Fig 1.
Mammarenavirus Z protein interacts with NMTases.
Evidence of Z matrix protein myristoylation and interaction with NMT1 and NMT2 during infection (A-B). A549 were infected with rMOPV WT, mCherryFlag or Z-Flag at MOI 0.01 for 48 h, WCE were collected and Flag immunoprecipitated. (A) In gel fluorescence for the detection by biorthogonal ligation (click reaction) of Alk-AF647 with the AzC12 analogue of myristoylated proteins in WCE (bottom) or Flag immunoprecipitated proteins (top). (B) WCE and IP products from identically set experiments were analyzed by Western Blotting for the presence of NMT1, NMT2, Z, Flag and β-actin. (C-D) Characterization of the Z/NMT interaction. HEK293T cells were cotransfected for 24 h with the indicated plasmids before cell lysis and Flag immunoprecipitation. WCE and IP products were analyzed by Western Blot for the presence of Flag (WT or Ala mutant of the Z protein of MOPV), HA (NMT1, NMT2 and ITCH) and β-actin. Results shown are representative of two (A-B) and three (C-D) independent experiments.
Fig 2.
NMT1 over NMT2 is a host dependency factor for Mammarenavirus.
(A) Western Blot analysis of WCE of HAP1 WT, NMT1ko or NMT2ko cells for the expression of NMT1, NMT2 and β-actin. (B) HAP1 WT, NMT1ko or NMT2ko cells were infected with MOPV, LASV, LCMV, LUJV, MACV, JUNV and YFV at MOI 0.01 for 48 h. Cell culture supernatants were titrated and the results of three independent experiments are represented as mean percentage of infectious titers +/- SEM of the HAP1 WTcells. P <0.05 (*), <0.01 (**) and <0.001 (***).
Fig 3.
Paucity of NMT2 drives NMT1 dependency.
(A) A549 cells were infected or not with rMOPV Z-Flag at MOI 0.01 and total RNA and WCE were collected at 0 h, 24 h and 48 h post infection. mRNA levels for NMT1 and NMT2 were quantified and standardized to GAPDH levels. WCE were analyzed by Western Blot for Z-Flag, NMT1, NMT2 and β-actin. Results are mean +/- SEM of three independent experiments. (B) HAP1 NMT1ko were transfected with the indicated control, HA-NMT1 or HA-NMT2 encoding plasmids for 24 h and then infected with MOPV at MOI 0.01 for 48 h. WCE collected at infection time were analyzed by Western Blot for the expression of HA tagged proteins and β-actin. HA-NMT1/2 lanes were brought closer from distant lanes. Cell culture supernatants were titrated and the results of four independent experiments are represented as mean +/- SEM and expressed as FFU/mL. (C) Endogenous NMT2 detection by Western Blot from the WCE of (B). P <0.05 (*) and <0.01 (**).
Fig 4.
IMP1088 and DDD85646 specifically inhibit Mammarenavirus multiplication.
(A-C) A549 cells were infected with YFV 17D, MOPV (A), LASV (B), or MACV (C) at MOI 0.01 for 1 h. Before, during and after infection, cells were treated or not with 10 fold dilutions of IMP1088, DDD85646 or C75 ranging from 1 nM to 10 μM. After 48 h of infection, cell culture supernatants were titrated (left panels) while cell viability was measured (right panels). The results of three independent experiments are represented as mean +/- SEM and expressed as FFU/mL for titrations and standardized to mock condition for viability. (D) Measure of the potential virucidal effect of IMP1088, DDD85646 and C75 on MOPV particles. 105 FFU of MOPV were incubated for 1 h with 10 μM of each compounds and were then titrated. The results of three independent experiments are represented as mean +/- SEM and expressed as FFU/mL. (E) Potency of IMP1088 and DDD85646 against MOPV, LASV, LCMV, LUJV, MACV or JUNV. A549 cells were infected (MOI 0.01) and treated or not with IMP1088 (20 nM), DDD85646 or C75 (500 nM) as described above. Cell culture supernatant were titrated and results of three independent experiments expressed as mean percentage +/- SEM to mock treated condition. P <0.05 (*) and <0.001 (***).
Fig 5.
IMP1088 inhibits myristoylation of Z through the inhibition of the interaction between the Z protein and NMTases.
HEK293T cells were transfected with a Z-Flag MOPV plasmid and HA-NMT1, HA-NMT2 or HA-ITCH plasmids (or control plasmids). Eight hours after transfection, cells were treated (A) or not (B) with AzC12 (10 μM) before mock or IMP1088 treatment (10 nM, 100 nM or 1 μM) for 24h. (A) WCE and IP eluates containing AzC12 incorporated proteins were subjected to “click reaction” with Alk-AF647 for in gel fluorescence detection of protein myristoylation and silver staining for total protein detection. (B) Non AzC12 treated WCE and IP eluates were analyzed by Western Blot as described in Fig 1. Results shown for A and B are representative of two independent experiments. Lane numbers [1–14] were added to match viewings between gels and panels.
Fig 6.
IMP1088 and DDD85646 block cell to cell spread through the inhibition of viral particle egress.
(A) A549 cells were infected with MOPV at MOI 0.01 and mock treated or treated with IMP1088, DDD85646 or C75 at 25, 100 or 250 nM. After 48 h of infection, cells were fixed and stained to detect intracellular NP (green channel) and counterstained with DAPI. Results shown are representative of two independent experiments. Scale bar 20 μm. (B) A549 cells were infected with rMOPV Z-Flag or rMOPV mCherryFlag at MOI 0.01 and treated with 10 fold dilutions of IMP1088, DDD85646 or C75 as in Fig 4A. After 48 h of infection, cells were collected, fixed and stained with an anti-Flag-APC conjugated mAb to detect intracellular Z-Flag or mCherryFlag and analyzed by FACS. The results of three independent experiments are represented as mean +/- SEM and expressed as percentage of infected cells. (C) Supernatants from A549 infected with rMOPV Z-Flag (MOI 1) and treated with IMP1088 (50 nM), DDD85646 or C75 (500 nM) were ultracentrifuged (sucrose cushion 20% w/v). Pellets were analyzed by Western Blot for the presence of GP2 or Z-Flag proteins. (D) HEK293T cells were transfected with indicated plasmids and treated or not with IMP1088, DDD85646 or C75 for 24 h. Cell culture supernatants were ultracentrifuged as in (C). WCE and pellets analyzed by Western Blot for the presence of Z-Flag, mCherryFlag and β-actin. Results in C-D are representative of two independent experiments. P <0.001 (***).
Fig 7.
IMP1088 and DDD85646 inhibit viral particle release and promote Z matrix protein disappearance from infected cells.
(A) A549 cells were infected with rMPOV Z-Flag at MOI 0.01 for 48 h before mock or IMP1088 treatment (50 nM) for the following 48 h. Cells were then stained for the expression of GP2, NP or Z proteins (green) and DAPI. Scale bar 10 μM. (B-C) A549 cells were infected at MOI 0.01 with rMPOV Z-Flag (left panel) or rMOPV NP-HA (right panel) for 48 h before mock or IMP1088 (50 nM), DDD85646 or C75 (500 nM) treatment for 48 h. Permeabilized cells were stained with anti-Flag APC or anti-HA FITC and analyzed by FACS. The results of five (rMPOV Z-Flag) and four (rMPOV NP-HA) independent experiments are expressed in (B) as the mean +/- SEM of the percentage of Fag or HA positive cells and in (C) as the mean of fluorescence intensity (MFI) +/- SEM of Flag or HA positive cells (percentages relative to the mock condition). (D) A549 cells were infected and treated as in (B-C) and WCE were analyzed by Western Blot for the presence of Z-Flag, NP-HA and β-actin. (E) Volcano plot representation of tandem mass spectrometry and comparative proteomics analyses of WCE from A549 cells infected with rMOPV Z-Flag at MOI 1 and treated or not with IMP1088 (50 nM) for 48 h (three independent experiments). (F) Quantification of intracellular mRNA encoding for NP (top) or Z (bottom) from A549 cells infected with rMOPV Z-Flag at MOI 0.01 and mock or IMP1088, DDD85646, C75 treated. mRNAs were quantified at 0 h, 24 h and 48 h post treatment. Results are mean +/- SEM of four independent experiments expressed as mRNA copies standardized to GAPDH levels. P <0.001 (***).
Fig 8.
The Z matrix protein is degraded in a post-translational manner.
(A) Reverse genetics strategy for the expression of Z-Flag and mCherryHA from the MOPV Z ORF and representative immunofluorescence of A549 cells infected with the rMOPV Zflag-P2A-mCherryHA depicting the expression of Z (green) and mCherry (red) and DAPI counterstaining (scale bar 5μm). (B-E) A549 cells were infected with the rMOPV Zflag-P2A-mCherryHA at MOI 2 and Mock treated or treated with IMP1088 (50 nM), DDD85646 or C75 (500 nM). Cells were collected at 5 h, 10 h, 14 h and 24 h post infection for intracellular staining with anti-Flag APC or anti-HA FITC and analyzed by FACS. The results of four independent experiments are in (B) the mean +/- SEM of the percentage of Fag positive cells and in (C) and (D) are the mean of fluorescence intensity (MFI) +/- SEM of Flag or HA positive cells respectively (percentages relative to the mock condition). (E) WCE of experiment set as in (B-D) analyzed by Western Blot for the presence of Z-Flag, mCherryHA and β-actin. Results shown are representative of three independent experiments. P <0.05 (*) and <0.001 (***).
Fig 9.
The IMP1088 driven degradation of the Z matrix protein is independent of the proteasome and the autophagy catabolic pathways.
A549 cells were infected with rMOPV Z-Flag at MOI 2 and subsequently treated or not with IMP1088 (50 nM). MG132, Chloroquine (1 μM or 10 μM) or a mix of both drugs was readily added and incubated for 24 h. WCE were analyzed by Western Blot for the presence of HSP70, LC3I/II, Z-Flag and β-actin. Results are representative of two independent experiments.