Principles of Virology, Volume 1. Jane Flint

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Principles of Virology, Volume 1 - Jane Flint

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specific sequences within it prior to cleavage of the mRNA or inhibition of its translation.

      To determine the role of a viral gene in the reproduction cycle, siRNA targeting the mRNA is introduced into cells. Reduced protein levels are verified (e.g., by immunoblot analysis) and the effect on virus reproduction is determined. The same approach is used to evaluate the role of cell proteins such as receptors or antiviral proteins.

      No matter which method is used to identify genes that affect viral reproduction, the most convincing confirmation of the result is restoration of the phenotype by expression of a gene containing a mutation that makes the mRNA resistant to silencing.

       Targeted Gene Editing with CRISPR-Cas9

      Bacteria and archaea possess an endogenous system of defense in which short single-stranded guide RNAs (sgRNAs) are used to target and destroy invading DNA (Volume II, Chapter 3, Box 3.9). One embodiment of this defense, the CRISPR-Cas9 (clustered regularly interspersed short palindromic repeat [CRISPR]-associated nuclease 9) system, has been adapted for effective and efficient targeting gene disruptions and mutations in any genome. The specificity depends on the ability of the sgRNAs to hybridize to the correct DNA sequence within the chromosome. Once annealed, the endonuclease Cas9 catalyzes formation of a double-strand break, which is then repaired, creating frameshifting insertion/deletion mutations within the gene. One advantage of using CRISPR-Cas9 methodology to modify cell genomes is that the method can be applied to any cell type. Like siRNAs, CRISPR-Cas9 can be used to affect individual mRNAs or to carry out genome-wide screens to identify cell genes that stimulate or block viral reproduction (Fig. 3.13). As with RNAi screens, the most convincing confirmation of the result is restoration of the phenotype by expression of a gene containing a mutation that makes it resistant to Cas9, via changes in the sgRNA target sequence.

       Haploid Cell Screening

      Haploid cell lines have been used to identify genes required for viral reproduction. These cells, which have only one copy of each chromosome, are infected with retroviruses under conditions where one integration event occurs per cell. The disruption of individual genes that are essential for viral replication can be identified by the isolation of cells resistant to infection (Fig. 3.13). Surviving cells are expanded and the site of proviral integration is determined by PCR and high-throughput sequencing. This approach has been used to identify receptors for viruses, including ebolavirus, Lassa virus, and hantavirus, and genes required for receptor modification and endosomal trafficking.

      While powerful, a drawback of this approach is that only a few haploid cell lines are available, and not all viruses can infect these cells.

      Naked DNA can be introduced into cultured animal cells as complexes with calcium phosphate or lipid-based reagents or directly by electroporation. Such DNA can direct synthesis of its gene products transiently or stably from integrated or episomal copies. Introduction of DNA into cells is a routine method in virological research and is also employed for certain clinical applications, such as the production of a therapeutic protein or a vaccine or the engineering of primary cells, progenitor cells, and stem cells for subsequent introduction into patients. However, this approach is not suitable for all applications. In some cases, gene delivery by viral vector is preferred. Viral vectors have also found widespread use in the research laboratory, including applications in which the delivery of a gene to specific cells, or at high efficiency, is desired. The use of viral vectors for gene therapy, the delivery of a gene to patients who either lack the gene or carry defective versions of it, or to destroy tumors typically employs viral vectors, not naked DNA (see Volume II, Chapter 9). In one application, DNA including the gene is introduced and expressed in cells obtained from the patient. After infusion into patients, the cells can become permanently established. If the primary cells to be used are limiting in a culture (e.g., stem cells), it is not practical to select and amplify the rare cells that receive naked DNA. Recombinant viruses carrying foreign genes can infect a greater percentage of cells and thus facilitate generation of the desired population. A complete understanding of the structure and function of viral vectors requires knowledge of viral genome replication, a topic discussed in subsequent chapters for selected viruses and summarized in the Appendix.

      Design requirements for viral vectors include the use of an appropriate promoter, maintenance of genome size within the packaging limit of the particle,

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