Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 151

Recombinant DNA techniques allow the introduction of many kinds of mutation at any desired site in cloned DNA (Box 3.10). Deletion mutations can be used to remove an entire gene to assess its role in reproduction, to produce truncated gene products, or to assess the functions of specific segments 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.