Figure 1.
Analysis of the de novo fatty acid biosynthesis pathway during vaccinia infection.
A) Schematic representation of the de novo fatty acid biosynthetic pathway and the biological functions of palmitate. Key enzymes in the pathway are highlighted in bold [acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN)] as well as their cognate inhibitors TOFA and C75, respectively. B) One-step growth analysis of BSC40 cells infected with WT vaccinia virus (MOI 5) in the presence of DMSO, TOFA (154 µM) or C75 (39 µM) in the absence (black bars) or presence (grey bars) of supplemental, exogenous palmitate (50 µM); viral yield at 16 hpi is shown (n = 6) (**, p<0.02; ***, p<0.001).
Figure 2.
Inhibition of palmitoylation or phospholipid synthesis has no impact on viral replication.
A) One-step growth analysis of BSC40 cells infected with WT vaccinia virus (MOI 5) in the presence of various concentrations (0, 25, 50 and 75 µM) of 2-bromopalmitate, a palmitate analogue and inhibitor of palmitoylation; viral yield at 16 hpi is shown (n = 6). B) One-step growth analysis of BSC40 cells infected with WT vaccinia virus (MOI 5) in the presence of various concentrations (0, 12.5 and 25 µM) of triacsin C, an inhibitor of fatty acid chain elongation and hence phospholipid synthesis; viral yield at 16 hpi is shown (n = 6).
Figure 3.
Palmitate is utilized within mitochondria for energy production via ß-oxidation.
A) One step growth analysis of BSC40 cells infected with WT vaccinia virus (MOI 5) in glucose-free media (gray bar) or glucose-free media supplemented with 4 mM glucose (black bar); viral yield at 16 hpi is shown (n = 6). B) One step growth analysis of BSC40 cells infected with WT vaccinia virus (MOI 5) in the presence (left bars) or absence (right bars) of 4 mM glucose supplemented with DMSO (vehicle control) (solid bars) or etomoxir (360 µM) (striped bars), an inhibitor of long-chain fatty acid import into the mitochondria; viral yield at 16 hpi is shown (n = 6) (***, p<0.001). C) One-step growth analysis of BSC40 cells infected with WT vaccinia virus (MOI 5) in the presence (black diamonds) or absence (gray circles) of 4 mM glucose and treated with various concentrations (0–8 mM) of trimetazidine, an inhibitor of ß-oxidation; viral yield at 16 hpi is shown (n = 6).
Figure 4.
Glutamine is essential for vaccinia infection and anaplerotically supplements the TCA cycle.
A) One step growth analysis of BSC40 cells infected with WT vaccinia virus (MOI 5) in glutamine-free media supplemented with various concentrations of glutamine; viral yield at 16 hpi is shown (n = 2). B) Grey bars: one step growth analysis of BSC40 cells infected with WT vaccinia virus (MOI 5) in glutamine-free media or glutamine-free media supplemented with 15 mM oxaloacetate (diagonal stripes) or 5 mM dimethyl-α-ketoglutaric acid (horizontal stripes). Black bars: one step growth analysis of BSC40 cells infected with WT vaccinia virus (MOI 5) in medium containing 200 µM glutamine, or media containing 200 µM glutamine supplemented with 15 mM oxaloacetate (diagonal stripes) or 5 mM dimethyl-α-ketoglutaric acid (horizontal stripes). Viral yield at 16 hpi is shown (n = 4); *** denotes p<0.001).
Figure 5.
Vaccinia infection is accompanied by an increase in ATP synthesis that is ablated by etomoxir.
Seahorse technology was employed for real-time, continual measurement of oxygen consumption rates (OCR) in cultures of uninfected or infected cells as a surrogate assessment of ATP synthesis. A) BSC40 cells were either mock infected (gray diamonds) or infected with WT vaccinia virus (MOI 5) (black triangles), and OCR was measured from 1.5 to 12 hpi (n = 12) (***, p<0.001). B) BSC40 cells were infected with WT vaccinia virus (MOI 5) in the presence of vehicle control (DMSO) (black triangle) or etomoxir (360 µM) (gray circles) and OCR was measured from 1.5 to 12 hpi (n = 12) (***, p<0.001).
Figure 6.
Immunofluorescence analysis of FASN localization in mock and virally infected cells.
BSC40 cells were either mock infected (left and middle columns) or infected with WT vaccinia virus (MOI 2) (right column) for seven hours and fixed with cold methanol∶acetone (1∶1). Samples were stained with primary antiserum recognizing FASN and blocking peptide (as appropriate) followed by AlexaFluor488 secondary antibody (top row) as well as the cell permeable dyes Mitotracker Red (to stain the mitochondria) (second row) and DAPI (to stain DNA) (third row). Merged images of FASN, Mitotracker and DAPI are shown in the fourth row. A single cell is shown at higher magnification in the bottom row and corresponds to the cell outlined in the dotted box (fourth row). Merged images show the ablation of FASN staining when a blocking peptide was included, and the significant colocalization of FASN with the mitochondria, both in mock and infected samples.
Figure 7.
Viral DNA replication is mildly impacted by TOFA, C75 or etomoxir.
A) BSC40 cells were infected with WT vaccinia virus (MOI 2) in the presence of 25 µg/ml BrdU, to track nascent DNA synthesis, as well as DMSO (left column), TOFA (154 µM) (middle column) or etomoxir (360 µM) (right column). At 7 hpi, cells were fixed and stained with primary antisera recognizing the viral I3 protein (top row), a marker of DNA replication sites, and BrdU (second row) as well as DAPI (third row). Merged images of I3, BrdU and DAPI are shown in the fourth row. A single cell is shown at higher magnification in the bottom row and corresponds to the cell shown in the dotted box (fourth row). Merged images show accumulation of replication foci under all treatments, although the foci seen in TOFA or etomoxir treated cells appear smaller and more numerous. B) Pulsed-field gel electrophoresis was employed to examine the accumulation and integrity of full length viral genomes. BSC40 cells were infected with WT vaccinia virus in presence of DMSO, TOFA (154 µM), C75 (39 µM) or etomoxir (360 µM). At 10 hpi, cells were processed for PFGE; DNA was visualized by ethidium bromide staining. Under all conditions tested, full length genomes accumulated; however in TOFA-, C75- and etomoxir-treated cells the levels of full-length genomic DNA that accumulated were 50, 70 and 83% (respectively) of that seen when cells were treated with vehicle (DMSO) alone.
Figure 8.
Viral protein synthesis is modestly reduced in the presence of TOFA, C75 and etomoxir.
Protein synthesis was examined by pulsing both mock treated (lanes 1–4 and 9–12) and WT-infected (lanes 5–8 and 13–16) cells with 100 µCi/ml [35S]-Methionine for 30 min. Cells were maintained in the presence of vehicle (DMSO), TOFA (154 µM), C75 (39 µM) or etomoxir (360 µM) throughout the time course. At 4.5 hpi, WT infected cells showed a characteristic decrease in host protein synthesis as well as a concomitant appearance of intermediate protein synthesis (open circles, lanes 5–8). At later time points, WT infected cells show the synthesis of late viral proteins (open stars, lanes 13–16). Protein standards are shown at left with the molecular masses indicated in kDa. Quantification of protein synthesis was normalized to DMSO treated cells within each bracketed grouping.
Figure 9.
Viral assembly is inhibited significantly by TOFA and C75.
A) BSC40 cells were infected with Cts28 (MOI 5) for 12 h at the non-permissive temperature (39.7°C) to synchronize the infectious cycle at the initiation of assembly. Cultures were then shifted to the permissive temperature (31.5°C) to relieve the block to assembly and infection was allowed to proceed for an additional 8 h. The shift-down was performed in the presence of rifampicin (rif) (100 µg/ml), a known inhibitor of assembly, DMSO (vehicle control), TOFA (154 µM) or C75 (39 µM). Viral yield is shown; a significant block to virus production is seen in the presence of rif, TOFA or C75 (n = 6) (***, p<0.001). B) BSC40 cells were infected with WT vaccinia virus (MOI 5) in the presence rif (100 µg/ml). At 12 hpi, cells were released from the rif block into media containing rif (100 µg/ml), DMSO, TOFA (154 µM) or C75 (39 µM) in the absence (black bars) or presence (gray bars) of exogenous palmitate (50 µM) for 8 h. Cells were harvested and viral yield analysis reveals that TOFA or C75 exert a significant impact on viral yield that can be partially rescued by the addition of palmitate (n = 6) (**, p<0.02). C) Electron microscopy was utilized to examine specific block in viral assembly seen when rif-arrested cells were released into media containing C75. Cells were treated as in (B) and processed for electron microscopy. Cells maintained in the presence of rif (upper left panel) accumulated large virosomes (V) that were surrounded by flaccid membranes (arrowheads). When cells were released into DMSO (upper right panel), assembly resumed and crescent membranes (C), immature virions (IV) and mature virions (not shown) were abundant. When cells were released into C75 (bottom panels), the virosomal contents dispersed into splotchy aggregates (star); IV, empty IV, and “peanut” shaped double-IV (open circle) were seen.
Figure 10.
Model for palmitate utilization during vaccinia virus infection.
The de novo fatty acid biosynthetic pathway is utilized to generate ATP during vaccinia virus infection. ACC (acetyl-CoA carboxylase) converts acetyl-CoA to malonyl-CoA; FASN (fatty acid synthease) catalyzes successive condensation reactions of malonyl CoA with acetyl-CoA to generate the 16-carbon fatty acid, palmitate. Palmitate is then imported into the mitochondria by CPT1 (carnitine palmitoyl transferase) 1. Within the mitochondria, palmitate undergoes β-oxidation to generate acetyl-CoA which drives the TCA cycle (and subsequent oxidative phosphorylation) to generate ATP. Glutaminolysis enables glutamine to anaplerotically fill in the TCA cycle. Glutamine is converted to glutamate by Glnase (glutaminase) which is further converted to α-ketoglutarate by GDH (glutamate dehydrogenase). Inhibitors of this pathway are shown in red; impairment of each of the steps in this pathway significantly diminishes vaccinia virus production. ATP production supports DNA replication and protein synthesis (gray dashed lines), but is most important for the assembly of nascent virions (black dashed line).