The C-terminal 42 residues of the Tula virus Gn protein regulate interferon induction - PubMed (original) (raw)

The C-terminal 42 residues of the Tula virus Gn protein regulate interferon induction

Valery Matthys et al. J Virol. 2011 May.

Abstract

Hantaviruses primarily infect the endothelial cell lining of capillaries and cause two vascular permeability-based diseases. The ability of pathogenic hantaviruses to regulate the early induction of interferon determines whether hantaviruses replicate in endothelial cells. Tula virus (TULV) and Prospect Hill virus (PHV) are hantaviruses which infect human endothelial cells but fail to cause human disease. PHV is unable to inhibit early interferon (IFN) responses and fails to replicate within human endothelial cells. However, TULV replicates successfully in human endothelial cells, suggesting that TULV is capable of regulating cellular IFN responses. We observed a >300-fold reduction in the IFN-stimulated genes (ISGs) MxA and ISG56 following TULV versus PHV infection of endothelial cells 1 day postinfection. Similar to results with pathogenic hantaviruses, expressing the TULV Gn protein cytoplasmic tail (Gn-T) blocked RIG-I- and TBK1-directed transcription from IFN-stimulated response elements (ISREs) and IFN-β promoters (>90%) but not transcription directed by constitutively active IFN regulatory factor-3 (IRF3). In contrast, expressing the PHV Gn-T had no effect on TBK1-induced transcriptional responses. Analysis of Gn-T truncations demonstrated that the C-terminal 42 residues of the Gn-T (Gn-T-C42) from TULV, but not PHV, inhibited IFN induction >70%. These findings demonstrate that the TULV Gn-T inhibits IFN- and ISRE-directed responses upstream of IRF3 at the level of the TBK1 complex and further define a 42-residue domain of the TULV Gn-T that inhibits IFN induction. In contrast to pathogenic hantavirus Gn-Ts, the TULV Gn-T lacks a C-terminal degron domain and failed to bind tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3), a TBK1 complex component required for IRF3 activation. These findings indicate that the nonpathogenic TULV Gn-T regulates IFN induction but accomplishes this via unique interactions with cellular TBK1 complexes. These findings fundamentally distinguish nonpathogenic hantaviruses, PHV and TULV, and demonstrate that IFN regulation alone is insufficient for hantaviruses to cause disease. Yet regulating the early IFN response is necessary for hantaviruses to replicate within human endothelial cells and to be pathogenic. Thus, in addition to IFN regulation, hantaviruses contain discrete virulence determinants which permit them to be human pathogens.

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Figures

Fig. 1.

Hantavirus infection of endothelial cells. (A) Hantavirus replication in HUVECs and Vero E6 cells. HUVECS and Vero E6 cells were infected with PHV and TULV at a multiplicity of infection (MOI) of 1. Viral titers were analyzed 1 to 3 days postinfection by infectious focus assay as previously described (7). (B) HUVECs were either infected with PHV or TULV (MOI of 1) or mock infected. Cells were methanol fixed at 1 to 3 days postinfection, and infected cells were detected by immunostaining using an antinucleocapsid antibody as previously described (7).

Fig. 2.

Induction of MxA and ISG56 following TULV and PHV infection. Induction of the ISGs MxA and ISG56 was monitored by quantitative reverse transcription-PCR. Endothelial cells were infected with either PHV or TULV at an MOI of 1. One to 3 days postinfection, total RNAs were isolated, and MxA and ISG56 mRNA levels were determined in duplicate using quantitative reverse transcription-PCR normalized to GAPDH mRNA levels and relative to mock-infected controls.

Fig. 3.

TULV Gn-T regulates RIG-I- and TBK1-directed ISRE and IFN-β transcription. HEK293 cells were transfected with an ISRE-driven luciferase reporter construct (A, B, and D) or an IFN-β-luciferase reporter construct (C) in the presence or absence of an activating (N)RIG-I (A), TBK1 (B and C), or IRF3-5D (D) expression vector. Cells were cotransfected with NY-1V Gn-T, PHV Gn-T, and TULV Gn-T (A and D) or increasing amounts of plasmid (0.5, 1, and 2 μg) expressing PHV Gn-T and TULV Gn-T (B and C) and the control empty vector (pBIND) to maintain constant DNA transfection levels. NY-1V Gn-T expression was used as a positive control. At 2 days posttransfection, cells were lysed, and luciferase activity was measured and normalized to Renilla luciferase levels. Luciferase activity is reported as the fold increase compared to that of controls lacking (N)RIG-I, TBK1, or IRF3-5D. Assays were performed in triplicate with similar results in at least three separate experiments. Western blot (WB) analysis indicates equal expression of TULV Gn-T and PHV Gn-T (A and B) and of TBK1 cotransfected with TULV Gn-T and PHV Gn-T (B). Cells were lysed at 48 h posttransfection, protein amounts were determined using bicinchoninic acid protein assay, and equal amounts were loaded onto a 10% SDS-PAGE gel. Proteins were detected by Western blotting using anti-Gal4 or anti-myc (Santa Cruz Biotechnology) and anti-mouse HRP-conjugated secondary antibody. Blots were stripped and reprobed with anti-β-actin (Sigma). IB, immunoblot.

Fig. 4.

TULV C42 inhibits TBK1-directed ISRE and IFN-β activation. (A) HEK293 cells were transfected with an ISRE-driven luciferase reporter construct in the presence or absence of an activating TBK1 expression vector. Cells were cotransfected with plasmids expressing PHV, TULV Gn-T-C42, or an empty vector control (pBIND expressing the Gal4 tag) to maintain DNA transfection levels. (B) HEK293 cells were transfected with an IFN-β-driven luciferase reporter construct and with or without an activating TBK1 expression vector. Cells were cotransfected with plasmids expressing PHV Gn-T-C42, increasing amounts of TULV Gn-T-C42, or an empty vector control (pBIND) to maintain DNA transfection levels. Two days posttransfection, luciferase activity was measured as described in the legends of previous figures, and values are reported as the fold increase compared to controls lacking TBK1. Assays were performed in triplicate with similar results in at least two separate experiments. Western blot (WB) analysis showing equal expression of TULV Gn-T-C42 and PHV Gn-T C42 (B) was performed. Cells were lysed at 48 h posttransfection, and equal amounts were loaded onto a 12% SDS-PAGE gel. Proteins were detected by Western blotting using anti-Gal4 (Santa Cruz Biotechnology) and anti-mouse HRP-conjugated secondary antibody. Blots were stripped and reprobed with anti-β-actin (Sigma).

Fig. 5.

TULV Gn-T inhibition of TBK1- and TRAF2-directed NF-κB activation. HEK293 cells transfected with an NF-κB promoter-luciferase reporter construct in the presence or absence of activating TBK1 (A) or TRAF2 (B) expression vectors and plasmids expressing NY-1V (positive control) or PHV or TULV Gn-T. Luciferase activity within lysates was determined at 48 h posttransfection and normalized to Renilla luciferase activity, and values are reported as the fold increase compared to that of controls lacking TBK1 or TRAF2. Assays were performed in triplicate with similar results in at least three separate experiments. Western Blot (WB) analysis indicates equal expression of TRAF2 in the presence of pBIND, TULV Gn-T and PHV Gn-T (B). Cells were lysed at 48 h posttransfection, and equal amounts were loaded onto a 10% SDS-PAGE gel. Proteins were detected by Western blotting using anti-Flag M2 (Stratagene) and anti-mouse HRP-conjugated secondary antibody. Blots were stripped and reprobed with anti-β-actin (Sigma).

Fig. 6.

The TULV Gn-T does not interact with TRAF3 N415. Cos7 cells were transfected with pBIND or pBIND-NY-1V Gn-T, -TULV Gn-T, and -PHV Gn-T and pRK-TRAF3 N415 as previously described (2). Briefly, at 6 h prior to lysis, transfected cells were treated with MG132 or mock treated as indicated, and proteins were analyzed at 48 h posttransfection. Hantavirus Gn-Ts were immunoprecipitated (IP) using an anti-Gal4 antibody (Santa Cruz Biotechnology) and protein A/G Plus-agarose beads (Santa Cruz Biotechnology). Coimmunoprecipitated TRAF3 N415 was detected by Western blotting using an anti-Flag M2 antibody (Stratagene). TRAF3 N415 expression was analyzed by anti-Flag Western blotting. IB, immunoblotting; *, IgG heavy chain.

References

1. Alff P. J., et al. 2006. The pathogenic NY-1 hantavirus G1 cytoplasmic tail inhibits RIG-I- and TBK-1-directed interferon responses. J. Virol. 80:9676–9686 - PMC - PubMed
1. Alff P. J., Sen N., Gorbunova E., Gavrilovskaya I. N., Mackow E. R. 2008. The NY-1 hantavirus Gn cytoplasmic tail coprecipitates TRAF3 and inhibits cellular interferon responses by disrupting TBK1-TRAF3 complex formation. J. Virol. 82:9115–9122 - PMC - PubMed
1. Borges E., Jan Y., Ruoslahti E. 2000. Platelet-derived growth factor receptor beta and vascular endothelial growth factor receptor 2 bind to the beta 3 integrin through its extracellular domain. J. Biol. Chem. 275:39867–39873 - PubMed
1. Brakenhielm E. 2007. Substrate matters: reciprocally stimulatory integrin and VEGF signaling in endothelial cells. Circ. Res. 101:536–538 - PubMed
1. Clement J. P. 2003. Hantavirus. Antiviral Res. 57:121–127 - PubMed

The C-terminal 42 residues of the Tula virus Gn protein regulate interferon induction - PubMed (original) (raw)