Targeting XPO1 enhances innate immune response and inhibits KSHV lytic replication during primary infection by nuclear stabilization of the p62 autophagy adaptor protein.
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ABSTRACT:
SUBMITTER: Meng W
PROVIDER: S-EPMC7790339 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:RSK1, a downstream kinase of the MAPK pathway, has been shown to regulate multiple cellular processes and is essential for lytic replication of a variety of viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV). Besides phosphorylation, it is not known whether other post-translational modifications play an important role in regulating RSK1 function. We demonstrate that RSK1 undergoes robust SUMOylation during KSHV lytic replication at lysine residues K110, K335, and K421. SUMO modification does not alter RSK1 activation and kinase activity upon KSHV ORF45 co-expression, but affects RSK1 downstream substrate phosphorylation. Compared to wild-type RSK1, the overall phosphorylation level of RxRxxS*/T* motif is significantly declined in RSK1K110/335/421R expressing cells. Specifically, SUMOylation deficient RSK1 cannot efficiently phosphorylate eIF4B. Sequence analysis showed that eIF4B has one SUMO-interacting motif (SIM) between the amino acid position 166 and 170 (166IRVDV170), which mediates the association between eIF4B and RSK1 through SUMO-SIM interaction. These results indicate that SUMOylation regulates the phosphorylation of RSK1 downstream substrates, which is required for efficient KSHV lytic replication.
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Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is causally associated with Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. The IFIT family of proteins inhibits replication of some viruses, but their effects on KSHV lytic replication was unknown. Here we show that KSHV lytic replication induces IFIT expression in epithelial cells. Depletion of IFIT1, IFIT2 and IFIT3 (IFITs) increased infectious KSHV virion production 25-32-fold compared to that in control cells. KSHV lytic gene expression was upregulated broadly with preferential activation of several genes involved in lytic viral replication. Intracellular KSHV genome numbers were also increased by IFIT knockdown, consistent with inhibition of KSHV DNA replication by IFITs. RNA seq demonstrated that IFIT depletion also led to downregulation of IFN β and several interferon-stimulated genes (ISGs), especially OAS proteins. OAS down-regulation led to decreased RNase L activity and slightly increased total RNA yield. IFIT immunoprecipitation also showed that IFIT1 bound to viral mRNAs and cellular capped mRNAs but not to uncapped RNA or trimethylated RNAs, suggesting that IFIT1 may also inhibit viral mRNA expression through direct binding. In summary, IFIT inhibits KSHV lytic replication through positively regulating the IFN β and OAS RNase L pathway to degrade RNA in addition to possibly directly targeting viral mRNAs.
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Project description:IFI16 (interferon gamma-inducible protein 16) recognizes nuclear episomal herpesvirus (Kaposi's sarcoma-associated herpesvirus [KSHV], Epstein-Barr virus [EBV], and herpes simplex virus 1 [HSV-1]) genomes and induces the inflammasome and interferon beta responses. It also acts as a lytic replication restriction factor and inhibits viral DNA replication (human cytomegalovirus [HCMV] and human papillomavirus [HPV]) and transcription (HSV-1, HCMV, and HPV) through epigenetic modifications of the viral genomes. To date, the role of IFI16 in the biology of latent viruses is not known. Here, we demonstrate that knockdown of IFI16 in the latently KSHV-infected B-lymphoma BCBL-1 and BC-3 cell lines results in lytic reactivation and increases in levels of KSHV lytic transcripts, proteins, and viral genome replication. Similar results were also observed during KSHV lytic cycle induction in TREX-BCBL-1 cells with the doxycycline-inducible lytic cycle switch replication and transcription activator (RTA) gene. Overexpression of IFI16 reduced lytic gene induction by the chemical agent 12-O-tetradecoylphorbol-13-acetate (TPA). IFI16 protein levels were significantly reduced or absent in TPA- or doxycycline-induced cells expressing lytic KSHV proteins. IFI16 is polyubiquitinated and degraded via the proteasomal pathway. The degradation of IFI16 was absent in phosphonoacetic acid-treated cells, which blocks KSHV DNA replication and, consequently, late lytic gene expression. Chromatin immunoprecipitation assays of BCBL-1 and BC-3 cells demonstrated that IFI16 binds to KSHV gene promoters. Uninfected epithelial SLK and osteosarcoma U2OS cells transfected with KSHV luciferase promoter constructs confirmed that IFI16 functions as a transcriptional repressor. These results reveal that KSHV utilizes the innate immune nuclear DNA sensor IFI16 to maintain its latency and repression of lytic transcripts, and a late lytic KSHV gene product(s) targets IFI16 for degradation during lytic reactivation.Like all herpesviruses, latency is an integral part of the life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent for many human cancers. Herpesviruses utilize viral and host factors to successfully evade the host immune system to maintain latency. Reactivation is a complex event where the latent episomal viral genome springs back to active transcription of lytic cycle genes. Our studies reveal that KSHV has evolved to utilize the innate immune sensor IFI16 to keep lytic cycle transcription in dormancy. We demonstrate that IFI16 binds to the lytic gene promoter, acts as a transcriptional repressor, and thereby helps to maintain latency. We also discovered that during the late stage of lytic replication, KSHV selectively degrades IFI16, thus relieving transcriptional repression. This is the first report to demonstrate the role of IFI16 in latency maintenance of a herpesvirus, and further understanding will lead to the development of strategies to eliminate latent infection.
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Project description:Duck Tembusu virus (DTMUV) has recently appeared in ducks in China and the key cellular determiners for DTMUV replication in host cells remain unknown. Autophagy is an evolutionarily conserved cellular process that has been reported to facilitate flavivirus replication. In this study, we utilized primary duck embryo fibroblast (DEF) as the cell model and found that DTMUV infection triggered LC3-II increase and polyubiquitin-binding protein sequestosome 1 (p62) decrease, confirming that complete autophagy occurred in DEF cells. The induction of autophagy by pharmacological treatment increased DTMUV replication in DEF cells, whereas the inhibition of autophagy with pharmacological treatments or RNA interference decreased DTMUV replication. Inhibiting autophagy enhanced the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) and interferon regulatory factor 7 (IRF7) pathways and increased the p62 protein level in DTMUV-infected cells. We further found that the overexpression of p62 decreased DTMUV replication and inhibited the activation of the NF-?B and IRF7 pathways, and changes in the NF-?B and IRF7 pathways were consistent with the level of phosphorylated TANK-binding kinase 1 (p-TBK1). Opposite results were found in p62 knockdown cells. In summary, we found that autophagy-mediated p62 degradation acted as a new strategy for DTMUV to evade host innate immunity.
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Project description:The nucleus is a highly structured environment containing multiple membrane-less bodies formed through liquid-liquid phase separation. These provide spatial separation and concentration of specific biomolecules enabling efficient and discrete processes to occur which regulate gene expression. One such nuclear body, paraspeckles, are comprised of multiple paraspeckle proteins (PSPs) built around the architectural RNA, NEAT1_2. Paraspeckle function is yet to be fully elucidated but has been implicated in a variety of developmental and disease scenarios. We demonstrate that Kaposi’s sarcoma-associated herpesvirus (KSHV) drives formation of structurally distinct paraspeckles with a dramatically increased size and altered protein composition that are essential for productive lytic replication. We highlight these virus-induced paraspeckle-like structures form adjacent to virus replication centres, functioning as RNA processing hubs for both viral and cellular transcripts during infection. Notably, we reveal that PSP sequestration into virus-induced paraspeckle-like structures results in increased genome instability during both KSHV and Epstein Barr virus (EBV) infection, implicating their formation in virus-mediated tumorigenesis.
2024-11-04 | PXD048834 | Pride
Project description:Previous studies have implicated autophagy in osteoclast differentiation. The aim of this study was to investigate the potential role of p62, a characterized adaptor protein for autophagy, in RANKL-induced osteoclastogenesis. Real-time quantitative PCR and western blot analyses were used to evaluate the expression levels of autophagy-related markers during RANKL-induced osteoclastogenesis in mouse macrophage-like RAW264.7 cells. Meanwhile, the potential relationship between p62/LC3 localization and F-actin ring formation was tested using double-labeling immunofluorescence. Then, the expression of p62 in RAW264.7 cells was knocked down using small-interfering RNA (siRNA), followed by detecting its influence on RANKL-induced autophagy activation, osteoclast differentiation, and F-actin ring formation. The data showed that several key autophagy-related markers including p62 were significantly altered during RANKL-induced osteoclast differentiation. In addition, the expression and localization of p62 showed negative correlation with LC3 accumulation and F-actin ring formation, as demonstrated by western blot and immunofluorescence analyses, respectively. Importantly, the knockdown of p62 obviously attenuated RANKL-induced expression of autophagy- and osteoclastogenesis-related genes, formation of TRAP-positive multinuclear cells, accumulation of LC3, as well as formation of F-actin ring. Our study indicates that p62 may play essential roles in RANKL-induced autophagy and osteoclastogenesis, which may help to develop a novel therapeutic strategy against osteoclastogenesis-related diseases.
| S-EPMC4244301 | biostudies-literature
Project description:BackgroundAccumulating evidence suggests that dysregulated expression of lytic genes plays an important role in KSHV (Kaposi's sarcoma associated herpesvirus) tumorigenesis. However, the molecular events leading to the dysregulation of KSHV lytic gene expression program are incompletely understood.Methodology/principal findingsWe have studied the effect of KSHV-encoded latent protein vFLIP K13, a potent activator of the NF-kappaB pathway, on lytic reactivation of the virus. We demonstrate that K13 antagonizes RTA, the KSHV lytic-regulator, and effectively blocks the expression of lytic proteins, production of infectious virions and death of the infected cells. Induction of lytic replication selects for clones with increased K13 expression and NF-kappaB activity, while siRNA-mediated silencing of K13 induces the expression of lytic genes. However, the suppressive effect of K13 on RTA-induced lytic genes is not uniform and it fails to block RTA-induced viral IL6 secretion and cooperates with RTA to enhance cellular IL-6 production, thereby dysregulating the lytic gene expression program.Conclusions/significanceOur results support a model in which ongoing KSHV lytic replication selects for clones with progressively higher levels of K13 expression and NF-kappaB activity, which in turn drive KSHV tumorigenesis by not only directly stimulating cellular survival and proliferation, but also indirectly by dysregulating the viral lytic gene program and allowing non-lytic production of growth-promoting viral and cellular genes. Lytic Replication-Induced Clonal Selection (LyRICS) may represent a general mechanism in viral oncogenesis.
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Project description:Non-coding RNA (ncRNA) regulatory networks are emerging as critical regulators of gene expression. These intricate networks of ncRNA:ncRNA interactions modulate multiple cellular pathways and impact the development and progression of multiple diseases. Herpesviruses, including Kaposi's sarcoma-associated herpesvirus, are adept at utilising ncRNAs, encoding their own as well as dysregulating host ncRNAs to modulate virus gene expression and the host response to infection. Research has mainly focused on unidirectional ncRNA-mediated regulation of target protein-coding transcripts; however, we identify a novel host ncRNA regulatory network essential for KSHV lytic replication in B cells. KSHV-mediated upregulation of the host cell circRNA, circHIPK3, is a key component of this network, functioning as a competing endogenous RNA of miR-30c, leading to increased levels of the miR-30c target, DLL4. Dysregulation of this network highlights a novel mechanism of cell cycle control during KSHV lytic replication in B cells. Importantly, disruption at any point within this novel ncRNA regulatory network has a detrimental effect on KSHV lytic replication, highlighting the essential nature of this network and potential for therapeutic intervention.
| S-SCDT-EMBOR-2021-54117V1 | biostudies-other
Project description:The ubiquitin-proteasome system and autophagy were long viewed as independent, parallel degradation systems with no point of intersection. By now we know that these degradation pathways share certain substrates and regulatory molecules and show coordinated and compensatory function. Two ubiquitin-like protein conjugation pathways were discovered that are required for autophagosome biogenesis: the Atg12-Atg5-Atg16 and Atg8 systems. Autophagy has been considered to be essentially a nonselective process, but it turned out to be at least partially selective. Selective substrates of autophagy include damaged mitochondria, intracellular pathogens, and even a subset of cytosolic proteins with the help of ubiquitin-binding autophagic adaptors, such as p62/SQSTM1, NBR1, NDP52, and Optineurin. These proteins selectively recognize autophagic cargo and mediate its engulfment into autophagosomes by binding to the small ubiquitin-like modifiers that belong to the Atg8/LC3 family.
| S-EPMC4075091 | biostudies-literature
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) belongs to the gamma herpesvirus family, which can cause human malignancies including Kaposi sarcoma, primary effusion lymphoma, and multicentric Castleman's diseases. KSHV typically maintains a persistent latent infection within the host. However, after exposure to intracellular or extracellular stimuli, KSHV lytic replication can be reactivated. The reactivation process of KSHV triggers the innate immune response to limit viral replication. Here, we found that the transcriptional regulator RUNX3 is transcriptionally upregulated by the NF-κB signaling pathway in KSHV-infected SLK cells and B cells during KSHV reactivation. Notably, knockdown of RUNX3 significantly promotes viral lytic replication as well as the gene transcription of KSHV. Consistent with this finding, overexpression of RUNX3 impairs viral lytic replication. Mechanistically, RUNX3 binds to the KSHV genome and limits viral replication through transcriptional repression, which is related to its DNA- and ATP-binding ability. However, KSHV has also evolved corresponding strategies to antagonize this inhibition by using the viral protein RTA to target RUNX3 for ubiquitination and proteasomal degradation. Altogether, our study suggests that RUNX3, a novel host-restriction factor of KSHV that represses the transcription of viral genes, may serve as a potential target to restrict KSHV transmission and disease development.IMPORTANCEThe reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) from latent infection to lytic replication is important for persistent viral infection and tumorigenicity. However, reactivation is a complex event, and the regulatory mechanisms of this process are not fully elucidated. Our study revealed that the host RUNX3 is upregulated by the NF-κB signaling pathway during KSHV reactivation, which can repress the transcription of KSHV genes. At the late stage of lytic replication, KSHV utilizes a mechanism involving RTA to degrade RUNX3, thus evading host inhibition. This finding helps elucidate the regulatory mechanism of the KSHV life cycle and may provide new clues for the development of therapeutic strategies for KSHV-associated diseases.
| S-EPMC10878072 | biostudies-literature