ABSTRACT
Viral infections of host cells cause multiple changes of cellular metabolism including immediate defense mechanisms as well as processes to support viral replication. Coxsackievirus B3 (CVB3) is a member of the Picornavirus family and is responsible for a wide variety of mild or severe infections including acute and chronic inflammations. Thereby, intracellular signaling can be changed very comprehensively. In order to compare the influence of CVB3 replication on gene expression pattern of two different cell lines, DNA microarray systems were used to study a set of 780 genes related to inflammation. Expression analysis of HeLa cells and HepG2 cells infected with CVB3 identified 34 genes whose mRNA levels were altered significantly upon infection. The expression of additional 16 genes in HepG2 cells and 31 genes in HeLa cells were found to be influenced during CVB3 replication as well. All genes expressed differentially were sorted with regard to their functions and interpreted in view of known contributors to the infection process. The activation of the tumor necrosis factor pathways by CVB3 represents one peculiar observation, including apoptosis, stress, and inflammation responses.
Subject(s)
Coxsackievirus Infections/genetics , Coxsackievirus Infections/metabolism , Enterovirus B, Human/genetics , RNA, Messenger/biosynthesis , Apoptosis/genetics , Cell Line, Tumor , Coxsackievirus Infections/virology , Enzyme Activation , Gene Expression , Gene Expression Profiling , HeLa Cells/virology , Hep G2 Cells/virology , Humans , Oligonucleotide Array Sequence Analysis , Signal Transduction/genetics , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Virus Replication/geneticsABSTRACT
Coxsackievirus is linked to a large variety of severe human and animal diseases such as myocarditis. The interplay between host factors and virus components is crucial for the fate of the infected cells. However, host proteins which may play a role in coxsackievirus-induced diseases are ill-defined. Two-dimensional gel electrophoresis of protein extracts obtained from coxsackievirus B3 (CVB3)-infected and uninfected HeLa or HepG2 cells combined with spot analysis revealed several proteins which are exclusively up-regulated in infected cells. One of these proteins was identified as the fatty acid synthase (FAS). By using cerulenin and C75, two known inhibitors of FAS we were able to significantly block CVB3 replication. FAS appears to be directly involved in CVB3-caused pathology and is therefore suitable as a therapeutic target in CVB3-induced diseases.
Subject(s)
Enterovirus B, Human/growth & development , Fatty Acid Synthases/biosynthesis , 4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/pharmacology , Amino Acid Sequence , Antiviral Agents/pharmacology , Cell Line , Cerulenin/pharmacology , Electrophoresis, Gel, Two-Dimensional , Enzyme Inhibitors , Fatty Acid Synthases/antagonists & inhibitors , Fatty Acid Synthases/chemistry , Humans , Molecular Sequence Data , Up-Regulation , Virus Replication/drug effectsABSTRACT
The induction of apoptosis during coxsackievirus B3 (CVB3) infection is well documented. In order to study whether the inhibition of apoptosis has an impact on CVB3 replication, the pan-caspase inhibitor Z-VAD-FMK was used. The decreased CVB3 replication is based on reduced accumulation of both viral RNA and viral proteins. These effects are due to an inhibitory influence of Z-VAD-FMK on the proteolytic activity of the CVB3 proteases 2A and 3C, which was demonstrated by using the target protein poly(A)-binding protein (PABP). The antiviral effect of the structurally different pan-caspase inhibitor Q-VD-OPH was independently of the viral protease inhibition and resulted in suppression of virus progeny production and impaired release of newly produced CVB3 from infected cells. A delayed release of cytochrome c into the cytoplasm was detected in Q-VD-OPH-treated CVB3-infected cells pointing to an involvement of caspases in the initial steps of mitochondrial membrane-permeabilization.
Subject(s)
Amino Acid Chloromethyl Ketones/pharmacology , Caspase Inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Enterovirus B, Human/physiology , Virus Replication/drug effects , Caspases/genetics , Caspases/metabolism , Cell Line , Coxsackievirus Infections , Cytochromes c/metabolism , Humans , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
Coxsackievirus B3 (CVB3) is a common factor in human myocarditis. The interplay between host factors and virus components is crucial for the fate of the infected cells. Despite that, host protein responses, which characterize CVB3-induced diseases, have not yet been determined in detail. To investigate the nature of modified protein patterns in infected human cells compared with uninfected cells, two-dimensional gel electrophoresis in combination with matrix-assisted laser desorption/ionization-mass spectrometry were used. The regulated proteins, e.g. nucleophosmin (nucleolar protein B23), lamin, the RNA-binding protein UNR and the p38 mitogen-activated protein kinase, were sorted according to their functional groups and interpreted in the context of the myocarditis process.
