Reovirus forms neo-organelles for progeny particle assembly within reorganized cell membranes.
ABSTRACT: 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 ?NS and an ERGIC marker inside inclusions. These findings suggest that cell membranes within reovirus inclusions form a scaffold to coordinate viral replication and assembly.Viruses alter the architecture of host cells to form an intracellular environment conducive to viral replication. This step in viral infection requires the concerted action of viral and host components and is potentially vulnerable to pharmacological intervention. Reoviruses form large cytoplasmic replication sites called inclusions, which have been described as membrane-free structures. Despite the importance of inclusions in the reovirus replication cycle, little is known about their formation and composition. We used light and electron microscopy to demonstrate that reovirus inclusions are membrane-containing structures and that the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment interact closely with these viral organelles. These findings enhance our understanding of the cellular machinery usurped by viruses to form inclusion organelles and complete an infectious cycle. This information, in turn, may foster the development of antiviral drugs that impede this essential viral replication step.
Project description:Like most viruses that replicate in the cytoplasm, mammalian reoviruses assemble membranous neo-organelles called inclusions that serve as sites of viral genome replication and particle morphogenesis. Viral inclusion formation is essential for viral infection, but how these organelles form is not well understood. We investigated the biogenesis of reovirus inclusions. Correlative light and electron microscopy showed that endoplasmic reticulum (ER) membranes are in contact with nascent inclusions, which form by collections of membranous tubules and vesicles as revealed by electron tomography. ER markers and newly synthesized viral RNA are detected in inclusion internal membranes. Live-cell imaging showed that early in infection, the ER is transformed into thin cisternae that fragment into small tubules and vesicles. We discovered that ER tubulation and vesiculation are mediated by the reovirus ?NS and ?NS proteins, respectively. Our results enhance an understanding of how viruses remodel cellular compartments to build functional replication organelles.IMPORTANCE Viruses modify cellular structures to build replication organelles. These organelles serve as sites of viral genome replication and particle morphogenesis and are essential for viral infection. However, how these organelles are constructed is not well understood. We found that the replication organelles of mammalian reoviruses are formed by collections of membranous tubules and vesicles derived from extensive remodeling of the peripheral endoplasmic reticulum (ER). We also observed that ER tubulation and vesiculation are triggered by the reovirus ?NS and ?NS proteins, respectively. Our results enhance an understanding of how viruses remodel cellular compartments to build functional replication organelles and provide functions for two enigmatic reovirus replication proteins. Most importantly, this research uncovers a new mechanism by which viruses form factories for particle assembly.
Project description:Progeny virions of mammalian reoviruses are assembled in the cytoplasm of infected cells at discrete sites termed viral inclusions. Studies of temperature-sensitive (ts) mutant viruses indicate that nonstructural protein sigmaNS and core protein mu2 are required for synthesis of double-stranded (ds) RNA, a process that occurs at sites of viral assembly. We used confocal immunofluorescence microscopy and ts mutant reoviruses to define the roles of sigmaNS and mu2 in viral inclusion formation. In cells infected with wild-type (wt) reovirus, sigmaNS and mu2 colocalize to large, perinuclear structures that correspond to viral inclusions. In cells infected at a nonpermissive temperature with sigmaNS-mutant virus tsE320, sigmaNS is distributed diffusely in the cytoplasm and mu2 is contained in small, punctate foci that do not resemble viral inclusions. In cells infected at a nonpermissive temperature with mu2-mutant virus tsH11.2, mu2 is distributed diffusely in the cytoplasm and the nucleus. However, sigmaNS localizes to discrete structures in the cytoplasm that contain other viral proteins and are morphologically indistinguishable from viral inclusions seen in cells infected with wt reovirus. Examination of cells infected with wt reovirus over a time course demonstrates that sigmaNS precedes mu2 in localization to viral inclusions. These findings suggest that viral RNA-protein complexes containing sigmaNS nucleate sites of viral replication to which other viral proteins, including mu2, are recruited to commence dsRNA synthesis.
Project description: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.
Project description:Cells infected with mammalian reoviruses often contain large perinuclear inclusion bodies, or "factories," where viral replication and assembly are thought to occur. Here, we report a viral strain difference in the morphology of these inclusions: filamentous inclusions formed in cells infected with reovirus type 1 Lang (T1L), whereas globular inclusions formed in cells infected with our laboratory's isolate of reovirus type 3 Dearing (T3D). Examination by immunofluorescence microscopy revealed the filamentous inclusions to be colinear with microtubules (MTs). The filamentous distribution was dependent on an intact MT network, as depolymerization of MTs early after infection caused globular inclusions to form. The inclusion phenotypes of T1L x T3D reassortant viruses identified the viral M1 genome segment as the primary genetic determinant of the strain difference in inclusion morphology. Filamentous inclusions were seen with 21 of 22 other reovirus strains, including an isolate of T3D obtained from another laboratory. When the mu2 proteins derived from T1L and the other laboratory's T3D isolate were expressed after transfection of their cloned M1 genes, they associated with filamentous structures that colocalized with MTs, whereas the mu2 protein derived from our laboratory's T3D isolate did not. MTs were stabilized in cells infected with the viruses that induced filamentous inclusions and after transfection with the M1 genes derived from those viruses. Evidence for MT stabilization included bundling and hyperacetylation of alpha-tubulin, changes characteristically seen when MT-associated proteins (MAPs) are overexpressed. Sequencing of the M1 segments from the different T1L and T3D isolates revealed that a single-amino-acid difference at position 208 correlated with the inclusion morphology. Two mutant forms of mu2 with the changes Pro-208 to Ser in a background of T1L mu2 and Ser-208 to Pro in a background of T3D mu2 had MT association phenotypes opposite to those of the respective wild-type proteins. We conclude that the mu2 protein of most reovirus strains is a viral MAP and that it plays a key role in the formation and structural organization of reovirus inclusion bodies.
Project description:Mammalian orthoreoviruses (reoviruses) are highly tractable experimental models for studies of double-stranded (ds) RNA virus replication and pathogenesis. Reoviruses infect respiratory and intestinal epithelium and disseminate systemically in newborn animals. Until now, a strategy to rescue infectious virus from cloned cDNA has not been available for any member of the Reoviridae family of dsRNA viruses. We report the generation of viable reovirus following plasmid transfection of murine L929 (L) cells using a strategy free of helper virus and independent of selection. We used the reovirus reverse genetics system to introduce mutations into viral capsid proteins sigma1 and sigma3 and to rescue a virus that expresses a green fluorescent protein (GFP) transgene, thus demonstrating the tractability of this technology. The plasmid-based reverse genetics approach described here can be exploited for studies of reovirus replication and pathogenesis and used to develop reovirus as a vaccine vector.
Project description:Bloodstream spread is a critical step in the pathogenesis of many viruses. However, mechanisms that promote viremia are not well understood. Reoviruses are neurotropic viruses that disseminate hematogenously to the central nervous system. Junctional adhesion molecule A (JAM-A) is a tight junction protein that serves as a receptor for reovirus. JAM-A is required for establishment of viremia in infected newborn mice and viral spread to sites of secondary replication. To determine how viruses gain access to the circulatory system, we examined reovirus infection of polarized human brain microvascular endothelial cells (HBMECs). Reovirus productively infects polarized HBMECs, but infection does not alter tight junction integrity. Apical infection of polarized HBMECs is more efficient than basolateral infection, which is attributable to viral engagement of sialic acid and JAM-A. Viral release occurs exclusively from the apical surface via a mechanism that is not associated with lysis or apoptosis of infected cells. These data suggest that infection of endothelial cells routes reovirus apically into the bloodstream for systemic dissemination in the host. Understanding how viruses invade the bloodstream may aid in the development of therapeutics that block this step in viral pathogenesis.Bloodstream spread of viruses within infected hosts is a critical but poorly understood step in viral disease. Reoviruses first enter the host through the oral or respiratory route and infect cells in the central nervous system. Spread of reoviruses to the brain occurs by blood or nerves, which makes reoviruses useful models for studies of systemic viral dissemination. In this study, we examined how reoviruses infect endothelial cells, which form the walls of blood vessels. We found that reovirus infection of endothelial cells allows the virus to enter blood vessels and serves as a means for the virus to reach high titers in the circulation. Understanding how reovirus is routed through endothelial cells may aid in the design of antiviral drugs that target this important step in systemic viral infections.
Project description:Replication of Mononegavirales occurs in viral factories which form inclusions in the host-cell cytoplasm. For rabies virus, those inclusions are called Negri bodies (NBs). We report that NBs have characteristics similar to those of liquid organelles: they are spherical, they fuse to form larger structures, and they disappear upon hypotonic shock. Their liquid phase is confirmed by FRAP experiments. Live-cell imaging indicates that viral nucleocapsids are ejected from NBs and transported along microtubules to form either new virions or secondary viral factories. Coexpression of rabies virus N and P proteins results in cytoplasmic inclusions recapitulating NBs properties. This minimal system reveals that an intrinsically disordered domain and the dimerization domain of P are essential for Negri bodies-like structures formation. We suggest that formation of liquid viral factories by phase separation is common among Mononegavirales and allows specific recruitment and concentration of viral proteins but also the escape to cellular antiviral response.Negative strand RNA viruses, such as rabies virus, induce formation of cytoplasmic inclusions for genome replication. Here, Nikolic et al. show that these so-called Negri bodies (NBs) have characteristics of liquid organelles and they identify the minimal protein domains required for NB formation.
Project description:Mammalian orthoreovirus attachment to target cells is mediated by the outer capsid protein ?1, which projects from the virion surface. The ?1 protein is a homotrimer consisting of a filamentous tail, which is partly inserted into the virion; a body domain constructed from ?-spiral repeats; and a globular head with receptor-binding properties. The ?1 tail is predicted to form an ?-helical coiled coil. Although ?1 undergoes a conformational change during cell entry, the nature of this change and its contributions to viral replication are unknown. Electron micrographs of ?1 molecules released from virions identified three regions of flexibility, including one at the midpoint of the molecule, that may be involved in its structural rearrangement. To enable a detailed understanding of essential ?1 tail organization and properties, we determined high-resolution structures of the reovirus type 1 Lang (T1L) and type 3 Dearing (T3D) ?1 tail domains. Both molecules feature extended ?-helical coiled coils, with T1L ?1 harboring central chloride ions. Each molecule displays a discontinuity (stutter) within the coiled coil and an unexpectedly seamless transition to the body domain. The transition region features conserved interdomain interactions and appears rigid rather than highly flexible. Functional analyses of reoviruses containing engineered ?1 mutations suggest that conserved residues predicted to stabilize the coiled-coil-to-body junction are essential for ?1 folding and encapsidation, whereas central chloride ion coordination and the stutter are dispensable for efficient replication. Together, these findings enable modeling of full-length reovirus ?1 and provide insight into the stabilization of a multidomain virus attachment protein.IMPORTANCE While it is established that different conformational states of attachment proteins of enveloped viruses mediate receptor binding and membrane fusion, less is understood about how such proteins mediate attachment and entry of nonenveloped viruses. The filamentous reovirus attachment protein ?1 binds cellular receptors; contains regions of predicted flexibility, including one at the fiber midpoint; and undergoes a conformational change during cell entry. Neither the nature of the structural change nor its contribution to viral infection is understood. We determined crystal structures of large ?1 fragments for two different reovirus serotypes. We observed an unexpectedly tight transition between two domains spanning the fiber midpoint, which allows for little flexibility. Studies of reoviruses with engineered changes near the ?1 midpoint suggest that the stabilization of this region is critical for function. Together with a previously determined structure, we now have a complete model of the full-length, elongated reovirus ?1 attachment protein.
Project description:Activating mutation of KRas is a genetic alteration that occurs in the majority of pancreatic tumors and is therefore an ideal therapeutic target. The ability of reoviruses to preferentially replicate and induce cell death in transformed cells that express activated Ras prompted the development of a reovirus-based formulation for cancer therapy called Reolysin. We hypothesized that Reolysin exposure would trigger heavy production of viral products leading to endoplasmic reticular (ER) stress-mediated apoptosis. Here, we report that Reolysin treatment stimulated selective reovirus replication and decreased cell viability in KRas-transformed immortalized human pancreatic duct epithelial cells and pancreatic cancer cell lines. These effects were associated with increased expression of ER stress-related genes, ER swelling, cleavage of caspase-4, and splicing of XBP-1. Treatment with ER stress stimuli including tunicamycin, brefeldin A, and bortezomib (BZ) augmented the anticancer activity of Reolysin. Cotreatment with BZ and Reolysin induced the simultaneous accumulation of ubiquitinated and viral proteins, resulting in enhanced levels of ER stress and apoptosis in both in vitro and in vivo models of pancreatic cancer. Our collective results demonstrate that the abnormal protein accumulation induced by the combination of Reolysin and BZ promotes heightened ER stress and apoptosis in pancreatic cancer cells and provides the rationale for a phase I clinical trial further investigating the safety and efficacy of this novel strategy.
Project description:Mammalian reoviruses replicate in a broad range of hosts, cells, and tissues. These viruses display strain-dependent variation in tropism for different types of cells in vivo and ex vivo. Early steps in the reovirus life cycle, attachment, entry, and disassembly, have been identified as pivotal points of virus-cell interaction that determine the fate of infection, either productive or abortive. However, in studies of the differential capacity of reovirus strains type 1 Lang and type 3 Dearing to replicate in Madin-Darby canine kidney (MDCK) cells, we found that replication efficiency is regulated at a late point in the viral life cycle following primary transcription and translation. Results of genetic studies using recombinant virus strains show that reovirus tropism for MDCK cells is primarily regulated by replication protein ?2 and further influenced by the viral RNA-dependent RNA polymerase protein, ?3, depending on the viral genetic background. Furthermore, ?2 residue 347 is a critical determinant of replication efficiency in MDCK cells. These findings indicate that components of the reovirus replication complex are mediators of cell-selective viral replication capacity at a post-entry step. Thus, reovirus cell tropism may be determined at early and late points in the viral replication program.