Principles of Virology, Volume 1. Jane Flint

Principles of Virology, Volume 1 - Jane Flint


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protocols differ in that FAIRE-seq does not require permeabilization of cells or the isolation of nuclei. The methylation state of DNA can be assessed using bisulfite sequencing. Treatment of DNA with bisulfite converts C to U but does not affect 5-methylated cytosines. A variety of sequencing methods that can use this change to provide single-nucleotide resolution information about DNA methylation have been developed. As might be expected, interpreting the growing sets of data on chromatin structure has required the development of new statistical and computational approaches.

      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 the methods described above for genome analysis can be used to identify proteins that participate in the regulation of gene expression.

      At one time the mass spectrometer was a very expensive instrument restricted to chemistry laboratories. Recent advances in the instrumentation, including cost reduction, as well as sample preparation and computational biology have propelled this technology into the virology research laboratory.

      A major goal of virology research is to understand how protein-protein interactions modulate reproduction cycles and pathogenesis. Consequently, multiple experimental approaches have been devised to identify the entire set of interactions among viral proteins and between viral and cell proteins. The yeast two-hybrid screen, a complementation assay which was designed to discover protein-protein interactions, has been adapted to high-throughput applications. In this assay, a transcriptional regulatory protein is split into two fragments, the DNA-binding domain and the activating domain. The coding sequences of two different proteins are fused with the two domains. If the two proteins interact, when the fusion proteins are produced in cells, transcriptional activation (leading to the transcription of a reporter gene) will take place. For high-throughput applications, libraries of protein-coding DNAs are screened against a single viral protein or all viral proteins. This method was used to describe the virus-host interactome of two herpesviruses.

      While these methods allow definition of virus-cell interactomes, they are not unambiguous. For at least one virus, interactomes determined in different laboratories are very diverse. Most importantly, the observation of a protein-protein interaction does not confirm biological relevance: the roles of such interactions in viral reproduction must be determined by other means (Box 2.12).

      Much of virology research is carried out by using populations of cells in culture or in animals. However, as discovered by virologists in the 1950s, individual cells of the same type can behave very differently with respect to susceptibility and permissiveness to infection and the kinetics of virus production.

      As early efforts to study virus infections in single cells were hampered by technical difficulties, the field failed to progress. This situation has changed with the development of flow cytometry and microfluidics and the adaptation of highthroughput methods, such as genome sequencing and mass spectrometry, to single cells.

      Initially, micropipettes were used to aspirate a single cell at a time from a population, using a microscope. This labor-intensive method was supplanted by fluorescence-activated cell sorting to allow isolation of up to millions of cells in a few hours, according to size, morphology, or synthesis of specific proteins. More recently, automated microfluidic devices have been developed to allow automated capture of single cells using integrated fluidic circuits. Infection, cell lysis, reverse transcription, and amplification are all performed in these systems before high-throughput sequencing.

      WARNING

       Determining a role for cellular proteins in viral reproduction can be quite difficult

      Understanding the roles of both viral and cellular proteins at various stages of viral reproduction is essential for elucidating molecular mechanisms and for developing strategies for blocking pathogenic infections. As viral genomes have a limited set of genes, the viral proteins or genetic elements that are essential at each step can be deduced by introducing mutations and observing phenotypes. Identifying


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