Hantaan virus nucleocapsid protein binds to importin alpha proteins and inhibits tumor necrosis factor alpha-induced activation of nuclear factor kappa B

Abstract

Hantaviruses such as Hantaan virus (HTNV) and Andes virus cause two human diseases, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome, respectively. For both, disease pathogenesis is thought to be immunologically mediated and there have been numerous reports of patients with elevated levels of proinflammatory and inflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), in their sera. Multiple viruses have developed evasion strategies to circumvent the host cell inflammatory process, with one of the most prevalent being the disruption of nuclear factor kappa B (NF-kappaB) activation. We hypothesized that hantaviruses might also moderate host inflammation by interfering with this pathway. We report here that the nucleocapsid (N) protein of HTNV was able to inhibit TNF-alpha-induced activation of NF-kappaB, as measured by a reporter assay, and the activation of endogenous p65, an NF-kappaB subunit. Surprisingly, there was no defect in the degradation of the inhibitor of NF-kappaB (IkappaB) protein, nor was there any alteration in the level of p65 expression in HTNV N-expressing cells. However, immunofluorescence antibody staining demonstrated that cells expressing HTNV N protein and a green fluorescent protein-p65 fusion had limited p65 nuclear translocation. Furthermore, we were able to detect an interaction between HTNV N protein and importin alpha, a nuclear import molecule responsible for shuttling NF-kappaB to the nucleus. Collectively, our data suggest that HTNV N protein can sequester NF-kappaB in the cytoplasm, thus inhibiting NF-kappaB activity. These findings, which were obtained using cells transfected with cDNA representing the HTNV N gene, were confirmed using HTNV-infected cells.

FIG. 1.

Effect of HTNV N protein on NF-κB gene expression. (A) 293T cells were cotransfected with 500 ng of pNF-κB-hrGFP and 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M. After 24 h, cells were incubated in medium with or without TNF-α (0 to 100 ng/ml) for 4 h. As a positive control for inhibition in our assay, cells transfected with only 500 ng of pNF-κB-hrGFP were pretreated with 50 μM MG132 for 2 h before the addition of TNF-α and throughout the experiment. (B) Cells were cotransfected with 500 ng of pNF-κB-hrGFP and 5 to 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M or transfected with 500 ng of pNF-κB-hrGFP and treated with 0 to 50 μM MG132. After 24 h, cells were incubated in medium with or without TNF-α (10 ng/ml) for 4 h. Following a 4-h treatment, medium was removed from all wells and replaced with medium lacking TNF-α for an additional 24 h. (C) Cells were cotransfected with 500 ng of pCAGGS-GFP and 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M. After 24 h, cells were incubated in medium with TNF-α (10 ng/ml) for an additional 24 h. GFP expression was examined by fluorescence microscopy.

FIG. 2.

Endogenous NF-κB transcription activation in cells expressing HTNV N protein. A549 cells were transfected with 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M for 24 h. After treatment of the cells with 50 ng/ml of TNF-α for 15 min, cytoplasmic and nuclear extracts from lysates were prepared. (A) Nuclear extracts were allowed to bind to NF-κB consensus sequence oligonucleotides on 96-well plates and then probed with antibodies specific for NF-κB p65 or NF-κB p50. The absorbance reading for each sample was determined using a spectrophotometer. OD, optical density; MUT oligo, mutated consensus oligonucleotide; WT oligo, wild-type consensus oligonucleotide. (B) Cytoplasmic extracts were used for immunoblotting to detect HTNV N protein and GAPDH. Each point represents an average ± standard deviation of results for six samples. The statistical significance of results for HTNV S with TNF and MG132 with TNF was determined by comparing the results to those for the empty vector with TNF. Asterisks indicate significant differences (P < 0.05) as determined by Student's t test. +, with; −, without.

FIG. 3.

Examination of NF-κB p50 and p65 levels and TNF-α-induced IκBα degradation in HTNV N-expressing cells. A549 cells were transfected with 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M for 24 h. To induce the degradation of IκBα, cells were treated with 50 ng/ml of TNF-α for 15 min, and lysates were prepared for immunoblotting. For uninduced samples, cells were left untreated. Proteins were transferred onto PVDF membranes and probed with antibodies against IκBα, p50, p65, HTNV N protein, or GAPDH. +, with; −, without.

FIG. 4.

Translocation of NF-κB p65 in HTNV N-expressing cells. 293T cells were cotransfected with 500 ng of pCAGGS-GFP-p65 and 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M for 24 h. After incubation, cells were fixed and stained with antibodies against HTNV N or Gc protein (red) and with DAPI to highlight nuclei (blue). Arrows indicate areas of protein accumulation.

FIG. 5.

Analysis of HTNV N protein interaction with importin α proteins. A549 cells were cotransfected with 0 or 1.25 μg of a FLAG-importin α1 (Iα1), FLAG-importin α2 (Iα2), FLAG-importin α3 (Iα3), or FLAG-importin α4 (Iα4) construct and 1.25 μg of pWRG7077-Empty or pWRG7077-HTNV-S plasmid for 24 h and left untreated (−TNF-α) or treated with 50 ng/ml TNF-α (+TNF-α) for 15 min. Immunoprecipitations (IP) were performed with anti-HTNV N protein antibody bound to Sepharose beads. The single asterisks indicate the location of HTNV N, whereas double asterisks identify the heavy chain. Immunoprecipitates and whole-cell lysates (WCL) were analyzed by Western blotting for the expression of importin α proteins and HTNV N protein. −, no importin α.

FIG. 6.

Examination of TNF-α-induced NF-κB p65 nuclear translocation and degradation of IκBα in HTNV-infected cells. A549 cells were mock infected (M) or infected with HTNV (V) at an MOI of 5. On day 5, mock- and HTNV-infected cells were left untreated (−) or treated (+) with 50 ng/ml of TNF-α for 15 min. (A) After fixation, cells were stained with antibodies against NF-κB p65 (green) and HTNV N protein (red) and stained with DAPI to highlight nuclei (blue). (B) Cell lysates were separated into cytoplasmic and nuclear fractions. (C) Total cell lysates were prepared for immunoblotting, and proteins were transferred onto PVDF membranes. Blots were probed with antibodies against IκBα, p50, p65, HTNV N protein, GAPDH, or histone H2B.