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1.
J Exp Med ; 218(5)2021 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-33630019

RESUMEN

Congenital human cytomegalovirus (cHCMV) infection of the brain is associated with a wide range of neurocognitive sequelae. Using infection of newborn mice with mouse cytomegalovirus (MCMV) as a reliable model that recapitulates many aspects of cHCMV infection, including disseminated infection, CNS infection, altered neurodevelopment, and sensorineural hearing loss, we have previously shown that mitigation of inflammation prevented alterations in cerebellar development, suggesting that host inflammatory factors are key drivers of neurodevelopmental defects. Here, we show that MCMV infection causes a dramatic increase in the expression of the microglia-derived chemokines CXCL9/CXCL10, which recruit NK and ILC1 cells into the brain in a CXCR3-dependent manner. Surprisingly, brain-infiltrating innate immune cells not only were unable to control virus infection in the brain but also orchestrated pathological inflammatory responses, which lead to delays in cerebellar morphogenesis. Our results identify NK and ILC1 cells as the major mediators of immunopathology in response to virus infection in the developing CNS, which can be prevented by anti-IFN-γ antibodies.

2.
Virus Res ; 293: 198260, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33316352

RESUMEN

Chronic infection with human immunodeficiency virus (HIV)-1 is characterized by accumulation of proviral sequences in the genome of target cells. Integration of viral DNA in patients on long-term antiretroviral therapy selectively persists at preferential loci, suggesting site-specific crosstalk of viral sequences and human genes. This crosstalk likely contributes to chronic HIV disease through modulation of host immune pathways and emergence of clonal infected cell populations. To systematically interrogate such effects, we undertook genome engineering to generate Jurkat cell models that replicate integration of HIV-1 long terminal repeat (LTR) sequences at the BTB and CNC Homolog 2 (BACH2) integration locus. This locus is a prominent HIV-1 integration gene in chronic infection, found in 30 % of long-term treated patients with mapped proviral integrations. Using five clonal models carrying an LTR-driven reporter at different BACH2 intergenic regions, we here show that LTR transcriptional activity is repressed in BACH2 regions associated with proviral-DNA integrations in vivo but not in a control region. Our data indicates that this repression is in part epigenetically regulated, particularly through DNA methylation. Importantly, we demonstrate that transcriptional activity of the LTR is independent of BACH2 gene transcription and vice versa in our models. This suggests no transcriptional interference of endogenous and HIV-1 promoters. Taken together, our study provides first insights into how activity of HIV-1 LTR sequences is regulated at the BACH2 locus as prominent example for a recurrently-detected integration gene in chronic infection. Given the importance of integration-site dependent virus/host crosstalk for chronic HIV disease, our findings for the BACH2 locus have potential implications for future therapeutic strategies.

3.
mBio ; 11(6)2020 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-33323517

RESUMEN

Viral noncoding RNAs have acquired increasing prominence as important regulators of infection and mediators of pathogenesis. Circular RNAs (circRNAs) generated by backsplicing events have been identified in several oncogenic human DNA viruses. Here, we show that Merkel cell polyomavirus (MCV), the etiologic cause of ∼80% of Merkel cell carcinomas (MCCs), also expresses circular RNAs. By RNase R-resistant RNA sequencing, four putative circRNA backsplice junctions (BSJs) were identified from the MCV early region (ER). The most abundantly expressed MCV circRNA, designated circMCV-T, is generated through backsplicing of all of ER exon II to form a 762-nucleotide (nt) circular RNA molecule. Curiously, circMCV-T, as well as two other less abundantly expressed putative MCV circRNAs, overlaps in a complementary fashion with the MCV microRNA (miRNA) locus that encodes MCV-miR-M1. circMCV-T is consistently detected in concert with linear T antigen transcripts throughout infection, suggesting a crucial role for this RNA molecule in the regulatory functions of the early region, known to be vital for viral replication. Knocking out the hairpin structure of MCV-miR-M1 in genomic early region expression constructs and using a new high-efficiency, recombinase-mediated, recircularized MCV molecular clone demonstrates that circMCV-T levels decrease in the presence of MCV-miR-M1, underscoring the interplay between MCV circRNA and miRNA. Furthermore, circMCV-T partially reverses the known inhibitory effect of MCV-miR-M1 on early gene expression. RNase R-resistant RNA sequencing of lytic rat polyomavirus 2 (RatPyV2) identified an analogously located circRNA, stipulating a crucial, conserved regulatory function of this class of RNA molecules in the family of polyomaviruses.IMPORTANCE Covalently closed circular RNAs were recently described in the human DNA tumor viruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV). Here, we show that MCV, another DNA tumor virus, generates circRNAs from its early regulatory region in concert with T antigen linear transcripts. MCV circMCV-T interacts with another MCV noncoding RNA, miR-M1, to functionally modulate early region transcript expression important for viral replication and long-term episomal persistence. This work describes a dynamic regulatory network integrating circRNA/miRNA/mRNA biomolecules and underscores the intricate functional modulation between several classes of polyomavirus-encoded RNAs in the control of viral replication.

4.
Kidney Int ; 2020 Dec 22.
Artículo en Inglés | MEDLINE | ID: mdl-33359499

RESUMEN

BK polyomavirus-associated nephropathy is a common complication after kidney transplantation leading to reduced graft function or loss. The molecular pathogenesis of BK polyomavirus-induced nephropathy is not well understood. A recent study had described a protective effect of the activating natural killer cell receptor KIR3DS1 in BK polyomavirus-associated nephropathy, suggesting a role of NK cells in modulating disease progression. Using an in vitro cell culture model of human BK polyomavirus infection and kidney biopsy samples from patients with BK polyomavirus-associated nephropathy, we observed significantly increased surface expression of the ligand for KIR3DS1, HLA-F, on BK polyomavirus-infected kidney tubular cells. Upregulation of HLA-F expression resulted in significantly increased binding of KIR3DS1 to BK polyomavirus-infected cells and activation of primary KIR3DS-positive natural killer cells. Thus, our data provide a mechanism by which KIR3DS-positive natural killer cells can control BK polyomavirus infection of the kidney, and rationale for exploring HLA-F/KIR3DS1 interactions for immunotherapeutic approaches in BK polyomavirus-associated nephropathy.

5.
Front Microbiol ; 11: 591778, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33193257

RESUMEN

The rhadinoviruses Kaposi's Sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus (MHV-68) persist in infected hosts in a latent state that is characterized by the absence of virus production and by restricted viral gene expression. Their major latency protein, the latency-associated nuclear antigen (kLANA for KSHV and mLANA for MHV-68), is essential for viral genome maintenance and replication and involved in transcriptional regulation. Both kLANA and mLANA interact with cellular chromatin-associated proteins, among them the Bromodomain and Extra Terminal domain (Brd/BET) proteins, which recruit cellular and viral proteins to acetylated histones through their bromodomains and modulate cellular gene expression. Brd/BET proteins also play a role in the tethering, replication, segregation or integration of a diverse group of viral DNA genomes. In this study we explored if Brd/BET proteins influence the localization of the LANAs to preferential regions in the host chromatin and thereby contribute to kLANA- or mLANA-mediated transcriptional regulation. Using ChIP-Seq, we revealed a genome-wide co-enrichment of kLANA with Brd2/4 near cellular and viral transcriptional start sites (TSS). Treatment with I-BET151, an inhibitor of Brd/BET, displaced kLANA and Brd2/4 from TSS in the viral and host chromatin, but did not affect the direct binding of kLANA to kLANA-binding sites (LBS) in the KSHV latent origin of replication. Similarly, mLANA, but not a mLANA mutant deficient for binding to Brd2/4, also associated with cellular TSS. We compared the transcriptome of KSHV-infected with uninfected and kLANA-expressing human B cell lines, as well as a murine B cell line expressing mLANA or a Brd2/4-binding deficient mLANA mutant. We found that only a minority of cellular genes, whose TSS are occupied by kLANA or mLANA, is transcriptionally regulated by these latency proteins. Our findings extend previous reports on a preferential deposition of kLANA on cellular TSS and show that this characteristic chromatin association pattern is at least partially determined by the interaction of these viral latency proteins with members of the Brd/BET family of chromatin modulators.

6.
Artículo en Inglés | MEDLINE | ID: mdl-33007476

RESUMEN

OBJECTIVES: Investigation whether in depth characterization of virus variant patterns can be used for epidemiological analysis of the first SARS-CoV-2 infection clusters in Hamburg, Germany. METHODS: Metagenomic RNA- and amplicon-sequencing and subsequent variant calling in 25 respiratory samples from SARS-CoV-2 infected patients involved in the earliest infection clusters in Hamburg. RESULTS: Amplikon sequencing and cluster analyses of these SARS-CoV-2 sequences allowed the identification of the first infection cluster and five non-related infection clusters occurring at the beginning of the viral entry of SARS-CoV-2 in the Hamburg metropolitan region. Viral genomics together with epidemiological analyses revealed that the index patient acquired the infection in Northern Italy and transmitted it to two out of 134 contacts. Single nucleotide polymorphisms clearly distinguished the virus variants of the index and other clusters and allowed to track in which sequences worldwide these mutations were first described. Minor variant analyses identified the transmission of intra-host variants in the index cluster and household clusters. CONCLUSIONS: SARS-CoV-2 variant tracing allows the identification of infection clusters and the follow up of infection chains occurring in the population. Furthermore, the follow up of minor viral variants in infection cluster can provide further resolution on transmission events indistinguishable on consensus sequence level.

7.
EMBO Mol Med ; 12(12): e13296, 2020 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-33012091

RESUMEN

We describe a multifactorial investigation of a SARS-CoV-2 outbreak in a large meat processing complex in Germany. Infection event timing, spatial, climate and ventilation conditions in the processing plant, sharing of living quarters and transport, and viral genome sequences were analyzed. Our results suggest that a single index case transmitted SARS-CoV-2 to co-workers over distances of more than 8 m, within a confined work area in which air is constantly recirculated and cooled. Viral genome sequencing shows that all cases share a set of mutations representing a novel sub-branch in the SARS-CoV-2 C20 clade. We identified the same set of mutations in samples collected in the time period between this initial infection cluster and a subsequent outbreak within the same factory, with the largest number of confirmed SARS-CoV-2 cases in a German meat processing facility reported so far. Our results indicate climate conditions, fresh air exchange rates, and airflow as factors that can promote efficient spread of SARS-CoV-2 via long distances and provide insights into possible requirements for pandemic mitigation strategies in industrial workplace settings.

8.
Viruses ; 12(9)2020 08 27.
Artículo en Inglés | MEDLINE | ID: mdl-32867368

RESUMEN

CRISPR activation (CRISPRa) has revealed great potential as a tool to modulate the expression of targeted cellular genes. Here, we successfully applied the CRISPRa system to trigger the Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation in latently infected cells by selectively activating ORF50 gene directly from the virus genome. We found that a nuclease-deficient Cas9 (dCas9) fused to a destabilization domain (DD) and 12 copies of the VP16 activation domain (VP192) triggered a more efficient KSHV lytic cycle and virus production when guided to two different sites on the ORF50 promoter, instead of only a single site. To our surprise, the virus reactivation induced by binding of the stable DD-dCas9-VP192 on the ORF50 promoter was even more efficient than reactivation induced by ectopic expression of ORF50. This suggests that recruitment of additional transcriptional activators to the ORF50 promoter, in addition to ORF50 itself, are needed for the efficient virus production. Further, we show that CRISPRa can be applied to selectively express the early lytic gene, ORF57, without disturbing the viral latency. Therefore, CRISPRa-based systems can be utilized to facilitate virus-host interaction studies by controlling the expression of not only cellular but also of specific KSHV genes.

9.
PLoS Pathog ; 16(8): e1008562, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32833988

RESUMEN

Merkel Cell Polyomavirus (MCPyV) is the etiological agent of the majority of Merkel Cell Carcinomas (MCC). MCPyV positive MCCs harbor integrated, defective viral genomes that constitutively express viral oncogenes. Which molecular mechanisms promote viral integration, if distinct integration patterns exist, and if integration occurs preferentially at loci with specific chromatin states is unknown. We here combined short and long-read (nanopore) next-generation sequencing and present the first high-resolution analysis of integration site structure in MCC cell lines as well as primary tumor material. We find two main types of integration site structure: Linear patterns with chromosomal breakpoints that map closely together, and complex integration loci that exhibit local amplification of genomic sequences flanking the viral DNA. Sequence analysis suggests that linear patterns are produced during viral replication by integration of defective/linear genomes into host DNA double strand breaks via non-homologous end joining, NHEJ. In contrast, our data strongly suggest that complex integration patterns are mediated by microhomology-mediated break-induced replication, MMBIR. Furthermore, we show by ChIP-Seq and RNA-Seq analysis that MCPyV preferably integrates in open chromatin and provide evidence that viral oncogene expression is driven by the viral promoter region, rather than transcription from juxtaposed host promoters. Taken together, our data explain the characteristics of MCPyV integration and may also provide a model for integration of other oncogenic DNA viruses such as papillomaviruses.


Asunto(s)
Carcinoma de Células de Merkel/patología , Reparación del ADN por Unión de Extremidades , Poliomavirus de Células de Merkel/genética , Infecciones por Polyomavirus/complicaciones , Infecciones Tumorales por Virus/complicaciones , Integración Viral , Replicación Viral , Antígenos Virales de Tumores , Neoplasias Óseas/genética , Neoplasias Óseas/secundario , Neoplasias Óseas/virología , Carcinoma de Células de Merkel/genética , Carcinoma de Células de Merkel/virología , Humanos , Infecciones por Polyomavirus/genética , Infecciones por Polyomavirus/virología , Recombinación Genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/virología , Infecciones Tumorales por Virus/genética , Infecciones Tumorales por Virus/virología , Proteínas Virales/genética
10.
mBio ; 11(4)2020 08 25.
Artículo en Inglés | MEDLINE | ID: mdl-32843547

RESUMEN

Kaposi's sarcoma-associated herpesvirus (KSHV) causes primary effusion lymphoma (PEL). The cellular transcription factor (TF) interferon (IFN) regulatory factor 4 (IRF4) is an essential oncogene in PEL, but its specific role in PEL and how KSHV deregulates IRF4 remain unknown. Here, we report that the KSHV latency protein viral interferon regulatory factor 3 (vIRF3) cooperates with IRF4 and cellular BATF (basic leucine zipper ATF-like TF) to drive a super-enhancer (SE)-mediated oncogenic transcriptional program in PEL. Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) experiments demonstrated that IRF4, vIRF3, and BATF cooccupy the SEs of key survival genes, in a pattern that is distinct from those seen with other IRF4-driven malignancies. All three proteins cooperatively drive SE-mediated IRF4 overexpression. Inactivation of vIRF3 and, to a lesser extent, BATF phenocopies the gene expression changes and loss of cellular viability observed upon inactivation of IRF4. In sum, this work suggests that KSHV vIRF3 and cellular IRF4 and BATF cooperate as oncogenic transcription factors on SEs to promote cellular survival and proliferation in KSHV-associated lymphomas.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) causes the aggressive disease primary effusion lymphoma (PEL). Here, we show that a viral transcription factor (vIRF3) cooperates with the cellular transcription factor IRF4 to control an oncogenic gene expression program in PEL cells. These proteins promote KSHV-mediated B cell transformation by activating the expression of prosurvival genes through super-enhancers. Our report thus demonstrates that this DNA tumor virus encodes a transcription factor that functions with cellular IRF4 to drive oncogenic transcriptional reprogramming.

11.
Cancer Res ; 80(15): 3116-3129, 2020 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-32518203

RESUMEN

Kaposi sarcoma is a tumor caused by Kaposi sarcoma herpesvirus (KSHV) infection and is thought to originate from lymphatic endothelial cells (LEC). While KSHV establishes latency in virtually all susceptible cell types, LECs support spontaneous expression of oncogenic lytic genes, high viral genome copies, and release of infectious virus. It remains unknown the contribution of spontaneous virus production to the expansion of KSHV-infected tumor cells and the cellular factors that render the lymphatic environment unique to KSHV life cycle. We show here that expansion of the infected cell population, observed in LECs, but not in blood endothelial cells, is dependent on the spontaneous virus production from infected LECs. The drivers of lymphatic endothelium development, SOX18 and PROX1, regulated different steps of the KSHV life cycle. SOX18 enhanced the number of intracellular viral genome copies and bound to the viral origins of replication. Genetic depletion or chemical inhibition of SOX18 caused a decrease of KSHV genome copy numbers. PROX1 interacted with ORF50, the viral initiator of lytic replication, and bound to the KSHV genome in the promoter region of ORF50, increasing its transactivation activity and KSHV spontaneous lytic gene expression and infectious virus release. In Kaposi sarcoma tumors, SOX18 and PROX1 expression correlated with latent and lytic KSHV protein expression. These results demonstrate the importance of two key transcriptional drivers of LEC fate in the regulation of the tumorigenic KSHV life cycle. Moreover, they introduce molecular targeting of SOX18 as a potential novel therapeutic avenue in Kaposi sarcoma. SIGNIFICANCE: SOX18 and PROX1, central regulators of lymphatic development, are key factors for KSHV genome maintenance and lytic cycle in lymphatic endothelial cells, supporting Kaposi sarcoma tumorigenesis and representing attractive therapeutic targets.

12.
Microbiol Resour Announc ; 9(23)2020 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-32499358

RESUMEN

Here, we describe the complete genome sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain isolated from an oropharyngeal swab sample from a female patient with COVID-19 who was infected in Hamburg, northern Germany.

13.
BMC Bioinformatics ; 21(1): 233, 2020 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-32513098

RESUMEN

BACKGROUND: The detection of known human papillomaviruses (PVs) from targeted wet-lab approaches has traditionally used PCR-based methods coupled with Sanger sequencing. With the introduction of next-generation sequencing (NGS), these approaches can be revisited to integrate the sequencing power of NGS. Although computational tools have been developed for metagenomic approaches to search for known or novel viruses in NGS data, no appropriate tool is available for the classification and identification of novel viral sequences from data produced by amplicon-based methods. RESULTS: We have developed PVAmpliconFinder, a data analysis workflow designed to rapidly identify and classify known and potentially new Papillomaviridae sequences from NGS amplicon sequencing with degenerate PV primers. Here, we describe the features of PVAmpliconFinder and its implementation using biological data obtained from amplicon sequencing of human skin swab specimens and oral rinses from healthy individuals. CONCLUSIONS: PVAmpliconFinder identified putative new HPV sequences, including one that was validated by wet-lab experiments. PVAmpliconFinder can be easily modified and applied to other viral families. PVAmpliconFinder addresses a gap by providing a solution for the analysis of NGS amplicon sequencing, increasingly used in clinical research. The PVAmpliconFinder workflow, along with its source code, is freely available on the GitHub platform: https://github.com/IARCbioinfo/PVAmpliconFinder.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Papillomaviridae/aislamiento & purificación , Interfaz Usuario-Computador , ADN Viral/química , ADN Viral/metabolismo , Humanos , Papillomaviridae/genética , Flujo de Trabajo
14.
Life Sci Alliance ; 3(8)2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32576602

RESUMEN

HIV and EBV are human pathogens that cause a considerable burden to worldwide health. In combination, these viruses are linked to AIDS-associated lymphomas. We found that EBV, which transforms B cells, renders them susceptible to HIV-1 infection in a CXCR4 and CD4-dependent manner in vitro and that CXCR4-tropic HIV-1 integrates into the genome of these B cells with the same molecular profile as in autologous CD4+ T cells. In addition, we established a humanized mouse model to investigate the in vivo interactions of EBV and HIV-1 upon coinfection. The respective mice that reconstitute human immune system components upon transplantation with CD34+ human hematopoietic progenitor cells could recapitulate aspects of EBV and HIV immunobiology observed in dual-infected patients. Upon coinfection of humanized mice, EBV/HIV dual-infected B cells could be detected, but were susceptible to CD8+ T-cell-mediated immune control.

15.
Microbiol Resour Announc ; 9(24)2020 Jun 11.
Artículo en Inglés | MEDLINE | ID: mdl-32527781

RESUMEN

Scenedesmus acuminatus, also known as Tetradesmus acuminatus, is a promising green microalga for sustainable production of microalga products, including valuable compounds such as astaxanthin, ß-carotene, and lutein, polysaccharides such as ß-glucan, and polyunsaturated fatty acids. Here, we report the draft whole-genome sequence of Scenedesmus acuminatus SAG 38.81.

16.
Cell Rep ; 31(3): 107549, 2020 04 21.
Artículo en Inglés | MEDLINE | ID: mdl-32320654

RESUMEN

Importin-α adaptor proteins orchestrate dynamic nuclear transport processes involved in cellular homeostasis. Here, we show that importin-α3, one of the main NF-κB transporters, is the most abundantly expressed classical nuclear transport factor in the mammalian respiratory tract. Importin-α3 promoter activity is regulated by TNF-α-induced NF-κB in a concentration-dependent manner. High-level TNF-α-inducing highly pathogenic avian influenza A viruses (HPAIVs) isolated from fatal human cases harboring human-type polymerase signatures (PB2 627K, 701N) significantly downregulate importin-α3 mRNA expression in primary lung cells. Importin-α3 depletion is restored upon back-mutating the HPAIV polymerase into an avian-type signature (PB2 627E, 701D) that can no longer induce high TNF-α levels. Importin-α3-deficient mice show reduced NF-κB-activated antiviral gene expression and increased influenza lethality. Thus, importin-α3 plays a key role in antiviral immunity against influenza. Lifting the bottleneck in importin-α3 availability in the lung might provide a new strategy to combat respiratory virus infections.

17.
J Virol ; 94(13)2020 06 16.
Artículo en Inglés | MEDLINE | ID: mdl-32295923

RESUMEN

The anaphase-promoting complex, or cyclosome (APC/C), is a large E3 ubiquitin ligase composed of 14 subunits. The activity of APC/C oscillates during the cell cycle to ensure a timely transition through each phase by promoting the degradation of important cell cycle regulators. Of the human herpesviruses, cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) both impair the activity of APC/C during their lytic replication cycle through virus-encoded protein kinases. Here, we addressed whether the oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) deregulates the activity of APC/C during the lytic replication cycle. To this end, we used the well-characterized iSLK.219 cell model of KSHV infection and established a new infection model of primary lymphatic endothelial cells (LECs) infected with a lytically replicating KSHV BAC16 mutant. In contrast to those of EBV and HCMV, the KSHV lytic cycle occurs while the APC/C is active. Moreover, interfering with the activity of APC/C did not lead to major changes in the production of infectious virus. We further investigated whether rereplication stress induced by the unscheduled activation of the APC/C-CDH1 complex affects the number and integrity of KSHV viral episomes. Deep sequencing of the viral episomes and host chromosomes in iSLK.219 cells revealed that, while distinct regions in the cellular chromosomes were severely affected by rereplication stress, the integrity of the viral episomes remained unaltered.IMPORTANCE DNA viruses have evolved complex strategies to gain control over the cell cycle. Several of them target APC/C, a key cellular machinery that controls the timely progression of the cell cycle, by either blocking or enhancing its activity. Here, we investigated the activity of APC/C during the lytic replication cycle of KSHV and found that, in contrast to that of KSHV's close relatives EBV and HCMV, KSHV lytic replication occurs while the APC/C is active. Perturbing APC/C activity by depleting a core protein or the adaptor proteins of the catalytic domain, and hence interfering with normal cell-cycle progression, did not affect virus replication. This suggests that KSHV has evolved to replicate independently of the activity of APC/C and in various cell cycle conditions.

18.
Front Immunol ; 11: 450, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32231671

RESUMEN

Deciphering complex virus-host interactions is crucial for pandemic preparedness. In this study, we assessed the impact of recently postulated cellular factors ANP32A and ANP32B of influenza A virus (IAV) species specificity on viral pathogenesis in a genetically modified mouse model. Infection of ANP32A-/- and ANP32A+/+ mice with a seasonal H3N2 IAV or a highly pathogenic H5N1 human isolate did not result in any significant differences in virus tropism, innate immune response or disease outcome. However, infection of ANP32B-/- mice with H3N2 or H5N1 IAV revealed significantly reduced virus loads, inflammatory cytokine response and reduced pathogenicity compared to ANP32B+/+ mice. Genome-wide transcriptome analyses in ANP32B+/+ and ANP32B-/- mice further uncovered novel immune-regulatory pathways that correlate with reduced pathogenicity in the absence of ANP32B. These data show that ANP32B but not ANP32A promotes IAV pathogenesis in mice. Moreover, ANP32B might possess a yet unknown immune-modulatory function during IAV infection. Targeting ANP32B or its regulated pathways might therefore pose a new strategy to combat severe influenza.

19.
mBio ; 11(2)2020 03 10.
Artículo en Inglés | MEDLINE | ID: mdl-32156811

RESUMEN

Merkel cell polyomavirus (MCPyV) is the only polyomavirus known to be associated with tumorigenesis in humans. Similarly to other polyomaviruses, MCPyV expresses a large tumor antigen (LT-Ag) that, together with a small tumor antigen (sT-Ag), contributes to cellular transformation and that is of critical importance for the initiation of the viral DNA replication. Understanding the cellular protein network regulated by MCPyV early proteins will significantly contribute to our understanding of the natural MCPyV life cycle as well as of the mechanisms by which the virus contributes to cellular transformation. We here describe KRAB-associated protein 1 (Kap1), a chromatin remodeling factor involved in cotranscriptional regulation, as a novel protein interaction partner of MCPyV T antigens sT and LT. Kap1 knockout results in a significant increase in the level of viral DNA replication that is highly suggestive of Kap1 being an important host restriction factor during MCPyV infection. Differently from other DNA viruses, MCPyV gene expression is unaffected in the absence of Kap1 and Kap1 does not associate with the viral genome. Instead, we show that in primary normal human dermal fibroblast (nHDF) cells, MCPyV DNA replication, but not T antigen expression alone, induces ataxia telangiectasia mutated (ATM) kinase-dependent Kap1 S824 phosphorylation, a mechanism that typically facilitates repair of double-strand breaks in heterochromatin by arresting the cells in G2 We show that MCPyV-induced inhibition of cell proliferation is mainly conferred by residues within the origin binding domain and thereby by viral DNA replication. Our data suggest that phosphorylation of Kap1 and subsequent Kap1-dependent G2 arrest/senescence represent host defense mechanisms against MCPyV replication in nHDF cells.IMPORTANCE We here describe Kap1 as a restriction factor in MCPyV infection. We report a novel, indirect mechanism by which Kap1 affects MCPyV replication. In contrast with from other DNA viruses, Kap1 does not associate with the viral genome in MCPyV infection and has no impact on viral gene expression. In MCPyV-infected nHDF cells, Kap1 phosphorylation (pKap1 S824) accumulates because of genomic stress mainly induced by viral DNA replication. In contrast, ectopic expression of LT or LT MCPyV mutants, previously shown to be important for induction of genotoxic stress, does not result in a similar extent of pKap1 accumulation. We show that cells actively replicating MCPyV accumulate pKap1 (in a manner dependent on the presence of ATM) and display a senescence phenotype reflected by G2 arrest. These results are supported by transcriptome analyses showing that LT antigen, in a manner dependent on the presence of Kap1, induces expression of secreted factors, which is known as the senescence-associated secretory phenotype (SASP).

20.
Semin Immunopathol ; 42(2): 143-157, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32219477

RESUMEN

Kaposi sarcoma-associated herpesvirus (KSHV) is the etiologic agent of several malignancies of endothelial and B-cell origin. The fact that latently infected tumor cells in these malignancies do not express classical viral oncogenes suggests that pathogenesis of KSHV-associated disease results from multistep processes that, in addition to constitutive viral gene expression, may require accumulation of cellular alterations. Heritable changes of the epigenome have emerged as an important co-factor that contributes to the pathogenesis of many non-viral cancers. Since KSHV encodes a number of factors that directly or indirectly manipulate host cell chromatin, it is an intriguing possibility that epigenetic reprogramming also contributes to the pathogenesis of KSHV-associated tumors. The fact that heritable histone modifications have also been shown to regulate viral gene expression programs in KSHV-infected tumor cells underlines the importance of epigenetic control during latency and tumorigenesis. We here review what is presently known about the role of epigenetic regulation of viral and host chromatin in KSHV infection and discuss how viral manipulation of these processes may contribute to the development of KSHV-associated disease.

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