Principles of Virology, Volume 1. Jane Flint

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Principles of Virology, Volume 1 - Jane Flint

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(Chapter 14) and can also be used to discover new viruses. In this method, millions of unique viral DNA sequences fixed to glass or silicon wafers are incubated with sequences complementary to DNAs or RNAs, which have been amplified from clinical and environmental samples by PCR. Binding is usually detected by using fluorescent molecules incorporated into amplified nucleic acids. Microarrays have been largely supplanted by high-throughput sequencing, which allows identification of transcripts and their quantification in an unbiased manner, e.g., without prior assumption of what genes are involved.

      In RNAseq, RNAs extracted from cells or tissues are converted by reverse transcription to complementary DNAs, which are then subjected to high-throughput DNA sequencing. The results provide insight into sequences and quantity of RNAs in a cell at a given time under specific conditions. It allows detection and quantification of transcripts that are not represented on microarrays. Information on transcriptional activity is provided by native elongating transcript sequencing (NET-seq), in which immunoprecipitation of RNA polymerase is followed by high-throughput sequencing of the 3′ ends of the associated RNAs. A method to study the association of RNAs with ribosomes is ribo-seq, in which polysomes are treated with RNases and the 20- to 30-nucleotide ribosome-protected fragments are sequenced. The information provides insight into translational control of gene expression and the mechanism of protein synthesis and allows annotation of translated sequences.

      Many protocols have been devised for genome-wide analysis of RNA-protein interactions that are based on cross-linking immunoprecipitation (CLIP). In CLIP-seq, RNA-protein complexes are cross-linked in cells in culture with UV light. Cells are lysed and proteins of interest are immunoprecipitated. Proteins are removed by digestion with protease, DNA is synthesized from the previously bound RNA with reverse transcriptase, and the product is subjected to high-throughput sequence analysis. Interaction sites are identified by mapping the nucleic acid sequence reads to the transcriptome. A modification of this technique is called photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation, PAR-CLIP. In this method, photoreactive ribonucleoside analogs such as 4-thiouridine are incorporated into RNA transcripts in living cells. Irradiation with UV light induces efficient cross-linking of RNAs containing these analogs to interacting proteins. Immunoprecipitation and sequencing are then carried out as in other CLIP methods.

      Other genome-wide mapping analyses that can be performed include identifying the binding sites for long noncoding RNAs (lncRNA) on chromatin using capture hybridization analysis of RNA targets (CHART). In this method, biotin-linked oligonucleotides that are complementary to the target RNA are designed. These are added to reversibly cross-linked chromatin extracts, and the target RNA is purified with streptavidin beads, which bind with high afnity to biotin. The sequences of the RNA targets identify the genomic binding sites of endogenous RNAs. A related method is chromatin isolation by RNA purification (ChIRP), in which tiled oligonucleotides labeled with biotin are used to retrieve specific lncRNA bound to protein and DNAs.

      Mass spectrometry (MS) is a technique that can identify the chemical constituents of complex and simple mixtures. It has emerged as a powerful tool for detecting and quantifying thousands of proteins in biological samples, including viruses and virus-infected cells.

      A mass spectrometer ionizes the chemical constituents of a mixture and then sorts the ions based on their mass-to-charge ratio. Identification of the components is done by comparison with the patterns generated by known materials.

      The total protein content of a cell or a virus particle is called the proteome. Human cells have been estimated to contain from 500,000 to 3,000,000 proteins per cubic micrometer, encoded by ∼20,000 open reading frames, and their products are further diversified by transcriptional, posttranscriptional, translational, and posttranslational regulation. The cell proteome may be further altered during virus infection. The proteome of virus particles is far less complex, but the very largest viruses can still contain hundreds of proteins. Mass spectrometry can be used to identify proteins and their concentrations in cells and in virus particles and also to reveal protein localization, protein-protein interactions, and posttranslational modifications in infected and uninfected cells.

      Mass spectrometry may be combined with biochemical and genomic techniques to provide global views of viral reproduction cycles. For example, changes in proteins secreted by host cells upon virus infection can be readily characterized by performing mass spectrometry on supernatants from infected cells. Another application is to identify protein-protein interactions in virus-infected cells: a promiscuous biotinylating enzyme can be directed to a subcellular compartment, where it biotinylates adjacent molecules. These can be purified by attachment to streptavidin-containing beads and identified by mass spectrometry. Integration of mass spectrometry with some of

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