Principles of Virology, Volume 1. Jane Flint

Principles of Virology, Volume 1 - Jane Flint


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shown to contain horsepox virus, as determined by viral genome sequencing. The rescued horsepox virus protected immunized mice against a lethal challenge with vaccinia virus.

      This work is the first complete synthesis of a poxvirus using synthetic biology methodology. Some argued that the work enabled the rescue of smallpox virus. However, these concerns are spurious, as no new methods were developed by this work. The infectivity of DNA copies of viral genomes had been known for many years when this work was undertaken.

       Noyce RS, Lederman S, Evans DH. 2018. Construction of an infectious horsepox virus vaccine from chemically synthesized DNA fragments. PLoS One 13:e0188453.

      (ii) () strand RNA viruses. Genomic RNA of (–) strand RNA viruses is not infectious, because it can be neither translated nor copied into (+) strand RNA by host cell RNA polymerases (Chapter 6). Two different experimental approaches have been used to develop infectious DNA clones of these viral genomes (Fig. 3.12B and C).

      The recovery of influenza virus from cloned DNA is achieved using an expression system in which cloned DNA copies of the eight RNA segments of the viral genome are inserted between two cellular promoters, so that complementary RNA strands can be synthesized (Fig. 3.12B). When all eight plasmids carrying DNA for each viral RNA segment are introduced into cells, infectious influenza virus is produced.

      When the full-length (–) strand RNA of viruses with a nonsegmented genome, such as vesicular stomatitis virus (a rhabdovirus), is introduced into cells containing plasmids that produce viral proteins required for production of mRNA, no infectious virus is recovered. Lack of infectivity is thought to be a consequence of the hybridization of fulllength (–) strand RNA with (+) strand mRNAs produced from plasmids encoding viral proteins. Such hybridization might interfere with association of the (–) strand RNA with the N protein, which is required for copying by the viral RNA-dependent RNA polymerase. In contrast, when a fulllength (+) strand RNA is transfected into cells that have been engineered to synthesize the vesicular stomatitis virus nucleocapsid protein, phosphoprotein, and polymerase, the (+) strand RNA is copied into (–) strand RNAs. These RNAs initiate an infectious cycle, leading to the production of new virus particles.

      dsRNA viruses. Genomic RNA of dsRNA viruses is not infectious because ribosomes cannot access the (+) strand in the duplex. The recovery of reovirus from cloned DNA is achieved by an expression system in which cloned DNA copies of the 10 RNA segments of the viral genome are inserted under the control of a promoter for bacteriophage T7 RNA polymerase (Fig. 3.12D). When all 10 plasmids carrying DNA for each viral dsRNA segment are introduced into cells, infectious reovirus is produced.

       Types of Mutation

       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


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