1.7 VIRUSES CAN BE MANIPULATED GENETICALLY

 

One of the easiest ways to understand the steps involved in a particular reaction within an organism is to isolate mutants which are unable to carry out that reaction. Like all other organisms, viruses sport mutants in the course of their growth, and these mutations can affect all properties including the type of plaque formed, the range of hosts which the virus can infect, and the physicochemical properties of the virus. One obvious caveat, however, is that many mutations will be lethal to the virus and remain undetected. This problem was overcome in 1963 by Epstein and Edgar and their collaborators with the discovery of conditional lethal mutants. One class of these mutants, the temperature-sensitive mutants, was able to grow at a lower temperature than normal, the permissive temperature, but not at a higher, restrictive temperature at which normal virus could grow. Another class of conditional lethal mutants was the amber mutant. In these mutants a DNA lesion converts a codon within transcribed RNA into a triplet which terminates protein synthesis. They can only grow on a permissive host cell, which has an amber-suppressor transfer RNA (tRNA) that can insert an amino acid at the mutation site during translation. 14 PART I WHAT IS A VIRUS? The drawback to conditional lethal mutants is that mutation is random, but the advent of recombinant DNA technology has facilitated controlled mutagenesis, known as reverse genetics, at least for those viruses for which infectious particles can be reconstituted from cloned genomic DNA or cDNA (DNA that has been transcribed from RNA). What happens is that a piece of a cloned viral DNA or cDNA genome containing the target sequence is excised from the plasmid using two different restriction enzymes, so that it forms a unique restriction fragment which can eventually be reinserted in the correct orientation. The fragment is then modified by oligonucleotide- or site-directed mutagenesis via the polymerase chain reaction (PCR) using appropriate mutagenic primers, is then inserted back into the original sequence in a plasmid, and then used to form a mutated virus particle.

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