RNA viruses. () strand RNA viruses. Te genomic RNA of retroviruses is copied into dsDNA by reverse transcriptase early during infection, a process described in Chapter 7. Such DNA is infectious when introduced into cells, as are molecularly cloned forms inserted into bacterial plasmids. Introduction of a plasmid containing cloned poliovirus DNA into cultured mammalian cells results in the production of progeny virus Te 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. Te 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. Te specific infectivity of such RNA transcripts resembles that of genomic RNA which is higher than that of cloned DNA (103 Infectious DNA clones have been constructed for many () 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, as discussed in 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 by an expression system in which cloned DNA copies of the eight RNA segments of the viral genome are inserted between two promoters, so that complementary RNA strands can be synthesized .
When the eight plasmids carrying DNA for each viral RNA segment are introduced into cells, infectious influenza virus is produced. Te full-length () strand RNA of viruses with a nonsegmented genome, such as vesicular stomatitis virus (a rhabdovirus), is not infectious, because it cannot be translated into protein or copied into mRNA by the host cell. When the full-length () strand is introduced into cells containing plasmids that produce viral proteins required for production of mRNA, no infectious virus is recovered. Unexpectedly, when a full-length () strand RNA is transfected into cells that synthesize the vesicular stomatitis virus nucleocapsid protein, phosphoprotein, and polymerase, the () strand RNA is copied into () strand RNAs. Tese RNAs initiate an infectious cycle, leading to the production of new virus particles.