Bub1 in Complex with LANA Recruits PCNA To Regulate Kaposi's Sarcoma-Associated Herpesvirus Latent Replication and DNA Translesion Synthesis

Abstract

Latent DNA replication of Kaposi's sarcoma-associated herpesvirus (KSHV) initiates at the terminal repeat (TR) element and requires trans-acting elements, both viral and cellular, such as ORCs, MCMs, and latency-associated nuclear antigen (LANA). However, how cellular proteins are recruited to the viral genome is not very clear. Here, we demonstrated that the host cellular protein, Bub1, is involved in KSHV latent DNA replication. We show that Bub1 constitutively interacts with proliferating cell nuclear antigen (PCNA) via a highly conserved PIP box motif within the kinase domain. Furthermore, we demonstrated that Bub1 can form a complex with LANA and PCNA in KSHV-positive cells. This strongly indicated that Bub1 serves as a scaffold or molecular bridge between LANA and PCNA. LANA recruited PCNA to the KSHV genome via Bub1 to initiate viral replication in S phase and interacted with PCNA to promote its monoubiquitination in response to UV-induced damage for translesion DNA synthesis. This resulted in increased survival of KSHV-infected cells. IMPORTANCEDuring latency in KSHV-infected cells, the viral episomal DNA replicates once each cell cycle. KSHV does not express DNA replication proteins during latency. Instead, KSHV LANA recruits the host cell DNA replication machinery to the replication origin. However, the mechanism by which LANA mediates replication is uncertain. Here, we show that LANA is able to form a complex with PCNA, a critical protein for viral DNA replication. Furthermore, our findings suggest that Bub1, a spindle checkpoint protein, serves as a scaffold or molecular bridge between LANA and PCNA. Our data further support a role for Bub1 and LANA in PCNA-mediated cellular DNA replication processes as well as monoubiquitination of PCNA in response to UV damage. These data reveal a therapeutic target for inhibition of KSHV persistence in malignant cells. K aposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8, an oncogenic member of the gammaherpesvirus subfamily, was first identified in 1994 from body cavity-based lymphoma (BCBL) and HIV-infected patients with Kaposi's sarcoma (KS) (1). KSHV is believed to be the etiological agent of several human cancers, including KS (1, 2), primary effusion lymphoma (PEL) or body cavity based lymphoma (3), and multicentric Castleman's disease (MCD) (4, 5). Additionally, there have been reports of KSHV-associated solid lymphomas in HIV-positive and -negative patients as well as KSHV-associated lymphomas in patients with primary immune deficiencies, such as common variable immune deficiency (6, 7). Similar to other members of the gammaherpesvirus family, KSHV establishes a predominantly latent infection in host cells after primary infection (8). During latent infections, the KSHV genome persists as a circular doublestranded DNA (episome) with most viral genes being silenced, except for a small number of latent genes, which include the viral cyclin (v-cyclin), the latency-associated nuclear antigen (LANA), and the viral FLICE inhibitory prote...

KSHV Genome Replication and Maintenance

“…A recent study showed that siRNA mediated depletion of RFC ATPase compromised the interaction between RFC and LANA and directly affected LANA-mediated replication and episome maintenance (Moldovan et al, 2007 ). Similarly, it has been shown that cellular mitotic checkpoint kinase, Bub1 plays a significant role in LANA mediated recruitment of PCNA to the TR to initiate viral replication in S phase (Sun et al, 2015 ). Bub1 establishes a molecular complex with PCNA and LANA and was found to be critical to promote LANA mediated mono-ubiquitination of PCNA (Sun et al, 2015 ).…”

“…Similarly, it has been shown that cellular mitotic checkpoint kinase, Bub1 plays a significant role in LANA mediated recruitment of PCNA to the TR to initiate viral replication in S phase (Sun et al, 2015 ). Bub1 establishes a molecular complex with PCNA and LANA and was found to be critical to promote LANA mediated mono-ubiquitination of PCNA (Sun et al, 2015 ). Two more cellular proteins shown to be critical for KSHV genome replication and maintenance are the components of Timeless-dependent DNA replication fork protection complex, Tim and Tipin (Dheekollu et al, 2013 ).…”

Shugoshin 1 is dislocated by KSHV-encoded LANA inducing aneuploidy

“…Immunoprecipitation (IP) and Western blotting were performed as described previously [ 70 ]. Briefly, cells were collected and were lysed in lysis buffer (10 mM Tris, 1% NP-40, 2 mM EDTA, 150 mM NaCl [pH 7.5]) with protease inhibitors.…”

Major Histocompatibility Complex Class II HLA-DRα Is Downregulated by Kaposi's Sarcoma-Associated Herpesvirus-Encoded Lytic Transactivator RTA and MARCH8

“…Immunofluorescence assays (IFA) were performed as previously described (39). Briefly, the cells were fixed with 4% paraformaldehyde and 0.1% Triton X-100 for 15 to 20 min.…”

“…pCDNA3-RTA, PGEX-RTA, His-Uba1, HisUbc5a, and His-ubiquitin were described previously (39,40). The coding sequences (CDS) of MHC-II molecules were generated by using PCR and cloned into pA3M vector using the following primers: for HLA-DR␣, 5=-AT AAGAATGCGGCCGCCAGAGGCCCCCTGCGTTCTG-3= and 5=-CG GGATCCATGGCCATAAGTGGAGTCCC-3=; for HLA-DR␤, 5=-CGGGA TCCATGGTGTGTCTGAAGTTCCC-3= and 5=-ATAAGAATGCGGCC GCGCTCAGGAATCCTGTTGGCT-3=; for HLA-DP␣, 5=-CGGGATCC ATGCGCCCTGAAGACAGAAT-3= and 5=-ATAAGAATGCGGCCGCC AGGGTCCCCTGGGCCCGGG-3=; for HLA-DP␤, 5=-CGGGATCCATG ATGGTTCTGCAGGTTTC-3= and 5=-ATAAGAATGCGGCCGCTGCA GATCCTCGTTGAACTT-3=; for HLA-DQ␣, 5=-CGGGATCCATGATC ACATTCCTGCCGCT-3= and 5=-ATAAGAATCGGCCGCCAATGGCC CTTGGTGTCTGG-3=; for HLA-DQ␤, 5=-CCCAAGCTTATGTCTTGG AAGAAGGCTTT-3= and 5=-ATAAGAATGCGGCCGCGTGCAGAAGC CCTTTCTGAC-3=.…”