Metabolic profiling during HIV-1 and HIV-2 infection of primary human monocyte-derived macrophages

Abstract

We evaluated cellular metabolism profiles of HIV-1 and HIV-2 infected primary human monocyte-derived macrophages (MDMs). First, HIV-2 GL-AN displays faster production kinetics and greater amounts of virus as compared to HIV-1s: YU-2, 89.6 and JR-CSF. Second, quantitative LC-MS/MS metabolomics analysis demonstrates very similar metabolic profiles in glycolysis and TCA cycle metabolic intermediates between HIV-1 and HIV-2 infected macrophages, with a few notable exceptions. The most striking metabolic change in MDMs infected with HIV-2 relative to HIV-1-infected MDMs was the increased levels of quinolinate, a metabolite in the tryptophan catabolism pathway that has been linked to HIV/AIDS pathogenesis. Third, both HIV-1 and HIV-2 infected MDMs showed elevated levels of ribose-5-phosphate, a key metabolic component in nucleotide biosynthesis. Finally, HIV-2 infected MDMs display increased dNTP concentrations as predicted by Vpx-mediated SAMHD1 degradation. Collectively, these data show differential metabolic changes during HIV-1 and HIV-2 infection of macrophages.

Keywords: Glycolysis; HIV-1; HIV-2; LC–MS/MS; Macrophages; Metabolites; TCA.

Fig. 1

HIV reverse transcription (RT) activity. (A) The time course for days 1–28 are plotted for virus generation. MDMs were infected GL-AN, JR-CSF, 89.6 and YU-2 viruses (triplicated wells). (B) Analysis of days 1–4 of the time course. Day 1 was set at 1 for all the donors and fold increases of RT-activity are plotted for each virus. Each sample was analyzed in triplicate wells for HIV RT activity to determine counts per minute (CPM) at each time point. Data was plotted as mean and S.E.M. for three independent human primary MDM donors.

Fig. 2

Characterization of MDMs used for metabolomic analysis. Three independent MDM donors were infected with VSV-G pseduotyped GL-AN, GL-ANÄVpx, JR-CSF, 89.6 and YU-2 viruses or not infected (NI). At day 4 after infection, MDMs and supernatant were collected for multiple analyses. (A) MDMs were intracellular stained for capsid protein and analyzed by FACS. The percentage of infected MDMs were graphed. (B) Cell-free supernatants were analyzed by ELISA for p24 antigen (HIV-1) or p27 antigen (HIV-2) levels. Significant differences (Student T test) between the different viruses are displayed. (C) Immunoblot analysis for SAMHD1 and actin protein levels was done for the different MDM samples. (D) SAMHD1 expression levels were quantitated and displayed for the three independent donors.

Fig. 3

Evaluation of cellular intermediates. HPLC–MS/MS analysis was used to determine changes in glycolysis intermediates for (A) hexose 6-P, (B) fructose 1,6 bisphosphate (FBP), (C) glyceraldehyde 3-phosphate (G3P), (D) 3-phosphoglycerate (3-PG), and (E) pyruvate. Significant increases are denoted by * (p < 0.05) and *** (p < 0.001). TCA cycle intermediates for (F) citrate, (G) á-ketoglutarate, (H) succinate and (I) malate are plotted. Normalized intensities are graphed as means and S.E.M. for three independent donors.

Fig. 4

Evaluation of UDP-sugars. We monitored changes in (A) UDP-glucose, (B) UDP-N-acetyl-glucosamine and (C) UDP-D-glucuronate. Data are graphed as means and S.E. M. are plotted for three independent donors. Significant increase is denoted by *** (p < 0.001).

Fig. 5

Ribose and dNTP concentrations. The normalized levels of (A) ribose-5-Phosphate is shown for the different viral infected MDMs. Means and S.E.M. are plotted for three independent donors. Significant increase is denoted by * (p < 0.05) when compared to NI MDMs. Determining dNTP concentrations in HIV-2 infected MDMs. Seven day maturated MDMs were infected with either GL-AN (filled squares) or GL-ANÄVpx (open squares). The dNTP levels were determined for (B) dATP, (C) dGTP, (D) dCTP and (E) dTTPþdUTP at days 0–3 after infection for four independent MDM donors. Significant difference* (p < 0.05; Student T test) are indicated. The data are graphed as means and S.E.M. for three independent donors.

Fig. 6

Evaluating metabolites in the kynurenine pathway. We evaluated the pool sizes for (A) quinolinate and (B) NADþ metabolites. Significant increase is denoted by * (p < 0.05) and *** (p < 0.001). Data are graphed as means and S.E.M. are plotted for three independent MDM donors.