The Presence of the Iron-Sulfur Motif Is Important for the Conformational Stability of the Antiviral Protein, Viperin

Viperin, an antiviral protein, has been shown to contain a CX3CX2C motif, which is conserved in the radical S-adenosyl-methionine (SAM) enzyme family. A triple mutant which replaces these three cysteines with alanines has been shown to have severe deficiency in antiviral activity. Since the crystal structure of Viperin is not available, we have used a combination of computational methods including multi-template homology modeling and molecular dynamics simulation to develop a low-resolution predicted structure. The results show that Viperin is an α -β protein containing iron-sulfur cluster at the center pocket. The calculations suggest that the removal of iron-sulfur cluster would lead to collapse of the protein tertiary structure. To verify these predictions, we have prepared, expressed and purified four mutant proteins. In three mutants individual cysteine residues were replaced by alanine residues while in the fourth all the cysteines were replaced by alanines. Conformational analyses using circular dichroism and steady state fluorescence spectroscopy indicate that the mutant proteins are partially unfolded, conformationally unstable and aggregation prone. The lack of conformational stability of the mutant proteins may have direct relevance to the absence of their antiviral activity

Radical SAM enzyme QueE defines a new minimal core fold and metal-dependent mechanism

7-carboxy-7-deazaguanine synthase (QueE) catalyzes a key S-adenosyl-L-methionine (AdoMet)- and Mg[superscript 2+]-dependent radical-mediated ring contraction step, which is common to the biosynthetic pathways of all deazapurine-containing compounds. QueE is a member of the AdoMet radical superfamily, which employs the 5′-deoxyadenosyl radical from reductive cleavage of AdoMet to initiate chemistry. To provide a mechanistic rationale for this elaborate transformation, we present the crystal structure of a QueE along with structures of pre- and post-turnover states. We find that substrate binds perpendicular to the [4Fe-4S]-bound AdoMet, exposing its C6 hydrogen atom for abstraction and generating the binding site for Mg[superscript 2+], which coordinates directly to the substrate. The Burkholderia multivorans structure reported here varies from all other previously characterized members of the AdoMet radical superfamily in that it contains a hypermodified ([β [subscript 6] over α [subscript 3]]) protein core and an expanded cluster-binding motif, CX[subscript 14]CX[subscript 2]C.United States. Dept. of Energy. Office of Biological and Environmental ResearchUnited States. Dept. of Energy. Office of Basic Energy SciencesNational Center for Research Resources (U.S.) (P41RR012408)National Institute of General Medical Sciences (U.S.) (P41GM103473)National Center for Research Resources (U.S.) (5P41RR015301-10)National Institute of General Medical Sciences (U.S.) (8 P41 GM 103403-10)United States. Dept. of Energy (Contract DE-AC02-06CH11357

Transcriptome profiling of immune responses to cardiomyopathy syndrome (CMS) in Atlantic salmon

<p>Abstract</p> <p>Background</p> <p>Cardiomyopathy syndrome (CMS) is a disease associated with severe myocarditis primarily in adult farmed Atlantic salmon (<it>Salmo salar </it>L.), caused by a double-stranded RNA virus named piscine myocarditis virus (PMCV) with structural similarities to the <it>Totiviridae </it>family. Here we present the first characterisation of host immune responses to CMS assessed by microarray transcriptome profiling.</p> <p>Results</p> <p>Unvaccinated farmed Atlantic salmon post-smolts were infected by intraperitoneal injection of PMCV and developed cardiac pathology consistent with CMS. From analysis of heart samples at several time points and different tissues at early and clinical stages by oligonucleotide microarrays (SIQ2.0 chip), six gene sets representing a broad range of immune responses were identified, showing significant temporal and spatial regulation. Histopathological examination of cardiac tissue showed myocardial lesions from 6 weeks post infection (wpi) that peaked at 8-9 wpi and was followed by a recovery. Viral RNA was detected in all organs from 4 wpi suggesting a broad tissue tropism. High correlation between viral load and cardiac histopathology score suggested that cytopathic effect of infection was a major determinant of the myocardial changes. Strong and systemic induction of antiviral and IFN-dependent genes from 2 wpi that levelled off during infection, was followed by a biphasic activation of pathways for B cells and MHC antigen presentation, both peaking at clinical pathology. This was preceded by a distinct cardiac activation of complement at 6 wpi, suggesting a complement-dependent activation of humoral Ab-responses. Peak of cardiac pathology and viral load coincided with cardiac-specific upregulation of T cell response genes and splenic induction of complement genes. Preceding the reduction in viral load and pathology, these responses were probably important for viral clearance and recovery.</p> <p>Conclusions</p> <p>By comparative analysis of gene expression, histology and viral load, the temporal and spatial regulation of immune responses were characterised and novel immune genes identified, ultimately leading to a more complete understanding of host-virus responses and pathology and protection in Atlantic salmon during CMS.</p

Cysteine Methylation Controls Radical Generation in the Cfr Radical AdoMet rRNA Methyltransferase

The 'radical S-adenosyl-L-methionine (AdoMet)' enzyme Cfr methylates adenosine 2503 of the 23S rRNA in the peptidyltransferase centre (P-site) of the bacterial ribosome. This modification protects host bacteria, notably methicillin-resistant Staphylococcus aureus (MRSA), from numerous antibiotics, including agents (e.g. linezolid, retapamulin) that were developed to treat such organisms. Cfr contains a single [4Fe-4S] cluster that binds two separate molecules of AdoMet during the reaction cycle. These are used sequentially to first methylate a cysteine residue, Cys338; and subsequently generate an oxidative radical intermediate that facilitates methyl transfer to the unreactive C8 (and/or C2) carbon centres of adenosine 2503. How the Cfr active site, with its single [4Fe-4S] cluster, catalyses these two distinct activities that each utilise AdoMet as a substrate remains to be established. Here, we use absorbance and electron paramagnetic resonance (EPR) spectroscopy to investigate the interactions of AdoMet with the [4Fe-4S] clusters of wild-type Cfr and a Cys338 Ala mutant, which is unable to accept a methyl group. Cfr binds AdoMet with high (∼ 10 µM) affinity notwithstanding the absence of the RNA cosubstrate. In wild-type Cfr, where Cys338 is methylated, AdoMet binding leads to rapid oxidation of the [4Fe-4S] cluster and production of 5'-deoxyadenosine (DOA). In contrast, while Cys338 Ala Cfr binds AdoMet with equivalent affinity, oxidation of the [4Fe-4S] cluster is not observed. Our results indicate that the presence of a methyl group on Cfr Cys338 is a key determinant of the activity of the enzyme towards AdoMet, thus enabling a single active site to support two distinct modes of AdoMet cleavage

Expression profiles of immune mediators in feline Coronavirus-infected cells and clinical samples of feline Coronavirus-positive cats

Abstract Background There are two biotypes of feline coronavirus (FCoV): the self-limiting feline enteric coronavirus (FECV) and the feline infectious peritonitis virus (FIPV), which causes feline infectious peritonitis (FIP), a fatal disease associated with cats living in multi-cat environments. This study provides an insight on the various immune mediators detected in FCoV-positive cats which may be responsible for the development of FIP. Results In this study, using real-time PCR and multiplex bead-based immunoassay, the expression profiles of several immune mediators were examined in Crandell-Reese feline kidney (CRFK) cells infected with the feline coronavirus (FCoV) strain FIPV 79–1146 and in samples obtained from FCoV-positive cats. CRFK cells infected with FIPV 79–1146 showed an increase in the expression of interferon-related genes and pro-inflammatory cytokines such as MX1, viperin, CXCL10, CCL8, RANTES, KC, MCP1, and IL8. In addition, an increase in the expression of the above cytokines as well as GM-CSF and IFNγ was also detected in the PBMC, serum, and peritoneal effusions of FCoV-positive cats. Although the expression of MX1 and viperin genes was variable between cats, the expression of these two genes was relatively higher in cats having peritoneal effusion compared to cats without clinically obvious effusion. Higher viral load was also detected in the supernatant of peritoneal effusions compared to in the plasma of FCoV-positive cats. As expected, the secretion of IL1β, IL6 and TNFα was readily detected in the supernatant of peritoneal effusions of the FCoV-positive cats. Conclusions This study has identified various pro-inflammatory cytokines and interferon-related genes such as MX1, viperin, CXCL10, CCL8, RANTES, KC, MCP1, IL8, GM-CSF and IFNγ in FCoV-positive cats. With the exception of MX1 and viperin, no distinct pattern of immune mediators was observed that distinguished between FCoV-positive cats with and without peritoneal effusion. Further studies based on definitive diagnosis of FIP need to be performed to confirm the clinical importance of this study