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

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. 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.