Principles of Virology. Jane Flint

the rescued virus.

       Allow synthesis of the wild-type protein in the mutant background. If the wild-type phenotype is restored (complemented), then the probability is high that the phenotype arises from the mutation. The merit of this method over marker rescue is that the latter shows only that unlinked mutations are probably not the cause of the phenotype.

      Each of these approaches has limitations, and it is therefore prudent to use more than one.

Some mutations within the origin of replication (Ori) of simian virus 40 reduce viral DNA replication and induce the formation of small plaques (see Chapter 9 for more information on the Ori). Pseudorevertants of Ori mutants were isolated by random mutagenesis of mutant viral DNA followed by introduction into cultured cells and screening for viruses that form large plaques. The second-site mutations that suppressed the replication defects were localized to a specific region within the gene for large T antigen. These results indicated that a specific domain of large T antigen interacts with the Ori sequence during viral genome replication.

      The 5′ untranslated region of the poliovirus genome contains elaborate RNA secondary-structural features, which are important for RNA replication and translation, as discussed in Chapters 6 and 11, respectively. Disruption of such features by substitution of a short nucleotide sequence produces a virus that replicates poorly and readily gives rise to pseudorevertants that reproduce more efficiently. Nucleotide sequence analysis of the genomes of two pseudorevertants revealed base changes that restore the disrupted secondary structure. These results confirm that the RNA secondary structure is important for the biological activity of this untranslated region.

       RNA Interference (RNAi)

      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.

       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.

Figure 3.13 Use of RNAi, haploid cells, and CRISPR-Cas9 to study virus-host interactions. In arrayed screens, siRNAs are introduced into cells growing in wells that are subsequently infected with virus. Production of infectious virus or a viral protein is quantified by plaque assay or measurement of a fluorescent protein. Individual siRNA with the desired effect can be identified based on their location in the multiwell plate. In pooled RNAi screens, collections of shRNA producing lentiviral vectors are used to infect cells. After selection for cells with integrated vectors, the cells are infected with the test virus and the production of a viral protein or infectious virus is monitored. In pooled haploid cell screens, cells are infected with lentiviruses at a low multiplicity of infection so that on average one viral genome integration per cell takes place. In pooled CRISPR-Cas9 screens, libraries of sgRNAs are introduced, via lentivirus vector, into cells that produce Cas9. After selection for lentiviral integration, cells are infected with virus. Cell survival and production of infectious virus or a viral protein may be measured depending on what types of genes are sought (e.g., those that are essential for reproduction). In each screen, the cell gene that is disrupted is identified by nucleotide sequencing.

While the experimental use of RNAi can lead to reduced protein production, genomic manipulation by CRISPR-Cas9 has advantages of complete depletion of the protein through the production of a homozygous null genotype and fewer off-target effects. With CRISPR-Cas9, the expression of a gene can be permanently extinguished. In contrast, the shRNA-expressing provirus must continually silence the product of ongoing transcription.

       Haploid Cell Screening