Neutralization Serotyping of BK Polyomavirus Infection in Kidney Transplant Recipients

BK polyomavirus (BKV or BKPyV) associated nephropathy affects up to 10% of kidney transplant recipients (KTRs). BKV isolates are categorized into four genotypes. It is currently unclear whether the four genotypes are also serotypes. To address this issue, we developed high-throughput serological assays based on antibody-mediated neutralization of BKV genotype I and IV reporter vectors (pseudoviruses). Neutralization-based testing of sera from mice immunized with BKV-I or BKV-IV virus-like particles (VLPs) or sera from naturally infected human subjects revealed that BKV-I specific serum antibodies are poorly neutralizing against BKV-IV and vice versa. The fact that BKV-I and BKV-IV are distinct serotypes was less evident in traditional VLP-based ELISAs. BKV-I and BKV-IV neutralization assays were used to examine BKV type-specific neutralizing antibody responses in KTRs at various time points after transplantation. At study entry, sera from 5% and 49% of KTRs showed no detectable neutralizing activity for BKV-I or BKV-IV neutralization, respectively. By one year after transplantation, all KTRs were neutralization seropositive for BKV-I, and 43% of the initially BKV-IV seronegative subjects showed evidence of acute seroconversion for BKV-IV neutralization. The results suggest a model in which BKV-IV-specific seroconversion reflects a de novo BKV-IV infection in KTRs who initially lack protective antibody responses capable of neutralizing genotype IV BKVs. If this model is correct, it suggests that pre-vaccinating prospective KTRs with a multivalent VLP-based vaccine against all BKV serotypes, or administration of BKV-neutralizing antibodies, might offer protection against graft loss or dysfunction due to BKV associated nephropathy

The methyltransferase inhibitor UNC0638 inhibits SARS-CoV2 proliferation in A549 cells.

A.) IGV plots of SARS-CoV-2 aligned reads from biological replicates of RNA-seq experiments from SARS-CoV-2 infected cells treated with DMSO or UNC0638. B.) Absolute numbers of RNA-seq reads aligned to the SARS-CoV-2 (left panel) or the human genome (right panel). C.) qPCR showing reduction of viral RNA and induction of IFNB1 and IFNL3 (n = 3 for each condition). D.) Gene set enrichment analysis (GSEA) showing that transcripts downregulated by FH-NSP1 transfection are induced upon treatment of SARS-CoV-2 infected A549 cells with UNC0638.</p

S2 Fig -

A. Spectral counts from SARS-CoV-2 host proximity interactome data (covid19interactome.org) B,C. Confocal Immunofluorescence microscopy analysis of A549 cells after introduction of a scrambled siRNA or an siRNA targeting PRRC2B expression. Green, anti PRRC2B; blue, DAPI. D. Meta-analysis of the distribution of di-methylated Histone 3 Lysin 9 chromatin (H3K9me2), obtained by H3K9me2 ChIP-seq, relative to genomic coordinates of protein-coding genes. (TIF)</p

UNC0638 abrogates NSP1-mediated downregulation of immune related.

A.) Volcano plots showing suppression of type I and III interferon genes by NSP1 (left panel) and attenuation of the suppression following UNC0638 treatment. RNA-seq experiments in each condition are biological triplicates. B.) Profiling of secreted cytokines showing restoration of antiviral genes suppressed by NSP1. C.) Cumulative distribution frequency plots showing the effect of UNC0638 on H3K9me2 marks (-0.26; p value p value = 0.075) and PolII occupancy (+0.09; p value < 0.001) on the top genes H3K9me2-enriched after NSP1 expression.</p

NSP1 affects host-cell transcription of immune-related genes via H3K9me2.

A.) Meta-analysis of the distribution of PolII associated chromatin (obtained by PolII ChIP-seq) relative to genomic coordinates of protein-coding genes (TSS, Transcription Start Site; TES, Transcription End Site). B.) GSEA showing that transcriptionally downregulated genes (PolII ChIP-seq, orange dots) show decreased transcript levels by RNA-seq after NSP1 expression. C.) Bar graph of log2Fold change values in RNA-Seq data of WT versus NSP1 K164A/H165A transfected A459 cells after scramble or PRRC2B silencing showing restoration of IFNB1 IFNL1 IFNL2 and IFNL3 expression. D.) GSEA showing that genes with increased H3K9me2 mark (blue dots) show decreased transcript levels by RNA-seq after NSP1 expression.</p

S1 Table -

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades the innate immune machinery through multiple viral proteins, including nonstructural protein 1 (NSP1). While NSP1 is known to suppress translation of host mRNAs, the mechanisms underlying its immune evasion properties remain elusive. By integrating RNA-seq, ribosome footprinting, and ChIP-seq in A549 cells we found that NSP1 predominantly represses transcription of immune-related genes by favoring Histone 3 Lysine 9 dimethylation (H3K9me2). G9a/GLP H3K9 methyltransferase inhibitor UNC0638 restored expression of antiviral genes and restricted SARS-CoV-2 replication. Our multi-omics study unravels an epigenetic mechanism underlying host immune evasion by SARS-CoV-2 NSP1. Elucidating the factors involved in this phenomenon, may have implications for understanding and treating viral infections and other immunomodulatory diseases.</div

NSP1 downregulates steady-state levels of mRNAs encoded by immune-related genes.

A.) A549 cells and their growth medium were collected 16 hours after mock, NSP1-GFP or FH-NSP1 transfection. Growth media and cell extracts were analyzed by western blot, and samples from FH-NSP1 cells were also used for immunoprecipitation with anti-FLAG antibodies prior to Western blot analysis using an anti-NSP1 antibody. B.) Confocal fluorescence microscopy of A549 cells transfected with NSP1-GFP or mock (same transfection and processing in the absence of DNA). C,D) Principal Component Analysis (PCA) of mRNA profiles after transient transfection of NSP1 and control GFP or the K164A/H165A NSP1 mutant, obtained by RNA-seq following rRNA depletion (C) or poly-A selection (D). E.) Gene ontology analysis of transcripts downregulated (≥2-fold) upon expression of NSP1. Size of bubble reflects the number of downregulated genes of the respective categories. F.) Venn diagram of genes downregulated by ≥ 2-fold upon NSP1 expression compared to expression of the GFP control, using either rRNA depletion or poly-A selection for mRNA enrichment. Genes shared by the two groups are listed below.</p

Ribosome footprints follow steady state mRNA abundance.

A.) Ribo-seq Ribosome-Protected Fragments (RPFs) are as expected aligned predominantly to protein-coding sequences in a sense-strand specific manner. B.) Metagene plot showing distribution of ribosome-protected mRNA footprints (RPF) relative to mRNA functional elements. C.) RPFs length distribution centers on 29 nucleotides (nt). D.) PCA of indicated Ribo-seq experiments. E.) Comparison of ribosome density (RPFs versus RPKM) after transient transfection of NSP1 and GFP. Ribo-seq experiments were performed in biological triplicates. F.) Gene set enrichment analysis (GSEA) showing that NSP1-mediated changes in RPFs correlate to changes in mRNA abundance.</p

S1 Raw images -

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades the innate immune machinery through multiple viral proteins, including nonstructural protein 1 (NSP1). While NSP1 is known to suppress translation of host mRNAs, the mechanisms underlying its immune evasion properties remain elusive. By integrating RNA-seq, ribosome footprinting, and ChIP-seq in A549 cells we found that NSP1 predominantly represses transcription of immune-related genes by favoring Histone 3 Lysine 9 dimethylation (H3K9me2). G9a/GLP H3K9 methyltransferase inhibitor UNC0638 restored expression of antiviral genes and restricted SARS-CoV-2 replication. Our multi-omics study unravels an epigenetic mechanism underlying host immune evasion by SARS-CoV-2 NSP1. Elucidating the factors involved in this phenomenon, may have implications for understanding and treating viral infections and other immunomodulatory diseases.</div