Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Apr;88(8):4572-85.
doi: 10.1128/JVI.03021-13. Epub 2014 Jan 29.

Hijacking of RIG-I signaling proteins into virus-induced cytoplasmic structures correlates with the inhibition of type I interferon responses

Felix W Santiago  1 , Lina M Covaleda, Maria T Sanchez-Aparicio, Jesus A Silvas, Ana C Diaz-Vizarreta, Jenish R Patel, Vsevolod Popov, Xue-jie Yu, Adolfo García-Sastre, Patricia V Aguilar
Affiliations

Hijacking of RIG-I signaling proteins into virus-induced cytoplasmic structures correlates with the inhibition of type I interferon responses

Felix W Santiago et al. J Virol. 2014 Apr.

Abstract

Recognition of viral pathogens by the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family results in the activation of type I interferon (IFN) responses. To avoid this response, most viruses have evolved strategies that target different essential steps in the activation of host innate immunity. In this study, we report that the nonstructural protein NSs of the newly described severe fever with thrombocytopenia syndrome virus (SFTSV) is a potent inhibitor of IFN responses. The SFTSV NSs protein was found to inhibit the activation of the beta interferon (IFN-β) promoter induced by viral infection and by a RIG-I ligand. Astonishingly, we found that SFTSV NSs interacts with and relocalizes RIG-I, the E3 ubiquitin ligase TRIM25, and TANK-binding kinase 1 (TBK1) into SFTSV NSs-induced cytoplasmic structures. Interestingly, formation of these SFTSV NSs-induced structures occurred in the absence of the Atg7 gene, a gene essential for autophagy. Furthermore, confocal microscopy studies revealed that these SFTSV NSs-induced structures colocalize with Rab5 but not with Golgi apparatus or endoplasmic reticulum markers. Altogether, the data suggest that sequestration of RIG-I signaling molecules into endosome-like structures may be the mechanism used by SFTSV to inhibit IFN responses and point toward a novel mechanism for the suppression of IFN responses.

Importance: The mechanism by which the newly described SFTSV inhibits host antiviral responses has not yet been fully characterized. In this study, we describe the redistribution of RIG-I signaling components into virus-induced cytoplasmic structures in cells infected with SFTSV. This redistribution correlates with the inhibition of host antiviral responses. Further characterization of the interplay between the viral protein and components of the IFN responses could potentially provide targets for the rational development of therapeutic interventions.

PubMed Disclaimer

Figures

FIG 1
FIG 1
SFTSV NSs inhibits type I IFN. (A and B) HEK293T cells were transfected with an IFN-β-CAT plasmid, along with the indicated expression plasmids. At 24 h posttransfection, cells were infected with SeV (A) or transfected with dsRNA (B), and at 24 h after infection or dsRNA treatment, cell lysates were collected and assessed for CAT activity. Error bars represent the standard deviation of the mean percentage in three independent experiments. Asterisks specify statistically significant differences (P < 0.05) between the indicated group and the pCAGGS-treated group. (C) HeLa cells were transfected with a plasmid expressing mCherry or SFTSV NSs fused to mCherry and infected with SeV. At 24 h postinfection, supernatants were collected and assessed for IFN-β by ELISA. Error bars represent the standard deviation of the mean percentage in three independent experiments. Asterisks indicate statistically significant differences (P < 0.05) in the production of IFN-β between the mCherry- and SFTSV NSs-mCherry-expressing cells. (D) HEK293T cells were transfected with plasmids carrying IRF3 and the indicated HA-tagged expression plasmids. At 24 h after transfection, cells were infected with SeV, and 10 h later cell lysates were collected and assayed for IRF3 phosphorylation by using phosphor and total IRF3 antibodies. Expression of the viral proteins was confirmed by using antibodies against the HA tag, and loading was verified with antibodies against β-actin. n.d., not detected.
FIG 2
FIG 2
SFTSV NSs inhibits activation of IFN-β induced by different signaling factors. HEK293T cells were transfected with the indicated viral protein expression plasmids and an IFN-β–CAT plasmid, along with the RIG-I N terminus (RIG-I N) (A), MAVS (B), TBK1 (C), or IRF3-5D (D), and at 16 h after transfection, cell lysates were collected and assessed for CAT activity. Error bars represent the standard deviation of the mean percentage in three independent experiments. Asterisks specify statistically significant differences (P < 0.05) between the indicated group and the pCAGGS-treated group. Expression of the proteins was confirmed by Western blotting using anti-FLAG, anti-HA, anti-IRF3, and anti-β-tubulin antibodies.
FIG 3
FIG 3
SFTSV NSs interacts with TBK1, RIG-I, and TRIM25. (A) HEK293T cells were transfected with the indicated expression plasmids, and at 24 h after transfection, cell lysates were collected and subjected to immunoprecipitation (IP) by using anti-HA antibodies. Immunoprecipitates were analyzed by immunoblotting (IB) using anti-FLAG and anti-HA antibodies. Whole-cell lysates (WCL) were immunoblotted by using anti-FLAG and anti-HA antibodies. (B) HEK293T cells were transfected with the indicated expression plasmids, and at 24 h after transfection, cell lysates were collected and subjected to immunoprecipitation by using anti-HA antibodies. Immunoprecipitates were analyzed by immunoblotting using anti-FLAG, anti-V5, and anti-HA antibodies. Whole-cell lysates were immunoblotted by using anti-FLAG, anti-V5, and anti-HA antibodies. Numbers to the left of the gels in panels A and B are molecular masses (in kilodaltons). (C) Densitometry analysis of immunoprecipitated RIG-I for panel B. The band signal intensity of the immunoprecipitated RIG-I protein was normalized to the signal intensity of the RIG-I protein expressed in the whole-cell lysate. Signal intensities were obtained by using Image Studio Lite software.
FIG 4
FIG 4
SFTSV NSs targets the RIG-I/TRIM25 complex to cytoplasmic structures. (A) HeLa cells were transfected with a plasmid carrying SFTSV NSs fused to mCherry, and at 24 h after transfection, cells were fixed and analyzed by confocal microscopy (top). In another set of experiments, the SFTSV NSs-induced structures were purified from cells stably expressing SFTSV NSs-mCherry using serial centrifugation and OptiPrep density gradient centrifugation and subjected to immunogold labeling and electron microscopy analyses (bottom). Red arrowhead, SFTSV NSs protein staining. (B) HeLa cells were transfected with BiFC RIG-I/TRIM25 (left) or BiFC RIG-I/MAVS (right) construct pairs along with an empty plasmid or a plasmid carrying SFTSV NSs-HA. Transfected cells were incubated for 24 h at 37°C and then incubated at 30°C for 2 h to promote fluorophore maturation. Cells were fixed and analyzed by confocal microscopy. Rabbit anti-HA and Alexa Fluor 633 were used for detection of SFTSV NSs. Cell nuclei were visualized by using Hoechst dye.
FIG 5
FIG 5
SFTSV NSs targets TBK1, RIG-I, and TRIM25 to cytoplasmic structures. (A) HeLa cells were transfected with a plasmid carrying YFP–RIG-I (A), TBK1-FLAG (B), or YFP-TRIM25 (C) along with an empty vector or a plasmid carrying SFTSV NSs-HA. Then, at 24 h after transfection, cells were fixed, stained with anti-FLAG and anti-HA antibodies, and analyzed by confocal microscopy. Cell nuclei were visualized by using Hoechst dye. Arrows, sections selected for the insets.
FIG 6
FIG 6
RIG-I and TRIM25 colocalize in SFTSV NSs-induced cytoplasmic structures following SFTSV infection. (A) HeLa and Vero cells were infected with SFTSV (MOI = 0.5), and at 24 h postinfection, cells were fixed and immunofluorescence staining was performed with antibodies against SFTSV NSs. Cell nuclei were visualized by using Hoechst dye. Results from HeLa cells are shown. (B and D) HeLa cells were transfected with YFP-RIG-I (B), YFP-TRIM25 (C), or TBK1-FLAG (D) expression plasmids and then mock infected or infected with SFTSV (MOI = 0.5). Then, at 24 h postinfection, cells were fixed and immunofluorescence staining was performed by using antibodies against SFTSV NSs and FLAG. Cell nuclei were visualized by using Hoechst dye. Arrows, sections selected for the insets.
FIG 7
FIG 7
SFTSV NSs-induced cytoplasmic structures colocalize with LC3 but are not autophagosomes. (A) HeLa cells were transfected with a plasmid carrying GFP-LC3, and at 24 h after transfection, cells were mock infected or infected with SFTSV (MOI = 0.5). Cells were fixed, and immunofluorescence staining was performed by using antibodies against SFTSV NSs. Cell nuclei were visualized by using Hoechst dye. (B and C) Atg7+/+ and Atg7+/+ MEF cells were transfected with plasmids carrying YFP-TRIM25 (B) or TBK1-FLAG (C), together with a plasmid carrying SFTSV NSs-HA. At 24 h posttransfection, cells were fixed and immunofluorescence staining was performed with antibodies against SFTSV NSs and FLAG. Cell nuclei were visualized by using Hoechst dye. Arrows, sections selected for the insets.
FIG 8
FIG 8
The early endosomal marker Rab5 is associated with the SFTSV NSs-induced cytoplasmic vesicles. HeLa cells were transfected with plasmid carrying the Golgi apparatus marker B4GalT1-RFP (A), the endosomal marker Rab5-RFP (B), or the a dominant negative (DN) variant of Rab5 fused to mCherry (C). At 24 h posttransfection, cells were infected with SFTSV (MOI = 0.5). At 24 h postinfection, the cells were fixed, stained with anti-SFTSV NSs antibody, and analyzed by confocal microscopy. Cell nuclei were visualized by using Hoechst dye. Arrows, sections selected for the insets.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Takeuchi O, Akira S. 2010. Pattern recognition receptors and inflammation. Cell 140:805–820. 10.1016/j.cell.2010.01.022 - DOI - PubMed
    1. Akira S, Uematsu S, Takeuchi O. 2006. Pathogen recognition and innate immunity. Cell 124:783–801. 10.1016/j.cell.2006.02.015 - DOI - PubMed
    1. Meylan E, Tschopp J, Karin M. 2006. Intracellular pattern recognition receptors in the host response. Nature 442:39–44. 10.1038/nature04946 - DOI - PubMed
    1. Yoneyama M, Fujita T. 2007. RIG-I family RNA helicases: cytoplasmic sensor for antiviral innate immunity. Cytokine Growth Factor Rev. 18:545–551. 10.1016/j.cytogfr.2007.06.023 - DOI - PubMed
    1. Baum A, García-Sastre A. 2010. Induction of type I interferon by RNA viruses: cellular receptors and their substrates. Amino Acids 38:1283–1299. 10.1007/s00726-009-0374-0 - DOI - PMC - PubMed

Publication types

MeSH terms