Tremendous progress has been made in understanding the molecular basis of the antiviral actions of interferons (IFNs), as well as strategies evolved by viruses to antagonize the actions of IFNs. Furthermore, advances made while elucidating the IFN system have contributed significantly to our understanding in multiple areas of virology and molecular cell biology, ranging from pathways of signal transduction to the biochemical mechanisms of transcriptional and translational control to the molecular basis of viral pathogenesis. IFNs are approved therapeutics and have moved from the basic research laboratory to the clinic. Among the IFN-induced proteins important in the antiviral actions of IFNs are the RNA-dependent protein kinase (PKR), the 2',5'-oligoadenylate synthetase (OAS) and RNase L, and the Mx protein GTPases. Double-stranded RNA plays a central role in modulating protein phosphorylation and RNA degradation catalyzed by the IFN-inducible PKR kinase and the 2'-5'-oligoadenylate-dependent RNase L, respectively, and also in RNA editing by the IFN-inducible RNA-specific adenosine deaminase (ADAR1). IFN also induces a form of inducible nitric oxide synthase (iNOS2) and the major histocompatibility complex class I and II proteins, all of which play important roles in immune response to infections. Several additional genes whose expression profiles are altered in response to IFN treatment and virus infection have been identified by microarray analyses. The availability of cDNA and genomic clones for many of the components of the IFN system, including IFN-alpha, IFN-beta, and IFN-gamma, their receptors, Jak and Stat and IRF signal transduction components, and proteins such as PKR, 2',5'-OAS, Mx, and ADAR, whose expression is regulated by IFNs, has permitted the generation of mutant proteins, cells that overexpress different forms of the proteins, and animals in which their expression has been disrupted by targeted gene disruption. The use of these IFN system reagents, both in cell culture and in whole animals, continues to provide important contributions to our understanding of the virus-host interaction and cellular antiviral response.

Antiviral Actions of Interferons

Motivation: DNA microarray experiments generating thousands of gene expression measurements, are being used to gather information from tissue and cell samples regarding gene expression differences that will be useful in diagnosing disease. We have developed a new method to analyse this kind of data using support vector machines (SVMs). This analysis consists of both classification of the tissue samples, and an exploration of the data for mis-labeled or questionable tissue results. Results: We demonstrate the method in detail on samples consisting of ovarian cancer tissues, normal ovarian tissues, and other normal tissues. The dataset consists of expression experiment results for 97 802 cDNAs for each tissue. As a result of computational analysis, a tissue sample is discovered and confirmed to be wrongly labeled. Upon correction of this mistake and the removal of an outlier, perfect classification of tissues is achieved, but not with high confidence. We identify and analyse a subset of genes from the ovarian dataset whose expression is highly differentiated between the types of tissues. To show robustness of the SVM method, two previously published datasets from other types of tissues or cells are analysed. The results are comparable to those previously obtained. We show that other machine learning methods also perform comparably to the SVM on many of those datasets. Availability: The SVM software is available at http:// www. cs.columbia.edu/ ∼bgrundy/ svm.

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Support vector machine classification and validation of cancer tissue samples using microarray expression data

Experimental genomics involves taking advantage of sequence information to investigate and understand the workings of genes, cells and organisms. We have developed an approach in which sequence information is used directly to design high-density, two-dimensional rays of synthetic oligonucleotides. The GeneChipe probe arrays are made using spatially patterned, light-directed combinatorial chemical synthesis and contain up to hundreds of thousands of different oligonucleotides on a small glass surface. The arrays have been designed and used for quantitative and highly parallel measurements of gene expression, to discover polymorphic loci and to detect the presence of thousands of alternative alleles. Here, we describe the fabrication of the arrays, their design and some specific applications to high-throughput genetic and cellular analysis.

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High density synthetic oligonucleotide arrays

Epstein-Barr virus.

正しい方法がわかる臨床基本手技 : from the New England journal of medicine

Human cytomegalovirus (HCMV), a ubiquitous herpesvirus, causes a lifelong subclinical infection in healthy adults but leads to significant morbidity and mortality in neonates and immunocompromised individuals. Its ability to grow in different cell types is responsible for HCMV-associated diseases, including mental retardation and retinitis, and vascular disorders. To globally assess viral gene function for replication in cells, we determined the genomic sequence of a bacterial artificial chromosome (BAC)-based clone of HCMV Towne strain and used this information to delete each of its 162 unique ORFs and generate a collection of viral mutants. The growth of these mutants in different cultured cells was examined to systematically investigate the necessity of each ORF for replication. Our results showed that 45 ORFs are essential for viral replication in fibroblasts and 117 are nonessential. Some genes were found to be required for viral replication in retinal pigment epithelial cells and microvascular endothelial cells, but not in fibroblasts, indicating their role as tropism factors. Interestingly, several viral mutants grew 10- to 500-fold better than the parental strain in different cell types, suggesting that the deleted ORFs encode replication temperance or repressing functions. Thus, HCMV encodes supportive and suppressive growth regulators for optimizing its replication in human fibroblasts, epithelial, and endothelial cells. Suppression of viral replication by virus-encoded temperance factors represents a novel mechanism for regulating the growth of an animal virus, and may contribute to HCMV's optimal infection of different tissues and successful proliferation among the human population.

https://www.pnas.org/content/pnas/100/24/14223.full.pdf

Functional profiling of a human cytomegalovirus genome

Human cytomegalovirus-infected fibroblasts are resistant to the induction of apoptosis by superinfection with a mutant adenovirus unable to produce the viral E1B 19-kDa protein that normally causes an E1A protein-mediated apoptotic response. Two cytomegalovirus gene products that block apoptosis were identified. The IE1 and IE2 proteins each inhibit the induction of apoptosis by tumor necrosis factor alpha or by the E1B 19-kDa-protein-deficient adenovirus but not by irradiation with UV light. Our results suggest a new physiological role for the IE1 and IE2 proteins in the human cytomegalovirus replication cycle.

Human cytomegalovirus IE1 and IE2 proteins block apoptosis.

Mechanistic insights to viral replication and pathogenesis generally have come from the analysis of viral gene products, either by studying their biochemical activities and interactions individually or by creating mutant viruses and analyzing their phenotype Now it is possible to identify and catalog the host cell genes whose mRNA levels change in response to a pathogen We have used DNA array technology to monitor the level of ≈6,600 human mRNAs in uninfected as compared with human cytomegalovirus-infected cells The level of 258 mRNAs changed by a factor of 4 or more before the onset of viral DNA replication Several of these mRNAs encode gene products that might play key roles in virus-induced pathogenesis, identifying them as intriguing targets for further study

Cellular gene expression altered by human cytomegalovirus: Global monitoring with oligonucleotide arrays

Much evidence suggests that the major immediate-early (IE) transactivator of human cytomegalovirus (HCMV), IE-2, is likely to be critical for efficient viral replication; however, the lack of an IE-2 mutant HCMV has precluded an experimental test of this hypothesis. As an initial step toward characterizing an IE-2 mutant, we first cloned the HCMV Towne genome as a bacterial artificial chromosome (BAC) and analyzed the ability of transfected Towne-BAC DNA (T-BACwt) to produce plaques following introduction into permissive human fibroblasts. Like Towne viral DNA, transfected T-BACwt DNA was infectious in permissive cells, and the resulting virus stocks were indistinguishable from Towne virus. We then used homologous recombination in Escherichia coli to delete the majority of UL122, the open reading frame encoding the unique portion of IE-2, from T-BACwt. From this deleted BAC, a third BAC clone in which the deletion was repaired with wild-type UL122 was created. In numerous transfections of permissive human foreskin fibroblast cells with these three BAC DNA clones, the rescued BAC and T-BACwt consistently yielded plaques, while the UL122 mutant BAC never generated plaques, even after 4 weeks. Protein and mRNA of other IE genes were readily detected from transfected UL122 mutant BAC DNA; however, reverse transcription-PCR failed to detect mRNA expression from any of five early genes examined. The generalized failure of this mutant to express early genes is consistent with expectations from in vitro assays which have demonstrated that IE-2 transactivates most HCMV promoters. These experiments provide the first direct demonstration that IE-2 is required for successful HCMV infection and indicate that virus lacking IE-2 arrests early in the replication cycle.

Human Cytomegalovirus with IE-2 (UL122) Deleted Fails To Express Early Lytic Genes

We used differential display analysis to identify mRNAs that accumulate to enhanced levels in human cytomegalovirus-infected cells as compared with mock-infected cells. RNAs were compared at 8 hr after infection of primary human fibroblasts. Fifty-seven partial cDNA clones were isolated, representing about 26 differentially expressed mRNAs. Eleven of the mRNAs were virus-coded, and 15 were of cellular origin. Six of the partial cDNA sequences have not been reported previously. All of the cellular mRNAs identified in the screen are induced by interferon α. The induction in virus-infected cells, however, does not involve the action of interferon or other small signaling molecules. Neutralizing antibodies that block virus infection also block the induction. These RNAs accumulate after infection with virus that has been inactivated by treatment with UV light, indicating that the inducer is present in virions. We conclude that human cytomegalovirus induces interferon-responsive mRNAs.

https://www.pnas.org/content/pnas/94/25/13985.full.pdf

Use of differential display analysis to assess the effect of human cytomegalovirus infection on the accumulation of cellular RNAs: Induction of interferon-responsive RNAs

Hua Zhu

Papers

Functional profiling of a human cytomegalovirus genome

Human cytomegalovirus IE1 and IE2 proteins block apoptosis.

Cellular gene expression altered by human cytomegalovirus: Global monitoring with oligonucleotide arrays

Human Cytomegalovirus with IE-2 (UL122) Deleted Fails To Express Early Lytic Genes

Use of differential display analysis to assess the effect of human cytomegalovirus infection on the accumulation of cellular RNAs: Induction of interferon-responsive RNAs