Single-Cell Transcriptome Identifies the Renal Cell Type Tropism of Human BK Polyomavirus.
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SUBMITTER: Yang F
PROVIDER: S-EPMC9861348 | biostudies-literature | 2023 Jan
REPOSITORIES: biostudies-literature
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Single-Cell Transcriptome Identifies the Renal Cell Type Tropism of Human BK Polyomavirus.
Yang Feng F Chen Xutao X Zhang Hui H Zhao Guo-Dong GD Yang Huifei H Qiu Jiang J Meng Siyan S Wu Penghan P Tao Liang L Wang Qin Q Huang Gang G
International journal of molecular sciences 20230110 2
BK polyomavirus (BKPyV) infection is the main factor affecting the prognosis of kidney transplant recipients, as no antiviral agent is yet available. A better understanding of the renal-cell-type tropism of BKPyV can serve to develop new treatment strategies. In this study, the single-cell transcriptomic analysis demonstrated that the ranking of BKPyV tropism for the kidney was proximal tubule cells (PT), collecting duct cells (CD), and glomerular endothelial cells (GEC) according to the signatu ...[more]
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Project description:BK polyomavirus (BKPyV) contributes to premature renal failure in 10%-20% of kidney transplant recipients. Current treatment relies on reducing immunosuppression to regain BKPyV-specific immune control. Subsequently, declining allograft function may result from persisting viral cytopathology, BKPyV-specific immune reconstitution, or alloimmunity/rejection, all being poorly distinguishable by current histological or molecular approaches. To reduce the complexity encountered in BKPyV-replicating kidneys, we analyzed differentially expressed genes (DEGs) in primary human renal proximal tubular epithelial cells at 24 and 48 h post-infection (hpi) using single-cell RNA-sequencing (10x-Genomics-3´ kit). At 24 hpi, viral transcript reads predominantly mapped to the early viral gene region (EVGR) and shifted to >100-fold higher late viral gene region (LVGR) levels at 48 hpi, matching the sequential bi-directional viral protein expression from the circular double-stranded BKPyV-DNA genome. Besides expected coverage "hills" at viral 3´-poly-A sites, unexpected "spike" and "pulse" reads resulted from off-target TSO priming. "Spike" and "pulse" patterns were rare for the mostly unidirectional reads mapping to the circular mitochondrial genome. Bioinformatic curation removed "spikes" and "pulses" and reclassified 10% of DEGs in renal proximal tubular epithelial cells (RPTECs). Up-regulated gene ontologies included S and G2/M phase, double-stranded DNA repair, proximal tubulopathy, and renal tubular dysfunction, whereas allograft rejection, antigen presentation, innate immunity, translation, and autophagy were down-regulated. BKPyV-LVGR expression induced a novel mitochondrial cell stress pattern consisting of discordant up-regulation and down-regulation of mitochondria-encoded and nucleus-encoded mitochondrial genes, respectively. We explored which top-scoring gene sets of late-phase BKPyV-replicating RPTECs can identify BKPyV-associated nephropathy in kidney transplant biopsies. The results should facilitate distinguishing BKPyV-associated pathology from other entities in kidney transplant biopsies.IMPORTANCEBK polyomavirus (BKPyV) infects more than 90% of the general population and then persists in the reno-urinary tract. Subsequently, low-level urinary shedding is seen in 10% of healthy BKPyV-seropositive persons, indicating that BKPyV replication occurs despite the presence of virus-specific cellular and humoral immunity. Notably, transplantation of donor kidneys with low-level BKPyV replication is a risk factor for progression to high-level BKPyV viruria, new-onset BKPyV-DNAemia and biopsy-proven BKPyV nephropathy. Here, we identify a short list of robust up- and down-regulated nucleus-encoded differentially expressed genes potentially allowing to discriminate viral from allograft immune damage. By carefully curating viral and mitochondrial transcriptomes, we identify a novel virus-associated mitochondrial stress pattern of up-regulated mitochondria-encoded and down-regulated nucleus-encoded mitochondrial transcripts which heralds the BKPyV-agnoprotein-mediated immune escape by breakdown of the mitochondrial membrane potential and network and mitophagy. The results may prove useful when assessing the role of BKPyV replication in kidney transplant patients with suspected acute rejection and/or BKPyV nephropathy.
| S-EPMC11657676 | biostudies-literature
Project description:BK polyomavirus (BKV) causes significant urinary tract pathogenesis in immunosuppressed individuals, including kidney and bone marrow transplant recipients. It is currently unclear whether BKV-neutralizing antibodies can moderate or prevent BKV disease. We developed reporter pseudoviruses based on seven divergent BKV isolates and performed neutralization assays on sera from healthy human subjects. The results demonstrate that BKV genotypes I, II, III, and IV are fully distinct serotypes. While nearly all healthy subjects had BKV genotype I-neutralizing antibodies, a majority of subjects did not detectably neutralize genotype III or IV. Surprisingly, BKV subgenotypes Ib1 and Ib2 can behave as fully distinct serotypes. This difference is governed by as few as two residues adjacent to the cellular glycan receptor-binding site on the virion surface. Serological analysis of mice given virus-like particle (VLP)-based BKV vaccines confirmed these findings. Mice administered a multivalent VLP vaccine showed high-titer serum antibody responses that potently cross-neutralized all tested BKV genotypes. Interestingly, each of the neutralization serotypes bound a distinct spectrum of cell surface receptors, suggesting a possible connection between escape from recognition by neutralizing antibodies and cellular attachment mechanisms. The finding implies that different BKV genotypes have different cellular tropisms and pathogenic potentials in vivo. Individuals who are infected with one BKV serotype may remain humorally vulnerable to other BKV serotypes after implementation of T cell immunosuppression. Thus, prevaccinating organ transplant recipients with a multivalent BKV VLP vaccine might reduce the risk of developing posttransplant BKV disease.
| S-EPMC3754014 | biostudies-literature
Project description:Most humans have a lifelong imperceptible BK Polyomavirus (BKPyV) infection in epithelial cells lining the reno-urinary tract. In kidney transplant recipients, unrestricted high-level replication of donor-derived BKPyV in the allograft underlies polyomavirus-associated nephropathy, a condition with massive epithelial cell loss and inflammation causing premature allograft failure. There is limited understanding on how BKPyV disseminates throughout the reno-urinary tract and sometimes causes kidney damage. Tubule epithelial cells are tightly connected and have unique apical and basolateral membrane domains with highly specialized functions but all in vitro BKPyV studies have been performed in non-polarized cells. We therefore generated a polarized cell model of primary renal proximal tubule epithelial cells (RPTECs) and characterized BKPyV entry and release. After 8 days on permeable inserts, RPTECs demonstrated apico-basal polarity. BKPyV entry was most efficient via the apical membrane, that in vivo faces the tubular lumen, and depended on sialic acids. Progeny release started between 48 and 58 hours post-infection (hpi), and was exclusively detected in the apical compartment. From 72 hpi, cell lysis and detachment gradually increased but cells were mainly shed by extrusion and the barrier function was therefore maintained. The decoy-like cells were BKPyV infected and could transmit BKPyV to uninfected cells. By 120 hpi, the epithelial barrier was disrupted by severe cytopathic effects, and BKPyV entered the basolateral compartment mimicking the interstitial space. Addition of BKPyV-specific neutralizing antibodies to this compartment inhibited new infections. Taken together, we propose that during in vivo low-level BKPyV replication, BKPyV disseminates inside the tubular system, thereby causing minimal damage and delaying immune detection. However, in kidney transplant recipients lacking a well-functioning immune system, replication in the allograft will progress and eventually cause denudation of the basement membrane, leading to an increased number of decoy cells, high-level BKPyV-DNAuria and DNAemia, the latter a marker of allograft damage.
| S-EPMC10491296 | biostudies-literature
Project description:PurposeTo construct a dynamic prediction model for BK polyomavirus (BKV) reactivation during the early period after renal transplantation and to provide a statistical basis for the identification of and intervention for high-risk populations.MethodsA retrospective study of 312 first renal allograft recipients was conducted between January 2015 and March 2022. The covariates were screened using univariable time-dependent Cox regression, and those with P<0.1 were included in the dynamic and static analyses. We constructed a prediction model for BKV reactivation from 2.5 to 8.5 months after renal transplantation using dynamic Cox regression based on the landmarking method and evaluated its performance using the area under the curve (AUC) value and Brier score. Monte-Carlo cross-validation was done to avoid overfitting. The above evaluation and validation process were repeated in the static model (Cox regression model) to compare the performance. Two patients were presented to illustrate the application of the dynamic model.ResultsWe constructed a dynamic prediction model with 18 covariates that could predict the probability of BKV reactivation from 2.5 to 8.5 months after renal transplantation. Elder age, basiliximab combined with cyclophosphamide for immune induction, acute graft rejection, higher body mass index, estimated glomerular filtration rate, urinary protein level, urinary leukocyte level, and blood neutrophil count were positively correlated with BKV reactivation, whereas male sex, higher serum albumin level, and platelet count served as protective factors. The AUC value and Brier score of the static model were 0.64 and 0.14, respectively, whereas those of the dynamic model were 0.79 ± 0.05 and 0.08 ± 0.01, respectively. In the cross-validation, the AUC values of the static and dynamic models decreased to 0.63 and 0.70 ± 0.03, respectively, whereas the Brier score changed to 0.11 and 0.09 ± 0.01, respectively.ConclusionDynamic Cox regression based on the landmarking method is effective in the assessment of the risk of BKV reactivation in the early period after renal transplantation and serves as a guide for clinical intervention.
| S-EPMC9428263 | biostudies-literature
Project description:Impaired BK virus (BKV)-specific immunity is a key risk factor of polyomavirus-associated nephropathy. We hypothesized that BKV agnoprotein might constitute an important immune target, as it is highly expressed after infection in vitro. We demonstrate abundant expression of BKV agnoprotein in vivo by immunostaining of kidney transplant (KT) biopsy specimens. Antibody responses to the recombinant affinity-purified BKV agnoprotein, large tumor (LT), and VP1 antigens in 146 sera from 38 KT patients and in 19 sera from 16 healthy donors (HD) were compared by enzyme immunoassay. In HD, low titers of anti-agnoprotein immunoglobulin G (IgG) were found in 15% of sera, compared to 41% for anti-LT antigen and 63% for anti-VP1. No anti-BKV IgM was detectable. In KT patients, anti-agnoprotein IgG and IgM were found in 8% and 3.6% of sera, compared to 63% and 18% for anti-LT IgG and IgM and 80% and 41% for anti-VP1 IgG and IgM, respectively. Anti-LT antigen and anti-VP1, but not anti-agnoprotein, activities increased during and after BKV viremia in KT patients. To investigate specific cellular immune responses, we compared levels of gamma interferon production in peripheral blood mononuclear cells (PBMC) of 10 HD and 30 KT patients by enzyme-linked immunospot assay. In HD, the median numbers of gamma interferon spot-forming units per million PBMC for the agnoprotein, LT antigen, and VP1 peptides were 1, 23, and 25, respectively, whereas the responses in KT patients were 2, 24, and 99, respectively. We conclude that BKV agnoprotein, though abundantly expressed in vivo, is poorly recognized immunologically.
| S-EPMC2044487 | biostudies-literature
Project description:UnlabelledThe human fetal glial cell line SVG was generated in 1985 by transfecting primary fetal brain cells with a plasmid containing an origin-defective mutant of simian virus 40 (SV40). The cells, which express SV40 large T-antigen, support the replication of human JC polyomavirus (JCPyV) and have been used for JCPyV studies but also for other studies in which cells of neural origin were desirable. We intended to use the SVG p12 cells from ATCC for antiviral drug studies with JCPyV. However, during initial experiments, immunofluorescence microscopy controls unexpectedly revealed cells expressing the late viral proteins VP1, VP2/VP3, and agno. This was confirmed by Western blotting. Since our agnoprotein antiserum is specific for BKPyV agnoprotein, infection with BKPyV was suspected. Indeed, specific BKPyV PCR of SVG p12 supernatants revealed a viral load of >1 × 10(10) genomic equivalents/ml. Negative-staining electron microscopy showed characteristic polyomavirus virions, and infectious BKPyV was transmitted from SVG p12 supernatant to other cells. Long-range PCR covering the viral genome, followed by DNA sequencing, identified BKPyV strain UT as well as deletion derivatives. This was confirmed by next-generation sequencing. JCPyV (MAD-4) was found to infect apparently uninfected and BKPyV-infected SVG p12 cells. In total, 4 vials from 2 different ATCC lots of SVG p12 cells dating back to 2006 contained BKPyV, whereas the subclone SVG-A was negative. In conclusion, SVG p12 cells from ATCC contain infectious BKPyV. This may have affected results and interpretations of previous studies, and caution should be taken in future experiments.ImportanceThis work reveals that one of the most frequently used cell lines for JC polyomavirus (JCPyV) research, the SV40-immortalized human fetal glial cell line SVG p12 obtained directly from ATCC, contains infectious BK polyomavirus (BKPyV) of strain UT and a spectrum of defective mutants. Strain UT has been previously found in urine and in tumors of different patients but is also frequently used for research. It is therefore not clear if BKPyV was present in the brain tissue used to generate the cell line or if this is a contamination. Although productive JCPyV infection of SVG cells was not dependent on prior BKPyV infection, the unnoticed presence of BKPyV may have influenced the results of studies using these cells. The interpretation of past results should therefore be reconsidered and cells tested for BKPyV before new studies are initiated. The frequently used subclone SVG-A did not contain BKPyV and could be a useful substitute.
| S-EPMC4054427 | biostudies-literature
Project description:BK polyomavirus (BKPyV) is a ubiquitous human virus that establishes a persistent infection in renal tubular epithelial cells and mainly causes disease in kidney transplant recipients. The closely related simian polyomavirus SV40 is known to cause cytoplasmic vacuolization in simian kidney cells, possibly increasing progeny release and cell death. This study aimed to determine whether BKPyV causes cytoplasmic vacuolization in primary human renal proximal tubule epithelial cells (RPTECs) and to investigate its potential role in the replication cycle. Using a large infectious dose (MOI 100-1000), a fraction of RPTECs (10-72%) showed early-wave vacuolization from 3 hours post-infection (hpi), which was mainly reversed by 36 hpi. Independent of the infectious dose, late-wave vacuolization occurred around the timepoint of progeny release. BKPyV receptor binding and internalization were required, as neuraminidase pretreatment and preincubation or treatment with a BKPyV-specific neutralizing antibody prevented early or late-occurring vacuolization. Microscopy revealed that the vacuoles were enlarged acidic endo-/lysosomal structures (dextran, EEA1, Rab5, Rab7, LAMP1, and/or Lysoview positive) that contained membrane-bound BKPyV. Time-lapse microscopy and quantitative PCR revealed that cell death and progeny release preceded late-wave vacuolization, mainly affecting cells directly neighboring the lysed cells. Thus, vacuolization had little impact on cell death or progeny release. Addition of the V-ATPase inhibitor Bafilomycin A1 at 0 hpi blocked vacuolization and BKPyV replication, but addition at 2 hpi only blocked vacuolization, suggesting that continuous endosomal acidification and maturation is needed for vacuole formation, but not for BKPyV replication. Our study shows that a massive uptake of BKPyV in RPTECs induces transient enlargement of endo-/lysosomes and is an early event in the viral replication cycle. Vacuolization gives no clear benefit for BKPyV and is possibly the result of a transiently overloaded endocytic pathway. Focal vacuolization around lysed cells suggests that the spread of BKPyV is preferably local.
| S-EPMC11581322 | biostudies-literature
Project description:Antiviral drugs for treating polyomavirus BK (BKV) replication in polyomavirus-associated nephropathy or hemorrhagic cystitis are of considerable clinical interest. Unlike cidofovir, the lipid conjugate 1-O-hexadecyloxypropyl cidofovir (CMX001) is orally available and has not caused detectable nephrotoxicity in rodent models or human studies to date. Primary human renal proximal tubular epithelial cells were infected with BKV-Dunlop, and CMX001 was added 2 h postinfection (hpi). The intracellular and extracellular BKV DNA load was determined by quantitative PCR. Viral gene expression was examined by quantitative reverse transcription-PCR, Western blotting, and immunofluorescence microscopy. We also examined host cell viability, proliferation, metabolic activity, and DNA replication. The titration of CMX001 identified 0.31 μM as the 90% effective concentration (EC(90)) for reducing the extracellular BKV load at 72 hpi. BKV large T antigen mRNA and protein expression was unaffected at 24 hpi, but the intracellular BKV genome was reduced by 90% at 48 hpi. Late gene expression was reduced by 70 and 90% at 48 and 72 hpi, respectively. Comparisons of CMX001 and cidofovir EC(90)s from 24 to 96 hpi demonstrated that CMX001 had a more rapid and enduring effect on BKV DNA and infectious progeny at 96 hpi than cidofovir. CMX001 at 0.31 μM had little effect on overall cell metabolism but reduced bromodeoxyuridine incorporation and host cell proliferation by 20 to 30%, while BKV infection increased cell proliferation in both rapidly dividing and near-confluent cultures. We conclude that CMX001 inhibits BKV replication with a longer-lasting effect than cidofovir at 400× lower levels, with fewer side effects on relevant host cells in vitro.
| S-EPMC2976161 | biostudies-literature
Project description:BK polyomavirus (BKV) is an emerging pathogen in immunocompromised individuals. BKV subtype III is rarely identified and has not previously been associated with disease. Here we provide the whole-genome sequence of a subtype III BKV from a pediatric kidney transplant patient with polyomavirus-associated nephropathy.
| S-EPMC3838085 | biostudies-literature
Project description:ObjectiveThis study aimed to explore the molecular mechanism of cytoplasmic vacuolation caused by BK polyomavirus (BKPyV) and thus search for potential target for drug repurposing.MethodsMorphological features of BK polyomavirus-associated nephropathy (BKPyVAN) were studied under light and electron microscopes. Microarray datasets GSE75693, GSE47199, and GSE72925 were integrated by ComBat, and differentially expressed genes (DEGs) were analyzed using limma. Furthermore, the endoplasmic reticulum (ER)-related genes obtained from GenCLiP 2.0 were intersected with DEGs. GO and KEGG enrichment pathways were performed with intersection genes by R package clusterProfiler. The single-cell RNA sequencing (scRNA-seq) from a BKPyVAN recipient was analyzed with a dataset (GSE140989) downloaded from Gene Expression Omnibus (GEO) as control for gene set variation analysis (GSVA). Immunohistochemistry and electron microscopy of kidney sections from drug-induced ERS mouse models were performed to explore the association of ERS and renal tubular vacuolation. Protein-protein interaction (PPI) network of the intersection genes was constructed to identify hub target. AutoDock was used to screen Food and Drug Administration (FDA)-approved drugs that potentially targeted hub gene.ResultsLight and electron microscopes exhibited obvious intranuclear inclusions, vacuoles, and virus particles in BKPyV-infected renal tubular cells. Transcriptome analysis revealed 629 DEGs between samples of BKPyVAN and stable transplanted kidneys, of which 16 were ER-associated genes. GO analysis with the intersection genes illustrated that ERS-related pathways were significantly involved, and KEGG analysis showed a prominent enrichment of MAPK, Toll-like receptor, and chemokine signaling pathways. GSVA analysis of the proximal tubule revealed similar pathways enrichment. An electron microscope image of the kidney from ERS mouse models showed an obvious renal tubular vacuolation with prominent activation of ERS markers verified by immunohistochemistry. Furthermore, DDIT3 was identified as the hub gene based on PPI analysis, and ZINCOOOOO1531009 (Risedronate) was indicated to be a potential drug for DDIT3.ConclusionERS was involved in renal tubular cytoplasmic vacuolation in BKPyVAN recipients. Risedronate was screened as a potential drug for BKPyVAN by targeting DDIT3.
| S-EPMC9027570 | biostudies-literature