Oligoadenylate synthetase (OAS) proteins are cellular, double-stranded RNA (dsRNA) sensors that function as part of the innate immune response to virus infections [1
]. In the human genome, the OAS family consists of one copy each of the OAS1, OAS2, OAS3 and OASL genes. The OAS1 protein contains a single OAS domain, while the OAS2 and OAS3 proteins are composed of two and three OAS domains, respectively [3
]. Human OASL contains an inactive OAS domain plus two domains of ubiquitin-like sequences [4
]. Interaction with dsRNA or base-paired regions in a single-stranded RNA (ssRNA) activates OAS proteins to polymerize ATP into 2′-5′-linked oligoadenylates (2-5A). Trimer and higher order 2-5As bind to endoribonuclease L (RNase L), inducing its dimerization and activation. Activated RNase L cleaves viral and cellular ssRNAs, including 28S and 18S ribosomal RNA (rRNA) [1
]. Although human OASL is catalytically inactive, upon dsRNA binding, it has an RNase L-independent antiviral activity that is mediated through enhancing RIG-I signaling [9
Multiple alternatively spliced human OAS1 gene mRNAs have previously been amplified from human cells [11
]. An A/G SNP (rs10774671) at the intron-5/exon-6 splice acceptor site alters hOAS1 pre-mRNA splicing [11
]. The p46 isoform mRNA is predominantly produced by individuals with the G allele, while those with the A allele produce p48 and p52.
Although all of these hOAS1 isoforms share the same 346 amino acid sequence in the N-terminal catalytic domain, they differ at the C-termini [15
]. Individuals with one or two copies of the A allele were previously found to have lower OAS activity [12
Antiviral activity for some hOAS1 isoforms, but not for others, was previously reported in response to infections with the mosquito-borne flaviviruses, West Nile virus (WNV) and Dengue virus, that are of public health importance. The p42 isoform was shown to be activated by interaction with a 5′ sequence of the WNV genome RNA, comprised of stem loops I, II and III [17
]. A Dengue virus infection only induced RNase L activation in cells that were overexpressing the hOAS1 p42 and p46 isoforms, but not the p44, p48 or p52 isoforms [18
In the present study, we analyzed the in vitro synthetase activities of seven bacterially-expressed recombinant hOAS1 isoform proteins, and all were found to synthesize 2-5A after incubation with poly(I:C). We then individually overexpressed five of the hOAS1 isoforms in HEK293 cells, and analyzed their ability to activate endogenous RNase L. Each of the hOAS1 isoforms activated RNase L in response to treatment of the cells with poly(I:C). The data indicate that all five of these isoforms are active synthetases, both in vitro and in mammalian cells. A yeast two-hybrid screen was utilized to identify novel binding partner candidates for the hOAS1 p44 and p42 isoforms. The data support a role for the unique C-terminal sequences of the different hOAS1 isoforms in recruiting unique partners that may facilitate additional functions and/or intracellular localization.
2. Materials and Methods
Human embryonic kidney HEK293 cells were cultured at 37 °C in a 5% CO2 atmosphere in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 1% L-glutamine, 5% fetal bovine serum (FBS) and 0.1% gentamicin. Human lung carcinoma A549 cells were cultured at 37 °C in a 5% CO2.atmosphere in F-12K nutrient media supplemented with 1% L-glutamine, 10% fetal bovine serum and 0.1% gentamicin.
2.2. Cloning and Expression of hOAS1 Isoform Proteins in Bacteria
Six plasmid DNAs each containing the cDNA for an individual human OAS1 isoform (p41, p42, p44, p46, p48 and p49) were provided by Dr. Andrey Perelygin (Georgia State University). A plasmid clone of the p52 cDNA was provided by Dr. Svetlana Scherbik (Georgia State University). The GenBank accession number for P49 is DQ914956.1, for p52 it is AY730627.1 and for p41 it is DQ823040.1. These cDNAs were previously amplified by reverse transcription--polymerase chain reaction (RT-PCR) amplification from RNA extracted from ATCC human fibroblasts CCL-110 or CCL-66 that had been treated with IFNβ for 24 h and cloned into a TOPO-TA vector. Individual clones were selected and sequenced to identify the different isoform cDNAs. Each hOAS1 isoform was then subcloned into the pET151-TOPO bacterial expression vector (Thermo Fisher Scientific, Waltham, MA, USA) with a 6 X His tag and a V5 epitope fused at the N-terminus.
The hOAS1 plasmid DNAs were transformed into One Shot TOP10 chemically competent Escherichia coli (E. coli) (Thermo Fisher Scientific, Waltham, MA, USA), re-isolated from colonies and sequenced. hOAS1 isoform proteins were expressed in transformed One Shot BL21-(DE3)-pLysS cells (Thermo Fisher Scientific, Waltham, MA, USA), grown in 125 mL of Luria-Bertani (LB) media containing 0.05% glucose and 100 μg/mL of carbenicillin (CRB). Expression was induced with 1 mM of isopropyl β-d-1-thiogalacto-pyranoside (IPTG) overnight at 16 °C. Cells were pelleted by centrifugation at 6000× g for 10 min at 4 °C. The cells were resuspended in 10 mL of 1 X Equilibration buffer [NaCl (300 mM), sodium phosphate (50 mM) and 1 X Complete Mini EDTA-Free Protease inhibitor cocktail (Roche, Indianapolis, IN, USA)] and frozen at −80 °C until use. CelLytic Express lysis powder (Sigma-Aldrich, St. Louis, MO, USA) was added to the thawed cell suspensions and incubated at 37 °C for 30 min with shaking. The cell lysates were clarified by centrifugation at 15,000× g at 4 °C for 10 min. The volume of the clarified supernatant was increased to 20 mL by addition of 1 X Equilibration buffer and transferred to a column containing 1 mL of TALON metal affinity nickel resin (Clontech, Mountain View, CA, USA).
After washing the column with 1 X washing buffer [50 mM sodium phosphate (pH 7.4), 300 mM NaCl and 5 mM imidazole], the bound proteins were eluted with 5 mL of 1 X Elution buffer [50 mM sodium phosphate (pH 7.4), 300 mM NaCl and 150 mM imidazole]. The eluted protein fractions were combined, and the buffer was first exchanged with 1 X Storage buffer [20 mM Hepes-KOH (pH7.5), 50 mM KCl, 25 mM Mg(OAc)2, 7 mM β-ME, 0.03 mM ethylenediaminetetraacetic acid (EDTA), 0.25% glycerol and 1 X Complete Mini EDTA-Free Protease inhibitor cocktail (Roche, Basel, Switzerland)] and then concentrated using a Microcon-10 kDa Centrifugal Filter Unit (Millipore, Burlington, MA, USA). The partially purified proteins were aliquoted and stored at −80 °C.
2.3. In Vitro 2′-5′ OAS Activity Assay
Each adenosine triphosphate (ATP) polymerization reaction mixture (50 µL) contained an individual hOAS1 isoform protein (22 µL), α32p-ATP (15 µCi) and poly(I:C) (50 ng/µL) in 1 X Assay buffer [20 mM HEPES-KOH pH 7.5, 50 mM KCl, 25 mM Mg(OAC)2, 10 mM creatine phosphate, 1 U/µL creatine kinase, 5 mM ATP and 7 mM β-ME]. After incubation at 30 °C for 18 h, the reaction was stopped by the addition of 50 µL of Gel Loading Buffer II [95% formamide, 18 mM EDTA, 0.025% SDS, xylene cyanol and bromophenol blue (Ambion, Austin, TX, USA)]. An aliquot of the reaction (2 µL) was separated on a 20% polyacrylamide/Urea gel at 800 V for 3.5 h, and the production of radiolabeled 2-5A was detected by autoradiography.
2.4. Functional Analysis of hOAS1 Isoforms in Mammalian Cells
The hOAS1 p42, p44, p46, p48 and p52 isoform cDNAs were subcloned into the p3xFlag-CMV mammalian expression vector with a 3 X Flag tag fused at the N-terminus. HEK293 cells were seeded in a 6-well plate and grown to ~70% confluence before transfection with either empty vector DNA or with a hOAS1 isoform plasmid DNA using Lipofectamine LTX/PLUS reagent (Thermo Fisher Scientific, Waltham, MA, USA). At different times after the initial transfection, the cells were transfected with 0.5 µg of poly(I:C) for 6 h using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Cell lysates were harvested in TRI reagent (Molecular Research Center. Inc., Cincinnati, OH, USA). Total intracellular RNA was extracted and purified according to the manufacturer’s protocol and then separated on a denaturing formaldehyde/3-(N-morpholino)propanesulfonic acid (MOPS) agarose gel. The RNA gel was stained with ethidium bromide and imaged under ultraviolet (UV) light.
For protein detection by Western blotting, hOAS1 cDNA-transfected HEK293 cells were lysed by incubation with 1 X RIPA buffer [1 X phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate,0.1% sodium dodecyl sulfate (SDS) and 1 X Halt™ protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA)].
2.5. Yeast Two Hybrid Assay
A yeast two hybrid assay was performed using the Matchmaker Gold Yeast Two-Hybrid System following the manufacturer’s protocol (Clontech, Mountain View, CA, USA). Briefly, the cDNA sequences of the full length hOAS1-p42 and hOAS1-p44 isoforms were fused to the GAL4-BD domain in the pGBKT7 vector, and separately transformed into the Y2HGold yeast strain as the bait. A Y187 yeast strain containing a universal human cDNA library fused to the GAL4-AD domain in a pGADT7-RecAB vector (Clontech, Mountain View, CA, USA) was used as the prey. The Y2HGold and Y187 yeast strains were mated at 30 °C for 24 h and plated on double dropout (DDO, minus Trp and Leu) plates for selection of mated diploid cells. The selected diploid cells were then plated on triple dropout plates supplemented with Aureobasidin A (TDO/A, minus His, Trp and Leu) and quadruple dropout plates supplemented with Aureobasidin A (QDO/A, minus Ade, His, Trp and Leu) for the selection of positive clones that contained interacting prey and bait under increasingly stringent conditions. When the prey and bait interact, in addition to the expression of the missing components in the media, the AUR1-C gene is also expressed, which confers strong resistance to the otherwise highly toxic drug Aureobasidin A, so that the yeast can grow on media containing Aureobasidin A.
The prey and bait plasmid constructs were extracted from yeast colonies that grew on QDO/A plates, and their cDNA inserts were sequenced. A basic local alignment search tool (BLAST) search was then performed to determine the identity of the putative binding peptide.
2.6. Yeast Co-Transformation
A plasmid expressing a putative binding peptide was transformed into the Y187 yeast strain. The transformed Y187 strain was then mated with the Y2HGold strain containing an hOAS1 bait at 30 °C for 24 h. The mated diploid cells were then plated on DDO, TDO/A and QDO/A plates to confirm the co-transformation, as well as the positive interaction, between the bait and the putative binding peptide.
2.7. In Vitro Transcription/Translation and Pull-Down Assay
The cDNAs of the full-length hOAS1 bait and the putative binding peptide were individually cloned into a pTNT expression vector (Promega, Madison, WI, USA) with a c-myc tag or an HA tag fused at the N-terminus, respectively. The resulting bait and prey constructs were then expressed in vitro in the presence of [35S]-methionine using a TnT-coupled transcription/translation system (Promega), according to the manufacture’s protocol. The in vitro translated bait and prey peptides were incubated together for 1 h at room temperature and then divided into three equal portions. One portion was stored at −20 °C and was used as a lysate sample, one portion was incubated with control agarose beads conjugated with a non-specific control IgG antibody, and one portion was incubated with agarose beads conjugated with either anti-c-myc or anti-HA antibody. After an overnight incubation at 4 °C with rotation, the beads were washed with 1 X Lysis buffer [Triton X-100 (1%), SDS (0.1%), NaCl (150 mM), Tris-HCl (50 mM) and Halt™ protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA)]. The washed beads were suspended in 2 X sample buffer [(SDS (20%), glycerol (25%), Tris-HCl (0.5 M), bromophenol blue (0.5%) and β-mercaptoethanol (5%)] and boiled for 5 min. The pulled-down protein complexes and the original lysate sample were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were first incubated in a fixing solution (10% acetic acid and 30% methanol), then in Autofluor (National Diagnostics, Atlanta, GA, USA) and finally in anti-cracking buffer (7% acetic acid, 7% methanol and 1% glycerol). The gels were dried onto 3 mm chromatography paper (Whatman, Cleves, OH, USA) and autoradiographed.
2.8. Mammalian Cell Co-Immunoprecipitation
A549 cells were seeded in a 10-cm plate and transiently transfected with the p3xFlag-CMV-OAS1-p44 construct. At 48 h after transfection, cell lysates were harvested in 1 X Lysis buffer [Triton X-100 (1%), SDS (0.1%), NaCl (150 mM), Tris-HCl (50 mM) and Halt™ protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA)]. The cell lysates were divided into three portions. One portion was stored at −20 °C and used as a lysate sample, one portion was incubated with control agarose beads conjugated with IgG antibody, and one portion was incubated with agarose beads conjugated with anti-Flag antibody. After an overnight incubation at 4 °C with rotation, the beads were washed with 1 X Lysis buffer and suspended in 2 X Sample buffer before denaturing at 95 °C for 5 min. The immunoprecipitated proteins were separated by 8% SDS-PAGE, transferred to a nitrocellulose membrane and blocked with 5% non-fat dry milk at room temperature for 1 h. The blocked membrane was then cut into strips and the strips were incubated with either anti-SVIL (Sigma-Aldrich, St. Louis, MO, USA, 1:1000 dilution) or anti-Flag (Sigma-Aldrich, St. Louis, MO, USA, 1:1000 dilution) antibody overnight at 4 °C. After washing with 1 X Tris-buffered saline containing 0.1% Tween 20, the membrane was incubated with an anti-mouse or anti-rabbit secondary antibody (Cell signaling, Danvers, MA, USA, 1:2000 dilution) for 1 h at room temperature, followed by washing and development with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA).
2.9. Western Blot Assay
hOAS1 proteins expressed in bacteria or in mammalian cells were separated on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane at 100 V for 1 h. The membrane was incubated in blocking buffer (1 X Tris-buffered saline containing 5% non-fat dry milk and 0.1% Tween 20) at room temperature for 1 h and then cut into strips. To detect the hOAS1 isoform proteins expressed in bacteria, the membrane strips were incubated with anti-V5 (Sigma-Aldrich, St. Louis, MO, USA; 1:1000) antibody at 4 °C overnight. To detect overexpressed hOAS1 isoforms in HEK293 cell lysates, the membrane strips were incubated at 4 °C overnight with mouse anti-Flag antibody (Sigma-Aldrich, St. Louis, MO, USA; 1:1000) or mouse anti-β actin antibody (Cell signaling, Danvers, MA, USA; 1: 10,000). The membranes were washed three times for 10 min with 1 X Tris-buffered saline containing 0.1% Tween 20, and then incubated with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. After washing, the membrane strips were processed for enhanced chemiluminescence using a Super-Signal West Pico detection kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.
The OAS/RNase L system is an important component of the IFN-dependent antiviral response in cells [27
]. The human genome encodes a single OAS1 gene, but multiple isoform proteins are produced due to alternative splicing, as well as to single nucleotide variations that affect splicing [6
]. The hOAS1 isoforms differ in their C-terminal sequences. The crystal structures of the porcine OAS1 either alone or bound to dsRNA and of hOAS1 p46 indicate that the N- and C-terminal regions of these proteins interact [20
]. The presence of alternative C-terminal ends in the different isoform proteins could affect protein folding, protein stability, synthetase activity, protein post-translational modification and/or protein intracellular localization.
When activated by dsRNA, OAS proteins produce 2-5A that induces dimerization and activation of host RNase L, that in turn cleaves both viral and cellular ssRNAs. In the present study, we showed that each of the seven bacterially-expressed hOAS1 isoform proteins tested could synthesize trimers and some higher order 2-5As in vitro, including p41 and p49. We also showed that poly(I:C) stimulation of hOAS1 p42, p44, p46, p48 or p52 expressed in HEK293 cells activated RNase L cleavage of cellular 18S/28S rRNA, indicating that each of these isoforms can be activated by poly(I:C) in mammalian cells. The data indicate that the alternate C-terminal sequences do not negatively affect 2-5A synthetase activity.
Antiviral activity has been previously reported to be associated with the expression of specific hOAS1 isoforms in cells. An A-allele in the intron-5/exon-6 splice acceptor site that leads to the expression of p48 and p52, but not p46, was reported to correlate with increased risk of WNV infections in humans [30
]. Another study reported that Dengue virus replication is blocked by an RNase L-dependent mechanism in human cells overexpressing p42 or p46, but not p44, p48 or p52 [18
]. In the present study, comparison of the expression efficiencies of different hOAS1 isoforms indicated that the p42 and p46 isoforms were expressed at much higher levels in HEK293 cells than the other isoforms tested, suggesting that these two isoforms may have a higher stability in a mammalian cell. A recent study also showed that p44 and p48 were expressed at lower levels compared to p42 and p46 after transfection of HEK 293 cells, even though the mRNA levels for all of the isoforms were similar [24
]. Sequential truncation of the C-terminal ends of p44 and p48 resulted in increased expression levels, indicating that the C-terminal regions are responsible for the decreased stability of these two isoforms.
Surprisingly, the high expression levels of the p42 and p46 isoforms resulted in the activation of RNase L cleavage of 18S/28S rRNA in the absence of poly(I:C) treatment. Interestingly, using less DNA for transfection and a shorter transfection time abrogated the poly(I:C)-independent activation of rRNA cleavage.
Although we found that each of the expressed isoforms was activated by transfection of poly(I:C), it is possible that the higher expression levels of the p42 and p46 isoforms in mammalian cells were the reason for the previous detection of RNase L activation by a Dengue virus infection only in cells overexpressing these two isoforms [18
]. However, a recent study showed that hOAS3 was the primary hOAS protein activated by infection with another flavivirus Zika virus [31
]. The means by which the hOAS1 p42 and p46 isoform proteins were activated to produce 2-5A after expression in HEK293 cells at high levels in the absence of poly(I:C), is not known. The conformational change induced by dsRNA has been shown to be closely linked to the catalytic activity of hOAS proteins [11
]. Although, it was previously reported that in vitro-synthesized p42 and p48 require tetramerization for their activity via a C-F-K motif that is conserved in all OAS1 isoforms [32
], it was subsequently demonstrated that monomeric p42 expressed in insect cells was fully active [11
]. It is possible that concentration-dependent multimerization may have increased the sensitivity of p42 and p46 to endogenous dsRNA, and that this was responsible for the dsRNA-independent rRNA cleavage that we observed.
Although the majority of the predicted post-translational modifications of the C-terminal sequences of the various hOAS1 isoforms (Figure 1
) would not be expected to be added by bacteria, these post translational modifications may differentially affect the stability of the various hOAS1 isoforms in mammalian cells. No post translational modification sites were predicted for the C-terminal region of p42, but multiple post translational modification sites were predicted for the p46 C terminal region, and p46 was previously predicted to associate with mitochondria [33
]. The different C-terminal sequences and post translational modifications of the individual hOAS1 isoforms may mediate hOAS1 isoform interactions with unique protein partners that facilitate novel functions and/or determine intracellular localization. Previous studies have identified novel binding partners for some of the hOAS1 isoform proteins. The intracellular domain of the prolactin receptor was identified as a binding partner of the p42 isoform, and this interaction was suggested to modulate prolactin-induced STAT1 binding at gene promoters regulated by interferon-regulatory factor 1 [26
]. The p48 isoform was predicted to contain a BH3 domain and to mediate cellular apoptosis by interacting with Bcl-2 and Bcl-XL
]. The hOAS1 p46 isoform was shown to associate with mitochondria [33
]. The identification of these novel interactions suggested that individual hOAS1 isoforms are likely to have additional functions beyond a role in the canonical OAS-RNase L pathway. It is possible that the isoform proteins that are present in low levels could still be functionally important.
In the present study, SVIL was identified as a binding partner for the hOAS1 p44 isoform, and FBN1 was identified as a putative binding partner for the hOAS1 p42 isoform. However, the prolactin receptor was not reidentified as a p42 binding partner in our yeast two hybrid screen. Additional IP assays with an anti-FBN1 antibody are needed to further test the interaction of p42 with endogenous FBN1. FBN1 is an extracellular matrix glycoprotein that can downregulate the transforming growth factor beta (TGF-β) signaling pathway [34
]. Previous publications have reported OAS activity in the serum, and showed that extracellular OAS1 could enter cells and exert an antiviral activity independent of RNase L [37
]. It is possible that FBN1 could function as a scaffold protein to tether extracellular OAS1 proteins on the cell surface. RNase L was recently reported to bind to actin-binding protein filamin A, and to function as an innate immune component maintaining a cellular barrier to viral entry [40
]. SVIL, the partner identified for p44, is an actin binding protein from the Villin family. It is possible that interaction between the hOAS1 p44 isoform and actin-bound SVIL could bring this isoform in close proximity to RNase L at the cytoskeleton barrier. The C-terminal headpiece domain of SVIL, which is located at the C-terminus of the identified peptide that interacts with p44, is conserved among many members in the Villin family [41
], suggesting the possibility that the hOAS1 p44 isoform might also interact with other members of this family.
In summary we show that the alternative C-terminal sequences of the hOAS1 isoforms have little effect on activation by poly(I:C) or on the ability to synthesize 2-5A. The identification of novel binding partners for some of the hOAS1 isoforms in this and previous studies suggests that the different C-terminal sequences of the different isoform proteins may mediate differential protein–protein interactions that are required for cellular localization and possible alternative functions of the different isoform proteins.