publicly available sequences in the Influenza Research database (www.fludb.org ) were downloaded, and the frequencies of H3N8 EIV and CIV NS1 sequences containing amino acid E186 or K186 are represented according to the years of virus isolation.
EIV Mi63 was the first EIV isolated after the virus was transmitted from an avian host to horses, suggesting that the ability to inhibit host gene expression was lost during the evolution of EIV in horses but before the virus was transmitted to dogs in 2004 (Fig. 9). To assess the timeline describing the period over which amino acid substitution E186K was incorporated into NS1, the percentages of H3N8 EIV and CIV strains whose NS1 sequence encoded the different amino acids were plotted (Fig. 9). Notably, database analysis suggests that amino acid substitution E186K occurred early in the 1970s in EIVs and has remained stable since then (Fig. 9). Moreover, all H3N8 CIV strains analyzed encode amino acid K186, indicating that since EIV transferred into dogs in 2004, K186 has been maintained (Fig. 9).
The avian-origin H3N2 CIV NS1 protein is able to block host gene expression.To determine if the inability to block general gene expression was a feature of influenza viruses of dogs, we next examined the NS1 sequences of H3N2 CIVs, as this lineage's origin is different from that of H3N8 CIV. Interestingly, all the H3N2 CIV NS1 proteins exhibit E186 (data not shown), suggesting that the NS1 proteins of this lineage would block host gene expression. To confirm this, we cotransfected MDCK cells with the reporter-expressing plasmids (Fig. 1), and the H3N2 CIV NS1 protein efficiently inhibited the expression of GFP and Gluc (Fig. 10A and B, respectively). Moreover, Western blot analysis further confirmed the ability of H3N2 CIV NS1 protein to inhibit host gene expression, including its own expression (Fig. 10C). Notably, this phenotype was reverted when the amino acid substitution E186K was introduced into the H3N2 CIV NS1 protein (Fig. 10). These data suggest that the ability to block general host gene expression is strain dependent, similar to what has been observed with human influenza viruses.
The avian-origin H3N2 CIV NS1 protein is able to efficiently block host gene expression. MDCK cells (12-well plate format, 3 × 105 cells/well, triplicates) were transiently cotransfected with expression plasmids encoding GFP and Gluc (50 ng) together with plasmids encoding the indicated NH2 HA-tagged H3N8 or H3N2 CIV NS1 proteins or an empty (E) plasmid as a control (2 μg). At 24 hpt, cells were analyzed for GFP (A) and Gluc (B) expression. NS1 protein expression levels from total cell lysates were determined by Western blotting using an anti-HA polyclonal antibody (HA-NS1), and actin was included as a loading control (C). Bar, 100 μm. Results represent the means and standard deviations of triplicate values. *, P < 0.05 using Student's t test. H3N8 CIV, A/canine/NY/dog23/2009 H3N8; H3N2 CIV, A/canine/IL/41915/2015 H3N2.
DISCUSSION
The continued interspecies transmission of IAVs to humans and other mammals is a constant threat (4, 39–41). As dogs are popular companion animals and can support the replication of multiple IAV subtypes such as H3N8, H3N1, H3N2, H5N1, H5N2, and H1N1 (10, 11, 42–53), they could act as intermediate hosts for IAV reassortment or human exposure (7). Various human IAVs, such as PR8, A/Udorn/307/72 H3N2, and pH1N1, replicate in the respiratory tract of the dog, and viable reassortant viruses between H3N8 CIV and pH1N1 IAV have been reported (54). In addition, it has been shown that dogs can be coinfected by pH1N1 and H3N2 CIV, leading to the generation of viable reassortants (48, 55). This highlights the need for monitoring the evolution of CIVs and studying the mechanism of IAV adaptation to new hosts.
IAVs possess mechanisms to antagonize host IFN and IFN-induced responses (56, 57). IAV NS1 is a multifunctional viral protein and is the main factor in counteracting host innate immune responses, allowing the virus to efficiently replicate during infection (18, 58). One mechanism of inhibition of innate immune responses is mediated through the binding of IAV NS1 to CPSF30, which blocks the processing of pre-mRNAs in the nucleus and suppresses the expression of host genes, including IFN and ISGs (25, 26, 28–34). However, CPSF30 binding is not essential for successful IAV infection, since some NS1 proteins do not bind CPSF30 (25, 26, 28–34). The reasons for such variation are not well understood. However, it is possible that in mammals, the lack of inhibition of important host genes (e.g., cytokines) prevents infection by other pathogens that could interfere and/or compete with viral replication and dissemination.
Our results show that H3N8 CIV NS1 protein inhibits the induction of IFN and ISGs (Fig. 5) but does not bind CPSF30 (Fig. 4) and is therefore unable to block host gene expression in either canine or human cells (Fig. 1). NS1 residue 186 proved to be a main determinant in blocking general gene expression (Fig. 2), and the K186E substitution introduced both the NS1-CPSF30 interaction (Fig. 4) and gene expression inhibition (Fig. 2). This inhibition of host gene expression was dose dependent and specific for polymerase II nuclear expressed transcripts (Fig. 3).
Our results are consistent with previous work using A/Udorn/72 H3N2 NS1 protein, in which residues around position 186 (amino acids 184 to 188 [GLEWN]) are important for binding to CPSF30 and inhibiting host gene expression (32), and were found to be important for efficient viral replication (32). Interestingly, we found that CIV NY09 K186E replicated better than the WT virus in two canine cell lines, but only at a high MOI (Fig. 6). Moreover, while the two viruses were able to inhibit induction of IFN-β to a similar extent, CIV NY09 mutant K186E inhibited the expression of ISG more efficiently than WT virus (Fig. 7). However, the different abilities of CIV NY09 NS1 WT and K186E proteins to block host gene expression could have a more significant role in the natural host, the dog.
Our study also confirms that NS1 binding to CPSF30 and the resulting block in general gene expression does not impair the ability of this NS1 to counteract IFN induction (Fig. 5). As indicated for other IAVs (18, 25, 27, 30, 33, 34) our results clearly show that the NS1 proteins of different IAV strains that infect the same host can differ in their strategy to overcome the innate immune response. We showed that the avian-origin H3N2 CIV NS1 protein—which contains E186—inhibits host gene expression (Fig. 10) and that the same is observed with A/equine/Miami/63, an early EIV (the ancestor of H3N8 CIV) that possesses E186 (Fig. 8). As both EIV H3N8 and H3N2 CIV are avian-origin viruses, E186 might be important during the initial stages of IAV adaptation in mammals (C. Chauché, A. Nogales, H. Zhu, D. Goldfarb, A. I. Ahmad Shanizza, Q. Gu, C. R. Parrish, L. Martínez-Sobrido, J. F. Marshall, and P. R. Murcia, submitted for publication) and that K186 provides increased fitness at later evolutionary stages. Notably, since introduced in EIV in the early 1970s, amino acid substitution E186K has remained stable in both EIV and CIV strains (Fig. 9).
It has been recently reported that the PA-X protein from both H3N8 EIVs and CIVs is able to suppress host gene expression (59). It is possible that EIV and CIV H3N8 NS1 proteins lost the ability to inhibit gene expression because of the ability of the viral PA-X to suppress host protein expression (59). In fact, we have recently described that inhibition of host protein expression by influenza virus is subject to a strict balance, which can determine the successful progression of viral infection (60). Thus, it is possible that H3N8 EIV and CIV NS1 proteins lost the ability to interact and inhibit CPSF30 to improve viral fitness, since this function is carried out by the viral PA-X protein (59), and viruses encoding PA-X and NS1 proteins able to inhibit host gene expression would not be as fitted as viruses when only one of the viral proteins is able to inhibit host gene expression (60). It remains to be determined whether or not CIV H3N2 PA-X does inhibit host gene expression to the same extent as H3N8 EIV and/or CIV PA-X proteins. In sum, our study provides insights into the mechanisms employed by IAVs to counteract the innate immune response in different hosts and also how those can vary depending on the virus origin and the strain involved. Future in vivo studies using these viruses will shed light on the role of NS1 protein in pathogenicity, transmission efficiency, and mammalian adaptation in natural hosts.
MATERIALS AND METHODS
Cell lines.Human embryonic kidney 293T (HEK293T; ATCC CRL-11268), MDCK (ATCC CCL-34), and BSR-T7 (kindly provided by Karl-Klaus Conzelmann) (37) cells were grown at 37°C with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM; Mediatech, Inc.), 10% fetal bovine serum (FBS), and 1% PSG (penicillin, 100 units/ml; streptomycin, 100 μg/ml; l-glutamine, 2 mM) (61, 62). A72 cells (ATCC CRL-1542) were grown in 50/50 McCoy's 5A (Gibco)/Leibovitz's L-15 (Gibco) with 5% FBS and 1% PSG.
Virus rescue.Virus rescues were performed as previously described (36, 62). Briefly, cocultures (1:1) of HEK293T/MDCK cells (6-well plate format, 106 cells/well, triplicates) were cotransfected in suspension, using Lipofectamine 2000 (LPF2000; Invitrogen), with 1 μg each of the seven A/canine/NY/dog23/2009 H3N8 ambisense wild-type (WT) plasmids (pDZ-PB2, -PB1, -PA, -NP, -NA, -M, -HA) (13) plus the ambisense WT (pDZ-NS) or mutant K186E (pDZ-NS K186E) plasmids. At 12 h posttransfection (hpt), the medium was replaced with DMEM containing 0.3% bovine serum albumin (BSA), 1% PSG, and 1 μg/ml tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma). At 48 hpt, tissue culture supernatants (TCS) were used to infect fresh monolayers of MDCK cells (6-well plate format, 106 cells/well, triplicates). At 3 days postinfection (dpi), recombinant viruses were plaque purified and scaled up in MDCK cells. Stocks were titrated by plaque assay on MDCK cells (36, 62), and the identity of the NS segment was confirmed by restriction analysis and sequencing.
Virus growth kinetics and immunostaining.To evaluate viral growth kinetics, MDCK cells or A72 cells (12-well plate format, 5 × 105 cells/well, triplicates) were infected at low (0.001) or high (3) multiplicity of infection (MOI) and the TCS were collected at the indicated time points. Viral titers were determined by immunofocus assay (focus-forming units [FFU]) in MDCK cells as previously described (36, 62). The mean values and standard deviations were calculated using Microsoft Excel software. For the immunostaining, confluent MDCK cell monolayers (6-well plate format, 106 cells/well) were infected with 10-fold serial dilutions of H3N8 CIV WT or K186E viruses. After infection, monolayers were overlaid with agar and incubated for 72 h at 33°C. Then, cells were fixed with 4% paraformaldehyde (PFA), and the overlays were removed. Fixed cells were then permeabilized (0.5% Triton X-100 in PBS for 15 min at room temperature) and used for immunostaining as previously described (36, 62) using anti-NP monoclonal antibody (MAb; HB-65; ATCC) and vector kits (Vectastain ABC kit and DAB HRP substrate kit; Vector), according to the manufacturer's specifications.
RT-PCRs.Total RNA from CIV- or EIV-infected (MOI, 3) MDCK cells (6-well plate format, 106 cells/well) was collected at 18 hpi and purified using TRIzol (Invitrogen) according to the manufacturer's specifications. cDNA synthesis for NS vRNAs or NS1 mRNAs was performed using SuperScript II reverse transcriptase (Invitrogen) and specific primers. cDNAs were used as the templates for semiquantitative PCR with primers specific for the NS vRNA (A/canine/NY/dog23/2009 H3N8) or NS1 mRNAs (A/canine/Florida/2004 H3N8, A/equine/Pennsylvania/1/2007 H3N8, A/equine/Miami/1963 H3N8, and A/canine/IL/41915/2015 H3N2). Primer sequences are available upon request. Influenza viruses A/equine/Pennsylvania/1/2007 and A/equine/Miami/1963 H3N8 were obtained from BEI Resources (NR-13426 and NR-3175, respectively). Influenza A/canine/Florida/2004 H3N8 and influenza A/canine/IL/41915/2015 H3N2 were provided by the Baker Institute for Animal Health at Cornell University.
For qRT-PCRs, MDCK cells (12-well plate format, 5 × 105 cells/well, triplicates) were mock infected or infected (MOI of 3) with NS1 WT and K186E H3N8 CIVs. At 12 or 24 hpi, total RNA was extracted using an RNeasy minikit (Qiagen) as per the manufacturer's recommendations. Reverse transcription reactions were performed at 37°C for 2 h using the high-capacity cDNA transcription kit and an oligo(dT) primer to amplify mRNAs. Quantitative PCRs were performed using TaqMan IFN-β and IFN-induced protein with tetratricopeptide repeats 2 (IFIT2) assays (Applied Biosystems) specific for the Canis familiaris (dog) genes (Cf03644503_s1 and Cf02645026_m1, respectively). Quantification was achieved using the 2-ΔΔCT method, where CT is threshold cycle (63).
Plasmids.To engineer polymerase II expression pCAGGS plasmids (64) containing the NS1 sequences fused to an HA epitope tag (YPYDVPDYA) at the N terminus (pCAGGS-HA-NH2) (23), we used standard molecular biology techniques. The different NS1 genes were amplified by RT-PCR using oligonucleotides with the appropriate flanking restriction sites (SmaI and XhoI) for cloning into pCAGGS-HA-NH2. NS1 open reading frames were also cloned in a pGEM-T plasmid (Promega) to introduce the different mutations by site-directed mutagenesis using specific primers. To abolish NS mRNA splicing, two silent mutations were introduced at nucleotides (nt) 501 and 504 of NS1 (30, 33). NS1 mutants were subcloned from the pGEM-T to the pCAGGS-HA-NH2 using SmaI and XhoI. Plasmids encoding NS1 variants under the control of the phage T7 polymerase were obtained by subcloning NS1 from the pCAGGS-HA-NH2 plasmids into pcDNA3 plasmid using EcoRI and XbaI restriction enzymes. pCAGGS and pDNA3 plasmids encoding the NS1 proteins of influenza A/Puerto Rico/8/34 (PR8) and influenza A/Brevig Mission/01/1918 (BM/18) H1N1 viruses were previously described (23, 28). To generate an H3N8 CIV containing the mutation K186E, the mutated NS viral segment was cloned into the ambisense pDZ plasmid for virus rescue. All the plasmid constructs were confirmed by sequencing (ACGT, Inc.). Sequences of the primers used for the construction of the different plasmids are available upon request.
Inhibition of host gene expression.To evaluate the effects of viral NS1 proteins on host protein synthesis, HEK293T or MDCK cells (12-well plate format, 3 × 105 cells/well, triplicates) were transiently cotransfected, using LPF2000, with 2 μg/well of pCAGGS-HA NH2 NS1 protein expression plasmids or an empty plasmid as a control, together with 50 ng/well of pCAGGS plasmids expressing GFP (28) and Gaussia luciferase (Gluc) (65). For T7-driven expression, BSR-T7 cells (12-well plate format, 3 × 105 cells/well, triplicates) were transiently cotransfected, as indicated above, with 2 μg/well of pCAGGS-HA NH2 NS1 protein expression plasmids, or an empty plasmid as a control, together with 0.5 μg/well of the reporter expression plasmids pCITE-FFLuc and pCITE-GFP, which express firefly luciferase (FFluc) or GFP under the control of the T7 promoter (36). At 24 hpt, cells were analyzed for GFP expression under a fluorescence microscope and Gluc (HEK293T and MDCK cells) or FFluc (BSR-T7 cells) activities were quantified from TCS (Gluc) or cell lysates (FFluc) using a Biolux Gaussia luciferase reagent (New England Bio-Labs) or a FFluc luciferase reporter buffer (Promega) and a Lumicount luminometer. The mean values and standard deviations were calculated using Microsoft Excel software.
Inhibition of IFN-β and ISRE promoters.The assays to evaluate the effects of NS1 proteins on the inhibition of IFN-β and IFN-stimulated response element (ISRE) promoters have been previously described (28). Briefly, HEK293T cells (12-well plate format, 3 × 105 cells/well, triplicates) were cotransfected with 1 μg/well of the pCAGGS-HA NH2 NS1-expressing plasmids or empty plasmid as a control, together with 0.5 μg/well of plasmids expressing FFluc under the control of the IFN-β or the ISRE promoters (pIFN-β-FFluc and pISRE-FFluc, respectively) (28) and 0.5 μg/well of plasmids expressing GFP or red fluorescent protein (RFP) fused to the chloramphenicol acetyltransferase (CAT) driven by the IFN-β or the ISRE promoter (pIFN-β-GFP/CAT and pISRE-RFP/CAT, respectively) using a calcium phosphate-based mammalian transfection kit (Stratagene). At 18 hpt, cells were infected (MOI, 3) with Sendai virus (SeV) strain Cantell (28), and at 18 hpi cells were analyzed for IFN-β or ISRE promoter activation by GFP (IFN-β) or RFP (ISRE) expression under a fluorescence microscope. FFluc activity was quantified from cell lysates using a FFluc luciferase reporter buffer (Promega) and a Lumicount luminometer (PacKard). Reporter gene activation is shown as relative light units (RLU) compared to SeV-infected cells in the absence of NS1 protein (empty plasmid). The mean values and standard deviations were calculated using Microsoft Excel software.
Protein gel electrophoresis and Western blot analysis.Total proteins from cell lysates were separated using 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked for 1 h with 5% dried skim milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (T-PBS) and incubated overnight at 4°C with anti-HA (for NS1; Sigma) or anti-FLAG (for F2/F3 CPSF30; Sigma) polyclonal antibodies (pAb). A monoclonal antibody specific for actin (Sigma) was used as an internal loading control. Bound primary antibodies were detected with horseradish peroxidase (HRP)-conjugated antibodies (GE Healthcare) against immunoglobulins of different species (mouse or rabbit). Proteins were detected by chemiluminescence using the SuperSignal West Femto maximum-sensitivity substrate (Thermo Scientific) as per the manufacturer's recommendations and photographed using a Kodak Image Station.
NS1 interaction with CPSF30.HA-tagged NS1 proteins were synthesized in vitro using pcDNA3 plasmids and the TNT7 transcription/translation kit (Promega) as per the manufacturer's recommendations. HEK293T cells (6-well format, 1.5 ×106 cells/well) were transiently transfected with 2 μg/well of a pCAGGS plasmid expressing a FLAG-tagged version of the F2/F3 region of the human CPSF30 (FLAG-F2/F3 CPSF30) (34). At 48 hpt, cells were lysed in 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5 mM EDTA, 5% glycerol, and 1% Triton X-100 supplemented with a complete Mini Protease inhibitor cocktail (Pierce). Cleared cell lysates were incubated overnight at 4°C with the in vitro-synthesized NS1 proteins and 20 μl of an anti-FLAG affinity resin (Sigma). After extensive washing, precipitated proteins were dissociated from the resin using Laemmli buffer and analyzed by Western blotting as described above.
ACKNOWLEDGMENTS
We thank Biodefense and Emerging Infectious Research Resources Repository (BEI Resources) for providing influenza viruses A/equine/Pennsylvania/1/2007 and A/equine/Miami/1963 H3N8 (NR-13426 and NR-3175, respectively). We also thank Karl-Klaus Conzelmann for kindly providing us with the BSR-T7 cells.
This research was partially funded by the New York Influenza Center of Excellence (NYICE, NIH 272201400005C), a member of the NIAID Centers of Excellence for Influenza Research and Surveillance (CEIRS), and by the University of Rochester Technology Development Fund. C.C. was funded by a Horserace Betting Levy Board PhD studentship (VET/RS/252). P.R.M. was funded by the Medical Research Council of the United Kingdom (grant number MC_UU_12014/9).
FOOTNOTES
- Received 28 May 2017.
- Accepted 20 August 2017.
- Accepted manuscript posted online 23 August 2017.
- Copyright © 2017 American Society for Microbiology.
