RNA-Dependent Synthesis of RNA and DNA:- Reverse Transcriptase Produces DNA from Viral RNA
Certain RNA viruses that infect animal cells carry within the viral particle an RNA-dependent DNA polymerase called reverse transcriptase. On infection, the single stranded RNA viral genome (~10,000 nucleotides) and the enzyme enter the host cell. The reverse transcriptase first catalyzes the synthesis of a DNA strand com plementary to the viral RNA (Fig. 1), then degrades the RNA strand of the viral RNA-DNA hybrid and re places it with DNA. The resulting duplex DNA often becomes incorporated into the genome of the eukaryotic host cell. These integrated (and dormant) viral genes can be activated and transcribed, and the gene products—viral proteins and the viral RNA genome itself— packaged as new viruses. The RNA viruses that contain reverse transcriptases are known as retroviruses (retro is the Latin prefix for “backward”).
FIGURE 1 Retroviral infection of a mammalian cell and integration of the retrovirus into the host chromosome. Viral particles entering the host cell carry viral reverse transcriptase and a cellular tRNA (picked up from a former host cell) already base-paired to the viral RNA. The tRNA facilitates immediate conversion of viral RNA to double-stranded DNA by the action of reverse transcriptase, as described in the text. Once converted to double-stranded DNA, the DNA enters the nucleus and is integrated into the host genome. The integration is catalyzed by a virally encoded integrase. Integration of viral DNA into host DNA is mechanistically similar to the insertion of transposons in bacterial chromosomes . For ex ample, a few base pairs of host DNA become duplicated at the site of integration, forming short repeats of 4 to 6 bp at each end of the inserted retroviral DNA (not shown).
FIGURE 2 Structure and gene products of an integrated retro viral genome. The long terminal repeats (LTRs) have sequences needed for the regulation and initiation of transcription. The sequence denoted ψ is required for packaging of retroviral RNAs into mature viral particles. Transcription of the retroviral DNA produces a primary transcript encompassing the gag, pol, and env genes. Translation produces a polyprotein, a single long polypeptide derived from the gag and pol genes, which is cleaved into six distinct proteins. Splicing of the primary transcript yields an mRNA derived largely from the env gene, which is also translated into a polyprotein, then cleaved to generate viral envelope proteins.
The existence of reverse transcriptases in RNA viruses was predicted by Howard Temin in 1962, and the enzymes were ultimately detected by Temin and, independently, by David Baltimore in 1970. Their discovery aroused much attention as dogma-shaking proof that genetic information can flow “backward” from RNA to DNA. Retroviruses typically have three genes: gag (de rived from the historical designation group associated antigen), pol, and env (Fig. 2). The transcript that contains gag and pol is translated into a long “polyprotein,” a single large polypeptide that is cleaved into six proteins with distinct functions. The proteins derived from the gag gene make up the interior core of the viral particle. The pol gene encodes the protease that cleaves the long polypeptide, an integrase that inserts the viral DNA into the host chromosomes, and reverse transcriptase. Many reverse transcriptases have two
Subunits, α and β. The pol gene specifies the subunit (Mr 90,000), and the α subunit (Mr 65,000) is simply a proteolytic fragment of the β subunit. The env gene en codes the proteins of the viral envelope. At each end of the linear RNA genome are long terminal repeat (LTR) sequences of a few hundred nucleotides. Transcribed into the duplex DNA, these sequences facilitate integration of the viral chromosome into the host DNA and contain promoters for viral gene expression.
ions: (1) RNA-dependent DNA synthesis, (2) RNA degradation, and (3) DNA-dependent DNA synthesis. Like many DNA and RNA polymerases, reverse transcriptases contain Zn2+. Each transcriptase is most active with the RNA of its own virus, but each can be used experimentally to make DNA complementary to a variety of RNAs. The DNA and RNA synthesis and RNA degradation activities use separate active sites on the protein. For DNA synthesis to begin, the reverse transcriptase requires a primer, a cellular tRNA obtained during an earlier infection and carried within the viral particle. This tRNA is base-paired at its 3end with a complementary sequence in the viral RNA. The new DNA strand is synthesized in the 5→3 direction, as in all RNA and DNA polymerase reactions. Reverse transcriptases, like RNA polymerases, do not have 3→5 proofreading exonucleases. They generally have error rates of about 1 per 20,000 nucleotides added. An error rate this high is extremely unusual in DNA replication and appears to be a feature of most enzymes that replicate the genomes of RNA viruses. A consequence is a higher mutation rate and faster rate of viral evolution, which is a factor in the frequent appearance of new strains of disease-causing retroviruses. Reverse transcriptases have become important reagents in the study of DNA-RNA relationships and in DNA cloning techniques. They make possible the syn thesis of DNA complementary to an mRNA template, and synthetic DNA prepared in this manner, called com plementary DNA (cDNA), can be used to clone cellular genes.
