- Retroviruses are RNA animal viruses that have a DNA stage.
- They have an enzyme called reverse transcriptase that carries out RNA ? cDNA transcription.
- Following replication, cDNA integrates into the host genome until viral reproduction occurs.
- After the RNA retrovirus enters a host cell, its genomic RNA will be transcribed into a double stranded DNA and then integrated into the host DNA. The RNA to DNA transcription is called reverse transcription.
Mechanism of reverse transcription 10 Stages
The entire process is catalyzed by reverse transcriptase which
has both DNA polymerase and RNase H activities.
Stages 1 - 6
- A retrovirus-specific cellular tRNA hybridizes with a complementary region called the primer-binding site (PBS).
- A DNA segment is extended from tRNA based on the sequence of the retroviral genomic RNA.
- The viral R and U5 sequences are removed by RNase H.
- First jump: DNA hybridizes with the remaining R sequence at the 3' end.
- A DNA strand is extended from the 3' end.
- Most viral RNA is removed by RNase H.
Stages 7 - 10
- A second DNA strand is extended from the viral RNA.
- Both tRNA and the remaining viral RNA are removed by RNase H.
- Second jump: The PBS region of the second strand hybridizes with the PBS region of the first strand.
- Extension on both DNA strands. LTR stands for "long terminal repeat".
Reproduction of HIV, a retrovirus An Overview
Reverse Transcription PCR
Reverse transcriptase polymerase chain reaction (RT-PCR) is a process
whereby DNA is amplified from RNA.1 It is an extremely useful technique
in RNA quantifying, cloning, cDNA library construction and signal amplification.
The technique consists of two main steps:
(1)In the first step of RT-PCR, called the "first strand reaction," complementary
DNA (cDNA) is made from an mRNA template using dNTP's and a reverse transcriptase.
There are two common reverse transcriptases. These components are combined
with a DNA primer in a reverse transcriptase buffer for an hour at 37°C. There
are three types of DNA primers commonly used to facilitate reverse transcription.
(2)After the reverse transcriptase reaction is complete, and cDNA has been generated from the
original single-stranded mRNA, standard PCR (called the "second strand reaction")
is initiated in the original reaction tube after addition of a thermostable DNA
polymerase, e.g., Taq polymerase, and upstream and downstream DNA primers. Heating
the reaction to temperatures above 37°C facilitates binding of DNA primers to the
cDNA, and subsequent higher temperatures allow the DNA polymerase to make double-stranded
DNA from the cDNA. Heating the reaction to ~95°C melts the two DNA strands apart,
enabling the primers to bind again at lower temperatures and begin the chain reaction
again. After ~30 cycles, millions of copies of the sequence of interest are generated.