Mechanistic and Kinetic Differences between Reverse Transcriptases of Vpx Coding and Non-coding Lentiviruses.
Among lentiviruses, HIV Type 2 (HIV-2) and many simian immunodeficiency virus (SIV) strains replicate rapidly in non-dividing macrophages, whereas HIV Type 1 (HIV-1) replication in this cell type is kinetically delayed. The efficient replication capability of HIV-2/SIV in non-dividing cells is induced by a unique, virally encoded accessory protein, Vpx, which proteasomally degrades the host antiviral restriction factor, SAM domain- and HD domain-containing protein 1 (SAMHD1). SAMHD1 is a dNTPase and kinetically suppresses the reverse transcription step of HIV-1 in macrophages by hydrolyzing and depleting cellular dNTPs. In contrast, Vpx, which is encoded by HIV-2/SIV, kinetically accelerates reverse transcription by counteracting SAMHD1 and then elevating cellular dNTP concentration in non-dividing cells. Here, we conducted the pre-steady-state kinetic analysis of reverse transcriptases (RTs) from two Vpx non-coding and two Vpx coding lentiviruses. At all three sites of the template tested, the two RTs of the Vpx non-coding viruses (HIV-1) displayed higher kpol values than the RTs of the Vpx coding HIV-2/SIV, whereas there was no significant difference in the Kd values of these two groups of RTs. When we employed viral RNA templates that induce RT pausing by their secondary structures, the HIV-1 RTs showed more efficient DNA synthesis through pause sites than the HIV-2/SIV RTs, particularly at low dNTP concentrations found in macrophages. This kinetic study suggests that RTs of the Vpx non-coding HIV-1 may have evolved to execute a faster kpol step, which includes the conformational changes and incorporation chemistry, to counteract the limited dNTP concentration found in non-dividing cells and still promote efficient viral reverse transcription.
From the Center for Drug Discovery, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322.
Gina M
GM
Lenzi
From the Center for Drug Discovery, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322.
Robert A
RA
Domaoal
the College of Pharmacy, Kyung-Hee University, Seoul 02447, South Korea.
Dong-Hyun
DH
Kim
From the Center for Drug Discovery, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322, the Veterans Affairs Medical Center, Decatur, Georgia 30033.
Raymond F
RF
Schinazi
From the Center for Drug Discovery, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322, the College of Pharmacy, Kyung-Hee University, Seoul 02447, South Korea, baek.kim@emory.edu.
Baek
B
Kim
D000818:Animals; D002478:Cells, Cultured; D004261:DNA Replication; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics*
C074286:VPX protein, Human immunodeficiency virus 2; D054334:Viral Regulatory and Accessory Proteins; C514824:reverse transcriptase, Human immunodeficiency virus 1; C072416:reverse transcriptase, Human immunodeficiency virus 2; D054303:HIV Reverse Transcriptase
DNA replication; SAMHD1; Vpx; dNTP; enzyme kinetics; lentivirus; macrophage; reverse transcription