Interactome analysis of the lymphocytic choriomeningitis virus nucleoprotein in infected cells reveals ATPase Na⁺/K⁺ transporting subunit Alpha 1 and prohibitin as host-cell factors involved in the life cycle of mammarenaviruses

<div><p>Several mammalian arenaviruses (mammarenaviruses) cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein (NP) containing an affinity tag (rLCMV/Strep-NP) and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na⁺/K⁺ transporting subunit alpha 1 (ATP1A1) and prohibitin (PHB) as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans.</p></div

A novel negative-stranded RNA virus mediates sex ratio in its parasitoid host

<div><p>Parasitoid wasps are important natural enemies of arthropod hosts in natural and agricultural ecosystems and are often associated with viruses or virion-like particles. Here, we report a novel negative-stranded RNA virus from a parasitoid wasp (<i>Pteromalus puparum</i>). The complete viral genome is 12,230 nucleotides in length, containing five non-overlapping, linearly arranged open reading frames. Phylogenetically, the virus clusters with and is a novel member of the mononegaviral family <i>Nyamiviridae</i>, here designated as Pteromalus puparum negative-strand RNA virus 1 (PpNSRV-1). PpNSRV-1 is present in various tissues and life stages of the parasitoid wasp, and is transmitted vertically through infected females and males. Virus infections in field populations of <i>P</i>. <i>puparum</i> wasps ranged from 16.7 to 37.5%, without linearly correlating with temperature. PpNSRV-1 increased adult longevity and impaired several fitness parameters of the wasp, but had no influence on successful parasitism. Strikingly, PpNSRV-1 mediated the offspring sex ratio by decreasing female offspring numbers. RNA interference knockdown of virus open reading frame I eliminated these PpNSRV-1-induced effects. Thus, we infer that PpNSRV-1 has complex effects on its insect host including sex ratio distortion towards males, as well as possible mutualistic benefits through increasing wasp longevity.</p></div

ATP1A1 and PHB are involved in different steps of the mammarenavirus life cycle.

<p><b>(A)</b> Influence of time of addition of ouabain (OUA) or rocaglamide (Roc-A) on virus multiplication. A549 cells seeded (2.5 x 10⁵ cells/well) in a 12-well plate and cultured overnight were infected (moi = 0.1) with rLCMV/eGFP or remained uninfected (mock). OUA (10 nM), Roc-A (100 nM), or DMSO (0.01%) was added to the culture media at the indicated time points and remained present throughout the end of the experiment. Ammonium chloride (20 mM) was added to culture medium at 4 h pi to prevent multiple rounds of virus infection. At 24 h pi, eGFP expression in infected cells was examined by flow cytometry. Data represent mean ± SD of the results of three independent experiments. <b>(B)</b> Effect of ouabain and Roc-A on LCMV replication. A549 cells seeded (1.25 x 10⁵ cells/well) in 24-well plates and cultured overnight were infected (MOI = 1) with rLCMVΔGPC/eGFP, followed by addition of the indicated concentrations of ouabain or Roc-A. At 72 h pi, total cell lysates were prepared, and eGFP expression levels were measured using a fluorescent plate reader. Data represent mean ± SD of three independent experiments. <b>(C-E)</b> Effect of ouabain and Roc-A on Z-mediated budding. Cells (HEK 293T) seeded (3.5 x 10⁵ cells/well) in a 12-well plate and cultured overnight were transfected with 0.5 μg of either pC-Empty or pC-LCMV-Z-Strep (LCMV-Z-Strep) <b>(C)</b> or pC-LASV-Z-FLAG (LASV-Z-FLAG) <b>(D, E)</b>. At 24 h post-transfection, cells were washed with fresh media to eliminate Z-mediated production of VLPs in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain or Roc-A at the indicated concentrations. VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag <b>(C)</b> and FLAG-tag <b>(D)</b>. Budding efficiency for each sample was estimated by dividing the signal intensity of the Z protein associated with VLPs by that of Z detected in the cell lysate. Numbers on the bottom of panel <b>C</b> correspond to LCMV Z budding efficiencies determined in a representative experiment. Results shown in panel <b>E</b> correspond to the average and SD from four independent experiments including the one shown in panel <b>D</b>. The mean budding efficiency of DMSO treated-samples was set to 100%. Data represent mean ± SD of four independent experiments. <b>(F)</b> Effect of ouabain on incorporation of viral glycoprotein into virions. 293T cells seeded (4.0 x 10⁵ cells/well) in a 12-well plate and cultured overnight were infected (MOI = 0.1) with scrLCMV/ZsG (1^st infection) for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM (OUA). At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells (2^nd infection) seeded (4.0 x 10⁵ cells/well) in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. ZsGreen signal intensity was measured by a fluorescent plate reader. GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate (2^nd) by that in 293T cell lysate (1^st). The mean GP-incorporation efficiency of DMSO treated samples was set to 100%. Data represent means ± SD from three independent experiments. <b>(G)</b> Effect of ouabain on the late stage of LCMV infection. A549 cells seeded (1.25 x 10⁵ cells/well) and cultured overnight were infected (MOI = 0.1) with rLCMV/eGFP. At 48 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh medium in the presence of ouabain (OUA) at indicated concentrations. At 72 h pi, TCS was collected and virus titers were determined by IFFA. Data represent means ± SD from three independent experiments.</p

Co-localization of NP with ATP1A1 and PHB.

<p><b>(A)</b> Intracellular distributions of NP and ATP1A1 or PHB. A549 cells seeded (5 x 10⁴ cells/well) in a 24-well plate and cultured overnight were infected (MOI = 0.1) with rLCMV Cl-13 or remained uninfected (mock). At 48 h pi, cells were fixed, stained with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluor 568 (anti-mouse IgG-AF568). Subsequently, cells were stained with VL-4-AF488 (anti-NP), and observed by a confocal microscope. Bars, 20 μm. <b>(B)</b> Comparison of non-weighted and weighted co-localization coefficients (CC). Non-weighted CC were determined by dividing the sum of both green and red positive pixels by the sum of green-positive pixels. Thresholds were determined based on the signal intensity of mock-infected sample stained with VL-4-AF488 and anti-mouse IgG-AF568. Weighted CC were determined by taking into consideration the brightness of each channel signal. <i>p</i> values were determined by a two-tailed paired <i>t</i> test using GraphPad Prism software.</p

Effect of knock-down of genes identified LC-MS/MS analysis on LCMV multiplication.

<p><b>(A)</b> Effect of siRNA-mediated knockdown expression of genes identified by proteomics analysis. A549 cells (1,000 cells/well) in a 384-well plate were reverse transfected with siRNA pools targeting each indicated gene. At 72 h post-transfection, cells were infected (MOI = 0.05) with rLCMV/ZsG. At 48 h pi, cells were fixed and stained with DAPI. ZsGreen and DAPI signals were measured by a fluorescence plate reader. ZsGreen signal was normalized to DAPI signal. Normalized values from cells transfected with the control (NC, black bar) non-specific siRNA was set to 100%. A panel of 154 siRNAs including NC siRNA was evaluated three independent times. Results from three independent experiments were sorted by mean values and assigned to three graphs with SD. Each graph includes identical NC results (black bar) for reference. <b>(B)</b> Reduction of protein expression of ATP1A1 and PHB by siRNA-mediated gene knockdown. A549 cells (3.0 x 10⁴ cells/well) were reverse transfected in a 24-well plate with siRNA pools against either ATP1A1 or PHB or with NC siRNA. At 72 h post-transfection, total cell lysate was prepared, and expression of ATP1A1 and PHB in cell lysates were determined by western blots. <b>(C)</b> Effect of siRNA-mediated kd of ATP1A1 on production of infectious LCMV progeny. A549 cells (1.5 x 10⁴ cells/well; 48-well plate) were reverse-transfected with siRNAs against ATP1A1 or with NC siRNA. 72 h later, cells were infected (MOI = 0.01) with rLCMV/ZsG. At 24 h and 48 h pi, TCSs were collected. At 48 h pi, cells were fixed and ZsGreen expression examined by fluorescence microscopy (i). Bar, 200 μm. Virus titers in TCSs were determined by IFFA (ii). Data represent means ± SD of results from three independent experiments. LoD, limit of detection.</p

Synergistic antiviral effect of ouabain and rocaglamide on rLCMV/eGFP multiplication.

<p>A549 cells seeded (2.0 x 10⁴ cells/well) in 96-well plates and cultured overnight were treated with combinations of ouabain and Roc-A at indicated concentrations for 2 h and then infected (MOI = 0.01) with rLCMV/eGFP. Compounds were present throughout the end of experiment. At 48 h pi, cells were fixed and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader. eGFP signal was normalized to DAPI signal, and the normalized data were used to analyze synergistic effect by MacSynergy II software. Data represent % synergy (% inhibition over the expected [additive effect]) at the 95% confidence interval from five independent experiments.</p

LC-MS/MS analysis of NP-binding proteins.

<p><b>(A)</b> Flow chart of the experimental approach to identify NP-interacting host-cell proteins in LCMV-infected cells. A549 cells prepared in six 15-cm dishes (total of 1.0 x 10⁸ cells) were infected (MOI = 0.1) with either rLCMV/Strep-NP or r3LCMV/eGFP. At 48 h pi, total cell lysates were prepared, and NP- or eGFP-interacting proteins were pulled down (PD) using Streptactin-coated sepharose resin. Protein complexes bound to the resin were eluted using 2.5 mM of desthiobiotin. Eluates were precipitated using TCA followed by trypsin digestion. Tryptic peptides were subjected to LC-MS/MS analysis. <b>(B)</b> Detection of proteins present in PD samples. Protein complexes present in PD samples were separated by SDS-PAGE and visualized by SYPRO staining. Some protein bands present only in the Strep-NP PD sample are indicated by asterisks. <b>(C)</b> Venn diagram of the NP- and eGFP-interacting proteins identified by LC-MS/MS analysis. <b>(D, E)</b> Gene Onthology (GO) analysis of the NP-interacting proteins identified by LC-MS/MS. Bioinformatic analysis by PANTHER was performed showing the number of genes of identified NP-interacting proteins classified by biological process <b>(D)</b> and protein class <b>(E)</b>.</p

Generation and characterization of rLCMV/Strep-NP and r3LCMV/eGFP-Strep.

<p><b>(A, B)</b> Schematic of the genome organization of rLCMVs expressing Strep-NP or eGFP-Strep <b>(A)</b> and amino acid composition of N- and C-terminus regions of Strep-NP and eGFP-strep, respectively <b>(B)</b>. <b>(C)</b> Expression of Strep-tagged proteins. A549 cells seeded (5.0 x 10⁵ cells/well) in a 6-well plate and cultured overnight were infected (MOI = 0.1) with the indicated rLCMVs. At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. <b>(D)</b> Growth properties of rLCMV expressing Strep-tagged proteins. BHK-21 (1.75 x 10⁵ cells/well), A549 (1.25 x 10⁵ cells/well), or Vero E6 (1.25 x 10⁵ cells/well) cells seeded in 24-well plates and cultured overnight were infected (MOI = 0.01) with the indicated rLCMVs. At the indicated times pi, TCSs were collected and virus titers determined by IFFA. Results represent means ± SD of the results of three independent experiments. <b>(E)</b> Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP. A549 cells were infected (MOI = 0.1) with the indicated rLCMV or mock-infected, and 36 h later infected with SeV (MOI = 1). At 24 h pi with SeV, TCS were collected and used, following virus inactivation by U.V., to treat Vero E6 cells for 16 h, followed by infection with rVSV (MOI = 0.1) [rVSV(+)] or mock-infection [rVSV(-)]. At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP(D382A) that contains mutation D382A within NP, which has been shown to abrogate the NP’s ability to counteract the induction of IFN-I production.</p

Effect of pharmacological inhibition of ATP1A1 and PHB on LCMV multiplication.

<p><b>(A)</b> A549 cells seeded (1.25 x 10⁵ cells/well) in 24-well plates and cultured overnight were treated with either ouabain (OUA) (i) or rocaglamide (Roc-A) (ii) at indicated concentrations or with DMSO (vehicle control) for 2 h and then infected (MOI = 0.01) with rLCMV/eGFP. Compounds were present throughout the experiment. At 24 and 48 h pi, TCSs were collected, and virus titers determined by IFFA. Data represent means ± SD of results from three independent experiments. LoD, the limit of detection. <b>(B)</b> Inhibitory effects of ouabain and Roc-A on virus propagation and cell viability. A549 cells seeded (2.0 x 10⁴ cells/well) in 96-well plates and cultured overnight were treated with 3-fold serial dilutions of either ouabain (i) or Roc-A (ii) for 2 h and then infected (MOI = 0.01) with rLCMV/eGFP. Compounds were present throughout the experiment. At 48 h pi, cells were fixed to examine eGFP expression and cell viability as determined by CellTiter 96 AQ_ueous one solution reagent. The data represent means ± SD of the results from four (cell viability assay) or six (virus spread assay) replicates. The therapeutic index (TI) was calculated by dividing CC₅₀ by IC₅₀. <b>(C)</b> Effect of ouabain or Roc-A treatment on rVSV/eGFP multiplication. A549 cells seeded (1.25 x 10⁵ cells/well) and cultured overnight were treated with either ouabain (10 nM) Roc-A (100 nM), or vehicle control (DMSO) for 2 h and infected (MOI = 0.01) with either rLCMV/eGFP or rVSV/eGFP. At 72 h pi, TCS was collected, and virus titers were determined by IFFA (rLCMV/eGFP, expressed as FFUs) or a plaque assay (rVSV/eGFP, expressed as PFUs). Compounds were present to study endpoint. Results represent means ± SD of the results of three independent experiments.</p

Inhibitory effects of ouabain, bufalin, and rocaglamide on LASV propagation.

<p>A549 cells seeded in a 96-well plate (3.0 x 10⁴ cells/well) and cultured overnight were treated with 2-fold serial compound dilutions at 37°C and 5% CO₂ for 2 h, followed by inoculation with rLASV/eGFP (MOI = 0.01). Compounds were present to study endpoint. At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader. Mean values obtained with DMSO-treated and rLASV/eGFP-infected cells were set to 100%. The data represent means ± SD of the results of three replicates.</p