Polypeptide 2CATPaseis one of the most thoroughly studied but least understood proteins in the life cycle of poliovirus. Within the protein, multiple functional domains important for uncoating, host cell membrane alterations, and RNA replication and encapsidation have previously been identified. In this study, charged to alanine-scanning mutagenesis was used to generate conditional-lethal mutations in hitherto-uncharacterized domains of the 2CATPasepolypeptide, particularly those involved in morphogenesis. Adjacent or clustered charged amino acids (2 to 4), scattered along the 2CATPasecoding sequence, were replaced with alanines. RNA transcripts of mutant poliovirus cDNA clones were transfected into HeLa cells. Subsequently, 10 lethal, 1 severely temperature-sensitive, 2 quasi-infectious, and 3 wild type-like mutants were identified. Using a luciferase-containing reporter virus, we demonstrated RNA replication defects in all lethal and quasi-infectious mutants. Temperature-sensitive mutants were defective in RNA replication only at the restricted temperatures. Furthermore, we characterized a quasi-infectious mutant (K6A/K7A) that produced a suppressor mutation (G1R) and a novel 2B∧2CATPasecleavage site (Q∧R). Surprisingly, this cleavage site mutation did not interfere with normal processing of the polyprotein. These mutants have led to the identification of several new sites within the 2CATPasepolypeptide that are required for RNA replication. In addition, analysis of the suppressor mutants has revealed a new domain near the C terminus of 2CATPasethat is involved in encapsidation, possibly achieved through interaction with an amino acid sequence between NTP binding motifs A and B of 2CATPase. Most importantly, the identification of suppressor mutations in both 2CATPaseand the capsid domains (VP1 and VP3) of poliovirus has confirmed that an interaction between 2CATPaseand capsid proteins is involved in viral morphogenesis.

Objective-Reactive oxygen species-generating nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase proteins (Noxs) are involved in cell differentiation, migration, and apoptosis. Nox4 is unique among Noxs in being constitutively active, and its subcellular localization may therefore be particularly important. In this study, we identified and characterized a novel nuclear-localized 28-kDa splice variant of Nox4 in vascular cells. Approach and Results-Nox4 immunoreactivity was noted in the nucleus and nucleolus of vascular smooth muscle cells and multiple other cell types by confocal microscopy. Cell fractionation, sequence analyses, and siRNA studies indicated that the nuclear-localized Nox4 is a 28-kDa splice variant, Nox4D, which lacks putative transmembrane domains. Nox4D overexpression resulted in significant NADPH-dependent reactive oxygen species production as detected by several different methods and caused increased phosphorylation of extracellular-signal-regulated kinase1/2 and the nuclear transcription factor Elk-1. Overexpression of Nox4D could also induce DNA damage as assessed by γ-H2AX phosphorylation. These effects were inhibited by a single amino acid substitution in the Nox4D NADPH-binding region. Conclusions-Nox4D is a nuclear-localized and functionally active splice variant of Nox4 that may have important pathophysiologic effects through modulation of nuclear signaling and DNA damage. Materials and MethodsMaterials and Methods are available in the online-only Supplement. Results Nuclear-Localized Nox4 ImmunoreactivityWe used a well-validated polyclonal antibody directed against the C-terminal region of Nox4 11,20,21 to investigate the localization of endogenous Nox4 by immunofluorescence in several cell types, including VSMC, endothelial cells, fibroblasts, and cardiomyocytes. A diffuse reticular staining pattern through the cell that colocalized with an ER marker, protein disulfide isomerase, was evident in most cell types ( Figures 1A and 2) as reported previously. 15,16 In addition, however, there was strong focal and granular Nox4 immunostaining within the nucleus in all cell types. Electron microscopy indicated that the focal Nox4 staining was in the nucleolus ( Figure 1C). We confirmed that the focal intranuclear staining was nucleolar by costaining with 2 different nucleolar markers, nucleophosmin and fibrillarin ( Figure 1B). Preincubation of the Nox4 antibody with the antigenic peptide against which it was raised completely abolished both the perinuclear and nucleolar staining, verifying the specificity of antigen-antibody interaction ( Figure 1D). Furthermore, treatment with a prevalidated Nox4 siRNA to knock down Nox4 protein expression also abolished staining as compared with a scrambled siRNA ( Figure 3A). Nuclear-Localized Nox4 Is a 28-kDa Nox4 Splice VariantTo confirm nuclear and nucleolar localization, VSMCs were fractionated into membrane and nuclear fractions. Immunoblotting for Nox4 demonstrated an ≈65-kDa band corresponding to the expected molecular size of Nox4 but ...

BackgroundThe effect of cold temperature on arthritis symptoms is unclear. The aim of this study was to investigate how environmental cold affects pain and blood flow in mono-arthritic mice, and examine a role for transient receptor potential ankyrin 1 (TRPA1), a ligand-gated cation channel that can act as a cold sensor.MethodsMono-arthritis was induced by unilateral intra-articular injection of complete Freund’s adjuvant (CFA) in CD1 mice, and in mice either lacking TRPA1 (TRPA1 KO) or respective wildtypes (WT). Two weeks later, nociception and joint blood flow were measured following exposure to 10 °C (1 h) or room temperature (RT). Primary mechanical hyperalgesia in the knee was measured by pressure application apparatus; secondary mechanical hyperalgesia by automated von Frey system; thermal hyperalgesia by Hargreaves technique, and weight bearing by the incapacitance test. Joint blood flow was recorded by full-field laser perfusion imager (FLPI) and using clearance of 99mTechnetium. Blood flow was assessed after pretreatment with antagonists of either TRPA1 (HC-030031), substance P neurokinin 1 (NK1) receptors (SR140333) or calcitonin gene-related peptide (CGRP) (CGRP8–37). TRPA1, TAC-1 and CGRP mRNA levels were examined in dorsal root ganglia, synovial membrane and patellar cartilage samples.ResultsCold exposure caused bilateral primary mechanical hyperalgesia 2 weeks after CFA injection, in a TRPA1-dependent manner. In animals maintained at RT, clearance techniques and FLPI showed that CFA-treated joints exhibited lower blood flow than saline-treated joints. In cold-exposed animals, this reduction in blood flow disappears, and increased blood flow in the CFA-treated joint is observed using FLPI. Cold-induced increased blood flow in CFA-treated joints was blocked by HC-030031 and not observed in TRPA1 KOs. Cold exposure increased TRPA1 mRNA levels in patellar cartilage, whilst reducing it in synovial membranes from CFA-treated joints.ConclusionsWe provide evidence that environmental cold exposure enhances pain and increases blood flow in a mono-arthritis model. These changes are dependent on TRPA1. Thus, TRPA1 may act locally within the joint to influence blood flow via sensory nerves, in addition to its established nociceptive actions.

Sepsis is a systemic inflammatory response triggered by microbial infection that can cause cardiovascular collapse, insufficient tissue perfusion and multi-organ failure. The cation channel transient receptor potential vanilloid 4 (TRPV4) is expressed in vascular endothelium and causes vasodilatation, but excessive TRPV4 activation leads to profound hypotension and circulatory collapse - key features of sepsis pathogenesis. We hypothesised that loss of TRPV4 signaling would protect against cardiovascular dysfunction in a mouse model of sepsis (endotoxaemia).Multi-parameter monitoring of conscious systemic haemodynamics (by radiotelemetry probe), mesenteric microvascular blood flow (laser speckle contrast imaging) and blood biochemistry (iSTAT blood gas analysis) was carried out in wild type (WT) and TRPV4 knockout (KO) mice. Endotoxaemia was induced by a single intravenous injection of lipopolysaccharide (LPS; 12.5 mg/kg) and systemic haemodynamics monitored for 24 h. Blood flow recording was then conducted under terminal anaesthesia after which blood was obtained for haematological/biochemical analysis. No significant differences were observed in baseline haemodynamics or mesenteric blood flow. Naïve TRPV4 KO mice were significantly acidotic relative to WT counterparts. Following induction of sepsis, all mice became significantly hypotensive, though there was no significant difference in the degree of hypotension between TRPV4 WT and KO mice. TRPV4 KO mice exhibited a higher sepsis severity score. While septic WT mice became significantly hypernatraemic relative to the naïve state, this was not observed in septic KO mice. Mesenteric blood flow was inhibited by topical application of the TRPV4 agonist GSK1016790A in naïve WT mice, but enhanced 24 h following LPS injection. Contrary to the initial hypothesis, loss of TRPV4 signaling (either through gene deletion or pharmacological antagonism) did not attenuate sepsis-induced cardiovascular dysfunction: in fact, pathology appeared to be modestly exaggerated in mice lacking TRPV4. Local targeting of TRPV4 signalling may be more beneficial than global inhibition in sepsis treatment.