Identification, design and synthesis of novel pyrazolopyridine influenza virus nonstructural protein 1 antagonists.

Nonstructural protein 1 (NS1) plays a crucial function in the replication, spread, and pathogenesis of influenza virus by inhibiting the host innate immune response. Here we report the discovery and optimization of novel pyrazolopyridine NS1 antagonists that can potently inhibit influenza A/PR/8/34 replication in MDCK cells, rescue MDCK cells from cytopathic effects of seasonal influenza A strains, reverse NS1-dependent inhibition of IFN-ß gene expression, and suppress the slow growth phenotype in NS1-expressing yeast. These pyrazolopyridines will enable researchers to investigate NS1 function during infection and how antagonists can be utilized in the next generation of treatments for influenza infection.

Novel inhibitor of influenza non-structural protein 1 blocks multi-cycle replication in an RNase L-dependent manner.

Influenza virus non-structural protein 1 (NS1) is the centrepiece of the viral response to the host interferon (IFN) system. NS1 has been demonstrated previously to be a potential therapeutic target for antiviral therapy by identification of specific small-molecule inhibitors. This study demonstrated the biological mechanism for a potent new NS1 antagonist. Compound JJ3297 inhibited virus replication by more than three orders of magnitude without affecting cell viability. Importantly, it efficiently reversed NS1-induced inhibition of IFN mRNA production. The hypothesis was tested that JJ3297 facilitates IFN production in infected cells, leading to protection of the surrounding uninfected cells. Accordingly, the compound efficiently prevented virus spread through a cell population during a 48 h multi-cycle infection initiated at a very low m.o.i. Consistent with the hypothesis, the compound had no detectable influence on a 6 h single-cycle infection initiated at a high m.o.i. The effect of JJ3297 on virus replication was not caused by inhibition of NS1 expression or its mislocalization in the cell. JJ3297 facilitated the induction of an IFN-like antiviral state, resulting in increased resistance to subsequent challenge with vesicular stomatitis virus. The activity of JJ3297 absolutely required the function of cellular RNase L, indicating that an intact IFN system is required for function of the compound. These results support a model in which inhibition of NS1 function results in restoration of the IFN-induced antiviral state and inhibition of virus replication and spread. This represents a new direction for anti-influenza virus drug development that exploits the IFN pathway to challenge virus replication.

A SPIKE1 signaling complex controls actin-dependent cell morphogenesis through the heteromeric WAVE and ARP2/3 complexes.

During morphogenesis, the actin cytoskeleton mediates cell-shape change in response to growth signals. In plants, actin filaments organize the cytoplasm in regions of polarized growth, and the filamentous arrays can be highly dynamic. Small GTPase signaling proteins termed Rho of plants (ROP)/RAC control actin polymerization. ROPs cycle between inactive GDP-bound and active GTP-bound forms, and it is the active form that interacts with effector proteins to mediate cytoskeletal rearrangement and cell-shape change. A class of proteins termed guanine nucleotide exchange factors (GEFs) generate GTP-ROP and positively regulate ROP signaling. However, in almost all experimental systems, it has proven difficult to unravel the complex signaling pathways from GEFs to the proteins that nucleate actin filaments. In this article, we show that the DOCK family protein SPIKE1 (SPK1) is a GEF, and that one function of SPK1 is to control actin polymerization via two heteromeric complexes termed WAVE and actin-related protein (ARP) 2/3. The genetic pathway was constructed by using a combination of highly informative spk1 alleles and detailed analyses of spk1, wave, and arp2/3 single and double mutants. Remarkably, we find that in addition to providing GEF activity, SPK1 associates with WAVE complex proteins and may spatially organize signaling. Our results describe a unique regulatory scheme for ARP2/3 regulation in cells, one that can be tested for widespread use in other multicellular organisms.

Novel influenza virus NS1 antagonists block replication and restore innate immune function.

The innate immune system guards against virus infection through a variety of mechanisms including mobilization of the host interferon system, which attacks viral products mainly at a posttranscriptional level. The influenza virus NS1 protein is a multifunctional facilitator of virus replication, one of whose actions is to antagonize the interferon response. Since NS1 is required for efficient virus replication, it was reasoned that chemical inhibitors of this protein could be used to further understand virus-host interactions and also serve as potential new antiviral agents. A yeast-based assay was developed to identify compounds that phenotypically suppress NS1 function. Several such compounds exhibited significant activity specifically against influenza A virus in cell culture but had no effect on the replication of another RNA virus, respiratory syncytial virus. Interestingly, cells lacking an interferon response were drug resistant, suggesting that the compounds block interactions between NS1 and the interferon system. Accordingly, the compounds reversed the inhibition of beta interferon mRNA induction during infection, which is known to be caused by NS1. In addition, the compounds blocked the ability of NS1 protein to inhibit double-stranded RNA-dependent activation of a transfected beta interferon promoter construct. The effects of the compounds were specific to NS1, because they had no effect on the ability of the severe acute respiratory syndrome coronavirus papainlike protease protein to block beta interferon promoter activation. These data demonstrate that the function of NS1 can be modulated by chemical inhibitors and that such inhibitors will be useful as probes of biological function and as starting points for clinical drug development.

Arabidopsis GNARLED encodes a NAP125 homolog that positively regulates ARP2/3.

In migrating cells, the actin filament nucleation activity of ARP2/3 is an essential component of dynamic cell shape change and motility. In response to signals from the small GTPase Rac1, alterations in the composition and/or subcellular localization of the WAVE complex lead to ARP2/3 activation. The human WAVE complex subunit, WAVE1/SCAR1, was first identified in Dictyostelium and is a direct ARP2/3 activator. In the absence of an intact WAVE complex, SCAR/WAVE protein is destabilized. Although the composition of the five-subunit WAVE complex is well characterized, the means by which individual subunits and fully assembled WAVE complexes regulate ARP2/3 in vivo are unclear. The molecular genetics of trichome distortion in Arabidopsis is a powerful system to understand how signaling pathways and ARP2/3 control multicellular development. In this paper we prove that the GNARLED gene encodes a homolog of the WAVE subunit NAP125. Despite the moderate level of amino acid identity between Arabidopsis and human NAP125, both homologs were functionally interchangeable in vivo and interacted physically with the putative Arabidopsis WAVE subunit ATSRA1. gnarled trichomes had nearly identical cell shape and actin cytoskeleton phenotypes when compared to ARP2/3 subunit mutants, suggesting that GRL positively regulates ARP2/3.

Requirements for Arabidopsis ATARP2 and ATARP3 during epidermal development.

Plant cells employ the actin cytoskeleton to stably position organelles, as tracks for long distance transport, and to reorganize the cytoplasm in response to developmental and environmental cues. While diverse classes of actin binding proteins have been implicated in growth control, the mechanisms of cytoskeletal reorganization and the cellular functions of specific actin filament arrays are unclear. Arabidopsis trichome morphogenesis includes distinct requirements for the microtubule and actin filament cytoskeletons. It also is a genetically tractable process that is providing new knowledge about cytoskeleton function in plants. The "distorted group" of mutants defines a class of at least eight genes that are required during the actin-dependent phase of trichome growth. Using map-based cloning and a candidate gene approach, we identified mutations in ARP3 (ATARP3) and ARP2 (ATARP2) genes as the cause of the distorted1 (dis1) and wurm (wrm) phenotypes, respectively. ARP2 and ARP3 are components of the evolutionarily conserved ARP2/3 complex that nucleates actin filament polymerization [3]. Mutations in DIS1 and WRM caused severe trichome growth defects but had relatively mild effects on shoot development. DIS1 rescued the phenotype of Deltaarp3 when overexpressed in S. cerevisiae. Developing dis1 trichomes had defects in cytoplasmic actin bundle organization and reduced relative amounts of cytoplasmic actin filaments in developing branches.

An integrated, valveless system for microfluidic purification and reverse transcription-PCR amplification of RNA for detection of infectious agents.

We describe the first miniaturized device capable of the front-end sample preparation essential for detecting RNA-based infectious agents. The microfluidic device integrates sample purification and reverse transcription PCR (RT-PCR) amplification for the identification and detection of influenza A. The device incorporates a chitosan-based RNA binding phase for the completely aqueous isolation of nucleic acids, avoiding the PCR inhibitory effects of guanidine and isopropanol used in silica-based extraction methods. The purified nucleic acids and the reagents needed for single-step RT-PCR amplification are fluidically mobilized simultaneously to a PCR chamber. Utilizing infrared (IR)-mediated heating allowed for a > 5-fold decrease in RT-PCR analysis time compared to a standard thermal cycling protocol used in a conventional thermal cycler. Influenza A virus [A/PR/8/34 (H1N1)] was used as a simulant in this study for virus-based infectious and biowarfare agents with RNA genomes, and was successfully detected in a mock nasal swab sample at clinically relevant concentrations. Following on-chip purification, a fragment specific to the influenza A nucleoprotein gene was first amplified via RT-PCR amplification using IR-mediated heating to achieve more rapid heating and cooling rates. This was initially accomplished on a two-chip system to optimize the SPE and RT-PCR, and then translated to an integrated SPE-RT-PCR device.

Yeast based small molecule screen for inhibitors of SARS-CoV.

Severe acute respiratory coronavirus (SARS-CoV) emerged in 2002, resulting in roughly 8000 cases worldwide and 10% mortality. The animal reservoirs for SARS-CoV precursors still exist and the likelihood of future outbreaks in the human population is high. The SARS-CoV papain-like protease (PLP) is an attractive target for pharmaceutical development because it is essential for virus replication and is conserved among human coronaviruses. A yeast-based assay was established for PLP activity that relies on the ability of PLP to induce a pronounced slow-growth phenotype when expressed in S. cerevisiae. Induction of the slow-growth phenotype was shown to take place over a 60-hour time course, providing the basis for conducting a screen for small molecules that restore growth by inhibiting the function of PLP. Five chemical suppressors of the slow-growth phenotype were identified from the 2000 member NIH Diversity Set library. One of these, NSC158362, potently inhibited SARS-CoV replication in cell culture without toxic effects on cells, and it specifically inhibited SARS-CoV replication but not influenza virus replication. The effect of NSC158362 on PLP protease, deubiquitinase and anti-interferon activities was investigated but the compound did not alter these activities. Another suppressor, NSC158011, demonstrated the ability to inhibit PLP protease activity in a cell-based assay. The identification of these inhibitors demonstrated a strong functional connection between the PLP-based yeast assay, the inhibitory compounds, and SARS-CoV biology. Furthermore the data with NSC158362 suggest a novel mechanism for inhibition of SARS-CoV replication that may involve an unknown activity of PLP, or alternatively a direct effect on a cellular target that modifies or bypasses PLP function in yeast and mammalian cells.

DISTORTED3/SCAR2 is a putative arabidopsis WAVE complex subunit that activates the Arp2/3 complex and is required for epidermal morphogenesis.

In a plant cell, a subset of actin filaments function as a scaffold that positions the endomembrane system and acts as a substrate on which organelle motility occurs. Other actin filament arrays appear to be more dynamic and reorganize in response to growth signals and external cues. The distorted group of trichome morphology mutants provides powerful genetic tools to study the control of actin filament nucleation in the context of morphogenesis. In this article, we report that DISTORTED3 (DIS3) encodes a plant-specific SCAR/WAVE homolog. Null alleles of DIS3, like those of other Arabidopsis thaliana WAVE and Actin-Related Protein (ARP) 2/3 subunit genes, cause trichome distortion, defects in cell-cell adhesion, and reduced hypocotyl growth in etiolated seedlings. DIS3 efficiently activates the actin filament nucleation and branching activity of vertebrate Arp2/3 and functions within a WAVE-ARP2/3 pathway in vivo. DIS3 may assemble into a WAVE complex via a physical interaction with a highly diverged Arabidopsis Abi-1-like bridging protein. These results demonstrate the utility of the Arabidopsis trichome system to understand how the WAVE and ARP2/3 complexes translate signaling inputs into a coordinated morphogenetic response.

DISTORTED2 encodes an ARPC2 subunit of the putative Arabidopsis ARP2/3 complex.

Arabidopsis trichomes are unicellular, branched structures that have highly constrained requirements for the cytoskeleton. The 'distorted group' genes function downstream from microtubule-based branch initiation, and are required during the actin-dependent phase of polarized stalk and branch expansion. Of the eight known 'distorted group' genes, a subset encode homologs of ARP2/3 complex subunits. In eukaryotic cells, the seven-protein ARP2/3 complex nucleates actin filament networks that push on the plasma membrane and organelles. In plants cells, the existence and function of an ARP2/3 complex is unclear. In this paper, we report that DISTORTED2 (DIS2) encodes a paralogue of the ARP2/3 complex subunit ARPC2. DIS2 has ARPC2 activity, based on its ability to rescue the growth defects of arpc2 (arc35Delta) null yeast cells. Like known ARPC2s, DIS2 physically interacts with ARPC4. Mutations in DIS2 cause a distorted trichome phenotype, defects in cell-cell adhesion, and a modest reduction in shoot FW. The actin cytoskeleton in dis2 trichomes is extensive, but developing branches fail to generate and maintain highly organized cytoplasmic actin bundles.

Interchangeable functions of Arabidopsis PIROGI and the human WAVE complex subunit SRA1 during leaf epidermal development.

The WAVE complex is an essential regulator of actin-related protein (ARP) 2/3-dependent actin filament nucleation and cell shape change in migrating cells. Although the composition of the WAVE complex is well characterized, the cellular mechanisms that control its activity and localization are not well known. The 'distorted group' defines a set of Arabidopsis genes that are required to remodel the actin cytoskeleton and maintain the polarized elongation of branched, hair-like cells termed trichomes. Several loci within this group encode homologs of ARP2/3 subunits. In addition to trichome distortion, ARP2/3 subunit mutants have reduced shoot fresh weight and widespread defects in epidermal cell-cell adhesion. The precise cellular function of plant ARP2/3, and the means by which it is regulated, is not known. In this paper, we report that the 'distorted group' gene PIROGI encodes a homolog of the WAVE complex subunit SRA1. The similar cell shape and actin phenotypes of pir and ARP2/3 complex subunit mutants suggest that PIROGI positively regulates ARP2/3. PIROGI directly interacts with the small GTPase ATROP2 with isoform specificity and with selectivity for active forms of the protein. PIROGI shares only 30% amino acid identity with its human homolog. However, both WAVE subunit homologs are functionally interchangeable and display identical physical interactions with RHO family GTPases and the Arabidopsis homolog of the WAVE complex subunit NAP125. These results demonstrate the utility of the 'distorted group' mutants to study ARP2/3 complex functions from signaling input to cell shape output.