Protein-mediated viral latency is a novel mechanism for Merkel cell polyomavirus persistence

Abstract

Viral latency, in which a virus genome does not replicate independently of the host cell genome and produces no infectious particles, is required for long-term virus persistence. There is no known latency mechanism for chronic small DNA virus infections. Merkel cell polyomavirus (MCV) causes an aggressive skin cancer after prolonged infection and requires an active large T (LT) phosphoprotein helicase to replicate. We show that evolutionarily conserved MCV LT phosphorylation sites are constitutively recognized by cellular Fbw7, βTrCP, and Skp2 Skp-F-box-cullin (SCF) E3 ubiquitin ligases, which degrade and suppress steady-state LT protein levels. Knockdown of each of these E3 ligases enhances LT stability and promotes MCV genome replication. Mutations at two of these phosphoreceptor sites [serine (S)220 and S239] in the full viral genome increase LT levels and promote MCV virion production and transmission, which can be neutralized with anti-capsid antibody. Virus activation is not mediated by viral gene transactivation, given that these mutations do not increase late gene transcription in the absence of genome replication. Mechanistic target of rapamycin inhibition by either nutrient starvation or use of an active site inhibitor reduces Skp2 levels and stabilizes LT, leading to enhanced MCV replication and transmission. MCV can sense stresses in its intracellular environment, such as nutrient loss, through SCF E3 ligase activities, and responds by initiating active viral transmission. Protein-mediated viral latency through cellular SCF E3 ligase targeting of viral replication proteins is a unique form of latency that may promote chronic viral persistence for some small DNA and RNA viruses.

Keywords: E3 ligase; Merkel cell polyomavirus; large T; latency; transmission.

Fig. 1.

LT is degraded by SCF E3 ligase recruitment. (A) Schematic diagram of MCV LT antigen (817 aa) with predicted Fbw7, Skp2, and βTrCP recognition phosphorylation sites. NetPhos 2.0 predicts 82 potential LT phosphorylation sites (59 Ser, 12 Thr, and 11 Tyr residues) exceeding a 0.5 threshold, including 15 minimal potential Fbw7/Skp2 (black circles) and βTrCP (white circles) recognition motifs. Nine of these are known phosphorylation sites (*) (–29). (B) LT alanine (A) substitution mutants at each of 15 potential Fbw7/Skp2 and βTrCP binding residues were generated and tested for stability by cycloheximide immunoblotting and replication efficiency using an MCV replicon assay. Testing was done in triplicate, and average values are shown. (C) Dose-dependent degradation of LT with increasing expression of Fbw7, βTrCP, and Skp2 SCF E3 ligases. LT was expressed in 293 cells with increasing plasmid amounts for each wild-type E3 ligase or its corresponding ubiquitylation-defective mutant (SI Appendix, Fig. S2). LT and tubulin were determined on the same blots; SCF proteins were determined on separate blots. (D) MCV LT interacts with each E3 ligase at specific phosphodegron sites. LT binding to each E3 ligase was assessed by immunoprecipitation. LT binding to βTrCP was lost with the S147A mutant, binding to Fbw7 was lost with the S239A mutant, and binding to Skp2 was lost with the S220A mutant. (E) SCF E3 ligase knockdown increases LT protein steady-state levels and replication of LT-dependent MCV origin replicon (mean ± SEM, n = 3).

Fig. 2.

SCF E3 ligase activity regulates MCV replication in 293 cells. (A) MCV genomes with SCF E3 ligase recognition site alanine substitution mutations at Fbw7 (S239)- or Skp2 (S220)- binding sites, or a double mutation (S220A and S239A), have increased replication (qPCR; mean ± SEM, n = 3) compared with wild-type (WT) or replication-defective (Rep−) genomes when transfected into 293 cells. Alanine substitution at the βTrCP-binding site (S147A) ablated virus replication. (B and C) MCV protein expression (B) and LT origin binding (by ChIP qPCR) (C) after genome transfection into 293 cells. VP1 and sT protein expression was detected only for viruses with Fbw7- or Skp2-binding site mutations. sT expression was prominent for the dual mutation virus MCV-HF_{LTS220A/S239A}. (D) MCV early and late gene transcription is replication-dependent. The MCV promoter region (nt 4928–195; GenBank accession no. EU375804) (20) was cloned into a bidirectional dual firefly (early) and Renilla (late) luciferase reporter, and promoter activity was measured by luciferase activity during cotransfection (0.2 μg) with wild-type or E3 ligase-binding mutant LT DNA plasmids (0.3 μg) into 293 cells. Relative luciferase activity was normalized to empty vector control (mean ± SEM, n = 3). Early gene reporter transcription was weakly activated by LT_S220A (by fivefold) or LT_S239A (by 1.4-fold) compared with wild-type LT (LT.wt) when the replication-competent reporter (Rep+) was used. LT_S147A repressed early transcription. When the replication-incompetent reporter (Rep−) was used, all cotransfected LT proteins suppressed early gene transcription to 10–20% 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 LT_S220A and LT_S239A. This increase was abolished for the replication-incompetent reporter, consistent with DNA template amplification being responsible for increased late gene expression.

Fig. 3.

mTOR inhibition increases MCV virus replication through Skp2 down-regulation. (A) 293 cells treated with the mTOR active site inhibitor PP242 (10 μM for 24 h) have down-regulated Skp2 mRNA levels compared with DMSO-treated control cells. Skp2 mRNA levels were standardized to GAPDH mRNA (RT qPCR, mean ± SEM, n = 3). (B) Levels of Skp2 protein expression decreased, whereas MCV LT protein expression increased, during PP242 treatment as in A. mTOR inhibition by serum starvation (24 h, 0.1% FCS) also increased LT and decreased Skp2 protein levels. This was reversed by serum replenishment (24 h, 10% FCS). (C) Serum starvation (24 h, 0.1% FCS) activates replication of MCV molecular clone (Methods). Viral DNA was determined by qPCR for MCV-HF and MCV-HF_Rep− and normalized to MCV-HF in 293 cells cultured with serum (mean ± SEM, n = 4). (D) mTOR inhibition by 10 μM PP242 stabilizes LT protein. LT stability was determined by quantitative cycloheximide immunoblotting. The graph plots LT expression relative to noncycloheximide-treated samples at time 0. n = 3, with a representative blot shown.

Fig. 4.

Inhibition of SCF E3 ligase targeting of LT causes a switch from latency to lytic MCV replication and permits virus transmission. (A) For transwell transmission assays, the integrity of transwell 0.4-μ membranes was monitored by placing eGFP-expressing 293 cells in the upper donor wells and untransfected 293 cells in the bottom receiver wells. (Upper) No fluorescence was detected among bottom recipient cells. (Lower) Phase-contrast microscopy. (B and C) Mutations to LT SCF E3 ligase recognition sites permit MCV encapsidation and transmission. Wild-type (MCV-HF), replication-deficient (MCV-HF_Rep−), or MCV-HF_{LTS220A/S239A} viruses were transfected into donor cells, and transmission to receiver cells was detected by LT (green) and capsid VP1 (red) immunofluorescence (n = 3, with representative result shown) (B) and by qPCR analysis (mean ± SEM, n = 3) (C). Virus DNA was determined per well. (D) MCV-HF_{LTS220A/S239A} replication in recipient cells induces apoptotic cell death. VP1⁺ virions (red) and LT (green) protein were detected in recipient cells. MCV-HF_{LTS220A/S239A} induced apoptotic nuclear fragmentation shown by DAPI (blue) staining at 24 h postinfection. (E) Transmissibility of MCV to cells in bottom of transwells was significantly increased by mTOR inhibition (serum starvation and PP242 treatment) compared with DMSO vehicle control. (F) MCV neutralization assay. Donor cells (Upper) and receiver cells (Lower) were seeded into the upper transwell insert and lower six-well plate, respectively. Receiver target cells were incubated for 24 h with VP1 antibody (CM9B2; 5 μg/mL), followed by collection for qPCR analysis. The relative infection rate was determined as the fold change qPCR inverse ΔΔCt values for MCV-HF and MCV-HF_LTS220A/239A in recipient cells (mean ± SEM, n = 4).

Fig. 5.

Model for MCV protein-mediated viral latency. MCV LT retains highly conserved phosphorylation sites recognized by cellular Fbw7, βTrCP, and Skp2 E3 ubiquitin ligases that cause degradation of LT, establishing viral latency. Cellular stresses, such as nutrient starvation, can reduce SCF E3 ligase activity, allowing LT accumulation to levels that permit assembly of the replication complex on the viral origin, which initiates virus DNA synthesis, capsid protein expression, and lytic replication. MCV sT protein, a replication accessory factor, enhances LT-dependent replication (17) by targeting SCF E3 ligases such as Fbw7 (22).