The C Terminus of Rotavirus VP4 Protein Contains an Actin Binding Domain Which Requires Cooperation with the Coiled-Coil Domain for Actin Remodeling

Abstract

The interactions between viruses and actin cytoskeleton have been widely studied. We showed that rotaviruses remodel microfilaments in intestinal cells and demonstrated that this was due to the VP4 spike protein. Microfilaments mainly occur in the apical domain of infected polarized enterocytes and favor the polarized apical exit of viral progeny. The present work aims at the identification of molecular determinants of actin-VP4 interactions. We used various deletion mutants of VP4 that were transfected into Cos-7 cells and analyzed interactions by immunofluorescence confocal microscopy. It has been established that the C-terminal part of VP4 is embedded within viral particles when rotavirus assembles. The use of specific monoclonal antibodies demonstrated that VP4 is expressed in different forms in infected cells: classically as spike on the outer layer of virus particles, but also as free soluble protein in the cytosol. The C terminus of free VP4 was identified as interacting with actin microfilaments. The VP4 actin binding domain is unable to promote microfilament remodeling by itself; the coiled-coil domain is also required in this process. This actin-binding domain was shown to dominate a previously identified peroxisomal targeting signal, located in the three last amino acids of VP4. The newly identified actin-binding domain is highly conserved in rotavirus strains from species A, B, and C, suggesting that actin binding and remodeling is a general strategy for rotavirus exit. This provides a novel mechanism of protein-protein interactions, not involving cell signaling pathways, to facilitate rotavirus exit.IMPORTANCE Rotaviruses are causal agents of acute infantile viral diarrhea. In intestinal cells, in vitro as well as in vivo, virus assembly and exit do not imply cell lysis but rely on an active process in which the cytoskeleton plays a major role. We describe here a novel molecular mechanism by which the rotavirus spike protein VP4 drives actin remodeling. This relies on the fact that VP4 occurs in different forms. Besides its structural function within the virion, a large proportion of VP4 is expressed as free protein. Here, we show that free VP4 possesses a functional actin-binding domain. This domain, in coordination with a coiled-coil domain, promotes actin cytoskeleton remodeling, thereby providing the capacity to destabilize the cell membrane and allow efficient rotavirus exit.

Keywords: VP4 protein; actin binding; actin remodeling; protein domain; rotavirus.

FIG 1

VP4 is expressed both within virions and as a free protein. MA104 cells were infected by rotavirus RF stain and analyzed by confocal microscopy and immunofluorescence at 6 h postinfection (h.p.i.) using two different monoclonal antibodies. MAb 7.7 (A and D) mainly recognizes VP8* either on assembled or unassembled VP4, and 2G4 (B and E) recognizes assembled VP5*. Panels C and F represent merged images of panels A and B and panels D and E, respectively. MAb 7.7 staining was revealed using FITC-labeled secondary antibody (green), and 2G4 staining was revealed using Alexa-547 dye (red) directly coupled to the antibody. Arrows point to some 2G4-labeled dots that colocalize faintly with 7.7 staining. Arrowheads point to some 7.7-labeled dots that do not colocalize with 2G4 staining. Scale bar, 5 µm. Images are representative of three independent experiments.

FIG 2

Summary of the VP4-GFP constructs used in the present study. Each construct was obtained using the backbone plasmid pEGFP-C1, and all truncated-VP4 constructs encoding amino acid stretches 481 to 776, 481 to 773, 481 to 757, 562 to 776, 641 to 776, 641 to 773, 641 to 757, 702 to 776, 713 to 776, 713 to 773, and 718 to 776, as well as the constructs containing deletions (481-776 del 574-670, 481-776 del 574-713, and 481-776 del 574-718), were generated by PCR and inserted within pEGFP-C1 vector via EcoRI and BamHI sites. Sp100 coiled-coil fused to VP4 (562-776) was made by insertion of PCR-amplified Sp100 coiled-coil 29-152 in VP4 (562-776). Note that GFP-truncated VP4 encoding amino acid stretches 713 to 776, 718 to 776, 718 to 773, and 733 to 776 were made by insertion of amplified segments from the SA11 strain in pEGFP-C1 via XhoI and BamHI sites.

FIG 3

2G4 antibody only recognizes assembled VP4. Cos-7 cells were transiently transfected using either a VP4-GFP construct or a VP8*-GFP construct. Three days later, cells were fixed, permeabilized, and analyzed by confocal microscopy and immunofluorescence using 7.7 or 2G4 MAbs. Transfected GFP constructs appear in green, and Alexa-547-labeled secondary antibodies (red) were used to reveal MAb 7.7 or 2G4. Scale bars, 5 µm. Images are representative of two independent experiments.

FIG 4

Effects of successive N-terminal deletions on the colocalization of VP4-GFP constructs with actin microfilaments. Cos-7 cells were transiently transfected using four RF VP4-GFP constructs (green) (A, D, G, and J) containing either the full-length VP4 (construct 1-776) (A to C), an N-terminal deletion keeping the coiled-coil domain (construct 481-776) (D to F), an N-terminal deletion removing the main part of the coiled-coil domain (construct 562-776) (G to I), or a construct containing only the last 59 C-terminal amino acids of VP4 (construct 718-776) (J to L). Three days after transfection, cells were fixed, permeabilized, labeled with Alexa-547-phalloidin (red) (B, E, H, and K), and analyzed by confocal fluorescence microscopy. Panels C, F, I, and L represent the corresponding merged images. Scale bar, 5 µm. Images are representative of four independent experiments.

FIG 5

Role of RV-ABD and coiled-coil domain in actin binding and remodeling. Cos-7 cells were transiently transfected using five of the RF VP4-GFP constructs described in the legend to Fig. 3, namely, constructs 481-776 (A to C), 670-776 (D to F), 481-776 del-574-670 (G to I), 481-776 del-574-713 (J to L), and the construct containing the exogenous coiled-coil domain Sp100 CC VP4 574-776 (M to O). Three days after transfection, cells were fixed, permeabilized, labeled with Alexa-547-phalloidin, and analyzed by confocal fluorescence microscopy. Panels A, D, G, J, and M represent GFP fluorescence (green); panels B, E, H, K, and N represent phalloidin fluorescence (red); panels C, F, I, L, and O correspond to the respective merged images. Scale bar, 5 µm. Images are representative of three to five independent experiments.

FIG 6

Homo-oligomerization of free VP4. Cos-7 cells were transiently transfected using plasmids containing the construct VP4 481-776, in line with either GFP or mCherry fluorescent proteins. Three days after transfection, Cos-7 cells were observed by confocal microscopy. (A) Typical image obtained with the GFP-containing construct; (B) typical image obtained with the mCherry-containing construct; (C) merged image. Scale bar represents 5 µm. (D) FRET experiment was performed using Cos-7 cells expressing both VP4-GFP and VP4-mCherry (upper) or GFP and mCherry alone (lower). Cells were only illuminated with a 488-nm laser beam, and fluorescence was recovered in the green range (500 to 530 nm) and in the red range (red filter, 570 to 650 nm). Eight different fields were analyzed. Blue color represents the background signal, green color the signal recorded for the GFP channel, and red color the signal for the mCherry channel. In the control lower line, the main signal detected was in the green range, whereas in the upper line a strong red fluorescence was detected in the eight fields analyzed, indicating a FRET signal. Scale bar represents 500 µm. (E) Images from panel D were quantified using ImageJ software, and the ratio of red fluorescence to green fluorescence was calculated for each field. Means and SD were calculated for each experiment and are reported as the percentage of red fluorescence over total fluorescence.

FIG 7

Functional peroxisome-targeting signal is present on VP4 but masked by RV-ABD. Cos-7 cells were transiently transfected using four VP4-GFP (A, D, G, and J) (green) constructs containing (i) the last four amino acids of SA11 strain VP4, namely, QCRL (a typical PTS-1 sequence), termed construct 773-776 (A to C), (ii) the last 59 amino acids of VP4 (construct 718-776) (D to F), (iii) the last 64 amino acids (construct 713-776) (G to I), or (iv) the same construct as construct ii but lacking the PTS signal (construct 718 to 773) (J to L). Three days after transfection, cells were fixed, permeabilized, labeled with Alexa-547-labeled anti-peroxisome antibody (D, E, H, K) (red), and analyzed by confocal fluorescence microscopy. (C, F, I, and L) Respective merged images. Scale bar, 5 µm. Images are representative of three independent experiments.

FIG 8

Sequence alignment of RV-ABD domains from several rotavirus strains. Sequences of VP4 proteins from different strains of species A, B, or C rotavirus were aligned using BLAST. An asterisk indicates amino acid identity, a colon indicates amino acid similarity, a period indicates amino acid divergence (also indicated by black color), and no sign indicates unrelated amino acids. Note that the line below species B and C rotavirus sequences summarizes the comparison of the eight strains studied here. Blue letter codes correspond to the PTS-1 signal. Amino acid numbering was aligned with the numbering of the RF strain.

FIG 9

Structural analysis of RV-ABD. (A and B) The 3D structure of a unit of assembled rotavirus particle was retrieved from the PDB (accession number 4V7Q) and is displayed in panel A. The sequence corresponding to RV-ABD was searched, and a zoomed image of this region is displayed in panel B, corresponding to the square in panel A. Mark 1 corresponds to aa 701 (Ile) and mark 2 to aa 776 (Leu) according to RF numbering. Only one of the three VP4 chains (the one annotated BX in PDB) is underlined in black between marks 1 and 2.