JC Virus Small t Antigen Binds Phosphatase PP2A and Rb Family Proteins and Is Required for Efficient Viral DNA Replication Activity

BACKGROUND: The human polyomavirus, JC virus (JCV) produces five tumor proteins encoded by transcripts alternatively spliced from one precursor messenger RNA. Significant attention has been given to replication and transforming activities of JCV's large tumor antigen (TAg) and three T' proteins, but little is known about small tumor antigen (tAg) functions. Amino-terminal sequences of tAg overlap with those of the other tumor proteins, but the carboxy half of tAg is unique. These latter sequences are the least conserved among the early coding regions of primate polyomaviruses. METHODOLOGY AND FINDINGS: We investigated the ability of wild type and mutant forms of JCV tAg to interact with cellular proteins involved in regulating cell proliferation and survival. The JCV P99A tAg is mutated at a conserved proline, which in the SV40 tAg is required for efficient interaction with protein phosphatase 2A (PP2A), and the C157A mutant tAg is altered at one of two newly recognized LxCxE motifs. Relative to wild type and C157A tAgs, P99A tAg interacts inefficiently with PP2A in vivo. Unlike SV40 tAg, JCV tAg binds to the Rb family of tumor suppressor proteins. Viral DNAs expressing mutant t proteins replicated less efficiently than did the intact JCV genome. A JCV construct incapable of expressing tAg was replication-incompetent, a defect not complemented in trans using a tAg-expressing vector. CONCLUSIONS: JCV tAg possesses unique properties among the polyomavirus small t proteins. It contributes significantly to viral DNA replication in vivo; a tAg null mutant failed to display detectable DNA replication activity, and a tAg substitution mutant, reduced in PP2A binding, was replication-defective. Our observation that JCV tAg binds Rb proteins, indicates all five JCV tumor proteins have the potential to influence cell cycle progression in infected and transformed cells. It remains unclear how these proteins coordinate their unique and overlapping functions

Mutation of tAg residues proline 99 and cysteine 157 reduce JCV DNA replication.

<p>PHFG cells in 60 mm plates were transfected in duplicate with 400 ng of wild type (T⁺/t⁺/T'⁺) or 8 different mutant (muT⁺/t⁻/T'⁺, T⁺/P99At⁺/T'⁺, T⁺/C157At⁺/T'⁺, T⁺/t⁺/T'⁻, T⁺/t⁻/T'⁻, muT⁺/t⁺/T'⁺, T⁻/t⁺/T'⁻, T⁺/t⁻/T'⁺) JCV genomes. Duplicate, independent samples representing each DNA construct were extracted by the method of Hirt <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010606#pone.0010606-Hirt1" target="_blank">[39]</a> on days 0, 7, 10 and 14 p.t. and analyzed using the Dpn1 assay as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010606#pone-0010606-g005" target="_blank">Figure 5</a>. The marker (M) shown in the first lane of each blot is 1 ng of linear JCV DNA (5130 bp), and the position of <i>Dpn</i>1-resistent replicating genomes is denoted by an arrow at days 7, 10 and 14 p.t.</p

Replication efficiencies of DNA constructs expressing combinations of JCV early proteins.

<p>PHFG cells in 60 mm plates were transfected in duplicate with 400 ng of wild type (T⁺/t⁺/T'⁺) or mutant (T⁺/t⁻/T'⁻, muT⁺/t⁺/T'⁺, T⁻/t⁺/T'⁻, T⁺/t⁻/T'⁺, or muT⁺/t⁻/T'⁺) JCV genomes. At each time point, viral DNA was extracted by the Hirt procedure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010606#pone.0010606-Hirt1" target="_blank">[39]</a>, purified, digested with <i>Dpn</i>I and <i>Eco</i>RI, electrophoresed on a 0.8% agarose gel, transferred to nitrocellulose membranes and hybridized to a [P³²]dCTP-labeled JCV DNA probe. Bands were visualized with a Typhoon phosphorimager and quantitated with ImageQuant software. The marker (M) shown in the first lane of each blot is 1 ng of linear JCV DNA (5130 bp). The duplicate, independent samples representing each DNA construct were analyzed, and the position of <i>Dpn</i>1-resistent replicating genomes on the Southern blots is denoted by an arrow at days 10, 14 and 21 p.t. <i>Dpn</i>1- and <i>Eco</i>RI-sensitive input DNAs are noted at the day 0 time point. Hirt extracts of uninfected PHFG cells (PHFG) were subjected to the same Dpn1 assay.</p

JCV tAg fails to complement replication of a tAg-deficient JCV genome in <i>trans</i>.

<p>PHFG cells in 60 mm plates were co-transfected in duplicate with 400 ng of a tAg-deficient (T⁺/t⁻/T'⁺) JCV genome and 400 ng of a JCV DNA construct expressing either wild type tAg (T⁻/t⁺/T′⁻) or a J domain mutant tAg (T⁻/H42Qt⁺/T′⁻) under the control of the JCV promoter-enhancer. Cells were also transfected with 400 ng of either a T′-deficient (T⁺/t⁺/T'⁻) JCV genome or a positive replication control (T⁺/t⁺/T'⁺). Duplicate, independent samples representing each DNA construct were extracted by the method of Hirt <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010606#pone.0010606-Hirt1" target="_blank">[39]</a> on days 0, 7, and 10 p.t. and analyzed using the Dpn1 assay as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010606#pone-0010606-g005" target="_blank">Figure 5</a>. The marker shown in the first lane of each blot is 1 ng of linear JCV DNA (5130 bp), and the position of <i>Dpn</i>1-resistent replicating genomes is denoted by an arrow at days 7 and 10 p.t. <i>Dpn</i>1- and <i>Eco</i>RI-sensitive input DNAs are noted at the day 0 time point.</p

Interaction of mutant JCV tAgs with PP2A.

<p>Interactions between PP2A and wild-type tAg and (A) tAg-mutant P99A or (B) C157A were compared. 3T3 cells were stably transfected with DNA constructs expressing JCV early proteins under the control of SV40 promoter-enhancer signals. Lysates of these cells were subjected to IP with the anti-T monoclonal antibody PAb 962 (α-T) or anti-PP2A antibody (α-PP2A). The amount of total cell protein subjected to IP with anti-PP2A antibody (lanes 5–8, Panel A; lanes 6–10, Panel B) was five times that used with the anti-T antibody (lanes 1–4, Panel A; lanes 1–5, Panel B). Samples were electrophoresed on 18% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. WB was performed using a cocktail of anti-T monoclonal antibodies. Results for two independently-derived cells lines (#1, #2) containing DNA constructs that either encode all 5 JCV wild type early proteins (T⁺/t⁺/T′⁺; Panel A, lanes 1,2,5,6, Panel B, lanes 1,2,6,7) or wild type TAg, T′₁₆₅, T′₁₃₆ and T′₁₃₅ plus tAg mutant P99A (T⁺/P99At⁺/T′⁺; Panel A, lanes 3,4,7,8) or C157A (T⁺/C157At⁺/T′⁺; Panel B, lanes 3,4,8,9) are shown. A single 3T3 cell line expressing tAg only (T⁻/t⁺/T′⁻) was isolated and tested for PP2A binding (Panel B, lanes 5, 10). Panels A and B of this figure each represent proteins electrophoresed on a single gel and transferred to a membrane, which was then cut in half and each half developed for different lengths of time.</p

Comparison of JCV and SV40 tAg amino acid sequences.

<p>The JCV tAg sequence containing 172 amino acids is indicated in red letters, and the 174 amino acid SV40 tAg is shown in black letters. The sequences following the vertical line comprise the unique C-terminal region of each protein. Two conserved cysteine (CxCxxC) clusters (Cluster 1, 2) that contribute to SV40 tAg binding to zinc ions, and possibly to PP2A, are noted in both sequences, as are two recently recognized LxCxE motifs in JCV tAg. Three amino acid residues are highlighted with a blue letter and arrow. Histidine 42 (H42) is a conserved residue in the HPDKGG hexapeptide motif of the J domain that spans most of the N-terminal region of tAg as well as the other JCV and SV40 early proteins; H42 is required for functional interaction with Hsc70. Proline 99 is within a conserved CxxxPxC sequence that influences SV40 tAg binding to PP2A. Cysteine 157 is the central residue of the newly recognized second LxCxE motif in JCV tAg.</p

Wild type tAg interacts with cellular phosphatase PP2A in cells expressing JCV early proteins.

<p>JCV early proteins, expressed in Rat 2 (R2) or MEF cells transformed with pSR:T⁺/t⁺/T'⁺ or in G418-selected 3T3 cells transfected with pSR:T⁺/t⁺/T'⁺ (encodes all 5 JCV early proteins) or pSR:T⁻/t⁺/T'⁻ (encodes JCV tAg only) were incubated with anti-T monoclonal antibody PAb 962 (α-T; lanes 4–7) or anti-PP2A antibody (α-PP2A; lanes 9–12). The amount of total cell protein subjected to IP in lanes 9–12 was four times that employed in the corresponding samples in lanes 4–7. Immunoprecipitated proteins were separated on a 20% SDS-polyacrylamide gel, and WB analysis was performed using a cocktail of anti-T monoclonal antibodies to detect JCV early proteins either expressed in the different cell lines (lanes 4–7) or expressed and bound to PP2A (lanes 9–12). Untransfected 3T3 and Rat 2 cells were included as negative controls (no JCV T proteins are present; lanes 1, 2), and α-mouse IgG was used in the IP step with the R2:T⁺/t⁺/T'⁺ cell extract to test for non-specific binding (lanes 3, 8). The asterisks denote antibody light and heavy chains. This figure represents proteins electrophoresed on a single gel and transferred to a membrane, which was then cut in half and each half developed for different lengths of time.</p

Identification and Characterization of Mefloquine Efficacy against JC Virus In Vitro▿ †

Progressive multifocal leukoencephalopathy (PML) is a rare but frequently fatal disease caused by the uncontrolled replication of JC virus (JCV), a polyomavirus, in the brains of some immunocompromised individuals. Currently, no effective antiviral treatment for this disease has been identified. As a first step in the identification of such therapy, we screened the Spectrum collection of 2,000 approved drugs and biologically active molecules for their anti-JCV activities in an in vitro infection assay. We identified a number of different drugs and compounds that had significant anti-JCV activities at micromolar concentrations and lacked cellular toxicity. Of the compounds with anti-JCV activities, only mefloquine, an antimalarial agent, has been reported to show sufficiently high penetration into the central nervous system such that it would be predicted to achieve efficacious concentrations in the brain. Additional in vitro experiments demonstrated that mefloquine inhibits the viral infection rates of three different JCV isolates, JCV(Mad1), JCV(Mad4), and JCV(M1/SVEΔ), and does so in three different cell types, transformed human glial (SVG-A) cells, primary human fetal glial cells, and primary human astrocytes. Using quantitative PCR to quantify the number of viral copies in cultured cells, we have also shown that mefloquine inhibits viral DNA replication. Finally, we demonstrated that mefloquine does not block viral cell entry; rather, it inhibits viral replication in cells after viral entry. Although no suitable animal model of PML or JCV infection is available for the testing of mefloquine in vivo, our in vitro results, combined with biodistribution data published in the literature, suggest that mefloquine could be an effective therapy for PML

Longitudinal patterns of health-related quality of life and dialysis modality: a national cohort study

Abstract Background Health-related quality of life (HrQoL) varies among dialysis patients. However, little is known about the association of dialysis modality with HrQoL over time. We describe longitudinal patterns of HrQoL among chronic dialysis patients by treatment modality. Methods National retrospective cohort study of adult patients who initiated in-center dialysis or a home modality (peritoneal or home hemodialysis) between 1/2013 and 6/2015. Patients remained on the same modality for the first 120 days of the first two years. HrQoL was assessed by the Kidney Disease and Quality of Life-36 (KDQOL) survey in the first 120 days of the first two years after dialysis initiation. Home modality patients were matched to in-center patients in a 1:5 fashion. Results In-center (n=4234) and home modality (n=880) patients had similar demographic and clinical characteristics. In-center dialysis patients had lower mean KDQOL scores across several domains compared to home modality patients. For patients who remained on the same modality, there was no change in HrQoL. However, there were trends towards clinically meaningful changes in several aspects of HrQoL for patients who switched modalities. Specifically, physical functioning decreased for patients who switched from home to in-center dialysis (p< 0.05). Conclusions Among a national cohort of chronic dialysis patients, there was a trend towards different patterns of HrQoL life that were only observed among patients who changed modality. Patients who switched from home to in-center modalities had significant lower physical functioning over time. Providers and patients should be mindful of HrQoL changes that may occur with dialysis modality change