Kaposi's Sarcoma-Associated Herpesvirus Nonstructural Membrane Protein pK15 Recruits the Class II Phosphatidylinositol 3-Kinase PI3K-C2α To Activate Productive Viral Replication

Abstract

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) causes the angiogenic tumor KS and two B-cell malignancies. The KSHV nonstructural membrane protein encoded by the open reading frame (ORF) K15 recruits and activates several cellular proteins, including phospholipase Cγ1 (PLCγ1), components of the NF-κB pathway, as well as members of the Src family of nonreceptor tyrosine kinases, and thereby plays an important role in the activation of angiogenic and inflammatory pathways that contribute to the pathogenesis of KS as well as KSHV productive (lytic) replication. In order to identify novel cellular components involved in the biology of pK15, we immunoprecipitated pK15 from KSHV-infected endothelial cells and identified associated proteins by label-free quantitative mass spectrometry. Cellular proteins interacting with pK15 point to previously unappreciated cellular processes, such as the endocytic pathway, that could be involved in the function of pK15. We found that the class II phosphatidylinositol 3-kinase (PI3K) PI3K-C2α, which is involved in the endocytosis of activated receptor tyrosine kinases and their signaling from intracellular organelles, interacts and colocalizes with pK15 in vesicular structures abundant in the perinuclear area. Further functional analysis revealed that PI3K-C2α contributes to the pK15-dependent phosphorylation of PLCγ1 and Erk1/2. PI3K-C2α also plays a role in KSHV lytic replication, as evidenced by the reduced expression of the viral lytic genes K-bZIP and ORF45 as well as the reduced release of infectious virus in PI3K-C2α-depleted KSHV-infected endothelial cells. Taken together, our results suggest a role of the cellular PI3K-C2α protein in the functional properties of the KSHV pK15 protein.IMPORTANCE The nonstructural membrane protein encoded by open reading frame K15 of Kaposi's sarcoma-associated herpesvirus (KSHV) (HHV8) activates several intracellular signaling pathways that contribute to the angiogenic properties of KSHV in endothelial cells and to its reactivation from latency. A detailed understanding of how pK15 activates these intracellular signaling pathways is a prerequisite for targeting these processes specifically in KSHV-infected cells. By identifying pK15-associated cellular proteins using a combination of immunoprecipitation and mass spectrometry, we provide evidence that pK15-dependent signaling may occur from intracellular vesicles and rely on the endocytotic machinery. Specifically, a class II PI3K, PI3K-C2α, is recruited by pK15 and involved in pK15-dependent intracellular signaling and viral reactivation from latency. These findings are of importance for future intervention strategies that aim to disrupt the activation of intracellular signaling by pK15 in order to antagonize KSHV productive replication and tumorigenesis.

Keywords: KSHV-K15; PI3K-C2α; lytic replication.

FIG 1

Experimental workflow and sample preparation. (A) Detailed scheme of the interaction proteomics workflow. The KSHV lytic cycle was induced by using RTA and SB in latently infected HuARLT2 cells or uninfected control parental cells. After 48 h, endogenous K15 protein was precipitated from both induced and uninduced samples by using a rat anti-K15 mAb (clone 6E7). (B) Western blot (WB) analysis, using antibody 10A6 to pK15, of a small aliquot of the IP preparation used for MS/MS analysis. Experiments were performed in two independent biological replicates.

FIG 2

Distribution of proteins identified after MS/MS analysis. (A and B) Scatter plot showing the ratio of the log₂ LFQ intensity of proteins from Hu-rKSHV cells to that of proteins from HuARLT2 cells, where experiment 1 is plotted against experiment 2, in uninduced cells (latent) (A) and after lytic induction (B). The names of proteins identified in both latent and lytic samples are shown in blue. The second dotted lines in panel B mark proteins enriched more than 64-fold for both experiments. The list of the pK15-interacting proteins identified in cells undergoing lytic KSHV replication (Table 2) was uploaded to the DAVID bioinformatics tool. (C and D) Gene Ontology (GO) annotations for enriched cellular compartments (C) and molecular functions (D), with a cutoff (P < 0.006) based on their fold enrichment, are shown.

FIG 3

pK15 interacts with PI3K-C2α through its C-terminal cytoplasmic tail. (A) Log₂-fold enrichment of PI3K-C2α using the LFQ intensity obtained from the induced and uninduced samples in the experiments in Fig. 1 and 2. (B to D) HEK-293T cells were cotransfected with plasmid vectors expressing GFP-tagged PI3K-C2α and Flag-tagged pK15. After 48 h, cellular lysates were collected, pulldown assays were performed by using anti-Flag M2 control IgG-conjugated beads, and coprecipitation of PI3K-C2α was analyzed by Western blotting (B and C), or pulldown assays were performed by using GFP-Trap or control beads, and coprecipitation of K15 was analyzed by Western blotting (D). The expression levels of the individual proteins in the cellular lysate are shown as input. (E) Endogenous PI3K-C2α protein from a HEK 293T cellular lysate was pulled down by using a purified GST fusion protein of the full-length K15 C-terminal cytoplasmic tail or a GST control. Results are representative of data from at least 3 experiments.

FIG 4

pK15 colocalizes with PI3K-C2α on vesicular structures in the perinuclear area. (A) Stably infected HuARLT2-rKSHV cells were stained with rat mAb 18E5 to pK15, followed by Cy3-conjugated anti-rat secondary antibody (red), with subsequent staining using a mouse anti-PI3K-C2α mAb and a FITC-conjugated anti-mouse secondary antibody (green). (B) HeLa-CNX cells cotransfected with vectors expressing GFP-tagged PI3K-C2α (green) and pK15 were stained with rat mAb 18E5 to pK15, followed by a Cy3-conjugated anti-rat secondary antibody (red). Images were acquired using a Leica confocal microscope. DIC, differential interference contrast.

FIG 5

Mapping the K15 region necessary for PI3K-C2α binding. (A) Schematic representation of the full-length K15 protein and its signaling motifs. (B) Purified GST fusion proteins of the full-length cytoplasmic tails of wild-type (wt) K15P and K15M or with mutations at either the SH3-binding site (PPxPP387–391 to AAxAA387–391/ΔSH3), the SH2-binding site (Y481 to F481 [YF] for K15P and Y489F for K15M), or a combination of both (ΔSH3 YF) as well as a GST control were used to pull down GFP-tagged PI3K-C2α that was transfected into HEK-293T cells. (C) Schematic representation of C-terminal or N-terminal deletion constructs of the pK15 cytoplasmic domain fused to GST. (D) GST-K15 fusion constructs in panel C were used to pull down GFP-tagged PI3K-C2α transfected into HEK-293T cells. (E) GST pulldown of endogenous PI3K-C2α protein from HEK-293T cells using a series of triple-alanine scanning mutants derived from the last C-terminal deletion mutant (C4 in panel C). (F) Pulldown of transiently expressed GFP-tagged PI3K-C2α using either the wild type or two triple-alanine scanning mutants in the full-length K15 C-terminal cytoplasmic tail fused to GST. Results are representative of data from at least three independent experiments.

FIG 6

PI3K-C2α is required for the pK15-induced phosphorylation of PLCγ1 and ERK1/2. HeLa-CNX cells were transfected with either a scrambled (Scr) siRNA as a control or a pool of siRNAs against PI3K-C2α. After 6 h, cells were transfected with a plasmid vector expressing wild-type K15P or an empty vector control. After 48 h, cells were collected, and the phosphorylation level of the indicated proteins was assessed by Western blotting using specific antibodies.

FIG 7

Depletion of PI3K-C2α from infected endothelial cells leads to reduced KSHV lytic replication. HuARLT2-rKSHV cells were microporated with either a control siRNA (Scr), four individual siRNAs or a pool of these siRNAs against PI3K-C2α, or a single siRNA against K15, and the KSHV lytic cycle was induced 24 h later using a cocktail of RTA and SB. (A to C) Forty-eight hours after lytic induction, images for GFP and RFP expression were acquired (A), and the number of RFP-positive cells from five or more fields was quantified; cells were then lysed, and the expression level of the indicated viral proteins was assessed by Western blotting (C). Results are representative of data from two independent experiments. The scatter plot (B) represents the means ± standard deviations (SD) for five or more fields. Ordinary one-way analysis of variance (ANOVA) was used to determine P values. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (D and E) Primary lymphatic endothelial cells infected with KSHV for 2 weeks (LEC-rKSHV) were transfected with either the pooled PI3K-C2α siRNA or a scrambled control. After 72 h, cells and the culture supernatant were collected. The expression levels of the indicated proteins were analyzed by Western blotting (D), and the amount of released infectious virus was assayed by titrating the culture supernatant on HEK-293 cells (E). A Mann-Whitney test was used to determine P values. **, P < 0.01.

FIG 8

Schematic representation of pK15-mediated signaling. The pK15 protein can recruit PLCγ1 and NF-κB as well as members of the Src family of tyrosine kinases, activating the PLCγ1, MAP kinase (MAPK), and NF-κB signaling pathways. The activation of these cellular signaling pathways in turn leads to the expression of angiogenic and inflammatory molecules, which are important for the pathogenesis of KSHV-mediated diseases and virus lytic replication. Additionally, the interaction of pK15 with the class II PI3K PI3K-C2α, which also contributes to KSHV lytic replication, might play a role in its intracellular localization (by mediating its internalization); it is possible that pK15 signals from intracellular membrane-bound organelles.