In vitro assembly of an empty picornavirus capsid follows a dodecahedral path.

The Picornaviridae are a large family of small, spherical RNA viruses that includes numerous pathogens. The picornavirus structural proteins VP0, VP1, and VP3 are believed to first form protomers, which then form 14S particles and subsequently assemble to form empty and RNA-filled particles. 14S particles have long been presumed to be pentamers. However, the structure of the 14S particles, their mechanism of assembly, and the role of empty particles during infection are all unknown. We established an in vitro assembly system for bovine enterovirus (BEV) by using purified baculovirus-expressed proteins. By Rayleigh scattering, we determined that 14S particles are 488 kDa, confirming they are pentamers. Image reconstructions based on negative-stain electron microscopy showed that 14S particles have 5-fold symmetry, and their structures correlate extremely well with the corresponding pentamer from crystal structures of mature BEV. Purified 14S particles readily assemble in response to increasing ionic strength or temperature to form 5.8-MDa 12-pentamer particles, indistinguishable from native empty particles. Surprisingly, empty particles were sufficiently stable that, under physiological conditions, dissociation is unlikely to be a biologically relevant reaction. This suggests that empty particles are not a storage form of 14S particles, at least for bovine enterovirus, but are either a dead-end product or direct precursor into which viral RNA is packaged by as-yet-unidentified machinery.

Suppression of ribosomal protein synthesis and protein translation factors by Peg-interferon alpha/ribavirin in HCV patients blood mononuclear cells (PBMC).

BACKGROUND: We have previously reported the induction of many interferon stimulated genes (ISGs) in PBMC collected from patients infected with HCV at various times after initiation of interferon-ribavirin treatment using DNA microarrays to identify changes in gene expression with time. Almost as many genes are down regulated (suppressed) during interferon-ribavirin treatment as are up regulated. METHODS: DNA microarrays were analyzed by different software, including MAS5 (Affymetrix-Kegg) and GSEA (gene set enrichment analysis) to identify specific pathways both up regulated and down regulated. Data was assessed from a clinical trial, which was a microarray analysis from 68 patients. RESULTS: Up regulated genes included genes associated with NF-kb, toll like receptor cytokine -cytokine interaction, and complement and adhesion pathways. The most prominent pathway down regulated was that for ribosomal structural proteins, and eukaryotic translational factors. Down regulation of ribosomal protein genes continued through the treatment up to the last measurement, which was at day 28. CONCLUSIONS: This suppression of the protein synthetic apparatus might explain the long-term side effects of interferon-ribavirin, and explain a non-specific effect of interferon-ribavirin on viral protein synthesis. There was no evidence for unique transcription factors or micro RNA involvement.

Natural killer inhibitory receptor expression associated with treatment failure and interleukin-28B genotype in patients with chronic hepatitis C.

UNLABELLED: Natural killer (NK) cells constitute a first line of defense against viral infections; their function is governed by the integration of signals from multiple activating and inhibitory surface receptors. We hypothesized that because NKs become rapidly activated by cytokines, response to anti-hepatitis C virus (HCV) therapy would be predicted by the phenotype and function of NKs. We used a cohort of 101 patients (55 African, 46 Caucasian-American) who received pegylated-interferon (IFN) and ribavirin for 48 weeks. Multiparameter FACS analysis was used to examine relative expression of 14 different inhibitory/activating receptors. Interleukin (IL)-28B genotyping (rs12979860) was also performed. Pretreatment levels of inhibitory receptors CD158a, CD158b, and CD158e were higher in patients who demonstrated poor viral decline within the first 28 days of therapy. Higher expression levels of inhibitory receptors NKG2A, CD158b, and CD158e were demonstrable in patients who failed to achieve sustained virologic response (SVR). Patients carrying the IL-28B T allele had higher NKG2A expression on effector NKs. We created a mathematical regression model incorporating race, viral level, and two inhibitory receptors. The area-under-the curve was 0.88, which is highly predictive of SVR. Moreover, the model performed complementarily with IL-28B across the CC, CT, and TT genotypes. Purified NKG2A(neg) NKs treated with pegylated-IFN-α for 4 hours demonstrated higher levels of IFN-γ-inducible protein-10 (IP-10) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) compared with their NKG2A(pos) counterparts. CONCLUSIONS: These results provide novel insights into the associations of NK phenotype with IL-28B genotype and gene expression patterns, as well as the role of NKs in mediating IFN-induced viral clearance of chronic HCV infection.

Cyclic changes in gene expression induced by Peg-interferon alfa-2b plus ribavirin in peripheral blood monocytes (PBMC) of hepatitis C patients during the first 10 weeks of treatment.

BACKGROUND AND AIMS: This study determined the kinetics of gene expression during the first 10 weeks of therapy with Pegylated-interferon-alfa2b (PegIntron) and ribavirin (administered by weight) in HCV patients and compared it with the recently completed Virahep C study 12 in which Peginterferon-alfa2a (Pegasys) and ribavirin were administered. METHODS: RNA was isolated from peripheral blood monocytes (PBMC) from twenty treatment-naïve patients just before treatment (day 1) and at days 3, 6, 10, 13, 27, 42 and 70 days after treatment. Gene expression at each time was measured using Affymetrix microarrays and compared to that of day 1. RESULTS: The expression of many genes differed significantly (p

Differential gene induction by type I and type II interferons and their combination.

Type I and type II interferons (IFNs) bind to different cell surface receptors but activate overlapping signal transduction pathways. We examined the effects of a type I IFN (IFN-alphacon1) and a type II IFN (IFN-gamma1b) on gene expression in A549 cells and demonstrate that there is a common set of genes modulated by both IFNs as well as a set of genes specifically regulated by each, reflecting the activation of different signaling pathways. In particular, IFN-gamma induced many more genes of the signaling pathways, apoptosis, and cytokine interactions than did IFN-alpha. Even with genes induced by both IFNs there were distinctive quantitative differences in expression. IFN-gamma1b plays a major role in the induction and regulation of the complement pathway. Previous work has shown a synergistic antiviral and antiproliferative effect of type I and type II IFNs in cell culture and in the treatment of tumors in mice. We demonstrate that a majority of genes showed an additive effect of IFN-alphacon1 and IFN-gamma1b, but a subset of genes is synergistically induced; these include ISG20, MX2, OAS2, and other genes known to be involved in the antiviral response, TRAIL (TNFSF10) and caspases involved in apoptosis and chemokine genes RANTES, CXCL10, and CXCL11. Greater than additive transcription of some of these genes in the presence of both IFNs was confirmed by real-time kinetic RT-PCR. Elevated induction of many of these genes may be sufficient to explain the synergistic antiviral and antitumor effects of this combination of IFNs in vivo.

REFINEMENT: a search framework for the identification of interferon-responsive elements in DNA sequences--a case study with ISRE and GAS.

Interferons (IFN) are a family of pleiotropic secreted proteins that play a key role in mediating antiviral and apoptotic responses, and in immune modulation. Interferons induce a large number of genes through activating the janus tyrosine kinase (JAK)-signal transducers and activators of transcription proteins (STAT) pathway, and the binding of transcription factors to upstream regions of the inducible genes (interferon-stimulated gene, ISG) at specific DNA regulatory elements known as interferon-stimulated response element (ISRE) and gamma-activated sequence (GAS). We have previously performed DNA micro-arrays on peripheral blood mononuclear cells (PBMC) treated with interferon-alpha in culture and showed that approximately 700 genes are significantly modulated (P < or = 0.001). In order to search for ISRE and GAS we have developed a framework called regulatory element finding with iteration and effective model refinement (REFINEMENT) using an existing program (HMMER) and a standard discriminating scoring technique. Although REFINEMENT uses existing programs, our framework itself is novel as it effectively discriminates occurrences using an iterative model refinement technique. REFINEMENT has detected either ISRE or GAS sequence in all of the genes shown to be induced at a P-value < or = 0.001. There were far more functional occurrences in ISRE than in GAS, suggesting that ISRE plays a greater role in response to interferon-alpha than GAS sequences. This method can be used to identify such sequences in any set of genes. REFINEMENT is non-commercial and is accessible at .

Induction of IL-1Ra in resistant and responsive hepatitis C patients following treatment with IFN-con1.

Hepatitis C virus (HCV) infection is resistant to interferon-alpha (IFN-alpha) in some patients. The mechanism of this resistance is unknown. Interleukin-1 receptor antagonist (IL-1Ra) is induced by IFN-alpha and is a good indicator of IFN activity. In the current study, we compared IL-1Ra levels in rapid virologic responders and flat responders who showed resistance to IFN. Three groups of patients were examined, including those who received a single dose of consensus IFN (IFN-con1), patients who received daily IFN-con1 for 1 week, and patients who received IFN-con1 daily for 24 weeks. Serum IL-1Ra, IL-6, and HCV RNA were measured serially in all groups. Serum IL-1Ra levels increased rapidly in all patients with hepatitis C after IFN-alpha administration, irrespective of their virologic response. IL-1Ra levels remained elevated at 1 week but were similar to baseline by week 2 of treatment in patients receiving continuous therapy. IL-6 levels also increased acutely but rose more slowly than IL-1Ra levels. The increase in IL-1Ra and IL-6 observed in both flat and rapid virologic responders indicates that IFN receptors are functioning in patients with IFN-resistant hepatitis C and that the lack of response is related to other virologic or immunologic factors.

Global effect of PEG-IFN-alpha and ribavirin on gene expression in PBMC in vitro.

Using oligonucleotide microarrays, we have examined the expression of 22,000 genes in peripheral blood cells treated with pegylated interferon-alpha2b (PEG-IFN-alpha) and ribavirin. Treatment with ribavirin had very little effect on gene expression, whereas treatment with PEG-IFN-alpha had a dramatic effect, modulating the expression of approximately 1000 genes (at p < 0.001). In addition to genes previously reported to be induced by type I or type II IFNs, many novel genes were found to be upregulated, including transcription factors, such as ATF3, ATF4, properdin, a key regulator of the complement pathway, a homeobox gene (HESX1), and an RNA editing enzyme (apobec3). Chemokines CXCL10 and CXCL11 were upregulated, whereas CXCL5 was downregulated. Cytokines interleukin-15 (IL-15) and IL-18 were also significantly induced, whereas IL-1alpha and IL-1beta were downregulated. Most other interleukins were not affected. The results of the microarrays were confirmed by kinetic real-time PCR. These data indicate that IFN treatment causes upregulation of genes associated with the stress response, apoptosis, and signaling, and an equal number of genes are downregulated, including those associated with protein synthesis, specific cytokines and chemokines and other biosynthetic functions.

The blood transcriptional signature of chronic hepatitis C virus is consistent with an ongoing interferon-mediated antiviral response.

Blood transcriptional profiling is a powerful tool for understanding global changes after infection, and may be useful for prognosis and prediction of drug treatment responses. This study characterizes the effects of chronic hepatitis C virus (HCV) infection on gene expression by analyzing blood samples from 10 treatment-naïve HCV patients and 6 healthy volunteers. Differential expression analysis of microarray data from peripheral blood mononuclear cells (PBMCs) identified a 136-gene signature, including 66 genes elevated in infected individuals. Most of the upregulated genes were associated with interferon (IFN) activity (including members of the OAS and MX families, ISG15, and IRF7), suggesting an ongoing immune response. This HCV signature was also found to be consistently enriched in many other viral infection and vaccination datasets. These genes were validated using a second cohort composed of 5 HCV patients and 5 healthy volunteers, confirming the upregulation of the IFN signature. In summary, this is the first study to directly compare blood transcriptional profiles from HCV patients with healthy controls. The results show that chronic HCV infection has a pronounced effect on gene expression in PBMCs of infected individuals, and significantly elevates the expression of a subset of IFN-stimulated genes.

Association of single nucleotide polymorphisms in interferon signaling pathway genes and interferon-stimulated genes with the response to interferon therapy for chronic hepatitis C.

BACKGROUND/AIMS: Interferon signaling pathway genes (IPGs) and interferon-stimulated genes (ISGs) are associated with the host response to hepatitis C virus (HCV) infection. We studied single nucleotide polymorphisms (SNPs) in IPGs and ISGs for their associations with response to pegylated interferon alpha-2a (Peg-IFN-alpha) plus ribavirin therapy in HCV genotype-1 infected patients. METHODS: A two-stage study design was used. First, out of 118 SNPs selected, 91 SNPs from 5 IPGs and 12 ISGs were genotyped in a cohort of 374 treatment-naïve HCV patients and assessed for association with sustained virologic response (SVR). Next, 14 potentially functional SNPs from the OASL gene were studied in this cohort. RESULTS: Three OASL SNPs (rs3213545 and rs1169279 from stage I, and rs2859398 from stage II), were significantly associated with SVR [rs3213545: p=0.03, RR=1.27 (1.03-1.58); rs1169279: p=0.02, RR=1.32 (1.05-1.65) p=0.02; rs2859398: p=0.02, RR=1.29 (1.04-1.61)] after adjusting for other covariates. Further analysis showed that these three SNPs independently associated with SVR. Additionally, a similar trend towards the associations of these three SNPs with SVR was observed in a smaller, independent HCV cohort consisting of subjects from a number of clinical practice settings. CONCLUSIONS: Our study suggests that OASL variants are involved in the host response to IFN-based therapy in HCV patients.

A novel unsupervised method to identify genes important in the anti-viral response: application to interferon/ribavirin in hepatitis C patients.

BACKGROUND: Treating hepatitis C with interferon/ribavirin results in a varied response in terms of decrease in viral titer and ultimate outcome. Marked responders have a sharp decline in viral titer within a few days of treatment initiation, whereas in other patients there is no effect on the virus (poor responders). Previous studies have shown that combination therapy modifies expression of hundreds of genes in vitro and in vivo. However, identifying which, if any, of these genes have a role in viral clearance remains challenging. AIMS: The goal of this paper is to link viral levels with gene expression and thereby identify genes that may be responsible for early decrease in viral titer. METHODS: Microarrays were performed on RNA isolated from PBMC of patients undergoing interferon/ribavirin therapy. Samples were collected at pre-treatment (day 0), and 1, 2, 7, 14 and 28 days after initiating treatment. A novel method was applied to identify genes that are linked to a decrease in viral titer during interferon/ribavirin treatment. The method uses the relationship between inter-patient gene expression based proximities and inter-patient viral titer based proximities to define the association between microarray gene expression measurements of each gene and viral-titer measurements. RESULTS: We detected 36 unique genes whose expressions provide a clustering of patients that resembles viral titer based clustering of patients. These genes include IRF7, MX1, OASL and OAS2, viperin and many ISG's of unknown function. CONCLUSION: The genes identified by this method appear to play a major role in the reduction of hepatitis C virus during the early phase of treatment. The method has broad utility and can be used to analyze response to any group of factors influencing biological outcome such as antiviral drugs or anti-cancer agents where microarray data are available.

Changes in gene expression during pegylated interferon and ribavirin therapy of chronic hepatitis C virus distinguish responders from nonresponders to antiviral therapy.

Treating chronic hepatitis C virus (HCV) infection using pegylated alpha interferon and ribavirin leads to sustained clearance of virus and clinical improvement in approximately 50% of patients. Response rates are lower among patients with genotype 1 than with genotypes 2 and 3 and among African-American (AA) patients compared to Caucasian (CA) patients. Using DNA microarrays, gene expression was assessed for a group of 33 African-American and 36 Caucasian American patients with chronic HCV genotype 1 infection during the first 28 days of treatment. Results were examined with respect to treatment responses and to race. Patients showed a response to treatment at the gene expression level in RNA isolated from peripheral blood mononuclear cells irrespective of degree of decrease in HCV RNA levels. However, gene expression responses were relatively blunted in patients with poor viral response (<1.5 log(10)-IU/ml decrease at 28 days) compared to those in patients with a marked (>3.5 log(10)-IU/ml decrease) or intermediate (1.5 to 3.5 log(10)-IU/ml decrease) response. The number of genes that were up- or down-regulated by pegylated interferon and ribavirin treatment was fewer in patients with a poor response than in those with an intermediate or marked viral response. However AA patients had a stronger interferon response than CA patients in general. The induced levels of known interferon-stimulated genes such as the 2'5'-oligoadenylate synthetase, MX1, IRF-7, and toll-like receptor TLR-7 genes was lower in poor-response patients than in marked- or intermediate-response patients. Thus, the relative lack of viral response to interferon therapy of hepatitis C virus infection is associated with blunted interferon cell signaling. No specific regulatory gene could be identified as responsible for this global blunting or the racial differences.

Pretreatment sequence diversity differences in the full-length hepatitis C virus open reading frame correlate with early response to therapy.

Pegylated alpha interferon and ribavirin therapy for hepatitis C virus (HCV) genotype 1 infection fails for half of Caucasian American patients (CA) and more often for African Americans (AA). The reasons for these low response rates are unknown. HCV is highly genetically variable, but it is unknown how this variability affects response to therapy. To assess effects of viral diversity on response to therapy, the complete pretreatment genotype 1 HCV open reading frame was sequenced using samples from 94 participants in the Virahep-C study. Sequences from patients with >3.5 log declines in viral RNA levels by day 28 (marked responders) were more variable than those from patients with declines of <1.4 log (poor responders) in NS3 and NS5A for genotype 1a and in core and NS3 for genotype 1b. These correlations remained when all T-cell epitopes were excluded, indicating that these differences were not due to differential immune selection. When the sequences were compared by race of the patients, higher diversity in CA patients was found in E2 and NS2 but only for genotype 1b. Core, NS3, and NS5A can block the action of alpha interferon in vitro; hence, these genetic patterns are consistent with multiple amino acid variations independently impairing the function of HCV proteins that counteract interferon responses in humans, resulting in HCV strains with variable sensitivity to therapy. No evidence was found for novel HCV strains in the AA population, implying that AA patients may be infected with a higher proportion of the same resistant strains that are found in CA patients.

Model-based identification of cis-acting elements from microarray data.

Identification of transcriptional regulatory motifs continues to be a challenging problem in computational biology. We report a model-based procedure, MotifModeler, that uses global gene expression data to (1) identify cis-acting elements (CAE) that regulate gene expression under a given condition and (2) estimate the effects of the CAE on gene expression. MotifModeler repeatedly tests random subsets of all possible motifs of a given size and selects those that best fit a combinatorial model of the expression levels. We tested MotifModeler using data from a microarray experiment on the effects of interferon-alpha in peripheral blood monocytes. Focusing on 6-bp motifs, we predicted 16 stimulatory and 4 inhibitory motifs. Motifs were extended and compared to known binding sites in the TRANSFAC database using position-specific scoring matrices. Many predicted CAE match sites known to be involved in interferon action. MotifModeler demonstrated the potential to computationally identify CAE important in gene regulation.

Identification of a novel promoter in the E2 open reading frame of the human papillomavirus type 18 genome.

Northern blot and RT-PCR analyses indicated that the human papillomavirus E4 open reading frame is expressed in HeLa cells. Because integration at the E1 or E2 open reading frame would place the viral upstream regulatory region downstream of the viral late genes, the expression of E4 in HeLa cells is most likely regulated by host cellular promoter(s) or unidentified viral promoter(s) in the E2 region. Primer extension analysis and transient transfection experiments with luciferase reporter constructs under the transcriptional control of various subgenomic fragments of HPV-18 were carried out to identify and characterize functional promoters within the E2 region. The in vivo activity of a novel promoter located within the 5'-end region of the E2 open reading frame of human papillomavirus type 18 is demonstrated.