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Exploiting bacterial effector proteins to uncover evolutionarily conserved antiviral host machinery [1]
['Aaron Embry', 'Department Of Microbiology', 'University Of Texas Southwestern Medical Center', 'Dallas', 'Texas', 'United State Of America', 'Nina S. Baggett', 'David B. Heisler', 'Addison White', 'Maarten F. De Jong']
Date: 2024-05
Arboviruses are a diverse group of insect-transmitted pathogens that pose global public health challenges. Identifying evolutionarily conserved host factors that combat arbovirus replication in disparate eukaryotic hosts is important as they may tip the balance between productive and abortive viral replication, and thus determine virus host range. Here, we exploit naturally abortive arbovirus infections that we identified in lepidopteran cells and use bacterial effector proteins to uncover host factors restricting arbovirus replication. Bacterial effectors are proteins secreted by pathogenic bacteria into eukaryotic hosts cells that can inhibit antimicrobial defenses. Since bacteria and viruses can encounter common host defenses, we hypothesized that some bacterial effectors may inhibit host factors that restrict arbovirus replication in lepidopteran cells. Thus, we used bacterial effectors as molecular tools to identify host factors that restrict four distinct arboviruses in lepidopteran cells. By screening 210 effectors encoded by seven different bacterial pathogens, we identify several effectors that individually rescue the replication of all four arboviruses. We show that these effectors encode diverse enzymatic activities that are required to break arbovirus restriction. We further characterize Shigella flexneri-encoded IpaH4 as an E3 ubiquitin ligase that directly ubiquitinates two evolutionarily conserved proteins, SHOC2 and PSMC1, promoting their degradation in insect and human cells. We show that depletion of either SHOC2 or PSMC1 in insect or human cells promotes arbovirus replication, indicating that these are ancient virus restriction factors conserved across invertebrate and vertebrate hosts. Collectively, our study reveals a novel pathogen-guided approach to identify conserved antimicrobial machinery, new effector functions, and conserved roles for SHOC2 and PSMC1 in virus restriction.
Microbial pathogens such as viruses and bacteria encounter diverse host cell responses during infection. While viruses possess antagonists to counter these responses in natural host species, their replication can be restricted in unnatural host cells where their antagonists are ineffective. Bacteria also employ a diverse repertoire of immune evasion proteins known as “effectors” that can inhibit antimicrobial responses found in invertebrate and vertebrate hosts. In this study, we hypothesized that some bacterial effectors may target host immunity proteins that restrict both bacteria and viruses. To test this hypothesis, we screened a bacterial effector library comprising 210 effectors from seven distinct bacterial pathogens for their ability to rescue the replication of four viruses in insect cells that are normally non-permissive to these viruses. Though numerous effectors were identified to rescue the replication of each virus, the uncharacterized IpaH4 protein encoded by the human pathogen Shigella flexneri was able to rescue all four viruses screened. We discovered that IpaH4 enhances arbovirus replication in both restrictive insect and permissive human cells by directly targeting two novel, evolutionarily conserved antiviral host proteins, SHOC2 and PSMC1, for degradation. Our study establishes bacterial effectors as valuable tools for identifying critical antimicrobial machinery employed by eukaryotic hosts.
Funding: This work was supported by grants to DBG from the National Institutes of Health [NIH;
https://www.nih.gov/ ] (1R35GM137978-01 and 1R21AI169558-01A1) and by funding to DBG from the UTSW Endowed Scholars Program. NMA was supported by NIH National Institute of Allergy and Infectious Diseases (R01AI083359), The Welch Foundation [
https://welch1.org/ ] (I-1704) and The Burroughs Welcome Fund [
https://www.bwfund.org/ ] (1011019). This research was also supported with training grant funding from the NIH to AE, NSB, DBH (T32 AI007520). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Here, we further explore the restricted infections of arboviruses in lepidopterans by infecting moth cells with the rhabdovirus, VSV, and the togaviruses: SINV, RRV, and ONNV. We develop a simple, yet innovative approach to uncover evolutionarily conserved antiviral factors through the identification of bacterial effectors that rescue arbovirus replication in LD652 cells. By expressing a library of 210 effector proteins encoded by seven distinct bacterial pathogens, we identify six effectors capable of rescuing all four restricted arboviruses in LD652 cells: SopB, IpgD, HopT1-2, HopAM1, Ceg10, and IpaH4. Using mutagenesis, we demonstrate the importance of diverse enzymatic functions for SopB, IpgD, HopAM1, and IpaH4 in breaking arbovirus restriction. Moreover, crystallography and cell cultures studies reveal Ceg10 to encode a putative cysteine protease function that is required for arbovirus rescue. By focusing on the Shigella flexneri-encoded effector IpaH4, we reveal this novel bacterial E3 ubiquitin ligase to directly target two conserved host proteins, SHOC2 and PSMC1, for degradation in moth and human cells. To our knowledge, roles for these host factors in virus restriction had not been reported in any eukaryotic system. However, we show that depletion of intracellular SHOC2 or PSMC1 levels in moth or human cells promotes arbovirus replication, suggesting they have ancient roles in combating viral infection across diverse eukaryotic host species. Together, our findings demonstrate the utility of using naturally abortive arbovirus infections in lepidopteran cells for the interrogation of arbovirus-host interactions and establish it as a model for identifying conserved host immunity proteins targeted by pathogens.
Bacterial pathogens encode a wide array of IEPs that can manipulate eukaryotic immune responses. Many of these bacterial IEPs are “effector” proteins that are injected into eukaryotic host cells through bacterial secretion systems [ 10 , 15 ]. These effectors can manipulate, usurp, and/or inhibit a variety of cellular processes once inside the host cell cytoplasm including cytoskeletal dynamics, host signaling cascades, and innate immune responses [ 10 , 15 ]. Interestingly, some bacterial effectors inhibit innate immune pathways that are also antagonized by viruses, such as the Type I interferon (IFN) response [ 16 ], suggesting that bacterial and viral pathogens may need to evade common eukaryotic defense mechanisms. Although significant advances have been made towards understanding effector biology, the function of many effectors remains unknown. Understanding bacterial effector function is important because these proteins can be critical drivers of bacterial pathogenesis [ 10 , 15 ]. However, defining the role of individual effectors during infection can be challenging due to functional redundancy among independent effectors encoded by a single bacterial pathogen [ 17 ]. Therefore, experimental strategies to study effector functions outside of bacterial infections may be useful for determining their role during natural infection.
Although arboviruses are well-adapted to replicate in dipteran and mammalian hosts, we have previously shown that several arboviruses, such as VSV and SINV, undergo abortive infections in cells derived from lepidopteran (moth and butterfly) hosts [ 12 , 13 ]. For example, in Lymantria dispar (spongy moth)-derived LD652 cells, VSV and SINV undergo abortive infections post-entry after limited gene expression. However, their replication can be rescued by global inhibition of host transcription or by expression of mammalian poxvirus-encoded IEPs termed “A51R proteins”, suggesting that innate antiviral defenses block VSV and SINV replication in LD652 cells [ 12 ]. However, the host immune responses that are at play during restricted arboviral infections in LD652 cells remain poorly defined. More recently, we have reported the full genomic sequence of L. dispar and the LD652 cell transcriptome [ 14 ], making virus-LD652 cell systems more amenable to uncovering pathogen-host interactions at the molecular level. Our finding that mammalian poxviral IEPs can retain immunosuppressive function in LD652 cells suggests that some pathogen-encoded IEPs target host machinery conserved between insects and mammals. Thus, we were interested in identifying IEPs from other mammalian pathogens that promote arbovirus replication in LD652 cells. Such IEPs might be useful molecular tools in identifying the conserved host immunity factors they target.
While genome-wide CRISPR-Cas9 and RNA interference (RNAi) screening platforms have been used to identify host immunity factors affecting arbovirus replication [ 6 – 8 ], these assays can be difficult, time-consuming, and cost prohibitive to set up, and are not easily applicable to non-model host systems. Moreover, these assays cannot provide insight into the strategies used by pathogens to combat host antiviral factors identified in these screens. Identification of pathogen-encoded immune evasion proteins (IEPs ) targeting host immunity factors is important for several reasons. First, the existence of such IEPs is strong evidence for the physiologic importance of these interactions during the “molecular arms race” between pathogen and host. Second, while some IEPs simply bind/sequester host factors to inhibit their function, others can alter post-translation modifications to modify stability or function [ 9 ]. Thus, IEPs can be used as “tools” to both identify the host immunity factors they target and uncover molecular mechanisms that regulate host factor function. Third, IEPs often drive virulence and thus their characterization can reveal pathogenesis mechanisms [ 10 , 11 ]. It is paramount to develop simplistic, functional assays that can both identify key antiviral factors restricting viral replication and that provide molecular tools to mechanistically dissect the function of such immunity factors.
Arboviruses comprise a diverse group of arthropod-borne viruses that are transmitted by dipteran (fly and mosquito) vectors to animal and human hosts. For example, vesicular stomatitis virus (VSV) is a negative-sense single-stranded (ss)RNA virus belonging to the Rhabdoviridae family that is the leading cause of vesicular disease in livestock in the United States, resulting in costly animal quarantines and trade embargoes [ 1 ]. Of the ~500 arboviruses that have been identified, ~150 are known to cause disease in humans [ 2 ]. Consequently, in 2022, the “Global Arbovirus Initiative” was launched by the World Health Organization to monitor and control arboviral disease [ 3 ]. Notable among arboviruses causing disease in humans are the positive-sense ssRNA viruses belonging to the Togaviridae family. This family includes chikungunya virus, the second-most prevalent arbovirus infecting humans worldwide [ 2 ]. However, the need for biosafety level (BSL)-3 facilities to culture wild-type strains of chikungunya virus poses significant challenges to studying this togavirus. In contrast, other less pathogenic togaviruses [e.g. Ross River virus (RRV), O’nyong’nyong virus (ONNV), Sindbis virus (SINV)], can be cultured under BSL-2 conditions and thus have become important models for understanding togavirus-host interactions [ 4 , 5 ]. However, we still lack vaccines and antiviral drugs to combat most human arbovirus infections, including those caused by togaviruses [ 5 ]. Thus, the identification of immune mechanisms that restrict arbovirus replication may provide additional avenues for the development of effective strategies to combat arboviral disease.
Results
Inhibition of host transcription rescues restrictive arbovirus replication in LD652 cells Previously, we showed that the normally abortive infection of VSV and SINV in LD652 cells can be rescued by treatment of cultures with actinomycin D (ActD), an inhibitor of transcription [12]. ActD globally blocks transcription by host DNA-dependent RNA polymerases by intercalating into GC-rich regions of cellular DNA and thus does not impede viral RNA-dependent RNA polymerase-mediated transcription [18,19]. The relief of arbovirus restriction by ActD treatment suggests that VSV and SINV undergo abortive infections in LD652 cells due to cellular antiviral responses that require active transcription [12]. To confirm these previous results and to determine if ActD treatment could relieve restriction of additional togaviruses related to SINV, such as RRV and ONNV, LD652 cells were infected with GFP reporter viruses (VSV-GFP [12], SINV-GFP [12], RRV-GFP [20], and ONNV-GFP [21]) in the absence or presence of ActD. Cells were then stained with CellTracker Orange Dye and imaged 72 h post-infection (hpi). Representative GFP fluorescence images and quantitative GFP signals (normalized to cell number with CellTracker signals) were used as a readout for viral replication and are shown in Fig 1A and 1B. As expected, 0.05 μg/mL ActD treatment increased GFP signal in VSV-GFP and SINV-GFP infections by ~10,000- and 100-fold, respectively (Fig 1B). Additionally, ActD treatment during VSV-GFP and SINV-GFP infection increased their viral titer by ~1,000-fold for both viruses (Fig 1C). During ONNV-GFP and RRV-GFP infections, ActD increased GFP signal and viral titer by ~100-fold and ~10-fold, respectively (Fig 1A–1C). Importantly, we have previously shown that LD652 cells treated with this dose of ActD retain ~90% viability [12], and thus enhanced virus replication is not due to a general decrease in cell viability. Together, these findings suggest that arbovirus infection of LD652 cells induces a restrictive immune response that requires active host transcription. PPT PowerPoint slide
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TIFF original image Download: Fig 1. Abortive arbovirus replication in LD652 cells can be relieved with ActD treatment. A. Representative fluorescence microscopy images (GFP channel) of LD652 cells treated with DMSO (vehicle) or 0.05 μg/mL ActD and infected with the indicated GFP reporter strains for 72 h. B. Fold-change in normalized GFP signals in ActD-treated cultures relative to DMSO treatments. Cells were stained 72 hpi with CellTracker Orange dye (not shown) and imaged in GFP and RFP channels to calculate fold-change in GFP signal after normalization of cell number using CellTracker (RFP) channel signals. C. Fold-change in titer of supernatants from LD652 cell cultures treated as in A-B 72 hpi relative to input inoculum (dotted line). Data in B-C are means ± SD; n = 3. Statistical significance was determined with unpaired student’s t-test; ns = P>0.1234, * = P<0.0332, ** = P<0.0021, *** = P<0.0002, **** = P<0.0001.
https://doi.org/10.1371/journal.ppat.1012010.g001
Specific bacterial effectors relieve arbovirus restriction in LD652 cells We have previously shown that poxvirus-encoded A51R proteins are IEPs that rescue restricted arbovirus replication when expressed from plasmids transfected into LD652 cells [12,22]. Therefore, we asked if bacterial effector proteins, which often function as IEPs, could also rescue arbovirus replication. To do this, we adapted and expanded a previously described effector library for expression in insect cells [23]. Briefly, 210 secreted bacterial effectors from seven pathogens (Shigella flexneri, Salmonella enterica serovar Typhimurium, Pseudomonas syringae, Enterohemorrhagic E. coli O157:H7, Yersinia pseudotuberculosis, Legionella pneumophila, and Bartonella henselae) were cloned into the pIB/V5-His insect expression vector and screened for their ability to alleviate arbovirus restriction in LD562 cells. The pIB/V5-His-based effector library was transfected into LD652 cells for 48 h and then cells were infected with either GFP reporter viruses (RRV-GFP and ONNV-GFP) or luciferase reporter strains (SINV-LUC and VSV-LUC [12,22]) for 72 h (Fig 2A). After infection, reporter read-outs were measured and the “fold change” in GFP or luciferase signals was calculated by dividing values in effector treatments by the mean values obtained in cultures transfected with empty vector (control) plasmids. Effector proteins that enhanced viral GFP signals by >2.5-fold or luciferase signals by >4-fold above empty vector-transfected cells, were considered “hits” in our screen (Fig 2B–2E). These cutoffs were chosen to avoid false positives stemming from experimental noise within our screening system and allowed us to focus on effectors that robustly rescued arbovirus replication. Of the 210 effector proteins screened, 10 effectors rescued RRV-GFP, 11 rescued ONNV-GFP, 18 rescued SINV-LUC, and 10 rescued VSV-LUC (Fig 2B–2F and S1 Table). Interestingly, effectors generally rescued in a virus-specific manner. For instance, 21 effectors only rescued one of the four arboviruses screened (Fig 2F). This suggests that these effectors may relieve virus-specific restrictions to replication. In contrast, seven effectors rescued three or more arboviruses: IpaH4, SopB, HopT1-2, HopAM1, Ceg10, EspK, and SidM (Fig 2F), suggesting that these effectors may target host restriction mechanisms that are active against a broader range of viral pathogens. Importantly, we also assessed LD652 cells for signs of toxicity due to effector expression using a lactate dehydrogenase (LDH)-based cytotoxicity assay (S1A–S1C Fig and S1 Table). Overall LDH activity in effector-transfected cultures was within 13–14% of that measured in empty vector-transfected cells, indicating that effector expression had minimal impact on cell viability. This suggests that enhanced arbovirus replication observed in effector treatments was unlikely due to generalized effects on cell viability. PPT PowerPoint slide
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TIFF original image Download: Fig 2. Specific Bacterial Effectors Relieve Arbovirus Restriction in LD652 Cells. A. Schematic outlining screen for bacterial effectors that rescue arbovirus restriction in LD652 cells. Cells were transfected with expression plasmids from a library consisting of 210 different effector proteins. After 48 h, cells were infected with either GFP or luciferase reporter strains. At 72 hpi, viral replication was quantified using fluorescence microscopy (RRV-GFP and ONNV-GFP) or luciferase assays (VSV-LUC and SINV-LUC). Image was created with BioRender.com. B-E. Fold-change in reporter readout, normalized to empty vector controls for all four screens. The cutoff for fold-change in GFP-based assays was set to >2.5, while the cutoff for luciferase reporters was set to >4-fold (represented by dotted horizontal lines). Data points are means. RLU = relative light units. F. Summary of bacterial effector proteins that rescued at least one virus. Green blocks indicate the effector rescued the virus indicated in the column header. The bacterium encoding each effector is noted to the right: Shigella flexneri (S. flexneri), Pseudomonas syringae (P. syringae), Salmonella enterica (S. enterica), Legionella pneumophila (L. pneumo.) Enterohemorrhagic Escherichia coli 0157:H7 (EHEC). Additional effector proteins from Yersinia pseudotuberculosis and Bartonella henselae were also screened but did not rescue arbovirus replication. The complete list of effectors screened and the raw results of the screens can be found in S1 Table.
https://doi.org/10.1371/journal.ppat.1012010.g002
Depletion of SHOC2 and PSMC1 rescues restrictive arbovirus replication in LD652 cells One prediction of our approach is that the host substrates of effector proteins secreted by mammalian and plant pathogens are key regulators of viral restriction in the moth. To then determine if endogenous SHOC2 proteins contributed to arbovirus restriction in moth cells as our data suggests, we adapted a previously described CRISPR-Cas9 system for disrupting gene expression in lepidopteran insects [57] to inhibit SHOC2 expression. We cloned two independent single-guide RNAs (sgRNAs) targeting L. dispar SHOC2 into pIE1-Cas9-SfU6-sgRNA-Puro [57], an “all-in-one” vector system that expresses Cas9 nuclease, sgRNA, and a puromycin resistance cassette for selection. As controls, cells were transfected with either empty vector or a sgRNA targeting the L. dispar relish gene, which encodes a Nuclear Factor-κB-like transcription factor that we have shown to contribute to VSV and SINV restriction in LD652 cells [12,22]. Transfected cells underwent three rounds of puromycin selection and were then challenged with reporter arboviruses. As expected, cells expressing sgRNA targeting relish were significantly more susceptible to VSV-GFP and SINV-GFP infection when compared to empty vector control treatments. RRV-GFP and ONNV-GFP replication was also elevated in cells expressing relish-targeted sgRNAs, indicating that these togaviruses are also restricted by Relish-dependent antiviral responses (Fig 6A). Interestingly, cells expressing either SHOC2 sgRNA-A or sgRNA-B were significantly more susceptible to infection with ONNV-GFP, SINV-GFP, and VSV-GFP infection (Fig 6A). While only cells expressing SHOC2 sgRNA-A displayed statistically-significant differences in RRV-GFP infection, SHOC2 sgRNA-B-expressing cells trended towards an increased susceptibility to this virus with an ~9-fold higher mean in GFP signal than empty vector controls (Fig 6A). We then sought to assess if viral titers were also enhanced after arbovirus infection in SHOC2 sgRNA-expressing cells. Thus, we repeated these infections and collected the supernatants following 72 h of infection before determining viral titers on BSC-40 cells. Overall, the trends observed in Fig 6A with increases in GFP signal in SHOC2 sgRNA-expressing cells correlated well with increases in viral titers across the four arboviruses (Fig 6B), although the magnitude of the increases sometimes differed between fluorescence and titer-based experiments, which likely reflects differences in the nature of the readout of these assays. These results indicate that SHOC2 is a broadly-acting restriction factor for multiple arboviruses in LD652 cells. However, the specific role of SHOC2 in arbovirus restriction requires further investigation. PPT PowerPoint slide
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TIFF original image Download: Fig 6. Depletion of IpaH4 substrates SHOC2 and PSMC1 enhances arbovirus replication in LD652 cells. A. Fold-change in normalized viral GFP signals in cells expressing gRNA targeting Relish or SHOC2 relative to empty vector controls 72 hpi. Cells were stained with CellTracker dye 72 hpi and imaged to calculate fold-change in normalized GFP signal over empty vector (control) treatments. Data are means ± SD; n = 3. Statistical significance was determined with unpaired student’s t-test. B. Titer of supernatants from LD652 cell cultures treated as described in A. C. Fold-change in normalized viral GFP signals relative to LacZ siRNA (control) treatments. Cells were stained with CellTracker dye 72 hpi and imaged to calculate fold-change in normalized GFP signal over LacZ (control) siRNA treatments. D. Titer of supernatants from LD652 cell cultures treated as described in C. Data are means ± SD; n = 3. Statistical significance was determined with unpaired student’s t-test; ns = P>0.1234, * = P<0.0332, ** = P<0.0021, *** = P<0.0002, **** = P<0.0001.
https://doi.org/10.1371/journal.ppat.1012010.g006 PSMC1 has been reported to be an essential component of the 19S cap of the 26S proteasome [58]. Consistent with this, our attempts to knock out PSMC1 in LD652 cells with CRISPR-Cas9 techniques resulted in complete cell death after 1–2 rounds of puromycin selection. However, in mammalian systems, transient PSMC1 depletion has been achieved by siRNA knockdown [59]. Therefore, we sought to deplete PSMC1 in LD652 cells in an analogous manner. However, the application of siRNA-based RNAi in L. dispar and other lepidopteran cell types has not been well-established. Thus, we took advantage of a prior study that developed guidelines for designing siRNAs to achieve efficient knockdown in another moth species, Bombyx mori, as a basis for our siRNA design for use in LD652 cells [60]. To evaluate the efficiency of siRNA-mediated RNAi in LD652 cells, we designed siRNA targeting the coding sequences of E. coli LacZ (negative control) and firefly luciferase. Cells were transfected with either an empty vector or a luciferase-encoding pDGOpIE2 plasmid for 48 h and then subsequently transfected with siRNAs targeting transcripts encoding LacZ or luciferase (S5A Fig). We then evaluated the relative expression of luciferase using luminescence assays 72 h later. Compared to luciferase signals observed in cells transfected with control LacZ siRNA, there was a significant ~75% reduction in luminescence signals in cells transfected with siRNA targeting transcripts encoding luciferase (S5B Fig), suggesting our siRNA design and transfection strategy was relatively efficient at reducing target gene expression. We next designed three independent siRNAs targeting L. dispar PSMC1 sequence in LD652 cells [14] and assessed their relative impact on PSMC1 levels compared to treatments where control siRNAs targeting LacZ were transfected. As a positive control for arbovirus rescue, siRNAs targeting transcripts encoding argonaute-2 (AGO2), which we have shown to restrict VSV and SINV replication in LD652 cells [61], were also transfected into cells. Compared to LacZ (negative control) siRNA treatments, at least 2/3 PSMC1-targeting siRNA transfections resulted in significant increases in viral GFP signals for all four arboviruses (Fig 6C). Importantly, we confirmed knockdown of PSMC1 in LD652 cells and found that PSMC1 siRNAs-A and -C were the most effective at reducing protein level (S5C Fig), which correlated with viral rescue phenotypes (Fig 6C). Following these observations, we repeated these knockdown experiments to determine impacts on viral titers. Again, the overall trends we observed in Fig 6C with increased GFP signals in PSMC1 depletion conditions correlated well with increases in viral titers across the four arboviruses (Fig 6D). These data indicate that, like SHOC2, PSMC1 may also play a role in restricting arbovirus replication in LD652 cells. Given that PSMC1 is a proteasome subunit, we asked if treatment of LD652 cells with the proteasome inhibitor bortezomib (Bort) would alter their susceptibility to arbovirus infection. Interestingly, addition of Bort to cell culture media 2 hpi resulted in significantly greater viral replication by 72 hpi (S6A and S6B Fig). These data suggest that depletion of a proteasome subunit or inhibition of proteasome activity sensitizes LD652 cells to arbovirus infection. However, the mechanism(s) by which PSMC1 and proteasome activity restrict arbovirus replication will require additional studies in the future.
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