Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 155

In genetic analysis of viruses, mutations are made in vitro by a variety of techniques, all of which can introduce unintended changes. Errors can be introduced during cloning, PCR, or sequencing and when the viral DNA or plasmid DNA is introduced into the cell.

With these potential problems in mind, how can it be concluded that a phenotype arises from the planned mutation? Here are some possible solutions.

 Test several independent DNA clones for the phenotype.

 Repeat the plasmid construct ion. It is unlikely that an unlinked mutation with the same phenotype would occur twice.

 Look for marker rescue. Replace the mutation and all adjacent DNA with parental DNA. If the mutation indeed causes the phenotype, the wild-type phenotype should be restored in 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.