Retroviruses contain an RNA genome that is replicated through a double-stranded DNA intermediate. DNA synthesis is initiated from a tRNA primer annealed to the 5’ untranslated region of the viral RNA catalyzed by the virus-encoded reverse transcriptase (RT). During the reverse transcription process, the 5’ unique (U5) and 3’ unique (U3) regions of the viral RNA are duplicated and juxtaposed to form the ends of the linear DNA. Each U3–R–U5 end is referred to as a “long terminal repeat” (LTR). The double-stranded DNA, flanked by the LTRs, is then integrated into the host chromosome through the action of a second virus-encoded enzyme, integrase (IN). Understanding of the mechanistic roles of these two enzymes in viral replication has vastly increased with the cloning of the retrovirus genome and its subsequent molecular genetic analysis. The chapter discusses methods both to introduce mutations into the retrovirus genome and to reconstitute in vitro the activities of RT and IN in the initiation of reverse transcription and integration of viral DNA, respectively. The efficiency of Rous sarcoma virus (RSV) RT to initiate DNA synthesis from the tRNA primer is dependent not only on the primer but also on secondary structures near the primer binding site (PBS) in the viral RNA.
ASJC Scopus subject areas
- Molecular Biology