Efficient pre-catalytic conformational change of reverse transcriptases from SAMHD1 non-counteracting primate lentiviruses during dNTP incorporation.

Unlike HIV-1, HIV-2 and some SIV strains replicate at high dNTP concentrations even in macrophages due to their accessory proteins, Vpx or Vpr, that target SAMHD1 dNTPase for proteasomal degradation. We previously reported that HIV-1 reverse transcriptase (RT) efficiently synthesizes DNA even at low dNTP concentrations because HIV-1 RT displays faster pre-steady state kpol values than SAMHD1 counteracting lentiviral RTs. Here, since the kpol step consists of two sequential sub-steps post dNTP binding, conformational change and chemistry, we investigated which of the two sub-steps RTs from SAMHD1 non-counteracting viruses accelerate in order to complete reverse transcription in the limited dNTP pools found in macrophages. Our study demonstrates that RTs of SAMHD1 non-counteracting lentiviruses have a faster conformational change rate during dNTP incorporation, supporting that these lentiviruses may have evolved to harbor RTs that can efficiently execute the conformational change step in order to circumvent SAMHD1 restriction and dNTP depletion in macrophages.

The 80's loop (residues 78 to 85) is important for the differential activity of retroviral proteases.

The abundance of structural data available for retroviral proteases affords a unique opportunity to investigate structure activity relationships. Our approach attempts to genetically engineer an HIV (human immunodeficiency virus)-1 protease that is functionally equivalent to the HIV-2 and the SIV (simian immunodeficiency virus) enzymes and conversely to engineer an HIV-2 protease that is functionally equivalent to the HIV-1 enzyme. For this purpose, the HIV-2 and SIV proteases were cloned and characterized in an Escherichia coli (E. coli) assay system along with 33 engineered HIV-1 and HIV-2 enzymes. The results of these experiments show that a relatively large S1 or S1' subsite volume, which is likely determined by the conformation of the 80's loop (residues 78 to 85), is necessary to fully accommodate the HIV-1 protease specificity site AETF*YCDG (the asterisk indicates the location scissile bond) during productive binding.