Autofagia como mecanismo de protección frente al virus de la septicemia hemorrágica vírica en eritrocitos de trucha arcoíris

La industria acuícola se encuentra amenazada por las enfermedades que producen los patógenos virales, causando graves pérdidas económicas en el sector. Es necesario encontrar diversas estrategias antivirales y es aquí donde los eritrocitos (glóbulos rojos, red blood cells, RBCs) pueden jugar un papel importante. En este trabajo, se ha estudiado como los RBCs de teleósteos son capaces de responder mediante distintos mecanismos antivirales a la exposición al virus de la septicemia hemorrágica vírica (VHSV). Tanto por PCR cuantitativa como por citometría de flujo, se pudo observar un aumento en la actividad autofágica en RBCs de trucha arcoíris tras ser expuestos a VHSV ex vivo. Según los resultados obtenidos, se ha podido observar que compuestos como 3-MA o niclosamida, capaces de modular la actividad autofágica, producen variaciones en la cantidad de VHSV intracelular. Además, observamos un aumento de ubiquitinación de proteínas en RBCs de trucha arcoíris expuestos al virus. Por otro lado, los resultados obtenidos sobre la cuantificación de proteína p62 (también denominada secuestosoma 1) indican que está involucrada en el transporte de proteínas ubiquitinadas hacia los autofagosomas. Además, VHSV es capaz de inducir la expresión de genes de respuesta antioxidante en los RBCs. De acuerdo con los resultados obtenidos, podemos concluir que los RBCs de trucha arcoíris son capaces de desarrollar respuestas antivirales frente a VHSV mediadas por el sistema de autofagia.Aquaculture industry is threaten by viral pathogen diseases, causing severe economic losses in this field. It is necessary to find novel antiviral strategies and here the erythrocytes (red blood cells, RBCs) play an important role. In this work, it has been studied how teleost RBCs are able to respond to the exposure to viral hemorrhagic septicemia virus (VHSV) by different antiviral mechanisms. An increament in autophagic activity could be observed in rainbow trout RBCs after being exposed to VHSV ex vivo, by means of quantitative PCR and flow cytometry. According to the obtained results, compounds such as 3-MA or niclosamide, were capable of modulating autophagic activity, producing variations in the amount of intracellular VHSV. In addition, we observed an increment in cellular proteins ubiquitination in rainbow trout RBCs exposed to the virus. On the other hand, the results obtained on the quantification of protein p62 (also called sequestosoma 1) indicated that this protein is involved in the transport of ubiquitinated proteins towards autophagosomes. Furthermore, VHSV is able to induce the expression of antioxidant response genes in RBCs. According to the obtained results, we could conclude that rainbow trout RBCs were able to develop antiviral responses against VHSV, by means of the autophagy system

Rainbow Trout Red Blood Cells Exposed to Viral Hemorrhagic Septicemia Virus Up-Regulate Antigen-Processing Mechanisms and MHC I&II, CD86, and CD83 Antigen-presenting Cell Markers

Nucleated teleost red blood cells (RBCs) are known to express molecules from the major histocompatibility complex and peptide-generating processes such as autophagy and proteasomes, but the role of RBCs in antigen presentation of viruses have not been studied yet. In this study, RBCs exposed ex vivo to viral hemorrhagic septicemia virus (VHSV) were evaluated by means of transcriptomic and proteomic approaches. Genes and proteins related to antigen presentation molecules, proteasome degradation, and autophagy were up-regulated. VHSV induced accumulation of ubiquitinated proteins in ex vivo VHSV-exposed RBCs and showed at the same time a decrease of proteasome activity. Furthermore, induction of autophagy was detected by evaluating LC3 protein levels. Sequestosome-1/p62 underwent degradation early after VHSV exposure, and it may be a link between ubiquitination and autophagy activation. Inhibition of autophagosome degradation with niclosamide resulted in intracellular detection of N protein of VHSV (NVHSV) and p62 accumulation. In addition, antigen presentation cell markers, such as major histocompatibility complex (MHC) class I & II, CD83, and CD86, increased at the transcriptional and translational level in rainbow trout RBCs exposed to VHSV. In summary, we show that nucleated rainbow trout RBCs can degrade VHSV while displaying an antigen-presenting cell (APC)-like profil

Nature of viruses and pandemics: Coronaviruses

Coronaviruses (CoVs) have the largest genome among RNA viruses and store large amounts of information without genome integration as they replicate in the cell cytoplasm. The replication of the virus is a continuous process, whereas the transcription of the subgenomic mRNAs is a discontinuous one, involving a template switch, which resembles a high frequency recombination mechanism that may favor virus genome variability. The origin of the three deadly human CoVs SARS-CoV, MERS-CoV and SARS-CoV-2 are zoonotic events. SARS-CoV-2 has incorporated in its spike protein a furine proteolytic site that facilitates the activation of the virus in any tissue, making this CoV strain highly polytropic and pathogenic. Using MERS-CoV as a model, a propagation-deficient RNA replicon was generated by removing E protein gene (essential for viral morphogenesis and involved in virulence), and accessory genes 3, 4a, 4b and 5 (responsible for antagonism of the innate immune response) to attenuate the virus: MERS-CoV-Δ[3,4a,4b,5,E]. This RNA replicon is strongly attenuated and elicits sterilizing protection after a single immunization in transgenic mice with the receptor for MERS-CoV, making it a promising vaccine candidate for this virus and an interesting platform for vector-based vaccine development. A strategy could be developed for the design of RNA replicon vaccines for other human pathogenic coronaviruses.This work was supported by grants from the Government of Spain (PID2019-107001RB-I00 AEI/FEDER, UE; SEV 2017-0712 and PIE_INTRAMURAL_LINEA 1-202020E079), the CSIC (PIE_INTRAMURAL-202020E043), the European Commission (ISOLDA_848166 H2020-SC1-2019-Two-Stage-RTD, RIA; MANCO_101003651 H2020-SC1-PHE-CORONAVIRUS-2020 RIA), and the U.S. National Institutes of Health (NIH_2P01AI060699).Peer reviewe

MERS-CoV ORF4b is a virulence factor involved in the inflammatory pathology induced in the lungs of mice

23 Pág. Centro de Investigación en Sanidad Animal (CISA)No vaccines or specific antiviral drugs are authorized against Middle East respiratory syndrome coronavirus (MERS-CoV) despite its high mortality rate and prevalence in dromedary camels. Since 2012, MERS-CoV has been causing sporadic zoonotic infections in humans, which poses a risk of genetic evolution to become a pandemic virus. MERS-CoV genome encodes five accessory proteins, 3, 4a, 4b, 5 and 8b for which limited information is available in the context of infection. This work describes 4b as a virulence factor in vivo, since the deletion mutant of a mouse-adapted MERS-CoV-Δ4b (MERS-CoV-MA-Δ4b) was completely attenuated in a humanized DPP4 knock-in mouse model, resulting in no mortality. Attenuation in the absence of 4b was associated with a significant reduction in lung pathology and chemokine expression levels at 4 and 6 days post-infection, suggesting that 4b contributed to the induction of lung inflammatory pathology. The accumulation of 4b in the nucleus in vivo was not relevant to virulence, since deletion of its nuclear localization signal led to 100% mortality. Interestingly, the presence of 4b protein was found to regulate autophagy in the lungs of mice, leading to upregulation of BECN1, ATG3 and LC3A mRNA. Further analysis in MRC-5 cell line showed that, in the context of infection, MERS-CoV-MA 4b inhibited autophagy, as confirmed by the increase of p62 and the decrease of ULK1 protein levels, either by direct or indirect mechanisms. Together, these results correlated autophagy activation in the absence of 4b with downregulation of a pathogenic inflammatory response, thus contributing to attenuation of MERS-CoV-MA-Δ4b.This work was supported by grants from the Government of Spain (BIO2016-75549-R; PID2019-107001RB-I00 AEI/FEDER, UE; SEV 2017-0712 and PIE_INTRAMURAL_LINEA 1-202020E079), CSIC (PIE_INTRAMURAL -202020E043), the European Zoonotic Anticipation and Preparedness Initiative (ZAPI) (IMI_JU_115760), the European Commission (H2020-SC1-2019, ISOLDA Project No. 848166-2), and the U.S. National Institutes of Health (NIH) (2P01AI060699 to I.S.). J.C. and J.H-T: received fellowships from the Ministry of Science an Innovation of Spain (BIO2013- 42869-R and PID2019-107001RB-I00 AEI/FEDER). M.B-P. received a contract from ISOLDA_848166 H2020-SC1-2019-Two-Stage-RTD.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Characterization of MERS-CoV-MA-Δ4b and MERS-CoV-MA-mNLS mutants.

(A) Huh-7 cells were infected with MERS-CoV-MA, MERS-CoV-MA-Δ4b or MERS-CoV-MA-mNLS at a m.o.i. of 0.1 for 24 h and the localization of 4b (in green) was analyzed by confocal microscopy. (B) Subconfluent monolayers of Huh-7 were infected with wild-type (black), Δ4b (red) or mNLS (purple) at a m.o.i. of 0.1 or 0.001. Culture supernatants were collected at 24, 48, 72 and 96 h p.i. and titrated by plaque assay. The average of two independent experiments is represented. (C) Pro-inflammatory response induced in Huh-7 by the infection with WT, Δ4b or mNLS. RNA was collected at 24 h p.i and quantified by RT-qPCR. Error bars represent standard deviations of the mean (n = 3). Differences with WT group were analyzed by Student’s t test: *, p-value < 0.05; **, p-value < 0.01.</p

Proposed mechanism of action of 4b protein in MERS-CoV virulence.

During MERS-CoV-WT infection, 4b protein accumulates in the nucleus, while in the absence of a functional nuclear localization signal (mNLS), 4b protein is retained in the cytoplasm. The presence of either nuclear or cytoplasmatic 4b is associated with activation of inflammation and inhibition of autophagy, which contribute to virulence in vivo, as shown for MERS-CoV- WT and MERS-CoV-mNLS in a mouse model of infection. According to this work, autophagy contributes to limit inflammation. In the absence of 4b protein, autophagy was activated and a lower inflammatory response was induced, which contributes to attenuation. Chemokine mRNAs increased both in MERS-CoV-WT and MERS-CoV-mNLS infection are indicated in orange. Cytokines mRNA differentially induced in MERS-CoV-WT infection are shown in red. Changes in protein levels are indicated in green.</p

Taqman assays.

Taqman assays.</p

Characterization of MERS-CoV-MA-WT and MERS-CoV-MA-Δ4b in MRC-5 cells.

(A) Subconfluent monolayers of MRC-5 were infected with wild-type or Δ4b at a m.o.i. of 0.1 or 0.001. Culture supernatants were collected at 24, 48 and 72 h p.i. and titrated by plaque assay. The average of two independent experiments is represented. (B) The mRNA expression levels of genes related to the IFN or the pro-inflammatory responses were quantified by RT-qPCR in MRC-5 cells either mock-infected or infected with WT or Δ4b viruses at m.o.i. 1 and 24 h p.i. Error bars represent standard deviations of the mean (n = 3). Differences were analyzed by Student’s t test: *, p-value < 0.05; **, p-value < 0.01.</p

Virulence of MERS-CoV-MA-Δ4b and MERS-CoV-MA-mNLS in mice.

30-week-old hDPP4-KI mice were intranasally inoculated with 5x104 pfu of MERS-CoV-MA-WT (WT), MERS-CoV-MA-Δ4b (Δ4b) or MERS-CoV-MA-mNLS (mNLS) viruses. Weight loss (A) and survival (B) were monitored for 12 days. Error bars represent standard deviations of the mean (n = 5 mice per group). (C) Representative images of pulmonary histopathological lesions (H&E staining; magnification 10x) observed in mice euthanized at 4 and 6 dpi. Alveolar septal thickening, perivascular and peribronchiolar cuffs populated by lymphocytes (black arrows) and alveolar edema (red arrowheads) were among the most characteristic inflammatory lesions observed. (D) Scores associated with edema and cell infiltrates at 4 and 6 dpi. (E) Viral titers in the lungs were determined by plaque assay at 4 and 6 dpi (n = 3).</p

Rainbow trout red blood cells exposed to viral hemorrhagic septicemia virus up-regulate antigen-processing mechanisms and MHC I&II, CD86, and CD83 antigen-presenting cell markers

Nucleated teleost red blood cells (RBCs) are known to express molecules from the major histocompatibility complex and peptide-generating processes such as autophagy and proteasomes, but the role of RBCs in antigen presentation of viruses have not been studied yet. In this study, RBCs exposed ex vivo to viral hemorrhagic septicemia virus (VHSV) were evaluated by means of transcriptomic and proteomic approaches. Genes and proteins related to antigen presentation molecules, proteasome degradation, and autophagy were up-regulated. VHSV induced accumulation of ubiquitinated proteins in ex vivo VHSV-exposed RBCs and showed at the same time a decrease of proteasome activity. Furthermore, induction of autophagy was detected by evaluating LC3 protein levels. Sequestosome-1/p62 underwent degradation early after VHSV exposure, and it may be a link between ubiquitination and autophagy activation. Inhibition of autophagosome degradation with niclosamide resulted in intracellular detection of N protein of VHSV (NVHSV) and p62 accumulation. In addition, antigen presentation cell markers, such as major histocompatibility complex (MHC) class I & II, CD83, and CD86, increased at the transcriptional and translational level in rainbow trout RBCs exposed to VHSV. In summary, we show that nucleated rainbow trout RBCs can degrade VHSV while displaying an antigen-presenting cell (APC)-like profile.This work was supported by the European Research Council (ERC Starting Grant GA639249). The proteomic analysis was performed in the Proteomics Facility of The Spanish National Center for Biotechnology (CNB-CSIC) belonging to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019.Peer reviewe