Targeting Kaposi's Sarcoma-Associated Herpesvirus ORF21 Tyrosine Kinase and Viral Lytic Reactivation by Tyrosine Kinase Inhibitors Approved for Clinical Use

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the cause of three human malignancies: Kaposi’s sarcoma, primary effusion lymphoma, and the plasma cell variant of multicentric Castleman disease. Previous research has shown that several cellular tyrosine kinases play crucial roles during several steps in the virus replication cycle. Two KSHV proteins also have protein kinase function: open reading frame (ORF) 36 encodes a serine-threonine kinase, while ORF21 encodes a thymidine kinase (TK), which has recently been found to be an efficient tyrosine kinase. In this study, we explore the role of the ORF21 tyrosine kinase function in KSHV lytic replication. By generating a recombinant KSHV mutant with an enzymatically inactive ORF21 protein, we show that the tyrosine kinase function of ORF21/TK is not required for the progression of the lytic replication in tissue culture but that it is essential for the phosphorylation and activation to toxic moieties of the antiviral drugs zidovudine and brivudine. In addition, we identify several tyrosine kinase inhibitors, already in clinical use against human malignancies, which potently inhibit not only ORF21 TK kinase function but also viral lytic reactivation and the development of KSHV-infected endothelial tumors in mice. Since they target both cellular tyrosine kinases and a viral kinase, some of these compounds might find a use in the treatment of KSHV-associated malignancies. IMPORTANCE Our findings address the role of KSHV ORF21 as a tyrosine kinase during lytic replication and the activation of prodrugs in KSHV-infected cells. We also show the potential of selected clinically approved tyrosine kinase inhibitors to inhibit KSHV TK, KSHV lytic replication, infectious virion release, and the development of an endothelial tumor. Since they target both cellular tyrosine kinases supporting productive viral replication and a viral kinase, these drugs, which are already approved for clinical use, may be suitable for repurposing for the treatment of KSHV-related tumors in AIDS patients or transplant recipients.

Recent Advances in Developing Treatments of Kaposi’s Sarcoma Herpesvirus-Related Diseases

“…Genes for herpesviral TKs occur in alpha-and gammaherpesviruses, but not in betaherpesviruses. In alpha-and gammaherpesviruses, the TK homologs are conserved: UL23 of herpes simplex virus 1 and 2 (HSV-1/2), ORF36 of varicella-zoster virus (VZV), BXLF1 of Epstein-Barr virus (EBV), and ORF21 of KSHV (52). Herpesviral TKs differ in their capacity to phosphorylate nucleoside analogs.…”

“…Herpesviral TKs differ in their capacity to phosphorylate nucleoside analogs. Pyrimidine nucleosides such as brivudine and azidothymidine (zidovudine, the anti-HIV nucleoside reverse transcriptase inhibitor) are efficiently phosphorylated by KSHV TK [40,52]. Instead, PK/ORF36 efficiently phosphorylates purine analogs like valganciclovir [53].…”

“…Of the more than 20 currently available inhibitors of cellular tyrosine kinases, five compounds (dasatinib, ponatinib, bosutinib, gefitinib, and nilotinib) were shown to also inhibit KSHV thymidine kinase (TK/pORF21), which, in contrast to its name, acts as an efficient protein tyrosine kinase [52,123]. Dasatinib and ponatinib also strongly inhibited KSHV early viral gene expression and the production of new viral progeny in B, endothelial and epithelial cells, most likely as a result of the inhibition of cellular tyrosine kinases, and dasatinib inhibited the growth of KSHV-driven endothelial tumors in a mouse xenograft model [52]. UNC3810A, a small molecule inhibitor of the receptor tyrosine kinase Tyro3, a member of the Tyro3/Axl/Mer (TAM) family of tyrosine kinases that promote the proliferation and survival of several cancers, was shown to be a potent inhibitor of PEL cell growth in a mouse xenograft model [124].…”

Manipulation of Focal Adhesion Signaling by Pathogenic Microbes

“…A more in-depth understanding of the mechanisms driving altered FA dynamics during infection offer exciting opportunities for therapeutic intervention. In fact, several FDA-approved kinase inhibitors have been shown to inhibit TK activity and lytic activation in vitro and to prevent KSHV-induction of tumors in mice [ 291 ]. Thus, targeting the ability of TK to modify FA proteins is a promising approach for preventing lytic reactivation of KSHV infection in AIDS patients.…”

3D culture conditions support Kaposi’s sarcoma herpesvirus (KSHV) maintenance and viral spread in endothelial cells

“…Notably, some KSHV-related features such as the sprouting of endothelial cells are noticeable only under 3D culture conditions [19,22]. Upon transplantation into mice, these KSHV-infected cells form lesions that exhibit KSHV-dependent growth and that histologically reflect early patch stages of KS [19,22,23].…”