Characterization of functional domains in the Merkel cell polyoma virus Large T antigen

Abstract

Merkel cell polyomavirus (MCPyV)-positive Merkel cell carcinoma (MCC) tumor cell growth is dependent on the expression of a viral Large T antigen (LT) with an intact retinoblastoma protein (RB)-binding site. This RB-binding domain in MCPyV-LT is-in contrast to other polyomavirus LTs (e.g., SV40)-embedded between two large MCPyV unique regions (MUR1 and MUR2). To identify elements of the MCPyV-LT necessary for tumor cell growth, we analyzed the rescue activity of LT variants following knockdown of the endogenous LT in MCC cells. These experiments demonstrate that nuclear localization is essential for LT function, but that a motif previously described to be a nuclear localization sequence is neither required for nuclear accumulation of truncated MCPyV-LT nor for promotion of MCC cell proliferation. Furthermore, large parts of the MURs distal to the RB binding domain as well as ALTO-a second protein encoded by an alternative reading frame in the MCPyV-LT mRNA-are completely dispensable for MCPyV-driven tumor cell proliferation. Notably, even MCPyV-LTs in which the entire MURs have been removed are still able to promote MCC cellular growth although rescue activity is reduced which may be due to MUR1 being required for stable LT expression in MCC cells. Finally, we provide evidence implying that-while binding to Vam6p is not essential-HSC-70 interaction is significantly involved in mediating MCPyV-LT function in MCC cells including growth promotion and induction of E2F target genes.Polyomaviruses are small DNA viruses with a circular double-stranded genome coding for 5-9 proteins. 1 The genome is divided into an early and a late region. From the early region the T antigens (TA) are derived by differential splicing of a common transcript while the late region encodes for capsid proteins into which the viral genome is packaged. 2 It has long been known that polyomaviruses-as the name indicates-can induce tumor formation. 3 Until recently, however, this had only been observed in animal models whiledespite extensive research and controversial discussion-no convincing evidence for a contribution of the Simian Virus 40 (SV40), a monkey polyomavirus that is also present in the human population, or the two well-studied human polyomaviruses JC and BK to human malignancies could be obtained. 4,5 In contrast, one of the seven newly identified human polyomaviruses discovered during the last 6 years, the Merkel cell polyomavirus (MCPyV), is now well established as a causal factor of Merkel cell carcinoma (MCC). 2,4,6 MCC is a rare but very aggressive skin cancer with high mortality rates. Immunosuppression, UV exposure and old age are risk factors for developing MCC. 7 The molecular pathogenesis of MCC, however, remained enigmatic until MCPyV was discovered as monoclonally integrated into the genome of most MCCs indicating that integration occurred prior to clonal expansion of the tumor cells. 8 In many different countries the presence of MCPyV in the vast majority of MCC tumors has been confirmed, 9-12 and a recent publication s...

RB1 is the crucial target of the Merkel cell polyomavirus Large T antigen in Merkel cell carcinoma cells

“…The only exception was the cell line LoKe for which no RB1 expression could be detected. Notably, LoKe, although encoding a functional truncated MCPyV-LT [20], is up to date the only MCPyV-positive MCC cell line tested which is not dependent on LT expression for cell growth [21]. Immunoblot analysis confirmed the expression of all PPs in all other cell lines as well as the lack of RB1 expression in LoKe (Figure 1b).…”

“…First, the TA shRNA induced growth arrest can be rescued to the same extent by an LT cDNA as by a TA gene (coding for sT and LT) indicating that with this experimental system we evaluate only LT functions although the applied TA shRNA also targets sT [20, 22]. Second, in the cell line WaGa the rescue by TA or LT is incomplete demonstrating a similar rescue activity as achieved by RB1 knockdown (Figure 2; [20, 22]). Hence, it is likely that the TA shRNA exerts growth inhibiting off-target effects in WaGa.…”

Large T and small T antigens of Merkel cell polyomavirus

“…The short NLS of MCV LT that lies between these two regions (277–280 aa) was initially believed to also be conserved in tumors [29], but recent evidence indicates that the NLS is not preferentially preserved in MCC [44,47]. In the cases where the NLS is eliminated by tumorspecific mutations to LT (as in cell lines MCC350, MCCL-11, and MCCL-12) nuclear localization is not lost, but rather both nuclear and cytoplasmic distribution are observed [44].…”

Epidemiology, biology and therapy of Merkel cell carcinoma: conclusions from the EU project IMMOMEC

“…A recent study aiming at the identification of the cellular interaction partner(s) for the PP-binding domain of LT identified the three PP family members RB1, p107 and p130 as the main candidates. Knockdown of these individually demonstrated that knockdown of RB1 alone was sufficient to rescue the effect of LT knockdown-mediated cellular growth impairment [44] (Fig. 1).…”