Principles of Virology. Jane Flint
of a coding sequence. Noncoding regions can be deleted to identify and characterize regulatory sequences such as promoters. Insertion mutations can be made by the addition of any desired sequences and may be used to produce fusion proteins. Substitution mutations, which can correspond to one or more nucleotides, are often made in coding or noncoding regions. Included in the former class are nonsense mutations, in which a termination codon is introduced, and missense mutations, in which a single nucleotide or a codon is changed, resulting in the synthesis of a protein with a single amino acid substitution. The introduction of a termination codon is frequently exploited to truncate a membrane protein so that it is secreted or to eliminate the synthesis of a protein without changing the size of the viral genome or mRNA. Substitutions are used to assess the roles of specific nucleotides in regulatory sequences or of amino acids in protein function, such as polymerase activity or binding of a viral protein to a cell receptor.
Figure 3.12 Recovery of infectivity from cloned DNA of RNA viruses. (A) The infectivity of cloned DNA of the (+) strand poliovirus RNA genome, which is infectious when introduced into cultured cells by transfection. A complete DNA clone of the viral RNA (blue strands), carried in a plasmid, is also infectious, as are RNAs derived by in vitro transcription of the full-length DNA. (B) Recovery of influenza viruses by transfection of cells with eight plasmids. Cloned DNA of each of the eight influenza virus RNA segments is inserted between an RNA polymerase I promoter (Pol I [green]) and terminator (brown), and an RNA polymerase II promoter (Pol II [yellow]) and a polyadenylation signal (red). When the eight plasmids are introduced into mammalian cells, (–) strand viral RNA (vRNA) molecules are synthesized from the RNA polymerase I promoter, and mRNAs are produced by transcription from the RNA polymerase II promoter. The mRNAs are translated into viral proteins, and infectious virus is produced from the transfected cells. For clarity, only one cloned viral RNA segment is shown. (C) Recovery of infectious virus from cloned DNA of viruses with a (–) strand RNA genome. Cells are infected with a vaccinia virus recombinant that synthesizes T7 RNA polymerase and transformed with plasmids that encode a full-length (+) strand copy of the viral genome RNA and proteins required for viral RNA synthesis (N, P, and L proteins). Production of RNA from these plasmids is under the control of the bacteriophage T7 RNA polymerase promoter (brown). Because bacteriophage T7 RNA transcripts are uncapped, an internal ribosome entry site (I) is included so the mRNAs will be translated. After the plasmids are transfected into cells, the (+) strand RNA is copied into (–) strands, which serve as templates for mRNA synthesis and genome replication. The example shown is for viruses with a single (–) strand RNA genome (e.g., rhabdoviruses and paramyxoviruses). A similar approach has been demonstrated for bunyamwera virus, with a genome comprising three (–) strand RNAs. (D) Recovery of infectious virus from cloned DNA of dsRNA viruses. Cloned DNA of each of the 10 reovirus dsRNA segments is inserted under the control of a bacteriophage T7 RNA polymerase promoter (brown). Because bacteriophage T7 RNA transcripts are uncapped, an internal ribosome entry site (I) is included so the mRNAs will be translated. Cells are infected with a vaccinia virus recombinant that synthesizes T7 RNA polymerase and transformed with all 10 plasmids. For clarity, only one cloned viral RNA segment is shown.
Introducing Mutations into the Viral Genome
Mutations can be introduced into a viral genome when it is cloned in its entirety. Mutagenesis is usually carried out on cloned subfragments, which are then substituted into full-length cloned DNA. This step can now be bypassed by using CRISPR/Cas9 to introduce mutations into complete DNA copies of viral genomes. Viruses are then recovered by introduction of the mutagenized DNA into cultured cells by transfection. This approach has been applied to cloned DNA copies of RNA and DNA viral genomes.
Introduction of mutagenized viral nucleic acid into cultured cells by transfection may have a variety of outcomes, ranging from no effect to a complete block of viral reproduction. Whether the introduced mutation is responsible for an observed phenotype deserves careful scrutiny (Box 3.10).
Reversion Analysis
The phenotypes caused by mutation can revert in one of two ways: by change of the mutation to the wild-type sequence or by acquisition of a mutation at a second site, either in the same gene or a different gene. Phenotypic reversion caused by second-site mutation is known as suppression, or pseudoreversion, to distinguish it from reversion at the original site of mutation. Reversion has been studied since the beginnings of classical genetic analysis. In the modern era of genetics, cloning and sequencing techniques can be used to demonstrate suppression and to identify the nature of the suppressor mutation (see below). The identification of suppressor mutations is a powerful tool for studying protein-protein and protein-nucleic acid interactions. Some mutations complement changes made at several sites, whereas allele-specific suppressor mutations complement only a specific change. The allele specificity of second-site mutations provides evidence for physical interactions among proteins and nucleic acids.
Phenotypic revertants can be isolated either by propagating the mutant virus under restrictive conditions or, in the case of mutants exhibiting phenotypes (e.g., small plaques), by searching for wild-type properties. Chemical mutagenesis may be required to produce revertants of DNA viruses but is not necessary for RNA viruses, which spawn mutants at a higher frequency. Nucleotide sequence analysis is then used to determine if the original mutation is still present in the genome of the revertant. The presence of the original mutation indicates that reversion has occurred by second-site mutation. The suppressor mutation is identified by nucleotide sequence analysis. The final step is introduction of the suspected suppressor mutation into the genome of the original mutant virus to confirm its effect. Several specific examples of suppressor analysis are provided below.
TERMINOLOGY
Operations on nucleic acids and proteins
A mutation is a change in DNA or RNA comprising base changes and nucleotide additions, deletions, and rearrangements. When mutations occur in open reading frames, they can be manifested as changes in the synthesized proteins. For example, one or more base changes in a specific codon may produce a single amino acid substitution, a truncated protein, or no protein. The terms “mutation” and “deletion” are often used incorrectly or ambiguously to describe alterations in proteins. In this textbook, these terms are used to describe genetic changes and the terms “amino acid substitution” and “truncation” to describe protein alterations.
BOX 3.11
DISCUSSION
Is the observed phenotype due to the mutation?
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