Question

You have a clone of HIV-1 RT that you transform into the a CD4⁺ T cell. Remember, HIV-1 RT needs to be heterodimeric form in order to work optimally. There are three ways in which you can get a functional enzyme. Discuss two of those ways.

Answer 1

HIV-1 RT is formed from a cleavage of Gag-Pol polyprotein with the viral PR (protease). The production of HIV-1 Gag-Pol is by a frameshift readthrough event in the region of p6 coding that happens about 5% of the time of translation of Gag from an unspliced full-length transcript of viral RNA. The Gag part of the Gag-Pol permits it to link with Gag at the time of virion assembly which ensures that the Pol part of the Gag-Pol containing RT, PR, and integrase is present inside the virion that is assembled.

A heterodimer, the mature form of HIV-1 RT consist two subunits- p66, larger subunit that is 560 amino acids long; and p51, smaller subunit that is composed of 440 amino acids of p66. The p66 subunit contains two domains- RNase H and polymerase. The p66 subunit of the mature HIV-1 RT heterodimer contains the active sites for the two enzymatic activities of RT. The crystallographic structures of the HIV-1 RT, mainly those that expose the structure of the complexes with incoming dNTPs and bound nucleic acids that have allowed the understanding of molecular mechanisms of working of the enzyme.

Two changes are made in the mature HIV-1 RT heterodimer when any mutation brings a change in the polymerase domain of HIV-1 RT. It is achievable to indicate forms of recombinant RT that possess changes only in one subunit. Both the RNase and polymerase H active sites are present in p66 but in some cases the change in p51 that interacts comprehensively with p66 and aids in forming the nucleic acid binding cleft that can contribute to the functioning of the recombinant enzyme.