Intracellular nucleotide levels and the control of retroviral infections

Abstract

Retroviruses consume cellular deoxynucleoside triphosphates (dNTPs) to convert their RNA genomes into proviral DNA through reverse transcription. While all retroviruses replicate in dividing cells, lentiviruses uniquely replicate in nondividing cells such as macrophages. Importantly, dNTP levels in nondividing cells are extremely low, compared to dividing cells. Indeed, a recently discovered anti-HIV/SIV restriction factor, SAMHD1, which is a dNTP triphosphohydrolase, is responsible for the limited dNTP pool of nondividing cells. Lentiviral reverse transcriptases (RT) uniquely stay functional even at the low dNTP concentrations in nondividing cells. Interestingly, Vpx of HIV-2/SIVsm proteosomally degrades SAMHD1, which elevates cellular dNTP pools and accelerates lentiviral replication in nondividing cells. These Vpx-encoding lentiviruses rapidly replicate in nondividing cells by encoding both highly functional RTs and Vpx. Here, we discuss a series of mechanistic and virological studies that have contributed to conceptually linking cellular dNTP levels and the adaptation of lentiviral replication in nondividing cells.

Figure 1. Cellular dNTP concentrations of human primary cells, human cancer cells and transformed cell lines

Red asterisks: The dNTP concentration difference between primary human macrophages and activated PBMCs. Blue asterisks: Effect of dN treatment on cellular dNTP concentrations in primary human lung fibroblasts. A549: human adenocarcinoma alveolar basal epithelial cell line, PANC1: human pancreas epithelioid carcinoma cell line, CHME5: transformed human microglia cell line, 293FT: transformed human embryonic kidney cell line. This figure was prepared by modifying the data previously presented (28).

Figure 2. Comparison of the Km values of HIV-1 RT and MuLV RT with respect to dNTP concentrations in primary human macrophages and T cell/PBMC

The dNTP concentrations of human macrophages (MDM) and activated T cells/PBMCs were determined by the primer-extension based assay (grey bars, (15)) and quantitative LCMS/ MS assay (black bars, (33)). The ranges of the RT Km values were marked by two longitudinal bars (red for HIV-1 RT and blue for MuLV RT). This figure was prepared by modifying the data presented previously (15, 33, 61).

Figure 3. Models for the direct interaction between the side chain of HIV-1 RT Q151 residue and 3’-OH of incoming dNTPs

This structural view of the active site of the HIV-1 RT complex with dNTP and template/primer was adapted from a 1D RT-based model previously reported (68). This figure shows the interaction between wild type residue Q151 (light green) or mutant residue N151 (orange) and the incoming dTTP (black for base and sugar and red for triphosphate), base-paring with the template nucleotide (dark blue). Template (blue line), primer (purple), and incoming dNTP are shown and the nearby R73 (gray) residue is also marked. While the Q151 side chain makes a hydrogen bond with the 3’OH (O3) of the incoming dTTP, the Q151N mutant residue has a shortened R side chain and cannot interact with the sugar on the incoming dTTP.

Figure 4. Effect of Vpx on cellular dNTP concentrations with respect to the Km values of HIV-1 RT and MuLV RT

The concentrations of four dNTPs in primary human macrophages treated with the virus like particles with or without Vpx protein as well as the dNTP concentrations of human primary activated PBMCs are presented (36). Blue arrow indicates the Km difference between HIV-1 RT and MuLV RT, while the red arrow indicates the elevation of dNTP concentration induced by the Vpx protein. The Km value of HIV-1 RT (red bar) stays above the dNTP concentration of macrophages. However, the Vpx treatment increases the dNTP concentration above the Km value of HIV-1 RT in macrophages.

Figure 5. Model for the mechanistic interplay among cellular dNTPs, SAMHD1, proviral DNA synthesis, and potential mutagenic consequences during HIV reverse transcription in macrophages

RT enzyme kinetics is controlled by the availability of cellular dNTPs, which significantly varies between dividing CD4⁺ T cells and macrophages. These kinetic disparities appears to be tightly regulated by cellular (SAMHD1) and viral (Vpx) players. The extremely low abundance of canonical dNTPs in macrophages can force RT to incorporate mutagenic non-canonical cellular nucleotides such as rNTPs and dUTP, generating a potentially mutagenic landscape in macrophages.