Abstract
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.
Highlights
Cells and terminally differentiated/non-dividing myeloid cells such as macrophages and microglia, whereas other retroviruses such as gammaretroviruses (i.e. murine leukemia virus (MuLV) and feline leukemia virus), alpharetroviruses, and spumavirus replicate only in dividing cells [1, 2]
Results deoxynucleotide triphosphate (dNTP) Concentration-dependent DNA Synthesis Efficiency of reverse transcriptases (RTs) from viral protein X (Vpx) Coding and Non-coding Lentiviruses—Because Vpx non-coding and coding lentiviruses are both able to replicate in macrophages with significant dNTP availability differences, we tested whether RTs from Vpx coding and non-coding lentiviruses display different concentration-dependent activity profiles
The HIV Type 2 (HIV-2) and simian immunodeficiency virus (SIV) RTs efficiently fully extended the primer in both T cell dNTP concentration and the dNTP concentration found in macrophages treated with Vpx (X, 500 nM [16])
Summary
Cells and terminally differentiated/non-dividing myeloid cells such as macrophages and microglia, whereas other retroviruses such as gammaretroviruses (i.e. murine leukemia virus (MuLV) and feline leukemia virus), alpharetroviruses (i.e. avian myeloblastosis virus), and spumavirus (i.e. foamy virus) replicate only in dividing cells [1, 2]. The HIV-2 and SIV RTs tested generated 5–10 times less fully extended product at low dNTP concentrations found in macrophages (M) as compared with HIV-1 RTs. Pausing (see Sites 2 and 3, * in Fig. 1, A and B) is generated by the kinetic delay of dNTP incorporation and is more significant in the HIV-2 and SIV RT proteins as compared with the HIV-1 RT enzymes (unpaired Student’s t test between RTs from Vpx non-coding and coding viruses, p Ͻ 0.05).
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