Kaposi’s Sarcoma-Associated Herpesvirus K3 and K5 Proteins Down Regulate Both DC-SIGN and DC-SIGNR

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of multicentric Castleman’s disease, primary effusion lymphoma and Kaposi’s sarcoma. In this study, we show that like the C-type lectin DC-SIGN, the closely related DC-SIGNR can also enhance KSHV infection. Following infection, they are both targeted for down modulation and our data indicate that the KSHV MARCH-family ubiquitin ligase K5 is mediating this regulation and subsequent targeting for degradation of DC-SIGN and DC-SIGNR in the context of the virus. The closely related viral K3 protein, is also able to target these lectins in exogenous expressions studies, but only weakly during viral infection. In addition to requiring a functional RING-CH domain, several protein trafficking motifs in the C-terminal region of both K3 and K5 are important in regulation of DC-SIGN and DC-SIGNR. Further exploration of this modulation revealed that DC-SIGN is endocytosed from the cell surface in THP-1 monocytes, but degraded from an internal location with minimal endocytosis in HEK-293 cells. Pull-down data indicate that both K3 and K5 preferentially associate with immature forms of the lectins, mediating their ubiquitylation and degradation. Together, these data emphasize the molecular complexities of K3 and K5, while expanding the repertoire of targets of these two viral proteins.

Kaposi's Sarcoma-Associated Herpesvirus K3 and K5 Ubiquitin E3 Ligases Have Stage-Specific Immune Evasion Roles during Lytic Replication

“…The characterization of KSHV K3 and K5 deletion mutants confirmed the importance of K5 in surface receptor modulation, but surprisingly, no obvious changes in MHC-I surface expression were apparent in cells infected with K3-deficient viruses (47), indicating that K5, but not K3, plays a dominant role in the context of infected cells. Similar observations were made for KSHV-mediated downregulation of DC-SIGN and DC-SIGNR, two substrates that can be downregulated by either K3 or K5 (32).…”

“…Subsequent studies revealed an increasing number of K5 substrates, including the NKT cell ligand CD1d (25); the MHC-Irelated molecule HFE (26); the adhesion molecules ICAM-1 (27,28), PECAM (29), VE-cadherin (30), ALCAM (31), DC-SIGN, and DC-SIGNR (32); the costimulatory molecule B7-2 (27,28); the NK cell-activating ligands, MICA, MICB, and AICL (33); the cellular restriction factor tetherin (34); the cytokine receptor interferon gamma receptor 1 (IFN-␥R1) (35); the plasma membrane t-SNARE syntaxin-4 (36); and the transforming growth factor ␤ (TGF-␤) family member BMPRII (37). In addition, Timms et al recently reported several novel K5 substrates, including 8 verified targets, CD32, CD33, CD99, EPHB4, Plexin A1, PMZL2, Kit (CD117), and IL9R (CD129), and 66 potential new targets, all of which were identified via a quantitative mass spectrometry approach (38).…”

Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission

“…DC-SIGN-expressing HEK 293 (DC-HEK) cells, as reported by Lang et al (31) were grown in DMEM-F12 (Life Technologies, UK) containing 10% v/v FCS and blasticidin (5 µg/mL) (Gibco). The cells were grown on 13 mm glass cover slips till a monolayer of cells was formed and then incubated with 15 µg/mL of recombinant ghA, ghB, and ghC (MBP as a negative control) separately in serum free medium and left to incubate for 30 min in 37°C.…”

Molecular Biology of KSHV Lytic Reactivation

“…Furthermore, KSHV K3 and K5 (also called modulator of the immune recognition (MIR) 1 and 2, respectively) are viral E3 ligases capable of ubiquitinating the MHC-I cytoplasmic tail to trigger internalization and proteasomal degradation of the MHC-I complex [ 205 , 242 , 243 ]. K3 and K5 proteins also have been shown to downregulate both C-type lectins, DC-SIGN, and DC-SIGNR by ubiquitin mediated degradation [ 206 ]. Similarly, KSHV K6, K4, and K4.1 encode three homologues of cellular chemokines: viral CC-chemokine ligand 1 vCCL1(vMIP1), vCCL2 (vMIP2), and vCCL3 (vMIP3), respectively [ 154 , 244 , 245 ].…”