BACKGROUND: Aberrant DNA methylation is a hallmark of cancer, and DNA methyltransferase inhibitors have demonstrated clinical efficacy in hematologic malignancies. On the basis of preclinical studies indicating that hypomethylating agents can reverse platinum resistance in ovarian cancer cells, the authors conducted a phase 1 trial of lowdose decitabine combined with carboplatin in patients with recurrent, platinum-resistant ovarian cancer. METHODS: Decitabine was administered intravenously daily for 5 days, before carboplatin (area under the curve, 5) on Day 8 of a 28-day cycle. By using a standard 3 þ 3 dose escalation, decitabine was tested at 2 dose levels: 10 mg/m

Necroptosis, a regulated form of necrotic cell death, requires the activation of the RIP3 kinase. Here, we identify that infection of host cells with reovirus can result in necroptosis. We find that necroptosis requires sensing of the genomic RNA within incoming virus particles via cytoplasmic RNA sensors to produce type I interferon (IFN). While these events that occur prior to the synthesis of viral RNA are required for the induction of necroptosis, they are not sufficient. The induction of necroptosis also requires late stages of reovirus infection. Specifically, efficient synthesis of double-stranded RNA (dsRNA) within infected cells is required for necroptosis. These data indicate that viral RNA interfaces with host components at two different stages of infection to induce necroptosis. This work provides new molecular details about events in the viral replication cycle that contribute to the induction of necroptosis following infection with an RNA virus. An appreciation of how cell death pathways are regulated following viral infection may reveal strategies to limit tissue destruction and prevent the onset of disease. Cell death following virus infection can occur by apoptosis or a regulated form of necrosis known as necroptosis. Apoptotic cells are typically disposed of without activating the immune system. In contrast, necroptotic cells alert the immune system, resulting in inflammation and tissue damage. While apoptosis following virus infection has been extensively investigated, how necroptosis is unleashed following virus infection is understood for only a small group of viruses. Here, using mammalian reovirus, we highlight the molecular mechanism by which infection with a dsRNA virus results in necroptosis.

The genus Alphavirus consists of a group of enveloped, single-stranded RNA viruses, many of which are transmitted by arthropods to a wide range of vertebrate host species. Here we report that Sindbis virus (SINV) produced from a representative mammalian cell line consists of at least two unique particle subpopulations, separable on the basis of virion density. In contrast, mosquito-derived SINV consists of a homogeneous population of particles. Our findings indicate that the denser particle subpopulation, SINV Heavy , is more infectious on a per-particle basis than SINV Light . SINV produced in mosquito cell lines (SINV C6/36 ) exhibited particle-to-PFU ratios similar to those observed for SINV Heavy . In mammalian cells, viral RNA was synthesized and accumulated more rapidly following infection with SINV Heavy or SINV C6/36 than following infection with SINV Light , due partly to enhanced translation of viral genomic RNA early in infection. Analysis of the individual particle subpopulations indicated that SINV Heavy and SINV C6/36 contain host-derived factors whose presence correlates with the enhanced translation, RNA synthesis, and infectivity observed for these particles. Members of the genus Alphavirus, of the family Togaviridae, are a group of enveloped positive-sense RNA viruses with a wide host range. For the mosquito-borne species, the virus is maintained in the enzootic cycle through transmission between a sylvatic reservoir and the mosquito host (1). The maintenance of this cycle directly affects the genetic fitness of the mosquito-borne alphaviruses. Prolonged disruption of this cycle leads to deleterious effects on viral transmission as the virus becomes adapted to a single host (2-5). Spillover from the enzootic cycle often results in the tangential infection of both humans and equines, which can result in significant outbreaks of disease. The outcome of alphaviral infection is dependent on the host system (6-12). Infection of mosquito cells does not result in the shutoff of host macromolecular synthesis and often culminates in persistent infection for the majority of mosquito cell lines (12-15). Nevertheless, cell death as a result of infection has been reported for several members of the genus in whole mosquitoes (16)(17)(18)(19)(20)(21). In contrast, infection of mammalian cells induces the shutoff of host macromolecular synthesis, resulting in a predominantly cytolytic infection. In vertebrates, the immune response to infection generally results in virus clearance. This is initiated by the recognition of viral doublestranded RNA and a rapid type I interferon (IFN-␣/␤) response (22)(23)(24)(25).Previously, we reported that the infectivity of Sindbis virus (SINV), as measured by the ratio of particles to infectious units, depends on the host cell line from which it is derived (26). SINV derived from mammalian cell lines exhibited a higher particle-to-PFU ratio, on average, than SINV generated from mosquito cell lines. This was due largely to differences in the quantity of total virus particles pr...

The mammalian orthoreovirus (reovirus) outer capsid is composed of 200 μ1-σ3 heterohexamers and a maximum of 12 σ1 trimers. During cell entry, σ3 is degraded by luminal or intracellular proteases to generate the infectious subviral particle (ISVP). When ISVP formation is prevented, reovirus fails to establish a productive infection, suggesting proteolytic priming is required for entry. ISVPs are then converted to ISVP*s, which is accompanied by μ1 rearrangements. The μ1 and σ3 proteins confer resistance to inactivating agents; however, neither the impact on capsid properties nor the mechanism (or basis) of inactivation is fully understood. Here, we utilized T1L/T3D M2 and T3D/T1L S4 to investigate the determinants of reovirus stability. Both reassortants encode mismatched subunits. When μ1-σ3 were derived from different strains, virions resembled wild-type particles in structure and protease sensitivity. T1L/T3D M2 and T3D/T1L S4 ISVPs were less thermostable than wild-type ISVPs. In contrast, virions were equally susceptible to heating. Virion associated μ1 adopted an ISVP*-like conformation concurrent with inactivation; σ3 preserves infectivity by preventing μ1 rearrangements. Moreover, thermostability was enhanced by a hyperstable variant of μ1. Unlike the outer capsid, the inner capsid (core) was highly resistant to elevated temperatures. The dual layered architecture allowed for differential sensitivity to inactivating agents.IMPORTANCENonenveloped and enveloped viruses are exposed to the environment during transmission to a new host. Protein-protein and/or protein-lipid interactions stabilize the particle and protect the viral genome. Mammalian orthoreovirus (reovirus) is composed of two concentric, protein shells. The μ1 and σ3 proteins form the outer capsid; contacts between neighboring subunits are thought to confer resistance to inactivating agents. We further investigated the determinants of reovirus stability. The outer capsid was disrupted concurrent with the loss of infectivity; virion associated μ1 rearranged into an altered conformation. Heat sensitivity was controlled by σ3; however, particle integrity was enhanced by a single μ1 mutation. In contrast, the inner capsid (core) displayed superior resistance to heating. These findings reveal structural components that differentially contribute to reovirus stability.

Cellular entry of nonenveloped and enveloped viruses is often accompanied by dramatic conformational changes within viral structural proteins. These rearrangements are triggered by a variety of mechanisms, such as low pH, virus-receptor interactions, and virus-host chaperone interactions. Reoviruses, a model system for entry of nonenveloped viruses, undergo a series of disassembly steps within the host endosome. One of these steps, infectious subviral particle (ISVP)-to-ISVP* conversion, is necessary for delivering the genome-containing viral core into host cells, but the physiological trigger that mediates ISVP-to-ISVP* conversion during cell entry is unknown. Structural studies of the reovirus membrane penetration protein, 1, predict that interactions between 1 and negatively charged lipid head groups may promote ISVP* formation; however, experimental evidence for this idea is lacking. Here, we show that the presence of polyanions (SO 4 2؊ and HPO 4 2؊ ) or lipids in the form of liposomes facilitates ISVP-to-ISVP* conversion. The requirement for charged lipids appears to be selective, since phosphatidylcholine and phosphatidylethanolamine promoted ISVP* formation, whereas other lipids, such as sphingomyelin and sulfatide, either did not affect ISVP* formation or prevented ISVP* formation. Thus, our work provides evidence that interactions with membranes can function as a trigger for a nonenveloped virus to gain entry into host cells. IMPORTANCECell entry, a critical stage in the virus life cycle, concludes with the delivery of the viral genetic material across host membranes. Regulated structural transitions within nonenveloped and enveloped viruses are necessary for accomplishing this step; these conformational changes are predominantly triggered by low pH and/or interactions with host proteins. In this work, we describe a previously unknown trigger, interactions with lipid membranes, which can induce the structural rearrangements required for cell entry. This mechanism operates during entry of mammalian orthoreoviruses. We show that interactions between reovirus entry intermediates and lipid membranes devoid of host proteins promote conformational changes within the viral outer capsid that lead to membrane penetration. Thus, this work illustrates a novel strategy that nonenveloped viruses can use to gain access into cells and how viruses usurp disparate host factors to initiate infection. N onenveloped and enveloped viruses undergo significant structural rearrangements that facilitate their entry into host cells. These conformational changes, which are required for delivering the viral genetic material across cellular membranes, can be triggered by one or more mechanisms. Low pH promotes genome release during rhinovirus infection (1, 2) and fusion between viral and host membranes during influenza virus infection (3-5). Virus-receptor interactions induce viral uncoating during poliovirus infection (6-8) and membrane fusion during herpesvirus infection (9-11). Avian retroviruses (enveloped) use both re...

The 80 trimeric, glycoprotein spikes that cover the surface of alphavirus particles are required for mediating viral entry into a host cell. Spike assembly is a regulated process that requires interactions between five structural proteins, E3, E2, 6K and its translational frameshift product TF, and E1. E3 is a small, ϳ65-amino-acid glycoprotein that has two known functions: E3 serves as the signal sequence for translocation of the E3-E2-6K-E1 polyprotein into the endoplasmic reticulum (ER), and cleavage of E3 from E2 is essential for virus maturation. Nonetheless, when E3 is replaced with an ER signal sequence, spikes do not form and infectious particles are not assembled, suggesting an additional role(s) for E3 in the viral life cycle. To further investigate the role of E3 in spike assembly, we made chimeric viruses in which E3 from one alphavirus species is replaced with E3 from another species. Our results demonstrate that when E3 is interchanged between alphavirus species that belong to the same virus clade, viral titers and particle morphologies and compositions are similar to what are observed for the parental virus. In contrast, for chimeras in which E3 is derived from a different clade than the parental virus, we observed reduced titers and the formation of particles with atypical morphologies and protein compositions. We further characterized the E3 chimeras using a combination of structure-function and revertant analyses. This work revealed two specific interactions between E3 and its cognate E2 glycoprotein that are important for regulating spike assembly.A lphavirus particles are characterized by the presence of 80 trimeric, glycoprotein spikes that protrude from the surface of the virion (5, 49). The spikes, which are composed of trimers of E2 and E1 heterodimers, are embedded in a host-derived lipid membrane and are required to initiate infection; E2 is responsible for receptor binding (4, 18, 39) and E1 mediates fusion between the viral and host cell membranes during entry (11,32,47). Internal to the viral membrane is the nucleocapsid core. The core consists of 240 copies of the capsid protein, which encapsidate the single-stranded, positive-sense RNA genome (5, 49). The glycoprotein spikes and the nucleocapsid core are linked through interactions between the cytoplasmic tails of E2 and the capsid protein (10,12,27,35). Thus, misfolded or misarranged spikes on the particle surface or aberrant interactions between the spikes and the nucleocapsid core may affect viral entry into a new host cell.Alphavirus spike assembly is a highly regulated process that requires interactions between both viral and host cell factors. The viral structural proteins are translated as a polyprotein, capsid-E3-E2-6K-E1 (or capsid-E3-E2-TF), from a subgenomic RNA (8, 43). Early in translation, capsid is autoproteolytically cleaved from the rest of the polyprotein (30). Following capsid cleavage, E3 serves as the signal sequence to translocate E3-E2-6K-E1 (or E3-E2-TF) into the endoplasmic reticulum (ER) (2, 3, 8, 23). Mul...

Virus-host interactions play a role in many stages of the viral lifecycle, including entry. Reovirus, a model system for studying the entry mechanisms of nonenveloped viruses, undergoes a series of regulated structural transitions that culminate in delivery of the viral genetic material. Lipids can trigger one of these conformational changes, infectious subviral particle (ISVP)-to-ISVP* conversion. ISVP* formation releases two virally encoded peptides, myristoylated μ1N (myr-μ1N) and Φ. Among these, myr-μ1N is sufficient to form pores within membranes. Released myr-μ1N can also promote ISVP* formation in trans Using thermal inactivation as a readout for ISVP-to-ISVP* conversion, we demonstrate that lipids render ISVPs less thermostable in a virus concentration-dependent manner. Under conditions in which neither lipids alone nor myr-μ1N alone promotes ISVP-to-ISVP* conversion, myr-μ1N induces particle uncoating when lipids are present. These data suggest that the pore-forming activity and the ISVP*-promoting activity of myr-μ1N are linked. Lipid-associated myr-μ1N interacts with ISVPs and triggers efficient ISVP* formation. The cooperativity between a reovirus component and lipids reveals a distinct virus-host interaction in which membranes can facilitate nonenveloped virus entry.

There are 80 trimeric, glycoprotein spikes that cover the surface of an alphavirus particle. The spikes, which are composed of three E2 and E1 glycoprotein heterodimers, are responsible for receptor binding and mediating fusion between the viral and host-cell membranes during entry. In addition, the cytoplasmic domain of E2 interacts with the nucleocapsid core during the last stages of particle assembly, possibly to aid in particle stability. During assembly, the spikes are nonfusogenic until the E3 glycoprotein is cleaved from E2 in the trans-Golgi network. Thus, a mutation in E2 potentially has effects on virus entry, spike assembly, or spike maturation. E2 is a highly conserved, cysteine-rich transmembrane glycoprotein. We made single cysteine-toserine mutations within two distinct regions of the E2 ectodomain in both Sindbis virus and Ross River virus. Each of the E2 Cys mutants produced fewer infectious particles than wild-type virus. Further characterization of the mutant viruses revealed differences in particle morphology, fusion activity, and polyprotein cleavage between Sindbis and Ross River virus mutants, despite the mutations being made at corresponding positions in E2. The nonconserved assembly defects suggest that E2 folding and function is species dependent, possibly due to interactions with a virus-specific chaperone.T he alphavirus particle is comprised of two concentric protein shells, the glycoprotein spikes and the nucleocapsid core, separated by a lipid membrane (7, 66). The outer protein shell consists of 80 trimeric, glycoprotein spikes embedded in a hostderived lipid membrane and arranged with icosahedral symmetry. The spikes are trimers of E2 and E1 protein heterodimers (7,26,66). The spike proteins are required to initiate infection; the E2 glycoprotein is responsible for receptor binding (5,24,53), and the E1 glycoprotein mediates fusion between the viral and hostcell membranes during entry (14,45,59). The nucleocapsid core is located in the center of the particle and is composed of 240 copies of the capsid protein arranged with icosahedral symmetry around the viral RNA genome (7, 66). The endodomain of E2 interacts with the nucleocapsid core, presumably to aid in particle stability (11,15,32,48).Alphavirus assembly is a highly regulated process comprised of multiple cleavage and oligomerization events. The alphavirus genome encodes five structural proteins that are translated as a single polyprotein, capsid-E3-E2-6K-E1 (where 6K indicates a 6,000-molecular-weight protein), from a subgenomic RNA (54). Capsid is cleaved from the rest of the polyprotein and remains in the cytoplasm, where nucleocapsid core formation occurs (38). The E3 glycoprotein contains a signal sequence that translocates the remaining polyprotein, E3-E2-6K-E1 (also referred to as the spike proteins), into the endoplasmic reticulum (ER) (1, 2, 38). Once inserted into the ER, the cellular enzyme signalase cleaves the polyprotein into E3-E2 (also referred to as pE2 or p62), 6K, and E1 (28, 68). In the ER, pE2 and E1 as...