Viruses recruit cellular membranes and subvert cellular proteins involved in lipid biosynthesis to build viral replicase complexes and replication organelles. Among the lipids, sterols are important components of membranes, affecting the shape and curvature of membranes. In this paper, the tombusvirus replication protein is shown to co-opt cellular Oxysterol-binding protein related proteins (ORPs), whose deletion in yeast model host leads to decreased tombusvirus replication. In addition, tombusviruses also subvert Scs2p VAP protein to facilitate the formation of membrane contact sites (MCSs), where membranes are juxtaposed, likely channeling lipids to the replication sites. In all, these events result in redistribution and enrichment of sterols at the sites of viral replication in yeast and plant cells. Using in vitro viral replication assay with artificial vesicles, we show stimulation of tombusvirus replication by sterols. Thus, co-opting cellular ORP and VAP proteins to form MCSs serves the virus need to generate abundant sterol-rich membrane surfaces for tombusvirus replication.

Assembling of the membrane-bound viral replicase complexes (VRCs) consisting of viral- and host-encoded proteins is a key step during the replication of positive-stranded RNA viruses in the infected cells. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host have revealed the involvement of eleven cellular ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. The ESCRT proteins are involved in endosomal sorting of cellular membrane proteins by forming multiprotein complexes, deforming membranes away from the cytosol and, ultimately, pinching off vesicles into the lumen of the endosomes. In this paper, we show an unexpected key role for the conserved Vps4p AAA+ ATPase, whose canonical function is to disassemble the ESCRT complexes and recycle them from the membranes back to the cytosol. We find that the tombusvirus p33 replication protein interacts with Vps4p and three ESCRT-III proteins. Interestingly, Vps4p is recruited to become a permanent component of the VRCs as shown by co-purification assays and immuno-EM. Vps4p is co-localized with the viral dsRNA and contacts the viral (+)RNA in the intracellular membrane. Deletion of Vps4p in yeast leads to the formation of crescent-like membrane structures instead of the characteristic spherule and vesicle-like structures. The in vitro assembled tombusvirus replicase based on cell-free extracts (CFE) from vps4Δ yeast is highly nuclease sensitive, in contrast with the nuclease insensitive replicase in wt CFE. These data suggest that the role of Vps4p and the ESCRT machinery is to aid building the membrane-bound VRCs, which become nuclease-insensitive to avoid the recognition by the host antiviral surveillance system and the destruction of the viral RNA. Other (+)RNA viruses of plants and animals might also subvert Vps4p and the ESCRT machinery for formation of VRCs, which require membrane deformation and spherule formation.

Transport of neo-synthesized influenza A virus (IAV) viral ribonucleoproteins (vRNPs) from the nucleus to the plasma membrane involves Rab 11 but the precise mechanism remains poorly understood. We used metal-tagging and immunolabeling to visualize viral proteins and cellular endomembrane markers by electron microscopy of IAV-infected cells. Unexpectedly, we provide evidence that the vRNP components and the Rab11 protein are present at the membrane of a modified, tubulated endoplasmic reticulum (ER) that extends all throughout the cell, and on irregularly coated vesicles (ICVs). Some ICVs are found very close to the ER and to the plasma membrane. ICV formation is observed only in infected cells and requires an active Rab11 GTPase. Against the currently accepted model in which vRNPs are carried onto Rab11-positive recycling endosomes across the cytoplasm, our findings reveal that the endomembrane organelle that is primarily involved in the transport of vRNPs is the ER.

We have performed a sequential study on the abundance of the mRNA for uncoupling protein (UCP), subunit I1 of cytochrome-c oxidase (COII) and lipoprotein lipase in brown adipose tissue during the fetal and postnatal periods. Moreover, we have determined whether these parameters can be modulated by ambient temperature in the early hours after birth, and at which point in development this sensitivity first appears.UCP gene expression in the fetal and neonatal period has particular features when compared with overall mitochondriogenesis (COII mRNA expression) or with the expression of lipoprotein lipase mRNA. There is a specific induction of UCP gene expression between days 18 and 19 of pregnancy followed by a specific increase of UCP gene expression in utero and a further increase after birth. The acquisition of the physiological apparatus capable of the response to UCP and lipoprotein lipase gene expression to the environmental temperature is not achieved until the last day of fetal development. This result suggests that mechanisms of P-adrenergic modulation of gene expression in brown fat are already established at birth.From an experiment on iopanoic acid treatment of pregnant mothers, it was concluded that iodothyronine 5'-deiodinase activity is not necessary for the expression of the mRNAs for UCP, COII and lipoprotein lipase in the fetus whereas it is necessary for the acquisition of temperature sensitivity to these parameters at birth.Brown fat is a specialized tissue of mammals responsible for facultative thermogenesis. Its physiological significance has long been recognized in newborns when the decrease in environmental temperature at birth requires an adaptative increase in heat production [l]. Brown adipose tissue possesses a unique mitochondrial protein (so called uncoupling protein, UCP) that uncouples oxidative phosphorylation from the respiratory chain. In this way, the energy of metabolic oxidation in this tissue is dissipated as heat (for a review see [2]).Noradrenaline regulates the overall thermogenic activity of brown adipose tissue [3] and specifically modulates the expression of the UCP gene at the transcriptional level [4]. However, noradrenaline is not the only factor involved in the modulation of brown fat thermogenesis. Since the discovery of a iodothyronine 5'-deiodinase activity in brown adipose tissue [5], capable of producing 3,3',5-triiodothyronine from systemic thyroxine, a strong correlation has been established between 5'-deiodinase and thermogenic activity in different situations of adaptative increase or decrease in brown fat thermogenesis [6 -81. A key role for 3,3'$triiodothyronine, generated in situ in the regulation of brown fat activity, has therefore been suggested.

Plus-stranded RNA viruses induce membrane deformations in infected cells in order to build viral replication complexes (VRCs).One of the intriguing aspects of VRC formation is the need for extensive subcellular membrane deformations. Accordingly, (ϩ)RNA viruses replicate in various membranous structures that could be formed from various membranes, such as endoplasmic reticulum, mitochondria, endosome, chloroplast, peroxisome, and plasma membranes, or induced de novo (1,(3)(4)(5). Membrane deformations are possibly induced by co-opted cellular phospholipid kinases, local enrichment of sterols, and subverted membrane-bending proteins, such as ESCRT factors, reticulons, and amphiphysins (6-12).A major type of subcellular membrane deformation induced by some (ϩ)RNA viruses is represented by vesicle-like small invaginations with single narrow openings toward the cytosol (13,14). These structures, called spherules, contain the membranebound VRCs consisting of viral and co-opted cellular proteins in the infected cells (1)(2)(3)(15)(16)(17)(18). The membranous spherule structures sequester all the replication factors into a confined cytosolic area and likely protect the fragile viral (ϩ)RNA from degradation