Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 92 (6)

Paramyxovirus V Proteins Interact With the RIG-I/TRIM25 Regulatory Complex and Inhibit RIG-I Signaling

Affiliations

Paramyxovirus V Proteins Interact With the RIG-I/TRIM25 Regulatory Complex and Inhibit RIG-I Signaling

Maria T Sánchez-Aparicio et al. J Virol.

Abstract

Paramyxovirus V proteins are known antagonists of the RIG-I-like receptor (RLR)-mediated interferon induction pathway, interacting with and inhibiting the RLR MDA5. We report interactions between the Nipah virus V protein and both RIG-I regulatory protein TRIM25 and RIG-I. We also observed interactions between these host proteins and the V proteins of measles virus, Sendai virus, and parainfluenza virus. These interactions are mediated by the conserved C-terminal domain of the V protein, which binds to the tandem caspase activation and recruitment domains (CARDs) of RIG-I (the region of TRIM25 ubiquitination) and to the SPRY domain of TRIM25, which mediates TRIM25 interaction with the RIG-I CARDs. Furthermore, we show that V interaction with TRIM25 and RIG-I prevents TRIM25-mediated ubiquitination of RIG-I and disrupts downstream RIG-I signaling to the mitochondrial antiviral signaling protein. This is a novel mechanism for innate immune inhibition by paramyxovirus V proteins, distinct from other known V protein functions such as MDA5 and STAT1 antagonism.IMPORTANCE The host RIG-I signaling pathway is a key early obstacle to paramyxovirus infection, as it results in rapid induction of an antiviral response. This study shows that paramyxovirus V proteins interact with and inhibit the activation of RIG-I, thereby interrupting the antiviral signaling pathway and facilitating virus replication.

Keywords: RIG-I; innate immunity; interferons; paramyxovirus.

Figures

FIG 1
FIG 1
The NiV V protein interacts with TRIM25 in a manner dependent on the V protein C terminus. (A) HeLa cells transiently expressing HA-NiV V, TRIM25-V5, or both, were fixed, and proteins were visualized by immunofluorescence assay and confocal microscopy. Representative images of at least five fields are shown. Nuclei were stained with DAPI. Scale bars: 20 μm. Enlarged images show details of the area in the white square. (B) 293T cells were transfected with plasmids expressing HA-NiV V or HA-NiV Vn, together with full-length (FL) TRIM25-V5, GST-V5, or TRIM25 CCD-V5. V5-tagged proteins were immunoprecipitated, and the eluant was immunoblotted against V5, HA, and TRIM25. An immunoblot (IB) assay of WCE done to confirm expression is shown. (C) 293T cells were transfected with plasmids expressing HA-NiV V or HA-NiV W. HA-tagged proteins were immunoprecipitated and immunoblotted against endogenous TRIM25. Immunoblot assays of WCE done to confirm expression are shown. (D) 293T cells were transfected with a plasmid expressing GST-NiV V CTD and TRIM25-V5 or TRIM25 CCD-V5 or with the empty vector. NiV V CTD was immunoprecipitated with a specific V CTD antibody, and the eluant was immunoblotted against the V5 and GST tags. An immunoblot assay of WCE done to confirm expression is shown. All of the experiments shown are representative of three repeats.
FIG 2
FIG 2
The NiV V protein interacts with RIG-I in a manner dependent on the V C terminus. (A) 293T cells were transfected with a plasmid expressing HA-NiV V and Flag–RIG-I, Flag-MDA5, or GFP-Flag. Flag-tagged proteins were immunoprecipitated, and eluants were immunoblotted against the Flag and HA tags. An immunoblot assay of WCE done to confirm expression is shown. (B) 293T cells were transfected with plasmids expressing HA-NiV V or HA-NiV W. HA-tagged proteins were immunoprecipitated and immunoblotted (IB) with antibodies against endogenous RIG-I. Immunoblot assays of WCE done to confirm expression are shown. (C) 293T cells were transfected with a plasmid expressing HA-NiV V, GST-NiV-V CTD, or HA-NiV Vn and either Flag–RIG-I or GFP-Flag as indicated. Flag-tagged proteins were immunoprecipitated and immunoblotted against the HA and GST tags. Immunoblot assays of WCE done to confirm expression are shown. All of the experiments shown are representative of three repeats.
FIG 3
FIG 3
V protein interaction with RIG-I and TRIM25 is conserved across paramyxovirus genera. (A) 293T cells were cotransfected with plasmids expressing V5-TRIM25 and the HA-tagged construct indicated (MeV V, SeV V, NiV V, NiV W). V5-tagged protein was immunoprecipitated, and the eluant was immunoblotted (IB) against the HA tag and TRIM25. An immunoblot assay of WCE done to confirm expression is shown. (B) 293T cells were cotransfected with plasmids expressing V5-TRIM25 and the HA-tagged construct indicated (PIV5 V, SeV V, NiV W). HA-tagged proteins were immunoprecipitated, and the eluant was immunoblotted against TRIM25. Immunoblot assays of WCE done to confirm expression are shown. (C) 293T cells were cotransfected with plasmids expressing Flag–RIG-I, GFP-Flag, or the empty vector and an HA-tagged construct (MeV V, SeV V). Flag-tagged proteins were immunoprecipitated, and the eluant was immunoblotted against the HA tag. Immunoblot assays of WCE done to confirm expression are shown. (D) As in panel C, but cells were transfected with a plasmid expressing HA-PIV5 V or HA-NiV V. All of the experiments shown are representative of three repeats.
FIG 4
FIG 4
The MeV V protein colocalizes with and interacts with RIG-I in infected cells. (A, part I) Mock-infected A549 cells were fixed; incubated with antibodies against MeV V and RIG-I, TRIM25, or MAVS protein; and then stained with Alexa Fluor 647 (for V) and 594 (for RIG-I, TRIM25, or MAVS protein) (magenta and red, respectively). Nuclei were stained with DAPI (blue channel). Scale bars: 20 μm. (A, part II) A549 cells were infected with MeV-GFP (green channel) at an MOI of 0.05, and cells were fixed and then incubated with anti-MeV V, anti-RIG-I (top), anti-TRIM25 (middle), or anti-MAVS protein (bottom) antibodies. Alexa Fluor 647- and 594 (magenta and red, respectively)-conjugated secondary antibodies were used to detect anti-V and anti-RIG-I, -TRIM25, and -MAVS protein antibodies. Nuclei were stained with DAPI (blue channel). Scale bars: 20 μm. Representative images of at least five fields are shown. On the right are enlargements of the areas in the white squares and histograms of fluorescence intensity profiles. Arrows point out the colocalization of V (MeV) and RIG-I in the top panel and V (MeV) and TRIM25 in the middle panel, respectively. There is no colocalization in the MAVS protein-stained panel. (B) A549 cells were infected with MeV-GFP (MOI of 0.05), influenza A/PR/8/34 virus (MOI of 2), or a deltaNS1 PR8 virus (MOI of 2). The cells were lysed, RIG-I was immunoprecipitated, and the eluant was immunoblotted (IB) against MeV V, RIG-I, TRIM25, NS1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Immunoblot assays of WCE done to confirm expression are shown on the right. (C) As in panel B, but lysed cells were immunoprecipitated with an antibody against the MeV V protein. The eluant was immunoblotted against V, RIG-I, TRIM25, and GAPDH. Immunoblot assays of WCE done to confirm expression are shown on the right. All of the experiments shown are representative of three repeats.
FIG 5
FIG 5
V protein interaction with RIG-I is distinct from V protein interaction with other RLRs. (A) Schematic of RIG-I and MDA5 showing the tandem CARDs, helicase domain, and RIG-I regulatory domain (RD). Boxes highlight the RIG-IN construct used in some experiments, as well as the MDA5 minimal binding site for all paramyxovirus V proteins. (B) 293T cells were cotransfected with plasmids expressing Flag–RIG-I, Flag–RIG-IN, Flag–RIG-IC, or Flag-GFP or the empty vector and an HA-tagged construct (NiV V or MeV V). Flag-tagged proteins were immunoprecipitated, and the eluant was immunoblotted (IB) against the HA tag. Immunoblot assays of WCE done to confirm expression are shown. (C) 293T cells were cotransfected with plasmids expressing HA-NiV V and V5-tagged full-length TRIM25 or the BBOX, CCD, SPRY, or RING domain of TRIM25. V5-tagged proteins were immunoprecipitated, and the eluant was immunoblotted against the HA and V5 tags. An immunoblot assay of WCE done to confirm expression is shown. The divider line indicates removal of irrelevant lanes. Experiments shown in panels B and C are representative of three repeats. (D) Binding model based on the interaction data collected. V proteins interact with RIG-I via the V C terminus and the RIG-I CARDs (top) and with TRIM25 via the V C terminus and the TRIM25 SPRY domain (middle). As RIG-I and TRIM25 interact with each other via SPRY-CARD interaction, this suggests a possible three-partner interaction of the V C terminus, the TRIM25 SPRY domain, and the RIG-I CARDs (bottom). NTD, N-terminal domain.
FIG 6
FIG 6
BiFC analysis shows interaction of V proteins with RIG-I, TRIM25, and RIG-I/TRIM25 complexes. (A) Cartoon depicting a BiFC pair used in this study. The BiFC technique involves the fusion of a split YFP construct to each of two potential interaction partners. If the two partners interact, the constructs will reconstitute YFP and yield yellow fluorescence, i.e., BiFC. (B) HeLa cells were transfected with NiV W fused to the N terminus of YFP (YN-NiV W) and RIG-I fused to the C terminus of YFP (YC–RIG-I). Cells were stained with anti-RIG-I and anti-YN antibodies. Alexa Fluor-conjugated secondary antibodies were used to detect RIG-I (Alexa Fluor 647, purple) and YN-NiV W (Alexa Fluor 594, red). The lack of yellow fluorescence (YFP) indicates no interaction between the RIG-I and NiV W proteins. (C) HeLa cells were transfected with YN-NiV V and YC–RIG-I. Cells were stained with RIG-I and YN antibodies to detect RIG-I and V, respectively. (D) HeLa cells were transfected with NiV V fused to the C terminus of YFP (YC-NiV V) and TRIM25 fused to the N terminus of YFP (YN-TRIM25). Cells were stained with TRIM25 and HA antibodies to detect TRIM25 and V, respectively. The inset is an enlargement of the area in the white rectangle. (E) HeLa cells were transfected with a validated pair of BiFC plasmids for TRIM25 and RIG-I together with HA-NiV V. NiV V localization was visualized with an HA antibody. All of the images shown here are representative of the analysis of at least three different fields, depict a single focal plane, and were obtained by confocal microscopy. The images were collected with a 63×/1.4 oil objective. Scale bars: 20 μm. (F) 293T cells were transfected with plasmids expressing V5-TRIM25 or V5-GST and cotransfected with plasmids expressing HA–RIG-I, HA-NiV V, or both as indicated. V5-tagged proteins were immunoprecipitated, and the eluant was immunoblotted (IB) against the HA tag. Immunoblot assays of WCE done to confirm expression are shown. All of the experiments shown are representative of three repeats.
FIG 7
FIG 7
V proteins inhibit TRIM25 activation of RIG-I signaling. (A) 293T cells were transfected with an IFN-β firefly luciferase reporter plasmid; a constitutive Renilla luciferase reporter plasmid; and a construct expressing NiV V, NiV W, or dominant negative RIG-I (RIG-IC) or the empty vector. Sixteen hours later, they were stimulated with total RNA purified from influenza virus-infected A549 cells, where indicated. Normalized firefly luciferase activity was determined 36 to 40 h after DNA transfection and is expressed as the mean fold induction ± the standard deviation of triplicates. (B) 293T cells were transfected in triplicate with the reporter plasmids described in panel A and the indicated combinations of empty vector, RIG-IN, TRIM25, and NiV V plasmids. The proportion of NiV V plasmid was increased (from 250 to 1,000 ng) to explore dose-dependent effects. Cells were harvested 24 h posttransfection, and reporter activity was measured as described for panel A. (C) 293T cells were transfected in triplicate with the plasmids described in panel B, except that full-length RIG-I was substituted for RIG-IN. Cells were also transfected with an empty vector, NiV V, or GST plasmid as indicated. Reporter activity was measured as described for panel A. Significance of TRIM25 enhancement was determined by Student t test (**, P < 0.01). (D) 293T cells were transfected in triplicate with the plasmids described in panel B and then cotransfected with either the empty vector or a plasmid expressing NiV V, MeV V, or NiV V G121E. Reporter activity was measured as described for panel A. Data are presented as the percentage by which TRIM25 enhances RIG-IN-mediated IFN-β promoter induction under each condition. The significance of TRIM25 enhancement with or without V was determined by Student t test (***, P < 0.001).
FIG 8
FIG 8
The ability of V proteins to interact with and inhibit RIG-I does not require other RLRs. (A) 293T cells were pretreated with IFN-α to induce RLR expression and then transfected with siRNA targeting LGP2 or MDA5 or with a nontargeting control (CTRL). Cells were harvested 48 h later and analyzed for MDA5 and LGP2 mRNA levels by qRT-PCR. Error bars indicate mean values of triplicates ± the standard deviations. (B) 293T cells were treated with siRNA as described for panel A but without exogenous IFN. At 48 h after siRNA transfection, cells were cotransfected with plasmids expressing Flag–RIG-IN or Flag-GFP and HA-NiV V. Flag-tagged proteins were immunoprecipitated, and the eluant was immunoblotted (IB) against the HA and Flag tags. An immunoblot assay of WCE against HA was performed to verify NiV V expression. The protein bands immunoprecipitated were quantified, and the efficiency of the V–RIG-IN interaction was determined by normalizing the V protein band to the RIG-IN band for each siRNA condition. (C) 293T cells were transfected with no siRNA, nontargeting siRNA, siMDA5, siLGP2, or siMAVS. At 48 h later, cells were transfected in triplicate with an IFN-β firefly luciferase reporter plasmid, a constitutive Renilla luciferase reporter plasmid, and the combinations of the empty vector, RIG-IN, TRIM25, or NiV V indicated. Cells were harvested 24 h after DNA transfection, and reporter activity was measured. Error bars indicate mean values of triplicates ± the standard deviations. Data were analyzed by two-way ANOVA to determine if individual siRNA treatments had significant global effects on reporter induction (in the absence of NiV V) and then to determine if there were any significant changes in the ability of NiV V to inhibit IFN-β–Luc induction by RIG-IN–TRIM25 under each siRNA condition. ***, P < 0.001. (D) 293T cells were transfected with the reporter plasmids as for panel C, as well as WT NiV V, NiV V R409A, NiV W, or RIG-IC. Cells were transfected with RNA from influenza virus-infected A549 cells 16 h after DNA transfection. Unstimulated cells received an equal amount of RNA from mock-infected A549 cells. Cells were harvested 36 to 40 h after DNA transfection, and reporter activity was measured. Error bars indicate mean values of triplicates ± the standard deviations.
FIG 9
FIG 9
V proteins inhibit RIG-I ubiquitination by TRIM25. (A) 293T cells were transfected with plasmids expressing HA-Ub, V5-TRIM25, GST, or GST–RIG-IN as indicated. Cells were also cotransfected with the empty vector or a plasmid expressing NiV V. A GST pulldown assay was performed, and eluants were immunoblotted (IB) against GST to show covalent ubiquitin-modified species of GST–RIG-IN as indicated. Immunoblot assays of WCE were performed to verify expression. (B) 293T cells were transfected as described for panel A but cotransfected with plasmids expressing HA-NiV V, HA-PIV5 V, and HA-MeV V as indicated. A GST pulldown assay was performed, and eluants were immunoblotted against GST to show covalent ubiquitin-modified species of GST–RIG-IN as indicated. Immunoblot assays of WCE were performed to verify expression. All of the experiments shown are representative of three repeats.
FIG 10
FIG 10
V protein inhibition of ubiquitination correlates with loss of RIG-I–MAVS protein interaction. (A) 293T cells were transfected with plasmids expressing HA-Ub, V5-TRIM25, GST, or GST–RIG-IN as indicated. Cells were also cotransfected with the empty vector or a plasmid expressing NiV V. A GST pulldown assay was performed, and eluants were immunoblotted (IB) against endogenous MAVS protein and against GST to assess pulldown efficiency, as well as show covalent ubiquitin-modified species of GST–RIG-IN. Immunoblot assays of WCE were performed to verify expression. (B) 293T cells were transfected as described for panel A, except that cells were cotransfected with a plasmid expressing NiV V, HA-PIV5 V, or HA-MeV V. Eluants were immunoblotted against endogenous MAVS protein and GST to assess pulldown efficiency, as well as show covalent ubiquitin-modified species of GST–RIG-IN. Immunoblot assays of WCE were performed to verify expression. All of the experiments shown are representative of three repeats.
FIG 11
FIG 11
Model showing how V proteins inhibit RIG-I signaling via antagonism of TRIM25-mediated RIG-I ubiquitination. Paramyxovirus V proteins disrupt RIG-I activation by binding to TRIM25 and RIG-I via their CTDs. This prevents RIG-I ubiquitination by TRIM25 such that RIG-I signaling does not progress to interaction with MAVS protein. RD, regulatory domain.

Similar articles

See all similar articles

Cited by 12 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback