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. Author manuscript; available in PMC: 2011 Feb 1.
Published in final edited form as: J Virol Methods. 2009 Nov 14;163(2):498–504. doi: 10.1016/j.jviromet.2009.11.010

Identification of the non-structural influenza A viral protein NS1A as a bona fide target of the Small Ubiquitin-like MOdifier by the use of dicistronic expression constructs

Sangita Pal 1,§, Juan M Rosas 1,§, Germán Rosas-Acosta 1,2,*
PMCID: PMC2834654  NIHMSID: NIHMS160258  PMID: 19917317

Abstract

The cellular SUMOylation system affects the function of numerous viral proteins. Hence, the identification of novel viral targets for the Small Ubiquitin-like MOdifier (SUMO) is key to our understanding of virus-host interactions. The data obtained in this study demonstrate that the non-structural influenza A viral protein NS1A is an authentic SUMO target through the use of a dicistronic expression plasmid containing SUMO (the modifier) and Ubc9 (the SUMO conjugating enzyme) separated by an Internal Ribosomal Entry Site (IRES). This dual expression plasmid produces a robust increase in cellular SUMOylation, therefore facilitating the characterization of cellular and viral SUMO targets. The identification of NS1A as a bona fide SUMO target suggests, for the first time, a role for SUMOylation during influenza virus infection.

Keywords: SUMO, Ubc9, Influenza, NS1A, IRES, SUMOylation

The post-translational conjugation of the Small Ubiquitin-like MOdifier (SUMO) to a target protein (SUMOylation) is now accepted widely to be an important mechanism to regulate protein activity, stability, cellular localization, and protein-protein interactions (Dohmen, 2004; Geiss-Friedlander and Melchior, 2007). Besides its various roles in the cell, SUMOylation has also been shown to play important roles during the life cycle of several viruses, and numerous viral targets for SUMO have been identified (Boggio and Chiocca, 2006; Rosas-Acosta and Wilson, 2004); however, no such targets have been identified for a ssRNA(-) virus, thus the relevance of this post-translational modification for these viruses remains undetermined. One common feature shared by viruses known to interact with the cellular SUMOylation system is their ability to replicate in the nucleus (Rosas-Acosta and Wilson, 2004), the cellular compartment where SUMOylation appears most active (Dohmen, 2004; Johnson, 2004). Influenza virus is one of only a few RNA viruses that replicates in the nucleus (Herz et al., 1981; Jackson et al., 1982) and several of its encoded proteins exhibit potential SUMOylation sites. The non-structural protein NS1A from H1N1 influenza A (A/PR/8/34) contains two predicted SUMOylation sites (based on SUMOplot analyses) and, due to its well characterized role as a down-regulator of cellular anti-viral responses (Gack et al., 2009; Li et al., 2006; Li et al., 2004; Lu et al., 1995; Ludwig et al., 2002; Min and Krug, 2006; Nemeroff et al., 1998; Noah et al., 2003; Qiu and Krug, 1994; Talon et al., 2000; Wang et al., 2000; Yuan and Krug, 2001), and the potential relevance of any post-translational modification on its functions, it was chosen as the initial target to determine whether SUMOylation played a role during influenza virus infection.

Studies aimed at establishing whether a given protein of interest is SUMOylated have been carried out using both in vitro and cell culture approaches. In vitro SUMOylation assays were first established during the initial characterization of the enzymatic activities involved in the SUMOylation pathway (Desterro et al., 1999; Desterro et al., 1997; Duprez et al., 1999; Mahajan et al., 1998; Matunis et al., 1998; Okuma et al., 1999), and have provided a convenient screening method for determining whether any given protein constitutes a target for SUMOylation. Thus, the first approach to determine whether NS1A constitutes a SUMO target was to verify its ability to be SUMOylated in vitro. To this end, NS1A was synthesized and labeled using a coupled transcription/translation system, and the 35S-labeled product was incubated for 90 minutes in the presence of either SUMOylation reaction buffer (SRB) alone or a mix of purified SUMO1, the E1 activating and E2 conjugating SUMOylation enzymes, and SRB, according to a previously described method (Rosas-Acosta et al., 2005a). To provide conclusive proof that any additional band observed in the latter reaction corresponded in fact to a SUMOylated form of the target protein, an additional sample was incubated with a mix of purified SUMO1, the E1 and E2 SUMOylation enzymes, and SRB (as above), but 30 minutes after the beginning of the reaction, a purified protein corresponding to the catalytic domain of the yeast de-SUMOylating enzyme Ulp1 (hereafter referred to as Ulp1403-621) was added to the sample. As a positive control, the same procedure was executed with C/EBP-β1, a well characterized SUMO target (Eaton and Sealy, 2003). In the presence of all components required for SUMOylation, a high molecular weight form of NS1A consistent with the expected molecular weight of SUMOylated NS1A was observed (Fig. 1, lane 5). Such high molecular weight form of NS1A disappeared upon incubation with Ulp1403-621 (Fig. 1, lane 6) in a manner equivalent to that observed for C/EBP-β1 (Fig.1, compare lanes 2 and 3).

Fig. 1.

Fig. 1

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NS1A is SUMOylated in vitro. C/EBP-β1 (positive control) and NS1A were 35S-labeled and synthesized in a coupled transcription/translation reticulocyte system. Subsequently, the proteins were incubated in the presence (+) or absence (-) of all components required for SUMOylation (i.e., SUMO1, the dimeric E1 SUMO-activating enzyme SAE2/SAE1, the E2 SUMO-conjugating enzyme Ubc9, and ATP), and the catalytic domain of the de-SUMOylating enzyme Ulp1. Diagonal arrows: SUMOylated forms of the proteins. Asterisk: Non-SUMOylated (unmodified) forms of the proteins.

Although the analysis above indicated that NS1A can be SUMOylated efficiently in vitro, similar evidence of SUMOylation in an in vivo system is essential to demonstrate that a given protein constitutes a bona fide SUMO target, as various observations suggest that the in vitro SUMOylation system does not display the fine specificity exhibited by the SUMOylation system in vivo. First, in vitro SUMOylation is very efficient even in the absence of ligases (Okuma et al., 1999), whereas ligases are thought to play an essential role in vivo. Second, SUMO1 chains are generated easily in vitro (Cooper et al., 2005; Pedrioli et al., 2006; Pichler et al., 2002), whereas their existence in vivo remains unproven (Matic et al., 2008). Lastly, for a few SUMO targets, mutations that abolish completely their SUMOylation in vivo still allow their SUMOylation in vitro (unpublished observations). This lack of specificity is likely associated with the substantial overdrive produced by the high concentration of both the SUMO modifier and the enzymatic components of the pathway in the in vitro system.

Despite being considered the gold standard, the cell culture approach to study of the SUMOylation of a given protein target is complicated by the high abundance of de-SUMOylating enzymes in the cell, which deconjugate SUMO from its target proteins and produce a very low steady state level of SUMO-modified forms for most SUMO targets in the cell (Yamaguchi et al., 2005). Most published reports on the characterization of a SUMO target using a cell culture approach involve the co-transfection of the model cells with either, two expression plasmids (one for the protein of interest and one for the SUMO modifier), or three expression plasmids (including one for the conjugating enzyme Ubc9 in addition to the two mentioned above), followed by the affinity purification of either, all SUMOylated proteins produced, or the protein of interest. Although these approaches are effective for the characterization of numerous viral SUMO substrates, such as HPV16-E2 (Wu et al., 2008), Apoptin (Janssen et al., 2007), and HHV-6 IE1 (Gravel et al., 2004), they proved ineffective to demonstrate the in vivo SUMOylation of NS1A. This failure was hypothesized to be due to limitations inherent to the experimental system employed, as it leads to the synthesis of either, large amounts of SUMO in the absence of any additional Ubc9 (for the two plasmid system), or similar amounts of SUMO and Ubc9 (for the three plasmid system), both likely to be suboptimal conditions for achieving the substantial increase in cellular SUMOylation required to allow the detection of transient SUMOylation events.

To test the hypothesis above and determine conclusively whether NS1A is an authentic SUMO target, it was deemed necessary to develop expression constructs that would induce robust increases in cellular SUMOylation with the two main types of SUMO modifiers produced in mammalian cells, SUMO1 and SUMO2/3. Internal Ribosomal Entry Sites (IRESs) have been used widely to create dicistronic expression constructs for the simultaneous expression of two different proteins from a single transcript. Importantly, under normal growth conditions, the protein encoded by the first open reading frame (ORF), translated by a 5′-cap binding/AUG scanning mechanism, is translated abundantly, whereas the protein encoded by the second ORF, translated by an IRES-driven mechanism, is translated poorly (Pestova et al., 2001). This property seemed optimal to produce the appropriate ratio of Ubc9 and SUMO required for a robust increase in cellular SUMOylation. Therefore, a series of dicistronic constructs were developed using the internal ribosomal entry site (IRES) from the encephalomyocarditis virus (EMCV) (residues 273-848 of the EMCV genome), a PCR approach to amplify and clone the desired inserts, and the pcDNA5/FRT/TO-based His-S-SUMO1 and His-S-SUMO3 constructs previously reported (Rosas-Acosta et al., 2005b) as parental plasmids. These constructs code for N-terminally His-tagged and S-peptide tagged SUMO proteins, thereby providing two tags for the affinity purification of the SUMOylated proteins.

To produce a substantial increase in overall SUMO1 SUMOylation, dicistronic constructs were developed carrying one copy each of SUMO1 and Ubc9 under the transcriptional control of the cytomegalovirus promoter. In these constructs, the placement of Ubc9 and SUMO1 was alternated between the first and second positions in the dicistron (Fig. 2). This allowed an unbiased assessment of the ideal placement of Ubc9 and SUMO1 in the dicistronic construct. Then, the SUMOylation-inducing activities of the newly developed constructs were compared with that achieved by co-transfecting single expression constructs into the same cell line. In each case, C/EBP-β1 was used as the substrate for SUMOylation. To this end, HEK293A cells were seeded in a 24 well plate, at a density of 1×105 cells/well, and co-transfected with various combinations of the different constructs and a C/EBP-β1 expression plasmid. All transfections were performed using TransIT®-LT1 transfection reagent (Mirus Bio LLC, Madison, WI) following the manufacturer's protocol. Total cell extracts were collected 24 h post-transfection using boiling 2× sample buffer and the samples were analyzed by SDS-PAGE and immunoblotting.

Fig. 2.

Fig. 2

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Dicistronic expression constructs used in this study. (A) Dicistronic constructs carrying either SUMO1 or SUMO3 in the first position. (B) Dicistronic constructs carrying Ubc9 in the first position. The relative positions of the poly-His tag (His-tag), the S-peptide tag (S-tag), the hemagglutinin tag (HA-tag), the Internal Ribosomal Entry Site (IRES) from the encephalomyocarditis virus (EMCV), the cytomegalovirus promoter (PCMV), and the bovine growth hormone poly-adenylation signal (BGH-pA) are indicated. The EMCV IRES was amplified and cloned in place by means of a PCR-based procedure, using the plasmid pIRES2-DsRed2 (Clontech Laboratories, Inc. Mountain View, CA) as template.

The SUMOylated form of C/EBP-β1 was not detected in cells transfected with C/EBP-β1 and an empty expression plasmid (Fig. 3A, lane 1). Co-transfection with the monocistronic expression plasmid for SUMO1 alone led to substantial SUMOylation of C/EBP-β1 (Fig. 3A, lane 2), and co-transfection with monocistronic expression plasmids for both SUMO1 and Ubc9 produced a further increase in the amount of SUMOylated C/EBP-β1 (Fig. 3A, lane 3). However, co-transfection with the dicistronic plasmid carrying SUMO1 in the first position (hereafter referred to as Dual S1/I/U) led to an even more efficient SUMOylation of C/EBP-β1, as indicated by the relative intensity of the SUMOylated band, and the appearance of a second SUMOylated form of C/EBP-β1 (Fig. 3A, lane4). Co-transfection with an amount of Dual S1/I/U equivalent to the combined total of the individual monocistronic plasmids for SUMO1 and Ubc9, produced a further increase in SUMOylated C/EBP-β1 (Fig. 3A, lane 5). In sharp contrast, co-transfection with the dicistronic plasmid carrying Ubc9 in the first position (hereafter referred to as Dual U/I/S1) led to only limited SUMOylation of C/EBP-β1 (Fig. 3A, lane 6).

Fig. 3.

Fig. 3

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The Dual S1/I/U construct induces the most robust SUMO1-SUMOylation of C/EBP-β1. HEK293A cells were transfected with (+) or without (-) the indicated expression constructs. At 24 h post-transfection, cell extracts were collected in boiling 2× sample buffer (50 mM Tris pH 6.8, 10% glycerol, 4% SDS, 0.01% bromophenol blue, 2% β-mercaptoethanol) and analyzed by SDS-PAGE and immunoblotting using the following antibodies: (A) anti-C/EBP-β1 mouse monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), diluted at 1:5,000. (B) anti-SUMO1 rabbit monoclonal antibody (Epitomics, Inc., Burlingame, CA), diluted at 1:5,000. (C) anti-Ubc9 rabbit monoclonal antibody (Epitomics, Inc.), diluted at 1:5,000. (D) anti-GAPDH mouse monoclonal antibody (Santa Cruz Biotech, Inc.), diluted at 1:5,000. Horseradish peroxidase conjugated goat antibodies directed against the appropriate antibody were used as secondary antibodies at a dilution of 1:10,000. Immunoblots were developed by chemiluminescence using Immobilon™ Western HRP substrate (Millipore Corp., Billerica, MA). The membrane was stripped for 10 minutes using a boiling solution containing 1% SDS and 0.2% β-mercaptoethanol between consecutive immunoblots. Brackets and arrows: SUMOylated and non-SUMOylated forms of the exogenous proteins expressed by transfection, respectively. Arrowhead: Endogenous GAPDH protein used as loading control. Asterisk: Residual C/EBP-β1 signal from the anti-C/EBP-β1 immunoblot.

The effects produced on total protein SUMOylation by co-transfection with the different expression plasmids mirrored the effects observed on C/EBP-β1 SUMOylation. Co-transfection with the monocistronic expression plasmids led to significant increases in overall SUMOylation, as evidenced by the appearance of high molecular weight SUMO1-specific bands (Fig. 3B, lanes 2 and 3); however, co-transfection with Dual S1/I/U produced an even larger increase in overall SUMOylation (Fig. 3B, lanes 4 and 5). Conversely, co-transfection with Dual U/I/S1 led to a very modest increase in overall SUMOylation (Fig. 3B, lane 6), surpassed by the increases observed in all other samples. Importantly, in spite of producing the smallest increase in overall SUMOylation, co-transfection with Dual U/I/S1 led to the production of the largest amount of Ubc9 (Fig. 3C, lane 6). A similar although slightly smaller increase in the amount of Ubc9 was also observed in cells co-transfected with the two monocistronic constructs, the ones for Ubc9 and SUMO1 (Fig. 3C, lane 3).

To produce a substantial increase in total SUMO3 SUMOylation, two dicistronic constructs were developed, each carrying one copy of SUMO3 and Ubc9 at alternating positions, as described above for SUMO1(Fig. 1), and their effects were tested in co-transfection assays with a C/EBP-β1 expression construct, as done previously with the SUMO1 dicistronic constructs. Co-transfection with SUMO3 alone (Fig. 4A, lane 2), but not Ubc9 alone (Fig. 4A, lane 3), produced a substantial increase in the SUMOylation of C/EBP-β1. However, co-transfection with the dicistronic construct carrying SUMO3 in the first position (hereafter referred to as Dual S3/I/U) led to the largest increase in SUMOylated C/EBP-β1 (Fig. 4A, lanes 4 and 5). Consistent with the amount of SUMOylated C/EBP-β1 observed, the largest increase in overall SUMOylation was also observed with Dual S3/I/U (Fig. 4B, lanes 4 and 5). Similarly as observed for SUMO1, the largest increase in the amount of Ubc9 was observed in cells transfected with the dicistronic construct carrying Ubc9 in the first position (hereafter referred to as Dual U/I/S3) (Fig. 4C, lane 7), although such increase in Ubc9 did not lead to comparable increases in overall SUMOylation nor in the amount of SUMOylated C/EBP-β1 (Fig. 4B, lane 7).

Fig. 4.

Fig. 4

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The Dual S3/I/U construct induces the most robust SUMO3-SUMOylation of C/EBP-β1. HEK293A cells were transfected with (+) or without (-) the indicated expression constructs and processed for immunoblotting as described in Fig. 2 using the following antibodies: (A) anti-C/EBP-β1 mouse monoclonal antibody (Santa Cruz Biotech, Inc.), diluted at 1:5,000. (B) anti-SUMO3 rabbit serum (Invitrogen Corp., Carlsbad, CA), diluted at 1:5,000. (C) anti-Ubc9 rabbit monoclonal antibody (Epitomics, Inc.), diluted at 1:5,000. (D) anti-GAPDH mouse monoclonal antibody (Santa Cruz Biotech, Inc.), diluted at 1:5,000. The membrane was stripped between consecutive immunoblots as described in Fig. 2. Brackets and arrows: SUMOylated and non-SUMOylated forms of the exogenous proteins expressed by transfection, respectively. Arrowheads: Endogenous proteins recognized by the corresponding antibodies. Asterisk: Residual C/EBP-β1 signal from the anti-C/EBP-β1 immunoblot. Notice that the differences in exogenous Ubc9 (arrow) observed among transfected samples correlate with the expression system used. The least additional Ubc9 was produced from a dicistronic construct carrying Ubc9 in the second position (lanes 4 and 5), the most Ubc9 was produced from a dicistronic construct carrying Ubc9 in the first position (lane 7), and intermediate levels were produced from a monocistronic construct (lanes 3 and 6).

Having developed a set of expression constructs that produced a more robust and reproducible increase in overall SUMOylation, co-transfection assays similar to those described above for C/EBP-β1, but replacing the C/EBP-β1 plasmid with an expression plasmid for H1N1 (A/PR/8/34) NS1A, were performed to determine whether NS1A constitutes a bona fide SUMO target in vivo. Co-transfection of the NS1A expression plasmid with an empty plasmid did not produce any high molecular weight forms of NS1A, as expected (Fig. 5A, lane 2). Similarly, co-transfection with monocistronic expression constructs for SUMO1 and Ubc9, alone or in combination, failed to stimulate the production of any high molecular weight forms NS1A (Fig. 5A, lanes 3-5). In sharp contrast, co-transfection with the Dual S1/I/U construct led to the production of a high molecular weight form of NS1A consistent with the expected molecular weight of a SUMOylated form of NS1A (Fig. 5A, lane 6). To substantiate further that the high molecular weight form of NS1A observed constituted a SUMOylated form, an additional sample co-transfected with the Dual S1/I/U construct and a monocistronic expression construct for the de-SUMOylating enzyme SENP1 was included. The expression of SENP1 produces a substantial increase in de-SUMOylating activity within the cell, able to reverse the SUMOylation of the target protein, thereby preventing the detection of the SUMOylated forms of the protein. As expected, the high molecular weight form of NS1A observed in the presence of Dual S1/I/U was not present in the sample co-transfected with the Dual S1/I/U construct and the SENP1 expression construct (Fig. 5A, lane 7), therefore supporting that the high molecular weight form of the protein is a SUMOylated form.

Fig. 5.

Fig. 5

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The influenza A virus non-structural protein NS1A is SUMOylated when over-expressed by transfection. Panels A-D: HEK293A cells were transfected with (+) or without (-) the indicated expression constructs and collected 24 h later in boiling 2× sample buffer and analyzed directly by SDS-PAGE and immunoblotting using the following antibodies: (A) anti-T7 mouse monoclonal antibody (Novagen, EMD Chemicals, Inc.), diluted at 1:5,000. (B) anti-SUMO1 rabbit monoclonal antibody (Epitomics, Inc.), diluted at 1:5,000. (C) anti-Ubc9 rabbit monoclonal antibody (Epitomics, Inc.), diluted at 1:5,000. (D) anti-GAPDH mouse monoclonal antibody (Santa Cruz Biotech, Inc.), diluted at 1:5,000. Panels E-F: To further confirm the identity of the high molecular weight form of NS1A as a SUMOylated form, HEK293A cells were transfected with (+) or without (-) the indicated expression constructs, and collected at 36 h post-transfection in a denaturing buffer containing 8M urea and 20 mM NEM (to inactivate SUMO proteases). The extracts were diluted down to 500 mM urea and the SUMOylated proteins were purified by affinity chromatography on S-Protein agarose beads (Novagen, EMD Chemicals, Inc.). Upon purification, the beads were treated with 2× sample buffer and analyzed by SDS-PAGE and immunoblotting using the following antibodies: (E) anti-T7 mouse monoclonal antibody (Novagen, EMD Chemicals, Inc.), diluted at 1:5,000. (F) anti-SUMO1 rabbit monoclonal antibody (Epitomics, Inc.), diluted at 1:5,000. The membranes were stripped between consecutive immunoblots as described in Fig. 2. The position of SUMO-conjugated and free forms of the different proteins is indicated. Notice the presence of a cross-reactive band in panel A, migrating slightly above NS1A, at around 35 kD. Also notice that, unlike the Ubc9 expression construct used in Figs. 2 and 3, the monocistronic Ubc9 expression construct used in this figure lacked an HA-tag.

To demonstrate conclusively that the high molecular form of NS1A represents SUMOylated NS1A, its ability to bind to S-protein agarose beads, owing to the presence of the dual His-tag S-peptide tag at the N-terminus of the SUMO1 encoded by the dicistronic Dual S1/I/U construct, was tested. To this end, HEK293A cells were co-transfected with different combinations of the expression constructs for NS1A, the dicistronic Dual S1/I/U construct, a monocistronic expression construct for the SUMO de-conjugating enzyme SENP1, and an empty plasmid. The transfected cells were lysed in denaturing buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris pH 8.0, 0.2% Triton X-100, 20 mM N-ethylmaleimide (NEM)), the resulting cell extract was diluted down to 500 mM Urea with dilution buffer (100 mM NaH2PO4, 10 mM Tris pH 8.0, 0.2% Triton X-100, 20 mM NEM), and the SUMOylated proteins were purified by affinity chromatography on S-Protein agarose beads (Novagen, EMD Chemicals, Inc., Madison, WI). The purified proteins were analyzed by SDS-PAGE and immunoblotting. Samples derived from cells transfected with the NS1A expression construct either without the dicistronic SUMO1 construct (Fig. 5E, lane 3) or with the SENP1 construct (Fig. 5E, lane 5) failed to show any high molecular weight forms of NS1A. In contrast, the sample derived from cells co-transfected with the NS1A expression construct and the dicistronic SUMO1 construct in the absence of SENP1 displayed a major high molecular weight form of NS1A consistent with the expected molecular weight of SUMOylated NS1A (Fig. 5E, lane 4). As only SUMOylated proteins are retained by the S-protein agarose beads, as previously reported (Rosas-Acosta et al., 2005b), this result demonstrated that the high molecular weight form of NS1A corresponds to SUMOylated NS1A.

To determine whether NS1A can also be SUMOylated during viral infection, recombinant adenoviruses carrying either, the dicistronic SUMO1 construct described previously (hereafter referred to as AdV-Dual S1/I/U), or a mutated version of it containing a deletion of the two C-terminal glycine residues in SUMO1 (hereafter referred to as Ad-Dual S1ΔGG/I/U), were developed using the pAd/CMV/V5-DEST vector and the ViraPower™ Adenoviral Gateway® expression system (Invitrogen). The deletion of the di-glycine motif in Ad-Dual S1ΔGG/I/U renders the over-expressed SUMO1 non-conjugatable but still able to bind to Ubc9, therefore producing a block in the cellular SUMOylation system. Lung epithelial human A549 cells were transduced with either AdV-Dual S1/I/U or Ad-Dual S1ΔGG/I/U at a multiplicity of infection (MOI) of 100 and 48 hours later the cells were infected with H1N1 influenza A virus (A/PR/8/34) at MOI 3. Fifteen hours post-infection, the cells were lysed in denaturing buffer and processed for affinity purification on S-Protein agarose beads as described above. The initial cell lysates and the purified proteins were analyzed by SDS-PAGE and immunoblotting. NS1A was detected efficiently in the cell extracts from influenza infected cells, and a high molecular weight form of NS1A was observed in cells transduced with the AdV-Dual S1/I/U, although other cross-reactive bands hindered its clear recognition. A high molecular weight form of NS1A consistent with the predicted molecular weight of SUMOylated NS1A was purified on the S-Protein beads from the influenza infected sample transduced with AdV-Dual S1/I/U but not from the influenza infected sample transduced with Ad-Dual S1ΔGG/I/U, therefore indicating conclusively that NS1A is SUMOylated during viral infection in a tissue culture model. Unexpectedly, a significant amount of non-SUMOylated NS1A was also detected on the beads. As NS1A was not detected at all on the beads from influenza infected cells transduced with the Ad-Dual S1ΔGG/I/U, and NS1A is known to form dimers (Nemeroff et al., 1995), the non-SUMOylated NS1A detected on the beads likely corresponds to a fraction of NS1A that interacts or dimerizes with the SUMOylated form.

Post-translational modifications provide a key functional regulatory link between viruses and their host cell. Thus, the identification of post-translational modification affecting viral proteins is fundamental to our understanding of virus-host interactions. The dicistronic constructs Dual S1/I/U and Dual S3/I/U developed in this work induced the largest increases in both overall and target-specific SUMOylation observed throughout the study, and therefore they provide a simple and efficient method to assess the SUMOylation of specific viral proteins in vivo. The sharp contrast in overall SUMOylation observed between cells transfected with the Dual S1/I/U or Dual S3/I/U constructs and cells transfected with the Dual U/I/S1 or Dual U/I/S3 constructs strongly supports that the most important limiting factor for the cellular SUMOylation system is the SUMO modifier itself. Thus, a substantial increase in the cellular concentration of SUMO (either SUMO1 or SUMO3) is critical to achieve maximum cellular SUMOylation. In contrast, a slight increase in the cellular concentration of Ubc9, probably equivalent to about a doubling of its normal cellular concentration as judged based on the band intensities of the endogenous and exogenous (HA-tagged) Ubc9 bands (Fig. 5C), is sufficient to achieve maximum cellular SUMOylation. The robust increase in overall SUMOylation provided by the dicistronic constructs facilitates enormously the identification of novel viral SUMO targets, as demonstrated by the identification of SUMOylated forms of the influenza A virus non-structural protein NS1A. Additionally, the epitope tags added to the SUMO modifiers allow the affinity purification of the SUMOylated proteins, providing an extra tool to assess conclusively the SUMOylation of any given viral protein.

Although in this work no direct comparison was executed with the Ubc9 fusion-directed SUMOylation (UFDS) method developed by Jakobs et al. (Jakobs et al., 2007a; Jakobs et al., 2007b), nor with the method developed by Tatham et al. (Tatham et al., 2009), the dicistronic constructs presented in this report provide two substantial advantages over those methods: First, they allow direct analysis of the SUMOylation of viral proteins under normal viral infection; second, thanks to their incorporation into recombinant Adenoviruses, the dicistronic constructs allow such analysis to be performed in any cell line deemed optimal for viral growth. These conditions are not possible using either of the other methods indicated above: The UFDS approach involves the development of an Ubc9 fusion of the target protein (Jakobs et al., 2007a; Jakobs et al., 2007b), which would require the development of a recombinant virus expressing an Ubc9 fusion of the target viral protein. The method reported by Tatham et al. (2009) is currently only available with HeLa cells, as it requires a stably transfected cell line. Therefore, the dicistronic constructs presented in this report have distinct advantages over previously reported methods that make them particularly appropriate to study the SUMOylation of viral proteins.

Current knowledge related to NS1A's potential regulation by post-translational modifications is limited to several reports indicating its ability to be phosphorylated (Hale et al., 2009; Mahmoudian et al., 2009; Petri et al., 1982; Privalsky and Penhoet, 1981). The data presented in this study shows that NS1A is SUMOylated, not only when overexpressed by transfection in mammalian cells, but also when expressed during viral infection in a lung epithelial human cell line, and therefore demonstrates conclusively that NS1A is a bona fide SUMO target. This finding adds SUMOylation to the list of post-translational modifications known to affect this viral protein. NS1A neutralizes multiple cellular antiviral responses and regulates cellular and viral gene expression (reviewed by Hale et al. (Hale et al., 2008) and Krug et al. (Krug et al., 2003)), and has been shown recently to be an important determinant of viral pathogenicity (Billharz et al., 2009; Jiao et al., 2008). As SUMO is known to exert numerous effects on its protein targets (Geiss-Friedlander and Melchior, 2007), the finding that NS1A is SUMOylated implies that the cellular SUMOylation system may play important roles during influenza virus infection, potentially affecting viral gene expression, multiplication, and pathogenicity. These possibilities are currently under investigation in our laboratory.

Fig. 6.

Fig. 6

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The influenza A virus non-structural protein NS1A is SUMOylated during viral infection in the A549 lung epithelial human cell line. A549 cells were transduced with (+) or without (-) AdV-Dual S1/I/U or Ad-Dual S1ΔGG/I/U at MOI 100. Two days later, the cells were infected with H1N1 influenza (A/PR/8/34) at MOI 3, and 15 hours later the cells were lysed in denaturing buffer and the SUMOylated proteins were purified as described in Fig. 5. The total cell extracts collected and the material purified on S-Protein beads were analyzed by SDS-PAGE and immunoblotting using antibodies against either NS1A or SUMO1. Diagonal arrow: SUMOylated NS1A. Asterisk: Non-SUMOylated (unmodified) NS1A.

Acknowledgments

This research was supported by grant #0765137Y from the American Heart Association (South-Central Filiate), grant #1SC2AI081377-01 from the National Institutes of Allergy and Infectious Diseases (NIAID) and the National Institute of General Medical Sciences (NIGMS), National Institutes of Health (NIH), and start-up funds provided by the University of Texas at El Paso, all to Dr. Rosas-Acosta. The Border Biomedical Research Center and its associated facilities is supported by grant #5G12RR008124 from the NIH. We are grateful to Dr. Van G. Wilson (Texas A&M Health Science Center - College of Medicine, College Station, TX) for his invaluable contributions to this work, Dr. Kyle L. Johnson (UTEP) for her critical review of this manuscript, and to Cinthia Gallegos for introducing the T7 tag into the NS1A expression construct.

Footnotes

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