What drug is used to treat congestive heart failure?
ouabain
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. 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. 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. Text: Introduction Mammarenaviruses Arenaviridae: Mammarenavirus cause chronic infections of rodents worldwide . Invasion of human dwellings by infected rodents can result in human infections through mucosal exposure to aerosols or by direct contact of abraded skin with infectious material. Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas  . Mammarenaviruses are classified into two main groups, Old World OW and New World NW . Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas  . Mammarenaviruses are classified into two main groups, Old World OW and New World NW . The OW Lassa virus LASV , causative agent of Lassa fever LF , is the most significant OW mammarenaviral pathogen. LASV is estimated to infect several hundred thousand individuals annually in Western Africa, resulting in a high number of LF cases associated with high morbidity and lethality. Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever . Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever . The worldwide-distributed OW mammarenavirus lymphocytic choriomeningitis virus LCMV is a neglected human pathogen of clinical significance especially in congenital infections  . Moreover, LCMV poses a particular threat to immunocompromised individuals, as has been illustrated by fatal cases of LCMV infection associated with organ transplants . However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina. However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina. Likewise, current anti-mammarenavirus therapy is limited to an offlabel use of the nucleoside analogue ribavirin that is only partially effective and can cause significant side effects . Development of effective anti-mammarenavirus drugs has been hampered by the lack of detailed understanding of virus/host-cell interactions required for mammarenavirus multiplication that could represent amenable targets for antiviral therapy. the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B . the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B . To facilitate the identification of specific PPI between NP and host cell proteins, we used our mammarenavirus tri-segmented r3 platform to design an r3LCMV expressing a C-terminus Strep-tag version of enhanced green fluorescent protein r3LCMV/eGFP-Strep that we used as a negative control Fig 1A and 1B . We rescued rLCMV/Strep-NP and r3LCMV/eGFP-Strep and confirmed the expression of strep-tagged NP and eGFP in rLCMV/ Strep-NP-and r3LCMV/eGFP-Strep-infected cells, respectively Fig 1C . Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV. Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV. However, both rLCMV/Strep-NP and r3LCMV/eGFP-Strep had WT-like growth kinetics and reached high titers. As with WT LCMV, infection with rLCMV/Strep-NP prevented production of bioactive IFN-I by cells in response to Sendai virus SeV infection as determined using an IFN bioassay based on protection against the cytopathic effect CPE induced by infection with vesicular stomatitis virus VSV Fig 1E . Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE. Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE. We selected the human A549 cell line because lung epithelial cells are one of the initial cell targets of humans following inhalation of mammarenavirions. We infected A549 cells multiplicity of infection MOI = 0.1 with either rLCMV/Strep-NP or r3LCMV/eGFP-Strep Fig 2A . At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining. At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining. We compared the pattern of stained protein bands detected between rLCMV/Strep-NP-and r3LCMV/eGFP-Strepinfected samples and confirmed the presence of Strep-NP and eGFP-Strep in pertinent samples Fig 2B . Protein complexes in the rest of eluates from the PD samples were concentrated by trichloroacetic acid TCA precipitation and subjected to trypsin digestion Fig 2A . Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 . Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 . Filtering the data using these criteria resulted in identification of 139 candidate host-cell proteins as NP-interacting partners. Among 53 proteins found present in both NP-and eGFP-PD samples, 36 had spectral counts in the NP-PD sample that were less than two-fold higher than their corresponding spectral counts in the eGFP-PD sample Fig 2C and S1 Table . Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV. Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV. The protein annotation through evolutionary relationship PANTHER protein family classification Biological Process of the NP-interacting protein candidates revealed that a large number of proteins were involved in metabolic and cellular processes Fig 2D . We also analyzed the molecular functions of the NP-interacting protein candidates according to the PAN-THER protein profile classification Fig 2E , which revealed diversified biochemical functions enriched for nucleic acid-binding proteins and chaperones. To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene . To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene . OTPs are latest generation of siRNAs and offer significant advantages over previous generations. Off-target effects are primarily driven by antisense strand microRNA miR -like seed activity. In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy. In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy. Numerous host-cell factors showed an anti-LCMV effect increased ZsG expression by kd of the genes , including microtubule-associated protein 1B MAP1B  , dengue virus 2 DENV-2 , and chikungunya virus CHIKV . To begin assessing the potential biological implications of the identified NP-host cell protein interactions, we selected ATP1A1 and PHB given the availability of reagents, existing knowledge about their roles in cell physiology, and evidence of participation in multiplication of other viruses. We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers. We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers. For this, we transfected A549 cells with siRNA targeting Expression of Strep-tagged proteins. A549 cells seeded 5.0 x 10 5 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. D Growth properties of rLCMV expressing Strep-tagged proteins. At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins. BHK-21 1.75 x 10 5 cells/well , A549 1.25 x 10 5 cells/well , or Vero E6 1.25 x 10 5 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. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP. Results represent means ± SD of the results of three independent experiments. E 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 mockinfection 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. 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. ATP1A1 or with NC siRNA 72 h prior to infection with rLCMV/ZsG. We found that siRNAmediated kd of ATP1A1 dramatically inhibited ZsGreen expression Fig 3Ci , which was associated with a significant reduction of infectious LCMV progeny Fig 3Cii . Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both. Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both. Another consideration is that only a minor fraction of NP might be engaged in the interaction with a given host cell protein, and therefore, detection of these interactions would require highly sensitive methods such as LC-MS/MS. To overcome this problem we used confocal microscopy to examine the co-localization of NP with ATP1A1 and PHB in LCMV-infected cells. Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries . Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries . The PHB inhibitor rocaglamide is a flavagline from an Aglaia tree used in traditional Chinese medicine that has potent anticancer activity . To examine whether pharmacological inhibition of ATP1A1 or PHB inhibited LCMV multiplication, we pretreated human A549 and HEK 293T , nonhuman primate Vero E6 , and rodent murine L929 and hamster BHK-21 cells with ouabain or rocaglamide and infected them with rLCMV/eGFP S1 Fig . Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition . Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition . Likewise, we observed a dose-dependent rocaglamide inhibition of eGFP expression in all cell lines infected with rLCMV/eGFP S1B Fig . Consistent with these findings, production of infectious LCMV progeny was reduced by treatment with either ouabain or rocaglamide Fig 5A within a concentration range that had minimal impact on cell viability Fig 5B . To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability. To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability. To gain insights about the mechanisms by which ouabain and rocaglamide exert their anti-LCMV activity, we examined effects of these compounds on distinct steps of the LCMV life cycle. First, we asked whether ouabain and rocaglamide affected cell entry of LCMV. We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection. We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection. The timing of compound addition did not significantly change the number of eGFP-positive cells, indicating that neither ouabain nor rocaglamide inhibited cell entry of LCMV. The number of eGFP + cells in ouabain-treated cells was reduced at all time-of-addition points compared to vehicle dimethyl sulfoxide DMSO -treated cells, but was similar to that observed in ammonium chloride-treated cells. Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription. Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription. To further investigate the effect of ouabain and rocaglamide on virus RNA synthesis, we infected A549 cells with a recombinant single-cycle infectious LCMV expressing eGFP rLCMVΔGPC/eGFP and treated cells with either ouabain or rocaglamide. Seventy-two hours later, we determined percent normalized eGFP expression in infected cells Fig 6B . Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis. Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis. We also examined the effect of ouabain and rocaglamide on the late step of the arenavirus life cycle, Z-mediated budding. For this experiment, we transfected cells with Z-Strep-and Z-FLAG DYKDDDDK epitope -expressing plasmids from LCMV and LASV, respectively. At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide. At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide. At 24 h, we determined by WB levels of Z protein in both whole cell lysates and associated with virus-like particles VLPs collected from TCS. Treatment with rocaglamide, but not with ouabain, caused a reduction in both LCMV and LASV Z budding efficiency Fig 6C and 6D . The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments. The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments. The first experiment involved the use of a newly generated single-cycle infectious recombinant LCMV expressing the reporter gene ZsGreen scrLCMV/ZsG-P2A-NP to infect MOI = 0.1 A549 cells 1 st infection . These cells were subsequently transfected with a plasmid expressing LCMV GPC. After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F . After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F . The second experiment involved infection MOI = 0.1 of cells with WT LCMV, and 48 h later we washed infected cells three times to remove the extracellular infectious progeny produced during the first 48 h of infection. Then, fresh media containing ouabain or DMSO vehicle control were added, and 24 h later we determined virus titers in TCS Fig 6G . Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections. Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections. Since ouabain and rocaglamide inhibited different steps of the LCMV life cycle, we examined whether their use in combination results in a synergistic anti-LCMV effect. For this experiment, we infected A549 cells with rLCMV/eGFP and treated them with ouabain and rocaglamide using different concentrations and combinations. At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide. At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide. We next asked whether the ATP1A1 and PHB host-cell factors contributed also to multiplication of viral hemorrhagic fever-causing LASV. We treated A549 cells with ouabain, bufalin, or rocaglamide and inoculated the treated cells with recombinant LASV expressing eGFP rLASV/eGFP . eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 . eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 . We also tested the effect of compounds targeting ATP1A1 and PHB on multiplication of JUNV. Consistent with our results obtained with LCMV and LASV, ouabain, bufalin, and rocaglamide strongly suppressed JUNV multiplication S3 Fig . These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses. These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses. Our approach using a recombinant LCMV expressing NP with an affinity tag facilitated defining the NP interactome in the context of LCMV infection. Recently, using an NP-specific monoclonal antibody mAb to precipitate NP and associated cellular protein partners in a mammarenavirus NP interactome, King et al. identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al. identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al. Differences in both experimental conditions and analysis methodologies used to generate the LCMV NP interactome documented by King et al. and ours likely 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 C and FLAG-tag D . 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 C and FLAG-tag D . 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 C correspond to LCMV Z budding efficiencies determined in a representative experiment. Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments. Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments. F Effect of ouabain on incorporation of viral glycoprotein into virions. 293T cells seeded 4.0 x 10 5 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 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 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 10 5 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%. 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. contributed to the observed differences between data sets. Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems. Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems. We used results of the eGFP-Strep interactome, determined in r3LCMV/eGFP-Strep-infected cells, as a control to filter out non-specific NP interactions, which may have resulted in a higher degree of stringency than in the study by King et al for selection of NP-interacting candidates. The combined information provided by the NP interactome reported by King et al. and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction . and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction . The anti-IFN activity of NP was mapped to its C-terminal part and linked to the 3'-5' exonuclease domain present with the NP C-terminal part . Inhibitor-B kinase ε IKKε was identified as an NP-binding protein using plasmid-mediated overexpression in transfected cells , and the NP-IKKε binding affinity correlated with NP's ability to inhibit IFN-I induction . We, as well as the work by King et al. , did not detect this NP-IKKε interaction. We, as well as the work by King et al. , did not detect this NP-IKKε interaction. This discrepancy may have been caused by very low expression of IKKε in LCMV-infected cells, which prevented detection of IKKε by LC-MS/MS. Alternatively, NP-IKKε interaction could possibility be temporarily regulated and take place at early times pi, but could be mostly absent at 48 h pi, the time at which we prepared the cell lysates for our proteomics studies. Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit . Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit . ATP1A1 represents one of four α subunits . Recent evidence has suggested that the Na + /K + -ATPase is involved in multiple cell signaling pathways that are independent of its ion-pumping function . Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase . Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase . Thus, a low concentration of ouabain induces a conformational change in ATP1A1 that results in activation and release of proto-oncogene tyrosine protein kinase, Src, from ATP1A1, followed by activation of as yet unknown downstream signaling that inhibits, for instance, cell entry of murine hepatitis virus MHV . However, our results indicated that ouabain did not interfere with LCMV cell entry. In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV. In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV. Moreover, cell entry of JUNV occurs also by clathrin-mediated endocytosis , a process affected by Src signaling. Ouabain has been clinically used in several European countries for the management of congestive heart failure, whereas bufalin has been tested in clinical trials for cancer treatments , and the CST digoxin has been FDA-approved since 1997 to treat heart failure and atrial fibrillation. Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry. Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry. In contrast, PHB was reported to be a cell entry receptor for DENV-2 and CHIKV . On the other hand, PHB did not act as a virus cell entry receptor for HCV. Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV . Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV . These findings indicate that PHB is involved in different steps of the life cycle of a variety of viruses, and thereby an attractive target for the development of broad-spectrum antiviral drugs. Rocaglate is a group of natural compounds, which includes rocaglamide, that inhibits protein synthesis by targeting the ATP-dependent DEAD-box RNA helicase eukaryotic initiation factor 4A eIF4A and exerts anti-tumor activity . The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication. The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication. These proteins, which included MAP1B, might have anti-LCMV activity. MAP1B has been shown to bind to nonstructural proteins 1 NS1 and 2 NS2 of human respiratory syncytial virus HRSV . NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined . NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined . Examining the role of NP-MAP1B interaction in modulating NP's ability to inhibit induction of type I IFN is of interest. We identified among the NP-interacting host cell proteins the RNA helicase Moloney leukemia virus 10 MOV10 , which has been reported to be an antiviral factor for FLUAV , retroviruses  , and DENV-2 . We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions. We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions. MOV10 was shown to enhance IRF-3-mediated IFN-I induction following SeV infection through a tank binding kinase 1 TBK1 -independent and IKKε-dependent manner. This finding was further supported by demonstrating MOV10-IKKε interaction by co-immunoprecipitation studies . We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined. We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined. Several members of the mammalian chaperonin-containing T-complex CCT were identified as prominent hits in our NP interactome. The mammalian CCT is critical for folding of many proteins with important functions in diverse cellular processes , and may protect complex protein topologies within its central cavity during biosynthesis and folding . The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV . The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV . However, the role of these CCT proteins in virus multiplication remains unknown and may involve functions other than acting as molecular chaperones. Previous studies documented the presence of several components of the of eIF4F, including 4A, within viral replication-transcription complexes RTC detected in cells infected with LCMV and TCRV . These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity. These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity. In this work, we documented the generation of rLCMV/Strep-NP and its use to define the NP-interactome in infected cells. We presented evidence that ATP1A1 and PHB contribute to efficient multiplication of mammarenaviruses using genetics and pharmacological inhibition of the genes. Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins. Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins. Future studies elucidating the roles of pro-and antiviral host-cell factors identified in this study in mammarenavirus multiplication will advance our understanding of the multiple functions of NP and uncover novel cellular targets for the development of antimammarenaviral drugs. In addition, by identifying proviral host-cell factors, drugs that are already approved can be repurposing as therapeutics to combat human pathogenic mammarenavirus infections. Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described . Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described . Generation of rLCMV/NP D382A and SeV, strain Cantell, was described 30, 82, 83 . An rLCMV lacking GPC and expressing eGFP rLCMVΔGPC/eGFP was generated by reverse genetics using procedures previously described . rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B . rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B . The rLCMV expressing eGFP rLCMV/eGFP was generated as described , and the rLCMV expressing ZsGreen rLCMV/ZsG instead of eGFP was generated by reverse genetics using similar procedures to generate rLCMV/eGFP. Generation of rLASV expressing eGFP rLASV/eGFP will be described elsewhere. A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG. A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG. scrLCMV/ZsG-P2A-NP was rescued by reverse genetics using similar procedures to generate rLCMVΔGPC/eGFP . LCMV titers were determined by immunofocus forming assay IFFA as described . Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA . Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA . VSV titers were determined by a plaque assay. Total cell lysates were prepared in PD lysis buffer + 250 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% TritonX-100, 10% glycerol, 1 mM of MgCl 2 , 1 μM of CaCl 2 , 1 μM of ZnCl 2 and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA . Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA . To detect Strep-tagged proteins, membranes were reacted with mouse monoclonal antibodies to Strep QIAGEN, Germantown, MD , eGFP Takara Bio USA, Mountain View, CA , GP 2 We33/ 36 , ATP1A1 TehrmoFisher Scientific, Rockford, IL , PHB Abcam, Cambridge, MA or rabbit polyclonal antibodies to α-tubulin Cell Signaling Technologies, Danvers, MA or glyceraldehyde-3-phosphate dehydrogenase GAPDH, Millipore , respectively, followed by incubation with appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G IgG antibodies Jackson ImmunoResearch Laboratories, West Grove, PA . SuperSignal West Pico or Femto chemiluminescent substrate Thermo Fisher Scientific was used to elicit chemiluminescent signals that were visualized using ImageQuant LAS 4000 Imager GE Healthcare Bio-Sciences, Pittsburgh, PA . Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1. Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1. At 48 h pi, cells were washed three times with ice-cold PBS, scraped into fresh ice-cold PBS, and centrifuged at 400 x g at 4˚C for 10 min. Supernatant was removed, and cells were lysed with 12 ml of PD lysis buffer + supplemented with halt protease and phosphatase inhibitor cocktail Thermo Fisher Scientific and 5 μg/ml of deoxyribonuclease I Worthington Biochemical Corporation, Lakewood, NJ . Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C. Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C. After 2 h of incubation, the resin was washed three times with PD lysis buffer + and once with PD lysis buffer without TritonX-100 PD lysis buffer - . After the centrifugation at 1,600 x g and 4˚C for 5 min, the last wash buffer was removed, and protein complexes associated with the resin were eluted into 2 ml of PD lysis buffer - containing 2.5 mM of desthiobiotin. The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn. The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn. A MudPIT microcolumn was prepared by first creating a Kasil frit at one end of an un-deactivated 250-μm outside diameter OD capillary tube interior diameter of 360 μm Agilent Technologies, Inc., Santa Clara, CA . The Kasil frit was prepared by briefly dipping a 20-30-cm capillary tube in 300 μl of Kasil 1624 potassium silicate well-mixed solution PQ Corporation, Malvern, PA and 100 μl of formamide, curing at 100˚C for 4 h, and cutting the frit to a length of %2 mm. Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading. Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading. MudPIT analysis. An analytical reversed-phase liquid chromatography column was generated by pulling a 100-μm interior diameter ID of 360 μm OD capillary tube Polymicro Technologies, Phoenix, AZ to 5-μm ID tip. Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid . Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid . MudPIT and analytical columns were assembled using a zero-dead volume union Upchurch Scientific, Oak Harbor, WA . LC-MS/MS analysis was performed with an Agilent high-pressure LC pump Agilent and linear quadrupole ion dual cell trap Orbitrap Velos Thermo using an in-house built electrospray stage. Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient. Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient. Data analysis. Protein and peptide identification were performed with Integrated Proteomics Pipeline-IP2 Integrated Proteomics Applications, San Diego, CA. integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in. integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in. Spectrum raw files were extracted into ms2 files from raw files using open source RawExtract 1.9.9 Scripps Research Institute, La Jolla, CA; and the tandem mass spectra were searched against a human protein database UniprotKB . To accurately estimate peptide probabilities and false discovery rates, we used a decoy database containing the reversed sequences of all the proteins appended to the target database. Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system. Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system. The search space included half and fully tryptic peptide candidates that fell within the mass tolerance window with no miscleavage constraint. Carbamidomethylation +57.02146 Da of cysteine was considered as a static modification. siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG. siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG. siRNA target host-cell proteins were selected based on availability of validated siRNA sequences. The siRNAs we used to examine the effects on LCMV multiplication of knockdown expression of NP-interacting host cell protein candidate hits corresponded to the Genome-wide ON TAR-GET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene; Dharmacon, Lafayette, CO . A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. The total protein concentration of clarified cell lysate was measured by Pierce BCA Protein Assay Kit Thermo Fisher Scientific . The same amount of protein from each sample was subjected to SDS-PAGE, and the protein expression of siRNA-targeted genes was analyzed by western blots. Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI . Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI . Green fluorescence eGFP or ZsGreen and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, Bio-Tek, Winooski, VT . Mock-and virus-infected cells were fixed with 4% PFA. After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488. After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488. In some samples, primary antibody against ATP1A1 or PHB was omitted to determine background fluorescence. To visualize nuclei, DAPI Fluoromount-G SouthernBiotech, Birmingham, AL was used to mount coverslips on a slide glass. Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss . Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss . Eight green NP-positive cells were marked and every pixel in the marked area was plotted in the scatter diagram based on its intensity level from each channel. Thresholds for green and red channels were determined using mock-infected cells stained with VL-4-AF488 anti-NP and anti-mouse IgG antibody conjugated with Alexa Fluor 568, without using anti-ATP1A1 or -PHB antibodies. Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels. Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels. This calculation was repeated for eight individual cells. To assess the specificity of co-localization, we determined weighted CC by taking into consideration the brightness of each channel signal. Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software. Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software. A549 or Vero E6 cells seeded 2.0 x 10 4 cells/well in a 96-well plate and cultured overnight were treated with 3-fold serial compound dilutions at 37˚C and 5% CO 2 for 2 h, followed by infection with rLCMV/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 Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%. At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%. The IC 50 concentrations were determined using GraphPad Prism. A549 or Vero E6 cells seeded in a 96-well plate 2.0 x 10 4 cells/well and cultured overnight were treated with 3-fold serial compound dilutions and cultured at 37˚C and 5% CO 2 for 48 h. Then, CellTiter 96 AQ ueous one solution reagent Promega, Madison, WI was added. Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%. Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%. The CC 50 concentrations were determined using GraphPad Prism. A plasmid expressing C-terminus Strep-tagged Z protein pC-LCMV-Z-Strep was generated using similar procedure to generate a plasmid expressing C-terminus FLAG-tagged LASV Z protein pC-LASV-Z-FLAG , and the budding assay was performed as previously described . Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium. Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium. After the removal of the last wash medium, cells were cultured in fresh medium containing ouabain 30 or 40 nM or rocaglamide 50 or 100 nM or equivalent concentration of DMSO, and 24 h later, virion-like particle VLP -containing TCS and cells were collected. Total cell lysate was prepared by lysing the cells with lysis buffer 1% NP-40, 50 mM of Tris-HCl pH 8.0 , 62.5 mM NaCl, 0.4% sodium deoxycholate . After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots. After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots. A549 cells infected with rLCMV/eGFP were harvested using Accutase cell detachment solution Innovative Cell Technologies, San Diego, CA and fixed with 4% PFA in PBS. eGFP expression was examined by flow cytometry using a BD LSR II Becton Dickson , and data were analyzed with FlowJo Tree Star, Inc., Ashland, OR . 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media 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 40 nM of ouabain or vehicle control DMSO . 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media 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 40 nM of ouabain or vehicle control DMSO . At 48 h pi, TCS was collected and used to infect fresh monolayers of BHK-21 cells seeded 4.0 x 10 5 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. Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . A549 cells seeded 2 x 10 5 cells/well in a 96-well plate and cultured overnight were treated with combinations of different concentrations of ouabain and rocaglamide for 2 h and then infected MOI = 0.01 with rLCMV/eGFP. Compounds were present in the culture medium throughout the experiment. At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . eGFP readouts were normalized by DAPI readouts, and normalized data were used to analyze synergistic effect of the two compounds by the MacSynergy II program . Data were analyzed for p values by a two-tailed unpaired t test using GraphPad Prism software.