Fig 1.
RVFV NSs interacts with LC3A and GABARAP in silico via LIR motifs.
(A) The linear sequence of RVFV NSs (UniProt ID P21698) was annotated based on mobility and disorder analysis by MobiDB [73] and regions labeled as structured, disordered, and observed, where “observed” refers to experimentally observed residues in the protein structure (i.e., X-ray, NMR, Cryo-EM). (B) The location of potential LIR motifs predicted by ELM and iLIR were mapped onto the amino acid sequence of NSs (green shading). (C-D) Representative structural models of RVFV NSs (orange) in complex with GABARAP (cyan) and LC3A (forest green) proteins, as calculated via AlphaFold and FoldX (Materials and Methods). The C-terminal LIR motif of NSs (i.e., NSs4; purple atoms) interacts with LC3 family members in all cases. (E-F) Representative structural models of NSs4 LIR motif-containing peptide (orange) in complex with the GABARAP (cyan) and LC3A (forest green) proteins, as calculated via AlphaFold and FoldX (Materials and Methods). In the models, the NSs4 LIR motif (purple atoms) interacts with LC3A and GABARAP in a similar manner.
Table 1.
Biophysical parameters of in silico interactions between NSs and LC3 family members.
Table 2.
NSs interacts with LC3 family members in vitro.
Table 3.
Phe 261 within NSs4 is important for the interaction with LC3A in vitro.
Fig 2.
Crystal structures of NSs4 in complex with LC3A and GABARAP.
(A) Cartoon representation of the crystal structure of the NSs4-LC3A complex highlighting the side chains of NSs4 (firebrick red) when bound to LC3A (forest green). (B) Cartoon representation of the crystal structure of the NSs4-GABARAP complex highlighting the side chains of NSs4 (firebrick red) when complexed with GABARAP (cyan). (C) Close-up and metrics (in Å) of the NSs4-LC3A complex highlighting the side chains of amino acids from HP1 of LC3A (forest green) that make either hydrophobic (F7, I23, P32, I34, L53, F108), anion-π (E19) or cation-π (K51) interactions with the side chain of F261 of NSs4 (firebrick red) at the binding interface. (D) Close-up and metrics (in Å) of the NSs4-GABARAP complex highlighting the side chains of amino acids from HP1 of GABARAP (cyan) that make either hydrophobic (Y5, I21, P30, I32, L50, F104), anion-π (E17) or cation-π (K48) interactions with the side chain of F261 of NSs4 (firebrick red) at the binding interface. The dashed lines (black) in panels C-D correspond to the distance measurements given in the text for the key interactions at the interfaces of the complexes.
Fig 3.
NSs interacts with LC3 family members in RVFV-infected cells.
(A) BSR-T7/5 cells were transfected with media alone (mock), GFP-tagged plasmid alone (GFP), or GFP-tagged LC3A, LC3B, or LC3C plasmids and 24 h post-transfection samples were infected with MOI 0.1 rMP-12 NSs-3XFlag. Samples were collected 24 h after infection and subjected to co-immunoprecipitation with an anti-GFP specific antibody followed by western blot analysis with FLAG and GFP antibodies. (B) BSR-T7/5 cells were transfected with media alone (mock), GFP-tagged plasmid alone (GFP), or GFP-tagged GABARAP, GABARAPL1, or GABRAPL2 plasmids and 24 h post-transfection samples were infected with MOI 0.1 rMP-12 NSs-3XFlag. Samples were collected 24 h after infection and subjected to co-immunoprecipitation with an anti-GFP specific antibody followed by western blot analysis with the Flag or GFP antibodies. Quantification of band intensity for each condition is shown on the right from four biological replicates. Statistical analysis was conducted using a one-way ANOVA and post-hoc sidak’s test with * = P ≤ 0.05 and ** = P ≤ 0.01.
Fig 4.
F261S substitution within the NSs4 LIR significantly reduces the interaction of NSs and LC3.
HSAEC cells or Vero cells were infected at an MOI of 3 with (r)MP12, rMP-12 NSs-3XFlag, rMP-12 NSs-3XFlag F261S, or media alone (mock). Samples were collected 24 hpi and subjected to co-immunoprecipitation with either an LC3A (panel A) or an LC3B (panels B and C) specific antibody followed by western blot analysis with the appropriate antibodies. Panels A and B displays results from HSAECs and panel C shows results from Vero cells. Quantification of band intensity for each condition is shown on the right from three biological replicates. rMP-12 NSs-3XFlag was set to 100% relative binding. Statistical analysis was conducted using a one-way ANOVA and post-hoc sidak’s test with *** = P ≤ 0.001 and **** = P ≤0.0001.
Fig 5.
LC3A is predominantly nuclear in RVFV infected cells and colocalizes with NSs.
(A) Vero cells were grown on coverslips and mock infected (media alone), infected with rMP-12 NSs-3XFlag, or infected with rMP-12 NSs-3XFlag F261S. Vero cells treated with serum starvation media is included as a positive control. The cells were stained for LC3A, NSs-Flag, and DAPI (nuclear), followed by confocal microscopy imaging. (B-C) Colocalization of LC3A and NSs was found in filaments and perinuclear regions. (D) Nuclear and cytoplasmic corrected fluorescence LC3A levels were quantified using FIJI ImageJ Software and statistically analyzed using one-way ANOVA with the GraphPad Prism Software. Twenty-five cells across three biological replicates per condition were utilized in the quantification. Statistical analysis was conducted using a one-way ANOVA and post-hoc sidak’s test with ns = not significant (P > 0.05), ** = P ≤ 0.01, and **** = P ≤0.0001. (E) Fluorescence intensity profiles of either a rMP-12 NSs-3XFlag or rMP-12 NSs-3XFlag F261S infected cell. The white arrows in panel A indicated the cells selected for analysis.
Fig 6.
NSs interacts with LC3B in the nucleus.
(A) HSAECs were infected with rMP-12 NSs-3XFlag (MOI 3), rMP-12 NSs-3XFlag F261S (MOI 3), or media alone (mock) and 24 hpi nuclear and cytoplasmic protein extracts were produced and immunoprecipitation performed with an anti-LC3B antibody. Western blot analysis was performed with anti-Flag and anti-LC3B antibodies. (B) Cytoplasmic and nuclear fraction purity was assessed via western blot analysis using anti-GAPDH and anti-Lamin A/C antibodies. (C) Quantification of band intensity for each condition is shown on the right from five biological replicates. Mock infected samples were set to a fold change of 1. Statistical analysis was conducted using a one-way ANOVA and post-hoc sidak’s test with *** = P ≤ 0.001.
Fig 7.
NSs suppresses autophagy through the F261 within the NSs4 LIR motif in Vero cells.
Cells were analyzed using the Abcam Autophagy Detection Kit Protocol, followed by confocal microscopy imaging. Green signal indicates autophagic vesicle formation. DAPI (blue) is the nuclear marker. Uninfected cells in complete media serve as negative controls. Mock infected, rMP-12 NSs-3XFlag (MOI 3), or rMP-12 NSs-3XFlag F261S (MOI 3) infected cells were subjected to serum starvation 4 hours prior to collection. Panel A is 8 hpi and Panel B is 24 hpi. The average foci per cell (50 cells/condition) is shown on the right of each panel. Statistical analysis was conducted using a one-way ANOVA and post-hoc sidak’s test with ns = not significant (P > 0.05) and **** = P ≤0.0001.
Fig 8.
NSs reduces LC3BII through the F261 within the NSs4 LIR motif.
Vero cells were infected with rMP-12 NSs-3XFlag (MOI 3) or rMP-12 NSs-3XFlag F261S (MOI 3). Uninfected (Mock) cells served as a control. In addition, mock infected, rMP-12 NSs-3XFlag, or rMP-12 NSs-3XFlag F261S infected cells were subjected to serum starvation 4 h prior to collection. Panel A is 8 hpi and Panel B is 24 hpi. Cell lysates were collected and analyzed for LC3B, NSs-3XFlag, and actin expression via western blot analysis. Quantification of LC3BII band intensity for each condition is shown on the right from six biological replicates. LC3II band intensity was normalized to actin, then mock infected cells without serum starvation were set to 100%, and all other samples normalized to this. Statistical analysis was conducted using a one-way ANOVA and post-hoc sidak’s test with ** = P ≤0.01 and **** = P ≤0.0001.
Fig 9.
rMP-12 NSs-3XFlag F261S has reduced replication capability in HSAECs, but not Vero cells.
HSAECs (panel A) or Vero cells (panel B) were infected with rMP-12 NSs-3XFlag or rMP-12 NSs-3XFlag F261S at an MOI of 3. Supernatants were collected at 4, 8, 16, 24, 48, and 72 h post infection and viral titers determined by plaque assay. Data presented are the averages of three biological replicates. Statistical analysis was conducted using a one-way ANOVA and post-hoc sidak’s test with ** = P ≤0.01 and **** = P ≤0.0001.
Table 4.
Imaging settings for the autophagy microscopy analysis.