bClonal integration of Merkel cell polyomavirus (MCV) DNA into the host genome has been observed in at least 80% of Merkel cell carcinoma (MCC). The integrated viral genome typically carries mutations that truncate the C-terminal DNA binding and helicase domains of the MCV large T antigen (LT), suggesting a selective pressure to remove this MCV LT region during tumor development. In this study, we show that MCV infection leads to the activation of host DNA damage responses (DDR). This activity was mapped to the C-terminal helicase-containing region of the MCV LT. The MCV LT-activated DNA damage kinases, in turn, led to enhanced p53 phosphorylation, upregulation of p53 downstream target genes, and cell cycle arrest. Compared to the N-terminal MCV LT fragment that is usually preserved in mutants isolated from MCC tumors, full-length MCV LT shows a decreased potential to support cellular proliferation, focus formation, and anchorage-independent cell growth. These apparently antitumorigenic effects can be reversed by a dominant-negative p53 inhibitor. Our results demonstrate that MCV LT-induced DDR activates p53 pathway, leading to the inhibition of cellular proliferation. This study reveals a key difference between MCV LT and simian vacuolating virus 40 LT, which activates a DDR but inhibits p53 function. This study also explains, in part, why truncation mutations that remove the MCV LT C-terminal region are necessary for the oncogenic progression of MCV-associated cancers. Merkel cell polyomavirus (MCV) is the first polyomavirus to be clearly associated with cancer in humans (1). Its genome was recently found integrated into the chromosomes of a highly aggressive skin cancer, Merkel cell carcinoma (MCC) (2). Subsequent analyses of a large number of MCC tumors have revealed that this polyomavirus is associated with at least 80% of all MCC cases (2-4). Integrated MCV genome has also been detected in non-small-cell lung cancer (5). Epidemiological surveys for MCV seropositivity (6, 7) and sequencing analyses of healthy human skin (8) have indicated that MCV represents a common component of the human skin microbial flora.As with other polyomaviruses, the MCV genome contains an early region that encodes the viral tumor antigens. Differential splicing of the early mRNA produces large tumor antigen (LT), small tumor antigen (sT), and 57kT proteins (9, 10). The highly multifunctional LT protein is involved in a variety of processes, including initiation of viral genome replication, as well as manipulation of the host cell cycle through a number of protein-protein interactions. It has been shown that MCV LT interacts with at least some of the same cellular factors as simian virus 40 (SV40) LT (11). SV40 LT interacts with classic partners including heat shock protein 70 (Hsc70) through the LT DnaJ domain and also interacts with retinoblastoma "pocket protein" (Rb) family members through a classic LxCxE motif in the N-terminal region of LT. SV40 LT binding of Rb abrogates its role as a repressor of E2F transcription factors,...

bReplication of the human papillomavirus (HPV) DNA genome relies on viral factors E1 and E2 and the cellular replication machinery. Bromodomain-containing protein 4 (Brd4) interacts with viral E2 protein to mediate papillomavirus (PV) genome maintenance and viral transcription. However, the functional role of Brd4 in the HPV life cycle remains to be clearly defined. In this study, we provide the first look into the E2-Brd4 interaction in the presence of other important viral factors, such as the HPV16 E1 protein and the viral genome. We show that Brd4 is recruited to actively replicating HPV16 origin foci together with HPV16 E1, E2, and a number of the cellular replication factors: replication protein A70 (RPA70), replication factor C1 (RFC1), and DNA polymerase ␦. Mutagenesis disrupting the E2-Brd4 interaction abolishes the formation of the HPV16 replication complex and impairs HPV16 DNA replication in cells. Brd4 was further demonstrated to be necessary for HPV16 viral DNA replication using a cell-free replication system in which depletion of Brd4 by small interfering RNA (siRNA) silencing leads to impaired HPV16 viral DNA replication and recombinant Brd4 protein is able to rescue viral DNA replication. In addition, releasing endogenous Brd4 from cellular chromatin by using the bromodomain inhibitor JQ1(؉) enhances HPV16 DNA replication, demonstrating that the role of Brd4 in HPV DNA replication could be uncoupled from its function in chromatin-associated transcriptional regulation and cell cycle control. Our study reveals a new role for Brd4 in HPV genome replication, providing novel insights into understanding the life cycle of this oncogenic DNA virus. Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that replicate in differentiating cutaneous and mucosal epithelia (1). They are one of the most prevalent sexually transmitted pathogens in the world. High-risk HPVs are known etiological agents of cervical, anogenital, and head and neck cancers (2), with HPV16 being responsible for over 50% of cervical cancer cases worldwide (3-5).HPVs specifically infect basal epithelial cells. HPV genome replication occurs during two different stages of the viral life cycle. In the infected basal epithelial cells, the viral genomes replicate an average of once per cell cycle during S phase, in synchrony with the host DNA replication (6). This allows the viral genome to be maintained as stable episomes at 50 to 100 copies per cell. This stage of DNA replication ensures a persistent infection in the basal layer of the epidermis. Terminal differentiation of infected cells triggers vegetative viral DNA replication, producing viral genomes, which can then be assembled into virions and be released from the surface of differentiated epithelium (7).Replication of the HPV genome is carried out by viral E1 and E2 proteins in combination with various components of the cellular DNA replication machinery (7). E2 binds to several consensus E2 binding sites near the HPV origin of replication (Ori) and recruits E1 to the...

Merkel cell polyomavirus (MCV or MCPyV) is the first human polyomavirus to be definitively linked to cancer. The mechanisms of MCV-induced oncogenesis and much of MCV biology are largely unexplored. In this study, we demonstrate that bromodomain protein 4 (Brd4) interacts with MCV large T antigen (LT) and plays a critical role in viral DNA replication. Brd4 knockdown inhibits MCV replication, which can be rescued by recombinant Brd4. Brd4 colocalizes with the MCV LT/replication origin complex in the nucleus and recruits replication factor C (RFC) to the viral replication sites. A dominant negative inhibitor of the Brd4-MCV LT interaction can dissociate Brd4 and RFC from the viral replication complex and abrogate MCV replication. Furthermore, obstructing the physiologic interaction between Brd4 and host chromatin with the chemical compound JQ1(+) leads to enhanced MCV DNA replication, demonstrating that the role of Brd4 in MCV replication is distinct from its role in chromatin-associated transcriptional regulation. Our findings demonstrate mechanistic details of the MCV replication machinery; providing novel insight to elucidate the life cycle of this newly discovered oncogenic DNA virus.

SUMMARY The intracellular microbial nucleic acid sensors, TLR3 and STING, recognize pathogen molecules and signal to activate the interferon pathway. The TIR-domain containing protein TRIF is the sole adaptor of TLR3. Here we report an essential role for TRIF in STING signaling: various activators of STING could not induce genes in the absence of TRIF. TRIF and STING interacted directly, through their carboxyl terminal domains, to promote STING dimerization, intermembrane translocation and signaling. Herpes simplex virus (HSV), which triggers the STING signaling pathway and is controlled by it, replicated more efficiently in the absence of TRIF and HSV-infected TRIF−/− mice displayed pronounced pathology. Our results indicate that defective STING signaling may be responsible for the observed genetic association between TRIF mutations and Herpes Simplex Encephalitis in patients.

IMPORTANCEMCPyV is the first polyomavirus to be clearly associated with human cancer. However, the MCPyV life cycle and its oncogenic mechanism remain poorly understood. In this report, we show that, in cells infected with native MCPyV virions, components of the ATM-and ATR-mediated DDR pathways accumulate in MCPyV LT-positive nuclear foci. Such a phenotype was recapitulated using our previously established system for visualizing MCPyV replication complexes in cells. By combining immunofluorescent staining, fluorescence in situ hybridization, and BrdU incorporation analysis, we demonstrate that DDR proteins are important for maintaining robust MCPyV DNA replication. This study not only provides the first look into the microscopic details of DDR factor/LT replication complexes at the MCPyV origin but also provides a platform for further studying the mechanistic role of host DDR factors in the MCPyV life cycle and virus-associated oncogenesis.