Merkel Cell Polyomavirus Large T Antigen Disrupts Host Genomic Integrity and Inhibits Cellular Proliferation

Abstract

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,...

Phosphorylation of Merkel Cell Polyomavirus Large Tumor Antigen at Serine 816 by ATM Kinase Induces Apoptosis in Host Cells

“…MCV LT Is Phosphorylated at Ser-816 -In our previous study, we found that either MCV infection or transfection of MCV genomes into cells activated both ATM and ATR kinases, whereas ectopic expression of only MCV LT primarily induced the ATR-Chk1 DDR pathway in U2OS cells (16). Interestingly, in these immunoblot experiments, we also discovered that the anti-phospho-Chk1 Ser-345 antibody not only detected phospho-Chk1 Ser-345 but also recognized protein bands with molecular masses that match those of transfected LT(1-817), LT(212-817), GFP-LT(441-817), and GFP-LT(1-817), respectively (Fig.…”

“…1B, asterisk). SV40 LT also activated phospho-Chk1 Ser-345 in U2OS cells (16), but there were no cross-reactive bands detected in the SV40 LT sample (Fig. 1A).…”

“…The identification of a tumor with intact full-length LT but a mutated viral origin sequence supports this hypothesis (26). Later studies have since suggested that the C-terminal helicase domain may contain other functions that oppose tumorigenesis (16,24). Our previous work indicated that expression of full-length MCV LT activates a dramatic DNA damage response (DDR) that is antagonistic to tumorigenesis; this activity activates p53 and induces a growthinhibiting phenotype (16).…”

“…Recombinant Plasmid Construction-Plasmids pcDNA-MCV LT(1-211), pcDNA4C-MCV LT(212-440), pcDNA4C-MCV LT(1-440), pcDNA4C-MCV LT(212-817), pcDNA4C-MCV LT(1-817), pEGFPC1-MCV LT(1-440), pEGFPC1-MCV LT(441-817), pEGFPC1-MCV LT(1-817), pcDNA4C-IIT-MCV LT(1-817), pLPCX-Cherry-LacI, pLPCX-MCV LT(1-817), and pGEX-MCV LT have been described previously (14,16). To generate pcDNA4C-MCV LT S816A, pEGFPC1-MCV LT S816A, or pLPCX-MCV LT S816A, site-directed mutagenesis was performed with QuikChange PCR (Stratagene) following the manufacturer's instructions using pcDNA4C-MCV LT(1-817), pEGFPC1-MCV LT(1-817), or pLPCX-MCV LT(1-817) as a template.…”

“…Later studies have since suggested that the C-terminal helicase domain may contain other functions that oppose tumorigenesis (16,24). Our previous work indicated that expression of full-length MCV LT activates a dramatic DNA damage response (DDR) that is antagonistic to tumorigenesis; this activity activates p53 and induces a growthinhibiting phenotype (16). Additionally, Cheng et al (24) reported that expression of the C-terminal 100 residues of MCV LT inhibits the growth of several different cell types.…”