Estudio de los virus de peces del banco pesquero Flemish Cap (Terranova)

En el presente estudio se analizaron un total de 250 peces sin síntomas aparentes de enfermedad, pertenecientes a 8 especies: gallineta (Sebastes mentella), fletán negro (Reinhardtius hippoglossoides), platija americana (Hippoglossoides platessoides), coreano (Glyptocephalus cynoglossus), bacalao (Gadus morhua), granadero (Macrourus berglax), perro del norte (Anarhichas lupus) y Antimora rostrata que se capturaron en el transcurso de una campaña de investigación pesquera realizada a bordo del buque oceanográfico Cornide de Saavedra en aguas de Terranova, en el banco pesquero Flemish Cap, durante los meses de junio y julio de 1999.In the present study we have analyzed a total of 250 apparently healthy fish, belonging to 8 different species: deepwater redfish (Sebastes mentella), Greenland halibut (Reinhardtius hippoglossoides), American plaice (Hippoglossoides platessoides), witch flounder (Glyptocephalus cynoglossus), Atlantic cod (Gadus morhua), onion-eye grenadier (Macrourus berglax), Atlantic wolf-fish (Anarhichas lupus) and blue antimora (Antimora rostrata). These fish were caught in the summer of 1999 during a marine research campaign carried out in the Flemish Cap fishery, close to Newfoundland

Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories

Membranous Replication Factories Induced by Plus-Strand RNA Viruses

In this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) RNA viruses. We discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. A general denominator is that two “morphotypes” of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (ER) and double membrane vesicles, representing extrusions also originating from the ER, respectively. We hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments

Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells

As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications

Endoplasmic Reticulum: The Favorite Intracellular Niche for Viral Replication and Assembly

The endoplasmic reticulum (ER) is the largest intracellular organelle. It forms a complex network of continuous sheets and tubules, extending from the nuclear envelope (NE) to the plasma membrane. This network is frequently perturbed by positive-strand RNA viruses utilizing the ER to create membranous replication factories (RFs), where amplification of their genomes occurs. In addition, many enveloped viruses assemble progeny virions in association with ER membranes, and viruses replicating in the nucleus need to overcome the NE barrier, requiring transient changes of the NE morphology. This review first summarizes some key aspects of ER morphology and then focuses on the exploitation of the ER by viruses for the sake of promoting the different steps of their replication cycles

Membrane alterations induced by nonstructural proteins of human norovirus.

Human noroviruses (huNoV) are the most frequent cause of non-bacterial acute gastroenteritis worldwide, particularly genogroup II genotype 4 (GII.4) variants. The viral nonstructural (NS) proteins encoded by the ORF1 polyprotein induce vesical clusters harboring the viral replication sites. Little is known so far about the ultrastructure of these replication organelles or the contribution of individual NS proteins to their biogenesis. We compared the ultrastructural changes induced by expression of norovirus ORF1 polyproteins with those induced upon infection with murine norovirus (MNV). Characteristic membrane alterations induced by ORF1 expression resembled those found in MNV infected cells, consisting of vesicle accumulations likely built from the endoplasmic reticulum (ER) which included single membrane vesicles (SMVs), double membrane vesicles (DMVs) and multi membrane vesicles (MMVs). In-depth analysis using electron tomography suggested that MMVs originate through the enwrapping of SMVs with tubular structures similar to mechanisms reported for picornaviruses. Expression of GII.4 NS1-2, NS3 and NS4 fused to GFP revealed distinct membrane alterations when analyzed by correlative light and electron microscopy. Expression of NS1-2 induced proliferation of smooth ER membranes forming long tubular structures that were affected by mutations in the active center of the putative NS1-2 hydrolase domain. NS3 was associated with ER membranes around lipid droplets (LDs) and induced the formation of convoluted membranes, which were even more pronounced in case of NS4. Interestingly, NS4 was the only GII.4 protein capable of inducing SMV and DMV formation when expressed individually. Our work provides the first ultrastructural analysis of norovirus GII.4 induced vesicle clusters and suggests that their morphology and biogenesis is most similar to picornaviruses. We further identified NS4 as a key factor in the formation of membrane alterations of huNoV and provide models of the putative membrane topologies of NS1-2, NS3 and NS4 to guide future studies

The Lipid Kinase Phosphatidylinositol-4 Kinase III Alpha Regulates the Phosphorylation Status of Hepatitis C Virus NS5A

<div><p>The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα) is an essential host factor of hepatitis C virus (HCV) replication. PI4KIIIα catalyzes the synthesis of phosphatidylinositol 4-phosphate (PI4P) accumulating in HCV replicating cells due to enzyme activation resulting from its interaction with nonstructural protein 5A (NS5A). This study describes the interaction between PI4KIIIα and NS5A and its mechanistic role in viral RNA replication. We mapped the NS5A sequence involved in PI4KIIIα interaction to the carboxyterminal end of domain 1 and identified a highly conserved PI4KIIIα functional interaction site (PFIS) encompassing seven amino acids, which are essential for viral RNA replication. Mutations within this region were also impaired in NS5A-PI4KIIIα binding, reduced PI4P levels and altered the morphology of viral replication sites, reminiscent to the phenotype observed by silencing of PI4KIIIα. Interestingly, abrogation of RNA replication caused by mutations in the PFIS correlated with increased levels of hyperphosphorylated NS5A (p58), indicating that PI4KIIIα affects the phosphorylation status of NS5A. RNAi-mediated knockdown of PI4KIIIα or pharmacological ablation of kinase activity led to a relative increase of p58. In contrast, overexpression of enzymatically active PI4KIIIα increased relative abundance of basally phosphorylated NS5A (p56). PI4KIIIα therefore regulates the phosphorylation status of NS5A and viral RNA replication by favoring p56 or repressing p58 synthesis. Replication deficiencies of PFIS mutants in NS5A could not be rescued by increasing PI4P levels, but by supplying functional NS5A, supporting an essential role of PI4KIIIα in HCV replication regulating NS5A phosphorylation, thereby modulating the morphology of viral replication sites. In conclusion, we demonstrate that PI4KIIIα activity affects the NS5A phosphorylation status. Our results highlight the importance of PI4KIIIα in the morphogenesis of viral replication sites and its regulation by facilitating p56 synthesis.</p></div

Triple alanine mutants induce ultrastructural changes similar to membranous web structures in PI4KIIIα knockdown cells.

<p>Huh7-Lunet T7 cells (A) or Huh7-Lunet T7 cells with stable PI4KIIIα knockdown (B) were transfected with pTM constructs expressing wt or mutant NS3 to NS5B polyproteins or eGFP. Cells were fixed and prepared for EM analysis 24 h post transfection. Consecutive enlargements of the boxed areas are shown from left to right. Note the heterogeneous membranous web (MW, yellow arrows) in cells expressing the wt polyprotein and the clusters of smaller double-membrane vesicles (DMVs) in shPI4KIIIα cells (B) and in cells expressing mutant polyproteins. Scale bars are given in the lower right of each panel. N, nucleus; LD, lipid droplet; rER, rough endoplasmic reticulum; m, mitochondrium. The number in the upper left of right panels shows the average diameter of 70 double-membranous vesicles (DMV) +/− SD measured for each condition. (C) Average diameter of DMVs detected in cells that had been transfected with constructs and conditions specified on the left and shown in panel A and B. Error bars indicate the mean +/− SD of seventy vesicles. Significance of differences in DMV sizes was measured by a paired t-test and is indicated ***, p<0.001.</p

Reduced PI4KIIIα interaction and RNA replication deficiency correlate with relative increase of NS5A hyperphosphorylation.

<p>A: Huh7-Lunet T7 cells were transfected with plasmids encoding the NS3 to NS5B polyprotein of genotype 2a (JFH-1) containing a wt sequence or triple alanine mutants as indicated or with empty plasmid (mock). Newly synthesized proteins were radiolabeled and cell lysates subjected to immunoprecipitation using NS5A specific antibodies. Immunocomplexes were analyzed by SDS-PAGE and autoradiography. B: Quantitative analysis of the NS5A p58/p56 ratio. Bands corresponding to NS5A p58 and p56, respectively, as shown in panel A were individually quantified by phosphoimaging to obtain a p58/p56 ratio. Error bars indicate mean values +/− SD of two independent experiments analyzed in duplicates. Significance was compared to the wt polyprotein and calculated by a paired t-test. *, p<0.05; **, p<0.01; ***, p<0.001. A blow up of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003359#ppat-1003359-g006" target="_blank">figure 6A</a> focusing on the NS5A phosphorylation of PFIS mutants in comparison to the wildtype polyprotein is shown below.</p