I. Fernández de Castro, R. Tenorio, P. Ortega-González, J.J. Knowlton, P.F. Zamora, C.H. Lee, J.J. Fernández, T.S. Dermody, C. Risco.
Abstract
Mammalian orthoreoviruses (reoviruses) are nonenveloped viruses that replicate in cytoplasmic membranous organelles called viral inclusions (VIs) where progeny virions are assembled. To better understand cellular routes of nonlytic reovirus exit, we imaged sites of virus egress in infected, nonpolarized human brain microvascular endothelial cells (HBMECs) and observed one or two distinct egress zones per cell at the basal surface. Transmission electron microscopy and 3D electron tomography (ET) of the egress zones revealed clusters of virions within membrane-bound structures, which we term membranous carriers (MCs), approaching and fusing with the plasma membrane. These virion-containing MCs emerged from larger, LAMP-1-positive membranous organelles that are morphologically compatible with lysosomes. We call these structures sorting organelles (SOs). Reovirus infection induces an increase in the number and size of lysosomes and modifies the pH of these organelles from ∼4.5-5 to ∼6.1 after recruitment to VIs and before incorporation of virions. ET of VI-SO-MC interfaces demonstrated that these compartments are connected by membrane-fusion points, through which mature virions are transported. Collectively, our results show that reovirus uses a previously undescribed, membrane-engaged, nonlytic egress mechanism and highlights a potential new target for therapeutic intervention.
DOI: 10.1083/jcb.201910131.
- The virus sequesters these cellular structures to egress new viral particles
- Cellular factors involved in this trafficking pathway may be key to design new antiviral drugs
Researchers from the National Centre for Biotechnology (CNB-CSIC), have described the mechanism used by human reovirus, a pathogen that causes respiratory and digestive diseases in children and young people, to exit infected cells. This virus, that has also been related to the development of celiac disease, is capable of sequestering cellular lysosomes (organelles attached to the cell membrane) to transport infectious viral particles from inside the cell to the surface. The results of this research that could lead to the design of new antiviral drugs, have been published in the Journal of Cell Biology.
Reoviruses build new organelles (viral factories) in the cytoplasm of infected cells to be able to replicate themselves. This study reveals that the human reovirus modifies and uses endoplasmic reticulum (ER) membranes to produce its replication organelles. The reovirus proteins σNS and μNS are in charge of ER remodeling and the use of this modified ER as scaffold for the assembly of viral factories.
MBio. 2018 Aug 7;9(4). pii: e01253-18. doi: 10.1128/mBio.01253-18.
Tenorio R, Fernández de Castro I, Knowlton JJ, Zamora PF, Lee CH, Mainou BA, Dermody TS, Risco C.
Annu Rev Virol 2014; 1: 453-473.
Risco C, Fernández de Castro I, Sanz-Sánchez L, Narayan K, Grandinetti G, Subramaniam S.
ReThree-dimensional (3D) imaging technologies are beginning to have significant impact in the field of virology, as they are helping us understand how viruses take control of cells.
In this article we review several methodologies for 3D imaging of cells and show how these technologies are contributing to the study of viral infections and the characterization of specialized structures formed in virus-infected cells. We include 3D reconstruction by transmission electron microscopy (TEM) using serial sections, electron tomography, and focused ion beam scanning electron microscopy (FIB-SEM).
We summarize from these methods selected contributions to our understanding of viral entry, replication, morphogenesis, egress and propagation, and changes in the spatial architecture of virus-infected cells. In combination with live-cell imaging, correlative microscopy, and new techniques for molecular mapping in situ, the availability of these methods for 3D imaging is expected to provide deeper insights into understanding the structural and dynamic aspects of viral infection.
Los virus ARN, entre los que se encuentran muchos patógenos para humanos, animales y plantas, replican sus genomas en membranas del interior de las células. En estas membranas los virus reclutan factores celulares que participan en el ensamblaje y actividades de los complejos replicativos. Estos agregados macromoleculares, que suelen ensamblarse en vesículas con aperturas al citosol o “esférulas”, se encargan de fabricar múltiples copias del genoma viral que se incorporarán posteriormente en las nuevas partículas virales infectivas. Aunque se han identificado numerosos factores necesarios para el ensamblaje funcional de los orgánulos de replicación viral se desconoce la función exacta de la mayoría de ellos.
En colaboración con investigadores de la Universidad de Kentucky en los Estados Unidos, el laboratorio del Centro Nacional de Biotecnología del CSIC (CNB) dirigido por Cristina Risco han encontrado evidencias de una función inesperada y sorprendente que desempeña la proteína celular Vps4 en la replicación de un virus ARN perteneciente a la familia de los Tombusvirus, patógenos que infectan plantas y que causan importantes pérdidas en cosechas.
Vps4 es un componente de las proteínas celulares ESCRT y de la familia de las ATPasas AAA+ que usan ATP para remodelar estructuras macromoleculares en las células. Vps4 participa en una variedad de procesos biológicos como por ejemplo la fusión de membranas para la formación de vesículas. Utilizando nuevas técnicas de imagen en microscopía electrónica y según acaban de publicar en la revista PLoS Pathogens, los autores han demostrado que Vps4 participa en la estabilización del poro o cuello de las esférulas a través del cual se produce el intercambio de materiales con el citosol. Esta apertura controlada es necesaria para que el complejo replicativo desempeñe sus funciones y para que el ARN viral quede protegido de la degradación por nucleasas.
A diferencia de lo que ocurre en los procesos celulares habituales en los que participa, Vps4 se incorpora de manera estable en las vesículas virales o esférulas y pasa a formar parte del complejo replicativo. De hecho cuando Vps4 no está presente se forman esférulas totalmente abiertas, sin constricción o cuello y en las que el ARN viral de nueva síntesis está desprotegido. Los autores proponen que Vps4 y otras proteínas ESCRT son indispensables para la deformación de las membranas y el ensamblaje de los complejos replicativos de Tombusvirus. Es muy probable que otros virus de plantas y animales que transforman membranas celulares para construir esférulas también utilicen estas proteínas para ensamblar sus replicasas y proteger sus genomas recién sintetizados.
- Barajas D, Martín IFdC, Pogany J, Risco C, Nagy PD. Noncanonical role for the host Vps4 AAA+ ATPase ESCRT protein in the formation of Tomato bushy stunt virus replicase. PLoS Pathog. 2014; 10(4): e1004087.
PLoS Pathog 2014; 10(4): e1004087.
Barajas D, Martín IFdC, Pogany J, Risco C, Nagy PD.
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.
MBio. 2014; 5(1). pii: e00931-13.
Fernández de Castro I, Zamora PF, Ooms L, Fernández JJ, Lai CM, Mainou BA, Dermody TS, Risco C.
Most viruses that replicate in the cytoplasm of host cells form neo-organelles that serve as sites of viral genome replication and particle assembly. These highly specialized structures concentrate viral replication proteins and nucleic acids, prevent the activation of cell-intrinsic defenses, and coordinate the release of progeny particles. Despite the importance of inclusion complexes in viral replication, there are key gaps in the knowledge of how these organelles form and mediate their functions.
Reoviruses are nonenveloped, double-stranded RNA (dsRNA) viruses that serve as tractable experimental models for studies of dsRNA virus replication and pathogenesis. Following reovirus entry into cells, replication occurs in large cytoplasmic structures termed inclusions that fill with progeny virions. Reovirus inclusions are nucleated by viral nonstructural proteins, which in turn recruit viral structural proteins for genome replication and particle assembly. Components of reovirus inclusions are poorly understood, but these structures are generally thought to be devoid of membranes. We used transmission electron microscopy and three-dimensional image reconstructions to visualize reovirus inclusions in infected cells.
These studies revealed that reovirus inclusions form within a membranous network. Viral inclusions contain filled and empty viral particles and microtubules and appose mitochondria and rough endoplasmic reticulum (RER). Immunofluorescence confocal microscopy analysis demonstrated that markers of the ER and ER-Golgi intermediate compartment (ERGIC) codistribute with inclusions during infection, as does dsRNA. dsRNA colocalizes with the viral protein σNS and an ERGIC marker inside inclusions. These findings suggest that cell membranes within reovirus inclusions form a scaffold to coordinate viral replication and assembly.
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