Kaposi's Sarcoma-Associated Herpesvirus ori - Lyt -Dependent DNA Replication: Involvement of Host Cellular Factors

Abstract

Herpesvirus lytic DNA replication requires both the cis-acting element, the origin, and trans-acting factors, including virally encoded origin-binding protein, DNA replication enzymes, and auxiliary factors. Two lytic DNA replication origins (ori-Lyt) of Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified, and two virally encoded proteins, namely, RTA and K8, have been shown to bind to the origins. In this study, we sought to identify cellular factors that associate with ori-Lyt by using DNA affinity purification and mass spectrometry. This approach led to identification of several cellular proteins that bind to KSHV ori-Lyt. They include topoisomerases (Topo) I and II, MSH2/6, RecQL, poly(ADP-ribose) polymerase I (PARP-1), DNA-PK, Ku86/70 autoantigens, and scaffold attachment factor A (SAF-A). RecQL appears to associate with prereplication complexes and be recruited to ori-Lyt through RTA and K8. Topoisomerases, MSH2, PARP-1, DNA-PK, and Ku86 were not detected in prereplication complexes but were present in replication initiation complexes on ori-Lyt. All these cellular proteins accumulate in viral replication compartments in the nucleus, indicating that these proteins may have a role in viral replication. Topo I and II appear to be essential for viral DNA replication as inhibition of their activities with specific inhibitors (camptothecin and ellipticine) blocked ori-Lyt-dependent DNA replication. Furthermore, inhibition of PARP-1 with chemical inhibitors (3-aminobenzamide and niacinamide) resulted in decreased ori-Lyt-dependent DNA replication, whereas hydroxyurea, which raises PARP-1 activity, caused an increase in the DNA replication, suggesting a positive role for PARP-1 in KSHV lytic DNA replication.Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8, is the etiologic agent of several AIDS-associated malignancies, including Kaposi's sarcoma (KS), primary effusion lymphoma, and multicentric Castleman's disease (1,17,33). In KS lesions, most spindle cells of endothelial origin are latently infected with KSHV, and a small percentage of these cells undergo spontaneous lytic replication (34,35,43). Increasing evidence suggests that the small percentage of cells experiencing viral lytic replication play important roles in viral pathogenicity. Treatment of KS patients or AIDS patients at risk for KS with antiherpesviral drugs, like foscarnet and ganciclovir, which are known to block lytic DNA replication, results in regression of KS lesions or a decease in the incidence of KS development. The lytic replication cycle directly contributes to viral tumorigenesis by spreading viruses to target cells and by providing paracrine regulation for KS development (11). A recent study by Grundhoff and Ganem (19) suggests a new role for lytic replication in sustaining the population of latently infected cells that otherwise would be quickly lost by segregation of latent viral episomes as spindle cells divide. Thus, unlike with other oncogenic viruses, where the latent life c...

Identification of Rep-Associated Factors in Herpes Simplex Virus Type 1-Induced Adeno-Associated Virus Type 2 Replication Compartments

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Tpl2/AP-1 Enhances Murine Gammaherpesvirus 68 Lytic Replication

“…For example, several cellular genes have been shown to mediate KSHV entry in different types of cells (1,31,46). Other cellular genes, such as those for topoisomerase I, topoisomerase II, and poly(ADP-ribose) polymerase 1 (PARP-1), were shown to function during KSHV lytic DNA replication (58).…”

Enhancement of Zta-activated lytic transcription of Epstein-Barr virus by Ku80

“…Earlier studies have shown the binding of DNA-PK and Ku70-Ku80 to the replication origins of herpes simplex virus type 1 (HSV-1) and Kaposi's sarcoma-associated herpesvirus (KSHV) (Taylor & Knipe, 2004;Wang et al, 2008), indicating that Ku70-Ku80 is critically involved in the replication of these two viruses. This study finds that Ku80 binds to the three ZRE in the BHLF1 promoter, which overlaps with oriLyt.…”

“…Furthermore, Zta is known to recruit basal transcription factors, including TFIID and TFIIA, to promoters by binding to ZRE (Lieberman & Berk, 1994). Accordingly, Zta and Ku80 may cooperate with each other to activate the transcription of EBV lytic genes.Earlier studies have shown the binding of DNA-PK and Ku70-Ku80 to the replication origins of herpes simplex virus type 1 (HSV-1) and Kaposi's sarcoma-associated herpesvirus (KSHV) (Taylor & Knipe, 2004;Wang et al, 2008), indicating that Ku70-Ku80 is critically involved in the replication of these two viruses. This study finds that Ku80 binds to the three ZRE in the BHLF1 promoter, which overlaps with oriLyt.…”