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Chemoproteomic profiling identifies changes in DNA-PK as markers of early dengue virus infection.

ABSTRACT: Many cellular factors are regulated via mechanisms affecting protein conformation, localization, and function that may be undetected by most commonly used RNA- and protein-based profiling methods that monitor steady-state gene expression. Mass-spectrometry-based chemoproteomic profiling provides alternatives for interrogating changes in the functional properties of proteins that occur in response to biological stimuli, such as viral infection. Taking dengue virus 2 (DV2) infection as a model system, we utilized reactive ATP- and ADP-acyl phosphates as chemical proteomic probes to detect changes in host kinase function that occur within the first hour of infection. The DNA-dependent protein kinase (DNA-PK) was discovered as a host enzyme with significantly elevated probe labeling within 60 min of DV2 infection. Increased probe labeling was associated with increased DNA-PK activity in nuclear lysates and localization of DNA-PK in nucleoli. These effects on DNA-PK were found to require a postfusion step of DV2 entry and were recapitulated by transfection of cells with RNA corresponding to stem loop B of the DV2 5' untranslated region. Upon investigation of the potential downstream consequences of these phenomena, we detected a modest but significant reduction in the interferon response induced by DV2 in cells partially depleted of the Ku80 subunit of DNA-PK. These findings identify changes in DNA-PK localization and activity as very early markers of DV2 infection. More broadly, these results highlight the utility of chemoproteomic profiling as a tool to detect changes in protein function associated with different cell states and that may occur on very short time scales.

SUBMITTER: Vetter ML

PROVIDER: S-EPMC3528803 | biostudies-other | 2012 Dec

REPOSITORIES: biostudies-other

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Chemoproteomic profiling identifies changes in DNA-PK as markers of early dengue virus infection.

Vetter Michael L ML Rodgers Mary A MA Patricelli Matthew P MP Yang Priscilla L PL

ACS chemical biology 20121002 12

Many cellular factors are regulated via mechanisms affecting protein conformation, localization, and function that may be undetected by most commonly used RNA- and protein-based profiling methods that monitor steady-state gene expression. Mass-spectrometry-based chemoproteomic profiling provides alternatives for interrogating changes in the functional properties of proteins that occur in response to biological stimuli, such as viral infection. Taking dengue virus 2 (DV2) infection as a model sys ...[more]

PMID: 22999307

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Project description:BackgroundInfection by chikungunya (CHIKV) and dengue virus (DENV) can cause a wide spectrum of clinical features, many of which are undifferentiated. Cytokines, which broadly also include chemokines and growth factors, have been shown to play a role in protective immunity as well as DENV and CHIKV pathogenesis. However, differences in cytokine response to both viruses remain poorly understood, especially in patients from countries where both viruses are endemic. Our study is therefore aimed to provide a comparative profiling of cytokine response induced by acute DENV and CHIKV infections in patients with similar disease stages and in experimental in vitro infections.MethodsBy using multiplex immunoassay, we compared host cytokine profiles between acute CHIKV and DENV infections by analysing serum cytokine levels of IL-1α, IL-4, IL-5, IL-8, IL-13, RANTES, MCP-3, eotaxin, PDGF-AB/BB, and FGF-2 from the sera of acute chikungunya and dengue fever patients. We further investigated the cytokine profile responses using experimental in vitro CHIKV and DENV infections of peripheral blood mononuclear cells (PBMCs).ResultsWe found that both CHIKV and DENV-infected patients had an upregulated level of IL-8 and IL-4, with the highest IL-4 level observed in DENV-2 infected patients. Higher IL-8 level was also correlated with lower platelet count in dengue patients. IL-13 and MCP-3 downregulation was observed only in chikungunya patients, while conversely PDGF-AB/BB and FGF-2 downregulation was unique in dengue patients. Age-associated differential expression of IL-13, MCP-3, and IL-5 was also observed, while distinct kinetics of IL-4, IL-8, and FGF-2 expression between CHIKV and DENV-infected patients were identified. Furthermore, the unique pattern of IL-8, IL-13 and MCP-3, but not IL-4 expression was also recapitulated using experimental in vitro infection in PBMCs.ConclusionsTaken together, our study identified common cytokine response profile characterized by upregulation of IL-8 and IL-4 between CHIKV and DENV infection. Downregulation of IL-13 and MCP-3 was identified as a unique cytokine response profile of acute CHIKV infection, while distinct downregulation of PDGF-AB/BB and FGF-2 characterized the response from acute DENV infection. Our study provides an important overview of the host cytokine responses between CHIKV and DENV infection, which is important to further understand the mechanism and pathology of these diseases.

Project description:BACKGROUND: Despite the seriousness of dengue-related disease, with an estimated 50-100 million cases of dengue fever and 250,000-500,000 cases of dengue hemorrhagic fever/dengue shock syndrome each year, a clear understanding of dengue pathogenesis remains elusive. Because of the lack of a disease model in animals and the complex immune interaction in dengue infection, the study of host response and immunopathogenesis is difficult. The development of genomics technology, microarray and high throughput quantitative PCR have allowed researchers to study gene expression changes on a much broader scale. We therefore used this approach to investigate the host response in dengue virus-infected cell lines and in patients developing dengue fever. METHODOLOGY/PRINCIPAL FINDINGS: Using microarray and high throughput quantitative PCR method to monitor the host response to dengue viral replication in cell line infection models and in dengue patient blood samples, we identified differentially expressed genes along three major pathways; NF-kappaB initiated immune responses, type I interferon (IFN) and the ubiquitin proteasome pathway. Among the most highly upregulated genes were the chemokines IP-10 and I-TAC, both ligands of the CXCR3 receptor. Increased expression of IP-10 and I-TAC in the peripheral blood of ten patients at the early onset of fever was confirmed by ELISA. A highly upregulated gene in the IFN pathway, viperin, was overexpressed in A549 cells resulting in a significant reduction in viral replication. The upregulation of genes in the ubiquitin-proteasome pathway prompted the testing of proteasome inhibitors MG-132 and ALLN, both of which reduced viral replication. CONCLUSION/SIGNIFICANCE: Unbiased gene expression analysis has identified new host genes associated with dengue infection, which we have validated in functional studies. We showed that some parts of the host response can be used as potential biomarkers for the disease while others can be used to control dengue viral replication, thus representing viable targets for drug therapy.

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Chemoproteomic profiling identifies changes in DNA-PK as markers of early dengue virus infection.

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Chemoproteomic profiling identifies changes in DNA-PK as markers of early dengue virus infection.

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