Retroviruses, also referred to as class VI ssRNA-RT viruses, are RNA reverse transcribing viruses with a DNA intermediate. Their genomes consist of two molecules of positive-sense single stranded RNA with a 5' cap and 3' polyadenylated tail. Examples of retroviruses include the human immunodeficiency virus (HIV) and the human T-lymphotropic virus (HTLV). Creation of double-stranded DNA occurs in the cytosol[7] as a series of these steps:
A specific cellular tRNA acts as a primer and hybridizes to a complementary part of the virus RNA genome called the primer binding site or PBS
Complementary DNA then binds to the U5 (non-coding region) and R region (a direct repeat found at both ends of the RNA molecule) of the viral RNA
A domain on the reverse transcriptase enzyme called RNAse H degrades the 5’ end of the RNA which removes the U5 and R region
The primer then ‘jumps’ to the 3’ end of the viral genome and the newly synthesised DNA strands hybridizes to the complementary R region on the RNA
The first strand of complementary DNA (cDNA) is extended and the majority of viral RNA is degraded by RNAse H
Once the strand is completed, second strand synthesis is initiated from the viral RNA
There is then another ‘jump’ where the PBS from the second strand hybridizes with the complementary PBS on the first strand
Both strands are extended further and can be incorporated into the hosts genome by the enzyme integrase
Creation of double-stranded DNA also involves strand transfer, in which there is a translocation of short DNA product from initial RNA dependent DNA synthesis to acceptor template regions at the other end of the genome, which are later reached and processed by the reverse transcriptase for its DNA-dependent DNA activity.[8]
Retroviral RNA is arranged in 5’ terminus to 3’ terminus. The site where the primer is annealed to viral RNA is called the primer-binding site (PBS). The RNA 5’end to the PBS site is called U5, and the RNA 3’ end to the PBS is called the leader. The tRNA primer is unwound between 14 and 22 nucleotides and forms a base-paired duplex with the viral RNA at PBS. The fact that the PBS is located near the 5’ terminus of viral RNA is unusual because reverse transcriptase synthesize DNA from 3’ end of the primer in the 5’ to 3’ direction (with respect to the RNA template).Therefore, the primer and reverse transcriptase must be relocated to 3’ end of viral RNA. In order to accomplish this reposition, multiple steps and various enzymes including DNA polymerase, ribonuclease H(RNase H) and polynucleotide unwinding are needed.[9]
The HIV reverse transcriptase also has ribonuclease activity that degrades the viral RNA during the synthesis of cDNA, as well as DNA-dependent DNA polymerase activity that copies the sense cDNA strand into an antisense DNA to form a double-stranded viral DNA intermediate (vDNA).