Gary K. Geiss

Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: The role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza

The NS1 protein of influenza A virus contributes to viral pathogenesis, primarily by enabling the virus to disarm the host cell type IFN defense system. We examined the downstream effects of NS1 protein expression during influenza A virus infection on global cellular mRNA levels by measuring expression of over 13,000 cellular genes in response to infection with wild-type and mutant viruses in human lung epithelial cells. Influenza A/PR/8/34 virus infection resulted in a significant induction of genes involved in the IFN pathway. Deletion of the viral NS1 gene increased the number and magnitude of expression of cellular genes implicated in the IFN, NF-κB, and other antiviral pathways. Interestingly, different IFN-induced genes showed different sensitivities to NS1-mediated inhibition of their expression. A recombinant virus with a C-terminal deletion in its NS1 gene induced an intermediate cellular mRNA expression pattern between wild-type and NS1 knockout viruses. Most significantly, a virus containing the 1918 pandemic NS1 gene was more efficient at blocking the expression of IFN-regulated genes than its parental influenza A/WSN/33 virus. Taken together, our results suggest that the cellular response to influenza A virus infection in human lung cells is significantly influenced by the sequence of the NS1 gene, demonstrating the importance of the NS1 protein in regulating the host cell response triggered by virus infection.

Cellular Gene Expression upon Human Immunodeficiency Virus Type 1 Infection of CD4+-T-Cell Lines

ABSTRACT The expression levels of ∼4,600 cellular RNA transcripts were assessed in CD4+-T-cell lines at different times after infection with human immunodeficiency virus type 1 strain BRU (HIV-1BRU) using DNA microarrays. We found that several classes of genes were inhibited by HIV-1BRU infection, consistent with the G2 arrest of HIV-1-infected cells induced by Vpr. These included genes involved in cell division and transcription, a family of DEAD-box proteins (RNA helicases), and all genes involved in translation and splicing. However, the overall level of cell activation and signaling was increased in infected cells, consistent with strong virus production. These included a subgroup of transcription factors, including EGR1 and JUN, suggesting they play a specific role in the HIV-1 life cycle. Some regulatory changes were cell line specific; however, the majority, including enzymes involved in cholesterol biosynthesis, of changes were regulated in most infected cell lines. Compendium analysis comparing gene expression profiles of our HIV-1 infection experiments to those of cells exposed to heat shock, interferon, or influenza A virus indicated that HIV-1 infection largely induced specific changes rather than simply activating stress response or cytokine response pathways. Thus, microarray analysis confirmed several known HIV-1 host cell interactions and permitted identification of specific cellular pathways not previously implicated in HIV-1 infection. Continuing analyses are expected to suggest strategies for impacting HIV-1 replication in vivo by targeting these pathways.

Discovery of Novel Methylation Biomarkers in Cervical Carcinoma by Global Demethylation and Microarray Analysis

A genome-wide screening study for identification of hypermethylated genes in invasive cervical cancer (ICC) was carried out to augment our previously discovered panel of three genes found to be useful for detection of ICC and its precursor neoplasia. Putatively hypermethylated and silenced genes were reactivated in four ICC cell lines by treatment with 5-aza-2′-deoxycytidine and trichostatin A and identified on expression microarrays. Thirty-nine of the 235 genes up-regulated in multiple ICC cell lines were further examined to determine the methylation status of associated CpG islands. The diagnostic use of 23 genes that were aberrantly methylated in multiple ICC cell lines were then analyzed in DNA from exfoliated cells obtained from patients with or without ICC. We show, for the first time, that aberrant methylation of six genes (SPARC, TFPI2, RRAD, SFRP1, MT1G, and NMES1) is present in a high proportion of ICC clinical samples but not in normal samples. Of these genes, SPARC and TFPI2 showed the highest frequency of aberrant methylation in ICC specimens (86.4% for either) and together were hypermethylated in all but one ICC cases examined. We conclude that expression profiling of epigenetically reactivated genes followed by methylation analysis in clinical samples is a powerful tool for comprehensive identification of methylation markers. Several novel genes identified in our study may be clinically useful for detection or stratification of ICC and/or of its precursor lesions and provide a basis for better understanding of mechanisms involved in development of ICC. (Cancer Epidemiol Biomarkers Prev 2006;(15)1:114–23)

Global Impact of Influenza Virus on Cellular Pathways Is Mediated by both Replication-Dependent and -Independent Events

ABSTRACT Influenza virus, the causative agent of the common flu, is a worldwide health problem with significant economic consequences. Studies of influenza virus biology have revealed elaborate mechanisms by which the virus interacts with its host cell as it inhibits the synthesis of cellular proteins, evades the innate antiviral response, and facilitates production of viral RNAs and proteins. With the advent of DNA array technology it is now possible to obtain a large-scale view of how viruses alter the environment within the host cell. In this study, the cellular response to influenza virus infection was examined by monitoring the steady-state mRNA levels for over 4,600 cellular genes. Infections with active and inactivated influenza viruses identified changes in cellular gene expression that were dependent on or independent of viral replication, respectively. Viral replication resulted in the downregulation of many cellular mRNAs, and the effect was enhanced with time postinfection. Interestingly, several genes involved in protein synthesis, transcriptional regulation, and cytokine signaling were induced by influenza virus replication, suggesting that some may play essential or accessory roles in the viral life cycle or the host cells stress response. The gene expression pattern induced by inactivated viruses revealed induction of the cellular metallothionein genes that may represent a protective response to virus-induced oxidative stress. Genome-scale analyses of virus infections will help us to understand the complexities of virus-host interactions and may lead to the discovery of novel drug targets or antiviral therapies.

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

ABSTRACT Alpha/beta interferons (IFN-α/β) 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.

A portable surface plasmon resonance sensor system for real-time monitoring of small to large analytes

Many environmental applications exist for biosensors capable of providing real-time analyses. One pressing current need is monitoring for agents of chemical- and bio-terrorism. These applications require systems that can rapidly detect small organics including nerve agents, toxic proteins, viruses, spores and whole microbes. A second area of application is monitoring for environmental pollutants. Processing of grab samples through chemical laboratories requires significant time delays in the analyses, preventing the rapid mapping and cleanup of chemical spills. The current state of development of miniaturized, integrated surface plasmon resonance (SPR) sensor elements has allowed for the development of inexpensive, portable biosensor systems capable of the simultaneous analysis of multiple analytes. Most of the detection protocols make use of antibodies immobilized on the sensor surface. The Spreeta 2000 SPR biosensor elements manufactured by Texas Instruments provide three channels for each sensor element in the system. A temperature-controlled two-element system that monitors for six analytes is currently in use, and development of an eight element sensor system capable of monitoring up to 24 different analytes will be completed in the near future. Protein toxins can be directly detected and quantified in the low picomolar range. Elimination of false positives and increased sensitivity is provided by secondary antibodies with specificity for different target epitopes, and by sensor element redundancy. Inclusion of more than a single amplification step can push the sensitivity of toxic protein detection to femtomolar levels. The same types of direct detection and amplification protocols are used to monitor for viruses and whole bacteria or spores. Special protocols are required for the detection of small molecules. Either a competition type assay where the presence of analyte inhibits the binding of antibodies to surface-immobilized analyte, or a displacement assay, where antibodies bound to analyte on the sensor surface are displaced by free analyte, can be used. The small molecule detection assays vary in sensitivity from the low micromolar range to the high picomolar.

Abstract 2441: NanoString 3D Biology™ technology: simultaneous digital counting of DNA, RNA and protein

Introduction: Development of improved cancer diagnostics and therapeutics requires detailed understanding of the genomic, transcriptomic, and proteomic profiles in the tumor microenvironment. Current technologies can excel at measuring a single analyte, but it remains challenging to simultaneously collect high-throughput DNA, RNA, and protein data from small samples. We have developed an approach that uses optical barcodes to simultaneously profile DNA, RNA, and protein from as little as 5ng DNA, 25ng RNA, and 250ng protein or just 2 5µm FFPE slides, and simplifies data analysis by generating digital counts for each analyte. Methods: The approach uses paired capture and reporter oligonucleotide probes and optical barcodes to enumerate up to 800 targets. The platform was initially developed to measure RNA, and we have adapted it to measure DNA single nucleotide variants (SNVs), proteins, and phospho-proteins. SNVs are detected by direct hybridization of sequence discriminating probes to the wild-type and mutant sequence of interest. Proteins are detected via binding of oligonucleotide-conjugated antibodies. Results: Combinations of DNA, RNA, and protein in biological and experimental contexts. SNV probes are able to detect variant alleles down to 5% abundance within a wild type population and can discriminate variants within mutation hotspots. It was >96% accurate at identifying variants from samples displaying a range of allele frequencies and DNA integrity when benchmarked against next-generation sequencing. Protein detection has been developed for cell surface, cytosolic, and nuclear proteins, as well as phospho-proteins. It was validated against flow cytometry, western blot, and mass spectrometry using cell lines with ectopic target expression and primary cells. To demonstrate concurrent measurement of DNA, RNA, and protein from a single system, BRAFWT or BRAFV600E cell lines were treated with the BRAFV600E inhibitor vemurafenib and the MEK inhibitor trametinib. We measured the allele usage at the BRAFV600 locus, as well as BRAFV600E dependent changes in mRNA expression, protein expression and protein phosphorylation in a single experiment. Conclusions: 3D Biology has several advantages over other analytical approaches. Direct, single-molecule digital counting allows detection over a broad dynamic range with high reproducibility, often over 98% concordance between technical replicates. The simultaneous interrogation of DNA, RNA, and protein maximizes the amount of data obtained from precious samples and minimizes instrumentation demands by leveraging a single detection platform. The 3D Biology approach allows holistic, digital analysis of biological samples with high specificity and precision. This technology is currently available for research use, but may also have clinical application in the future. Citation Format: Chris Lausted, Yong Zhou, Jinho Lee, Christopher Vellano, Karina A. Eterovic, Ping Song, Lin-ya Tang, Gloria Fawcett, Tae-Beom Kim, Ken Chen, Gary Geiss, Gavin Meredith, Qian Mei, Gokhan Demirkan, Dwayne Dunaway, Dae Kim, P. Martin Ross, Elizabeth Manrao, Nathan Elliott, Sarah Warren, Christina Bailey, Chung-Ying Huang, Joseph Beechem, Gordon Mills, Leroy Hood. NanoString 3D Biology™ technology: simultaneous digital counting of DNA, RNA and protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2441. doi:10.1158/1538-7445.AM2017-2441