Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 148

Recombinant DNA techniques have made it possible to introduce any kind of mutation anywhere in the genome of most animal viruses, whether that genome comprises DNA or RNA. The quintessential tool in virology today is the infectious DNA clone, a dsDNA copy of the viral genome that is carried on a bacterial vector such as a plasmid. Infectious DNA clones, or in vitro transcripts derived from them, can be introduced into cultured cells by transfection (Box 3.8) to recover infectious virus. This approach is a modern validation of the Hershey-Chase experiment described in Chapter 1. The availability of site-specific bacterial restriction endonucleases, DNA ligases, and an array of methods for mutagenesis has made it possible to manipulate these infectious clones at will. Infectious DNA clones also provide a stable repository of the viral genome, a particularly important advantage for vaccine strains. As oligonucleotide synthesis has become more efficient and less costly, the assembly of viral DNA genomes up to 212 kbp has become possible (Box 3.9).

DNA viruses. Current genetic methods for the study of most viruses with DNA genomes are based on the infectivity of viral DNA. When deproteinized viral DNA molecules are introduced into permissive cells by transfection, they generally initiate a complete infectious cycle, although the infectivity (number of plaques per microgram of DNA) may be low. For example, the infectivity of deproteinized human adenoviral DNA is between 10 and 100 PFU per μg. When the genome is isolated by procedures that do not degrade the covalently attached terminal protein, infectivity is increased by 2 orders of magnitude, probably because this protein facilitates the assembly of initiation complexes on the viral origins of replication.

The complete genomes of polyomaviruses, papillomaviruses, and adenoviruses can be cloned in plasmid vectors, and such DNA is infectious under appropriate conditions. The DNA genomes of herpesviruses and poxviruses are too large to insert into conventional bacterial plasmid vectors, but they can be cloned into vectors that accept larger insertions (e.g., cosmids and bacterial artificial chromosomes). The plasmids containing such cloned herpesvirus genomes are infectious. In contrast, poxvirus DNA is not infectious, because the viral promoters cannot be recognized by cellular DNA-dependent RNA polymerase. Poxvirus DNA is infectious when early functions (viral DNA-dependent RNA polymerase and transcription proteins) are provided by complementation with a helper virus.

RNA viruses. (i) (+) strand RNA viruses. The genomic RNA of retroviruses is copied into dsDNA by reverse transcriptase early during infection, a process described in Chapter 10. Such DNA is infectious when introduced into cells, as are molecularly cloned forms inserted into bacterial plasmids.

Infectious DNA clones have been constructed for many (+) strand RNA viruses. An example is the introduction of a plasmid containing cloned poliovirus DNA into cultured mammalian cells, which leads to the production of progeny virus (Fig. 3.12A). The mechanism by which cloned poliovirus DNA initiates infection is not known, but it has been suggested that the DNA enters the nucleus, where it is transcribed by cellular DNA-dependent RNA polymerase from cryptic, promoter-like sequences on the plasmid. The resulting (+) strand RNA transcripts initiate an infectious cycle. During genome replication, the extra terminal nucleotide sequences transcribed from the vector must be removed or ignored, because the virus particles that are produced contain RNA with the authentic 5′ and 3′ termini.

By incorporating promoters for bacteriophage T7 DNA-dependent RNA polymerase in plasmids containing poliovirus DNA, full-length (+) strand RNA transcripts can be synthesized in vitro. The specific infectivity of such RNA transcripts resembles that of genomic RNA (10⁶ PFU per μg), which is higher than that of cloned DNA (10³ PFU per μg).