Biomedical subjects

Marcus J Korth

Publications and source records attributed to Marcus J Korth.

13 recordsLinked to original sources

PubMed2006

The cellular protein P58IPK regulates influenza virus mRNA translation and replication through a PKR-mediated mechanism.

We previously hypothesized that efficient translation of influenza virus mRNA requires the recruitment of P58(IPK), the cellular inhibitor of PKR, an interferon-induced kinase that targets the eukaryotic translation initiation factor eIF2alpha. P58(IPK) also inhibits PERK, an eIF2alpha kinase that is localized in the endoplasmic reticulum (ER) and induced during ER stress. The ability of P58(IPK) to interact with and inhibit multiple eIF2alpha kinases suggests it is a critical regulator of both cellular and viral mRNA translation. In this study, we sought to definitively define the role of P58(IPK) during viral infection of mammalian cells. Using mouse embryo fibroblasts from P58(IPK-/-) mice, we demonstrated that the absence of P58(IPK) led to an increase in eIF2alpha phosphorylation and decreased influenza virus mRNA translation. The absence of P58(IPK) also resulted in decreased vesicular stomatitis virus replication but enhanced reovirus yields. In cells lacking the P58(IPK) target, PKR, the trends were reversed-eIF2alpha phosphorylation was decreased, and influenza virus mRNA translation was increased. Although P58(IPK) also inhibits PERK, the presence or absence of this kinase had little effect on influenza virus mRNA translation, despite reduced levels of eIF2alpha phosphorylation in cells lacking PERK. Finally, we showed that influenza virus protein synthesis and viral mRNA levels decrease in cells that express a constitutively active, nonphosphorylatable eIF2alpha. Taken together, our results support a model in which P58(IPK) regulates influenza virus mRNA translation and infection through a PKR-mediated mechanism which is independent of PERK.

Animals↗

PubMed2006

Hijacking of the host-cell response and translational control during influenza virus infection.

Influenza virus is a major public health problem with annual deaths in the US of 36,000 with pandemic outbreaks, such as in 1918, resulting in deaths exceeding 20 million worldwide. Recently, there is much concern over the introduction of highly pathogenic avian influenza H5N1 viruses into the human population. Influenza virus has evolved complex translational control strategies that utilize cap-dependent translation initiation mechanisms and involve the recruitment of both viral and host-cell proteins to preferentially synthesize viral proteins and prevent activation of antiviral responses. Influenza virus is a member of the Orthomyxoviridae family of negative-stranded, segmented RNA viruses and represents a particularly attractive model system as viral replication strategies are closely intertwined with normal cellular processes including the host defense and stress pathways. In this chapter, we review the parallels between translational control in influenza virus infected cells and in stressed cells with a focus on selective translation of viral mRNAs and the antagonism of the dsRNA and host antiviral responses. Moreover, we will discuss how the use of genomic technologies such as DNA microarrays and high through-put proteomics can be used to gain new insights into the control of protein synthesis during viral infection and provide a near comprehensive view of virus-host interactions.

Animals↗

PubMed2006

Functional gene analysis of individual response to challenge of SIVmac239 in M. mulatta PBMC culture.

It has previously been shown in macaques that individual animals exhibit varying responses to challenge with the same strain of SIV. We attempted to elucidate these differences using functional genomics and correlate them to biological response. Unfractionated PBMC from three rhesus macaques were isolated, activated, and infected with SIVmac239. Interestingly, one of the three animals used for these experiments exhibited a completely unique response to infection relative to the other two. After repeated attempts to infect the PBMC from this animal, little or no infectivity was seen across the time points considered, and corresponding to this apparent lack of infection, few genes were seen to be differentially expressed when compared to mock-infected cells. For the remaining two animals, gene expression analysis showed that while they exhibited responses for the same groups of pathways, these responses included differences specific to the individual animal at the gene level. In instances where the patterns of differential gene expression differed between these animals, the genes being differentially expressed were associated with the same categories of biological process, mainly immune response and cell signaling. At the pathway level, these animals again exhibited similar responses that could be predicted based on the experimental conditions. Even in these expected results, the degree of response and the specific genes being regulated differed greatly from animal to animal. The differences in gene expression on an individual level have the potential to be used as markers in identification of animals suitable for lentiviral infection experiments. Our results highlight the importance of individual variation in response to viral challenge.

Animals↗

PubMed2006

Gene expression patterns that correlate with hepatitis C and early progression to fibrosis in liver transplant recipients.

BACKGROUND & AIMS: Liver transplant recipients infected with hepatitis C virus (HCV) develop recurrent hepatitis soon after transplantation and, in some cases, progress to fibrosis within the first 2 years. Our goals were to identify molecular processes influencing the liver disease progression and to find potential gene markers of early fibrosis. METHODS: We performed gene expression profiling on serial liver biopsy specimens obtained from 13 (11 infected and 2 uninfected) transplant recipients within the first year after transplantation at 0, 3, 6, and 12 months. The data were compared with clinical observations and with a gene expression database obtained for 55 nontransplant HCV-infected and uninfected liver samples. RESULTS: We identified several specific gene expression patterns. The first pattern was unique for the transplant recipients regardless of their infection status. The corresponding genes encoded stress response proteins and blood proteins involved in coagulation that were differentially expressed in response to posttransplantation graft recovery. The second pattern was specific to HCV-infected samples and included up-regulation of genes encoding components of the interferon-mediated antiviral response and immune system (antigen presentation, cytotoxic response). This up-regulation pattern was absent or suppressed in the patients who developed early fibrosis, indicating that the disease progression might result from an impaired liver response to infection. Finally, we identified gene expression patterns that were specific for 12-month biopsy specimens in all 4 HCV-infected patients who developed early fibrosis. CONCLUSIONS: The identified gene expression patterns may prove useful for diagnostic and prognostic applications in HCV-infected patients, including predicting early progression to fibrosis.

Adult↗

PubMed2005

Analysis of the Macaca mulatta transcriptome and the sequence divergence between Macaca and human.

We report the initial sequencing and comparative analysis of the Macaca mulatta transcriptome. Cloned sequences from 11 tissues, nine animals, and three species (M. mulatta, M. fascicularis, and M. nemestrina) were sampled, resulting in the generation of 48,642 sequence reads. These data represent an initial sampling of the putative rhesus orthologs for 6,216 human genes. Mean nucleotide diversity within M. mulatta and sequence divergence among M. fascicularis, M. nemestrina, and M. mulatta are also reported.

Animals↗

PubMed2005

Virus infection and the interferon response: a global view through functional genomics.

The primary focus of this chapter is on providing an overview of how we use the tools of functional genomics to study virus infection, the interferon response, and the mechanisms by which viruses attenuate or evade this response to ensure successful replication. We provide examples of the types of analyses we perform, experimental design considerations, and the tools and techniques we use for data processing and mining. We have not attempted to provide detailed protocols for performing microarray or proteomics experiments because even individual components of such analyses, for example, techniques for the isolation and amplification of ribonucleic acid, could easily be the subject of an entire chapter. Rather, our goal is to show how data obtained from global gene expression and protein profiling can be used to gain new insights into virus-host interactions, with particular emphasis on the interferon response and its modulation by virus infection.

Animals↗

PubMed2005

Pancreatic beta-cell failure and diabetes in mice with a deletion mutation of the endoplasmic reticulum molecular chaperone gene P58IPK.

The endoplasmic reticulum (ER) transmits apoptotic signals in the pancreas during ER stress, implicating ER stress-mediated apoptosis in the development of diabetes. P58(IPK) (DNAJC3) is induced during ER stress and functions as a negative feedback component to inhibit eIF-2alpha signaling and attenuate the later phases of the ER stress response. To gain insight into a more comprehensive role of P58(IPK) function, we generated deletion mutant mice that showed a gradual onset of glucosuria and hyperglycemia associated with increasing apoptosis of pancreatic islet cells. Lack of P58(IPK) had no apparent effect on the functional integrity of viable beta-cells. A set of genes associated with apoptosis showed altered expression in pancreatic islets from P58(IPK)-null mice, further substantiating the apoptosis phenotype. The data provide in vivo evidence to support the concept that P58(IPK) functions as a signal for the downregulation of ER-associated proteins involved in the initial ER stress response, thus preventing excessive cell loss by degradation pathways. Insulin deficiency associated with the absence of P58(IPK) mimics beta-cell failure associated with type 1 and late-stage type 2 diabetes. P58(IPK) function and activity may therefore provide a novel area of investigation into ER-mediated mechanistic and therapeutic approaches for diabetes.

Animals↗

PubMed2004

High-throughput screening of the yeast kinome: identification of human serine/threonine protein kinases that phosphorylate the hepatitis C virus NS5A protein.

The hepatitis C virus NS5A protein plays a critical role in virus replication, conferring interferon resistance to the virus through perturbation of multiple intracellular signaling pathways. Since NS5A is a phosphoprotein, it is of considerable interest to understand the role of phosphorylation in NS5A function. In this report, we investigated the phosphorylation of NS5A by taking advantage of 119 glutathione S-transferase-tagged protein kinases purified from Saccharomyces cerevisiae to perform a global screening of yeast kinases capable of phosphorylating NS5A in vitro. A database BLAST search was subsequently performed by using the sequences of the yeast kinases that phosphorylated NS5A in order to identify human kinases with the highest sequence homologies. Subsequent in vitro kinase assays and phosphopeptide mapping studies confirmed that several of the homologous human protein kinases were capable of phosphorylating NS5A. In vivo phosphopeptide mapping revealed phosphopeptides common to those generated in vitro by AKT, p70S6K, MEK1, and MKK6, suggesting that these kinases may phosphorylate NS5A in mammalian cells. Significantly, rapamycin, an inhibitor commonly used to investigate the mTOR/p70S6K pathway, reduced the in vivo phosphorylation of specific NS5A phosphopeptides, strongly suggesting that p70S6 kinase and potentially related members of this group phosphorylate NS5A inside the cell. Curiously, certain of these kinases also play a major role in mRNA translation and antiapoptotic pathways, some of which are already known to be regulated by NS5A. The findings presented here demonstrate the use of high-throughput screening of the yeast kinome to facilitate the major task of identifying human NS5A protein kinases for further characterization of phosphorylation events in vivo. Our results suggest that this novel approach may be generally applicable to the screening of other protein biochemical activities by mechanistic class.

Animals↗

PubMed2003

Hepatitis C virus and liver disease: global transcriptional profiling and identification of potential markers.

Microarray analysis of RNA from hepatitis C virus (HCV)-infected cirrhotic livers was performed to identify a gene expression signature of liver disease. The expression levels of approximately 13600 genes were analyzed using surgical material and core biopsy specimens from HCV-infected cirrhotic liver explants in comparison with reference samples of normal nondiseased liver. In addition, normal liver samples were compared with each other to determine normal physiologic variation in gene expression. A set of genes, including some associated with stress, acute-phase immune response, and hepatic stellate cell activation, had variable expression levels in normal livers. These genes were subtracted from the sets of genes differentially expressed in cirrhotic livers. To exclude cancer-related genes from our marker sets, we subtracted genes that also were expressed differentially in hepatocellular carcinomas. The resultant HCV- and liver disease-associated gene set provided a molecular portrait of several processes occurring in the HCV-infected liver. It included (1). genes expressed in activated lymphocytes infiltrating the cirrhotic liver, and activated liver macrophages; (2). genes involved in remodeling of extracellular matrix-cell and cell-cell interactions associated with cytoskeleton rearrangements; (3). genes related to the anti-apoptotic pathway of Bcl-2 signaling; and (4). genes involved with the interferon response and virus-host interactions. In conclusion, our microarray analysis identified several potential gene markers of HCV-associated liver disease and contributed to our rapidly expanding database of experiments describing HCV pathogenesis.

Apoptosis↗

PubMed2003

Gene expression profiling of the cellular transcriptional network regulated by alpha/beta interferon and its partial attenuation by the hepatitis C virus nonstructural 5A protein.

Alpha/beta interferons (IFN-alpha/beta) induce potent antiviral and antiproliferative responses and are used to treat a wide range of human diseases, including chronic hepatitis C virus (HCV) infection. However, for reasons that remain poorly understood, many HCV isolates are resistant to IFN therapy. To better understand the nature of the cellular IFN response, we examined the effects of IFN treatment on global gene expression by using several types of human cells, including HeLa cells, liver cell lines, and primary fetal hepatocytes. In response to IFN, 50 of the approximately 4,600 genes examined were consistently induced in each of these cell types and another 60 were induced in a cell type-specific manner. A search for IFN-stimulated response elements (ISREs) in genomic DNA located upstream of IFN-stimulated genes revealed both previously identified and novel putative ISREs. To determine whether HCV can alter IFN-regulated gene expression, we performed microarray analyses on IFN-treated HeLa cells expressing the HCV nonstructural 5A (NS5A) protein and on IFN-treated Huh7 cells containing an HCV subgenomic replicon. NS5A partially blocked the IFN-mediated induction of 14 IFN-stimulated genes, an effect that may play a role in HCV resistance to IFN. This block may occur through repression of ISRE-mediated transcription, since NS5A also inhibited the IFN-mediated induction of a reporter gene driven from an ISRE-containing promoter. In contrast, the HCV replicon had very little effect on IFN-regulated gene expression. These differences highlight the importance of comparing results from multiple model systems when investigating complex phenomena such as the cellular response to IFN and viral mechanisms of IFN resistance.

Cell Line↗

PubMed2002

Control of PERK eIF2alpha kinase activity by the endoplasmic reticulum stress-induced molecular chaperone P58IPK.

P58(IPK) is an Hsp40 family member known to inhibit the interferon (IFN)-induced, double-stranded RNA-activated, eukaryotic initiation factor 2alpha (eIF2alpha) protein kinase R (PKR) by binding to its kinase domain. We find that the stress of unfolded proteins in the endoplasmic reticulum (ER) activates P58(IPK) gene transcription through an ER stress-response element in its promoter region. P58(IPK) interacts with and inhibits the PKR-like ER-localized eIF2alpha kinase PERK, which is normally activated during the ER-stress response to protect cells from ER stress by attenuating protein synthesis and reducing ER client protein load. Levels of phosphorylated eIF2alpha were lower in ER-stressed P58(IPK)-overexpressing cells and were enhanced in P58(IPK) mutant cells. In the ER-stress response, PKR-like ER kinase (PERK)-mediated translational repression is transient and is followed by translational recovery and enhanced expression of genes that increase the capacity of the ER to process client proteins. The absence of P58(IPK) resulted in increased expression levels of two ER stress-inducible genes, BiP and Chop, consistent with the enhanced eIF2alpha phosphorylation in the P58(IPK) deletion cells. Our studies suggest that P58(IPK) induction during the ER-stress response represses PERK activity and plays a functional role in the expression of downstream markers of PERK activity in the later phase of the ER-stress response.

Animals↗

PubMed2002

P52rIPK regulates the molecular cochaperone P58IPK to mediate control of the RNA-dependent protein kinase in response to cytoplasmic stress.

The 52 kDa protein referred to as P52(rIPK) was first identified as a regulator of P58(IPK), a cellular inhibitor of the RNA-dependent protein kinase (PKR). P52(rIPK) and P58(IPK) each possess structural domains implicated in stress signaling, including the charged domain of P52(rIPK) and the tetratricopeptide repeat (TPR) and DnaJ domains of P58(IPK). The P52(rIPK) charged domain exhibits homology to the charged domains of Hsp90, including the Hsp90 geldanamycin-binding domain. Here we present an in-depth analysis of P52(rIPK) function and expression, which first revealed that the 114 amino acid charged domain was necessary and sufficient for interaction with P58(IPK). This domain bound specifically to P58(IPK) TPR domain 7, the domain adjacent to the TPR motif required for P58(IPK) interaction with PKR, thus providing a mechanism for P52(rIPK) inhibition of P58(IPK) function. Both the charged domain of P52(rIPK) and the TPR 7 domain of P58(IPK) were required for P52(rIPK) to mediate downstream control of PKR activity, eIF2alpha phosphorylation, and cell growth. Furthermore, we found that P52(rIPK) and P58(IPK) formed a stable intracellular complex during the acute response to cytoplasmic stress induced by a variety of stimuli. We propose a model in which the P52(rIPK) charged domain functions as a TPR-specific signaling motif to directly regulate P58(IPK) within a larger cytoplasmic stress signaling cascade culminating in the control of PKR activity and cellular mRNA translation.

3T3 Cells↗

PubMed2002

Unlocking the mysteries of virus-host interactions: does functional genomics hold the key?

The interactions between viruses and the cells they infect are complex and multifaceted. While viruses strive to usurp cellular functions to their advantage, the cell strives to thwart these efforts by mounting a variety of defensive responses. These responses may include the induction of interferon, stress response, or apoptotic pathways, all of which are accompanied by changes in gene expression. Some viruses consistently win this tug of war, whereas others succumb to cellular defense mechanisms. The viral and cellular factors that determine the outcome are for the most part still unknown. With the advent of functional genomics, potent new technologies are now available to probe the complexities of virus-host interactions in ever increasing depth and detail. We describe here our efforts to use microarrays, proteomics, and bioinformatics to focus in on the changes in gene expression and protein production that occur in a virus-infected cell and to use these technologies to unlock the mysteries of virus-host interactions.

Animals↗