Vaithilingaraja Arumugaswami

The heat shock protein inhibitor Quercetin attenuates hepatitis C virus production.

The hepatitis C viral (HCV) genome is translated through an internal ribosome entry site (IRES) as a single polyprotein precursor that is subsequently cleaved into individual mature viral proteins. Nonstructural protein 5A (NS5A) is one of these proteins that has been implicated in regulation of viral genome replication, translation from the viral IRES and viral packaging. We sought to identify cellular proteins that interact with NS5A and determine whether these interactions may play a role in viral production. Mass spectrometric analysis of coimmunoprecipitated NS5A complexes from cell extracts identified heat shock proteins (HSPs) 40 and 70. We confirmed an NS5A/HSP interaction by confocal microscopy demonstrating colocalization of NS5A with HSP40 and with HSP70. Western analysis of coimmunoprecipitated NS5A complexes further confirmed interaction of HSP40 and HSP70 with NS5A. A transient transfection, luciferase‐based, tissue culture IRES assay demonstrated NS5A augmentation of HCV IRES‐mediated translation, and small interfering RNA (siRNA)‐mediated knockdown of HSP70 reduced this augmentation. Treatment with an inhibitor of HSP synthesis, Quercetin, markedly reduced baseline IRES activity and its augmentation by NS5A. HSP70 knockdown also modestly reduced viral protein accumulation, whereas HSP40 and HSP70 knockdown both reduced infectious viral particle production in an HCV cell culture system using the J6/JFH virus fused to the Renilla luciferase reporter. Treatment with Quercetin reduced infectious particle production at nontoxic concentrations. The marked inhibition of virus production by Quercetin may partially be related to reduction of HSP40 and HSP70 and their potential involvement in IRES translation, as well as viral morphogenesis or secretion. Conclusion: Quercetin may allow for dissection of the viral life cycle and has potential therapeutic use to reduce virus production with low associated toxicity. (HEPATOLOGY 2009.)

High-Resolution Functional Profiling of Hepatitis C Virus Genome

Hepatitis C virus is a leading cause of human liver disease worldwide. Recent discovery of the JFH-1 isolate, capable of infecting cell culture, opens new avenues for studying HCV replication. We describe the development of a high-throughput, quantitative, genome-scale, mutational analysis system to study the HCV cis-elements and protein domains that are essential for virus replication. An HCV library with 15-nucleotide random insertions was passaged in cell culture to examine the effect of insertions at each genome location by insertion-specific fluorescent-PCR profiling. Of 2399 insertions identified in 9517 nucleotides of the genome, 374, 111, and 1914 were tolerated, attenuating, and lethal, respectively, for virus replication. Besides identifying novel functional domains, this approach confirmed other functional domains consistent with previous studies. The results were validated by testing several individual mutant viruses. Furthermore, analysis of the 3′ non-translated variable region revealed a spacer role in virus replication, demonstrating the utility of this approach for functional discovery. The high-resolution functional profiling of HCV domains lays the foundation for further mechanistic studies and presents new therapeutic targets as well as topological information for designing vaccine candidates.

ORF18 Is a Transfactor That Is Essential for Late Gene Transcription of a Gammaherpesvirus

ABSTRACT Lytic replication of the tumor-associated human gammaherpesviruses Epstein-Barr virus and Kaposis sarcoma-associated herpesvirus has important implications in pathogenesis and tumorigenesis. Herpesvirus lytic genes have been temporally classified as exhibiting immediate-early (IE), early, and late expression kinetics. Though the regulation of IE and early gene expression has been studied extensively, very little is known regarding the regulation of late gene expression. Late genes, which primarily encode virion structural proteins, require viral DNA replication for their expression. We have identified a murine gammaherpesvirus 68 (MHV-68) early lytic gene, ORF18, essential for viral replication. ORF18 is conserved in both beta- and gammaherpesviruses. By generating an MHV-68 ORF18-null virus, we characterized the stage of the virus lytic cascade that requires the function of ORF18. Gene expression profiling and quantitation of viral DNA synthesis of the ORF18-null virus revealed that the expression of early genes and viral DNA replication were not affected; however, the transcription of late genes was abolished. Hence, we have identified a gammaherpesvirus-encoded factor essential for the expression of late genes independently of viral DNA synthesis.

Divergent antiviral effects of bioflavonoids on the hepatitis C virus life cycle.

We have previously demonstrated that quercetin, a bioflavonoid, blocks hepatitis C virus (HCV) proliferation by inhibiting NS5A-driven internal ribosomal entry site (IRES)-mediated translation of the viral genome. Here, we investigate the mechanisms of antiviral activity of quercetin and six additional bioflavonoids. We demonstrate that catechin, naringenin, and quercetin possess significant antiviral activity, with no associated cytotoxicity. Infectious virion secretion was not significantly altered by these bioflavonoids. Catechin and naringenin demonstrated stronger inhibition of infectious virion assembly compared to quercetin. Quercetin markedly blocked viral translation whereas catechin and naringenin demonstrated mild activity. Similarly quercetin completely blocked NS5A-augmented IRES-mediated translation in an IRES reporter assay, whereas catechin and naringenin had only a mild effect. Moreover, quercetin differentially inhibited HSP70 induction compared to catechin and naringenin. Thus, the antiviral activity of these bioflavonoids is mediated through different mechanisms. Therefore combination of these bioflavonoids may act synergistically against HCV.

Identification and comparative analysis of hepatitis C virus–host cell protein interactions

Hepatitis C virus (HCV) alters the global behavior of the host cell to create an environment conducive to its own replication, but much remains unknown about how HCV proteins elicit these changes. Thus, a better understanding of the interface between the virus and host cell is required. Here we report the results of a large-scale yeast two-hybrid screen to identify protein-protein interactions between HCV genotype 2a (strain JFH1) and cellular factors. Our study identified 112 unique interactions between 7 HCV and 94 human proteins, over 40% of which have been linked to HCV infection by other studies. These interactions develop a more complete picture of HCV infection, providing insight into HCV manipulation of pathways, such as lipid and cholesterol metabolism, that were previously linked to HCV infection and implicating novel targets within microtubule-organizing centers, the complement system and cell cycle regulatory machinery. In an effort to understand the relationship between HCV and related viruses, we compared the HCV 2a interactome to those of other HCV genotypes and to the related dengue virus. Greater overlap was observed between HCV and dengue virus targets than between HCV genotypes, demonstrating the value of parallel screening approaches when comparing virus-host cell interactomes. Using siRNAs to inhibit expression of cellular proteins, we found that five of the ten shared targets tested (CUL7, PCM1, RILPL2, RNASET2, and TCF7L2) were required for replication of both HCV and dengue virus. These shared interactions provide insight into common features of the viral life cycles of the family Flaviviridae.

Systematic Analysis of Enhancer and Critical cis-Acting RNA Elements in the Protein-Encoding Region of the Hepatitis C Virus Genome

ABSTRACT Hepatitis C virus (HCV) causes chronic hepatitis, cirrhosis, and liver cancer. cis-acting RNA elements of the HCV genome are critical for translation initiation and replication of the viral genome. We hypothesized that the coding regions of nonstructural proteins harbor enhancer and essential cis-acting replication elements (CRE). In order to experimentally identify new cis RNA elements, we utilized an unbiased approach to introduce synonymous substitutions. The HCV genome coding for nonstructural proteins (nucleotide positions 3872 to 9097) was divided into 17 contiguous segments. The wobble nucleotide positions of each codon were replaced, resulting in 33% to 41% nucleotide changes. The HCV genome containing one of each of 17 mutant segments (S1 to S17) was tested for genome replication and infectivity. We observed that silent mutations in segment 13 (S13) (nucleotides [nt] 7457 to 7786), S14 (nt 7787 to 8113), S15 (nt 8114 to 8440), S16 (nt 8441 to 8767), and S17 (nt 8768 to 9097) resulted in impaired genome replication, suggesting CRE structures are enriched in the NS5B region. Subsequent high-resolution mutational analysis of NS5B (nt 7787 to 9289) using approximately 51-nucleotide contiguous subsegment mutant viruses having synonymous mutations revealed that subsegments SS8195-8245, SS8654-8704, and SS9011-9061 were required for efficient viral growth, suggesting that these regions act as enhancer elements. Covariant nucleotide substitution analysis of a stem-loop, JFH-SL9098, revealed the formation of an extended stem structure, which we designated JFH-SL9074. We have identified new enhancer RNA elements and an extended stem-loop in the NS5B coding region. Genetic modification of enhancer RNA elements can be utilized for designing attenuated HCV vaccine candidates.

Mitochondrial NDUFS3 regulates the ROS-mediated onset of metabolic switch in transformed cells.

Summary Aerobic glycolysis in transformed cells is an unique metabolic phenotype characterized by a hyperactivated glycolytic pathway even in the presence of oxygen. It is not clear if the onset of aerobic glycolysis is regulated by mitochondrial dysfunction and, if so, what the metabolic windows of opportunity available to control this metabolic switch (mitochondrial to glycolytic) landscape are in transformed cells. Here we report a genetically-defined model system based on the gene-silencing of a mitochondrial complex I subunit, NDUFS3, where we demonstrate the onset of metabolic switch in isogenic human embryonic kidney cells by differential expression of NDUFS3. By means of extensive metabolic characterization, we demonstrate that NDUFS3 gene silencing systematically introduces mitochondrial dysfunction thereby leading to the onset of aerobic glycolysis in a manner dependent on NDUFS3 protein levels. Furthermore, we show that the sustained imbalance in free radical dynamics is a necessary condition to sustain the observed metabolic switch in cell lines with the most severe NDUFS3 suppression. Together, our data reveal a novel role for mitochondrial complex I subunit NDUFS3 in regulating the degree of mitochondrial dysfunction in living cells, thereby setting a “metabolic threshold” for the observation of aerobic glycolysis phenotype within the confines of mitochondrial dysfunction.

Nuclear localization and dynamic properties of the Marek's disease virus oncogene products Meq and Meq/vIL8.

ABSTRACT Mareks disease virus (MDV) is an avian herpesvirus that causes T-cell lymphomas and immune suppression in susceptible chickens. At least one gene product, MDV Eco Q-encoded protein (Meq), is essential for the oncogenicity of MDV. Alternative splicing permits the meq gene to give rise to two major transcripts encoding proteins designated Meq and Meq/vIL8. Meq is a basic leucine zipper protein capable of modulating transcription. The Meq/vIL8 protein retains a modified leucine zipper, along with the mature receptor-binding portion of vIL8, but lacks the domain of Meq responsible for transcriptional modulation. In this report, we describe studies using fusions between either Meq or Meq/vIL8 and fluorescent proteins to characterize the distribution and properties of these products in chicken embryo fibroblasts (CEFs). Meq and Meq/vIL8 both localized to the nucleoplasm, nucleoli, and Cajal bodies of transfected cells. Similar distributions were found for fluorescent fusion proteins and native Meq or Meq/vIL8. Fluorescence recovery after photobleaching and photoactivatable green fluorescent protein revealed that Meq exhibited mobility properties similar to those of other transcription factors, while Meq/vIL8 was far less mobile. In addition, fluorescence resonance energy transfer studies indicated the formation of Meq/vIL8 homodimers in CEFs. Time lapse studies revealed the coordinated elimination of a portion of Meq and Meq/vIL8 from the nucleus. Our data provide new insight regarding the dynamic cellular properties of two forms of a herpesvirus-encoded oncoprotein and suggest that these forms may have fundamentally different functions in MDV-infected cells.

A cell‐permeable hairpin peptide inhibits hepatitis C viral nonstructural protein 5A–mediated translation and virus production

NS5A is a key regulator of the hepatitis C virus (HCV) life cycle including RNA replication, assembly, and translation. We and others have shown that NS5A augments HCV internal ribosomal entry site (IRES)‐mediated translation. Furthermore, Quercetin treatment and heat shock protein (HSP) 70 knockdown inhibit the NS5A‐driven augmentation of IRES‐mediated translation and infectious virus production. We have also coimmunoprecipitated HSP70 with NS5A and demonstrated cellular colocalization, leading to the hypothesis that the NS5A/HSP70 complex formation is important for IRES‐mediated translation. Here, we have identified the NS5A region responsible for complex formation through in vitro deletion analyses. Deletion of NS5A domains II and III failed to reduce HSP70 binding, whereas domain I deletion eliminated complex formation. NS5A domain I alone also bound HSP70. Deletion mapping of domain I identified the C‐terminal 34 amino acids (C34) as the interaction site. Furthermore, addition of C34 to domains II and III restored complex formation. C34 expression significantly reduced intracellular viral protein levels, in contrast to same‐size control peptides from other NS5A domains. C34 also competitively inhibited NS5A‐augmented IRES‐mediated translation, whereas controls did not. Triple‐alanine scan mutagenesis determined that an exposed beta‐sheet hairpin in C34 was primarily responsible for NS5A‐augmented IRES‐mediated translation. Moreover, treatment with a 10–amino acid peptide derivative of C34 suppressed NS5A‐augmented IRES‐mediated translation and significantly inhibited intracellular viral protein synthesis, with no associated cytotoxicity. Conclusion: These results support the hypothesis that the NS5A/HSP70 complex augments viral IRES‐mediated translation, identify a sequence‐specific hairpin element in NS5A responsible for complex formation, and demonstrate the functional significance of C34 hairpin–mediated NS5A/HSP70 interaction. Identification of this element may allow for further interrogation of NS5A‐mediated IRES activity, sequence‐specific HSP recognition, and rational drug design. (HEPATOLOGY 2012;55:1662–1672)

A Repetitive Region of Gammaherpesvirus Genomic DNA Is a Ligand for Induction of Type I Interferon

ABSTRACT Innate immune responses against viral infection, especially the induction of type I interferon, are critical for limiting the replication of the virus. Although it has been shown that DNA can induce type I interferon, to date no natural DNA ligand of a virus that induces type I interferon has been described. Here we screened the genome of murine gammaherpesvirus 68 with mutations at various genomic locations to map the region of DNA that induces type I interferon. A repetitive region termed the 100-base-pair repeat region is a ligand that is both necessary and sufficient for the viral genomic DNA to induce type I interferon. A region colinear with this ligand in the genome of Kaposis sarcoma-associated herpesvirus also induces type I interferon. We have thus defined a repetitive region of the genomes of gammaherpesviruses as the first natural DNA virus ligand that induces type I interferon.