Enhanced enzyme kinetics of reverse transcriptase variants cloned from animals infected with SIVmac239 lacking viral protein X

Abstract

HIV Type 1 (HIV-1) and simian immunodeficiency virus (SIV) display differential replication kinetics in macrophages. This is because high expression levels of the active host deoxynucleotide triphosphohydrolase sterile alpha motif domain and histidine-aspartate domain-containing protein 1 (SAMHD1) depletes intracellular dNTPs, which restricts HIV-1 reverse transcription, and results in a restrictive infection in this myeloid cell type. Some SIVs overcome SAMHD1 restriction using viral protein X (Vpx), a viral accessory protein that induces proteasomal degradation of SAMHD1, increasing cellular dNTP concentrations and enabling efficient proviral DNA synthesis. We previously reported that SAMHD1 non-counteracting lentiviruses may have evolved to harbor reverse transcriptase (RT) proteins that efficiently polymerize DNA, even at low dNTP concentrations, to circumvent SAMHD1 restriction. Here we investigated whether RTs from SIVmac239 virus lacking a Vpx protein evolve during the course of in vivo infection to more efficiently synthesize DNA at the low dNTP concentrations found in macrophages. Sequence analysis of RTs cloned from Vpx (+) and Vpx (-) SIVmac239 infected animals revealed that Vpx (-) RTs contained more extensive mutations than Vpx (+) RTs. While the amino acid substitutions were dispersed indiscriminately across the protein, steady state and pre-steady state analysis demonstrated that selected SIVmac239 Vpx (-) RTs are characterized by higher catalytic efficiency and incorporation efficiency values than RTs cloned from SIVmac239 Vpx (+) infections. Overall, this study supports the possibility that the loss of Vpx may generate in vivo SIVmac239 RT variants that can counteract the limited availability of dNTP substrate in macrophages.

SAMHD1 … and Viral Ways around It

“…The authors compared RT sequences isolated from these monkeys at 6 to 7 months postinfection (mpi) and at 36 mpi, and found an enhanced accumulation of mutations in RT genes from samples infected with virus lacking Vpx. Biochemical analysis revealed that at 36 mpi, RT variants from (−)Vpx viruses displayed higher catalytic and nucleotide incorporation efficacies compared to RTs from (+)Vpx virus-infected animals or compared to samples from early time points [106]. These findings corroborate the idea that lentiviruses without a Vpx-like function may have evolved more efficient RT molecules to enable cDNA synthesis in nondividing cells.…”

“…In 2011, it became clear that lentiviruses, such as HIV-2 and several SIV strains, counteract SAMHD1 with the help of their accessory proteins Vpx and Vpr, resulting in the proteasomal degradation of SAMHD1 [54][55][56]89,96]. Another strategy to evade the dNTPase activity of SAMHD1, found in HIV-1 and other SIVs, is the use of highly efficient RTs that function at low dNTP concentrations in the presence of SAMHD1 [104][105][106]. In contrast, herpesviruses employ their conserved serine/threonine kinases to phosphorylate and inactivate SAMHD1 [73][74][75].…”

“…However, not all lentiviruses encode Vpx, or a protein with a Vpx-like function, to inactivate SAMHD1. Instead, to overcome the SAMHD1-driven shortage of dNTPs in nondividing cells, viruses lacking Vpx harbor very efficient reverse transcriptases that polymerize viral cDNA at low dNTP concentrations [104][105][106][107]. For instance, Lenzi and colleagues biochemically characterized RTs from HIV-1, HIV-2, and SIV isolates and determined the Km values of HIV-1 RTs to be very low in general and in the range of the low dNTP concentrations found in nondividing macrophages.…”

“…In contrast, enzymes encoded by viruses that trigger SAMHD1 degradation, such as HIV-2, displayed significantly higher Km values and required higher dNTP concentrations to efficiently generate cDNA [104]. Interestingly, the Kim laboratory also cloned and analyzed the sequences of RT genes derived from Rhesus macaques infected with either SIVmac WT virus encoding Vpx or a genetically engineered variant lacking the accessory protein [106]. The authors compared RT sequences isolated from these monkeys at 6 to 7 months postinfection (mpi) and at 36 mpi, and found an enhanced accumulation of mutations in RT genes from samples infected with virus lacking Vpx.…”

Mechanistic Interplay between HIV-1 Reverse Transcriptase Enzyme Kinetics and Host SAMHD1 Protein: Viral Myeloid-Cell Tropism and Genomic Mutagenesis

“…This increased efficiency allows the virus to bypass SAMHD1 restriction and replicate in the low-dNTP-pool conditions of macrophages [ 166 ]. Furthermore, RTs from SIVmac239 (Vpx-) infections acquire numerous amino-acid mutations that result in enhanced RT kinetics compared to RTs from SIVmac239 (Vpx+) infections [ 167 ]. This suggests that the loss of Vpx during an in vivo SIVmac239 infection can drive RT variations to counteract the limited availability of dNTPs in macrophages [ 167 ].…”