Constitutive expression of simian virus 40 large T antigen in monkey cells activates their capacity to support polyomavirus replication

The regulatory DNA (enhancer) of polyomavirus (Py) is a major determinant of tissue-specific DNA replication during acute infection of newborn mice. Previously, we reported that the combination of one of the two Py enhancers (A enhancer) and the repeated Moloney murine leukemia virus (Mo-MuLV) enhancer gave a chimeric Py genome (Py-MuLV) that replicates predominantly in the acinar cells of the pancreas, a tissue not permissive for wild-type PyA2 replication (R. Rochford, B. A. Campbell, and L. P. Villareal, Proc. Nat. Acad. Sci. USA 84: [449][450][451][452][453] 1987). In this report, we further examine the combined enhancer requirements for acinar cell-specific Py replication. We also compare enhancer requirements for Py replication in the acinar cells of the pancreas with those of a transformed acinar cell line (266-6 cells). The deletion of sequences within the A enhancer of Py-MuLV (nucleotides 5098 to 5132) results in a virus with 10-fold-reduced levels of pancreasspecific replication. The deletion, however, of one of the 72-bp repeated Mo-MuLV enhancer sequences from Py-MuLV results in a complete loss of pancreas-specific DNA replication. Thus, the Py A enhancer is required for efficient replication of Py in the pancreas without otherwise altering organ specificity, but both of the repeated copies of the Mo-MuLV enhancer are essential for pancreas-specific Py replication. In contrast to the enhancer requirements for in vivo pancreas replication, in transformed acinar cells (266-6), PyA2 wild-type replicated efficiently and the Py-MuLV recombinant replicated inefficiently. These data suggest that the cellspecific control of DNA replication is different between normal pancreas cells and their transformed cell line counterparts and that this difference is apparent in the enhancer requirement of cell-specific Py DNA replication.

mentioning

confidence: 98%

Polyomavirus DNA replication in the pancreas and in a transformed pancreas cell line has distinct enhancer requirements

Rochford

Villarreal

1991

“…Yet, the NCCR defines a second level of host cell tropism after delivery of the viral genome into the nucleus, which is critical for coordinating and directing decisions regarding latency/persistence as well as progression through the viral life cycle (33). To experimentally overcome the NCCR bottleneck, researchers have resorted to viral recombinants carrying genomes with hybrid NCCRs, e.g., between SV40 and JCPyV (41,42), or have provided SV40 EVGR proteins like the LTag in trans (43)(44)(45)(46). To study the role of specific TFBS in archetype and rearranged HPyV NCCRs, we have chosen the archetype BKPyV NCCR as a model and introduced inactivating point mutations in 28 common TFBS (47).…”

mentioning

confidence: 99%

Novel Human Polyomavirus Noncoding Control Regions Differ in Bidirectional Gene Expression according to Host Cell, Large T-Antigen Expression, and Clinically Occurring Rearrangements

Ajuh

Kraus

et al. 2018

Human polyomavirus (HPyV) DNA genomes contain three regions denoted the early viral gene region (EVGR), encoding the regulatory T-antigens and one microRNA, the late viral gene region (LVGR), encoding the structural Vp capsid proteins, and the noncoding control region (NCCR). The NCCR harbors the origin of viral genome replication and bidirectional promoter/enhancer functions governing EVGR and LVGR expression on opposite DNA strands. Despite principal similarities, HPyV NCCRs differ in length, sequence, and architecture. To functionally compare HPyV NCCRs, sequences from human isolates were inserted into a bidirectional reporter vector using dsRed2 for EVGR expression and green fluorescent protein (GFP) for LVGR expression. Transfecting HPyV NCCR reporter vectors into human embryonic kidney 293 (HEK293) cells and flow cytometry normalized to archetype BKPyV NCCR revealed a hierarchy of EVGR expression levels with MCPyV, HPyV12, and STLPyV NCCRs conferring stronger levels and HPyV6, HPyV9, and HPyV10 NCCRs weaker levels, while LVGR expression was less variable and showed comparable activity levels. Transfection of HEK293T cells expressing simian virus 40 (SV40) large T antigen (LTag) increased EVGR expression for most HPyV NCCRs, which correlated with the number of LTag-binding sites (Spearman's , 0.625; < 0.05) and decreased following SV40 LTag small interfering RNA (siRNA) knockdown. LTag-dependent activation was specifically confirmed for two different MCPyV NCCRs in 293MCT cells expressing the cognate MCPyV LTag. HPyV NCCR expression in different cell lines derived from skin (A375), cervix (HeLaNT), lung (A549), brain (Hs683), and colon (SW480) demonstrated that host cell properties significantly modulate the baseline HPyV NCCR activity, which partly synergized with SV40 LTag expression. Clinically occurring NCCR sequence rearrangements of HPyV7 PITT-1 and -2 and HPyV9 UF1 were found to increase EVGR expression compared to the respective HPyV archetype, but this was partly host cell type specific. HPyV NCCRs integrate essential viral functions with respect to host cell specificity, persistence, viral replication, and disease. Here, we show that HPyV NCCRs not only differ in sequence length, number, and position of LTag- and common transcription factor-binding sites but also confer differences in bidirectional viral gene expression. Importantly, EVGR reporter expression was significantly modulated by LTag expression and by host cell properties. Clinical sequence variants of HPyV7 and HPyV9 NCCRs containing deletions and insertions were associated with increased EVGR expression, similar to BKPyV and JCPyV rearrangements, emphasizing that HPyV NCCR sequences are major determinants not only of host cell tropism but also of pathogenicity. These results will help to define secondary HPyV cell tropism beyond HPyV surface receptors, to identify key viral and host factors shaping the viral life cycle, and to develop preclinical models of HPyV persistence and replication and suitable antiviral targets.

“…Interestingly, a recent study has shown that polyomavirus DNA replication in monkey cells is not unconditionally limited. DNAs containing the polyomavirus origin replicate efficiently in monkey cells which constitutively express the SV40 large T-Ag (46).…”

Section: Discussionmentioning

confidence: 99%

Intrachromosomal recombination mediated by papovavirus large T antigens

St‐Onge

Bouchard

Laurent

et al. 1990

To investigate the mechanism by which the large T antigen (T-Ag) of polyomavirus and simian virus 40 can promote recombination in mammalian cells, we analyzed homologous recombination events occurring between two defective copies of the polyomavirus middle T (pmt) oncogene lying in close proximity on the same chromosome in a rat cell line. Reconstitution of a functional pmt gene by spontaneous recombination occurred at a rate of about 2 x 10-7 per cell generation. Introduction of the polyomavirus large T (plt) oncogene into the cell line by DNA transfection promoted recombination very efficiently, with rates in the range of 10-1 to 10-2 per cell generation. Recombination was independent of any amplification of viral sequences and could even be promoted by the large TAg from simian virus 40, which cannot activate polyomavirus DNA replication. To explain the role of large TAg , we propose a novel mechanism of nonconservative recombination involving slipped-strand mispairing between the two viral repeats followed by gap repair synthesis.