Enhanced SARS-CoV-2 entry via UPR-dependent AMPK-related kinase NUAK2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remodels the endoplasmic reticulum (ER) to form replication organelles, leading to ER stress and unfolded protein response (UPR). However, the role of specific UPR pathways in infection remains unclear. Here, we found that SARS-CoV-2 infection causes marginal activation of signaling sensor IRE1α leading to its phosphorylation, clustering in the form of dense ER-membrane rearrangements with embedded membrane openings, and XBP1 splicing. By investigating the factors regulated by IRE1α-XBP1 during SARS-CoV-2 infection, we identified stress-activated kinase NUAK2 as a novel host-dependency factor for SARS-CoV-2, HCoV-229E, and MERS-CoV entry. Reducing NUAK2 abundance or kinase activity impaired SARS-CoV-2 particle binding and internalization by decreasing cell surface levels of viral receptors and viral trafficking likely by modulating the actin cytoskeleton. IRE1α-dependent NUAK2 levels were elevated in SARS-CoV-2-infected and bystander non-infected cells, promoting viral spread by maintaining ACE2 cell surface levels and facilitating virion binding to bystander cells.

Evidence of SARS-CoV-2 infection in postmortem lung, kidney, and liver samples, revealing cellular targets involved in COVID-19 pathogenesis.

There is an urgent need to understand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-host interactions involved in virus spread and pathogenesis, which might contribute to the identification of new therapeutic targets. In this study, we investigated the presence of SARS-CoV-2 in postmortem lung, kidney, and liver samples of patients who died with coronavirus disease (COVID-19) and its relationship with host factors involved in virus spread and pathogenesis, using microscopy-based methods. The cases analyzed showed advanced stages of diffuse acute alveolar damage and fibrosis. We identified the SARS-CoV-2 nucleocapsid (NC) in a variety of cells, colocalizing with mitochondrial proteins, lipid droplets (LDs), and key host proteins that have been implicated in inflammation, tissue repair, and the SARS-CoV-2 life cycle (vimentin, NLRP3, fibronectin, LC3B, DDX3X, and PPARγ), pointing to vimentin and LDs as platforms involved not only in the viral life cycle but also in inflammation and pathogenesis. SARS-CoV-2 isolated from a patient´s nasal swab was grown in cell culture and used to infect hamsters. Target cells identified in human tissue samples included lung epithelial and endothelial cells; lipogenic fibroblast-like cells (FLCs) showing features of lipofibroblasts such as activated PPARγ signaling and LDs; lung FLCs expressing fibronectin and vimentin and macrophages, both with evidence of NLRP3- and IL1ß-induced responses; regulatory cells expressing immune-checkpoint proteins involved in lung repair responses and contributing to inflammatory responses in the lung; CD34+ liver endothelial cells and hepatocytes expressing vimentin; renal interstitial cells; and the juxtaglomerular apparatus. This suggests that SARS-CoV-2 may directly interfere with critical lung, renal, and liver functions involved in COVID-19-pathogenesis.

Ultrastructural and biochemical basis for hepatitis C virus morphogenesis.

Chronic infection with HCV is a leading cause of cirrhosis, hepatocellular carcinoma and liver failure. One of the least understood steps in the HCV life cycle is the morphogenesis of new viral particles. HCV infection alters the lipid metabolism and generates a variety of microenvironments in the cell cytoplasm that protect viral proteins and RNA promoting viral replication and assembly. Lipid droplets (LDs) have been proposed to link viral RNA synthesis and virion assembly by physically associating these viral processes. HCV assembly, envelopment, and maturation have been shown to take place at specialized detergent-resistant membranes in the ER, rich in cholesterol and sphingolipids, supporting the synthesis of luminal LDs-containing ApoE. HCV assembly involves a regulated allocation of viral and host factors to viral assembly sites. Then, virus budding takes place through encapsidation of the HCV genome and viral envelopment in the ER. Interaction of ApoE with envelope proteins supports the viral particle acquisition of lipids and maturation. HCV secretion has been suggested to entail the ion channel activity of viral p7, several components of the classical trafficking and autophagy pathways, ESCRT, and exosome-mediated export of viral RNA. Here, we review the most recent advances in virus morphogenesis and the interplay between viral and host factors required for the formation of HCV virions.

The microglial NLRP3 inflammasome is involved in human SARS-CoV-2 cerebral pathogenicity: A report of three post-mortem cases.

We herein report, by using confocal immunofluorescence, the colocalization of the SARS-CoV-2 nucleocapsid within neurons, astrocytes, oligodendrocytes and microglia in three deceased COVID-19 cases, of between 78 and 85 years of age at death. The viral nucleocapsid was detected together with its ACE2 cell entry receptor, as well as the NLRP3 inflammasome in cerebral cortical tissues. It is noteworthy that NLRP3 was colocalized with CD68 + macrophages in the brain and lung of the deceased, suggesting the critical role of this type of inflammasome in SARS-CoV-2 lesions of the nervous system/lungs and supporting its potential role as a therapeutic target.

Ratio of HCV structural antigens in protein-based vaccine formulations is critical for functional immune response induction.

HCV (hepatitis C virus) infection is among the leading causes of chronic liver disease, but currently there is no vaccine available. Data have accumulated about the importance of targeting different HCV antigens in vaccine candidate preparations. Here, a surface response study to select the optimal ratio of recombinant HCV structural antigens in a vaccine preparation, capable of generating in vivo functional cellular immune response in mice, was performed. The immunogenicity of the selected HCV structural protein mixture (Co-E1-E2) in mice and African green monkeys, after five doses of immunization, was also demonstrated. Specific T-cell proliferative response against HCV structural antigens was induced in vaccinated mice. Moreover, on challenge with recombinant HCV VV (vaccinia virus), all mice controlled the viraemia and 80% were protected. On the other hand, monkeys immunized with Co-E1-E2 developed antibodies, specifically directed to region 412-438 of E2 protein, that include an epitope implicated in HCV neutralization, in addition to a specific proliferative response against HCV Core and E2 proteins. These results indicated that the optimal amount and ratio of HCV recombinant proteins should be taken into account to elicit a successful immune response against HCV and therefore have important implications for vaccine design.

Recombinant in vitro assembled hepatitis C virus core particles induce strong specific immunity enhanced by formulation with an oil-based adjuvant.

In the present work, immunogenicity of recombinant in vitro assembled hepatitis C virus core particles, HCcAg.120-VLPs, either alone or in combination with different adjuvants was evaluated in BALB/c mice. HCcAg.120-VLPs induced high titers of anti-HCcAg.120 antibodies and virus-specific cellular immune responses. Particularly, HCcAg.120-VLPs induced specific delayed type hypersensitivity, and generated a predominant T helper 1 cytokine pro file in immunized mice. In addition, HCcAg.120-VLPs prime splenocytes proliferate in vitro against different HCcAg.120-specific peptides, depending on either the immunization route or the adjuvant used. Remarkably, immunization with HCcAg.120-VLPs/Montanide ISA888 formulation resulted in a significant control of vaccinia virus titer in mice after challenge with a recombinant vaccinia virus expressing HCV core protein, vvCore. Animals immunized with this formulation had a marked increase in the number of IFN-gamma producing spleen cells, after stimulation with P815 cells infected with vvCore. These results suggest the use of recombinant HCV core particles as components of therapeutic or preventive vaccine candidates against HCV.

Immunization with a recombinant fowlpox virus expressing a hepatitis C virus core-E1 polyprotein variant, protects mice and African green monkeys (Chlorocebus aethiops sabaeus) against challenge with a surrogate vaccinia virus.

HCV (hepatitis C virus) is a worldwide health problem nowadays. No preventive vaccine is available against this pathogen, and therapeutic treatments currently in use have important drawbacks, including limited efficacy. In the present work a recombinant fowlpox virus, FPCoE1, expressing a truncated HCV core-E1 polyprotein, was generated. FPCoE1 virus generally failed to elicit a humoral immune response against HCV antigens in BALB/c mice. By contrast, mice inoculated with FPCoE1 elicited a positive interferon-gamma secretion response against HCV core in ex-vivo ELISPOT (enzyme-linked immunospot) assays. Remarkably, mice inoculated with FPCoE1 significantly controlled viraemia in a surrogate challenge model with vvRE, a recombinant vaccinia virus expressing HCV structural antigens. In fact, 40% of the mice had no detectable levels of vvRE in their ovaries. Administration of FPCoE1 in vervet monkeys [Chlorocebus (formerly Cercophitecus) aethiops sabaeus] induced lymphoproliferative response against HCV core and E1 proteins in 50% of immunized animals. Monkeys immunized with FPCoE1 had no detectable levels of vvRE in their blood, whereas monkeys inoculated with FP9, the negative control virus, had detectable levels of vvRE in blood up to 7 days after challenge. In conclusion, recombinant fowlpox virus FPCoE1 is able to induce an anti-HCV immune response in mice and monkeys. This ability could be rationally employed to develop effective strategies against HCV infection by using FPCoE1 in combination with other vaccine candidates or antiviral treatments.

High prevalence of a variety of autoantibodies in a population of hepatitis C virus-infected individuals.

Hepatitis C virus (HCV) infection has been related to self-reactivity, extrahepatic manifestations and autoimmune diseases. The main goals of this work were to study the prevalence of autoantibodies and their relationship with viral titers and biochemical markers of hepatic damage in patients infected with HCV. Autoantibodies (ANA, AMA, SMA, APC, LKM, DNAds, ANCA, ATG and RF) were determined in 73 individuals with chronic HCV infection and 44 healthy volunteers. The presence of these antibodies was related to demographic variables, viral titers and biochemical parameters. A high prevalence of autoantibodies, particularly for RF, that was associated with female gender was observed in HCV-infected patients. In addition, SMA, ANA and ATG showed increased frequencies in HCV infection. Interestingly, the concurrent detection of SMA and more than one autoantibody was associated with high gGT levels. Notably, concurrent higher gGT, HCV and SMA levels were observed in male patients as compared to their female counterparts. These results indicate a relationship between HCV infection and the concurrent detection of various autoantibodies in the absence of symptoms of autoimmune diseases. They also suggest a link among the presence of a variety of autoantibodies simultaneously with SMA, increased gGT levels and HCV titers in a population of male patients.

Multimeric HCV E2 protein obtained from Pichia pastoris cells induces a strong immune response in mice.

Production of immunogenic hepatitis C virus (HCV) envelope proteins will assist in the future development of preventive or therapeutics applications. Only properly folded monomeric E2 protein is able to bind a putative cellular co-receptor CD81, but this interaction may modulate cell immune function. Recombinant E2 proteins, similar to the native form, but lacking undesirable immunoregulatory features, might be promising components of vaccine candidates against HCV. To obtain E2 suitable for structural as well as functional studies, a recombinant E2 variant (E2680) was produced in Pichia pastoris cells. E2680, comprising amino acids 384 to 680 of the HCV polyprotein, was secreted into the culture supernatant in the N-glycosilated form and was mainly composed of disulfide-linked multimers. Both monomeric and oligomeric forms of E2680 were recognized by conformational-sensitive MAb H53. In addition, antibodies in sera from 70% of HCVpositive patients were reactive against E2680. By immunizing E2680 in BALB/c mice, both a specific cellular immune response and anti-E2680 IgG antibody titers of 1:200,000 were induced. Our data suggest that recombinant E2680 could be useful to successfully induce strong anti-HCV immunity.

Enhancement of the immune response generated against hepatitis C virus envelope proteins after DNA vaccination with polyprotein-encoding plasmids.

Plasmids expressing variants of the hepatitis C virus (HCV) core, E1 and E2 proteins individually or as polyproteins were administered to BALB/c mice. All plasmids induced a detectable and specific antibody response. Antibody titres against core, E1 and E2 proteins, 19 weeks after primary immunization, ranged from 1:50 to 1:4500 depending on the inoculated plasmid and the HCV antigen evaluated. Constructs expressing HCV envelope proteins as polyprotein variants including the core amino acid region induced statistically stronger antibody responses than plasmids encoding individual E1 and E2 proteins. Particularly, the pIDKE2 plasmid, expressing the first 650 amino acids in the viral polyprotein, induced a potent and multispecific antibody and lymphoproliferative response against HCV core, E1 and E2 proteins. Anti-E2 antibodies generated by pIDKE2 immunization were cross-reactive to hypervariable region-1 peptides from different genotypes. Immunization with the pIDKE2 also generated a positive cellular immune response against the core antigen, determined by interferon-gamma enzyme-linked immunospot (ELISPOT) assay, and induced detectable levels of interferon-gamma but not interleukin-4 in vaccinated mice. The detection of both antibody and cytotoxic T-lymphocyte responses, potentially targeted to circulating or cell-infecting virions respectively, in mice vaccinated with the pIDKE2 plasmid is very attractive for the effective eradication of HCV infection.

Biología molecular del virus de la hepatitis C / Molecular biology of hepatitis C virus

Resumen La infección por el virus de la hepatitis C (VHC) se distribuye en todo el mundo, frecuentemente se convierte en hepatitis crónica, cirrosis y hepatocarcinoma. El genoma del VHC es una molécula de ARN monocatenario, de polaridad positiva, de aproximadamente 9.6 kb de longitud. Esta revisión resume el conocimiento actual y los avances recientes en la investigación de la biología molecular del VHC.

Abstract Infection with hepatitis C virus (HCV), which is distributed worldwide, often becomes in chronic hepatitis, cirrhosis and hepatocellular carcinoma. The HCV genome is a single-stranded RNA molecule of positive polarity approximately 9.6 kb in length. This review summarizes the current knowledge of recent advances in the investigation of the molecular biology of HCV.

Hepatitis C virus (HCV) core protein enhances the immunogenicity of a co-delivered DNA vaccine encoding HCV structural antigens in mice.

In the present study, recombinant HCV (hepatitis C virus) core proteins enhanced the immune response elicited by a co-delivered DNA vaccine encoding HCV core and envelope proteins. A mixture of the plasmid pIDKE2 and Co.173, a protein comprising the first 173 amino acids of HCV core, in particular induces a strong humoral response, including antibodies that recognized peptides representing hypervariable region I from different viral isolates. Moreover, positive lymphoproliferative responses against the HCV structural proteins, encoded by the plasmid, were detected after two doses with this mixture. When the HCV core protein used in the mixture with pIDKE2 was Co.120, a protein comprising the first 120 amino acids of the viral antigen, a strong humoral response and a positive lymphoproliferative response were also detected. The effectiveness of this formulation was tested in vivo by measuring the protection against infection with a recombinant vaccinia virus expressing HCV core protein. A 2 log reduction in vaccinia-virus titre was observed in mice immunized with the mixture of pIDKE2 and Co.120. Humoral and cellular immune responses elicited for the mixture of pIDKE2 with either Co.173 or Co.120 was stronger and more diverse than those generated by individual components. In conclusion, our results indicate that formulations comprising both DNA constructs and protein subunit vaccine candidates are able to elicit strong humoral and cellular immunity against several antigens. Particularly, HCV core protein might be used as a feasible vehicle/adjuvant for DNA vaccines.

Expression and processing of hepatitis C virus structural proteins in Pichia pastoris yeast.

Development of heterologous systems to produce useful HCV vaccine candidates is an important part of HCV research. In this study different HCV structural region variants were designed to express the first 120 aa, 176 aa, 339 aa, and 650 aa of HCV polyprotein, and aa 384 to 521, or aa 384-605 or aa 384-746 of HCV E2 protein fused to the leader sequence of sucrose invertase 2 allowing the secretion of recombinant E2 proteins. Low expression levels were observed for HCV core protein (HCcAg) variants expressing the first 120 aa and 176 aa (HCcAg.120 and HCcAg.176, respectively). Higher expression levels were observed when HCcAg was expressed as a polypeptide with either E1 or E1 and E2 proteins. In addition, HCcAg was processed to produce two antigenic bands with 21 and 23kDa (P21 and P23, respectively) when expressed as a polypeptide with HCV E1 and E2 proteins. Results also suggest E1 processing in the context of HCcAg.E1.E2 polyprotein. On the other hand, E2.521, E2.605, and E2.680 were efficiently excreted to the culture medium. However, the entire E2.746 variant predominantly localized in the insoluble fraction of ruptured cells. Results suggest that the hydrophobic C-terminal E2 region from aa 681 to 746 is critical for intracellular retention of recombinant E2.746 protein in Pichia pastoris cells. Endo H or PNGase F treatment suggests that E2.746 was modified with high-mannose type oligosaccharides in P. pastoris. These data justify the usefulness of P. pastoris expression system to express HCV structural viral proteins which may be useful targets for HCV vaccine candidates.

A C-terminal truncated hepatitis C virus core protein variant assembles in vitro into virus-like particles in the absence of structured nucleic acids.

Little is known about the assembly pathway or structure of the hepatitis C virus (HCV). In this work a truncated HCcAg variant covering the first 120 aa (HCcAg.120) with a 32 aa N-terminal fusion peptide (6x Histag-Xpress epitope) was purified as a monomer under strong denaturing conditions. In addition, minor HCcAg.120 peaks exhibiting little different molecular mass by SDS-PAGE which possibly represents alternative forms harboring the N-termini of HCcAg.120 were detected. Analysis using gel filtration chromatography showed that HCcAg.120 assembled into high molecular weight structures in vitro in the absence of structured nucleic acids. The negative-stain electron microscopy analysis revealed that these structures correspond with spherical VLPs of uniform morphology and size distribution. The diameters of these particles ranged from 20 to 43nm with an average diameter of approximately 30 nm and were specifically immunolabelled with a mouse monoclonal antibody against the residues 5-35 of HCcAg. Results presented in this work showed that HCcAg.120 assembled in vitro into VLPs in the absence of structured nucleic acids with similar morphology and size distribution to those found in sera and hepatocytes from HCV-infected patients. Therefore, these VLPs would be important to elucidate the mechanisms behind the ability of HCcAg to assemble into a nucleocapsid structure.

HCV core protein localizes in the nuclei of nonparenchymal liver cells from chronically HCV-infected patients.

Understanding the mechanism of hepatitis C virus (HCV) pathogenesis is an important part of HCV research. Recent experimental evidence suggests that the HCV core protein (HCcAg) has numerous functional activities. These properties suggest that HCcAg, in concert with cellular factors, may contribute to pathogenesis during persistent HCV infection. HCV is capable of infecting cells other than hepatocytes. Although the extrahepatic cellular tropism of HCV may play a role in the pathophysiology of this infection, the precise biological significance of the presence of HCV components in different liver cell types presently remains to be established. In this study, HCcAg was detected in nonparenchymal liver cells of six patients out of eight positive for serum HCV RNA. Immunostaining with anti-HCcAg mAbs revealed the presence of this protein in different liver cell types such as lymphocytes, Kupffer, polymorphonuclear, pit, endothelial, stellate, and fibroblast-like cells. Interestingly, HCcAg was immunolabeled not only in the cytoplasm but also in the nucleus of these cells. Remarkably, HCcAg co-localized with large lipid droplets present in stellate cells and with collagen fibers in the extracellular matrix. Moreover, HCcAg was immunolabeled in bile canaliculus suggesting the involvement of the biliary system in the pathobiology of HCV. Data suggest that nonparenchymal liver cells may constitute a reservoir for HCV replication. Besides, HCcAg may contribute to modulate immune function and fibrosis in the liver as well as steatosis.

Improvement of human interferon HUIFNalpha2 and HCV core protein expression levels in Escherichia coli but not of HUIFNalpha8 by using the tRNA(AGA/AGG).

High-level expression from one particular heterologous gene in Escherichia coli generally requires the optimization of codon usage. Genes encoding for Hepatitis C virus core protein (HCcAg), human interferon alpha2 and 8 subtypes (HUIFNalpha2 and HUIFNalpha8) show a high content of AGA/AGG codons. These are encoded by the product of the dnaY gene in E. coli. The proteins used in this work have a high therapeutic value and were used as models for studying the effects of these rare codons on the efficiency of heterologous gene expression in E. coli. Expression plasmids were constructed to express any of these proteins and the dnaY gene product simultaneously in E. coli. After dnaY gene expression, HCcAg, and HUIFNalpha2 expression levels increased 5 and 3 times, respectively. However, HUIFNalpha8 expression was barely detected either supplying or not the additional dnaY gene product. These results suggest that the high frequency of AGA/AGG codons present in the HCcAg and HUIFNalpha2 genes could be one of the factors limiting its expression in E. coli. Nevertheless, for HUIFNalpha8 it seems that other factors prevail upon the lack of dnaY product. Data presented here for HCcAg and HUIFNalpha2 expressions proved the value of this approach to obtain therapeutic proteins in E. coli.

HCV core protein-expressing DNA vaccine induces a strong class I-binding peptide DTH response in mice.

The hepatitis C virus (HCV) core protein-encoding sequence (HCcAg) is the most conserved gene in HCV genome and therefore may be useful to study broadly reacting T-cell epitopes. In this study BALB/c and C57BL/6 mice were immunized with a DNA based vaccine expressing the first 176 aa of HCcAg (pIDKCo). After i.m or i.p injection of pIDKCo in BALB/c mice, a detectable INF-gamma secreting response to the relevant class I-binding peptide DLMGYIPLVGA (P1) (aa 132-142) was detected suggesting the induction of HCcAg specific CD8(+) T-cell effectors. CD8(+) T-cell responses were also monitored in vivo by T-cell-mediated DTH reactions after subcutaneous injection of class I-binding viral peptide P1. pIDKCo induced a strong P1-specific DTH response in both i.m and i.p immunized mice. To evaluate the T-cell response induced by pIDKCo in C57BL/6 mice, an HCcAg epitope was predicted based upon it containing the H-2K(b) binding motif XXXXF/YXXL (DLMGYIPL (P2)). pIDKCo induced a strong P2-specific DTH response with similar kinetics of swelling response to that observed in BALB/c mice. Previously, it had been demonstrated that only activated and protective CD8(+) effector T cells could mediate a specific DTH in footpads of virally infected mice after local injection of viral class I-binding peptides. Hence, pIDKCo could prime a strong HCcAg-specific T-cell response in mice with the potential capacity to exert their specific effector functions in peripheral tissues.

Processing of the Hepatitis C virus precursor protein expressed in the methylotrophic yeast Pichia pastoris.

The expression and processing of the Hepatitis C virus core protein (HCcAg) were analyzed in the methylotrophic yeast Pichia pastoris. Two proteins with 21 (p21) and 23 kDa (p23) were detected in immunoblot with a serum from a chronic carrier patient, as the major products for HCcAg. Both proteins, p21 and p23, produced by proteolytic processing in P. partoris, share the same N-terminal region and reacted with a monoclonal antibody towards the first 35 amino acids of HCcAg. The proteolytic processing of the recombinant polypeptide, having the HCcAg and the first 148 aa of E1 protein, was also confirmed by immunoblot analysis using mAbs with HCcAg and E1 specificities, respectively. The 32 kDa glycosilated E1 protein was then immuno-identified, as well as the processed HCcAg. These data demonstrated the usefulness of P. pastoris, as expression system, to study the processing of HCV structural proteins.