Importance of accessibility to the extracellular juxtamembrane stalk region of membrane protein for substrate recognition by viral ubiquitin ligase K5.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a carcinogenic virus that latently infects B cells and causes malignant tumors in immunocompromised patients. KSHV utilizes two viral E3 ubiquitin ligases, K3 and K5, in KSHV-infected cells to mediate the polyubiquitination-dependent down-regulation of several host membrane proteins involved in the immune system. Although K3 and K5 are members of the same family and have similar structural topologies, K3 and K5 have different substrate specificities. Hence, K5 may have a different substrate recognition mode than K3; however, the molecular basis of substrate recognition remains unclear. Here, we investigated the reason why human CD8α, which is known not to be a substrate for both K3 and K5, is not recognized by them, to obtain an understanding for molecular basis of substrate specificity. CD8α forms a disulfide-linked homodimer under experimental conditions to evaluate the viral ligase-mediated down-regulation. It is known that two interchain disulfide linkages in the stalk region between each CD8α monomer (Cys164-Cys164 and Cys181-Cys181) mediate homodimerization. When the interchain disulfide linkage of Cys181-Cys181 was eliminated, CD8α was down-regulated by K5 with a functional RING variant (RINGv) domain via polyubiquitination at the cytoplasmic tail. Aspartic acid, located at the stalk/transmembrane interface of CD8α, was essential for K5-mediated down-regulation of the CD8α mutant without a Cys181-Cys181 linkage. These results suggest that disulfide linkage near the stalk/transmembrane interface critically inhibits substrate targeting by K5. Accessibility to the extracellular juxtamembrane stalk region of membrane proteins may be important for substrate recognition by the viral ubiquitin ligase K5.

Kaposi's sarcoma-associated herpesvirus ubiquitin ligases downregulate cell surface expression of l-selectin.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic etiological factor for Kaposi's sarcoma and primary effusion lymphoma in immunocompromised patients. KSHV utilizes two immune evasion E3 ubiquitin ligases, namely K3 and K5, to downregulate the expression of antigen-presenting molecules and ligands of natural killer (NK) cells in the host cells through an ubiquitin-dependent endocytic mechanism. This allows the infected cells to evade surveillance and elimination by cytotoxic lymphocytes and NK cells. The number of host cell molecular substrates reported for these ubiquitin ligases is limited. The identification of novel substrates for these ligases will aid in elucidating the mechanism underlying immune evasion of KSHV. This study demonstrated that K5 downregulated the cell surface expression of l-selectin, a C-type lectin-like adhesion receptor expressed in the lymphocytes. Tryptophan residue located at the centre of the E2-binding site in the K5 RINGv domain was essential to downregulate l-selectin expression. Additionally, the lysine residues located at the cytoplasmic tail of l-selectin were required for the K5-mediated downregulation of l-selectin. K5 promoted the degradation of l-selectin through polyubiquitination. These results suggest that K5 downregulates l-selectin expression on the cell surface by promoting polyubiquitination and ubiquitin-dependent endocytosis, which indicated that l-selectin is a novel substrate for K5. Additionally, K3 downregulated l-selectin expression. The findings of this study will aid in the elucidation of a novel immune evasion mechanism in KSHV.