T antigen mutations are a human tumor-specific signature for Merkel cell polyomavirus

Abstract

Merkel cell polyomavirus (MCV) is a virus discovered in our laboratory at the University of Pittsburgh that is monoclonally integrated into the genome of approximately 80% of human Merkel cell carcinomas (MCCs). Transcript mapping was performed to show that MCV expresses transcripts in MCCs similar to large T (LT), small T (ST), and 17kT transcripts of SV40. Nine MCC tumor-derived LT genomic sequences have been examined, and all were found to harbor mutations prematurely truncating the MCV LT helicase. In contrast, four presumed episomal viruses from nontumor sources did not possess this T antigen signature mutation. Using coimmunoprecipitation and origin replication assays, we show that tumor-derived virus mutations do not affect retinoblastoma tumor suppressor protein (Rb) binding by LT but do eliminate viral DNA replication capacity. Identification of an MCC cell line (MKL-1) having monoclonal MCV integration and the signature LT mutation allowed us to functionally test both tumor-derived and wild type (WT) T antigens. Only WT LT expression activates replication of integrated MCV DNA in MKL-1 cells. Our findings suggest that MCV-positive MCC tumor cells undergo selection for LT mutations to prevent autoactivation of integrated virus replication that would be detrimental to cell survival. Because these mutations render the virus replication-incompetent, MCV is not a "passenger virus" that secondarily infects MCC tumors.

Fig. 1.

Transcript mapping of the multiply spliced MCV T antigen locus. (A) RACE analysis on MCC tumors shows four major MCV T antigen transcripts: T1 (LT, blue arrow), T2 (ST, yellow), T3 (ST, white), and T4 (17kT analog, red). Straight and dotted lines indicate noncoding and spliced regions, respectively. All four transcripts encode CR1 (pink, LXXLL) and DnaJ (light green, HPDKGG) domains. ST protein contains two PP2A binding motifs (white, CXCXXC). LT protein contains Rb binding (green, LXCXE), origin binding (red), zinc finger (yellow), leucine zipper (blue), and helicase (cyan)/ATPase (orange) motifs. The 17kT analog protein also encodes the Rb-binding motif. (B) Total RNA extracted from MCV T antigen-transfected 293 cells was Northern-hybridized to probes corresponding to mapped exons and introns (I–V in A). Each colored triangle indicates the corresponding transcript shown in A. An MCV339 deletion shortens TAg 339 T1 and T2 transcripts but is spliced out of T3 and T4 transcripts, which are similar in length to TAg350 and TAg206 T3 and T4 transcripts.

Fig. 2.

MCC-derived viruses have multiple mutations resulting in prematurely truncated LT protein. (A) Direct PCR sequencing of MCV LT antigen gene from control tissues (blue), MCC tissues (red), and MKL-1 cells (black) reveals LT mutations that prematurely truncate LT only in tumor-derived viruses. Circles are nucleotide polymorphisms observed among different MCV isolates (open, synonymous; pink, nonsynonymous; filled, premature stop codon). Multiple primers failed to amplify the 3′ portion of the MCV345 gene, suggesting a large deletion or integration site. Base-pair designations correspond to the prototype MCV350 genome (GenBank accession no. EU375803). (B) Diagrammatic representation of the MCV LT protein. Boxes show conserved CR1, DnaJ, and Rb-binding domains aligned against other human polyomaviruses and SV40. Arrows indicate premature stop codon mutation sites in tumor-derived viruses (● in A). (C) Both tumor-derived and nontumor-derived LT bind to Rb protein through a conserved LXCXE motif. V5-tagged WT TAg206.wt or tumor-derived TAg350 harboring LXCXE or its mutant LXCXK were cotransfected with HA-tagged Rb expression vector into 293 cells. Lysates were immunoprecipitated with antiV5 antibody and immunoblotted with antiHA antibody. Some 2.5% of the lysate was used as input control. SV40 LT antigen expression vectors with LXCXE or LXCXK were used as controls. Solid arrows show coprecipitated Rb protein with T antigens.

Fig. 3.

Identification of MCC line MKL-1 with monoclonal MCV-integration. (A) Uninfected variant (UISO, MCC26) MCC cells have a flattened, adherent morphology, whereas MCV-positive classic (MKL-1) MCC cells are rounded and loosely adherent. Phase-contrast images are at ×100 magnification. (B) Southern blot for MCV genomic DNA in cell lines (MCC352 tumor as positive control). Closed triangle (5.1 kb) represents viral genome in head-to-tail concatemers, and open triangles are viral-host fusion fragments. (C) Northern hybridization of UISO and MKL-1 total RNA with probes shown in Fig. 1A demonstrates expression of T1 (LT) and T2 (ST) transcripts in MLK-1 cells.

Fig. 4.

Initiation of MCV replication by WT but not tumor-derived LT. (A) T antigen expression vectors (TAg339, TAg350, and TAg206.wt) or pcDNA vector (Empty) were cotransfected with MCV replication origin plasmid (pMCV-Ori) in UISO and 293 cells, and replication was detected by Southern blotting. The positions of replicated DNA (DpnI-resistant) and unreplicated DNA (DpnI-sensitive) are indicated by solid and open arrows, respectively. (B) SV40 LT expression vector and SV40 origin plasmid were used as a positive control for the replication assay. Neither MCV nor SV40 LT initiates replication from each others' viral origin. (C) Southern blot of MKL-1 cells for MCV VP1 gene shows WT 206LT, but not tumor-derived 339LT or 350LT, induces endogenous integrated MCV DNA replication (arrow) after transfection. MCC26 was used as negative control for this replication assay. EtBr-stained agarose gel is shown as a loading control. (D) MCV integration into host chromosomes can be expected to lead to autonomous viral origin DNA replication when WT T antigen is expressed. Newly replicated virus DNA strands may collide with cellular replication forks unless secondary mutations eliminate viral LT antigen helicase activity.