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Contrasting effects of filamin A and B proteins in modulating filovirus entry [1]

['Ariel Shepley-Mctaggart', 'Department Of Pathobiology', 'School Of Veterinary Medicine', 'University Of Pennsylvania', 'Philadelphia', 'Pennsylvania', 'United States Of America', 'Jingjing Liang', 'Yang Ding', 'Marija A. Djurkovic']

Date: 2023-10

Abstract Ebola (EBOV) and Marburg viruses (MARV) cause severe hemorrhagic fever associated with high mortality rates in humans. A better understanding of filovirus-host interactions that regulate the EBOV and MARV lifecycles can provide biological and mechanistic insight critical for therapeutic development. EBOV glycoprotein (eGP) and MARV glycoprotein (mGP) mediate entry into host cells primarily by actin-dependent macropinocytosis. Here, we identified actin-binding cytoskeletal crosslinking proteins filamin A (FLNa) and B (FLNb) as important regulators of both EBOV and MARV entry. We found that entry of pseudotype psVSV-RFP-eGP, infectious recombinant rVSV-eGP-mCherry, and live authentic EBOV and MARV was inhibited in filamin A knockdown (FLNaKD) cells, but was surprisingly enhanced in filamin B knockdown (FLNbKD) cells. Mechanistically, our findings suggest that differential regulation of macropinocytosis by FLNa and FLNb likely contributes to their specific effects on EBOV and MARV entry. This study is the first to identify the filamin family of proteins as regulators of EBOV and MARV entry. These findings may provide insight into the development of new countermeasures to prevent EBOV and MARV infections.

Author summary Filoviruses utilize host proteins to successfully infect cells and complete their lifecycles. Identification of host proteins that regulate the EBOV and MARV lifecycles may allow us to find possible therapeutic targets to protect against these deadly viruses. Here, we identified actin-binding cytoskeletal proteins filamin A and B as regulators of EBOV and MARV entry. We show that filamin A is a positive regulator while filamin B is a negative regulator of both EBOV and MARV entry. In sum, our findings reveal a role for host cytoskeletal-associated filamin proteins in the entry stage of the filovirus lifecycle, potentially identifying filamin proteins as broad spectrum therapeutic targets for prevention and treatment of EBOV and MARV.

Citation: Shepley-McTaggart A, Liang J, Ding Y, Djurkovic MA, Kriachun V, Shtanko O, et al. (2023) Contrasting effects of filamin A and B proteins in modulating filovirus entry. PLoS Pathog 19(8): e1011595. https://doi.org/10.1371/journal.ppat.1011595 Editor: Alexander Bukreyev, University of Texas Medical Branch / Galveston National Laboratory, UNITED STATES Received: December 22, 2022; Accepted: August 2, 2023; Published: August 16, 2023 Copyright: © 2023 Shepley-McTaggart et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its supporting information files. Funding: This work was supported by the National Institutes of Health grants AI153815, AI139392, and the National Eye Institute grant EY031465 awarded to RNH. The National Institutes of Health grants AI154336, AI151717, and the Texas Biomed Forum Grant 2017 awarded to OS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction EBOV and MARV remain global public health threats that warrant urgent development of antiviral therapeutics [1–5]. The filoviruses target many cell types, including monocytes, macrophages, and dendritic cells, and viral entry and uptake is mediated by the surface glycoprotein via the process of macropinocytosis [4,6–10]. As host proteins also play critical roles in regulating (positively or negatively) filoviral entry, a better understanding of the interplay between the virus and host may enable us to identify therapeutic targets to combat these deadly pathogens. While much has been learned regarding the molecular aspects of filovirus entry through the use of surrogate viruses and assays, a more comprehensive understanding of the role of host proteins in both positive and negative regulation of early stages of the filovirus lifecycle is needed. For example, positive regulators such as T-cell Ig mucin domain 1 (TIM-1) signal to trigger the macropinocytosis program required to internalize EBOV virions after eGP attachment, while host receptor NPC1 is required to trigger fusion of the viral and endolysosomal membranes. Additionally, the Tyro3 receptor tyrosine kinase family-Axl, Dtk, and Mer-in have been shown to be involved in entry of both EBOV and MARV [11–14]. The plasma membrane is the site of entry for both EBOV and MARV, and thus the dynamics of the plasma membrane and its associated cytoskeletal proteins will likely play key roles in regulating this early stage of filovirus infection. The filamin proteins comprise a family of three members, filamin A (FLNa), B (FLNb) and C (FLNc), that localize in part to the plasma membrane. The N-terminal region of filamins contains an actin-binding domain, followed by a rod-like domain consisting of 24 tandem repeats that function in crosslinking cortical actin filaments into a dynamic, three-dimensional structure. These proteins also function as molecular scaffolds by connecting numerous functionally diverse proteins [15–17]. The filamin proteins are known to connect the actin cytoskeleton with several cellular receptors, such as dopamine D2 and androgen receptors on the plasma membrane, as well as numerous ß integrin proteins to the cytoskeleton for cell mechanoprotection [18]. FLNa and FLNb also differentially regulate the RhoA GTPase, which is directly involved in promoting macropinocytosis, the process in which EBOV enters cells [7,19–21]. Additionally, while FLNa is ubiquitously expressed in many cell types, FLNb is highly expressed in vascular endothelial cells, a cell type often targeted during filovirus infection [15,22–26]. Although the actin cytoskeleton plays a key role in filoviral entry [7,20,27,28], the role of the actin-binding filamin proteins in the EBOV and MARV lifecycles remains to be determined. Here, we report on a previously undescribed role for FLNa and FLNb in the filovirus lifecycle: eGP and mGP-meditated entry of EBOV and MARV, respectively. We used pseudotype VSV-RFP-eGP, replication-competent recombinant rVSV-mCherry-eGP, and authentic EBOV and MARV to transduce/infect HT-1080 WT, filamin A knockdown (FLNaKD), and filamin B knockdown (FLNbKD) cells to assess the role of filamin A and B proteins in entry/infectivity. Our findings indicated that knockdown of FLNa inhibited viral infectivity, suggesting FLNa is an important positive regulator of viral entry. In contrast, knockdown of FLNb enhanced viral infectivity, suggesting that expression of FLNb may restrict viral entry. We corroborated these findings using an siRNA approach to knockdown endogenous FLNa or FLNb in HEK293T cells followed by quantification of pseudotype VSV-RFP-eGP entry. Importantly, we showed that siRNA knockdown of endogenous FLNa in primary human macrophages resulted in a significant decrease in both EBOV and MARV infectivity. Toward the mechanism of action, we used flow cytometry and confocal microscopy to show that the filamin proteins regulate macropinocytosis to potentially impact macropinocytosis-mediated entry of EBOV and MARV. Our findings imply that FLNa and FLNb proteins affect filovirus entry inversely by modulating macropinocyctosis. In sum, we identified filamin proteins as novel regulators of both EBOV and MARV entry, and thus filamins may serve as broad-spectrum therapeutic targets to prevent infectivity and reduce transmission.

Discussion Filoviruses rely on the integrity and dynamics of the host cytoskeletal architecture at the plasma membrane to facilitate entry into host cells. EBOV and MARV GPs are the viral proteins responsible for mediating attachment, entry, and fusion into target cells. However, a growing list of host cell proteins recently identified may also be important facilitators of these early events of the filovirus lifecycle. For example, DC-SIGN/L-SIGN, LSECtin, hMGL, β1-integrin and Tyro3 family receptors have been implicated as attachment factors, and cellular receptors like TIM-1 may function as a signal to trigger the macropinocytosis program required to internalize EBOV virions following GP attachment [7,13,14,21,39–41]. Macropinocytosis is characterized by actin-dependent membrane ruffling associated with the formation of macropinosomes of approximately 0.5–10 μm in diameter. Macropinocytosis and the dynamic flexibility of the actin cytoskeleton beneath the plasma membrane enable the cell to take up large substances, such as the long, filamentous filovirus virions [21]. Here we have identified actin-crosslinking filamin proteins A and B as novel regulators of filovirus entry/infectivity. Intriguingly, FLNa and FLNb appear to have opposing effects on EBOV and MARV entry, such that expression of FLNa is important for efficient filovirus entry whereas expression of FLNb may act as a barrier to restrict or negatively regulate entry. When we investigated the roles of FLNa and FLNb in regulating macropinosome formation in HT-1080 cells, we observed the same opposing effects on macropinocytosis as we did for EBOV and MARV entry. Notably, knockdown of endogenous FLNa reduced the efficiency of cellular uptake of two dextran markers, whereas knockdown of endogenous FLNb enhanced uptake of the same markers of macropinocytosis. Mechanistically, these findings suggest that FLNa and FLNb likely modulate filovirus entry by regulating the process of macropinocyctosis. The opposite effects of FLNa and FLNb on entry and infectivity of EBOV and MARV was somewhat surprising since the filamin proteins share a high degree of homology between the conserved exon/intron structure. These two filamin isoforms physically interact and heterodimerize, suggesting a mechanism to regulate each other’s function, with loss of expression of one leading to upregulation of the other [19,42]. However, an antagonistic relationship between FLNa and FLNb occurs during the process of cell migration and spreading, whereby they differentially regulate the RhoA GTPase. RhoA is directly involved in promoting cell protrusions and macropinocytosis, with increased activity at the cell plasma during vesicle closure [19,43]. Additionally, we know from the literature that FLNa co-localizes with host protein Phafin2 on forming macropinosomes and dissociates together with actin. Phafin2 is expressed abundantly in dendritic cells and helps to strip away the dense actin/filamin coat from internalized macropinosomes allowing them to efficiently traffic through the cell to form the mature endosomal form [34]. Thus, the potential role and opposing effects of FLNa and FLNb on filovirus entry may indeed be linked to the process of macropinocytosis. Alternatively, there are other filamin interactors that could be linked to the filovirus lifecycle and affect entry and infectivity of the virus. For example, FLNa promotes entry of HIV-1 by linking viral receptors to the actin cytoskeleton and by modulating the antiviral factor tetherin [44,45]. In addition, FLNa interacts with viral NS3 and NS5A proteins in chronic hepatitis C virus (HCV) infection, and can act as an adapter protein to the adenoviral and coxsackieviral receptor, Integrin β1 [44,46,47]. It is tempting to speculate that a FLNa-β1 integrin interaction may play a role in filovirus entry since β1 integrin expression has been linked to eGP-mediated entry of EBOV [41,48,49]. Alternatively, RNAse-L is a well-known member of the host innate immune system that interacts with FLNa to form a barrier to entry of Sendai virus [50,51]. Based on our findings, one could speculate that a similar restrictive barrier to filovirus entry may result from an RNAse-L/FLNb interaction. Furthermore, it is known that FLNb, but not FLNa, plays a scaffolding role in IFN signaling and can be modified by IFN-stimulated gene 15 (ISG15) [23]. Whether or not the effect of FLNb knockdown on enhancing EBOV and MARV infectivity may be due, in part, to FLNb-mediated regulation of antiviral immune defenses remains to be determined [22,23,52]. Our identification of FLNa and FLNb proteins as novel regulators of plasma membrane driven stage of EBOV and MARV entry provide new insights into the complex roles that host proteins play in regulating the filovirus lifecycle. A better understanding of this interplay between filoviral proteins and FLN proteins will be critical for our overall understanding of the biology and pathogenesis of filoviruses and other emerging pathogens, as well as for the future development of effective, host-directed antiviral therapies. Further investigation into possible pharmalogical inhibition of FLNa activity during early filovirus infection, for example, may benefit the development of a broad-spectrum, multifunctional antiviral therapeutic.

Material and methods Cell lines, plasmids, and reagents HeLa, HEK293T, HT-1080 WT, FLNaKD and FLNbKD cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Corning) supplemented with 10% fetal bovine serum (FBS) (Gibco), penicillin (100 U/mL)/streptomycin (100 μg/mL) (Invitrogen). Cells were grown at 37°C in a humidified 5% CO 2 incubator. FLNaKD and FLNbKD cells were generated from parental HT-1080 cells as described previously [15]. The primary antibodies used in this study include mouse anti-FLNa (Santa Cruz), rabbit anti-FLNb antibody (Millipore), rabbit anti-LaminB1 (Abcam), rabbit anti-eGP (Invitrogen), mouse anti-GFP (Rosche) and mouse anti-β-actin (Proteintech). Dextran-Alexa568 (MW 10K) and Dextran-TRITC (MW 70K) were purchased from Invitrogen. VSV M protein was detected using mouse anti-VSV-M monoclonal antibody 23H12 (kindly provided by D. Lyles, Wake Forest, Winston-Salem, NC, USA). FLNa-specific siRNA, FLNb-specific siRNA and control siRNA pools were purchased from Santa Cruz and Origene. The rVSV-eGP-mCherry virus [53] was kindly provided by P. Bates (UPenn School of Medicine). MTT assay Cell proliferation was determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Briefly, cells were seeded in 96-well plates and incubated for 24, 48, and 72 hours, and cells were washed with phosphate-buffered saline (PBS) and then incubated in MTT solution for 3 hours. After dimethyl sulfoxide was added into each well, the absorbance was measured at 490 nm to determine cell viability with a microplate reader. EBOV and MARV infection and staining Experiments with live viruses were performed in the BSL-4 laboratory at Texas Biomedical Research Institute (Texas Biomed, San Antonio, TX) in accordance with standard operating procedures and protocols approved by the Institute’s Biohazard & Safety and Recombinant DNA Committees. The NCBI accession numbers for filoviruses used in these studies were NC_002549 (EBOV variant Mayinga), KF990213 (recombinant EBOV variant Mayinga, encoding GFP), and NC_001608 (Marburg virus strain Musoke). The virus stocks were obtained from the Texas Biomed repository, grown as before [54,55], and viral titers were determined using standard plaque assays. Briefly, HT-1080 WT and FLNKD cells grown in 96-well plates were incubated with EBOV or MARV at MOI = 0.1 in triplicate for 1 hour, then washed and overlaid with fresh medium. After 24 hours, cells were fixed and were treated with an antibody to viral GP (clone 4F3; IBT Bioservices) or mVP40 (IBT Bioservices) to detect infection and Hoechst 33342 dye (Thermofisher) to stain nuclei. Sample photographs acquired by Nikon Ti-Eclipse microscope (Nikon, Tokyo, Japan) were analyzed by CellProfiler software (Broad Institute) to determine infection efficiency, calculated as the number of infected (GP-positive or mVP40-positive) cells over the total number of cells (nuclei), for each condition. Each experiment was repeated three times. A one-way ANOVA followed by Dunnett’s multiple comparison test for one variable was used to assess a statistical difference between infection efficiencies in WT and FLNKD cells. A p value was determined for each sample pair analyzed, and the difference was considered statistically significant if p<0.05. psVSV-RFP-eGP propagation psVSV-RFP-eGP pseudotyped particles were generated using a VSV platform that incorporates the EBOV GP protein into VSV envelopes by transfection of HEK293T cells with pCG1 EBOV GP expression plasmid (kindly provided by P. Bates, UPenn School of Medicine). At 30 hours post transfection, the eGP expressing cells were transduced for 4 hours with psVSV-RFP-deltaG pseudotyped with G (kindly provided by P.Bates, UPenn School of Medicine). At 28–30 hours post transduction, the media containing the psVSV-RFP-eGP pseudotype particles was harvested and clarified by centrifugation twice at 4,000 rpm for 15 minutes. Viral particles were additionally clarified by ultracentrifugation at 36,000rpm for 2 hours and then stored at −80°C until use. Both psVSV-RFP-deltaG pseudotyped with G and psVSV-RFP-eGP express RFP. rVSV-eGP-mCherry infection HT-1080 WT and FLNKD cells were seeded at 1 ×106 cells overnight in 6-well plates. rVSV-eGP-mCherry (MOI = 0.1 ~5 ×107 RFU /ml) was added to cells for 1 hour at 37°C, virus inoculum was removed, and the cells were washed once with 1× DPBS. DMEM with 1% methylcellulose was added to the cells at 37°C for 10 hours. Cells were washed 3 times with DPBS and either harvested for Western blotting or fixed with 4%PFA for 15mins at room temperature. Unfixed cell extracts were harvested with RIPA and the indicated proteins were detected by Western blotting. Briefly, unfixed cells were harvested and lysed RIPA buffer and clarified for 5 min at 15,000 rpm. Cell lysates were suspended in loading buffer with boiling, fractionated by SDS-PAGE and EBOV GP and actin proteins were detected using specific antisera. For fixed cells, mCherry expression was visualized and quantified on a fluorescent microscope. siRNA knockdown and psVSV-RFP-eGP transduction HeLa cells in Opti-MEM (Thermofisher) in 6-well plates were transfected twice with either control siRNAs or FLNa-specific or FLNb-specific siRNAs (siRNA pools purchased from Santa Cruz Biotechnology) at a final concentration of 50 nM by using Lipofectamine Invitrogen at 2-day intervals. A total of 1.0 μg of eVP40 DNA was transfected with the second round of siRNAs. HeLa cells were then transduced with psVSV-RFP-eGP pseudotypes at MOI = 1 (~1×106 red fluorescent units per well) added to Opti-MEM and incubated with cells for 1 hour at 37°C. Pseudovirus inoculum was removed, and the cells were washed once with 1× DPBS. RFP expression was visualized and quantified on a fluorescent microscope at 24 hours post transduction. Cell extracts were then harvested and the indicated proteins were detected in cell samples by Western blotting. Primary human macrophages Peripheral blood was collected from healthy adult human donors according to the University of Texas Health-approved IRB protocol 20180013HU to prepare monocyte-derived macrophages (MDMs) as we described previously [56]. MDMs were cultured in RPMI medium supplemented with 10% autologous serum at 37°C in a humidified 5% CO2 incubator. siRNA treatments in MDMs and immunoblotting To deplete MDMs of FLNa, cells plated in 96-well or 12-well plates were transfected with three individual siRNA duplexes targeting the gene to a final concentration of 25 nM of FLNa siRNAs or the same concentration of a non-specific siRNA control (Origene). The transfections were performed in triplicate, using TransIT-X2 reagent (Mirus), following the manufacturer’s recommendations. After 48 hours, the siRNA treatments in 96-well plates were removed, and the MDMs were infected with EBOV-GFP or MARV at a MOI of 0.2 for 24 hours. The cells were stained and analyzed as above. FLNa protein depletion was assessed by immunoblotting as previously described [55]. Macropinocytosis assay HT-1080 WT and FLNKD cells were seeded overnight in 12-well plates and then media was replaced with Opti-MEM without phenol-red for 3 hours at 37°C. 5ul of DMSO was added to negative control 30 mins prior to addition of 0.5 mg/ml of 10 K dextran-Alexa568 (Invitrogen) with 100 ng/ml of endothelial growth factor (EGF) for 5 minutes at room temperature. Cells were washed twice with cold 1× DPBS and fixed with IC Fixation buffer (Invitrogen) for 10 minutes at room temperature. Cells were collected in DMEM with 1% FBS at 4°C overnight. Flow cytometry was performed with LSRFortessa (BD Biosciences) and data analyzed using FlowJo software (FlowJo, LLC). For confocal microscopy, cells were seeded on 35mm MatTek dishes and treated as previously described with 0.1mg/ml of dextran-Alexa568 (MW 10K) and 100ng/ml of EGF. Cells were fixed with 4% PFA and stained with Hoechst. Images were acquired using a laser scanning confocal microscope (Leica SP5-FLIM Inverted), equipped with 63× oil immersion objective. Images were processed using Fiji and 3–5 fields per sample were randomly selected with 5–10 cells per visual field for dextran particle analysis. HT1080-WT and FLNKD cells were fixed in 4 well chamber cover glass slides (Cellvis) after 30 minutes of co-incubation with 1.0 mg/ml dextran-TRITC (MW 70K) and 20 ng/ml EGF. Images were acquired using a laser scanning confocal microscope with 100× oil immersion objective and 6–7 fields per sample were randomly selected with 5–25 cells per visual field for dextran particle analysis by Fiji. In brief, red channel was converted to 8-bit grey scale and background subtraction was used after threshold fluorescence was set. Size filter was set to an area of 0.2–20.0 μm2 to exclude non-macropinosome structures for particle counts [33], and Hoechst staining of nuclei was used to calculate average number of dextran+ particles per cell within an image.

Acknowledgments The authors would like to thank Dr. David Calderwood (Yale University) for the generous gift of the HT-1080 WT, FLNaKD and FLNbKD cell lines. We would also like to thank members of the Harty and Bates labs for fruitful discussions, and the Penn Vet Imaging Core, especially Dr. Gordon Ruthel, for their advice and encouragement of this work.

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