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1.  Functional genomics approach for the identification of human host factors supporting dengue viral propagation 
Dengue virus (DENV) is endemic throughout tropical regions around the world and there are no approved treatments or anti-transmission agents currently available. Consequently, there exists an enormous unmet need to treat the human diseases caused by DENV and block viral transmission by the mosquito vector. RNAi screening represents an efficient method to expand the pool of known host factors that could become viable targets for treatments or provide rationale to consider available drugs as anti-DENV treatments. We developed a high throughput siRNA-based screening protocol that can identify human DENV host factors. The protocol herein describes the materials and the procedures necessary to screen a human cell line in order to identify genes which are either necessary for or restrict DENV propagation at any stage in the viral life cycle.
PMCID: PMC4075997  PMID: 24696344
RNA interference (RNAi); dengue virus; yellow fever virus; whole genome RNAi screening; whole genome siRNA screening; dengue virus host factors; flavivirus
2.  Transcription elongation regulator 1 (TCERG1) regulates competent RNA polymerase II-mediated elongation of HIV-1 transcription and facilitates efficient viral replication 
Retrovirology  2013;10:124.
Control of RNA polymerase II (RNAPII) release from pausing has been proposed as a checkpoint mechanism to ensure optimal RNAPII activity, especially in large, highly regulated genes. HIV-1 gene expression is highly regulated at the level of elongation, which includes transcriptional pausing that is mediated by both viral and cellular factors. Here, we present evidence for a specific role of the elongation-related factor TCERG1 in regulating the extent of HIV-1 elongation and viral replication in vivo.
We show that TCERG1 depletion diminishes the basal and viral Tat-activated transcription from the HIV-1 LTR. In support of a role for an elongation mechanism in the transcriptional control of HIV-1, we found that TCERG1 modifies the levels of pre-mRNAs generated at distal regions of HIV-1. Most importantly, TCERG1 directly affects the elongation rate of RNAPII transcription in vivo. Furthermore, our data demonstrate that TCERG1 regulates HIV-1 transcription by increasing the rate of RNAPII elongation through the phosphorylation of serine 2 within the carboxyl-terminal domain (CTD) of RNAPII and suggest a mechanism for the involvement of TCERG1 in relieving pausing. Finally, we show that TCERG1 is required for HIV-1 replication.
Our study reveals that TCERG1 regulates HIV-1 transcriptional elongation by increasing the elongation rate of RNAPII and phosphorylation of Ser 2 within the CTD. Based on our data, we propose a general mechanism for TCERG1 acting on genes that are regulated at the level of elongation by increasing the rate of RNAPII transcription through the phosphorylation of Ser2. In the case of HIV-1, our evidence provides the basis for further investigation of TCERG1 as a potential therapeutic target for the inhibition of HIV-1 replication
PMCID: PMC3874760  PMID: 24165037
TCERG1; Transcription elongation; RNA polymerase II; Pausing
3.  G Protein-Coupled Receptor Kinase 2 Promotes Flaviviridae Entry and Replication 
Flaviviruses cause a wide range of severe diseases ranging from encephalitis to hemorrhagic fever. Discovery of host factors that regulate the fate of flaviviruses in infected cells could provide insight into the molecular mechanisms of infection and therefore facilitate the development of anti-flaviviral drugs. We performed genome-scale siRNA screens to discover human host factors required for yellow fever virus (YFV) propagation. Using a 2×2 siRNA pool screening format and a duplicate of the screen, we identified a high confidence list of YFV host factors. To find commonalities between flaviviruses, these candidates were compared to host factors previously identified for West Nile virus (WNV) and dengue virus (DENV). This comparison highlighted a potential requirement for the G protein-coupled receptor kinase family, GRKs, for flaviviral infection. The YFV host candidate GRK2 (also known as ADRBK1) was validated both in siRNA-mediated knockdown HuH-7 cells and in GRK−/− mouse embryonic fibroblasts. Additionally, we showed that GRK2 was required for efficient propagation of DENV and Hepatitis C virus (HCV) indicating that GRK2 requirement is conserved throughout the Flaviviridae. Finally, we found that GRK2 participates in multiple distinct steps of the flavivirus life cycle by promoting both entry and RNA synthesis. Together, our findings identified GRK2 as a novel regulator of flavivirus infection and suggest that inhibition of GRK2 function may constitute a new approach for treatment of flavivirus associated diseases.
Author Summary
The Flavivirus genus includes several emergent and reemergent viruses, such as dengue and yellow fever viruses, which cause severe diseases in humans for which there is no approved treatment. Flaviviruses are transmitted to humans by arthropods and they rely on scores of vertebrate and invertebrate factors to replicate in these disparate hosts. Identifying the host factors involved in viral propagation is critical to understanding the molecular mechanisms of infection and the development of new therapeutics. To identify human host factors required for yellow fever virus propagation, we completed two genome-scale siRNA screens. Among the candidates discovered were the G protein-coupled receptor kinases GRK2 and GRK4. We focused on the protein GRK2, a kinase first identified for its role in cellular signal transduction. We found that GRK2 was a host factor needed for productive infection by yellow fever, dengue and hepatitis C viruses and was required for both viral entry and efficient replication of the viral genome. GRKs, which are considered druggable, may be used as targets to develop broadspectrum anti-flavivirals.
PMCID: PMC3441407  PMID: 23029581
4.  Factors affecting reproducibility between genome-scale siRNA-based screens 
Journal of biomolecular screening  2010;15(7):735-747.
RNA interference-based screening is a powerful new genomic technology which addresses gene function en masse. To evaluate factors influencing hit list composition and reproducibility, we performed two identically designed small interfering RNA (siRNA)-based, whole genome screens for host factors supporting yellow fever virus infection. These screens represent two separate experiments completed five months apart and allow the direct assessment of the reproducibility of a given siRNA technology when performed in the same environment. Candidate hit lists generated by sum rank, median absolute deviation, z-score, and strictly standardized mean difference were compared within and between whole genome screens. Application of these analysis methodologies within a single screening dataset using a fixed threshold equivalent to a p-value ≤ 0.001 resulted in hit lists ranging from 82 to 1,140 members and highlighted the tremendous impact analysis methodology has on hit list composition. Intra- and inter-screen reproducibility was significantly influenced by the analysis methodology and ranged from 32% to 99%. This study also highlighted the power of testing at least two independent siRNAs for each gene product in primary screens. To facilitate validation we conclude by suggesting methods to reduce false discovery at the primary screening stage.
In this study we present the first comprehensive comparison of multiple analysis strategies, and demonstrate the impact of the analysis methodology on the composition of the “hit list”. Therefore, we propose that the entire dataset derived from functional genome-scale screens, especially if publicly funded, should be made available as is done with data derived from gene expression and genome-wide association studies.
PMCID: PMC3149892  PMID: 20625183
RNA interference; analysis; RNAi screen analysis; siRNA; RNAi; siRNA screening; sum rank; median absolute deviation; strictly standardized mean difference; genome-wide; whole-genome; comparison; overlap; hit list
5.  PTB Regulates the Processing of a 3′-Terminal Exon by Repressing both Splicing and Polyadenylation 
Molecular and Cellular Biology  2005;25(21):9595-9607.
The polypyrimidine tract binding protein (PTB) has been described as a global repressor of regulated exons. To investigate PTB functions in a physiological context, we used a combination of morpholino-mediated knockdown and transgenic overexpression strategies in Xenopus laevis embryos. We show that embryonic endoderm and skin deficient in PTB displayed a switch of the α-tropomyosin pre-mRNA 3′ end processing to the somite-specific pattern that results from the utilization of an upstream 3′-terminal exon designed exon 9A9′. Conversely, somitic targeted overexpression of PTB resulted in the repression of the somite-specific exon 9A9′ and a switch towards the nonmuscle pattern. These results validate PTB as a key physiological regulator of the 3′ end processing of the α-tropomyosin pre-mRNA. Moreover, using a minigene strategy in the Xenopus oocyte, we show that in addition to repressing the splicing of exon 9A9′, PTB regulates the cleavage/polyadenylation of this 3′-terminal exon.
PMCID: PMC1265821  PMID: 16227608

Results 1-5 (5)