Mayaro Virus Non-Structural Protein 2 Circumvents the Induction of Interferon in Part by Depleting Host Transcription Initiation Factor IIE Subunit 2

Mayaro virus (MAYV) is an emerging mosquito-transmitted virus that belongs to the genus Alphavirus within the family Togaviridae. Humans infected with MAYV often develop chronic and debilitating arthralgia and myalgia. The virus is primarily maintained via a sylvatic cycle, but it has the potential to adapt to urban settings, which could lead to large outbreaks. The interferon (IFN) system is a critical antiviral response that limits replication and pathogenesis of many different RNA viruses, including alphaviruses. Here, we investigated how MAYV infection affects the induction phase of the IFN response. Production of type I and III IFNs was efficiently suppressed during MAYV infection, and mapping revealed that expression of the viral non-structural protein 2 (nsP2) was sufficient for this process. Interactome analysis showed that nsP2 interacts with DNA-directed RNA polymerase II subunit A (Rpb1) and transcription initiation factor IIE subunit 2 (TFIIE2), which are host proteins required for RNA polymerase II-mediated transcription. Levels of these host proteins were reduced by nsP2 expression and during infection by MAYV and related alphaviruses, suggesting that nsP2-mediated inhibition of host cell transcription is an important aspect of how some alphaviruses block IFN induction. The findings from this study may prove useful in design of vaccines and antivirals, which are currently not available for protection against MAYV and infection by other alphaviruses.


Introduction
Mosquito-transmitted viruses within the genus Alphavirus infect hundreds of thousands of people and animals each year, causing enormous morbidity and mortality [1,2]. Mayaro virus (MAYV) is an emerging alphavirus that is currently circulating in South America and the Caribbean and is maintained primarily via the sylvatic cycle [3]. Acute MAYV infection manifests with flu-like symptoms, but a significant proportion of patients develop chronic arthritis that can persist for years [4]. MAYV belongs to the Semliki Forest virus complex, which comprises other arthritogenic alphaviruses [5] including the chikungunya virus (CHIKV), which because it can spread via an urban cycle, has caused multiple large outbreaks across the globe [6,7]. There are justified concerns regarding large MAYV outbreaks because the virus can be transmitted by Aedes sp. and Anopheles sp. of mosquitoes, which are prevalent in urban settings across a wide geographical range [8][9][10]. Imported cases of MAYV infection have already been reported in Europe and North America and are expected to increase in the future [11][12][13]. While therapeutics and vaccine candidates against MAYV are being investigated [14][15][16][17][18], currently there are no specific antiviral measures that could be used in the case of a MAYV outbreak. As such, further understanding of how this virus affects vital host cell pathways may facilitate development of effective antivirals.
The interferon (IFN) response is a critical first line of defense against viral infections [19][20][21]. During the induction phase, viral RNAs are detected by cytoplasmic RNA helicases such as RIG-I (retinoic acid-inducible gene) and MDA5 (melanoma differentiationassociated protein 5), leading to activation of MAVS (mitochondrial antiviral signaling protein) followed by recruitment of TBK1 (TANK-binding kinase 1)/IKKε (IκB kinase ε) or IKKα/IKKβ. These kinases then, respectively, activate the transcription factors IRF3 or NFκB, leading to induction of IFNs [22]. In the signaling phase of the IFN response, secreted IFNs bind to cognate receptors on the cell surface, which then activate JAK (Janus kinase)-STAT (signal transducer and activator of transcription factor) signaling and ultimately expression of interferon-stimulated genes (ISGs). ISG expression creates an anti-viral state, as evidenced by the fact that replication of alphaviruses is dramatically inhibited in cells pre-treated with IFNs [23][24][25]. The fact that many viruses have evolved strategies to inhibit both IFN induction and signaling further illustrates the importance of this antiviral response [26].
In this study, we investigated how MAYV infection affects the IFN induction pathway. Similar to what has been reported for other alphaviruses [28,29,38], MAYV infection efficiently suppressed induction and secretion of IFN. Mapping studies indicated that nsP2 of MAYV blocks a step in the IFN induction pathway that is downstream of IRF3 phosphorylation. Interactome analysis revealed that nsP2 interacts with and causes depletion of the host protein TFIIE2, which is required for pol II-mediated transcription [39]. Loss of TFIIE2 was observed in cells infected with other alphaviruses within the Semliki Forest complex, suggesting that targeting this host protein is a common strategy used by these viruses to inhibit the IFN response.

Plasmids and Transfection
MAYV protein expression plasmids were generated from cDNA templates prepared by reverse transcription and polymerase chain reaction (RT-PCR) using RNA isolated from MAYV infected Vero cells. Viral gene-specific cDNAs including C-terminal 3×FLAG tag cassettes were cloned between NheI and FseI restriction sites in pcDNA 3.1(−) 3×FLAG plasmid [40]. Due to the auto-protease activity of capsid protein, which cleaved off the C-terminal 3×FLAG tag, a 3×FLAG sequence was added in frame to the N-terminal region of the protein. The primers used for the cloning are listed in Table S1. The sequence integrity of all constructs was confirmed by Sanger sequencing.
For indirect immunofluorescence analysis, transfection of the appropriate expression plasmids into A549 cells was performed using TransIT-LT1 from Mirus Bio (Madison, WI, USA). For luciferase reporter assays in A549 or HEK 293T cells, plasmid transfection was performed using Lipofectamine 2000 from Invitrogen (Waltham, MA, USA).

Immunoblotting
HEK293T cells (5.0 × 10 5 ) or A549 cells (1.5 × 10 5 ) seeded into 12-well plates were infected the next day with MAYV, SFV, or SINV, or transfected with protein expression plasmids. At experimental endpoints, cells were washed twice with phosphate-buffered saline (PBS) and lysed using 2× SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 50% (v/v) glycerol, 2% (w/v) SDS, 0.01% (w/v) Bromophenol blue) with 100 mM dithiothreitol (DTT). Cell lysates were incubated at 98 • C for 10 min to ensure denaturation of proteins. Samples were separated by molecular weight using SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated in blocking buffer (PBS with 5% bovine serum albumin (BSA; Sigma Aldrich) and 0.05% Tween 20) for 30 min, after which they were incubated with primary antibodies diluted in blocking buffer for 60 min. Following three 10 min washes with PBS-0.05% Tween 20, the membranes were incubated with secondary antibodies, diluted in blocking buffer for 60 min, and protected from the light. The membranes were then subjected to three washes with PBS-0.05% Tween 20 and once with PBS. Protein bands were imaged using an Odyssey Infrared Imaging system. Quantification of proteins was performed using Odyssey Image Studio Lite Software Version 5.2.

Immunoprecipitation
HEK293T cells (3.6 × 10 5 ) in T25 were transfected with 6 µg of indicated plasmids using Lipofectamine 2000 (Life Technologies; Carlsbad, CA, USA), according to the manufacturer's protocol. Twenty-four hours later, cells were harvested and resuspended in ice-cold IP buffer (150 mM NaCl, 50 mM Tris (pH 7.5), 1% Triton X-100, 1 mM NaF, 1 mM DTT, and protease inhibitor cocktail (Roche; Basel, Switzerland)). After 30 min, lysates were clarified by centrifugation at 16,000× g for 15 min and then incubated with 10 µL of anti-FLAG M2 magnetic beads (Millipore; Burlington, MA, USA) at 4 • C for 2 h. After washes with IP buffer, bound proteins were analyzed by mass spectrometry (LC-MS/MS) or SDS sample buffer was added to the beads, which were then incubated 98 • C for 10 min to denature proteins, followed by SDS-PAGE.

Mass Spectrometry and Mass Spectrometry Interaction Statistics (MiST)
Trypsin digestions of proteins immunoprecipitated using anti-FLAG magnetic resins were performed on a KingFisher Duo Prime (Thermo Scientific; Waltham, MA, USA). Briefly, the samples were reduced (10 mM DTT in 50 mM ammonium bicarbonate) and alkylated (50 mM iodoacetamide in 50 mM ammonium bicarbonate) before digestion for 5 h at 37 • C using sequencing grade modified trypsin (Promega; Madison, WI, USA). The tryptic peptides were separated using an Easy-nLC 1000 liquid chromatograph (Thermo Scientific) and an EASY-Spray capillary column (ES900, Thermo Scientific). Mass spectrometry was performed on a Q Exactive Orbitrap mass spectrometer (Thermo Scientific). The mass spectrometer was operated in data-dependent acquisition mode with a resolution of 35,000 and m/z range of 300-1700. The 12 most intense multiply charged ions were sequentially fragmented by HCD dissociation, and spectra of their fragments were recorded in the orbitrap at a resolution of 17,500, followed by a 30 s dynamic exclusion. Data were processed using Proteome Discoverer 1.4 (Thermo Scientific), and databases were searched using SEQUEST (Thermo Scientific). Search parameters included a strict false discovery rate (FDR) of 0.01, a relaxed FDR of 0.05, a precursor mass tolerance of 10 ppm, and a fragment mass tolerance of 0.01 Da. Peptides were searched with carbamidomethyl cysteine as a static modification and oxidized methionine, and deamidated glutamine and asparagine as dynamic modifications.
The results from mass spectrometry were analyzed for protein-protein interaction scoring with MiST (mass spectrometry interaction statistics) pipeline to generate the interactome map of each viral protein. Protein names, functions, and putative cellular localizations were extracted from the Uniprot database [41].

Biological Process Enrichment Analysis
Biological process enrichment analysis and generation of resulting heatmaps were performed using Metascape (http://metascape.org, accessed on 30 June 2021]) [42]. Host proteins found to interact with MAYV nsP2 with a MiST score of ≥0.80 were analyzed. The ontology sources GO biological processes, GO cellular components, KEGG structural complexes, and CORUM were used for the enrichment analysis. Metascape analyses generated accumulative hypergeometric p-values, q-values using the Benjamini-Hochberg procedure, and enrichment factors for filtering of the gene list. The following thresholds were used: p-value < 0.01, enrichment factor > 1.5, and a minimum overlap set at a count of 3. Statistically significant enriched terms were then hierarchically clustered using Kappa similarity scoring; a Kappa score > 0.3 was used to define clusters. Within each cluster, the most significantly enriched term was chosen and displayed in a heatmap.

Confocal Immunofluorescence (IF) Microscopy
A549 cells on coverslips were fixed with 4% electron microscopy-grade paraformaldehyde (Electron Microscopy Sciences; Hatfield, PA, USA) in PBS for 10 min at room temperature and permeabilized with 0.5% Triton X-100 in PBS for 10 min. Coverslips were then washed three times with PBS, after which they were incubated in blocking buffer for 30 min at room temperature. Coverslips were then incubated with primary antibodies diluted in blocking buffer for 45 min at room temperature. Coverslips were washed three times with PBS, and then they were incubated with secondary antibodies (1:1000; Invitrogen) and DAPI (4 ,6-diamidino-2-phenylindole; 1 µg/mL; Sigma Aldrich) in blocking buffer for 45 min at room temperature. Samples were then washed three times with PBS and once with de-ionized water; then, they were mounted onto microscope slides with Prolong Gold anti-fade mounting reagent (Life Technologies). Samples were imaged using an Olympus IX-81 spinning-disk confocal microscope equipped with a 60× PlanApo N oil objective. Images were analyzed using Volocity 6.2.1 software (PerkinElmer; Waltham, MA, USA).

Quantitative Real Time PCR (qRT-PCR)
Intracellular RNA was extracted from cells using the RNA NucleoSpin Kit (Macherey Nagel; Duren, Germany) and reverse transcribed into cDNAs by incubating the isolated RNA with random primers (Invitrogen) and Improm-II reverse transcriptase (Promega) at 42 • C for 1.5 h. The cDNAs were diluted 1:5 with pure water, and 5% of the volume was mixed with the appropriate primers (Integrated DNA Technologies; Coralville, IA, USA) and the PerfecTa SYBR green SuperMix with Low ROX (Quanta Biosciences; Gaithersburg, MD, USA) and amplified for 40 cycles (30 s at 94 • C, 40 s at 55 • C, and 20 s at 68 • C) in a Biorad CFX96 qRT-PCR machine. The target genes and primer sequences used are listed in Table S2. The CT values were normalized with Actb mRNA as the internal control or by cell count (∆CT). The ∆CT values were further normalized to ∆CT values of control samples (∆∆CT). The relative mRNA levels were calculated using the formulas 2 (−∆∆CT) .

Enzyme-Linked Immunosorbent Assay (ELISA)
Levels of human IFN-β in the cell culture supernatant were measured using Quantikine ® Human IFN-β Immunoassay kit (R&D Systems, Inc.; Minneapolis, MN, USA), as per the manufacturer's instructions. The total fluorescence was measured using a Synergy HTX plate reader (Biotek).

Statistical Analyses
All statistical analyses were performed using Microsoft Excel. Two-tailed paired Student's t-test was performed for pair-wise statistical comparison. One-way ANOVA followed by two-tailed paired Student's t-test was used for comparison of multiple samples. The individual data points from each independent experiment and mean ± standard error of the mean are shown in the bar and line graphs.

MAYV Suppressed Production of Type I and III IFNs Downstream of IRF3 Nuclear Translocation
To determine if/how IFN induction is affected during MAYV infection, we quantitated type I IFN (Ifnb) and viral genomic RNAs by qRT-PCR at 4, 8, 12, and 24 h after A549 cells were infected with MAYV or Sendai virus (SeV). Despite robust virus replication, induction of Ifnb mRNA in MAYV-infected cells was delayed and dramatically suppressed compared to cells infected with SeV ( Figure 1A,B), a potent inducer of IFN production [43]. However, treatment of cells with type I IFN prior to infection significantly inhibited MAYV in a dose-dependent manner ( Figure 1C), indicating that the virus is sensitive to IFN.
Next, we assessed whether the relatively low levels of Ifnb mRNA in MAYV-infected cells were due to active suppression of IFN induction pathways. MAYV-infected cells were transfected with poly(I:C), a dsRNA analog that induces IFN following detection by RIG-I-like receptors [44]. Transcripts encoding type I (Ifnb) and type III (Ifnl2) IFNs were then quantified by qRT-PCR. Poly(I:C) transfection robustly induced Ifnb and Ifnl2, but levels of these transcripts were ≈50-fold lower in MAYV-infected cells compared to mock-infected cells ( Figure 1D,E). MAYV infection alone resulted in a relatively modest increase (≈100-fold) in Ifnb and Ifnl2, which were further increased ≈10-fold by poly(I:C) challenge. In comparison, SeV infection resulted in ≈10,000-fold increased levels of Ifnb, but this effect was dampened in cells that were first infected with MAYV ( Figure 1F). IFN-β was not detected in the media of MAYV-infected cells, regardless of whether they were challenged with SeV or not ( Figure 1G). The observation that levels of SeV genomic RNA were higher in MAYV-infected cells suggests that impaired IFN induction during MAYV infection was not due to poor replication of SeV in those cells ( Figure 1H).
Next, we examined whether nuclear translocation of the antiviral transcription factor IRF3, which is required for production of IFN-β mRNA, was affected by MAYV infection. In contrast to SeV infection, which induced translocation of IRF3 into nuclei, IRF3 remained in the cytoplasm of MAYV-infected cells ( Figure 1I). However, when MAYV-infected cells were subsequently infected with SeV, IRF3 localized to the nucleus ( Figure 1I). This indicates that the IFN induction pathway is intact in MAYV infected cells up to and including the IRF3 nuclear translocation step. Taken together, these data are compatible with a scenario where MAYV efficiently avoids detection by RIG-I-like receptors, and/or partially blocks a step upstream of IRF3 nuclear transport to prevent IFN production. Because SeV-induced IRF3 nuclear translocation is unaffected in MAYV-infected cells ( Figure 1I), but production of IFN mRNA and secretion of the cytokines are blocked ( Figure 1D-G), the virus likely targets additional steps in the IFN induction pathway downstream of IRF3 nuclear translocation.

MAYV nsP2 Abrogated Interferon Induction Downstream of IRF3 Activation
To identify the viral protein(s) responsible for suppression of the IFN induction pathway during MAYV infection, we generated expression plasmids encoding individual epitope-tagged MAYV proteins. With the exception of the capsid protein, which was tagged on the N-terminus, all MAYV proteins were constructed with a 3×FLAG epitope at their C-termini. It was not feasible to tag the C-terminus of MAYV capsid because, like other alphavirus capsid proteins, it has auto-protease activity that cleaves the hydrophobic signal peptide at its C-terminus [45]. Expression of the tagged viral proteins in transfected HEK293T cells was authenticated by immunoblotting ( Figure 2A). and Ifnb mRNA (B) were quantitated by qRT-PCR and expressed as fold mock infected cells normalized to Actb mRNA levels. Data shown are the mean ± SEM from three independent experiments. (C) A549 cells were treated with IFN-α (0, 10, 100, and 100 U/mL) for 6 h and infected with MAYV (MOI = 1). At 24 hpi, relative levels of MAYV RNA in total cellular RNA samples were quantitated by qRT-PCR (normalized to Actb) and expressed as folds of replication to cells not treated with IFN. Data represent the mean ± SEM from three independent experiments and were analyzed by Student's t-test. *** p < 0.001, **** p < 0.0001. (D,E) A549 cells were infected with MAYV (MOI = 3) for 24 h, then treated with 2 µ g/mL of poly(I:C). After 16 h, total RNA was collected, and Ifnb (D) and Ifnl2 (E) mRNA were measured by qRT-PCR and normalized to Actb mRNA level and expressed as folds to mock infected cells. Data represent the mean ± SEM from three independent experiments and were analyzed by one-way ANOVA and Student's t-test. ** p < 0.01, *** p < 0.001, **** p < 0.0001. (F,G) A549 cells were infected with MAYV (MOI = 3) for 24 h, then challenged with (SeV) (50 HAU/mL) for 16 h. Ifnb transcripts in cells and IFN-β in cell culture supernatants was measured by qRT-PCR (F) and enzyme-linked immunosorbent assay (G), respectively. Data represent the mean ± SEM from three independent experiments and were analyzed by one-way ANOVA and Student's t-test. *** p < 0.001, **** p < 0.  Next, the effect of MAYV protein expression on IFN induction in response to SeV infection was evaluated using an IFN-β-promoter-based luciferase reporter assay. The only MAYV protein whose expression significantly blocked IFN-β promoter activity was nsP2 ( Figure 2B). Despite its relatively low expression compared to the other FLAG-tagged MAYV proteins ( Figure 1A), nsP2 suppressed IFN-β promoter activity almost as efficiently as the NS1 protein of influenza A virus, a known suppressor of the IFN induction [46] and the positive control for these experiments. Because activation of IRF3 and NFκB is critical for induction of Ifnb [21], we assessed whether nsP2 abrogates the activities of these two transcription factors using IRF3 and NFκB promoter-based luciferase reporters. Compared to the vector control and MAYV capsid protein, nsP2 reduced IRF3-and NFκB-dependent luciferase expression by as much as 10-fold ( Figure 2C,D). To determine the step in the IFN induction pathway targeted by nsP2, we assessed how overexpressing individual components in the pathway (RIG-I (2xCARD), TBK1, IKKε, IRF3, and constitutively active IRF3 (IRF3-5D)) affected nsP2-dependent inhibition. None of these components rescued the nsP2 blockade of the IFN induction pathway ( Figure 2E), suggesting that this viral protein acts downstream of the IRF3 phosphorylation step. Interestingly, IRF3 nuclear translocation in response to SeV infection was not affected by nsP2 expression (Figure 2F,G). These data are consistent with those shown in Figure 1 in which MAYV infection was able to block IFN induction downstream of IRF3 nuclear translocation.

The Effect of nsP2 on Interferon Induction Was Found to Be Partially Mediated by Transcriptional Shutoff
Previous studies have shown that other alphavirus nsP2 proteins translocate to the nucleus and abrogate host transcription by depleting Rbp1, a component of the RNA polymerase II complex [33,51]. To determine if these processes were important for the ability of MAYV nsP2 to antagonize IFN induction, we used site-directed mutagenesis to generate nuclear localization signal (NLS)-deficient (nsP2 RK653AA ) [36] and transcriptional shutoffdeficient (nsP2 P722S ) [51] mutants of nsP2 ( Figure 3A). Confocal microscopy analyses confirmed that nsP2 RK653AA was not able to translocate into the nucleus, whereas wildtype and nsP2 P722S were detected in nuclei and the cytoplasm ( Figure 3B). However, while both nsP2 RK653AA and nsP2 P722S significantly blocked IFN induction, nsP2 P722S was around fourfold less effective than wildtype nsP2 ( Figure 3C). Furthermore, in cells over-expressing a constitutively active form of IRF3 (IRF3-5D) and nsP2 RK653AA or nsP2 P722S , levels of IFN transcripts were higher than in cells expressing wild type nsP2 ( Figure 3E). Unlike, wildtype nsP2 and nsP2 RK653AA , however, nsP2 P722S did not reduce IRF3-5D expression in co-transfected cells ( Figure 3F,G). While these data suggested that nsP2 suppresses IFN induction in part by mediating host cell transcriptional shutoff, it also appears to function through a second mechanism that is independent from its nuclear function or ability to abrogate transcription.

nsP2 Interacted with and Downregulated the Levels of Host Transcription Mediators Rpb1 and TFIIE2
To further investigate how MAYV affects the host antiviral response, we used coimmunoprecipitation (co-IP) followed by tandem mass spectrometry (LC-MS/MS) to identify host cell proteins that interact with nsP2. Putative nsP2-binding host proteins were subjected to analyses using Molecular Interaction Search Tool (MiST) (Table S3), and key cellular processes associated with nsP2 interactors were assessed through Gene Ontology (GO) enrichment analysis. Surprisingly, we did not detect any protein directly involved in the IFN induction pathway interacting with nsP2 of MAYV. One of the key enriched GO terms was "RNA polymerase II holoenzyme complex" ( Figure 4A, Table S4), which consists of proteins involved in transcription by RNA polymerase II. Two members of this complex were identified in the nsP2 co-IP: specifically, Rpb1 (also known as POLR2A), a component of RNA polymerase II that was previously shown to interact with other alphavirus nsP2 proteins [33,51], and TFIIE2, an integral factor that functions in transcription initiation [39]. Until this study, TFIIE2 was not known to interact with alphavirus nsP2. Interestingly, the transcriptional shutoff mutant nsP2 P722S formed a stable interaction with Rpb1 but not TFIIE2 ( Figure 4B,C). Confocal microscopy analyses revealed that in cells transfected with nsP2, expression levels of Rpb1 and TFIIE2 were significantly reduced ( Figure 4D-G). The NLS mutant nsP2 RK653AA reduced TFIIE2 but not Rpb1 levels, whereas the transcriptional shutoff mutant nsP2 P722S did not affect levels of either protein ( Figure 4D-G). Accordingly, nuclear localization of nsP2 appears to be important for depleting Rpb1 but not for suppressing TFIIE2 expression.

Some but Not All Alphaviruses Induced Loss of TFIIE2
Quantitative confocal analyses confirmed that MAYV infection resulted in significant loss of Rpb1 and TFIIE2 proteins at 24 hpi ( Figure 5A-D). To elucidate how this occurs, we treated MAYV-infected cells with inhibitors of proteasomal-(epoxomicin) and lysosomal-(bafilomycin A1) dependent degradation followed by immunoblot analyses. Loss of Rpb1 protein during MAYV infection was significantly inhibited by epoxomicin ( Figure 5E,F), which is consistent with previous findings that old world alphavirus nsP2-dependent depletion of Rpb1 involves the proteasome [51]. Conversely, neither epoxomicin nor bafilomycin blocked degradation of TFIIE2 protein during infection with MAYV ( Figure 5E-G). Next, we examined the levels of Rpb1 and TFIIE2 proteins in SFV-and Sindbis virus (SINV)infected cells. Similar to what was observed during MAYV infection, Rpb1 and TFIIE2 protein levels were both reduced at 24 hpi in SFV-infected cells ( Figure 5H-J). However, in cells infected with SINV, Rpb1 but not TFIIE2 levels were lower ( Figure 5H-J).

Figure 3. Inhibition of interferon induction by nsP2 was found to be partially mediated by transcriptional shutoff. (A)
Schematic of 3×FLAG-tagged wildtype nsP2, NLS-deficient nsP2 RK653AA , and host transcription shutoff deficient nsP2 P722S . (B) A549 cells were transfected with indicated MAYV nsP2 constructs. After 24 h, cells were fixed and stained using α-FLAG antibody and imaged visualized with a confocal microscope using 60× oil objective. Nuclei are stained with DAPI. Scale bar = 12 µ m. (C) HEK293T cells were transfected with the indicated viral nsP2 constructs, IFN-β Firefly luciferase reporter, and a control Renilla reporter. After 24 h, cells were infected with 50 HAU/mL of SeV for 16 h, after which relative Firefly and Renilla luciferase activities were measured. Data represent the mean ± SEM from three independent experiments and were analyzed by one-way ANOVA and Student's t-test. ** p < 0.01, *** p < 0.001, **** p < 0.0001. (D-F HEK293T cells were transfected with indicated viral nsP2 constructs, FLAG-IRF3-5D, IRF3-promotor Firefly luciferase reporter, and a control Renilla reporter. After 24 h, Firefly and Renilla luciferase activities were measured (D). Cell whole lysate were also analyzed by immunoblotting using antibodies against FLAG and actin (E,F). Data represent the mean ± SEM from three independent experiments and were analyzed by one-way ANOVA and Student's t-test. * p < 0.05, ** p < 0.01, **** p < 0.0001, NS = not significant.

nsP2 Interacted with and Downregulated the Levels of Host Transcription Mediators Rpb1 and TFIIE2
To further investigate how MAYV affects the host antiviral response, we used coimmunoprecipitation (co-IP) followed by tandem mass spectrometry (LC-MS/MS) to identify host cell proteins that interact with nsP2. Putative nsP2-binding host proteins were subjected to analyses using Molecular Interaction Search Tool (MiST) (Table S3), and key cellular processes associated with nsP2 interactors were assessed through Gene On- . Cell whole lysate were also analyzed by immunoblotting using antibodies against FLAG and actin (E,F). Data represent the mean ± SEM from three independent experiments and were analyzed by one-way ANOVA and Student's t-test. * p < 0.05, ** p < 0.01, **** p < 0.0001, NS = not significant. (A) Heatmap of GO term enrichment analysis. HEK293T cells were transfected with pcDNA empty vector or nsP2 for 24 h, after which whole cell lysates were immunoprecipitated using magnetic beads coated with anti-FLAG. nsP2-interacting proteins were identified by liquid chromatography-tandem mass spectrometry, and the protein-protein interactions from three independent experiments were scored by MiST analysis. nsP2-interacting host proteins with MiST score of ≥0.8 were selected to perform the GO enrichment analysis using the Metascape software. (B,C) HEK293T cells were transfected with the indicated viral protein constructs, and 24 h later, whole cell lysates were immunoprecipitated with magnetic beads coated with anti-FLAG and then subjected to immunoblot analysis using antibodies against FLAG, Rpb1, and actin (B), or FLAG, TFIIE2, and actin (C). Representative blots from three independent experiments are shown. (D,E) WT MAYV nsP2 downregulated Rpb1. A549 cells were transfected with nsP2 WT or nsP2 RK653AA (NLS-deficient), or nsP2 P722S (host transcription shutoff deficient) constructs. After 24 h, cells were fixed and stained using α-FLAG and α-Rpb1 antibodies and imaged using a confocal microscope using 60× oil objective. The total Rpb1 fluorescent intensities were quantified using Volocity software. Scale bar = 12 µm. Data represent the mean ± SEM from three independent experiments (n = 11) and were analyzed by one-way ANOVA and Student's t-test. ** p < 0.01, NS = not significant. (F,G) A549 cells were transfected with indicated nsP2 constructs. After 24 h, cells were fixed, stained using antibodies to FLAG and TFIIE2, and visualized with a confocal microscope using 60× oil objective. The total TFIIE2 fluorescent intensities were quantified using Volocity software. Scale bar = 12 µm. Data represent the mean ± SEM from three independent experiments (n = 11) and were analyzed by one-way ANOVA and Student's t-test. ** p < 0.01, NS = not significant. , and then images were acquired using confocal microscope equipped with 60× oil lens. The total Rpb1 and TFIIE2 fluorescent intensities were quantified using Volocity software. Scale bar = 12 µ m. Data represent the mean ± SEM from three independent experiments (n = 20) and were analyzed by Student's t-test. **** p < 0.0001. (E-G) A549 cells were infected with MAYV (MOI = 3) for 8 h, then treated with epoxomicin or bafilomycin (100 µ M) for 24 h. Cell lysates were then subjected to immunoblotting using antibodies to Rpb1, TFIIE2, actin, CHIKV-capsid, and SFV-capsid proteins. The intensities of the protein bands were quantified using Image Studio software and then normalized to actin levels and expressed as folds of mock infected control. Data represent the mean ± SEM from three independent experiments and were analyzed by one-way ANOVA and Student's t-test. * p < 0.05, NS = not significant. (H-J) A549 cells were infected with either MAYV, SFV, or SINV (MOI = 1) for 24 h, after which cell lysates were processed for immunoblotting using antibodies against Rpb1, TFIIE2, actin, CHIKV-capsid, and SFV-capsid proteins. The intensities of the protein bands were quantified using Image Studio software and then normalized to actin levels and expressed as folds of mock infected control. Data presented represent the mean ± SEM from three independent experiments and were analyzed by Student's t-test. * p < 0.05, ** p < 0.01, *** p < 0.001. Cell lysates were then subjected to immunoblotting using antibodies to Rpb1, TFIIE2, actin, CHIKV-capsid, and SFV-capsid proteins. The intensities of the protein bands were quantified using Image Studio software and then normalized to actin levels and expressed as folds of mock infected control. Data represent the mean ± SEM from three independent experiments and were analyzed by one-way ANOVA and Student's t-test. * p < 0.05, NS = not significant. (H-J) A549 cells were infected with either MAYV, SFV, or SINV (MOI = 1) for 24 h, after which cell lysates were processed for immunoblotting using antibodies against Rpb1, TFIIE2, actin, CHIKV-capsid, and SFV-capsid proteins. The intensities of the protein bands were quantified using Image Studio software and then normalized to actin levels and expressed as folds of mock infected control. Data presented represent the mean ± SEM from three independent experiments and were analyzed by Student's t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.
Finally, as a first step toward understanding how MAYV infection affects host cell transcription, we assessed global levels of RNA as well as transcripts of housekeeping genes following infection. As expected, total cellular RNA was greatly reduced at 48 hpi ( Figure 6A), while housekeeping gene transcripts were lower at 24 hpi ( Figure 6B-D). One possibility to explain the lack of substantial decrease in total RNA level at 24 hpi may be due to increasing levels of viral RNA during this time period. Indeed, we observed a significant increase in viral RNA from 8 to 24 hpi ( Figure 6E). Together, these results suggested that MAYV infection abrogates IFN induction in part by blocking global transcription through nsP2-mediated depletion of transcription factors TFIIE and Rpb1.

Discussion
The IFN response is critical for controlling alphavirus infections, including the emerging mosquito-transmitted pathogen MAYV [52][53][54][55]. While considerable efforts have been directed at understanding how alphaviruses affect the downstream signaling phase of the IFN response [24,33,37], relatively little is known regarding how production of IFN is dampened during infection. Our analyses revealed that compared to SeV infection, induction of type I IFN (ifnb) transcripts in MAYV-infected cells was dramatically suppressed and significantly delayed. These findings are consistent with earlier studies on other alphaviruses such as SINV, CHIKV, Ross River virus, Venezuelan Equine Encephalitis virus, and Eastern Equine Encephalitis virus [28,38]. Similarly, production of type III IFN transcripts was inhibited, and secretion of IFN-β was completely abrogated by MAYV infection. These suggest that MAYV suppresses induction of transcripts and may further impede translation and/or secretion of IFNs. Impaired IFN secretion from alphavirus-infected epithelial cells has been reported [28][29][30], but interestingly, this does not appear to be the case during infection of monocytes with Venezuelan Equine Encephalitis virus [29]. Accordingly, it will be of interest to study the IFN response during MAYV infection of key target cells, such as synovial fibroblasts, monocytes, and macrophages in future.
Consistent with earlier studies showing that alphavirus nsP2 proteins are involved in antagonizing the IFN response [24,32,33,36,37,56], we observed that nsP2 of MAYV is highly effective at blocking IFN induction. Of note, Bae et al. showed that CHIKV envelope protein also inhibited IFN induction [56], but in our assays, nsP2 was the only MAYV

Discussion
The IFN response is critical for controlling alphavirus infections, including the emerging mosquito-transmitted pathogen MAYV [52][53][54][55]. While considerable efforts have been directed at understanding how alphaviruses affect the downstream signaling phase of the IFN response [24,33,37], relatively little is known regarding how production of IFN is dampened during infection. Our analyses revealed that compared to SeV infection, induction of type I IFN (ifnb) transcripts in MAYV-infected cells was dramatically suppressed and significantly delayed. These findings are consistent with earlier studies on other alphaviruses such as SINV, CHIKV, Ross River virus, Venezuelan Equine Encephalitis virus, and Eastern Equine Encephalitis virus [28,38]. Similarly, production of type III IFN transcripts was inhibited, and secretion of IFN-β was completely abrogated by MAYV infection. These suggest that MAYV suppresses induction of transcripts and may further impede translation and/or secretion of IFNs. Impaired IFN secretion from alphavirusinfected epithelial cells has been reported [28][29][30], but interestingly, this does not appear to be the case during infection of monocytes with Venezuelan Equine Encephalitis virus [29].
Accordingly, it will be of interest to study the IFN response during MAYV infection of key target cells, such as synovial fibroblasts, monocytes, and macrophages in future.
Consistent with earlier studies showing that alphavirus nsP2 proteins are involved in antagonizing the IFN response [24,32,33,36,37,56], we observed that nsP2 of MAYV is highly effective at blocking IFN induction. Of note, Bae et al. showed that CHIKV envelope protein also inhibited IFN induction [56], but in our assays, nsP2 was the only MAYV protein that blocked this process. This may be due to inherent differences between MAYV and CHIKV envelope proteins themselves and/or the fact that our study employed constructs comprised of original viral sequences rather than codon optimized plasmids as used by Bae et al. [56]. Finally, SINV transframe protein, which is produced when a frameshift occurs during translation of 6K protein, has been reported to antagonize the IFN response [57]. However, whether the analogous protein of MAYV or other alphaviruses functions in a similar manner remains to be determined.
Our analysis showed that MAYV nsP2 suppresses IFN induction downstream of IRF3 phosphorylation and nuclear translocation, which may indicate that transcription and/or translation of IFN mRNAs is affected. Transcriptional shutoff by other alphavirus nsP2 proteins is important for circumventing the IFN response, particularly the signaling arm of this pathway [32][33][34][35]. The fact that the transcriptional shutoff defective mutant of MAYV nsP2 (nsP2 P722S ) was less effective at blocking IFN induction is consistent with these previous studies. While a few studies have indicated that alphavirus nsP2 is responsible for hindering host translation during infection [34,58], others suggest that the translational shutoff observed during infection is due to sequestration of translational machinery by viral RNA [59,60]. Nevertheless, since we observed that MAYV completely blocked the translation and or/secretion of IFNs, future studies are required to determine whether nsP2 suppresses translation of IFNs. Alphavirus nsP2 proteins have been shown to abrogate host transcription in part by depleting Rpb1, a component of the RNA polymerase II holoenzyme [33,51]. Interactome analyses showed that MAYV nsP2 binds Rbp1 and TFIIE2, host proteins that are important for initiation of RNA polymerase II-dependent transcription [39]. Loss of TFIIE2 was observed in MAYV-infected cells as well as cells expressing nsP2 alone. Interestingly, while cells infected with SFV also had lower levels of this protein, SINV infection had no such effect on TFIIE2. Reducing levels of TFIIE2 would be expected to reduce cellular transcription during MAYV infection, but it is unclear as to how this occurs, as neither inhibition of proteasome-nor lysosome-dependent degradation blocked virus-induced turnover of this host protein. Conversely, we observed that nsP2-dependent loss of Rpb1 is proteasomedependent, similar to what has been reported for SFV and SINV infection [33,51]. If TFIIE2 degradation is important for blocking IFN induction during MAYV, it is unlikely that the protease activity of nsP2 is required for this process since the protease-dead nsP2 C478A mutant suppressed IFN induction just as well as wildtype nsP2. Moreover, TFIIE2 lacks the consensus cleavage sequence that is normally targeted by alphavirus nsP2 [61]. Thus, elucidating the mechanism by which nsP2 depletes TFIIE2 requires further investigations.
The observation that MAYV nsP2 P722S still inhibited IFN production, albeit not as well as wild type nsP2, may indicate that other mechanisms are at play. Interestingly, nsP2 P722S was able to form a stable complex with Rpb1, but not TFIIE2. This could mean that nsP2-TFIIE2 interaction is important for blocking host transcription or IFN induction by other means. Although expression of MAYV nsP2 resulted in depletion of both Rpb1 and TFIIE2, nuclear translocation of nsP2 was only required to reduce levels of Rpb1 but not TFIIE2. Prominent loss of TFIIE2 protein was also observed during SFV infection but not in cells infected with SINV. Additional studies are required to determine the impact of each Rpb1 and TFIIE2 depletion on host transcription and IFN induction.
In summary, the present study has revealed novel mechanisms by which MAYV subverts the innate immune response. Given that this emerging alphavirus has the potential to cause large multicontinental outbreaks similar to CHIKV [8,10,62], it is critical to understand how it manipulates host pathways during infection. Indeed, there is consider-able interest in developing therapeutic and prophylactic therapies against MAYV [14][15][16][17][18]. The findings from the present study may be of use for development and application of therapeutics against MAYV and other alphaviruses.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/cells10123510/s1, Figure S1: Full blot image from Figure 3E, Figure S2: Full blot image from Figure 4B, Figure S3: Full blot image from Figure 4C, Figure S4: Full blot image from Figure 5E, Figure S5: Full blot image from Figure 5H, Table S1: Primers used for cloning viral proteins, Table S2: Primers used for qRT-PCR, Table S3: MiST analysis-based sorting of nsP2-interacting cellular proteins, Table S4: GO enrichment analysis of nsP2-interacting cellular proteins.  Data Availability Statement: All relevant data generated during this study are included in this article or supplementary materials. Raw data and relevant information are available from the corresponding author upon request.