Molecular Mechanisms of Interferon Resistance Mediated by Viral-Directed Inhibition of PKR, the Interferon-Induced Protein Kinase
ABSTRACT The interferon (IFN)-induced cellular antiviral response is the first line of defense against viral infection within an animal host. In order to establish a productive infection, eukaryotic viruses must first overcome the IFN-induced blocks imposed on viral replication. The double-stranded RNA-activated protein kinase (PKR) is a key component mediating the antiviral actions of IFN. This IFN-induced protein kinase can restrict viral replication through its ability to phosphorylate the protein synthesis initiation factor eukaryotic initiation factor-2 α-subunit and reduce levels of viral protein synthesis. Viruses, therefore, must block the function of PKR in order to avoid these deleterious antiviral effects associated with PKR activity. Indeed, many viruses have developed effective measures to repress PKR activity during infection. This review will focus primarily on an overview of the different molecular mechanisms employed by these viruses to meet a common goal: the inhibition of PKR function, uncompromised viral protein synthesis, and unrestricted virus replication. The past few years have seen exciting new advances in this area. Rather unexpectedly, this area of research has benefited from the use of the yeast system to study PKR. Other recent advances include studies on PKR regulation by the herpes simplex viruses and data from our laboratory on the medically important hepatitis C viruses. We speculate that IFN is ineffective as a therapeutic agent against hepatitis C virus because the virus can effectively repress PKR function. Finally, we will discuss briefly the future directions of this PKR field. pharmacol. ther. 78(1):29–46, 1998.
SourceAvailable from: Pavan Kumar Kakumani[Show abstract] [Hide abstract]
ABSTRACT: The host-mediated RNA interference pathways (RNAi) restrict replication of viruses in plant, invertebrate and vertebrate systems. However, comparatively little is known about the interplay between RNAi and various viral infections in mammalian hosts. We show here that the siRNA mediated silencing of Drosha, Dicer and Ago2 transcripts in Huh7 cells resulted in elevated levels of HBV specific RNAs, and conversely, we observed a decrease in mRNA and protein levels of same RNAi components in HepG2 cells infected with HBV. Similar reductions were also detectable in chronic hepatitis B (CHB) patients. Analysis of CHB liver biopsy samples, with high serum HBV DNA load (>log108 IU/ml), revealed a reduced mRNA and protein levels of Drosha, Dicer and Ago2. The low expression levels of key RNAi pathway components in CHB patient samples as well as hepatic cells established a link between HBV replication and RNAi components. The HBV proteins were also examined for RNA silencing suppressor properties. Using the GFP-based reversion of silencing assays, it's been identified here that HBx protein is an RSS protein. Through a series of deletions as well as substitution mutants, we found that the full-length HBx protein is required for the optimum RSS activity. The in vitro dicing assays revealed that the HBx protein inhibited the human Dicer mediated processing of dsRNAs into siRNAs. Together, our results suggest that the HBx protein might function as RSS to manipulate host RNAi defense especially by abrogating the Dicer function. The study may have implications in the development of newer strategies to combat HBV infection.Biochemical Journal 06/2014; 462(2):347-358. DOI:10.1042/BJ20140316 · 4.78 Impact Factor
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ABSTRACT: Dicer plays an important role in the course of RNA interference (RNAi), i.e., it digests long double-stranded RNAs into 21–25 nucleotide small-interfering RNA (siRNA) duplexes functioning as sequence-specific RNAi mediators. In this study, we investigated the expression levels of Dicer and eIF2C1∼4, which, like Dicer, appear to participate in mammalian RNAi, in various mouse tissues. Results indicate that the levels of eIF2C1∼4 as well as Dicer are lower in skeletal muscle and heart than in other tissues. To see if RNAi could occur under such a condition with low levels of expression of Dicer and eIF2C1∼4, we examined RNAi activity in mouse skeletal muscle fibers. The results indicate that RNAi can be induced by synthetic siRNA duplexes in muscle fibers. We further examined RNAi activity during myogenic differentiation of mouse C2C12 cells. The data indicate that although the expression levels of Dicer and eIF2C1∼4 decrease during the differentiation, RNAi can be induced in the cells. Altogether, the data presented here suggest that muscle cells retain the ability to induce RNAi, although Dicer and eIF2C1∼4 appear to be barely expressed in them.Biochemical and Biophysical Research Communications 06/2004; 319(1):50-57. DOI:10.1016/S0006-291X(04)00878-2 · 2.28 Impact Factor
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ABSTRACT: Rationale: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. Objective: This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. Methods and Results: Human iPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were utilized to characterize virally-infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferon beta 1 (IFNβ1), ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after IFNβ1 treatment. Conclusions: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.Circulation Research 07/2014; 115(6). DOI:10.1161/CIRCRESAHA.115.303810 · 11.09 Impact Factor