Host Dependency Factors Research Articles

High-throughput approaches to unravel hepatitis C virus-host interactions

Hepatitis C virus (HCV) remains a major global health burden, with more than 130 million individuals chronically infected and at risk for the development of hepatocellular carcinoma (HCC). The recent clinical licensing of direct-acting antivirals enables viral cure. However, limited access to therapy and treatment failure in patient subgroups warrants a continuing effort to develop complementary antiviral strategies. Furthermore, once fibrosis is established, curing HCV infection does not eliminate the risk for HCC. High-throughput approaches and screens have enabled the investigation of virus-host interactions on a genome-wide scale. Gain- and loss-of-function screens have identified essential host-dependency factors in the HCV viral life cycle, such as host cell entry factors or regulatory factors for viral replication and assembly. Network analyses of systems-scale data sets provided a comprehensive view of the cellular state following HCV infection, thus improving our understanding of the virus-induced responses of the target cell. Interactome, metabolomics and gene expression studies identified dysregulated cellular processes potentially contributing to HCV pathogenesis and HCC. Drug screens using chemical libraries led to the discovery of novel antivirals. Here, we review the contribution of high-throughput approaches for the investigation of virus-host interactions, viral pathogenesis and drug discovery.

An integrative approach for a network based meta-analysis of viral RNAi screens

BackgroundBig data is becoming ubiquitous in biology, and poses significant challenges in data analysis and interpretation. RNAi screening has become a workhorse of functional genomics, and has been applied, for example, to identify host factors involved in infection for a panel of different viruses. However, the analysis of data resulting from such screens is difficult, with often low overlap between hit lists, even when comparing screens targeting the same virus. This makes it a major challenge to select interesting candidates for further detailed, mechanistic experimental characterization.ResultsTo address this problem we propose an integrative bioinformatics pipeline that allows for a network based meta-analysis of viral high-throughput RNAi screens. Initially, we collate a human protein interaction network from various public repositories, which is then subjected to unsupervised clustering to determine functional modules. Modules that are significantly enriched with host dependency factors (HDFs) and/or host restriction factors (HRFs) are then filtered based on network topology and semantic similarity measures. Modules passing all these criteria are finally interpreted for their biological significance using enrichment analysis, and interesting candidate genes can be selected from the modules.ConclusionsWe apply our approach to seven screens targeting three different viruses, and compare results with other published meta-analyses of viral RNAi screens. We recover key hit genes, and identify additional candidates from the screens. While we demonstrate the application of the approach using viral RNAi data, the method is generally applicable to identify underlying mechanisms from hit lists derived from high-throughput experimental data, and to select a small number of most promising genes for further mechanistic studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13015-015-0035-7) contains supplementary material, which is available to authorized users.

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors. We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions. Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected naïve cells in a receptor-dependent fashion. To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection. Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

Normalizing for individual cell population context in the analysis of high-content cellular screens

BackgroundHigh-content, high-throughput RNA interference (RNAi) offers unprecedented possibilities to elucidate gene function and involvement in biological processes. Microscopy based screening allows phenotypic observations at the level of individual cells. It was recently shown that a cell's population context significantly influences results. However, standard analysis methods for cellular screens do not currently take individual cell data into account unless this is important for the phenotype of interest, i.e. when studying cell morphology.ResultsWe present a method that normalizes and statistically scores microscopy based RNAi screens, exploiting individual cell information of hundreds of cells per knockdown. Each cell's individual population context is employed in normalization. We present results on two infection screens for hepatitis C and dengue virus, both showing considerable effects on observed phenotypes due to population context. In addition, we show on a non-virus screen that these effects can be found also in RNAi data in the absence of any virus. Using our approach to normalize against these effects we achieve improved performance in comparison to an analysis without this normalization and hit scoring strategy. Furthermore, our approach results in the identification of considerably more significantly enriched pathways in hepatitis C virus replication than using a standard analysis approach.ConclusionsUsing a cell-based analysis and normalization for population context, we achieve improved sensitivity and specificity not only on a individual protein level, but especially also on a pathway level. This leads to the identification of new host dependency factors of the hepatitis C and dengue viruses and higher reproducibility of results.

A functional genomics approach to viral-host interactions for HIV

New screening methodologies developed over the past five years, based on a revolution in understanding the biology of double-stranded DNA and RNA interference, provide the tools for genetic screening of mammalian cells. HIV is a straightforward RNA virus, and the challenge our team has tackled is to identify the host proteins the virus needs to replicate using short strands of small interfering RNA (siRNA) to block their function. The siRNA screen is an arrayed format—siRNAs to one gene per well—using liquid-handling robots. Cells are transiently transfected with siRNAs, producing protein knock-down in a gene-specific manner that is effective for about a week. Using a SMARTpool library produced by Dharmacon with four siRNAs per pool and 21,121 genes, we completed a validation round with the four individual siRNAs. Seventy-two hours after transecting the cells with siRNA, infected cells and supernatant were harvested, and the viral load measured. The cellular analysis indicated how well the virus had infected the cell and expressed a key capsid protein p24 protein; the supernatant indicated how efficiently the virus budded out of the cell in a functional form. We discovered several proteins that appear to be genetically important to the HIV lifecycle. Using bioinformatics, we were able to map out the role the host factors played in the viral life cycle, from the virus getting into the cell, to its integration into the DNA of the cell’s nucleus, to the messenger RNA (mRNA) processing of the virus; and to its export into the cytoplasm and out of the cell. Genetic screening results provided a rediscovery of all known host dependency factors in the HIV lifecycle states and confirmation of Host Dependency Factors (Rab6, Vps53, TNPO3, Med28), suggesting the model is effective. One of the proteins identified in the screen (TPNO3), a karyopherin, is likely to be the host factor that permits HIV access to our nucleus for integration into our genome. Our research demonstrates that when TPNO3 was knocked out by any one of eight siRNAs, the virus was prevented from infecting the cell and integrating into the genome. Utilizing unbiased whole genome screens which can be used for any pathogen, to identify host proteins that actively support replication could provide new drug targets. The way the screen is designed can profoundly influence which genes are uncovered and different screening platforms will yield different results, making the technique potentially very flexible.

Abstract 2592: Host dependency factors for binding and internalization of oncolytic Seneca Valley Virus

Abstract Background Seneca Valley Virus (SVV-001), an experimental oncolytic picornavirus, is the prototype member of the genus Senecavirus of the family Picornaviridae. SVV-001 is remarkable for its exquisite tumor selectivity for human cancers with neuroendocrine features including small cell lung carcinoma (SCLC) and numerous pediatric cancers while being non-cytotoxic in normal human cells in vitro and in vivo. The mechanism of tropism and neuroendocrine tumor specificity of SVV-001 has not been elucidated previously. Identification of host factors involved in infection would have immediate benefit for ongoing clinical trials by providing potential biomarker candidates to predict efficacy. Here we present data implicating α4 integrin as a key component of SVV-001 infection and demonstrating that the virus is dependent on caveloae for internalization and trafficking. Results Microarray analysis comparing permissive with non-permissive cell lines and membrane sub-proteome analysis of permissive H446 SCLC cells predict α4 integrin is a potential receptor candidate for SVV-001. To test SVV-001 dependency on α4 integrin, stable shRNA knockdown cell lines were generated from the permissive parental SCLC cell line H446. Viral infection was studied by single virus particle tracking, GFP reporter virus infection, and FACS analysis. α4 integrin knockdown cells showed reduced viral internalization and intracellular motility and were resistant to infection. Heterologous expression of human α4 integrin in chinese hamster ovary (CHO) cells significantly increased binding of fluorescently-labeled virus to the cell surface, but did not result in productive infection. Tracking of fluorescently-labeled SVV-001 in permissive cells expressing either clathrin-GFP, caveolin-1-GFP, or flotillin-1-GFP revealed a striking and long-lasting colocalization of internalized viral particles with caveolin-rich vesicles. Dependency on caveolin-1 for internalization was supported by results from pharmacological inhibition experiments. Pre-treatment of cells with nystatin, a cholesterol sequestration drug, resulted in a reduction in virus-induced cytopathic effect when compared to untreated cells. Stable shRNA caveolin-1 knockdown cells showed significantly reduced internalization, reduced infection by recombinant GFP reporter virus and significant impairment in SVV-001 genomic RNA release. Conclusions Defining the mechanism of tropism for SVV-001 is a key factor in successful clinical application. α4 integrin and caveolin-1 represent the first host cell factors identified in the mechanism of tropism of SVV-001 leading to a basic understanding of how the virus binds and enters permissive cells. These findings may have direct relevance for current clinical trials of SVV-001 in adult and pediatric settings and for development of second-generation derivatives of this agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2592.

Harnessing RNA interference for the treatment of viral infections

Exploiting the RNA interference (RNAi) pathway to inhibit viral gene expression has become an active field of research. The approach has potential for therapeutic application and several viruses are susceptible to RNAi-mediated knockdown. Differences in the characteristics of individual viruses require that viral gene silencing be tailored to specific infections. Important considerations are viral tissue tropism, acute or chronic nature of the infection and the efficiency with which antiviral sequences can be delivered to affected tissue. Both synthetic short interfering RNAs (siRNAs) and expressed RNAi activators are being developed for viral therapy. The sustained silencing of expressed antiviral sequences is useful for countering chronic viral infection. siRNAs, which may be chemically modified to improve specificity and stability, are being developed for knockdown of viruses that cause acute or chronic infections. Preventing viral escape from silencing is important and overcoming this problem using combinatorial RNAi or through silencing of host dependency factors is promising. Although improving delivery efficiency and limiting off-target effects remain obstacles, rapid progress continues to be made in the field and it is likely that the goal of achieving licensed RNAi-based viral therapies will soon be realized.

Realizing the Promise of RNAi

The convergence of the discovery of RNA interference (RNAi) with the completion of the human genome sequencing program has revolutionized our methods of analyzing gene function in mammalian cells.1,2 The task of studying gene function can be relatively easily accomplished by the use of small interfering RNAs (siRNAs) for targeted inhibition of gene expression. Furthermore, use of RNAi library screens to identify specified phenotypes can be applied to drug target development.3 As such, a recent publication in Science by Brass et al.4 has stimulated a great deal of interest in the AIDS research community. This group, led by Stephen Elledge and Judith Lieberman, published an exciting application of RNAi that used an siRNA library screen to identify cellular factors required for HIV entry, replication, and infectious virus production. A handful of essential cellular host factors had previously been determined by other techniques, but this latest screen revealed more than 250 new cellular proteins required for HIV-1 to complete its replication cycle. The team termed these genes host dependency factors (HDFs). It is exciting to speculate that small-molecule drugs may be developed for some of these HDF targets in the next few years and that there may even be existing drugs that inhibit some of these HDFs that can now be developed for treating HIV-1 infection.

AeTIOLOGY AND PATHOGENESIS OF ACUTE SPORADIC DISSEMINATED ENCEPHALOMYELITIS AND MULTIPLE SCLEROSIS

<h3>Objective</h3> Hepatitis D virus (HDV) is a circular RNA virus coinfecting hepatocytes with hepatitis B virus. Chronic hepatitis D results in severe liver disease and an increased risk of liver cancer. Efficient therapeutic approaches against HDV are absent. <h3>Design</h3> Here, we combined an RNAi loss-of-function and small molecule screen to uncover host-dependency factors for HDV infection. <h3>Results</h3> Functional screening unravelled the hypoxia-inducible factor (HIF)-signalling and insulin-resistance pathways, RNA polymerase II, glycosaminoglycan biosynthesis and the pyrimidine metabolism as virus-hepatocyte dependency networks. Validation studies in primary human hepatocytes identified the carbamoyl-phosphatesynthetase 2, aspartate transcarbamylase and dihydroorotase (CAD) enzyme and estrogen receptor alpha (encoded by <i>ESR1</i>) as key host factors for HDV life cycle. Mechanistic studies revealed that the two host factors are required for viral replication. Inhibition studies using N-(phosphonoacetyl)-L-aspartic acid and fulvestrant, specific CAD and ESR1 inhibitors, respectively, uncovered their impact as antiviral targets. <h3>Conclusion</h3> The discovery of HDV host-dependency factors elucidates the pathogenesis of viral disease biology and opens therapeutic strategies for HDV cure.

A NOTE ON THE DOG'S TAPETUM IN EARLY LIFE

Understanding how host factors and hundreds of viral genes or-chestrate the complex life cycle of herpesviruses represents a fundamental problem in virology. Here, we use CRISPR/Cas9-based screening to scan at high-resolution for functional elements in the genome of human cytomegalovirus (HCMV), and to generate a genome-wide mapping of host dependency and restriction factors. Our data reveal an architecture of functional modules in the HCMV genome, and host factor pathways involved in virus adhesion and entry, membrane trafficking, and innate immune response. Single-cell analysis shows that the large majority of cells follow a stereotypical trajectory in viral gene expression space. Perturbation of host factors does not alter this trajectory, but can accelerate or stall progression. Conversely, perturbation of viral factors creates discrete alternate ‘dead-end’ trajectories. Our results reveal a fundamental dichotomy between the roles of host and viral factors in orchestrating viral replication and more generally provide a road map for high-resolution dissection of host-pathogen interactions.