Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis

ABSTRACT: BACKGROUND: Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp) as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated. RESULTS: Using an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1) as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs) of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3[PRIME] poly(A) sequence identifying the 3[PRIME] end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA. CONCLUSIONS: NS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed replacement of cellular protein synthesis with viral protein synthesis during infection.     

Meningococcal factor H-binding protein: implications for disease susceptibility,virulence, and vaccines

Neisseria meningitidis is a human-adapted pathogen that causes meningitis and sepsis worldwide. N. meningitidis factor H-binding protein (fHbp) provides a mechanism for immune evasion by binding human complement factor H (CFH) to protect it from complement-mediated killing. Here, we discuss features of fHbp which enable it to engage human CFH (hCFH), and the regulation of fHbp expression. Studies of host susceptibility and bacterial genome-wide association studies (GWAS) highlight the importance of the interaction between fHbp and CFH and other complement factors, such as CFHR3, on the development of invasive meningococcal disease (IMD). Understanding the basis of fHbp:CFH interactions has also informed the design of next-generation vaccines as fHbp is a protective antigen. Structure-informed refinement of fHbp vaccines will help to combat the threat posed by the meningococcus, and accelerate the elimination of IMD.     

Molecular design, functional characterization and structural basis of a protein inhibitor against the HIV-1 pathogenicity factor Nef

Increased spread of HIV-1 and rapid emergence of drug resistance warrants development of novel antiviral strategies. Nef, a critical viral pathogenicity factor that interacts with host cell factors but lacks enzymatic activity, is not targeted by current antiviral measures. Here we inhibit Nef function by simultaneously blocking several highly conserved protein interaction surfaces. This strategy, referred to as "wrapping Nef", is based on structure-function analyses that led to the identification of four target sites: (i) SH3 domain interaction, (ii) interference with protein transport processes, (iii) CD4 binding and (iv) targeting to lipid membranes. Screening combinations of Nef-interacting domains, we developed a series of small Nef interacting proteins (NIs) composed of an SH3 domain optimized for binding to Nef, fused to a sequence motif of the CD4 cytoplasmic tail and combined with a prenylation signal for membrane association. NIs bind to Nef in the low nM affinity range, associate with Nef in human cells and specifically interfere with key biological activities of Nef. Structure determination of the Nef-inhibitor complex reveals the molecular basis for binding specificity. These results establish Nef-NI interfaces as promising leads for the development of potent Nef inhibitors.     

Expression, purification and characterization of recombinant severe acute respiratory syndrome coronavirus non-structural protein 1

The coronavirus (CoV) responsible for severe acute respiratory syndrome (SARS), SARS-CoV, encodes two large polyproteins (pp1a and pp1ab) that are processed by two viral proteases to yield mature non-structural proteins (nsps). Many of these nsps have essential roles in viral replication, but several have no assigned function and possess amino acid sequences that are unique to the CoV family. One such protein is SARS-CoV nsp1, which is processed from the N-terminus of both pp1a and pp1ab. The mature SARS-CoV protein is present in cells several hours post-infection and co-localizes to the viral replication complex, but its function in the viral life cycle remains unknown. Furthermore, nsp1 sequences are highly divergent across the CoV family, and it has been suggested that this is due to nsp1 possessing a function specific to viral interactions with its host cell or acting as a host specific virulence factor. In order to initiate structural and biophysical studies of SARS-CoV nsp1, a recombinant expression system and a purification protocol have been developed, yielding milligram quantities of highly purified SARS-CoV nsp1. The purified protein was characterized using circular dichroism, size exclusion chromatography, and multi-angle light scattering.     

Frp1 is a Fusarium oxysporum F-box protein required for pathogenicity on tomato

Fusarium oxysporum f. sp. lycopersici is the causal agent of tomato wilt disease. In order to identify genes involved in its pathogenicity, we performed insertional mutagenesis. Mutant N40 had lost its pathogenicity completely, when tested in bioassays with tomato seedlings. Molecular characterization of mutant N40 revealed that the plasmid insertion had occurred in a gene that codes for a 60.2 kDa protein containing an F-box motif. The gene was therefore designated as FRP1 (F-box protein required for pathogenicity). Targeted FRP1 disruptants had lost their pathogenicity completely, and became fully virulent again upon re-introduction of the FRP1 gene. This confirmed that the FRP1 gene is required for pathogenesis. In a yeast two-hybrid assay Frp1 interacts with Skp1, suggesting involvement of an SCF ubiquitin ligase complex in pathogenicity. FRP1 is constitutively expressed during infection and under different culture conditions. Although growth, spore formation and germination on artificial media were not impaired, confocal laser scanning microscopy of a GFP-marked mutant N40 and a GFP-marked targeted FRP1 disruptant revealed that they were unable to colonize the roots.     

NUT1, a major nitrogen regulatory gene inMagnaporthe grisea,is dispensable for pathogenicity

NUT1, a gene homologous to the major nitrogen regulatory genesnit-2 ofNeurospora crassa andareA ofAspergillus nidulans, was isolated from the rice blast fungus,Magnaporthe grisea. NUT1 encodes a protein of 956 amino acid residues and, likenit-2 andareA, has a single putative zinc finger DNA-binding domain. Functional equivalence ofNUT1 toareA was demonstrated by introducing theNUT1 gene by DNA-mediated transformation into anareA loss-of-function mutant ofA. nidulans. The introducedNUT1 gene fully complemented theareA null mutation, restoring to the mutant the ability to utilize a variety of nitrogen sources. In addition, the sensitivity ofAspergillus NUT1 transformants to ammonium repression of extracellular protease activity was comparable to that of wild-typeA. nidulans. Thus,NUT1 andareA encode functionally equivalent gene products that activate expression of nitrogen-regulated genes. A one-step gene disruption strategy was used to generatenutl ^? mutants ofM. grisea by transforming a rice-infecting strain with a disruption vector in which a gene for hygromycin B phosphotransferase (Hyg) replaced the zinc-finger DNA-binding motif ofNUT1. Of 31 hygromycin B (hyg B)-resistant transformants shown by Southern hybridization to contain a disruptedNUT1 gene (nut1::Hyg), 26 resulted from single-copy replacement events at theNUT1 locus. Althoughnut1 ^? transformants ofM. grisea failed to grown on a variety of nitrogen sources, glutamate, proline and alanine could still be utilized. This contrasts withA. nidulans where disruption of the zinc-finger region ofareA prevents utilization of nitrogen sources other than ammonium and glutamine. The role ofNUT1 and regulation of nitrogen metabolism in the disease process was evaluated by pathogenicity assays. The infection efficiency ofnut1 ^? transformants on susceptible rice plants was similar to that of the parental strain, although lesions were reduced in size. These studies demonstrate that theM. grisea NUT1 gene activates expression of nitrogen-regulated genes but is dispensable for pathogenicity.     

The endocytic protein intersectin is a major binding partner for the Ras exchange factor mSos1 in rat brain

We recently identified intersectin, a protein containing two EH and five SH3 domains, as a component of the endocytic machinery. The N-terminal SH3 domain (SH3A), unlike other SH3 domains from intersectin or various endocytic proteins, specifically inhibits intermediate events leading to the formation of clathrin-coated pits. We have now identified a brain-enriched, 170 kDa protein (p170) that interacts specifically with SH3A. Screening of combinatorial peptides reveals the optimal ligand for SH3A as Pp(V/I)PPR, and the 170 kDa mammalian son-of-sevenless (mSos1) protein, a guanine-nucleotide exchange factor for Ras, con- tains two copies of the matching sequence, PPVPPR. Immunodepletion studies confirm that p170 is mSos1. Intersectin and mSos1 are co-enriched in nerve terminals and are co-immunoprecipitated from brain extracts. SH3A competes with the SH3 domains of Grb2 in binding to mSos1, and the intersectin-mSos1 complex can be separated from Grb2 by sucrose gradient centrifugation. Overexpression of the SH3 domains of intersectin blocks epidermal growth factor-mediated Ras activation. These results suggest that intersectin functions in cell signaling in addition to its role in endocytosis and may link these cellular processes.     

TRPC1 protein channel is major regulator of epidermal growth factor receptor signaling

TRP channels have been associated with cell proliferation and aggressiveness in several cancers. In particular, TRPC1 regulates cell proliferation and motility, two processes underlying cancer progression. We and others have described the mechanisms of TRPC1-dependent cell migration. However, the involvement of TRPC1 in cell proliferation remains unexplained. In this study, we show that siRNA-mediated TRPC1 depletion in non small cell lung carcinoma cell lines induced G(0)/G(1) cell cycle arrest resulting in dramatic decrease in cell growth. The expression of cyclins D1 and D3 was reduced after TRPC1 knockdown, pointing out the role of TRPC1 in G(1)/S transition. This was associated with a decreased phosphorylation and activation of EGFR and with a subsequent disruption of PI3K/Akt and MAPK downstream pathways. Stimulation of EGFR by its natural ligand, EGF, induced Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry through TRPC1. Ca(2+) entry through TRPC1 conversely activated EGFR, suggesting that TRPC1 is a component of a Ca(2+)-dependent amplification of EGF-dependent cell proliferation.     

Toward rational protein crystallization: A Web server for the design of crystallizable protein variants

Growing well-diffracting crystals constitutes a serious bottleneck in structural biology. A recently proposed crystallization methodology for "stubborn crystallizers" is to engineer surface sequence variants designed to form intermolecular contacts that could support a crystal lattice. This approach relies on the concept of surface entropy reduction (SER), i.e., the replacement of clusters of flexible, solvent-exposed residues with residues with lower conformational entropy. This strategy minimizes the loss of conformational entropy upon crystallization and renders crystallization thermodynamically favorable. The method has been successfully used to crystallize more than 15 novel proteins, all stubborn crystallizers. But the choice of suitable sites for mutagenesis is not trivial. Herein, we announce a Web server, the surface entropy reduction prediction server (SERp server), designed to identify mutations that may facilitate crystallization. Suggested mutations are predicted based on an algorithm incorporating a conformational entropy profile, a secondary structure prediction, and sequence conservation. Minor considerations include the nature of flanking residues and gaps between mutation candidates. While designed to be used with default values, the server has many user-controlled parameters allowing for considerable flexibility. Within, we discuss (1) the methodology of the server, (2) how to interpret the results, and (3) factors that must be considered when selecting mutations. We also attempt to benchmark the server by comparing the server's predictions with successful SER structures. In most cases, the structure yielding mutations were easily identified by the SERp server. The server can be accessed at http://www.doe-mbi.ucla.edu/Services/SER.     

Trk is a calmodulin-binding protein: implications for receptor processing

The tyrosine kinase receptors for the neurotrophins (Trk) are a family of transmembrane receptors that regulate the differentiation and survival of different neuronal populations. Neurotrophin binding to Trk leads to the activation of several signalling pathways including a rapid, but moderate, increase in intracellular calcium levels. We have previously described the role of calcium and its sensor protein, calmodulin, in Trk-activated intracellular pathways. Here we demonstrate that calmodulin is able to precipitate TrkA from PC12 cell lysates. Using recombinant GST-fusion proteins containing the complete intracellular domain of TrkA, or fragments of this region, we show that calmodulin binds directly to the C-terminal domain of TrkA in a Ca2+-dependent manner. We have also co-immunoprecipitated endogenous Trk and calmodulin in primary cultures of cortical neurones. Moreover, we provide evidence that calmodulin is involved in the regulation of TrkA processing in PC12 cells. Calmodulin inhibition results in the generation of a TrkA-derived p41 fragment from the cytosolic portion of the protein. This fragment is autophosphorylated in tyrosines and can recruit PLCgamma and Shc adaptor proteins. These results suggest that calmodulin binding to Trk may be important for the regulation of Trk intracellular localization and cleavage.     

Rinderpest virus phosphoprotein gene is a major determinant of species-specific pathogenicity

We previously demonstrated that the rinderpest virus (RPV) hemagglutinin (H) protein plays an important role in determining host range but that other viral proteins are clearly required for full RPV pathogenicity to be manifest in different species. To examine the effects of the RPV nucleocapsid (N) protein and phosphoprotein (P) genes on RPV cross-species pathogenicity, we constructed two new recombinant viruses in which the H and P or the H, N, and P genes of the cattle-derived RPV RBOK vaccine were replaced with those from the rabbit-adapted RPV-Lv strain, which is highly pathogenic in rabbits. The viruses rescued were designated recombinant RPV-lapPH (rRPV-lapPH) and rRPV-lapNPH, respectively. Rabbits inoculated with RPV-Lv become feverish and show leukopenia and a decrease in body weight gain, while clinical signs of infection are never observed in rabbits inoculated with RPV-RBOK or with rRPV-lapH. However, rabbits inoculated with either rRPV-lapPH or rRPV-lapNPH became pyrexic and showed leukopenia. Further, histopathological lesions and high virus titers were clearly observed in the lymphoid tissues from animals infected with rRPV-lapPH or rRPV-lapNPH, although they were not observed in rabbits infected with RPV-RBOK or rRPV-lapH. The clinical, virological, and histopathological signs in rabbits infected with the two new recombinant viruses did not differ significantly; therefore, the RPV P gene was considered to be a key determinant of cross-species pathogenicity.     

Modeling coronavirus spike protein dynamics: implications for immunogenicity and immune escape

The ongoing COVID-19 pandemic is a global public health emergency requiring urgent development of efficacious vaccines. While concentrated research efforts have focused primarily on antibody-based vaccines that neutralize SARS-CoV-2, and several first-generation vaccines have either been approved or received emergency use authorization, it is forecasted that COVID-19 will become an endemic disease requiring updated second-generation vaccines. The SARS-CoV-2 surface spike (S) glycoprotein represents a prime target for vaccine development because antibodies that block viral attachment and entry, i.e., neutralizing antibodies, bind almost exclusively to the receptor-binding domain. Here, we develop computational models for a large subset of S proteins associated with SARS-CoV-2, implemented through coarse-grained elastic network models and normal mode analysis. We then analyze local protein domain dynamics of the S protein systems and their thermal stability to characterize structural and dynamical variability among them. These results are compared against existing experimental data and used to elucidate the impact and mechanisms of SARS-CoV-2 S protein mutations and their associated antibody binding behavior. We construct a SARS-CoV-2 antigenic map and offer predictions about the neutralization capabilities of antibody and S mutant combinations based on protein dynamic signatures. We then compare SARS-CoV-2 S protein dynamics to SARS-CoV and MERS-CoV S proteins to investigate differing antibody binding and cellular fusion mechanisms that may explain the high transmissibility of SARS-CoV-2. The outbreaks associated with SARS-CoV, MERS-CoV, and SARS-CoV-2 over the last two decades suggest that the threat presented by coronaviruses is ever-changing and long term. Our results provide insights into the dynamics-driven mechanisms of immunogenicity associated with coronavirus S proteins and present a new, to our knowledge, approach to characterize and screen potential mutant candidates for immunogen design, as well as to characterize emerging natural variants that may escape vaccine-induced antibody responses.     

The contribution of immunology to the rational design of novel antibacterial vaccines

In most cases, a successful vaccine must induce an immune response that is better than the response invoked by natural infection. Vaccines are still unavailable for several bacterial infections and vaccines to prevent such infections will be best developed on the basis of our increasing insights into the immune response. Knowledge of the signals that determine the best possible acquired immune response against a given pathogen - comprising a profound T- and B-cell memory response as well as long-lived plasma cells - will provide the scientific framework for the rational design of novel antibacterial vaccines.     

X-ray structures of the N- and C-terminal domains of a coronavirus nucleocapsid protein: implications for nucleocapsid formation

Coronaviruses cause a variety of respiratory and enteric diseases in animals and humans including severe acute respiratory syndrome. In these enveloped viruses, the filamentous nucleocapsid is formed by the association of nucleocapsid (N) protein with single-stranded viral RNA. The N protein is a highly immunogenic phosphoprotein also implicated in viral genome replication and in modulating cell signaling pathways. We describe the structure of the two proteolytically resistant domains of the N protein from infectious bronchitis virus (IBV), a prototype coronavirus. These domains are located at its N- and C-terminal ends (NTD and CTD, respectively). The NTD of the IBV Gray strain at 1.3-A resolution exhibits a U-shaped structure, with two arms rich in basic residues, providing a module for specific interaction with RNA. The CTD forms a tightly intertwined dimer with an intermolecular four-stranded central beta-sheet platform flanked by alpha helices, indicating that the basic building block for coronavirus nucleocapsid formation is a dimeric assembly of N protein. The variety of quaternary arrangements of the NTD and CTD revealed by the analysis of the different crystal forms delineates possible interfaces that could be used for the formation of a flexible filamentous ribonucleocapsid. The striking similarity between the dimeric structure of CTD and the nucleocapsid-forming domain of a distantly related arterivirus indicates a conserved mechanism of nucleocapsid formation for these two viral families.