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Direct observation of membrane insertion by enveloped virus matrix proteins by phosphate displacement
Enveloped virus release is driven by poorly understood proteins that are functional analogs of the coat protein assemblies that mediate intracellular vesicle trafficking. We used differential electron density mapping to detect membrane integration by membrane-bending proteins from five virus families. This demonstrates that virus matrix proteins replace an unexpectedly large portion of the lipid content of the inner membrane face, a generalized feature likely to play a role in reshaping cellular membranes
Alternate SlyA and H-NS nucleoprotein complexes control hlyE expression in Escherichia coli K-12
Haemolysin E is a cytolytic pore-forming toxin found in several Escherichia coli and Salmonella enterica strains. Expression of hlyE is repressed by the global regulator H-NS (histone-like nucleoid structuring protein), but can be activated by the regulator SlyA. Expression of a chromosomal hlyE–lacZ fusion in an E. coli slyA mutant was reduced to 60% of the wild-type level confirming a positive role for SlyA. DNase I footprint analysis revealed the presence of two separate SlyA binding sites, one located upstream, the other downstream of the hlyE transcriptional start site. These sites overlap AT-rich H-NS binding sites. Footprint and gel shift data showed that whereas H-NS prevented binding of RNA polymerase (RNAP) at the hlyE promoter (PhlyE), SlyA allowed binding of RNAP, but inhibited binding of H-NS. Accordingly, in vitro transcription analyses showed that addition of SlyA protein relieved H-NS-mediated repression of hlyE. Based on these observations a model for SlyA/H-NS regulation of hlyE expression is proposed in which the relative concentrations of SlyA and H-NS govern the nature of the nucleoprotein complexes formed at PhlyE. When H-NS is dominant RNAP binding is inhibited and hlyE expression is silenced; when SlyA is dominant H-NS binding is inhibited allowing RNAP access to the promoter facilitating hlyE transcription
ICTV Virus Taxonomy Profile : Arenaviridae
Members of the family Arenaviridae produce enveloped virions containing genomes consisting of two or three single-stranded RNA segments totalling about 10.5 kb. Arenaviruses can infect mammals, including humans and other primates, snakes, and fish. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Arenaviridae, which is available at www.ictv.global/report/arenaviridae.Non peer reviewe
ICTV Virus Taxonomy Profile: Arenaviridae 2023
Arenaviridae is a family for ambisense RNA viruses with genomes of about 10.5 kb that infect mammals, snakes, and fish. The arenavirid genome consists of two or three single-stranded RNA segments and encodes a nucleoprotein (NP), a glycoprotein (GP) and a large (L) protein containing RNA-directed RNA polymerase (RdRP) domains; some arenavirids encode a zinc-binding protein (Z). This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family Arenaviridae, which is available at www.ictv.global/report/arenaviridae
Accelerated and Improved Quantification of Lymphocytic Choriomeningitis Virus (LCMV) Titers by Flow Cytometry
Lymphocytic choriomeningitis virus (LCMV), a natural murine pathogen, is a member of the Arenavirus family, may cause atypical meningitis in humans, and has been utilized extensively as a model pathogen for the study of virus-induced disease and immune responses. Historically, viral titers have been quantified by a standard plaque assay, but for non-cytopathic viruses including LCMV this requires lengthy incubation, so results cannot be obtained rapidly. Additionally, due to specific technical constraints of the plaque assay including the visual detection format, it has an element of subjectivity along with limited sensitivity. In this study, we describe the development of a FACS-based assay that utilizes detection of LCMV nucleoprotein (NP) expression in infected cells to determine viral titers, and that exhibits several advantages over the standard plaque assay. We show that the LCMV-NP FACS assay is an objective and reproducible detection method that requires smaller sample volumes, exhibits a ∼20-fold increase in sensitivity to and produces results three times faster than the plaque assay. Importantly, when applied to models of acute and chronic LCMV infection, the LCMV-NP FACS assay revealed the presence of infectious virus in samples that were determined to be negative by plaque assay. Therefore, this technique represents an accelerated, enhanced and objective alternative method for detection of infectious LCMV that is amenable to adaptation for other viral infections as well as high throughput diagnostic platforms
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
Functional diversity of chemokines and chemokine receptors in response to viral infection of the central nervous system.
Encounters with neurotropic viruses result in varied outcomes ranging from encephalitis, paralytic poliomyelitis or other serious consequences to relatively benign infection. One of the principal factors that control the outcome of infection is the localized tissue response and subsequent immune response directed against the invading toxic agent. It is the role of the immune system to contain and control the spread of virus infection in the central nervous system (CNS), and paradoxically, this response may also be pathologic. Chemokines are potent proinflammatory molecules whose expression within virally infected tissues is often associated with protection and/or pathology which correlates with migration and accumulation of immune cells. Indeed, studies with a neurotropic murine coronavirus, mouse hepatitis virus (MHV), have provided important insight into the functional roles of chemokines and chemokine receptors in participating in various aspects of host defense as well as disease development within the CNS. This chapter will highlight recent discoveries that have provided insight into the diverse biologic roles of chemokines and their receptors in coordinating immune responses following viral infection of the CNS
Mutagenesis-Mediated Virus Extinction: Virus-Dependent Effect of Viral Load on Sensitivity to Lethal Defection
Background: Lethal mutagenesis is a transition towards virus extinction mediated by enhanced mutation rates during viral genome replication, and it is currently under investigation as a potential new antiviral strategy. Viral load and virus fitness are known to influence virus extinction. Here we examine the effect or the multiplicity of infection (MOI) on progeny production of several RNA viruses under enhanced mutagenesis. Results: The effect of the mutagenic base analogue 5-fluorouracil (FU) on the replication of the arenavirus lymphocytic choriomeningitis virus (LCMV) can result either in inhibition of progeny production and virus extinction in infections carried out at low multiplicity of infection (MOI), or in a moderate titer decrease without extinction at high MOI. The effect of the MOI is similar for LCMV and vesicular stomatitis virus (VSV), but minimal or absent for the picornaviruses foot-and-mouth disease virus (FMDV) and encephalomyocarditis virus (EMCV). The increase in mutation frequency and Shannon entropy (mutant spectrum complexity) as a result of virus passage in the presence of FU was more accentuated at low MOI for LCMV and VSV, and at high MOI for FMDV and EMCV. We present an extension of the lethal defection model that agrees with the experimental results. Conclusions: (i) Low infecting load favoured the extinction of negative strand viruses, LCMV or VSV, with an increase of mutant spectrum complexity. (ii) This behaviour is not observed in RNA positive strand viruses, FMDV or EMCV. (iii) The accumulation of defector genomes may underlie the MOI-dependent behaviour. (iv) LCMV coinfections are allowed but superinfection is strongly restricted in BHK-21 cells. (v) The dissimilar effects of the MOI on the efficiency of mutagenic-based extinction of different RNA viruses can have implications for the design of antiviral protocols based on lethal mutagenesis, presently under development. © 2012 Moreno et al.Centro de BiologÃa Molecular Severo Ochoa; Ministerio de Ciencia e Innovación (MICINN); Fundación Ramón ArecesPeer Reviewe
The Regulation of Sulfur Metabolism in Mycobacterium tuberculosis
Mycobacterium tuberculosis (Mtb) has evolved into a highly successful human pathogen. It deftly subverts the bactericidal mechanisms of alveolar macrophages, ultimately inducing granuloma formation and establishing long-term residence in the host. These hallmarks of Mtb infection are facilitated by the metabolic adaptation of the pathogen to its surrounding environment and the biosynthesis of molecules that mediate its interactions with host immune cells. The sulfate assimilation pathway of Mtb produces a number of sulfur-containing metabolites with important contributions to pathogenesis and survival. This pathway is regulated by diverse environmental cues and regulatory proteins that mediate sulfur transactions in the cell. Here, we discuss the transcriptional and biochemical mechanisms of sulfur metabolism regulation in Mtb and potential small molecule regulators of the sulfate assimilation pathway that are collectively poised to aid this intracellular pathogen in its expert manipulation of the host. From this global analysis, we have identified a subset of sulfur-metabolizing enzymes that are sensitive to multiple regulatory cues and may be strong candidates for therapeutic intervention
Characterization of MgtC, a Virulence Factor of Salmonella enterica Serovar Typhi
The MgtC is a virulence factor in Salmonella Typhimurium that is required for growth at low-Mg2+ concentrations and intramacrophage survival. This gene is codified in a conserved region of the Salmonella pathogenicity island 3 (SPI-3), and is also present in the chromosome of other Salmonella serovars. In this study we characterized the MgtC factor in S. Typhi, a human specific pathogen, by using mgtC and SPI-3 mutant strains. We found that MgtC is the most important factor codified in the SPI-3 of S. Typhi for growth in low-Mg2+ media and survival within human cells. In addition, by using reporter genes we determined that the low-Mg2+ concentration, acidic media and PhoP regulator induce mgtC expression in S. Typhi. We suggest that MgtC is the most important virulence factor codified in the SPI-3 of S. Typhi
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