Suppression of viral RNA polymerase activity is necessary for persistent infection during the transformation of measles virus into SSPE virus.

Subacute sclerosing panencephalitis (SSPE) is a fatal neurodegenerative disease caused by measles virus (MV), which typically develops 7 to 10 years after acute measles. During the incubation period, MV establishes a persistent infection in the brain and accumulates mutations that generate neuropathogenic SSPE virus. The neuropathogenicity is closely associated with enhanced propagation mediated by cell-to-cell fusion in the brain, which is principally regulated by hyperfusogenic mutations of the viral F protein. The molecular mechanisms underlying establishment and maintenance of persistent infection are unclear because it is impractical to isolate viruses before the appearance of clinical signs. In this study, we found that the L and P proteins, components of viral RNA-dependent RNA polymerase (RdRp), of an SSPE virus Kobe-1 strain did not promote but rather attenuated viral neuropathogenicity. Viral RdRp activity corresponded to F protein expression; the suppression of RdRp activity in the Kobe-1 strain because of mutations in the L and P proteins led to restriction of the F protein level, thereby reducing cell-to-cell fusion mediated propagation in neuronal cells and decreasing neuropathogenicity. Therefore, the L and P proteins of Kobe-1 did not contribute to progression of SSPE. Three mutations in the L protein strongly suppressed RdRp activity. Recombinant MV harboring the three mutations limited viral spread in neuronal cells while preventing the release of infectious progeny particles; these changes could support persistent infection by enabling host immune escape and preventing host cell lysis. Therefore, the suppression of RdRp activity is necessary for the persistent infection of the parental MV on the way to transform into Kobe-1 SSPE virus. Because mutations in the genome of an SSPE virus reflect the process of SSPE development, mutation analysis will provide insight into the mechanisms underlying persistent infection.

Brain tropism acquisition: The spatial dynamics and evolution of a measles virus collective infectious unit that drove lethal subacute sclerosing panencephalitis.

It is increasingly appreciated that pathogens can spread as infectious units constituted by multiple, genetically diverse genomes, also called collective infectious units or genome collectives. However, genetic characterization of the spatial dynamics of collective infectious units in animal hosts is demanding, and it is rarely feasible in humans. Measles virus (MeV), whose spread in lymphatic tissues and airway epithelia relies on collective infectious units, can, in rare cases, cause subacute sclerosing panencephalitis (SSPE), a lethal human brain disease. In different SSPE cases, MeV acquisition of brain tropism has been attributed to mutations affecting either the fusion or the matrix protein, or both, but the overarching mechanism driving brain adaptation is not understood. Here we analyzed MeV RNA from several spatially distinct brain regions of an individual who succumbed to SSPE. Surprisingly, we identified two major MeV genome subpopulations present at variable frequencies in all 15 brain specimens examined. Both genome types accumulated mutations like those shown to favor receptor-independent cell-cell spread in other SSPE cases. Most infected cells carried both genome types, suggesting the possibility of genetic complementation. We cannot definitively chart the history of the spread of this virus in the brain, but several observations suggest that mutant genomes generated in the frontal cortex moved outwards as a collective and diversified. During diversification, mutations affecting the cytoplasmic tails of both viral envelope proteins emerged and fluctuated in frequency across genetic backgrounds, suggesting convergent and potentially frequency-dependent evolution for modulation of fusogenicity. We propose that a collective infectious unit drove MeV pathogenesis in this brain. Re-examination of published data suggests that similar processes may have occurred in other SSPE cases. Our studies provide a primer for analyses of the evolution of collective infectious units of other pathogens that cause lethal disease in humans.

Fitness selection of hyperfusogenic measles virus F proteins associated with neuropathogenic phenotypes.

Measles virus (MeV) is resurgent and caused >200,000 deaths in 2019. MeV infection can establish a chronic latent infection of the brain that can recrudesce months to years after recovery from the primary infection. Recrudescent MeV leads to fatal subacute sclerosing panencephalitis (SSPE) or measles inclusion body encephalitis (MIBE) as the virus spreads across multiple brain regions. Most clinical isolates of SSPE/MIBE strains show mutations in the fusion (F) gene that result in a hyperfusogenic phenotype in vitro and allow for efficient spread in primary human neurons. Wild-type MeV receptor-binding protein is indispensable for manifesting these mutant F phenotypes, even though neurons lack canonical MeV receptors (CD150/SLAMF1 or nectin-4). How such hyperfusogenic F mutants are selected and whether they confer a fitness advantage for efficient neuronal spread is unresolved. To better understand the fitness landscape that allows for the selection of such hyperfusogenic F mutants, we conducted a screen of ≥3.1 × 105 MeV-F point mutants in their genomic context. We rescued and amplified our genomic MeV-F mutant libraries in BSR-T7 cells under conditions in which MeV-F-T461I (a known SSPE mutant), but not wild-type MeV, can spread. We recovered known SSPE mutants but also characterized at least 15 hyperfusogenic F mutations with an SSPE phenotype. Structural mapping of these mutants onto the prefusion MeV-F trimer confirm and extend our understanding of the F regulatory domains in MeV-F. Our list of hyperfusogenic F mutants is a valuable resource for future studies into MeV neuropathogenesis and the regulation of paramyxovirus F.

Host 5'-3' Exoribonuclease XRN1 Acts as a Proviral Factor for Measles Virus Replication by Downregulating the dsRNA-Activated Kinase PKR.

Many negative-sense RNA viruses, including measles virus (MeV), are thought to carry out much of their viral replication in cytoplasmic membraneless foci known as inclusion bodies (IBs). The mechanisms by which IBs facilitate efficient viral replication remain largely unknown but may involve an intricate network of regulation at the host-virus interface. Viruses are able to modulate such interactions by a variety of strategies including adaptation of their genomes and "hijacking" of host proteins. The latter possibility broadens the molecular reservoir available for a virus to enhance its replication and/or antagonize host antiviral responses. Here, we show that the cellular 5'-3' exoribonuclease, XRN1, is a host protein hijacked by MeV. We found that upon MeV infection, XRN1 is translocated to cytoplasmic IBs where it acts in a proviral manner by preventing the accumulation of double-stranded RNA (dsRNA) within the IBs. This leads to the suppression of the dsRNA-induced innate immune responses mediated via the protein kinase R (PKR)-integrated stress response (ISR) pathway. IMPORTANCE Measles virus remains a major global health threat due to its high transmissibility and significant morbidity in children and immunocompromised individuals. Although there is an effective vaccine against MeV, a large population in the world remains without access to the vaccine, contributing to more than 7,000,000 measles cases and 60,000 measles deaths in 2020 (CDC). For negative-sense RNA viruses including MeV, one active research area is the exploration of virus-host interactions occurring at cytoplasmic IBs where viral replication takes place. In this study we present evidence suggesting a model in which MeV IBs antagonize host innate immunity by recruiting XRN1 to reduce dsRNA accumulation and subsequent PKR kinase activation/ISR induction. In the absence of XRN1, the increased dsRNA level acts as a potent activator of the antiviral PKR/ISR pathway leading to suppression of global cap-dependent mRNA translation and inhibition of viral replication.

Measles virus exits human airway epithelia within dislodged metabolically active infectious centers.

Measles virus (MeV) is the most contagious human virus. Unlike most respiratory viruses, MeV does not directly infect epithelial cells upon entry in a new host. MeV traverses the epithelium within immune cells that carry it to lymphatic organs where amplification occurs. Infected immune cells then synchronously deliver large amounts of virus to the airways. However, our understanding of MeV replication in airway epithelia is limited. To model it, we use well-differentiated primary cultures of human airway epithelial cells (HAE) from lung donors. In HAE, MeV spreads directly cell-to-cell forming infectious centers that grow for ~3-5 days, are stable for a few days, and then disappear. Transepithelial electrical resistance remains intact during the entire course of HAE infection, thus we hypothesized that MeV infectious centers may dislodge while epithelial function is preserved. After documenting by confocal microscopy that infectious centers progressively detach from HAE, we recovered apical washes and separated cell-associated from cell-free virus by centrifugation. Virus titers were about 10 times higher in the cell-associated fraction than in the supernatant. In dislodged infectious centers, ciliary beating persisted, and apoptotic markers were not readily detected, suggesting that they retain functional metabolism. Cell-associated MeV infected primary human monocyte-derived macrophages, which models the first stage of infection in a new host. Single-cell RNA sequencing identified wound healing, cell growth, and cell differentiation as biological processes relevant for infectious center dislodging. 5-ethynyl-2'-deoxyuridine (EdU) staining located proliferating cells underneath infectious centers. Thus, cells located below infectious centers divide and differentiate to repair the dislodged infected epithelial patch. As an extension of these studies, we postulate that expulsion of infectious centers through coughing and sneezing could contribute to MeV's strikingly high reproductive number by allowing the virus to survive longer in the environment and by delivering a high infectious dose to the next host.

The Unstructured Paramyxovirus Nucleocapsid Protein Tail Domain Modulates Viral Pathogenesis through Regulation of Transcriptase Activity.

The paramyxovirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxovirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the Morbillivirus genus, a series of measles virus (MeV) and canine distemper virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbillivirus disease. The recombinant viruses displayed different stages of attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual virus attenuation for next-generation paramyxovirus vaccine design.IMPORTANCE Investigating the role of the paramyxovirus N protein tail domain (Ntail) in virus replication, we demonstrated in this study that the structurally disordered central Ntail region is a determinant for viral pathogenesis. We show that internal deletions in this Ntail region of up to 55 amino acids in length are compatible with efficient replication of recombinant viruses in cell culture but result in gradual viral attenuation in a lethal canine distemper virus (CDV)/ferret model. Mechanistically, we demonstrate a role of the intact Ntail region in the regulation of viral transcriptase activity. Recombinant viruses with Ntail truncations induce protective immunity against lethal challenge of ferrets with pathogenic CDV. This identification of the unstructured central Ntail domain as a nonessential paramyxovirus pathogenesis factor establishes a foundation for harnessing Ntail truncations for vaccine engineering against emerging and reemerging members of the paramyxovirus family.

Cell-to-Cell Measles Virus Spread between Human Neurons Is Dependent on Hemagglutinin and Hyperfusogenic Fusion Protein.

Measles virus (MV) usually causes acute infection but in rare cases persists in the brain, resulting in subacute sclerosing panencephalitis (SSPE). Since human neurons, an important target affected in the disease, do not express the known MV receptors (signaling lymphocyte activation molecule [SLAM] and nectin 4), how MV infects neurons and spreads between them is unknown. Recent studies have shown that many virus strains isolated from SSPE patients possess substitutions in the extracellular domain of the fusion (F) protein which confer enhanced fusion activity. Hyperfusogenic viruses with such mutations, unlike the wild-type MV, can induce cell-cell fusion even in SLAM- and nectin 4-negative cells and spread efficiently in human primary neurons and the brains of animal models. We show here that a hyperfusogenic mutant MV, IC323-F(T461I)-EGFP (IC323 with a fusion-enhancing T461I substitution in the F protein and expressing enhanced green fluorescent protein), but not the wild-type MV, spreads in differentiated NT2 cells, a widely used human neuron model. Confocal time-lapse imaging revealed the cell-to-cell spread of IC323-F(T461I)-EGFP between NT2 neurons without syncytium formation. The production of virus particles was strongly suppressed in NT2 neurons, also supporting cell-to-cell viral transmission. The spread of IC323-F(T461I)-EGFP was inhibited by a fusion inhibitor peptide as well as by some but not all of the anti-hemagglutinin antibodies which neutralize SLAM- or nectin-4-dependent MV infection, suggesting the presence of a distinct neuronal receptor. Our results indicate that MV spreads in a cell-to-cell manner between human neurons without causing syncytium formation and that the spread is dependent on the hyperfusogenic F protein, the hemagglutinin, and the putative neuronal receptor for MV.IMPORTANCE Measles virus (MV), in rare cases, persists in the human central nervous system (CNS) and causes subacute sclerosing panencephalitis (SSPE) several years after acute infection. This neurological complication is almost always fatal, and there is currently no effective treatment for it. Mechanisms by which MV invades the CNS and causes the disease remain to be elucidated. We have previously shown that fusion-enhancing substitutions in the fusion protein of MVs isolated from SSPE patients contribute to MV spread in neurons. In this study, we demonstrate that MV bearing the hyperfusogenic mutant fusion protein spreads between human neurons in a cell-to-cell manner. Spread of the virus was inhibited by a fusion inhibitor peptide and antibodies against the MV hemagglutinin, indicating that both the hemagglutinin and hyperfusogenic fusion protein play important roles in MV spread between human neurons. The findings help us better understand the disease process of SSPE.

Both type I and type III interferons are required to restrict measles virus growth in lung epithelial cells.

Measles virus (MeV) first infects immune cells in the respiratory tract of a human host, spreads to lymphoid organs throughout the body, and finally enters and grows in respiratory epithelial cells before being released and transmitted to the next host. Thus, efficient growth in respiratory epithelial cells is important for the person-to-person transmission of MeV. Upon viral entry, host cells detect viral nucleic acids and produce interferons (IFNs) to control viral growth. Type I (IFN-α/ß) and type III (IFN-λ) IFNs have largely common induction and signaling mechanisms and stimulate expression of similar target genes but utilize distinct receptors. To determine the relative contributions of type I and type III IFNs to the control of MeV growth in epithelial cells, we examined the growth of MeV and that of its mutants lacking either type I or type III IFN receptor in the human lung epithelial cell line H358. Our results revealed that both type I and type III IFNs are required to restrict MeV growth in H358 cells and that the induction of type III as well as type I IFNs was increased in the absence of the MeV nonstructural V protein.

Measles Virus Defective Interfering RNAs Are Generated Frequently and Early in the Absence of C Protein and Can Be Destabilized by Adenosine Deaminase Acting on RNA-1-Like Hypermutations.

UNLABELLED: Defective interfering RNAs (DI-RNAs) of the viral genome can form during infections of negative-strand RNA viruses and outgrow full-length viral genomes, thereby modulating the severity and duration of infection. Here we document the frequent de novo generation of copy-back DI-RNAs from independent rescue events both for a vaccine measles virus (vac2) and for a wild-type measles virus (IC323) as early as passage 1 after virus rescue. Moreover, vaccine and wild-type C-protein-deficient (C-protein-knockout [CKO]) measles viruses generated about 10 times more DI-RNAs than parental virus, suggesting that C enhances the processivity of the viral polymerase. We obtained the nucleotide sequences of 65 individual DI-RNAs, identified breakpoints and reinitiation sites, and predicted their structural features. Several DI-RNAs possessed clusters of A-to-G or U-to-C transitions. Sequences flanking these mutation sites were characteristic of those favored by adenosine deaminase acting on RNA-1 (ADAR1), which catalyzes in double-stranded RNA the C-6 deamination of adenosine to produce inosine, which is recognized as guanosine, a process known as A-to-I RNA editing. In individual DI-RNAs the transitions were of the same type and occurred on both sides of the breakpoint. These patterns of mutations suggest that ADAR1 edits unencapsidated DI-RNAs that form double-strand RNA structures. Encapsidated DI-RNAs were incorporated into virus particles, which reduced the infectivity of virus stocks. The CKO phenotype was dominant: DI-RNAs derived from vac2 with a CKO suppressed the replication of vac2, as shown by coinfections of interferon-incompetent lymphatic cells with viruses expressing different fluorescent reporter proteins. In contrast, coinfection with a C-protein-expressing virus did not counteract the suppressive phenotype of DI-RNAs. IMPORTANCE: Recombinant measles viruses (MVs) are in clinical trials as cancer therapeutics and as vectored vaccines for HIV-AIDS and other infectious diseases. The efficacy of MV-based vectors depends on their replication proficiency and immune activation capacity. Here we document that copy-back defective interfering RNAs (DI-RNAs) are generated by recombinant vaccine and wild-type MVs immediately after rescue. The MV C protein interferes with DI-RNA generation and may enhance the processivity of the viral polymerase. We frequently detected clusters of A-to-G or U-to-C transitions and noted that sequences flanking individual mutations contain motifs favoring recognition by the adenosine deaminase acting on RNA-1 (ADAR1). The consistent type of transitions on the DI-RNAs indicates that these are direct substrates for editing by ADAR1. The ADAR1-mediated biased hypermutation events are consistent with the protein kinase R (PKR)-ADAR1 balancing model of innate immunity activation. We show by coinfection that the C-defective phenotype is dominant.

In vivo ligands of MDA5 and RIG-I in measles virus-infected cells.

RIG-I-like receptors (RLRs: RIG-I, MDA5 and LGP2) play a major role in the innate immune response against viral infections and detect patterns on viral RNA molecules that are typically absent from host RNA. Upon RNA binding, RLRs trigger a complex downstream signaling cascade resulting in the expression of type I interferons and proinflammatory cytokines. In the past decade extensive efforts were made to elucidate the nature of putative RLR ligands. In vitro and transfection studies identified 5'-triphosphate containing blunt-ended double-strand RNAs as potent RIG-I inducers and these findings were confirmed by next-generation sequencing of RIG-I associated RNAs from virus-infected cells. The nature of RNA ligands of MDA5 is less clear. Several studies suggest that double-stranded RNAs are the preferred agonists for the protein. However, the exact nature of physiological MDA5 ligands from virus-infected cells needs to be elucidated. In this work, we combine a crosslinking technique with next-generation sequencing in order to shed light on MDA5-associated RNAs from human cells infected with measles virus. Our findings suggest that RIG-I and MDA5 associate with AU-rich RNA species originating from the mRNA of the measles virus L gene. Corresponding sequences are poorer activators of ATP-hydrolysis by MDA5 in vitro, suggesting that they result in more stable MDA5 filaments. These data provide a possible model of how AU-rich sequences could activate type I interferon signaling.

Differential induction of type I interferons in macaques by wild-type measles virus alone or with the hemagglutinin protein of the Edmonston vaccine strain.

Measles vaccines are highly effective and safe; however, the mechanism(s) underlying their attenuation has not been well understood. In this study, type I IFNs (IFN-α and IFN-ß) induction in macaques infected with measles virus (MV) strains was examined. Type I IFNs were not induced in macaques infected with wild-type MV. However, IFN-α was sharply induced in most macaques infected with recombinant wild-type MV bearing the hemagglutinin (H) protein of the Edmonston vaccine strain. These results indicate that the H protein of MV vaccine strains may have a role in MV attenuation.

Sustained autophagy contributes to measles virus infectivity.

The interplay between autophagy and intracellular pathogens is intricate as autophagy is an essential cellular response to fight against infections, whereas numerous microbes have developed strategies to escape this process or even exploit it to their own benefit. The fine tuned timing and/or selective molecular pathways involved in the induction of autophagy upon infections could be the cornerstone allowing cells to either control intracellular pathogens, or be invaded by them. We report here that measles virus infection induces successive autophagy signallings in permissive cells, via distinct and uncoupled molecular pathways. Immediately upon infection, attenuated measles virus induces a first transient wave of autophagy, via a pathway involving its cellular receptor CD46 and the scaffold protein GOPC. Soon after infection, a new autophagy signalling is initiated which requires viral replication and the expression of the non-structural measles virus protein C. Strikingly, this second autophagy signalling can be sustained overtime within infected cells, independently of the expression of C, but via a third autophagy input resulting from cell-cell fusion and the formation of syncytia. Whereas this sustained autophagy signalling leads to the autophagy degradation of cellular contents, viral proteins escape from degradation. Furthermore, this autophagy flux is ultimately exploited by measles virus to limit the death of infected cells and to improve viral particle formation. Whereas CD150 dependent virulent strains of measles virus are unable to induce the early CD46/GOPC dependent autophagy wave, they induce and exploit the late and sustained autophagy. Overall, our work describes distinct molecular pathways for an induction of self-beneficial sustained autophagy by measles virus.

Single-nucleotide polymorphism associations in common with immune responses to measles and rubella vaccines.

Single-nucleotide polymorphisms (SNPs) in candidate immune response genes were evaluated for associations with measles- and rubella-specific neutralizing antibodies, interferon (IFN)-γ, and interleukin (IL)-6 secretion in two separate association analyses in a cohort of healthy immunized subjects. We identified six SNP associations shared between the measles-specific and rubella-specific immune responses, specifically neutralizing antibody titers (DDX58), secreted IL-6 (IL10RB, IL12B), and secreted IFN-γ (IFNAR2, TLR4). An intronic SNP (rs669260) in the antiviral innate immune receptor gene, DDX58, was significantly associated with increased neutralizing antibody titers for both measles and rubella viral antigens post-MMR vaccination (p values 0.02 and 0.0002, respectively). Significant associations were also found between IL10RB (rs2284552; measles study p value 0.006, rubella study p value 0.00008) and IL12B (rs2546893; measles study p value 0.005, rubella study p value 0.03) gene polymorphisms and variations in both measles- and rubella virus-specific IL-6 responses. We also identified associations between individual SNPs in the IFNAR2 and TLR4 genes that were associated with IFN-γ secretion for both measles and rubella vaccine-specific immune responses. These results are the first to indicate that there are SNP associations in common across measles and rubella vaccine immune responses and that SNPs from multiple genes involved in innate and adaptive immune response regulation may contribute to the overall human antiviral response.

The receptor attachment function of measles virus hemagglutinin can be replaced with an autonomous protein that binds Her2/neu while maintaining its fusion-helper function.

Cell entry of enveloped viruses is initiated by attachment to the virus receptor followed by fusion between the virus and host cell membranes. Measles virus (MV) attachment to its receptor is mediated by the hemagglutinin (H), which is thought to produce conformational changes in the membrane fusion protein (F) that trigger insertion of its fusion peptide into the target cell membrane. Here, we uncoupled receptor attachment and the fusion-helper function of H by introducing Y481A, R533A, S548L, and F549S mutations into the viral attachment protein that made it blind to its normal receptors. An artificial receptor attachment protein specific for Her2/neu was incorporated into the membranes of pseudotyped lentivirus particles as a separate transmembrane protein along with the F protein. Surprisingly, these particles entered efficiently into Her2/neu-positive SK-OV-3 as well as CHO-Her2 cells. Cell entry was independent of endocytosis but strictly dependent on the presence of H. H-specific monoclonal antibodies, as well as a mutation in H interfering with H/F cooperation, blocked cell entry. The particles mediated stable and specific transfer of reporter genes into Her2/neu-positive human tumor cells also in vivo, while exhibiting improved infectivity and higher titers than Her2/neu-targeted vectors displaying the targeting domain on H. Extending the current model of MV cell entry, the data suggest that receptor binding of H is not required for its fusion-helper function but that particle-cell contact in general may be sufficient to induce the conformational changes in the H/F complex and activate membrane fusion.

Measles virus fusion machinery activated by sialic acid binding globular domain.

Paramyxoviruses, including the human pathogen measles virus (MV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral envelope with the target cell membrane. This fusion is driven by the concerted action of two viral envelope glycoproteins: the receptor binding protein and the fusion protein (F). The MV receptor binding protein (hemagglutinin [H]) attaches to proteinaceous receptors on host cells, while the receptor binding protein of NDV (hemagglutinin-neuraminidase [HN]) interacts with sialic acid-containing receptors. The receptor-bound HN/H triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. The mechanism of fusion activation has been proposed to be different for sialic acid-binding viruses and proteinaceous receptor-binding viruses. We report that a chimeric protein containing the NDV HN receptor binding region and the MV H stalk domain can activate MV F to fuse, suggesting that the signal to the stalk of a protein-binding receptor binding molecule can be transmitted from a sialic acid binding domain. By engineering the NDV HN globular domain to interact with a proteinaceous receptor, the fusion activation signal was preserved. Our findings are consistent with a unified mechanism of fusion activation, at least for the Paramyxovirinae subfamily, in which the receptor binding domains of the receptor binding proteins are interchangeable and the stalk determines the specificity of F activation.

Dual RNA-seq Analysis of Patients' Cells and Viral Genome After Measles Infection.

During the infection of a host cell by an infectious agent, a series of gene expression changes occurs as a consequence of host-pathogen interactions. Unraveling this complex interplay is the key for understanding of microbial virulence and host response pathways, thus providing the basis for new molecular insights into the mechanisms of pathogenesis and the corresponding immune response. Dual RNA sequencing (dual RNA-seq) has been developed to simultaneously determine pathogen and host transcriptomes enabling both differential and coexpression analyses between the two partners as well as genome characterization in the case of RNA viruses. Here, we provide a detailed laboratory protocol and bioinformatics analysis guidelines for dual RNA-seq experiments focusing on - but not restricted to - measles virus (MeV) as a pathogen of interest. The application of dual RNA-seq technologies in MeV-infected patients can potentially provide valuable information on the structure of the viral RNA genome and on cellular innate immune responses and drive the discovery of new targets for antiviral therapy.

Adenosine deaminase acting on RNA 1 (ADAR1) suppresses the induction of interferon by measles virus.

ADAR1, the interferon (IFN)-inducible adenosine deaminase acting on RNA, catalyzes the C-6 deamination of adenosine (A) to produce inosine (I) in RNA substrates with a double-stranded character. Because double-stranded RNA is a known inducer of IFN, we tested the role of ADAR1 in IFN induction following virus infection. HeLa cells made stably deficient in ADAR1 (ADAR1(kd)) were compared to vector control (CON(kd)) and protein kinase PKR-deficient (PKR(kd)) cells for IFN-ß induction following infection with either parental (wild-type [WT]) recombinant Moraten vaccine strain measles virus (MV) or isogenic knockout mutants deficient for either V (V(ko)) or C (C(ko)) protein expression. We observed potent IFN-ß transcript induction in ADAR1(kd) cells by all three viruses; in contrast, in ADAR1-sufficient CON(kd) cells, only the C(ko) mutant virus was an effective inducer and the IFN-ß RNA induction was amplified by PKR. The enhanced IFN-ß transcript-inducing capacity of the WT and V(ko) viruses seen in ADAR1-deficient cells correlated with the enhanced activation of PKR, IFN regulatory factor IRF3, and activator of transcription ATF2, reaching levels similar to those seen in C(ko) virus-infected cells. However, the level of IFN-ß protein produced was not proportional to the level of IFN-ß RNA but rather correlated inversely with the level of activated PKR. These results suggest that ADAR1 functions as an important suppressor of MV-mediated responses, including the activation of PKR and IRF3 and the induction of IFN-ß RNA. Our findings further implicate a balanced interplay between PKR and ADAR1 in modulating IFN-ß protein production following virus infection.

Measles virus C protein interferes with Beta interferon transcription in the nucleus.

Transcriptional induction of beta interferon (IFN-ß) through pattern recognition receptors is a key event in the host defense against invading viruses. Infection of cells by paramyxoviruses, like measles virus (MV) (genus Morbillivirus), is sensed predominantly by the ubiquitous cytoplasmic helicase RIG-I, recognizing viral 5'-triphosphate RNAs, and to some degree by MDA5. While MDA5 activation is effectively prevented by the MV V protein, the viral mechanisms for inhibition of MDA5-independent induction of IFN-ß remained obscure. Here, we identify the 186-amino-acid MV C protein, which shuttles between the nucleus and the cytoplasm, as a major viral inhibitor of IFN-ß transcription in human cells. Activation of the transcription factor IRF3 by upstream kinases and nuclear import of activated IRF3 were not affected in the presence of C protein, suggesting a nuclear target. Notably, C proteins of wild-type MV isolates, which are poor IFN-ß inducers, were found to comprise a canonical nuclear localization signal (NLS), whereas the NLSs of all vaccine strains, irrespective of their origins, were mutated. Site-directed mutagenesis of the C proteins from an MV wild-type isolate and from the vaccine virus strain Schwarz confirmed a correlation of nuclear localization and inhibition of IFN-ß transcription. A functional NLS and efficient nuclear accumulation are therefore critical for MV C to retain its potential to downregulate IFN-ß induction. We suggest that a defect in efficient nuclear import of C protein contributes to attenuation of MV vaccine strains.

CNS recruitment of CD8+ T lymphocytes specific for a peripheral virus infection triggers neuropathogenesis during polymicrobial challenge.

Although viruses have been implicated in central nervous system (CNS) diseases of unknown etiology, including multiple sclerosis and amyotrophic lateral sclerosis, the reproducible identification of viral triggers in such diseases has been largely unsuccessful. Here, we explore the hypothesis that viruses need not replicate in the tissue in which they cause disease; specifically, that a peripheral infection might trigger CNS pathology. To test this idea, we utilized a transgenic mouse model in which we found that immune cells responding to a peripheral infection are recruited to the CNS, where they trigger neurological damage. In this model, mice are infected with both CNS-restricted measles virus (MV) and peripherally restricted lymphocytic choriomeningitis virus (LCMV). While infection with either virus alone resulted in no illness, infection with both viruses caused disease in all mice, with ∼50% dying following seizures. Co-infection resulted in a 12-fold increase in the number of CD8+ T cells in the brain as compared to MV infection alone. Tetramer analysis revealed that a substantial proportion (>35%) of these infiltrating CD8+ lymphocytes were LCMV-specific, despite no detectable LCMV in CNS tissues. Mechanistically, CNS disease was due to edema, induced in a CD8-dependent but perforin-independent manner, and brain herniation, similar to that observed in mice challenged intracerebrally with LCMV. These results indicate that T cell trafficking can be influenced by other ongoing immune challenges, and that CD8+ T cell recruitment to the brain can trigger CNS disease in the apparent absence of cognate antigen. By extrapolation, human CNS diseases of unknown etiology need not be associated with infection with any particular agent; rather, a condition that compromises and activates the blood-brain barrier and adjacent brain parenchyma can render the CNS susceptible to pathogen-independent immune attack.