MCV sT is generated from alternative splicing of the T antigen encoding gene to form an iron-sulfur protein with an unclear function for the virus (20, 42). genomes when transfected into 293 cells. Alanine substitution at the TrCP-binding site (S147A) ablated virus replication. (and (late) luciferase reporter, and promoter activity was measured by luciferase activity during cotransfection (0.2 g) with wild-type or E3 ligase-binding mutant LT DNA plasmids (0.3 g) into 293 cells. Relative luciferase activity was normalized to empty vector control (mean SEM, = 3). Early gene reporter transcription was weakly activated by LTS220A (by fivefold) or LTS239A (by 1.4-fold) compared with wild-type LT (LT.wt) when the replication-competent reporter (Rep+) was used. LTS147A repressed early transcription. When the replication-incompetent reporter (Rep?) was used, all cotransfected LT proteins suppressed early gene transcription to 10C20% of empty vector control. Rep+ late gene expression was not significantly increased by wild-type LT protein coexpression but was increased by twofold to threefold by LTS220A and LTS239A. This increase was abolished for the replication-incompetent reporter, consistent with DNA template amplification being responsible for increased late gene expression. To measure the SCF E3 Bglap ligase effects on viral transcription, we generated a reporter containing the MCV NCCR in which the luciferase gene was substituted for VP2 at the late gene start site and the firefly luciferase gene was cloned in the opposite, reverse-sense direction at the MCV T antigen early gene start site (Fig. 2and = 3). (= 4). (= 3, with a representative blot shown. We surveyed 1-(3,4-Dimethoxycinnamoyl)piperidine MCV LT steady-state level responses to 1-(3,4-Dimethoxycinnamoyl)piperidine eight different kinase inhibitors. Treatment with the five PI-3K/Akt/mTOR pathway small-molecule inhibitors that down-regulate mTOR activity (LY294002, MK2206, Torin1, PP242, and rapamycin) significantly increased steady-state LT protein levels, whereas treatment with other kinase inhibitors (MEK1/2, CDK1/2, and GSK) did not (and = 3, with representative result shown) (= 3) (= 4). The receiver cells infected by MCV-HFLTS220A/S239A that became abundantly positive for capsid protein had DNA fragmentation and annexin A1 positivity typical for an apoptotic cytopathic effect occurring during lytic viral replication (Fig. 4and em SI Appendix /em , Fig. S7). The transmissibility of wild-type MCV was significantly increased when MCV-HFCtransfected 293 cells were treated with PP242 or were serum-starved (Fig. 4 em E /em ), consistent with Skp2 suppression resulting in replication permissivity. MCV transmission was neutralized by an anti-VP1 monoclonal antibody (Fig. 4 em F /em ), demonstrating 1-(3,4-Dimethoxycinnamoyl)piperidine that MCV transmission requires encapsidation, as would be expected for a productive polyomavirus infection. Discussion This study reveals that MCV relies on SCF E3 ligases to maintain latent persistence in cells as a nonreplicating viral plasmid. This is a different mechanism from herpesvirus and retrovirus latency, which rely on viral transactivator proteins to initiate lytic replication. The major MCV replication protein, LT, is continually transcribed and translated, but is also rapidly degraded by constitutive SCF E3 ligase activities in most cell lines so that LT levels do not surpass the threshold concentration required to assemble the multimeric LT helicase complex on the viral origin (Fig. 5). Furthermore, LT protein autoinhibits its own transcription, establishing a negative feedback loop that further buffers against LT accumulation to levels sufficient to activate viral genome replication. Nonetheless, point mutations that prevent Fbw7 or Skp2 recognition of LT allow the accumulation of LT protein, which can assemble on the origin to allow full genome MCV replication. We found no evidence indicating that LT protein specifically transactivates the transcription of late virion protein genes (VP1, VP2), but instead observed increased expression of these genes as the viral DNA genome was amplified by DNA replication. Open in a separate window Fig. 5. Model for MCV protein-mediated viral latency. MCV LT retains highly conserved phosphorylation sites recognized by.