Fig 1.
KSHV K1 mutant infected cells exhibit decreased survival following nutrient deprivation.
(A) KSHV-K1WT and KSHV-K1REV (revertant) harbor a wild type K1 gene. The KSHV-K15XSTOP has a K1 mutant gene that contains 5 stop codons. Three of the stop codons follow the first start codon of the K1 gene. The other two stop codons replace two downstream ATG codons at positions 481 and 763 in the K1 gene. The KSHVΔK1 mutant has had the K1 gene replaced by a RpsL-Neo cassette. Hence, the KSHV-K15XSTOP and KSHVΔK1 are not able to express any portion of the K1 protein. The grey box represents FLAG and horizontal lines represent the stop codons in KSHV-K15XSTOP. HUVEC infected with KSHV-K1WT, KSHV-K15XSTOP, KSHVΔK1 or KSHV-K1REV were starved of serum and growth factors for 24, 48 and 72 hours. (B) The proportion of metabolically active cells within each group was determined using an MTS assay. Error bars are the standard deviation of biological triplicates. GraphPad Prism was used to determine one-way ANOVA and Tukey’s post-test. *P<0.05, **P<0.005 (C) The percent of viable cells/well for each group was determined by trypan blue exclusion assay. Percent was calculated by normalization to total number of cells plated. Error bars are the standard deviation of biological triplicates. (D) KSHV-K1WT, KSHV-K15XSTOP, KSHVΔK1, and KSHV-K1REV infected iSLK cells were cultured without serum. Cell viability was determined by trypan blue exclusion. Error bars are the standard deviation of biological triplicates.
Fig 2.
Cells infected with KSHV containing WT K1 are more resistant to AMPK inhibition than cells infected with KSHV K1 mutants.
HUVEC infected with viruses containing a WT or mutant K1 gene were treated with 20 μM of compound C for 48 hours. Metabolically active cells were determined by an MTS assay. (A) The percent of metabolically active cells in HUVEC stably infected with the recombinant viruses KSHV-K1WT, KSHV-K15XSTOP, and KSHV-K1REV. (B) The percent of metabolically active cells of HUVEC stably infected with KSHV- K1WT, KSHVΔK1, and KSHV-K1REV. The percent was determined by normalizing to DMSO (0.2%) control. Error bars represent the standard deviation of biological triplicates. GraphPad Prism was used to determine one-way ANOVA and Tukey’s post-test. *P<0.02, **P<0.01, ***P<0.001.
Fig 3.
K1 expression provides a survival advantage in cells treated with compound C.
(A) HEK-293 cells stably expressing empty vector (EV) or FLAG-K1 (K1) were treated in duplicate with 0 μM/ DMSO (0.2%), 20 μM compound C (CC) or 10 μM compound C for 6–8 hours. (B) HEK-293 cells stably expressing EV or K1 were treated in duplicate with 0 μM/DMSO (0.1%) or 10 μM compound C (CC) for 8 and 24 hours. Cell viability was determined by trypan blue exclusion assay. Percentages were derived from normalization to vehicle treated samples. (C) The level of caspase-3 activity was determined in duplicate samples and normalized to cell number of each sample. (D) EV or K1 HEK-293 cells treated with either 0 μM/DMSO (0.1%) or 10 μM compound C for 6 hours and immunoblotted for K1 and tubulin. Statistical significance was evaluated by Student’s t test. *P<0.05, **P<0.005.
Fig 4.
(A) Number of PRKAG1 (AMPKγ1) and HSP90 peptides identified in association with K1 by mass spectrometry. (B) HEK-293 cells stably expressing empty vector (EV) or FLAG-K1 (K1) were transfected with V5-AMPKγ1 (AMPKγ1). AMPKγ1 was immunoprecipitated, and immunoblotted for K1 or AMPKγ1. (C) HEK-293 cells stably expressing EV or K1 were transfected with AMPKγ1 or EV (pcDNA3). K1 was immunoprecipitated and immunoblotted for AMPKγ1 or K1.
Fig 5.
The K1 N-terminus is important for association with AMPKγ1.
(A) HEK-293 cells stably expressing empty vector (EV) or FLAG-K1 (K1) were transfected with V5-AMPKγ1 (AMPKγ1). AMPKγ1 was immunoprecipitated, and blots were probed for AMPKγ1, endogenous AMPKα1, endogenous AMPKβ1 and K1. (B) Endogenous AMPKβ1 was immunoprecipitated from EV or K1 expressing HEK-293 cells. The blots were probed for K1 or AMPKβ1. (C) HEK-293 cells were transiently transfected with AMPKγ1 and with one of the following: K1WT, K1ΔCT or K1ΔNT. An equivalent amount of EV (pcDNA3) was also transfected along with K1WT. We immunoprecipitated the various K1 domain mutants, and immunoblotted for AMPKγ1 or K1.
Fig 6.
K1 and AMPK associate in membranes in the perinuclear area of the cell.
(A) HEK-293 cells stably expressing empty vector (EV) or FLAG-K1 (K1) were lysed and fractionated into cytoplasmic, membrane and nuclear fractions. Immunoblots were probed for endogenous AMPK subunit and isoforms. (B) HUVEC stably expressing empty (EV) or FLAG-K1 (K1) were fixed and permeabilized. Cells were then stained with anti-FLAG directly conjugated to FITC antibody, and an AMPKβ1/2 antibody, followed by an anti-rabbit conjugated to Alexa Fluor 647. Nuclei were stained with DAPI. Stained HUVEC were evaluated with a Zeiss 700 confocal microscope using a 63X oil objective. Mander’s overlap coefficient (R = 0.83) was generated using ImageJ. (C) HEK-293 cells were transiently transfected with EV (pcDNA3), K1WT, K1ΔCT and K1ΔNT. Cell lysates were separated into membrane, cytoplasmic and nuclear fractions. We evaluated the membrane fraction for expression of the K1 domain deleted mutants by immunoblot. We also immunoblotted for AIF (membrane), MEK1/2 (cytoplasm) and Histone H3 (nucleus). (D) HEK-293 cells were transiently transfected with the constructs described in (C) with the addition of AMPKγ1. Cell lysates were separated into membrane, cytoplasmic and nuclear fractions. We immunoprecipitated FLAG K1 and immunoblotted for AMPKγ1 or FLAG using only the membrane fraction.
Fig 7.
K1 and AMPK association is important for cell survival following exposure to stress.
(A) HEK-293 cells stably expressing FLAG-tagged K1WT, K1ΔCT, K1ΔNT or empty vector (EV = pLenti CMV) were treated with 10 or 7.5 μM compound C for 48 hours. The percent of metabolically active cells was determined using the Promega MTS assay. The percent was derived by normalization to the DMSO (0.1%) control. Error bars represent the standard deviation of biological triplicates. GraphPad Prism was used to determine one-way ANOVA and Tukey’s post-test. **P<0.005 (B) Western blot showing expression of EV, K1WT, K1ΔCT, and K1ΔNT using an anti-FLAG antibody. (C) Western blot from (Fig 7B) was stripped and probed for K1 using an anti-K1 antibody.
Fig 8.
K1-expression facilitates AMPK activity in stressed cells.
(A) HUVEC stably expressing empty vector (EV) or FLAG-K1 (K1) were deprived of serum and growth factors (starve) for 24 hours. At the time of starvation, 5 μM compound C or DMSO (0.05%) was added. Each condition for EV and K1 was performed in triplicate. AMPK-specific activity was determined by subtracting counts per minute (cpm) derived for each sample incubated without SAMS peptide from cpm derived from each sample incubated with SAMS peptide. These values were then normalized to total protein for each sample. The error bars represent the standard deviation of triplicates. *P<0.05 Student’s T test. (B) Western blot of K1 and tubulin using lysate from the AMPK activity assay.