Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 156

RNA interference (Chapter 8) has become a powerful and widely used tool because it enables targeted loss of gene function. In such analyses, duplexes of 21-nucleotide RNA molecules, called small interfering RNAs (siRNAs), which are complementary to small regions of the mRNA, are synthesized chemically or by transcription reactions. siRNAs or plasmids or viral vectors that encode them are then introduced into cultured cells by transformation or infection. The small molecules then block the production of specific proteins by inducing sequence-specific mRNA degradation or inhibition of translation. Duplex siRNAs are unwound from one 5′ end, and one strand becomes tightly associated with a member of the argonaute (Ago) family of proteins in the RNA-induced silencing complex, RISC. The small RNA acts as a “guide,” identifying the target mRNA by base-pairing to 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.

In another application of this technology, libraries of thousands of siRNAs directed at all cellular mRNAs or a specific subset can be introduced into cells to identify genes that stimulate or block viral reproduction. The siRNAs are produced from lentiviral vectors as short hairpin RNAs (shRNAs) that are processed into dsRNAs that are then targeted to mRNAs by RISC. In one approach, cells are infected with pools of shRNA-containing lentivirus vectors (Fig. 3.13). The cells are placed under selection and infected with virus to identify changes in reproduction caused by the integrated vector. If necessary, pools of vectors that have an effect on virus reproduction can be further subdivided and rescreened. Enriched shRNAs are detected by high-throughput sequencing and bioinformatic programs that quantitate the number of reads per shRNA compared with the starting population. The likelihood that knockdown of a specific mRNA is a valid result increases as the number of enriched orthologous shRNAs for the targeted gene increases. In other words, a gene targeted by three different shRNAs established by sequencing data is more likely to be a true positive than a gene targeted by only one. Another approach, arrayed RNAi screening, uses transfection of siRNAs into cells grown in a multiwell format (Fig. 3.13). As a record is kept of which siRNAs are added to each well, targeted genes can be readily identified after their effect on virus infection has been ascertained.

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.