Biology subjects

Forlenza, M.

Publications and source records attributed to Forlenza, M..

5 recordsLinked to original sources

bioRxiv2026

Cowpea Chlorotic Mottle Virus virus-like particles as DNA or RNA delivery vehicle to fish cells

Cowpea Chlorotic Mottle Virus (CCMV) is an icosahedral plant virus composed of 180 monomeric units of the capsid protein. Virions encapsulate the viral RNA genome, which can be removed from the virion by changing pH conditions, after which the capsid proteins can be reversibly assembled to form empty CCMV virus-like particles (VLPs), or VLPs that have encapsulated either heterologous DNA or RNA. Earlier research has shown that CCMV VLPs are suitable as delivery vehicles for heterologous DNA/RNA molecules into mammalian cells, leading to subsequent expression of their encoded genes. In this study, we evaluated the potential of CCMV VLPs to encapsulate nucleic acids and transfer them to fish cells in vitro, and to zebrafish larvae and juveniles in vivo. VLPs containing pVax-GFP plasmid (3690 bp) were stable for up to 5 weeks in buffer at pH 7.2 and pH 5, whereas VLPs containing GFP-mRNA (1168 nt) were stable for up to 3 weeks, but only when stored in a buffer at pH 4.5. When administered directly to the culture medium, VLPs were readily taken up by fish as well as mammalian cell lines but could not unpack and transfer nucleic acids to the cells. VLPs delivered by transfection however, could transfer nucleic acids to the cells, leading to reporter gene expression. When VLPs were administered directly to the culture medium along with the cell-penetrating peptide dfTAT, only VLPs containing pVax-GFP in pH 7.2 buffer were able to transfer DNA to mammalian but not fish cells. In vivo however, upon intramuscular injection, the same VLPs were able to transfer plasmid DNA to myocytes of zebrafish larvae and juveniles as efficiently as when injecting naked plasmid DNA. Altogether, these findings provide the foundation for future studies on the use of CCMV VLPs as delivery vehicles for DNA or RNA to fish cells.

molecular biology↗

bioRxiv2025

During an inflammatory response, zebrafish tnfa and tnfb are expressed by different cell types and have distinct expression kinetics

Tumour necrosis factor (TNF, TNFSF2) is a key orchestrator of inflammation. Though mammalian TNF biology is well studied, the pleotropic nature of this cytokine during inflammation or infection in other vertebrates remains elusive. Interestingly, zebrafish possess two homologues of mammalian TNF, Tnfa and Tnfb. Previous studies have predominantly focused on the role of tnfa during infection or inflammation, while tnfb may also have important roles, yet has remained largely unexplored. Here, we generated and characterized two transgenic reporter lines, Tg(-3.2tnfb:eGFP-F)ump21Tg and Tg(-6.2tnfb:mCherry-F)ump22Tg, marking tnfb expression in zebrafish. By combining our tnfb reporters with other available reporter lines, with high-resolution in vivo microscopy, and with public scRNAseq datasets, we define, in a cell type-specific manner, the expression kinetics of tnfb compared to those of tnfa and il1b. We report on constitutive tnfb expression in neuromast mantle cells from 32 hours post-fertilization onwards, and inducible tnfb expression in subpopulations of macrophages and neutrophils after caudal fin fold amputation and E. coli injection. After amputation, tnfb kinetics of expression are cell-dependent, with expression in macrophages detected prior to that in neutrophils. When analysing the kinetics of expression of tnfb, tnfa and il1b after wounding, our data indicate that, at least in macrophages, a sequential expression occurs, with tnfb being expressed first, followed by tnfa and il1b. Combined, these data show that tnfb has distinct characteristics from tnfa, indicative of a differential role of these cytokines. Our new tnfb reporter lines, set the stage for future studies in which both TNF homologs can be investigated placing zebrafish as an especially suited model to dissect the pleiotropic nature of Tnf in non-mammalian vertebrates.

immunology↗

bioRxiv2023

Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

The endoribonuclease (EndoU) nsp15 of coronaviruses plays an important role in evasion of host innate immune responses by reducing the abundance of viral double-stranded RNA, whereas less is known about potential host cellular targets of nsp15. In this study, we show that cellular protein synthesis is inhibited upon over-expression of nsp15 from four genera of coronaviruses and this is accompanied by nuclear retention of the poly(A) binding protein cytoplasmic 1 (PABPC1). We also show that the EndoU activity of nsp15 is indispensable for both, inhibition of protein synthesis and PABPC1 nuclear relocation. FISH analysis using oligo-dT probes, revealed an overlap between the localization of cellular mRNA and that of overexpressed nsp15 in some cells, suggesting that, when expressed alone, nsp15 may target host mRNA. When investigating the association of nsp15 on protein shut off in the context of a viral infection, we observed that the{gamma} -coronavirus infectious bronchitis virus (IBV), induced host translation shutoff in an p-eIF2-independent manner and mainly retained PABPC1 in the cytoplasm, whereas the nsp15 EndoU-deficient IBV accumulated viral dsRNA and caused p-PKR-p-eIF2-dependent host protein translation shutoff, accompanied with PABPC1 nuclear relocation or stress granule (SG) localization. This phenomenon suggests that during infection with wild type IBV, although the cellular translation is inhibited, initiation of viral mRNA translation leads to PABPC1 binding to viral mRNA, thereby preventing its nuclear entry; during infection with nsp15 EndoU-deficient IBV however, the eIF2-dependent host protein translation shutoff prevents both host and viral mRNA translation initiation, releasing PABPC1 from binding to cytosolic and viral mRNA, thereby relocating it to the nucleus or to SG. Altogether, this study reveals unique yet conserved mechanisms of host protein shutoff that add to our understanding of how coronaviruses regulate host protein expression through a mechanism that involves catalytically active nsp15 EndoU, and describes how nsp15 may target both, viral and host mRNA. Author summaryIt has been reported that coronavirus infection suppresses host protein translation, - and {beta}- coronavirus nsp1 is responsible for inhibition of host gene expression. However, for{gamma} - and{delta} -coronavirus, there is no nsp1 and the underlying mechanisms by which virus regulates host translation are not well characterized. Here, we show that coronavirus endoribonuclease nsp15 is responsible for the inhibition of host translation by targeting to host factors, meanwhile it helps virus bypass the PKR-eIF2 mediated host translation shutoff, which is harmful for virus gene expression, by reducing the accumulation of viral dsRNA. This novel finding gives insight how does nsp15 targets to both host factors and viral RNA, to facilitate virus replication. Moreover, the novel function of nsp15 is found to be conserved among coronaviruses, revealing the essential role of this endoribonuclease in hijacking host translation machinery for virus replication.

microbiology↗

bioRxiv2020

Differential response of macrophages and neutrophils to trypanosome infections in zebrafish: occurrence of foamy macrophages

A tightly regulated innate immune response to trypanosome infections is critical to strike a balance between parasite control and inflammation-associated pathology. In the present study, we make use of the recently established Trypanosoma carassii infection model in larval zebrafish to study the early response of macrophages and neutrophils to trypanosome infections in vivo. We consistently identified high- and low-infected individuals and were able to simultaneously characterize their differential innate response. Not only did macrophage and neutrophil number and distribution differ between the two groups, but also macrophage morphology and activation state. Exclusive to high-infected zebrafish, was the appearance of macrophages rich in lipid droplets, confirmed to be foamy macrophages and characterized by a strong pro-inflammatory profile. Altogether, we provide an in vivo characterization of the differential response of macrophage and neutrophil to trypanosome infection and identify foamy macrophages as potentially associated with an exacerbated immune response and susceptibility to the infection. To our knowledge this is the first report of the occurrence of foamy macrophages during an extracellular trypanosome infection.

microbiology↗

bioRxiv2020

Inhibition of Anti-viral Stress Granule Formation by infectious bronchitis virus endoribonuclease nsp15 Ensures Efficient Virus Replication

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA virus, coronavirus, mutates frequently and circulates among animals, imposing great threat to public safety and animal health; however, the significance of SGs in coronavirus infections is largely unknown. Infectious bronchitis virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provide evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We show that the intrinsic endoribonuclease activity of nsp15 is responsible for the inhibition of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A triggered transcription of IFN-{beta} which in turn greatly affected recombinant virus replication. Further analysis showed that SGs function as antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of PRRs and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV suppresses the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We speculate that coronaviruses employ similar mechanisms to antagonize the host anti-viral SGs formation for efficient virus replication, as the endoribonuclease function of nsp15 is conserved in all coronaviruses. Author summaryIt has been reported that stress granules (SGs) are part of the host cell antiviral response. Not surprisingly, viruses in turn produce an array of antagonists to counteract such host response. Here, we show that IBV inhibits the formation of SGs through its endoribonuclease nsp15, by reducing the accumulation of viral dsRNA, evading the activation of PKR, and by subsequently inhibiting eIF2 phosphorylation and SGs formation. Nsp15 also inhibits SG formation independent of the eIF2 pathway, probably by targeting host mRNA. Depletion of SG scaffold proteins decreases IRF3-IFN response and increases the production of IBV. All coronaviruses encode a conserved endoribonuclease nsp15, and it will be important to determine whether also other (non-avian) coronaviruses limit the formation of anti-viral SGs in a similar manner.

microbiology↗