Lack of an appropriate animal model for dengue virus (DEN), which causes dengue fever and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), has impeded characterization of the mechanisms underlying the disease pathogenesis. The cardinal feature of DHF/DSS, the severe form of DEN infection, is increased vascular permeability. To develop a murine model that is more relevant to DHF/DSS, a novel DEN strain, D2S10, was generated by alternately passaging a non-mouse-adapted DEN strain between mosquito cells and mice, thereby mimicking the natural transmission cycle of the virus between mosquitoes and humans. After infection with D2S10, mice lacking interferon receptors died early without manifesting signs of paralysis, carried infectious virus in both non-neuronal and neuronal tissues, and exhibited signs of increased vascular permeability. In contrast, mice infected with the parental DEN strain developed paralysis at late times after infection, contained detectable levels of virus only in the central nervous system, and displayed normal vascular permeability. In the mice infected with D2S10, but not the parental DEN strain, significant levels of serum tumor necrosis factor alpha (TNF-␣) were produced, and the neutralization of TNF-␣ activity prevented early death of D2S10-infected mice. Sequence analysis comparing D2S10 to its parental strain implicated a conserved region of amino acid residues in the envelope protein as a possible source for the D2S10 phenotype. These results demonstrate that D2S10 causes a more relevant disease in mice and that TNF-␣ may be one of several key mediators of severe DEN-induced disease in mice. This report represents a significant advance in animal models for severe DEN disease, and it begins to provide mechanistic insights into DEN-induced disease in vivo.Dengue virus (DEN) causes dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), with an estimated 2.5 billion people at risk for infection in subtropical and tropical regions of the world (6). DEN is a positivesense, single-stranded RNA virus that belongs to the family Flaviviridae and the genus Flavivirus, which includes yellow fever (YFV), West Nile, Japanese encephalitis, and St. Louis encephalitis viruses. Primary infection with any one of the four DEN serotypes typically leads to DF, a debilitating but selflimited acute febrile illness. However, some primary infections and a larger percentage of secondary infections with a different serotype result in the severe, life-threatening DHF/DSS, characterized by increased vascular permeability and hemorrhagic manifestations (6). Despite its name, bleeding manifestations are minor and major hemorrhages are unusual; instead, increased vascular permeability is the hallmark of DHF/DSS. In rare cases of DHF/DSS, neurologic abnormalities, including encephalitis, may also occur (32). DEN is transmitted to humans by the mosquitoes Aedes aegypti and Aedes albopictus. Due to uncontrolled urbanization, globalization, and dissemination of DEN-transmitting mosquito...

Defining specific pathways for efficient heat transfer from protein–solvent interfaces to their active sites represents one of the compelling and timely challenges in our quest for a physical description of the origins of enzyme catalysis. Enzymatic hydrogen tunneling reactions constitute excellent systems in which to validate experimental approaches to this important question, given the inherent temperature independence of quantum mechanical wave function overlap. Herein, we present the application of hydrogen–deuterium exchange coupled to mass spectrometry toward the spatial resolution of protein motions that can be related to an enzyme’s catalytic parameters. Employing the proton-coupled electron transfer reaction of soybean lipoxygenase as proof of principle, we first corroborate the impact of active site mutations on increased local flexibility and, second, uncover a solvent-exposed loop, 15–34 Å from the reactive ferric center whose temperature-dependent motions are demonstrated to mirror the enthalpic barrier for catalytic C–H bond cleavage. A network that connects this surface loop to the active site is structurally identified and supported by changes in kinetic parameters that result from site-specific mutations.

Bacterial growth and cell division are coordinated with hydrolysis of the peptidoglycan (PG) layer of the cell wall, but the mechanisms of regulation of extracellular PG hydrolases are not well understood. Here we report the biochemical, structural, and genetic analysis of the Mycobacterium tuberculosis homolog of the transmembrane PG-hydrolase regulator, FtsX. The purified FtsX extracellular domain binds the PG peptidase Rv2190c/RipC N-terminal segment, causing a conformational change that activates the enzyme. Deletion of ftsEX and ripC caused similar phenotypes in Mycobacterium smegmatis, as expected for genes in a single pathway. The crystal structure of the FtsX extracellular domain reveals an unprecedented fold containing two lobes connected by a flexible hinge. Mutations in the hydrophobic cleft between the lobes reduce RipC binding in vitro and inhibit FtsX function in M. smegmatis. These studies suggest how FtsX recognizes RipC and support a model in which a conformational change in FtsX links the cell division apparatus with PG hydrolysis.bacterial cell wall | extracellular signaling | divisome | long-range conformational change

The four serotypes of dengue virus (DENV1 to DENV4) cause extensive morbidity and mortality. A major obstacle to studying disease pathogenesis and developing therapies has been the lack of a small-animal model. We previously reported isolation of a DENV2 strain, obtained by passaging a clinical isolate between mosquito cells and mice, that caused severe DENV disease in mice and contained multiple mutations, including many in domain II of the envelope (E) protein. Here, we describe a recombinant virus, differing from the non-mouse-passaged virus by two mutations in the E protein, that induces vascular leakage and tumor necrosis factor alpha (TNF-␣)-mediated lethality, while the non-mouse-passaged virus causes paralysis. This recombinant virus has a weaker affinity for heparan sulfate, resulting in an increased serum half-life, higher systemic viral loads, and high levels of TNF-␣ in the serum of infected mice. These results exemplify the role of the E protein in modulating virion clearance and connect the effect of clearance on the systemic viral loads responsible for severe disease manifestations.The four serotypes of dengue virus (DENV1 to DENV4) are the etiologic agents of dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). DF is an acute, self-limited febrile illness. The more life-threatening DHF/DSS is characterized by a transient increase in vascular permeability, resulting in the leakage of plasma to the interstitium. DENV is transmitted to humans primarily by the mosquitoes Aedes aegypti and Aedes albopictus. It belongs to the Flaviviridae family and is related to the viruses that cause yellow fever (YFV), hepatitis C, and the West Nile (WNV), Japanese (JEV), and St. Louis encephalitides (5). DENV is an enveloped virus with a single-stranded, 10.7-kb, positive-sense RNA genome that is translated as a single polyprotein and cleaved into three structural proteins (C, prM/M, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by both viral and host proteases. At present, no effective antiviral therapy or vaccine exists. An estimated 2.5 billion people are at risk for DENV infection worldwide, and 50 million new cases of DF, 250,000 to 500,000 cases of DHF/DSS, and 25,000 deaths are reported per year (14,16).Despite the significant public health threat posed by DENV, the mechanisms of viral pathogenesis remain unclear. To better understand DENV disease, several mouse models have been developed. Mouse models of DENV infection include (i) intracerebral injection of suckling mice (50); (ii) mouse brainadapted DENV strains that rapidly induce encephalitis (12); (iii) A/J (20, 51) and AG129 (26, 52) mice that are infected with a high dose of a non-mouse-adapted DENV strain; (iv) chimeric SCID mice that are transplanted with either human peripheral blood mononuclear cells (58); CD34 ϩ cord blood cells (2), erythroleukemic K562 cells (36), or HepG2 hepatocarcinoma cells (1); and (v) chimeric RAG2 Ϫ/Ϫ ␥ c Ϫ/Ϫ mice transplanted with human fetal liver-de...

Dengue virus (DEN), a flavivirus, causes dengue fever and dengue hemorrhagic fever/dengue shock syndrome, the most common mosquito-borne viral illnesses in humans worldwide. In this study, using STAT1−/− mice bearing two different mutant stat1 alleles in the 129/Sv/Ev background, we demonstrate that IFNR-dependent control of primary DEN infection involves both STAT1-dependent and STAT1-independent mechanisms. The STAT1 pathway is necessary for clearing the initial viral load, whereas the STAT1-independent pathway controls later viral burden and prevents DEN disease in mice. The STAT1-independent responses in mice with primary DEN infection included the early activation of B and NK cells as well as the up-regulation of MHC class I molecules on macrophages and dendritic cells. Infection of bone marrow-derived dendritic cell cultures with either DEN or Sindbis virus, another positive-strand RNA virus, confirmed the early vs late natures of the STAT1-dependent and STAT1-independent pathways. Collectively, these data begin to define the nature of the STAT1-dependent vs the STAT1-independent pathway in vivo.

The HUSH complex represses retroviruses, transposons and genes to maintain the integrity of vertebrate genomes. HUSH regulates deposition of the epigenetic mark H3K9me3, but how its three core subunits — TASOR, MPP8 and Periphilin — contribute to assembly and targeting of the complex remains unknown. Here, we define the biochemical basis of HUSH assembly and find that its modular architecture resembles the yeast RNA-induced transcriptional silencing complex. TASOR, the central HUSH subunit, associates with RNA processing components. TASOR is required for H3K9me3 deposition over LINE-1 repeats and repetitive exons in transcribed genes. In the context of previous studies, this suggests that an RNA intermediate is important for HUSH activity. We dissect the TASOR and MPP8 domains necessary for transgene repression. Structure-function analyses reveal TASOR bears a catalytically-inactive PARP domain necessary for targeted H3K9me3 deposition. We conclude that TASOR is a multifunctional pseudo-PARP that directs HUSH assembly and epigenetic regulation of repetitive genomic targets.

The evolution of recognition specificities by the immune system depends on the generation of receptor diversity and on connecting the binding of new antigens with the initiation of downstream signaling. In plant immunity, the innate Nucleotide-Binding Leucine Rich Repeat (NLR) receptor family enables antigen binding and immune signaling. In this study, we surveyed the NLR complements of 62 ecotypes of Arabidopsis thaliana and 54 lines of Brachypodium distachyon and identified a limited number of NLR subfamilies that show high allelic diversity. We show that the predicted specificity-determining residues cluster on the surfaces of Leucine Rich Repeat domains, but the locations of the clusters vary among NLR subfamilies. By comparing NLR phylogeny, allelic diversity, and known functions of the Arabidopsis NLRs, we formulate a hypothesis for the emergence of direct and indirect pathogen-sensing receptors and of the autoimmune NLRs. These findings reveal the recurring patterns of evolution of innate immunity and can inform NLR engineering efforts.

Background: Peptidoglycan hydrolases help elongate, divide, and recycle bacterial cell walls. Results: The structure and activity of Rv3717, a peptidoglycan hydrolase of Mycobacterium tuberculosis, are described. Conclusion: Our data suggest a peptidoglycan-recycling role for Rv3717. Significance: Functional specialization of homologous peptidoglycan hydrolases can be achieved by altering their substrate range via structural variation.