Principles of Virology, Volume 1. Jane Flint

or entire organisms. Data obtained from high-throughput measurements are integrated and analyzed using mathematical algorithms to generate models that are predictive of the system. For example, virus infections of different animals are characterized by the induction of distinct sets of cytokine genes, a property that can be correlated with different pathogenic outcomes. When a model has been developed, it can be further refined by the use of viral mutants or targeted inhibition of host genes or pathways. Global analysis is therefore a holistic, host-directed approach that complements traditional methods for studying viruses.

Figure 2.19 One-step growth curves of animal viruses. (A) Growth of a nonenveloped virus, adenovirus type 5. The inset illustrates the concept that viruses multiply by assembly of preformed components into particles. (B) Growth of an enveloped virus, Western equine encephalitis virus, a member of the Togaviridae. This virus acquires infectivity after maturation at the plasma membrane, and therefore, little intracellular virus can be detected. The small quantities observed at each time point probably represent released virus contaminating the cell extract.

      Examples of global analyses include genome-wide transcriptional profiling to study the host response to infection. Introduction of the 1918 strain of influenza virus into mice leads to a rapidly fatal disease characterized by sustained induction of proinflammatory cytokine and chemokine genes. Understanding the gene expression signature that correlates with lethality is one goal of these studies. Global analysis can also predict signatures of vaccine efficacy. In one study, transcriptional profiling of peripheral blood mononuclear cells from vaccinated subjects revealed that the yellow fever virus vaccine induces the expression of genes encoding members of the complement system and stress response proteins. This pattern accurately predicts CD8⁺ T cell and antibody responses that are thought to mediate protection from infection with yellow fever virus. A separate signature that accurately predicts neutralizing antibody synthesis during infection was also identified.

      Some of the methods used in global analysis are described below.

DNA Microarrays

      An early staple of global analyses, this method enables the study of the gene expression profile of a cell in response to virus infection (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.

A number of methods yield global views of protein-nucleic acid interactions at unprecedented levels of resolution. Chromatin-immunoprecipitation sequencing (ChiP-seq) can localize protein-DNA interactions with single-nucleotide precision (Fig. 2.20). In this method, protein-DNA complexes are immunoprecipitated with antibodies to DNA binding proteins, such as transcription proteins, histones, or even specific methyl groups on histones. The DNAs are then subjected to high-throughput sequencing to identify the sites on DNA to which these proteins bind. An early variant called ChiP on chip employed microarrays to identify protein binding sites on DNA.

Figure 2.20 Chromatin immunoprecipitation and DNA sequencing, ChiP-seq. This technique is used to identify the precise binding sites of proteins on DNA. DNA is cross-linked to proteins by treating cells with formaldehyde, followed by sonication to shear DNA to 200 to 1,000 bp. Beads coated with antibody to the DNA binding protein of interest are added and precipitated. The protein is removed and DNA purified and subjected to high-throughput sequencing to identify protein binding sites on the DNA.

      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.

How DNA is organized in virus particles and in the cell nucleus is being studied using chromosome conformation capture technology, abbreviated as 3C, 4C, 5C, and Hi-C, which differ in scope. For example, 3C identifies interactions between a single pair of genomic loci. Chromosome conformation capture on chip (4C) studies the interaction of one genomic locus and all other genomic loci, while chromosome conformation capture carbon copy (5C) detects interactions between all restriction fragments in a given region. In HiC, high-throughput sequencing is used to identify the restriction fragments studied. These methods begin with cross-linking of cell genomes with formaldehyde and digestion with restriction endonucleases, followed by random ligation under conditions where joining of cross-linked fragments is favored over those that are not. PCR is then used to amplify ligated junctions and identify interacting loci. The open or closed state of chromatin can be measured by DNaseI-seq (DNaseI hypersensitive sites sequencing) and FAIRE-seq (formaldehyde-assisted isolation of regulatory elements). These protocols are based on the use of formaldehyde to cross-link DNA: this reaction is more efficient in nucleosome-rich regions than in nucleosome-poor areas. The non-cross-linked DNA, typically from open chromatin, is then purified and its sequence is determined. The