Identification of putative interactions between swine and human influenza A virus nucleoprotein and human host proteins

Influenza A viruses (IAVs) are important pathogens that affect the health of humans and many additional animal species. In humans, seasonal IAV infection presents as a non-fatal, uncomplicated, acute infection characterized by the presence of upper respiratory symptoms as well as fever, headache, soreness and fatigue lasting 2–5 days [1]. However, deaths from seasonal IAV infections often arise when the normal flu symptoms are exacerbated by compromised immunity or age [1]. In addition to seasonal influenza A viruses, pandemic strains periodically appear causing increased mortality rates. For example, the 1918 Spanish flu resulted in approximately 50 million human deaths [2]. The most recent pandemic resulted from the emergence of the swine-origin H1N1 virus [3],[4]. Due to influenza A’s potential for mortality, high mutation rates (resulting in genetic drift) and pandemic potential (resulting from genetic reassortment), it is critical to learn more about the virus, especially as it pertains to virulence, transmissibility, and identification of potential targets for development of therapeutics.

Influenza A viruses exhibit a broad host range beyond humans [5]. Waterfowl serve as the central reservoir species. In addition to humans, various IAV subtypes circulate in pigs, poultry, horses, dogs as well as other species. Subtypes (H1N1 and H3N2) that circulate in the human population also circulate in the swine populations [6]. In addition to the concern for IAV in terms of swine health [7], IAV infection in swine is also important due to the potential for zoonotic infections as well as the potential for serving as a mixing vessel for the generation of human pandemic viruses [5]. The majority of inter-species transmission research has focused on amino acids in the hemagglutinin affecting viral attachment and entry [8]. Although known to be important, other than amino acids associated with temperature sensitivity (e.g., PB2 627 [9] or 701 [10]), the effects of differences in amino acid residues in proteins of the replication complex on species specificity is less well understood. Previous evidence suggests that there may be a role in some of these replication complex proteins in allowing for zoonotic infections [11].

The genome of the enveloped influenza A virion consists of eight segments of negative-sense single-stranded RNA that encode at least ten viral genes: Hemagglutinin (HA), Neuraminidase (NA), Matrix 2 protein (M2), Matrix 1 protein (M1), Non-structural Protein 1 (NS1), Non-structural Protein 2 (NS2), Nucleoprotein (NP), Polymerase Basic 1 (PB-1), Polymerase Basic 2 (PB-2), and Polymerase Acidic (PA) [1]. The viral envelope contains HA, NA, and M2 proteins, and M1 proteins form a layer inside the envelope. The IAV RNA genome located inside the virion is coated with NP and is bound by the replication complex consisting of PB1, PB2, and PA. The NP, the focus of this study, encoded by the fifth IAV RNA segment, binds with high affinity to viral RNA [12]. NP plays a role in viral RNA replication and transcription [12]. More recent data suggests that NP binds the polymerase as well as the newly replicated RNA and may act as a processivity factor that is necessary for replication of the viral RNA to be completed [13]. Phylogenic analysis of IAV NP shows distinct lineages of NP based on the host species [14],[15]. Within the NP, there are amino acid signatures found within different host species [16],[17]. These host-specific amino acid residues may result in differences in affinities for the various host proteins with which they interact (e.g., importin α1 [18], F- actin [19], nuclear factor 90 [20], cyclophilin E [21], exportin 1 [22], HMGB1 [23], HMGB2 [23], MxA/Mx1 [24],[25], HSP40 [26], karyopherin alpha [27],[28], clusterin [29], Raf-2p48/BAT1/UAP56 [30], Aly/REF [31], Tat-SF1 [32], TRIM22 [33], and alpha actinin 4 [34]) or they may result in differences in how the NP interacts with other viral proteins that have also made host-specific adaptations [11]. Given the suggestion that NP plays a role in determining host range [5],[35]-[37], it is important to identify all host proteins that interact with NP.

Replication of the IAV is dependent on the host cell machinery and interactions between host proteins and viral proteins. Therefore any inquiry attempting to investigate the life cycle of IAV must incorporate host-pathogen protein interactions in order to truly provide a mechanistic understanding of the process. Previous screens have identified important protein-protein interactions between IAV and host proteins, but often results are not consistent between screens. Therefore, corroborating evidence with additional screens is crucial to accumulating the best understanding of critical pathogen-host interactions in viral replication. As examples, several genome-wide screens have been performed using RNAi to systematically knockdown host genes and evaluate the effect on various stages of the IAV life cycle [38]-[41]. An integrated approach carried out by Shapira and colleagues involved transcription profiling and yeast two hybrid screens using specific viral proteins as baits [28]. Proteomics approaches have revealed host proteins found within viral particles [42],[43]. The Random Homozygous Gene Perturbation strategy was used to identify host factors that prevent influenza-mediated killing of host cells [44].

In order to better understand the role of NP-host protein interactions in IAV replication, a Gal4-based yeast two-hybrid (Y2H) assay was used in this study. The Y2H system allows for the identification of unique human binding partners with NP. A “bait” plasmid encoding the binding domain (BD) of the Gal4 transcriptional activator fused to NP and a “prey” plasmid encoding Gal4’s activation domain (AD) fused to a protein encoded by a human cDNA are introduced into a yeast strain containing Gal4-responsive reporter genes. Interaction of the bait and prey brings together Gal4’s BD and AD, resulting in transcriptional activation of reporter genes [45]. The Y2H approach has successfully identified cellular factors (e.g., Raf-2p48/BAT1/UAP56, Hsp40, KPNA1, KNPA3, KPNA6, C16orf45, GMCL1, MAGED1, MLH1, USHBP1, ZBTB25, CLU, Aly/REF, and ACTN4) that interact with the IAV NP [26]-[31],[34], (Table 1). In this study, the nucleoprotein from a classical swine H1N1 IAV (Sw/NC/44173/00) was used as the “bait” in a Y2H screen against a “prey” HeLa cDNA library. To investigate if the origin of NP affects the ability of the interactions to take place, the nucleoprotein from a contemporary human H3N2 IAV (A/Ca/07/04) was also used as the “bait” in a Y2H screen against the same prey HeLa cDNA library. By determination of the putative interaction partners between NP and human proteins, the possible functions of the newfound protein-protein interactions or verification and perhaps elucidation of previously identified host factors may be further investigated.

Reported herein are ten potential human host cell proteins that interact with IAV NP in a Y2H screen. The relative strengths of these protein-protein interactions were characterized by a plating assay and beta-galactosidase assay.

This Y2H screen identified multiple candidate host proteins that were previously identified in genome-wide screens as playing a role in the IAV infection cycle (PAICS, ATP1B1, SLC30A9, and TRA2B), while also identifying new potential interactors with IAV nucleoprotein (ACOT13, CKAP5, KCTD9, PA28B, MSANDTD3, and FLJ30306). These results complement previous reports by suggesting the mechanism for involvement of the host proteins with the IAV (i.e. interaction with the NP). The yeast screen is internally validated by the observation that half of the prey proteins identified were independently isolated multiple times during the screen. All host prey proteins identified interact in yeast with IAV NP from both human and swine isolates. Although there can be variability in gene expression among yeast colonies containing the same combination of bait/prey, both a qualitative growth assay and a quantitative β-gal assay yielded similar results in terms of the strength of interaction between IAV NP bait and human prey proteins. Notably, CKAP5 displayed good growth at the more stringent 37°C in the qualitative assay and also had high β-gal activity. The putative interacting host proteins represent a variety of cellular processes, consistent with the diversity of processes required during the IAV infection cycle.

Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) is an enzyme necessary to catalyze the sixth and seventh steps of the de novo purine biosynthesis pathway [46]. PAICS has been identified by two separate RNAi screens as being crucial to IAV replication [40],[47]. Furthermore, proteins involved in purine biosynthesis, including PAICS, were found to be significantly up-regulated during an IAV infection [48]. The data presented herein suggest that the requirement for PAICS during IAV infection may be through the interaction with NP.

The sodium/potassium-transporting ATPase subunit beta-1 (ATP1B1) represents another host protein that interacts in yeast with NP and that has been previously shown to be important for influenza virus replication in host cells. It belongs to the family of Na+/K + −ATPase beta chain proteins. Na+/K + −ATPase is necessary for the creation and maintenance of electrochemical gradients of sodium and potassium ions on either side of the plasma membrane [49]. This gradient is used by cells to permit functional nervous and muscular excitability, osmoregulation, and active transport of numerous molecules [49]. The beta subunit functions to regulate the quantity of sodium pumps transported to the plasma membrane [49]. Using a quantitative proteomics approach, ATP1B1 was one of 43 proteins found to be significantly up-regulated in IAV-infected primary human alveolar macrophages [50]. In a Y2H study, Mi and colleagues [51] isolated ATP1B1 as a protein that binds the cytoplasmic domain of IAV M2 and subsequently showed that knockdown of ATP1B1 in MDCK cells suppressed IAV replication [51]. These studies suggest that ATP1B1 may be interacting with multiple IAV proteins.

The solute carrier family 30 (zinc transporter), member 9 (SLC30A9) is involved in nucleotide-excision repair of human DNA [52] and has an efflux motif characteristic of proteins in the SLC30 zinc efflux transporter family [53]. SLC30A9 protein found in the cytoplasm can bind nuclear receptors and/or nuclear receptor coactivators and translocate to the nucleus where it regulates gene transcription [54]. SLC30A9 was identified as a possible host target that confers upon the host cell resistance to IAV infection [44]. How SLC30A9 might interact with NP and be necessary for IAV replication is unclear.

Another protein identified by this screen is TRA2B, a tissue-specific splicing factor [55]. TRA2B protein binds to AGAA and CAA RNA sequences affecting the inclusion of introns in the processed transcript [55]. With respect to influenza, expression of TRA2B protein was reported to be down-regulated in porcine alveolar macrophage cells infected by two swine IAVs [56]. Because influenza genes M2 and NS2 are spliced, it is possible that TRA2B is important for the splicing of these genes. However, how NP might be involved in TRA2B’s function in IAV replication is unknown.

Another binding partner identified is Acyl CoA thioesterase 13 (ACOT13), a eukaryotic protein also known as thioesterase superfamily member 2 (Them2) [57],[58]. ACOT13 catalyzes fatty acyl-CoA hydrolysis that exists primarily in association with mitochondria [57]. ACOT13/Them2 was identified in a Y2H screen focused on phosphatidylcholine transfer protein, suggestive of a role in fatty acid metabolism [57]. Although ACOT13 has not previously been reported to play a role in the IAV life cycle, another member of the ACOT family (ACOT9) which has two hot dog fold domains (ACOT13 has one) [59] has been shown to physically interact with the IAV PA protein [28]. How ACOT13 may be interacting with NP during IAV replication is unclear.

Cytoskeletal associated protein 5 (CKAP5, also known as TOG) binds microtubules and is important for the process of centrosomal microtubule assembly especially during mitosis [60]. The microtubule network of the cell plays a role in movement of materials throughout the cell, including during infection [61]. TOG2 binds to a ribonucleoprotein and plays a role in RNA trafficking [62]. While a specific role for CKAP5 in the IAV life cycle is unknown, a genome-wide siRNA screen revealed CKAP5 as one of 96 human genes that supports replication of another RNA virus- the hepatitis C virus [63]. It’s possible that CKAP5 functions during the IAV infection cycle to organize microtubules for trafficking of viral components.

Another interactor identified in this screen, KCTD9, has been previously shown to interact with a subunit of the Mediator complex, acting as a scaffold between regulatory proteins and the RNA polymerase II [64]. In addition, KCTD9 homolog FIP2 acts as a cytoskeletal rearrangement protein suspected to be involved in nuclear export in some plants through the organization of actin cables [64]. Perhaps KCTD9 aids in translocating NP to the nucleus for replication and transcription of the viral RNA. It has been shown that the translocation of NF-E2 related factor 2 (Nrf2) is mediated by cytoskeletal rearrangement in the oxidative stress response [65], and the level of Nrf2 expression is associated with IAV entry and replication in nasal epithelial cells [66].

Also identified by this screen was PA28B, a protein associated with the proteasome complex. In the typical proteasome, there is a 19S regulator, which is replaced by the 11S regulator in the modified immunoproteasome [67]. PA28B is part of the proteasome complex and acts as a subunit in the 11S alternate regulator. It is referred to as the beta subunit and is comprised of three beta and three alpha subunits arranged in a heterohexameric ring [68]. The proteasome, as a whole, is used for the cleaving of peptides and the 11S regulator, in particular, induces the degradation of short peptides, but not entire proteins [68]. Induction of 11S regulator expression is the responsibility of interferon gamma. PA28B is involved in cleavage of the peptides which bind to the major histocompatibility complex (MHC). It is possible that NP interacts with PA28B to prevent MHC I presentation of IAV antigens.

Myb/SANT-like DNA-binding domain containing 3 (MSANTD3) is a member of the MSANTD3 family which contains DNA binding domains for Myb proteins and the SANT domain family. Myb proteins are proto-oncogenes which are necessary for hematopoiesis and possibly involved in tumorigenesis [69]. The SANT domain permits the interaction of chromatin remodeling proteins with histones and is found in chromatin-remodeling complexes as well as nuclear receptor co-repressors [70]. NP interactions with MSANTD3 could have an effect on gene transcription of the host through chromatin modification, presumably to inhibit antiviral genes or up-regulate genes beneficial to viral replication.

The screen also identified FLJ30306 as a potential interactor with NP. FLJ30306 maintains a sequence similarity to retroviral elements, resulting in its classification as endogenous retrovirus group K3, member 1 [Homo sapiens] [71]. The endogenous retrovirus sequence was revealed by sequence analysis of the mRNA, not by direct protein sequencing. It is unclear whether the protein carries out a normal cellular effect, such as endogenous retrovirus 1’s (ERV-1) use in placental syncytia formation or if it is a result of mis-regulation due to influenza infection [71]. In Hodgkin’s lymphoma it has been shown that many endogenous retroviruses are reactivated and produce virus-like particles [71]. It may be that instead of directly utilizing FLJ30306 influenza may simply be up-regulating its expression due to its pathogenesis. Alternatively, FLJ30306 may contain useful enzymatic activity for influenza replication through an unknown mechanism. A characterization of normal FLJ30306 function would allow for appropriately testing either hypothesis.

All human host proteins identified in this Y2H screen interact in yeast with NP from both swine and human IAV strains. While the data suggest there may be differences in the strength of bait-prey interactions in yeast based on NP host origin, these differences should be interpreted carefully given the nature of the assay. Given the suggestion that NP affects species specificity, additional screens using NP from other IAV strains as the bait and/or cDNA libraries from other susceptible species may shed light on amino acids within the NP and specific host factors that are responsible for host restrictions. As is the case with other published screens (e.g., [72]), it will be important to verify the putative interactions described here in infected cells by co-immunoprecipitation, co-localization, or RNA interference (e.g., [34]) and to investigate the role of the identified host proteins in the IAV life cycle.

A Y2H screen using a classical swine influenza A virus nucleoprotein as a bait resulted in isolation of ten different putative interacting human host proteins involved in a variety of cellular processes and structures: purine biosynthesis (PAICS), metabolism (ACOT13), proteasome (PA28B), DNA-binding (MSANTD3), cytoskeleton (CKAP5), potassium channel formation (KCTD9), zinc transporter function (SLC30A9), Na+/K+ ATPase function (ATP1B1), and RNA splicing (TRA2B). All of the identified human host proteins interacted in yeast with NP from both human and swine origin. Proteins PAICS, ATP1B1, TRA2B, and SLC30A9 have been previously identified in studies related to host response to the IAV. Host proteins SLC30A9 and CKAP5 displayed the strongest interactions with IAV NP in yeast in a quantitative β-gal assay.