Figure 1.
SH3 domains of amphiphysin-1 and -2 bind to conserved proline-rich regions shared by alphaviral nsP3 proteins.
(A) Under a schematic representation of the overall structural organization of SFV nsP3 are shown carboxyterminal regions of selected alphaviral nsP3 proteins aligned based on a shared proline-rich region, dubbed the “PIPPPR motif” (boxed in red). Indicated on the right is the amino acid numbering of the aligned nsP3 regions of Semliki Forest (SFV), Sindbis (SINV), Chikungunya (CHKV), Barmah Forest (BAR), Aura, O'nyong-nyong (ONYO), Mayaro viruses, and Western equine encephalomyelitis virus (WEEV). The amino acids identical in at least four sequences are shown in bold. The regions targeted by deletions (P1 and P2) are indicated by horizontal square brackets above the sequences. Another proline-rich region found in the P2 region in the SFV and WEEV is underlined in blue. Of note, WEEV nsP3 contains only this second motif but is lacking the PIPPPR motif. (B) Binding of phages displaying the SH3 domain of amphiphysin-1 or -2 bind to wild-type or deletion mutants of SIN, CHKV, or SFV nsP3 proteins. The numbers indicate relative phage binding compared to a parallel mock experiment without nsP3.
Figure 2.
The SH3 binding site of alphaviral nsP3 proteins mediates binding to amphiphysin-1 and -2 proteins in transfected and in infected cells.
Expression vectors for complete or proline rich region-deleted versions of SINV, CHKV, and SFV nsP3 expressed as fusion proteins with a biotin acceptor domain were cotransfected to 293 cells together with the Myc-tagged amphiphysin-1 (A) or amphiphysin-2 (B). Lysates of the transfected cells were subjected to a precipitation with streptavidin-coated beads, followed by Western blot analysis using labeled streptavidin or anti-Myc antibodies to detect associated nsP3 (top panels) and amphiphysin (bottom panels) proteins, respectively. To confirm uniform expression of the amphiphysin proteins in transfected cells, total lysates were also examined by anti-Myc Western blot analysis (middle panels). (C) HeLa cells were infected with wild-type SFV or a modified virus carrying the ΔP1+2 mutant of nsP3. Lysates of the infected cells were examined by anti-nsP3 Western blot analysis (bottom panel), or subjected to immunoprecipitation with anti-nsP3 antibodies followed by Western blotting analysis of the immunocomplexes using antibodies against amphiphysin-2 (top panel) or nsP3 (middle panel). A lysate prepared from mock-infected cells was included as a negative control.
Figure 3.
Identification of the critical amphiphysin-binding residues in the P1 region of SINV nsP3.
Association of amphiphysin-1 (A) and amphiphysin-2 (B) with SINV nsP3 proteins carrying indicated single amino acid substitutions in the P1 region was examined as in Figure 2. The mutant indicated as ΔPC is lacking the complete P1 region plus the rest of the SINV nsP3 carboxyterminus.
Figure 4.
Recruitment of endogenous amphiphysin-2 to SFV replication sites in HeLa cells.
Cells were infected with SFV at m.o.i. 50 (B, C, and D) or mock infected (A), fixed at the indicated time points and stained with specific antibodies against nsP3, amphiphysin-2, or dsRNA. Bound antibodies were detected with fluorophore-linked secondary antibodies, and the images were pseudocolored for visualization (nsP3 – magenta, dsRNA – red and amphiphysin – green). Each channel is shown in a separate image, and the overlay of dsRNA (indicating the localization of RCs) and amphiphysin-2 staining is shown on the right (colocalization seen in yellow). A representative image of the main phenotype detected is shown at each time point. Arrowheads: the localization of RCs matches with nsP3 staining. Single confocal sections are shown, and the position of each section is indicated in the bottom left corner of each row. The scale bars are 10 µm.
Figure 5.
Dynamic changes in amphiphysin-1 localization during SFV infection of neuronal cells.
N2A cells were infected with SFV (B, C, and D) or mock infected (A), and examined for nsP3, amphiphysin-1, or dsRNA staining at the indicated time points as in Figure 4.
Figure 6.
Amphiphysin is recruited by nsP3 in infected cells in an SH3-binding motif-dependent manner.
HeLa cells were infected with wild-type SFV (A) or SINV (C) or the corresponding mutant viruses with SH3 binding motif-deficient nsP3 proteins, ΔP1+2 mutant of SFV nsP3 (B) and R426E mutant of SINV nsP3 (D) at m.o.i. 50, fixed at 10 h p.i. (6 h p.i. for wild-type SFV) and stained with specific antibodies for nsP3 (SFV), amphiphysin-2, or dsRNA. Bound antibodies were detected with fluorophore-linked secondary antibodies and the images were pseudocolored for visualization. SINV nsP3 was detected via its fluorescent fusion partner (pseudocolored in magenta). The overlay of nsP3 and amphiphysin-2 staining is shown on the right (colocalization seen in white). The images were chosen to show the phenotype representing nsP3 granules, which are present in a subpopulation of the cells. Arrowheads indicate nsP3 granules devoid of viral dsRNA. The position of each confocal section is indicated in the left bottom corner of each row. The scale bars are 10 µm.
Figure 7.
Deletion of the SH3 binding motif impairs amphiphysin-2 recruitment to RCs, and delays CPV formation.
HeLa cells were infected with SFV wild-type (A) or SFV ΔP1+2 (B) at m.o.i. 50, fixed at the indicated time points and stained with specific antibodies. Colocalization of RCs (dsRNA staining) and amphiphysin-2 is shown in yellow. The scale bars are 10 µm. (C) Deletion of SH3 binding motif impaired amphiphysin-2 recruitment to replication complexes, as indicated by Pearson's coefficients measured for colocalization of amphiphysin-2 with dsRNA and nsP3 in CPVs at 10 h p.i. (n = 8-13 fields examined (at least 34 cells); *p<0.05; ** p<0.005).
Figure 8.
A functional SH3 binding motif in nsP3 is required for optimal SFV and SINV replication.
(A-D) BHK and HeLa cells were infected with SFV (wild-type ▪ or ΔP1+2 ▴) and SIN (wild-type ▪ or R426E ▴) at m.o.i. 5. The total RNA was collected at 2 h intervals until 8 h p.i., and the viral RNA levels were detected by RT-qPCR. In each experiment wild-type 8 h sample was set as 100%, and the RNA amounts for each time point are shown relative to this. The measurements correspond to the mean value of three biological replicates. Standard deviations are indicated by error bars (n = 3; * P<0.05; ** P<0.001). (E) Aliquots of growth medium were withdrawn at indicated time points and the virus production was measured by plaque titration. The data shown are representative of two independent experiments.
Figure 9.
Amphiphysin silencing impairs viral RNA replication.
(A) HeLa cells were treated for 68 h with amphiphysin-2 or control siRNAs. Levels of amphiphysin-2 isoforms expressed were detected by Western blot. Beta-actin staining is shown as a loading control. (B) Viability of the siRNA-transfected cells was analyzed by measuring the cellular ATP levels using a luminescence based assay. Cell cultures that were left untreated are included for comparison (n = 3). (C) Cells treated with siRNA were infected in parallel with wild-type or mutant viruses at m.o.i. 5, and RNA replication was measured at 5 h p.i. by RT-qPCR (n = 3; * p<0.05). (D) Cells were infected with SFV-RLuc at m.o.i. 0.5 (□ control Δ amphiphysin-2 siRNA) or m.o.i 5 (▪ control ▴ amphiphysin-2 siRNA), and viral replication was assayed based on Renilla luciferase activity (RLuc) at the indicated time points. The data shown represent the average of two independent experiments.
Figure 10.
Neurologic symptoms and mortality in Balb/c mice following SFV infection.
Female 6-week-old Balb/c mice were infected i.p. with 1×106 pfu of (A) SFV4 or (B) SFVΔP1+2 mutant virus. Bars indicate the number of surviving mice, and grading of the clinical status of the mice at each day during a period of two weeks after the infection. The complete grading system used was: 0 = no symptoms; 1 = weakness of limbs, or hunched back, or ruffled fur, or any combinations of these; 2 = partial paralysis of hind limbs; 3 = paralysis of limbs, limited moving or abnormal moving behavior; 4 = moribund or dead.