Optimization of potent hepatitis C virus NS3 helicase inhibitors isolated from the yellow dyes thioflavine S and primuline.

PubMed

Li, Kelin; Frankowski, Kevin J; Belon, Craig A; Neuenswander, Ben; Ndjomou, Jean; Hanson, Alicia M; Shanahan, Matthew A; Schoenen, Frank J; Blagg, Brian S J; Aubé, Jeffrey; Frick, David N

2012-04-12

A screen for hepatitis C virus (HCV) NS3 helicase inhibitors revealed that the commercial dye thioflavine S was the most potent inhibitor of NS3-catalyzed DNA and RNA unwinding in the 827-compound National Cancer Institute Mechanistic Set. Thioflavine S and the related dye primuline were separated here into their pure components, all of which were oligomers of substituted benzothiazoles. The most potent compound (P4), a benzothiazole tetramer, inhibited unwinding >50% at 2 ± 1 μM, inhibited the subgenomic HCV replicon at 10 μM, and was not toxic at 100 μM. Because P4 also interacted with DNA, more specific analogues were synthesized from the abundant dimeric component of primuline. Some of the 32 analogues prepared retained ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors (compound 17) was active against the replicon and inhibited the helicase more than 50% at 2.6 ± 1 μM. © 2012 American Chemical Society

Establishment of a robust dengue virus NS3-NS5 binding assay for identification of protein-protein interaction inhibitors.

PubMed

Takahashi, Hirotaka; Takahashi, Chikako; Moreland, Nicole J; Chang, Young-Tae; Sawasaki, Tatsuya; Ryo, Akihide; Vasudevan, Subhash G; Suzuki, Youichi; Yamamoto, Naoki

2012-12-01

Whereas the dengue virus (DENV) non-structural (NS) proteins NS3 and NS5 have been shown to interact in vitro and in vivo, the biological relevance of this interaction in viral replication has not been fully clarified. Here, we first applied a simple and robust in vitro assay based on AlphaScreen technology in combination with the wheat-germ cell-free protein production system to detect the DENV-2 NS3-NS5 interaction in a 384-well plate. The cell-free-synthesized NS3 and NS5 recombinant proteins were soluble and in possession of their respective enzymatic activities in vitro. In addition, AlphaScreen assays using the recombinant proteins detected a specific interaction between NS3 and NS5 with a robust Z' factor of 0.71. By employing the AlphaScreen assay, we found that both the N-terminal protease and C-terminal helicase domains of NS3 are required for its association with NS5. Furthermore, a competition assay revealed that the binding of full-length NS3 to NS5 was significantly inhibited by the addition of an excess of NS3 protease or helicase domains. Our results demonstrate that the AlphaScreen assay can be used to discover novel antiviral agents targeting the interactions between DENV NS proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

The C-terminal 50 amino acid residues of dengue NS3 protein are important for NS3-NS5 interaction and viral replication.

PubMed

Tay, Moon Y F; Saw, Wuan Geok; Zhao, Yongqian; Chan, Kitti W K; Singh, Daljit; Chong, Yuwen; Forwood, Jade K; Ooi, Eng Eong; Grüber, Gerhard; Lescar, Julien; Luo, Dahai; Vasudevan, Subhash G

2015-01-23

Dengue virus multifunctional proteins NS3 protease/helicase and NS5 methyltransferase/RNA-dependent RNA polymerase form part of the viral replication complex and are involved in viral RNA genome synthesis, methylation of the 5'-cap of viral genome, and polyprotein processing among other activities. Previous studies have shown that NS5 residue Lys-330 is required for interaction between NS3 and NS5. Here, we show by competitive NS3-NS5 interaction ELISA that the NS3 peptide spanning residues 566-585 disrupts NS3-NS5 interaction but not the null-peptide bearing the N570A mutation. Small angle x-ray scattering study on NS3(172-618) helicase and covalently linked NS3(172-618)-NS5(320-341) reveals a rigid and compact formation of the latter, indicating that peptide NS5(320-341) engages in specific and discrete interaction with NS3. Significantly, NS3:Asn-570 to alanine mutation introduced into an infectious DENV2 cDNA clone did not yield detectable virus by plaque assay even though intracellular double-stranded RNA was detected by immunofluorescence. Detection of increased negative-strand RNA synthesis by real time RT-PCR for the NS3:N570A mutant suggests that NS3-NS5 interaction plays an important role in the balanced synthesis of positive- and negative-strand RNA for robust viral replication. Dengue virus infection has become a global concern, and the lack of safe vaccines or antiviral treatments urgently needs to be addressed. NS3 and NS5 are highly conserved among the four serotypes, and the protein sequence around the pinpointed amino acids from the NS3 and NS5 regions are also conserved. The identification of the functionally essential interaction between the two proteins by biochemical and reverse genetics methods paves the way for rational drug design efforts to inhibit viral RNA synthesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Development of chemical inhibitors of the SARS coronavirus: viral helicase as a potential target.

PubMed

Keum, Young-Sam; Jeong, Yong-Joo

2012-11-15

Severe acute respiratory syndrome (SARS) was the first pandemic in the 21st century to claim more than 700 lives worldwide. However, effective anti-SARS vaccines or medications are currently unavailable despite being desperately needed to adequately prepare for a possible SARS outbreak. SARS is caused by a novel coronavirus, and one of its components, a viral helicase, is emerging as a promising target for the development of chemical SARS inhibitors. In the following review, we describe the characterization, family classification, and kinetic movement mechanisms of the SARS coronavirus (SCV) helicase-nsP13. We also discuss the recent progress in the identification of novel chemical inhibitors of nsP13 in the context of our recent discovery of the strong inhibition of the SARS helicase by natural flavonoids, myricetin and scutellarein. These compounds will serve as important resources for the future development of anti-SARS medications. Copyright © 2012 Elsevier Inc. All rights reserved.

Computational Study on the Inhibitor Binding Mode and Allosteric Regulation Mechanism in Hepatitis C Virus NS3/4A Protein

PubMed Central

Xue, Weiwei; Yang, Ying; Wang, Xiaoting; Liu, Huanxiang; Yao, Xiaojun

2014-01-01

HCV NS3/4A protein is an attractive therapeutic target responsible for harboring serine protease and RNA helicase activities during the viral replication. Small molecules binding at the interface between the protease and helicase domains can stabilize the closed conformation of the protein and thus block the catalytic function of HCV NS3/4A protein via an allosteric regulation mechanism. But the detailed mechanism remains elusive. Here, we aimed to provide some insight into the inhibitor binding mode and allosteric regulation mechanism of HCV NS3/4A protein by using computational methods. Four simulation systems were investigated. They include: apo state of HCV NS3/4A protein, HCV NS3/4A protein in complex with an allosteric inhibitor and the truncated form of the above two systems. The molecular dynamics simulation results indicate HCV NS3/4A protein in complex with the allosteric inhibitor 4VA adopts a closed conformation (inactive state), while the truncated apo protein adopts an open conformation (active state). Further residue interaction network analysis suggests the communication of the domain-domain interface play an important role in the transition from closed to open conformation of HCV NS3/4A protein. However, the inhibitor stabilizes the closed conformation through interaction with several key residues from both the protease and helicase domains, including His57, Asp79, Asp81, Asp168, Met485, Cys525 and Asp527, which blocks the information communication between the functional domains interface. Finally, a dynamic model about the allosteric regulation and conformational changes of HCV NS3/4A protein was proposed and could provide fundamental insights into the allosteric mechanism of HCV NS3/4A protein function regulation and design of new potent inhibitors. PMID:24586263

The Enigmatic Alphavirus Non-Structural Protein 3 (nsP3) Revealing Its Secrets at Last

PubMed Central

Götte, Benjamin; Liu, Lifeng

2018-01-01

Alphaviruses encode 4 non-structural proteins (nsPs), most of which have well-understood functions in capping and membrane association (nsP1), polyprotein processing and RNA helicase activity (nsP2) and as RNA-dependent RNA polymerase (nsP4). The function of nsP3 has been more difficult to pin down and it has long been referred to as the more enigmatic of the nsPs. The protein comprises three domains, an N-terminal macro domain, a central zinc-binding domain and a C-terminal hypervariable domain (HVD). In this article, we review old and new literature about the functions of the three domains. Much progress in recent years has contributed to a picture of nsP3, particularly through its HVD as a hub for interactions with host cell molecules, with multiple effects on the biology of the host cell at early points in infection. These and many future discoveries will provide targets for anti-viral therapies as well as strategies for modification of vectors for vaccine and oncolytic interventions. PMID:29495654

Molecular models of NS3 protease variants of the Hepatitis C virus.

PubMed

da Silveira, Nelson J F; Arcuri, Helen A; Bonalumi, Carlos E; de Souza, Fátima P; Mello, Isabel M V G C; Rahal, Paula; Pinho, João R R; de Azevedo, Walter F

2005-01-21

Hepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed. The atomic coordinates of crystallographic structure 1CU1 and 1DY9 were used as starting model for modeling of the NS3 protease variant structures. The NS3 protease variant structures are composed of six subdomains, which occur in sequence along the polypeptide chain. The protease domain exhibits the dual beta-barrel fold that is common among members of the chymotrypsin serine protease family. The helicase domain contains two structurally related beta-alpha-beta subdomains and a third subdomain of seven helices and three short beta strands. The latter domain is usually referred to as the helicase alpha-helical subdomain. The rmsd value of bond lengths and bond angles, the average G-factor and Verify 3D values are presented for NS3 protease variant structures. This project increases the certainty that homology modeling is an useful tool in structural biology and that it can be very valuable in annotating genome sequence information and contributing to structural and functional genomics from virus. The structural models will be used to guide future efforts in the structure-based drug design of a new generation of NS3 protease variants

Crystal structure of a novel conformational state of the flavivirus NS3 protein: implications for polyprotein processing and viral replication.

PubMed

Assenberg, René; Mastrangelo, Eloise; Walter, Thomas S; Verma, Anil; Milani, Mario; Owens, Raymond J; Stuart, David I; Grimes, Jonathan M; Mancini, Erika J

2009-12-01

The flavivirus genome comprises a single strand of positive-sense RNA, which is translated into a polyprotein and cleaved by a combination of viral and host proteases to yield functional proteins. One of these, nonstructural protein 3 (NS3), is an enzyme with both serine protease and NTPase/helicase activities. NS3 plays a central role in the flavivirus life cycle: the NS3 N-terminal serine protease together with its essential cofactor NS2B is involved in the processing of the polyprotein, whereas the NS3 C-terminal NTPase/helicase is responsible for ATP-dependent RNA strand separation during replication. An unresolved question remains regarding why NS3 appears to encode two apparently disconnected functionalities within one protein. Here we report the 2.75-A-resolution crystal structure of full-length Murray Valley encephalitis virus NS3 fused with the protease activation peptide of NS2B. The biochemical characterization of this construct suggests that the protease has little influence on the helicase activity and vice versa. This finding is in agreement with the structural data, revealing a single protein with two essentially segregated globular domains. Comparison of the structure with that of dengue virus type 4 NS2B-NS3 reveals a relative orientation of the two domains that is radically different between the two structures. Our analysis suggests that the relative domain-domain orientation in NS3 is highly variable and dictated by a flexible interdomain linker. The possible implications of this conformational flexibility for the function of NS3 are discussed.

Structural determinants for membrane association and dynamic organization of the hepatitis C virus NS3-4A complex

PubMed Central

Brass, Volker; Berke, Jan Martin; Montserret, Roland; Blum, Hubert E.; Penin, François; Moradpour, Darius

2008-01-01

Hepatitis C virus (HCV) NS3-4A is a membrane-associated multifunctional protein harboring serine protease and RNA helicase activities. It is an essential component of the HCV replication complex and a prime target for antiviral intervention. Here, we show that membrane association and structural organization of HCV NS3-4A are ensured in a cooperative manner by two membrane-binding determinants. We demonstrate that the N-terminal 21 amino acids of NS4A form a transmembrane α-helix that may be involved in intramembrane protein–protein interactions important for the assembly of a functional replication complex. In addition, we demonstrate that amphipathic helix α0, formed by NS3 residues 12–23, serves as a second essential determinant for membrane association of NS3-4A, allowing proper positioning of the serine protease active site on the membrane. These results allowed us to propose a dynamic model for the membrane association, processing, and structural organization of NS3-4A on the membrane. This model has implications for the functional architecture of the HCV replication complex, proteolytic targeting of host factors, and drug design. PMID:18799730

Structural features of NS3 of Dengue virus serotypes 2 and 4 in solution and insight into RNA binding and the inhibitory role of quercetin.

PubMed

Pan, Ankita; Saw, Wuan Geok; Subramanian Manimekalai, Malathy Sony; Grüber, Ardina; Joon, Shin; Matsui, Tsutomu; Weiss, Thomas M; Grüber, Gerhard

2017-05-01

Dengue virus (DENV), which has four serotypes (DENV-1 to DENV-4), is the causative agent of the viral infection dengue. DENV nonstructural protein 3 (NS3) comprises a serine protease domain and an RNA helicase domain which has nucleotide triphosphatase activities that are essential for RNA replication and viral assembly. Here, solution X-ray scattering was used to provide insight into the overall structure and flexibility of the entire NS3 and its recombinant helicase and protease domains for Dengue virus serotypes 2 and 4 in solution. The DENV-2 and DENV-4 NS3 forms are elongated and flexible in solution. The importance of the linker residues in flexibility and domain-domain arrangement was shown by the compactness of the individual protease and helicase domains. Swapping of the 174 PPAVP 179 linker stretch of the related Hepatitis C virus (HCV) NS3 into DENV-2 NS3 did not alter the elongated shape of the engineered mutant. Conformational alterations owing to RNA binding are described in the protease domain, which undergoes substantial conformational alterations that are required for the optimal catalysis of bound RNA. Finally, the effects of ATPase inhibitors on the enzymatically active DENV-2 and DENV-4 NS3 and the individual helicases are presented, and insight into the allosteric effect of the inhibitor quercetin is provided.

Helicases as Prospective Targets for Anti-Cancer Therapy

PubMed Central

Gupta, Rigu; Brosh, Robert M.

2008-01-01

It has been proposed that selective inactivation of a DNA repair pathway may enhance anti-cancer therapies that eliminate cancerous cells through the cytotoxic effects of DNA damaging agents or radiation. Given the unique and critically important roles of DNA helicases in the DNA damage response, DNA repair, and maintenance of genomic stability, a number of strategies currently being explored or in use to combat cancer may be either mediated or enhanced through the modulation of helicase function. The focus of this review will be to examine the roles of helicases in DNA repair that might be suitably targeted by cancer therapeutic approaches. Treatment of cancers with anti-cancer drugs such as small molecule compounds that modulate helicase expression or function is a viable approach to selectively kill cancer cells through the inactivation of helicase-dependent DNA repair pathways, particularly those associated with DNA recombination, replication restart, and cell cycle checkpoint. PMID:18473724

Biochemical Activities of Minute Virus of Mice Nonstructural Protein NS1 Are Modulated In Vitro by the Phosphorylation State of the Polypeptide

PubMed Central

Nüesch, Jürg P. F.; Corbau, Romuald; Tattersall, Peter; Rommelaere, Jean

1998-01-01

NS1, the 83-kDa major nonstructural protein of minute virus of mice (MVM), is a multifunctional nuclear phosphoprotein which is required in a variety of steps during progeny virus production, early as well as late during infection. NS1 is the initiator protein for viral DNA replication. It binds specifically to target DNA motifs; has site-specific single-strand nickase, intrinsic ATPase, and helicase activities; trans regulates viral and cellular promoters; and exerts cytotoxic stress on the host cell. To investigate whether these multiple activities of NS1 depend on posttranslational modifications, in particular phosphorylation, we expressed His-tagged NS1 in HeLa cells by using recombinant vaccinia viruses, dephosphorylated it at serine and threonine residues with calf intestine alkaline phosphatase, and compared the biochemical activities of the purified un(der)phosphorylated (NS1O) and the native (NS1P) polypeptides. Biochemical analyses of replicative functions of NS1O revealed a severe reduction of intrinsic helicase activity and, to a minor extent, of ATPase and nickase activities, whereas its affinity for the target DNA sequence [ACCA]2–3 was enhanced compared to that of NS1P. In the presence of endogenous protein kinases found in replication extracts, NS1O showed all functions necessary for resolution and replication of the 3′ dimer bridge, indicating reactivation of NS1O by rephosphorylation. Partial reactivation of the helicase activity was found as well when NS1O was incubated with protein kinase C. PMID:9733839

Crystal structures of the methyltransferase and helicase from the ZIKA 1947 MR766 Uganda strain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bukrejewska, Malgorzata; Derewenda, Urszula; Radwanska, Malwina

2017-08-15

Two nonstructural proteins encoded byZika virusstrain MR766 RNA, a methyltransferase and a helicase, were crystallized and their structures were solved and refined at 2.10 and 2.01 Å resolution, respectively. The NS5 methyltransferase contains a boundS-adenosyl-L-methionine (SAM) co-substrate. The NS3 helicase is in the apo form. Comparison with published crystal structures of the helicase in the apo, nucleotide-bound and single-stranded RNA (ssRNA)-bound states suggests that binding of ssRNA to the helicase may occur through conformational selection rather than induced fit.

Purification and crystallization of Kokobera virus helicase

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Colibus, Luigi; Speroni, Silvia; Coutard, Bruno

2007-03-01

Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method and exhibit a diffraction limit of 2.3 Å. Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. The flavivirus genus is characterized by a positive-sense single-stranded RNA genome. The unique open reading frame of the viral RNA is transcribed and translated as a single polyprotein which is post-translationally cleaved to yield three structural and seven nonstructural proteins, one of which ismore » the NS3 gene that encodes a C-terminal helicase domain consisting of 431 amino acids. Helicase inhibitors are potential antiviral drugs as the helicase is essential to viral replication. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P3{sub 1}21 (or P3{sub 2}21), with unit-cell parameters a = 88.6, c = 138.6 Å, and exhibit a diffraction limit of 2.3 Å.« less

Assessment of Dengue virus helicase and methyltransferase as targets for fragment-based drug discovery.

PubMed

Coutard, Bruno; Decroly, Etienne; Li, Changqing; Sharff, Andrew; Lescar, Julien; Bricogne, Gérard; Barral, Karine

2014-06-01

Seasonal and pandemic flaviviruses continue to be leading global health concerns. With the view to help drug discovery against Dengue virus (DENV), a fragment-based experimental approach was applied to identify small molecule ligands targeting two main components of the flavivirus replication complex: the NS3 helicase (Hel) and the NS5 mRNA methyltransferase (MTase) domains. A library of 500 drug-like fragments was first screened by thermal-shift assay (TSA) leading to the identification of 36 and 32 fragment hits binding Hel and MTase from DENV, respectively. In a second stage, we set up a fragment-based X-ray crystallographic screening (FBS-X) in order to provide both validated fragment hits and structural binding information. No fragment hit was confirmed for DENV Hel. In contrast, a total of seven fragments were identified as DENV MTase binders and structures of MTase-fragment hit complexes were solved at resolution at least 2.0Å or better. All fragment hits identified contain either a five- or six-membered aromatic ring or both, and three novel binding sites were located on the MTase. To further characterize the fragment hits identified by TSA and FBS-X, we performed enzymatic assays to assess their inhibition effect on the N7- and 2'-O-MTase enzymatic activities: five of these fragment hits inhibit at least one of the two activities with IC50 ranging from 180μM to 9mM. This work validates the FBS-X strategy for identifying new anti-flaviviral hits targeting MTase, while Hel might not be an amenable target for fragment-based drug discovery (FBDD). This approach proved to be a fast and efficient screening method for FBDD target validation and discovery of starting hits for the development of higher affinity molecules that bind to novel allosteric sites. Copyright © 2014 Elsevier B.V. All rights reserved.

A Point Mutation in the N-Terminal Amphipathic Helix α0 in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding

PubMed Central

Yan, Yu; He, Ying; Boson, Bertrand; Wang, Xuesong; Cosset, François-Loïc

2017-01-01

ABSTRACT The assembly of hepatitis C virus (HCV), a complicated process in which many viral and cellular factors are involved, has not been thoroughly deciphered. NS3 is a multifunctional protein that contains an N-terminal amphipathic α helix (designated helix α0), which is crucial for the membrane association and stability of NS3 protein, followed by a serine protease domain and a C-terminal helicase/NTPase domain. NS3 participates in HCV assembly likely through its C-terminal helicase domain, in which all reported adaptive mutations enhancing virion assembly reside. In this study, we determined that the N-terminal helix α0 of NS3 may contribute to HCV assembly. We identified a single mutation from methionine to threonine at amino acid position 21 (M21T) in helix α0, which significantly promoted viral production while having no apparent effect on the membrane association and protease activity of NS3. Subsequent analyses demonstrated that the M21T mutation did not affect HCV genome replication but rather promoted virion assembly. Further study revealed a shift in the subcellular localization of core protein from lipid droplets (LD) to the endoplasmic reticulum (ER). Finally, we showed that the M21T mutation increased the colocalization of core proteins and viral envelope proteins, leading to a more efficient envelopment of viral nucleocapsids. Collectively, the results of our study revealed a new function of NS3 helix α0 and aid understanding of the role of NS3 in HCV virion morphogenesis. IMPORTANCE HCV NS3 protein possesses the protease activity in its N-terminal domain and the helicase activity in its C-terminal domain. The role of NS3 in virus assembly has been mainly attributed to its helicase domain, because all adaptive mutations enhancing progeny virus production are found to be within this domain. Our study identified, for the first time to our knowledge, an adaptive mutation within the N-terminal helix α0 domain of NS3 that significantly enhanced

A Point Mutation in the N-Terminal Amphipathic Helix α0 in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding.

PubMed

Yan, Yu; He, Ying; Boson, Bertrand; Wang, Xuesong; Cosset, François-Loïc; Zhong, Jin

2017-03-15

The assembly of hepatitis C virus (HCV), a complicated process in which many viral and cellular factors are involved, has not been thoroughly deciphered. NS3 is a multifunctional protein that contains an N-terminal amphipathic α helix (designated helix α 0 ), which is crucial for the membrane association and stability of NS3 protein, followed by a serine protease domain and a C-terminal helicase/NTPase domain. NS3 participates in HCV assembly likely through its C-terminal helicase domain, in which all reported adaptive mutations enhancing virion assembly reside. In this study, we determined that the N-terminal helix α 0 of NS3 may contribute to HCV assembly. We identified a single mutation from methionine to threonine at amino acid position 21 (M21T) in helix α 0 , which significantly promoted viral production while having no apparent effect on the membrane association and protease activity of NS3. Subsequent analyses demonstrated that the M21T mutation did not affect HCV genome replication but rather promoted virion assembly. Further study revealed a shift in the subcellular localization of core protein from lipid droplets (LD) to the endoplasmic reticulum (ER). Finally, we showed that the M21T mutation increased the colocalization of core proteins and viral envelope proteins, leading to a more efficient envelopment of viral nucleocapsids. Collectively, the results of our study revealed a new function of NS3 helix α 0 and aid understanding of the role of NS3 in HCV virion morphogenesis. IMPORTANCE HCV NS3 protein possesses the protease activity in its N-terminal domain and the helicase activity in its C-terminal domain. The role of NS3 in virus assembly has been mainly attributed to its helicase domain, because all adaptive mutations enhancing progeny virus production are found to be within this domain. Our study identified, for the first time to our knowledge, an adaptive mutation within the N-terminal helix α 0 domain of NS3 that significantly enhanced

The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis.

PubMed

Aoubala, M; Holt, J; Clegg, R A; Rowlands, D J; Harris, M

2001-07-01

Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. The HCV non-structural protein NS3 complexes with NS4A and has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). Here we demonstrate that full-length, catalytically active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunit in vitro. This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved helicase motif II, both of which also abolished NTPase activity. As PKA C-subunit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase activity), we hypothesized that PKA C-subunit inhibition could be due to NS3/4A-mediated ATP hydrolysis. This was confirmed by experiments in which a constant ATP concentration was maintained by addition of an ATP regeneration system--under these conditions PKA C-subunit inhibition was not observed. Interestingly, the mutations also abrogated the ability of wild-type NS3/4A to inhibit the PKA-regulated transcription factor CREB in transiently transfected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit. However, in vivo effects of NS3/4A suggest that ATPase activity may play a role in viral pathology in the infected liver.

Viperin Restricts Zika Virus and Tick-Borne Encephalitis Virus Replication by Targeting NS3 for Proteasomal Degradation.

PubMed

Panayiotou, Christakis; Lindqvist, Richard; Kurhade, Chaitanya; Vonderstein, Kirstin; Pasto, Jenny; Edlund, Karin; Upadhyay, Arunkumar S; Överby, Anna K

2018-04-01

Flaviviruses are arthropod-borne viruses that constitute a major global health problem, with millions of human infections annually. Their pathogenesis ranges from mild illness to severe manifestations such as hemorrhagic fever and fatal encephalitis. Type I interferons (IFNs) are induced in response to viral infection and stimulate the expression of interferon-stimulated genes (ISGs), including that encoding viperin (virus-inhibitory protein, endoplasmic reticulum associated, IFN inducible), which shows antiviral activity against a broad spectrum of viruses, including several flaviviruses. Here we describe a novel antiviral mechanism employed by viperin against two prominent flaviviruses, tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV). Viperin was found to interact and colocalize with the structural proteins premembrane (prM) and envelope (E) of TBEV, as well as with nonstructural (NS) proteins NS2A, NS2B, and NS3. Interestingly, viperin expression reduced the NS3 protein level, and the stability of the other interacting viral proteins, but only in the presence of NS3. We also found that although viperin interacted with NS3 of mosquito-borne flaviviruses (ZIKV, Japanese encephalitis virus, and yellow fever virus), only ZIKV was sensitive to the antiviral effect of viperin. This sensitivity correlated with viperin's ability to induce proteasome-dependent degradation of NS3. ZIKV and TBEV replication was rescued completely when NS3 was overexpressed, suggesting that the viral NS3 is the specific target of viperin. In summary, we present here a novel antiviral mechanism of viperin that is selective for specific viruses in the genus Flavivirus , affording the possible availability of new drug targets that can be used for therapeutic intervention. IMPORTANCE Flaviviruses are a group of enveloped RNA viruses that cause severe diseases in humans and animals worldwide, but no antiviral treatment is yet available. Viperin, a host protein produced in response to

CHD3 facilitates vRNP nuclear export by interacting with NES1 of influenza A virus NS2.

PubMed

Hu, Yong; Liu, Xiaokun; Zhang, Anding; Zhou, Hongbo; Liu, Ziduo; Chen, Huanchun; Jin, Meilin

2015-03-01

NS2 from influenza A virus mediates Crm1-dependent vRNP nuclear export through interaction with Crm1. However, even though the nuclear export signal 1 (NES1) of NS2 does not play a requisite role in NS2-Crm1 interaction, there is no doubt that NES1 is crucial for vRNP nuclear export. While the mechanism of the NES1 is still unclear, it is speculated that certain host partners might mediate the NES1 function through their interaction with NES1. In the present study, chromodomain-helicase-DNA-binding protein 3 (CHD3) was identified as a novel host nuclear protein for locating NS2 and Crm1 on dense chromatin for NS2 and Crm1-dependent vRNP nuclear export. CHD3 was confirmed to interact with NES1 in NS2, and a disruption to this interaction by mutation in NES1 significantly delayed viral vRNPs export and viral propagation. Further, the knockdown of CHD3 would affect the propagation of the wild-type virus but not the mutant with the weakened NS2-CHD3 interaction. Therefore, this study demonstrates that NES1 is required for maximal binding of NS2 to CHD3, and that the NS2-CHD3 interaction on the dense chromatin contributed to the NS2-mediated vRNP nuclear export.

DNA mechanics as a tool to probe helicase and translocase activity.

PubMed

Lionnet, Timothée; Dawid, Alexandre; Bigot, Sarah; Barre, François-Xavier; Saleh, Omar A; Heslot, François; Allemand, Jean-François; Bensimon, David; Croquette, Vincent

2006-01-01

Helicases and translocases are proteins that use the energy derived from ATP hydrolysis to move along or pump nucleic acid substrates. Single molecule manipulation has proved to be a powerful tool to investigate the mechanochemistry of these motors. Here we first describe the basic mechanical properties of DNA unraveled by single molecule manipulation techniques. Then we demonstrate how the knowledge of these properties has been used to design single molecule assays to address the enzymatic mechanisms of different translocases. We report on four single molecule manipulation systems addressing the mechanism of different helicases using specifically designed DNA substrates: UvrD enzyme activity detection on a stretched nicked DNA molecule, HCV NS3 helicase unwinding of a RNA hairpin under tension, the observation of RecBCD helicase/nuclease forward and backward motion, and T7 gp4 helicase mediated opening of a synthetic DNA replication fork. We then discuss experiments on two dsDNA translocases: the RuvAB motor studied on its natural substrate, the Holliday junction, and the chromosome-segregation motor FtsK, showing its unusual coupling to DNA supercoiling.

A Proline-Rich N-Terminal Region of the Dengue Virus NS3 Is Crucial for Infectious Particle Production.

PubMed

Gebhard, Leopoldo G; Iglesias, Néstor G; Byk, Laura A; Filomatori, Claudia V; De Maio, Federico A; Gamarnik, Andrea V

2016-06-01

Dengue virus is currently the most important insect-borne viral human pathogen. Viral nonstructural protein 3 (NS3) is a key component of the viral replication machinery that performs multiple functions during viral replication and participates in antiviral evasion. Using dengue virus infectious clones and reporter systems to dissect each step of the viral life cycle, we examined the requirements of different domains of NS3 on viral particle assembly. A thorough site-directed mutagenesis study based on solvent-accessible surface areas of NS3 revealed that, in addition to being essential for RNA replication, different domains of dengue virus NS3 are critically required for production of infectious viral particles. Unexpectedly, point mutations in the protease, interdomain linker, or helicase domain were sufficient to abolish infectious particle formation without affecting translation, polyprotein processing, or RNA replication. In particular, we identified a novel proline-rich N-terminal unstructured region of NS3 that contains several amino acid residues involved in infectious particle formation. We also showed a new role for the interdomain linker of NS3 in virion assembly. In conclusion, we present a comprehensive genetic map of novel NS3 determinants for viral particle assembly. Importantly, our results provide evidence of a central role of NS3 in the coordination of both dengue virus RNA replication and particle formation. Dengue virus is an important human pathogen, and its prominence is expanding globally; however, basic aspects of its biology are still unclear, hindering the development of effective therapeutic and prophylactic treatments. Little is known about the initial steps of dengue and other flavivirus particle assembly. This process involves a complex interplay between viral and cellular components, making it an attractive antiviral target. Unpredictably, we identified spatially separated regions of the large NS3 viral protein as determinants for

Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

PubMed

Morohashi, Kengo; Sahara, Hiroeki; Watashi, Koichi; Iwabata, Kazuki; Sunoki, Takashi; Kuramochi, Kouji; Takakusagi, Kaori; Miyashita, Hiroki; Sato, Noriyuki; Tanabe, Atsushi; Shimotohno, Kunitada; Kobayashi, Susumu; Sakaguchi, Kengo; Sugawara, Fumio

2011-04-29

Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

Cyclosporin A Associated Helicase-Like Protein Facilitates the Association of Hepatitis C Virus RNA Polymerase with Its Cellular Cyclophilin B

PubMed Central

Sahara, Hiroeki; Iwabata, Kazuki; Sunoki, Takashi; Kuramochi, Kouji; Takakusagi, Kaori; Miyashita, Hiroki; Sato, Noriyuki; Tanabe, Atsushi; Shimotohno, Kunitada; Kobayashi, Susumu; Sakaguchi, Kengo; Sugawara, Fumio

2011-01-01

Background Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. Principal Findings Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. Conclusions We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology. PMID:21559518

Telomere and ribosomal DNA repeats are chromosomal targets of the bloom syndrome DNA helicase

PubMed Central

Schawalder, James; Paric, Enesa; Neff, Norma F

2003-01-01

Background Bloom syndrome is one of the most cancer-predisposing disorders and is characterized by genomic instability and a high frequency of sister chromatid exchange. The disorder is caused by loss of function of a 3' to 5' RecQ DNA helicase, BLM. The exact role of BLM in maintaining genomic integrity is not known but the helicase has been found to associate with several DNA repair complexes and some DNA replication foci. Results Chromatin immunoprecipitation of BLM complexes recovered telomere and ribosomal DNA repeats. The N-terminus of BLM, required for NB localization, is the same as the telomere association domain of BLM. The C-terminus is required for ribosomal DNA localization. BLM localizes primarily to the non-transcribed spacer region of the ribosomal DNA repeat where replication forks initiate. Bloom syndrome cells expressing the deletion alleles lacking the ribosomal DNA and telomere association domains have altered cell cycle populations with increased S or G2/M cells relative to normal. Conclusion These results identify telomere and ribosomal DNA repeated sequence elements as chromosomal targets for the BLM DNA helicase during the S/G2 phase of the cell cycle. BLM is localized in nuclear bodies when it associates with telomeric repeats in both telomerase positive and negative cells. The BLM DNA helicase participates in genomic stability at ribosomal DNA repeats and telomeres. PMID:14577841

The influenza virus NS1 protein as a therapeutic target.

PubMed

Engel, Daniel A

2013-09-01

Nonstructural protein 1 (NS1) of influenza A virus plays a central role in virus replication and blockade of the host innate immune response, and is therefore being considered as a potential therapeutic target. The primary function of NS1 is to dampen the host interferon (IFN) response through several distinct molecular mechanisms that are triggered by interactions with dsRNA or specific cellular proteins. Sequestration of dsRNA by NS1 results in inhibition of the 2'-5' oligoadenylate synthetase/RNase L antiviral pathway, and also inhibition of dsRNA-dependent signaling required for new IFN production. Binding of NS1 to the E3 ubiquitin ligase TRIM25 prevents activation of RIG-I signaling and subsequent IFN induction. Cellular RNA processing is also targeted by NS1, through recognition of cleavage and polyadenylation specificity factor 30 (CPSF30), leading to inhibition of IFN-β mRNA processing as well as that of other cellular mRNAs. In addition NS1 binds to and inhibits cellular protein kinase R (PKR), thus blocking an important arm of the IFN system. Many additional proteins have been reported to interact with NS1, either directly or indirectly, which may serve its anti-IFN and additional functions, including the regulation of viral and host gene expression, signaling pathways and viral pathogenesis. Many of these interactions are potential targets for small-molecule intervention. Structural, biochemical and functional studies have resulted in hypotheses for drug discovery approaches that are beginning to bear experimental fruit, such as targeting the dsRNA-NS1 interaction, which could lead to restoration of innate immune function and inhibition of virus replication. This review describes biochemical, cell-based and nucleic acid-based approaches to identifying NS1 antagonists. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

The influenza virus NS1 protein as a therapeutic target

PubMed Central

Engel, Daniel A.

2015-01-01

Nonstructural protein 1 (NS1) of influenza A virus plays a central role in virus replication and blockade of the host innate immune response, and is therefore being considered as a potential therapeutic target. The primary function of NS1 is to dampen the host interferon (IFN) response through several distinct molecular mechanisms that are triggered by interactions with dsRNA or specific cellular proteins. Sequestration of dsRNA by NS1 results in inhibition of the 2’-5’ oligoadenylate synthetase/RNase L antiviral pathway, and also inhibition of dsRNA-dependent signaling required for new IFN production. Binding of NS1 to the E3 ubiquitin ligase TRIM25 prevents activation of RIG-I signaling and subsequent IFN induction. Cellular RNA processing is also targeted by NS1, through recognition of cleavage and polyadenylation specificity factor 30 (CPSF30), leading to inhibition of IFN- mRNA processing as well as that of other cellular mRNAs. In addition NS1 binds to and inhibits cellular protein kinase R (PKR), thus blocking an important arm of the IFN system. Many additional proteins have been reported to interact with NS1, either directly or indirectly, which may serve its anti-IFN and additional functions, including the regulation of viral and host gene expression, signaling pathways and viral pathogenesis. Many of these interactions are potential targets for small-molecule intervention. Structural, biochemical and functional studies have resulted in hypotheses for drug discovery approaches that are beginning to bear experimental fruit, such as targeting the dsRNA-NS1 interaction, which could lead to restoration of innate immune function and inhibition of virus replication. This review describes biochemical, cell-based and nucleic acid-based approaches to identifying NS1 antagonists. PMID:23796981

Structural basis for MTR4-ZCCHC8 interactions that stimulate the MTR4 helicase in the nuclear exosome-targeting complex.

PubMed

Puno, M Rhyan; Lima, Christopher D

2018-06-12

The nuclear exosome-targeting (NEXT) complex functions as an RNA exosome cofactor and is involved in surveillance and turnover of aberrant transcripts and noncoding RNAs. NEXT is a ternary complex composed of the RNA-binding protein RBM7, the scaffold zinc-knuckle protein ZCCHC8, and the helicase MTR4. While RNA interactions with RBM7 are known, it remains unclear how NEXT subunits collaborate to recognize and prepare substrates for degradation. Here, we show that MTR4 helicase activity is enhanced when associated with RBM7 and ZCCHC8. While uridine-rich substrates interact with RBM7 and are preferred, optimal activity is observed when substrates include a polyadenylated 3' end. We identify a bipartite interaction of ZCCHC8 with MTR4 and uncover a role for the conserved C-terminal domain of ZCCHC8 in stimulating MTR4 helicase and ATPase activities. A crystal structure reveals that the ZCCHC8 C-terminal domain binds the helicase core in a manner that is distinct from that observed for Saccharomyces cerevisiae exosome cofactors Trf4p and Air2p. Our results are consistent with a model whereby effective targeting of substrates by NEXT entails recognition of elements within the substrate and activation of MTR4 helicase activity. Copyright © 2018 the Author(s). Published by PNAS.

Fitting CRISPR-associated Cas3 into the helicase family tree.

PubMed

Jackson, Ryan N; Lavin, Matthew; Carter, Joshua; Wiedenheft, Blake

2014-02-01

Helicases utilize NTPs to modulate their binding to nucleic acids and many of these enzymes also unwind DNA or RNA duplexes in an NTP-dependent fashion. These proteins are phylogenetically related but functionally diverse, with essential roles in virtually all aspects of nucleic acid metabolism. A new class of helicases associated with RNA-guided adaptive immune systems in bacteria and archaea has recently been identified. Prokaryotes acquire resistance to invading genetic parasites by integrating short fragments of foreign nucleic acids into repetitive loci in the host chromosome known as CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats). CRISPR-associated gene 3 (cas3) encodes a conserved helicase protein that is essential for phage defense. Here we review recent advances in Cas3 biology, and provide a new phylogenetic framework that positions Cas3 in the helicase family tree. We anticipate that this Cas3 phylogeny will guide future biochemical and structural studies. Copyright © 2014. Published by Elsevier Ltd.

Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen

PubMed Central

2011-01-01

Background The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4). Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. Results We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. Conclusions We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy. PMID:22014111

Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen.

PubMed

Le Breton, Marc; Meyniel-Schicklin, Laurène; Deloire, Alexandre; Coutard, Bruno; Canard, Bruno; de Lamballerie, Xavier; Andre, Patrice; Rabourdin-Combe, Chantal; Lotteau, Vincent; Davoust, Nathalie

2011-10-20

The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4). Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy.

DNA Unwinding Functions of Minute Virus of Mice NS1 Protein Are Modulated Specifically by the Lambda Isoform of Protein Kinase C

PubMed Central

Dettwiler, Sabine; Rommelaere, Jean; Nüesch, Jürg P. F.

1999-01-01

The parvovirus minute virus of mice NS1 protein is a multifunctional protein involved in a variety of processes during virus propagation, ranging from viral DNA replication to promoter regulation and cytotoxic action to the host cell. Since NS1 becomes phosphorylated during infection, it was proposed that the different tasks of this protein might be regulated in a coordinated manner by phosphorylation. Indeed, comparing biochemical functions of native NS1 with its dephosphorylated counterpart showed that site-specific nicking of the origin and the helicase and ATPase activities are remarkably reduced upon NS1 dephosphorylation while site-specific affinity of the protein to the origin became enhanced. As a consequence, the dephosphorylated polypeptide is deficient for initiation of DNA replication. By adding fractionated cell extracts to a kinase-free in vitro replication system, the combination of two protein components containing members of the protein kinase C (PKC) family was found to rescue the replication activity of the dephosphorylated NS1 protein upon addition of PKC cofactors. One of these components, termed HA-1, also stimulated NS1 helicase function in response to acidic lipids but not phorbol esters, indicating the involvement of atypical PKC isoforms in the modulation of this NS1 function (J. P. F. Nüesch, S. Dettwiler, R. Corbau, and J. Rommelaere, J. Virol. 72:9966–9977, 1998). The present study led to the identification of atypical PKCλ/ι as the active component of HA-1 responsible for the regulation of NS1 DNA unwinding and replicative functions. Moreover, a target PKCλ phosphorylation site was localized at S473 of NS1. By site-directed mutagenesis, we showed that this residue is essential for NS1 helicase activity but not promoter regulation, suggesting a possible modulation of NS1 functions by PKCλ phosphorylation at residue S473. PMID:10438831

Unzippers, Resolvers and Sensors: A Structural and Functional Biochemistry Tale of RNA Helicases

PubMed Central

Leitão, Ana Lúcia; Costa, Marina C.; Enguita, Francisco J.

2015-01-01

The centrality of RNA within the biological world is an irrefutable fact that currently attracts increasing attention from the scientific community. The panoply of functional RNAs requires the existence of specific biological caretakers, RNA helicases, devoted to maintain the proper folding of those molecules, resolving unstable structures. However, evolution has taken advantage of the specific position and characteristics of RNA helicases to develop new functions for these proteins, which are at the interface of the basic processes for transference of information from DNA to proteins. RNA helicases are involved in many biologically relevant processes, not only as RNA chaperones, but also as signal transducers, scaffolds of molecular complexes, and regulatory elements. Structural biology studies during the last decade, founded in X-ray crystallography, have characterized in detail several RNA-helicases. This comprehensive review summarizes the structural knowledge accumulated in the last two decades within this family of proteins, with special emphasis on the structure-function relationships of the most widely-studied families of RNA helicases: the DEAD-box, RIG-I-like and viral NS3 classes. PMID:25622248

Dengue virus NS2 and NS4: Minor proteins, mammoth roles.

PubMed

Gopala Reddy, Sindhoora Bhargavi; Chin, Wei-Xin; Shivananju, Nanjunda Swamy

2018-04-17

Despite the ever-increasing global incidence of dengue fever, there are no specific chemotherapy regimens for its treatment. Structural studies on dengue virus (DENV) proteins have revealed potential drug targets. Major DENV proteins such as the envelope protein and non-structural (NS) proteins 3 and 5 have been extensively investigated in antiviral studies, but with limited success in vitro. However, the minor NS proteins NS2 and NS4 have remained relatively underreported. Emerging evidence indicating their indispensable roles in virus propagation and host immunomodulation should encourage us to target these proteins for drug discovery. This review covers current knowledge on DENV NS2 and NS4 proteins from structural and functional perspectives and assesses their potential as targets for antiviral design. Antiviral targets in NS2A include surface-exposed transmembrane regions involved in pathogenesis, while those in NS2B include protease-binding sites in a conserved hydrophilic domain. Ideal drug targets in NS4A include helix α4 and the PEPEKQR sequence, which are essential for NS4A-2K cleavage and NS4A-NS4B association, respectively. In NS4B, the cytoplasmic loop connecting helices α5 and α7 is an attractive target for antiviral design owing to its role in dimerization and NS4B-NS3 interaction. Findings implicating NS2A, NS2B, and NS4A in membrane-modulation and viroporin-like activities indicate an opportunity to target these proteins by disrupting their association with membrane lipids. Despite the lack of 3D structural data, recent topological findings and progress in structure-prediction methods should be sufficient impetus for targeting NS2 and NS4 for drug design. Copyright © 2018 Elsevier Inc. All rights reserved.

Assessment of Drug Binding Potential of Pockets in the NS2B/NS3 Dengue Virus Protein

NASA Astrophysics Data System (ADS)

Amelia, F.; Iryani; Sari, P. Y.; Parikesit, A. A.; Bakri, R.; Toepak, E. P.; Tambunan, U. S. F.

2018-04-01

Every year an endemic dengue fever estimated to affect over 390 million cases in over 128 countries occurs. However, the antigen types which stimulate the human immune response are variable, as a result, neither effective vaccines nor antiviral treatments have been successfully developed for this disease. The NS2B/NS3 protease of the dengue virus (DENV) responsible for viral replication is a potential drug target. The ligand-enzyme binding site determination is a key role in the success of virtual screening of new inhibitors. The NS2B/NS3 protease of DENV (PDB ID: 2FOM) has two pockets consisting of 37 (Pocket 1) and 27 (Pocket 2) amino acid residues in each pocket. In this research, we characterized the amino acid residues for binding sites in NS3/NS2B based on the hydrophobicity, the percentage of charged residues, volume, depth, ΔGbinding, hydrogen bonding and bond length. The hydrophobic percentages of both pockets are high, 59 % (Pocket 1) and 41% (Pocket 2) and the percentage of charged residues in Pocket 1 and 2 are 22% and 48%, and the pocket volume is less than 700 Å3. An interaction analysis using molecular docking showed that interaction between the ligand complex and protein in Pocket 1 is more negative than Pocket 2. As a result, Pocket 1 is the better potential target for a ligand to inhibit the action of NS2B/NS3 DENV.

Substrate inhibition kinetic model for West Nile virus NS2B-NS3 protease.

PubMed

Tomlinson, Suzanne M; Watowich, Stanley J

2008-11-11

West Nile virus (WNV) has recently emerged in North America as a significant disease threat to humans and animals. Unfortunately, no approved antiviral drugs exist to combat WNV or other members of the genus Flavivirus in humans. The WNV NS2B-NS3 protease has been one of the primary targets for anti-WNV drug discovery and design since it is required for virus replication. As part of our efforts to develop effective WNV inhibitors, we reexamined the reaction kinetics of the NS2B-NS3 protease and the inhibition mechanisms of newly discovered inhibitors. The WNV protease showed substrate inhibition in assays utilizing fluorophore-linked peptide substrates GRR, GKR, and DFASGKR. Moreover, a substrate inhibition reaction step was required to accurately model kinetic data generated from protease assays with a peptide inhibitor. The substrate inhibition model suggested that peptide substrates could bind to two binding sites on the protease. Reaction product analogues also showed inhibition of the protease, demonstrating product inhibition in addition to and distinct from substrate inhibition. We propose that small peptide substrates and inhibitors may interact with protease residues that form either the P3-P1 binding surface (i.e., the S3-S1 sites) or the P1'-P3' interaction surface (i.e., the S1'-S3' sites). Optimization of substrate analogue inhibitors that target these two independent sites may lead to novel anti-WNV drugs.

Computer Aided Screening of Phytochemicals from Garcinia against the Dengue NS2B/NS3 Protease.

PubMed

Qamar, Tahir Ul; Mumtaz, Arooj; Ashfaq, Usman Ali; Azhar, Samia; Fatima, Tabeer; Hassan, Muhammad; Hussain, Syed Sajid; Akram, Waheed; Idrees, Sobia

2014-01-01

Dengue virus NS2/NS3 protease because of its ability to cleave viral proteins is considered as an attractive target to screen antiviral agents. Medicinal plants contain a variety of phytochemicals that can be used as drug against different diseases and infections. Therefore, this study was designed to uncover possible phytochemical of different classes (Aromatic, Carbohydrates, Lignin, Saponins, Steroids, Tannins, Terpenoids, Xanthones) that could be used as inhibitors against the NS2B/NS3 protease of DENV. With the help of molecular docking, Garcinia phytochemicals found to be bound deeply inside the active site of DENV NS2B/NS3 protease among all tested phytochemicals and had interactions with catalytic triad (His51, Asp75, Ser135). Thus, it can be concluded from the study that these Gracinia phytochemicals could serve as important inhibitors to inhibit the viral replication inside the host cell. Further in-vitro investigations require confirming their efficacy.

Computer Aided Screening of Phytochemicals from Garcinia against the Dengue NS2B/NS3 Protease

PubMed Central

Qamar, Tahir ul; Mumtaz, Arooj; Ashfaq, Usman Ali; Azhar, Samia; Fatima, Tabeer; Hassan, Muhammad; Hussain, Syed Sajid; Akram, Waheed; Idrees, Sobia

2014-01-01

Dengue virus NS2/NS3 protease because of its ability to cleave viral proteins is considered as an attractive target to screen antiviral agents. Medicinal plants contain a variety of phytochemicals that can be used as drug against different diseases and infections. Therefore, this study was designed to uncover possible phytochemical of different classes (Aromatic, Carbohydrates, Lignin, Saponins, Steroids, Tannins, Terpenoids, Xanthones) that could be used as inhibitors against the NS2B/NS3 protease of DENV. With the help of molecular docking, Garcinia phytochemicals found to be bound deeply inside the active site of DENV NS2B/NS3 protease among all tested phytochemicals and had interactions with catalytic triad (His51, Asp75, Ser135). Thus, it can be concluded from the study that these Gracinia phytochemicals could serve as important inhibitors to inhibit the viral replication inside the host cell. Further in-vitro investigations require confirming their efficacy. PMID:24748749

Flavonoids as noncompetitive inhibitors of Dengue virus NS2B-NS3 protease: inhibition kinetics and docking studies.

PubMed

de Sousa, Lorena Ramos Freitas; Wu, Hongmei; Nebo, Liliane; Fernandes, João Batista; da Silva, Maria Fátima das Graças Fernandes; Kiefer, Werner; Kanitz, Manuel; Bodem, Jochen; Diederich, Wibke E; Schirmeister, Tanja; Vieira, Paulo Cezar

2015-02-01

NS2B-NS3 is a serine protease of the Dengue virus considered a key target in the search for new antiviral drugs. In this study flavonoids were found to be inhibitors of NS2B-NS3 proteases of the Dengue virus serotypes 2 and 3 with IC50 values ranging from 15 to 44 μM. Agathisflavone (1) and myricetin (4) turned out to be noncompetitive inhibitors of dengue virus serotype 2 NS2B-NS3 protease with Ki values of 11 and 4.7 μM, respectively. Docking studies propose a binding mode of the flavonoids in a specific allosteric binding site of the enzyme. Analysis of biomolecular interactions of quercetin (5) with NT647-NHS-labeled Dengue virus serotype 3 NS2B-NS3 protease by microscale thermophoresis experiments, yielded a dissociation constant KD of 20 μM. Our results help to understand the mechanism of inhibition of the Dengue virus serine protease by flavonoids, which is essential for the development of improved inhibitors. Copyright © 2014 Elsevier Ltd. All rights reserved.

Saccharomyces cerevisiae Hrq1 requires a long 3'-tailed DNA substrate for helicase activity.

PubMed

Kwon, Sung-Hun; Choi, Do-Hee; Lee, Rina; Bae, Sung-Ho

2012-10-26

RecQ helicases are well conserved proteins from bacteria to human and function in various DNA metabolism for maintenance of genome stability. Five RecQ helicases are found in humans, whereas only one RecQ helicase has been described in lower eukaryotes. However, recent studies predicted the presence of a second RecQ helicase, Hrq1, in fungal genomes and verified it as a functional gene in fission yeast. Here we show that 3'-5' helicase activity is intrinsically associated with Hrq1 of Saccharomyces cerevisiae. We also determined several biochemical properties of Hrq1 helicase distinguishable from those of other RecQ helicase members. Hrq1 is able to unwind relatively long duplex DNA up to 120-bp and is significantly stimulated by a preexisting fork structure. Further, the most striking feature of Hrq1 is its absolute requirement for a long 3'-tail (⩾70-nt) for efficient unwinding of duplex DNA. We also found that Hrq1 has potent DNA strand annealing activity. Our results indicate that Hrq1 has vigorous helicase activity that deserves further characterization to expand our understanding of RecQ helicases. Copyright © 2012 Elsevier Inc. All rights reserved.

Conformational flexibility of DENV NS2B/NS3pro: from the inhibitor effect to the serotype influence

NASA Astrophysics Data System (ADS)

Piccirillo, Erika; Merget, Benjamin; Sotriffer, Christoph A.; do Amaral, Antonia T.

2016-03-01

The dengue virus (DENV) has four well-known serotypes, namely DENV1 to DENV4, which together cause 50-100 million infections worldwide each year. DENV NS2B/NS3pro is a protease recognized as a valid target for DENV antiviral drug discovery. However, NS2B/NS3pro conformational flexibility, involving in particular the NS2B region, is not yet completely understood and, hence, a big challenge for any virtual screening (VS) campaign. Molecular dynamics (MD) simulations were performed in this study to explore the DENV3 NS2B/NS3pro binding-site flexibility and obtain guidelines for further VS studies. MD simulations were done with and without the Bz-nKRR-H inhibitor, showing that the NS2B region stays close to the NS3pro core even in the ligand-free structure. Binding-site conformational states obtained from the simulations were clustered and further analysed using GRID/PCA, identifying four conformations of potential importance for VS studies. A virtual screening applied to a set of 31 peptide-based DENV NS2B/NS3pro inhibitors, taken from literature, illustrated that selective alternative pharmacophore models can be constructed based on conformations derived from MD simulations. For the first time, the NS2B/NS3pro binding-site flexibility was evaluated for all DENV serotypes using homology models followed by MD simulations. Interestingly, the number of NS2B/NS3pro conformational states differed depending on the serotype. Binding-site differences could be identified that may be crucial to subsequent VS studies.

Human SUV3 helicase regulates growth rate of the HeLa cells and can localize in the nucleoli.

PubMed

Szewczyk, Maciej; Fedoryszak-Kuśka, Natalia; Tkaczuk, Katarzyna; Dobrucki, Jurek; Waligórska, Agnieszka; Stępień, Piotr P

2017-01-01

The human SUV3 helicase (SUV3, hSUV3, SUPV3L1) is a DNA/RNA unwinding enzyme belonging to the class of DexH-box helicases. It localizes predominantly in the mitochondria, where it forms an RNA-degrading complex called mitochondrial degradosome with exonuclease PNP (polynucleotide phosphorylase). Association of this complex with the polyA polymerase can modulate mitochondrial polyA tails. Silencing of the SUV3 gene was shown to inhibit the cell cycle and to induce apoptosis in human cell lines. However, since small amounts of the SUV3 helicase were found in the cell nuclei, it was not clear whether the observed phenotypes of SUV3 depletion were of mitochondrial or nuclear origin. In order to answer this question we have designed gene constructs able to inhibit the SUV3 activity exclusively in the cell nuclei. The results indicate that the observed growth rate impairment upon SUV3 depletion is due to its nuclear function(s). Unexpectedly, overexpression of the nuclear-targeted wild-type copies of the SUV3 gene resulted in a higher growth rate. In addition, we demonstrate that the SUV3 helicase can be found in the HeLa cell nucleoli, but it is not detectable in the DNA-repair foci. Our results indicate that the nucleolar-associated human SUV3 protein is an important factor in regulation of the cell cycle.

Inhibitor Bound Dengue NS2B-NS3pro Reveals Multiple Dynamic Binding Modes.

PubMed

Gibbs, Alan C; Steele, Ruth; Liu, Gaohua; Tounge, Brett A; Montelione, Gaetano T

2018-03-13

Dengue virus poses a significant global health threat as the source of increasingly deleterious dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. As no specific antiviral treatment exists for dengue infection, considerable effort is being applied to discover therapies and drugs for maintenance and prevention of these afflictions. The virus is primarily transmitted by mosquitoes, and infection occurs following viral endocytosis by host cells. Upon entering the cell, viral RNA is translated into a large multisubunit polyprotein which is post-translationally cleaved into mature, structural and nonstructural (NS) proteins. The viral genome encodes the enzyme to carry out cleavage of the large polyprotein, specifically the NS2B-NS3pro cofactor-protease complex-a target of high interest for drug design. One class of recently discovered NS2B-NS3pro inhibitors is the substrate-based trifluoromethyl ketone containing peptides. These compounds interact covalently with the active site Ser135 via a hemiketal adduct. A detailed picture of the intermolecular protease/inhibitor interactions of the hemiketal adduct is crucial for rational drug design. We demonstrate, through the use of protein- and ligand-detected solution-state 19 F and 1 H NMR methods, an unanticipated multibinding mode behavior of a representative of this class of inhibitors to dengue NS2B-NS3pro. Our results illustrate the highly dynamic nature of both the covalently bound ligand and protease protein structure, and the need to consider these dynamics when designing future inhibitors in this class.

[Bioinformatics analysis of mosquito densovirus nostructure protein NS1].

PubMed

Dong, Yun-qiao; Ma, Wen-li; Gu, Jin-bao; Zheng, Wen-ling

2009-12-01

To analyze and predict the structure and function of mosquito densovirus (MDV) nostructual protein1 (NS1). Using different bioinformatics software, the EXPASY pmtparam tool, ClustalX1.83, Bioedit, MEGA3.1, ScanProsite, and Motifscan, respectively to comparatively analyze and predict the physic-chemical parameters, homology, evolutionary relation, secondary structure and main functional motifs of NS1. MDV NS1 protein was a unstable hydrophilic protein and the amino acid sequence was highly conserved which had a relatively closer evolutionary distance with infectious hypodermal and hematopoietic necrosis virus (IHHNV). MDV NS1 has a specific domain of superfamily 3 helicase of small DNA viruses. This domain contains the NTP-binding region with a metal ion-dependent ATPase activity. A virus replication roller rolling-circle replication(RCR) initiation domain was found near the N terminal of this protein. This protien has the biological function of single stranded incision enzyme. The bioinformatics prediction results suggest that MDV NS1 protein plays a key role in viral replication, packaging, and the other stages of viral life.

The Bacteroides sp. 3_1_23 Pif1 protein is a multifunctional helicase.

PubMed

Liu, Na-Nv; Duan, Xiao-Lei; Ai, Xia; Yang, Yan-Tao; Li, Ming; Dou, Shuo-Xing; Rety, Stephane; Deprez, Eric; Xi, Xu-Guang

2015-10-15

ScPif1 DNA helicase is the prototypical member of a 5'-to-3' helicase superfamily conserved from bacteria to human and plays various roles in the maintenance of genomic homeostasis. While many studies have been performed with eukaryotic Pif1 helicases, including yeast and human Pif1 proteins, the potential functions and biochemical properties of prokaryotic Pif1 helicases remain largely unknown. Here, we report the expression, purification and biochemical analysis of Pif1 helicase from Bacteroides sp. 3_1_23 (BsPif1). BsPif1 binds to a large panel of DNA substrates and, in particular, efficiently unwinds partial duplex DNAs with 5'-overhang, fork-like substrates, D-loop and flap-like substrates, suggesting that BsPif1 may act at stalled DNA replication forks and enhance Okazaki fragment maturation. Like its eukaryotic homologues, BsPif1 resolves R-loop structures and unwinds DNA-RNA hybrids. Furthermore, BsPif1 efficiently unfolds G-quadruplexes and disrupts nucleoprotein complexes. Altogether, these results highlight that prokaryotic Pif1 helicases may resolve common issues that arise during DNA transactions. Interestingly, we found that BsPif1 is different from yeast Pif1, but resembles more human Pif1 with regard to substrate specificity, helicase activity and mode of action. These findings are discussed in the context of the possible functions of prokaryotic Pif1 helicases in vivo. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Hmi1p from Saccharomyces cerevisiae mitochondria is a structure-specific DNA helicase.

PubMed

Kuusk, Silja; Sedman, Tiina; Jõers, Priit; Sedman, Juhan

2005-07-01

Hmi1p is a Saccharomyces cerevisiae mitochondrial DNA helicase that is essential for the maintenance of functional mitochondrial DNA. Hmi1p belongs to the superfamily 1 of helicases and is a close homologue of bacterial PcrA and Rep helicases. We have overexpressed and purified recombinant Hmi1p from Escherichia coli and describe here the biochemical characteristics of its DNA helicase activities. Among nucleotide cofactors, the DNA unwinding by Hmi1p was found to occur efficiently only in the presence of ATP and dATP. Hmi1p could unwind only the DNA substrates with a 3'-single-stranded overhang. The length of the 3'-overhang needed for efficient targeting of the helicase to the substrate depended on the substrate structure. For substrates consisting of duplex DNA with a 3'-single-stranded DNA overhang, at least a 19-nt 3'-overhang was needed. In the case of forked substrates with both 3'- and 5'-overhangs, a 9-nt 3'-overhang was sufficient provided that the 5'-overhang was also 9 nt in length. In flap-structured substrates mimicking the chain displacement structures in DNA recombination process, only a 5-nt 3'-single-stranded DNA tail was required for efficient unwinding by Hmi1p. These data indicate that Hmi1p may be targeted to a specific 3'-flap structure, suggesting its possible role in DNA recombination.

Dengue Virus NS2B/NS3 Protease Inhibitors Exploiting the Prime Side.

PubMed

Lin, Kuan-Hung; Ali, Akbar; Rusere, Linah; Soumana, Djade I; Kurt Yilmaz, Nese; Schiffer, Celia A

2017-05-15

The mosquito-transmitted dengue virus (DENV) infects millions of people in tropical and subtropical regions. Maturation of DENV particles requires proper cleavage of the viral polyprotein, including processing of 8 of the 13 substrate cleavage sites by dengue virus NS2B/NS3 protease. With no available direct-acting antiviral targeting DENV, NS2/NS3 protease is a promising target for inhibitor design. Current design efforts focus on the nonprime side of the DENV protease active site, resulting in highly hydrophilic and nonspecific scaffolds. However, the prime side also significantly modulates DENV protease binding affinity, as revealed by engineering the binding loop of aprotinin, a small protein with high affinity for DENV protease. In this study, we designed a series of cyclic peptides interacting with both sides of the active site as inhibitors of dengue virus protease. The design was based on two aprotinin loops and aimed to leverage both key specific interactions of substrate sequences and the entropic advantage driving aprotinin's high affinity. By optimizing the cyclization linker, length, and amino acid sequence, the tightest cyclic peptide achieved a K i value of 2.9 μM against DENV3 wild-type (WT) protease. These inhibitors provide proof of concept that both sides of DENV protease active site can be exploited to potentially achieve specificity and lower hydrophilicity in the design of inhibitors targeting DENV. IMPORTANCE Viruses of the flaviviral family, including DENV and Zika virus transmitted by Aedes aegypti , continue to be a threat to global health by causing major outbreaks in tropical and subtropical regions, with no available direct-acting antivirals for treatment. A better understanding of the molecular requirements for the design of potent and specific inhibitors against flaviviral proteins will contribute to the development of targeted therapies for infections by these viruses. The cyclic peptides reported here as DENV protease inhibitors

Identification of novel small molecule inhibitors against NS2B/NS3 serine protease from Zika virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Hyun; Ren, Jinhong; Nocadello, Salvatore

Zika flavivirus infection during pregnancy appears to produce higher risk of microcephaly, and also causes multiple neurological problems such as Guillain–Barré syndrome. The Zika virus is now widespread in Central and South America, and is anticipated to become an increasing risk in the southern United States. With continuing global travel and the spread of the mosquito vector, the exposure is expected to accelerate, but there are no currently approved treatments against the Zika virus. The Zika NS2B/NS3 protease is an attractive drug target due to its essential role in viral replication. Our studies have identified several compounds with inhibitory activitymore » (IC50) and binding affinity (KD) of ~5–10 μM against the Zika NS2B-NS3 protease from testing 71 HCV NS3/NS4A inhibitors that were initially discovered by high-throughput screening of 40,967 compounds. Competition surface plasmon resonance studies and mechanism of inhibition analyses by enzyme kinetics subsequently determined the best compound to be a competitive inhibitor with a Ki value of 9.5 μM. We also determined the X-ray structure of the Zika NS2B-NS3 protease in a “pre-open conformation”, a conformation never observed before for any flavivirus proteases. This provides the foundation for new structure-based inhibitor design.« less

DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors

NASA Astrophysics Data System (ADS)

Deka, Jashmini; Mojumdar, Aditya; Parisse, Pietro; Onesti, Silvia; Casalis, Loredana

2017-03-01

Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors.

MAVS dimer is a crucial signaling component of innate immunity and the target of hepatitis C virus NS3/4A protease.

PubMed

Baril, Martin; Racine, Marie-Eve; Penin, François; Lamarre, Daniel

2009-02-01

The mitochondrial antiviral signaling (MAVS) protein plays a central role in innate antiviral immunity. Upon recognition of a virus, intracellular receptors of the RIG-I-like helicase family interact with MAVS to trigger a signaling cascade. In this study, we investigate the requirement of the MAVS structure for enabling its signaling by structure-function analyses and resonance energy transfer approaches in live cells. We now report the essential role of the MAVS oligomer in signal transduction and map the transmembrane domain as the main determinant of dimerization. A combination of mutagenesis and computational methods identified a cluster of residues making favorable van der Waals interactions at the MAVS dimer interface. We also correlated the activation of IRF3 and NF-kappaB with MAVS oligomerization rather than its mitochondrial localization. Finally, we demonstrated that MAVS oligomerization is disrupted upon expression of HCV NS3/4A protease, suggesting a mechanism for the loss of antiviral signaling. Altogether, our data suggest that the MAVS oligomer is essential in the formation of a multiprotein membrane-associated signaling complex and enables downstream activation of IRF3 and NF-kappaB in antiviral innate immunity.

Novel dengue virus NS2B/NS3 protease inhibitors.

PubMed

Wu, Hongmei; Bock, Stefanie; Snitko, Mariya; Berger, Thilo; Weidner, Thomas; Holloway, Steven; Kanitz, Manuel; Diederich, Wibke E; Steuber, Holger; Walter, Christof; Hofmann, Daniela; Weißbrich, Benedikt; Spannaus, Ralf; Acosta, Eliana G; Bartenschlager, Ralf; Engels, Bernd; Schirmeister, Tanja; Bodem, Jochen

2015-02-01

Dengue fever is a severe, widespread, and neglected disease with more than 2 million diagnosed infections per year. The dengue virus NS2B/NS3 protease (PR) represents a prime target for rational drug design. At the moment, there are no clinical PR inhibitors (PIs) available. We have identified diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and noncompetitive inhibition of the serotype 2 and 3 dengue virus PR in vitro and in cells by benzothiazole derivatives exhibiting 50% inhibitory concentrations (IC50s) in the low-micromolar range. Inhibition of replication of DENV serotypes 1 to 3 was specific, since all substances influenced neither hepatitis C virus (HCV) nor HIV-1 replication. Molecular docking suggests binding at a specific allosteric binding site. In addition to the in vitro assays, a cell-based PR assay was developed to test these substances in a replication-independent way. The new compounds inhibited the DENV PR with IC50s in the low-micromolar or submicromolar range in cells. Furthermore, these novel PIs inhibit viral replication at submicromolar concentrations. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Crystal structure of the SF3 helicase from adeno-associated virus type 2.

PubMed

James, J Anson; Escalante, Carlos R; Yoon-Robarts, Miran; Edwards, Thomas A; Linden, R Michael; Aggarwal, Aneel K

2003-08-01

We report here the crystal structure of an SF3 DNA helicase, Rep40, from adeno-associated virus 2 (AAV2). We show that AAV2 Rep40 is structurally more similar to the AAA(+) class of cellular proteins than to DNA helicases from other superfamilies. The structure delineates the expected Walker A and B motifs, but also reveals an unexpected "arginine finger" that directly implies the requirement of Rep40 oligomerization for ATP hydrolysis and helicase activity. Further, the Rep40 AAA(+) domain is novel in that it is unimodular as opposed to bimodular. Altogether, the structural connection to AAA(+) proteins defines the general architecture of SF3 DNA helicases, a family that includes simian virus 40 (SV40) T antigen, as well as provides a conceptual framework for understanding the role of Rep proteins during AAV DNA replication, packaging, and site-specific integration.

Molecular targets for flavivirus drug discovery

PubMed Central

Sampath, Aruna; Padmanabhan, R.

2009-01-01

Flaviviruses are a major cause of infectious disease in humans. Dengue virus causes an estimated 50 million cases of febrile illness each year, including an increasing number of cases of hemorrhagic fever. West Nile virus, which recently spread from the Mediterranean basin to the Western Hemisphere, now causes thousands of sporadic cases of encephalitis annually. Despite the existence of licensed vaccines, yellow fever, Japanese encephalitis and tick-borne encephalitis also claim many thousands of victims each year across their vast endemic areas. Antiviral therapy could potentially reduce morbidity and mortality from flavivirus infections, but no effective drugs are currently available. This article introduces a collection of papers in Antiviral Research on molecular targets for flavivirus antiviral drug design and murine models of dengue virus disease that aims to encourage drug development efforts. After reviewing the flavivirus replication cycle, we discuss the envelope glycoprotein, NS3 protease, NS3 helicase, NS5 methyltransferase and NS5 RNA-dependent RNA polymerase as potential drug targets, with special attention being given to the viral protease. The other viral proteins are the subject of individual articles in the journal. Together, these papers highlight current status of drug discovery efforts for flavivirus diseases and suggest promising areas for further research. PMID:18796313

Helicase-dependent amplification of nucleic acids.

PubMed

Cao, Yun; Kim, Hyun-Jin; Li, Ying; Kong, Huimin; Lemieux, Bertrand

2013-10-11

Helicase-dependent amplification (HDA) is a novel method for the isothermal in vitro amplification of nucleic acids. The HDA reaction selectively amplifies a target sequence by extension of two oligonucleotide primers. Unlike the polymerase chain reaction (PCR), HDA uses a helicase enzyme to separate the deoxyribonucleic acid (DNA) strands, rather than heat denaturation. This allows DNA amplification without the need for thermal cycling. The helicase used in HDA is a helicase super family II protein obtained from a thermophilic organism, Thermoanaerobacter tengcongensis (TteUvrD). This thermostable helicase is capable of unwinding blunt-end nucleic acid substrates at elevated temperatures (60° to 65°C). The HDA reaction can also be coupled with reverse transcription for ribonucleic acid (RNA) amplification. The products of this reaction can be detected during the reaction using fluorescent probes when incubations are conducted in a fluorimeter. Alternatively, products can be detected after amplification using a disposable amplicon containment device that contains an embedded lateral flow strip. Copyright © 2013 John Wiley & Sons, Inc.

Purification and crystallization of dengue and West Nile virus NS2B–NS3 complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

D’Arcy, Allan, E-mail: allan.darcy@novartis.com; Chaillet, Maxime; Schiering, Nikolaus

Crystals of dengue serotype 2 and West Nile virus NS2B–NS3 protease complexes have been obtained and the crystals of both diffract to useful resolution. Sample homogeneity was essential for obtaining X-ray-quality crystals of the dengue protease. Controlled proteolysis produced a crystallizable fragment of the apo West Nile virus NS2B–NS3 and crystals were also obtained in the presence of a peptidic inhibitor. Both dengue and West Nile virus infections are an increasing risk to humans, not only in tropical and subtropical areas, but also in North America and parts of Europe. These viral infections are generally transmitted by mosquitoes, but maymore » also be tick-borne. Infection usually results in mild flu-like symptoms, but can also cause encephalitis and fatalities. Approximately 2799 severe West Nile virus cases were reported this year in the United States, resulting in 102 fatalities. With this alarming increase in the number of West Nile virus infections in western countries and the fact that dengue virus already affects millions of people per year in tropical and subtropical climates, there is a real need for effective medicines. A possible therapeutic target to combat these viruses is the protease, which is essential for virus replication. In order to provide structural information to help to guide a lead identification and optimization program, crystallizations of the NS2B–NS3 protease complexes from both dengue and West Nile viruses have been initiated. Crystals that diffract to high resolution, suitable for three-dimensional structure determinations, have been obtained.« less

A Genetic Interaction between the Core and NS3 Proteins of Hepatitis C Virus Is Essential for Production of Infectious Virus▿†

PubMed Central

Jones, Daniel M.; Atoom, Ali M.; Zhang, Xiaozhen; Kottilil, Shyamasundaran; Russell, Rodney S.

2011-01-01

By analogy to other members of the Flaviviridae family, the hepatitis C virus (HCV) core protein is presumed to oligomerize to form the viral nucleocapsid, which encloses the single-stranded RNA genome. Core protein is directed to lipid droplets (LDs) by domain 2 (D2) of the protein, and this process is critical for virus production. Domain 1 (D1) of core is also important for infectious particle morphogenesis, although its precise contribution to this process is poorly understood. In this study, we mutated amino acids 64 to 75 within D1 of core and examined the ability of these mutants to produce infectious virus. We found that residues 64 to 66 are critical for generation of infectious progeny, whereas 67 to 75 were dispensable for this process. Further investigation of the defective 64 to 66 mutant (termed JFH1T-64–66) revealed it to be incapable of producing infectious intracellular virions, suggesting a fault during HCV assembly. Furthermore, isopycnic gradient analyses revealed that JFH1T-64–66 assembled dense intracellular species of core, presumably representing nucleocapsids. Thus, amino acids 64 to 66 are seemingly not involved in core oligomerization/nucleocapsid assembly. Passaging of JFH1T-64–66 led to the emergence of a single compensatory mutation (K1302R) within the helicase domain of NS3 that completely rescued its ability to produce infectious virus. Importantly, the same NS3 mutation abrogated virus production in the context of wild-type core protein. Together, our results suggest that residues 64 to 66 of core D1 form a highly specific interaction with the NS3 helicase that is essential for the generation of infectious HCV particles at a stage downstream of nucleocapsid assembly. PMID:21957313

Cancer-Associated Mutants of RNA Helicase DDX3X Are Defective in RNA-Stimulated ATP Hydrolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Epling, Leslie B.; Grace, Christy R.; Lowe, Brandon R.

The DEAD-box RNA helicase DDX3X is frequently mutated in pediatric medulloblastoma. We dissect how these mutants affect DDX3X function with structural, biochemical, and genetic experiments. We identify an N-terminal extension (“ATP-binding loop”, ABL) that is critical for the stimulation of ATP hydrolysis by RNA. We present crystal structures suggesting that the ABL interacts dynamically with ATP and confirming that the interaction occurs in solution by NMR chemical shift perturbation and isothermal titration calorimetry. DEAD-box helicases require interaction between two conserved RecA-like helicase domains, D1 and D2 for function. We use NMR chemical shift perturbation to show that DDX3X interacts specificallymore » with double-stranded RNA through its D1 domain, with contact mediated by residues G302 and G325. Mutants of these residues, G302V and G325E, are associated with pediatric medulloblastoma. These mutants are defective in RNA-stimulated ATP hydrolysis. We show that DDX3X complements the growth defect in a ded1 temperature-sensitive strain of Schizosaccharomyces pombe, but the cancer-associated mutants G302V and G325E do not complement and exhibit protein expression defects. In conclusion, taken together, our results suggest that impaired translation of important mRNA targets by mutant DDX3X represents a key step in the development of medulloblastoma.« less

Cancer-Associated Mutants of RNA Helicase DDX3X Are Defective in RNA-Stimulated ATP Hydrolysis

DOE PAGES

Epling, Leslie B.; Grace, Christy R.; Lowe, Brandon R.; ...

2015-02-25

The DEAD-box RNA helicase DDX3X is frequently mutated in pediatric medulloblastoma. We dissect how these mutants affect DDX3X function with structural, biochemical, and genetic experiments. We identify an N-terminal extension (“ATP-binding loop”, ABL) that is critical for the stimulation of ATP hydrolysis by RNA. We present crystal structures suggesting that the ABL interacts dynamically with ATP and confirming that the interaction occurs in solution by NMR chemical shift perturbation and isothermal titration calorimetry. DEAD-box helicases require interaction between two conserved RecA-like helicase domains, D1 and D2 for function. We use NMR chemical shift perturbation to show that DDX3X interacts specificallymore » with double-stranded RNA through its D1 domain, with contact mediated by residues G302 and G325. Mutants of these residues, G302V and G325E, are associated with pediatric medulloblastoma. These mutants are defective in RNA-stimulated ATP hydrolysis. We show that DDX3X complements the growth defect in a ded1 temperature-sensitive strain of Schizosaccharomyces pombe, but the cancer-associated mutants G302V and G325E do not complement and exhibit protein expression defects. In conclusion, taken together, our results suggest that impaired translation of important mRNA targets by mutant DDX3X represents a key step in the development of medulloblastoma.« less

Development and evaluation of a truncated recombinant NS3 antigen-based indirect ELISA for detection of pestivirus antibodies in sheep and goats.

PubMed

Kalaiyarasu, Semmannan; Mishra, Niranjan; Rajukumar, Katherukamem; Nema, Ram Kumar; Behera, Sthita Pragnya

2015-01-01

The aim of this study was to develop an indirect ELISA using the helicase domain of bovine viral diarrhoea virus (BVDV) NS3 protein instead of full-length NS3 protein for detection of BVDV and BDV antibodies in sheep and goats and its validation by comparing its sensitivity and specificity with virus neutralization test (VNT) as the reference test. The purified 50 kDa recombinant NS3 protein was used as the coating antigen in the ELISA. The optimal concentration of antigen was 320 ng/well at a serum dilution of 1:20 and the optimal positive cut-off optical density value was 0.40 based on test results of 418 VNT negative sheep and goat sera samples. When 569 serum samples from sheep (463) and goats (106) were tested, the ELISA showed a sensitivity of 91.71% and specificity of 94.59% with BVDV VNT. A good correlation (93.67%) was observed between the two tests. It showed a sensitivity of 85% and specificity of 86.6% with VNT in detecting BDV antibody positive or negative samples. This study demonstrates the efficacy of truncated recombinant NS3 antigen based ELISA for seroepidemiological study of pestivirus infection in sheep and goats.

The xeroderma pigmentosum group B protein ERCC3 produced in the baculovirus system exhibits DNA helicase activity.

PubMed Central

Ma, L; Siemssen, E D; Noteborn, H M; van der Eb, A J

1994-01-01

The XPB/ERCC3 gene corrects the nucleotide excision-repair defect in the human hereditary disease xeroderma pigmentosum group B and encodes the largest subunit of the basal transcription factor BTF2/TFIIH. The primary sequence of the XPB/ERCC3 protein features the hallmarks of seven helicase motifs found in many known and putative helicases or helicase-related proteins. Recently, the multiprotein BTF2/TFIIH complex has been found to be associated with DNA helicase activity. To explore the properties and functions of XPB/ERCC3, we have used the baculovirus/insect-cell expression system to produce recombinant protein. We report here the construction and analysis of recombinant baculovirus expressing XPB/ERCC3. The XPB/ERCC3 protein is synthesized at a relatively high level in baculovirus-infected insect cells. While the majority of XPB/ERCC3 end up in the insoluble fraction of insect cell lysates, a minor fraction of recombinant protein is present in soluble form which can be purified under native conditions. We have found that a DNA helicase activity is associated with the purified XPB/ERCC3 protein, suggesting that XPB/ERCC3 may function as a DNA helicase in local unwinding of DNA template both in the context of transcription and nucleotide excision repair. Images PMID:7937133

Extended substrate specificity and first potent irreversible inhibitor/activity-based probe design for Zika virus NS2B-NS3 protease.

PubMed

Rut, Wioletta; Zhang, Linlin; Kasperkiewicz, Paulina; Poreba, Marcin; Hilgenfeld, Rolf; Drąg, Marcin

2017-03-01

Zika virus is spread by Aedes mosquitoes and is linked to acute neurological disorders, especially to microcephaly in newborn children and Guillan-Barré Syndrome. The NS2B-NS3 protease of this virus is responsible for polyprotein processing and therefore considered an attractive drug target. In this study, we have used the Hybrid Combinatorial Substrate Library (HyCoSuL) approach to determine the substrate specificity of ZIKV NS2B-NS3 protease in the P4-P1 positions using natural and a large spectrum of unnatural amino acids. Obtained data demonstrate a high level of specificity of the S3-S1 subsites, especially for basic amino acids. However, the S4 site exhibits a very broad preference toward natural and unnatural amino acids with selected D-amino acids being favored over L enantiomers. This information was used for the design of a very potent phosphonate inhibitor/activity-based probe of ZIKV NS2B-NS3 protease. Copyright © 2016 Elsevier B.V. All rights reserved.

Anthracene-based Inhibitors of Dengue Virus NS2B-NS3 Protease†

PubMed Central

Tomlinson, Suzanne M.; Watowich, Stanley J.

2010-01-01

Summary Dengue virus (DENV) is a mosquito-borne flavivirus that has strained global healthcare systems throughout tropical and subtropical regions of the world. In addition to plaguing developing nations, it has re-emerged in several developed countries with recent outbreaks in the USA (CDC, 2010), Australia (Hanna et al., 2009), Taiwan (Kuan et al., 2010) and France (La Ruche et al., 2010). DENV infection can cause significant disease, including dengue fever, dengue hemorrhagic fever, dengue shock syndrome, and death. There are no approved vaccines or antiviral therapies to prevent or treat dengue-related illnesses. However, the viral NS2B-NS3 protease complex provides a strategic target for antiviral drug development since NS3 protease activity is required for virus replication. Recently, we reported two compounds with inhibitory activity against the DENV protease in vitro and antiviral activity against dengue 2 (DEN2V) in cell culture (Tomlinson et al., 2009a). Analogs of one of the lead compounds were purchased, tested in protease inhibition assays, and the data evaluated with detailed kinetic analyses. A structure activity relationship (SAR) identified key atomic determinants (i.e. functional groups) important for inhibitory activity. Four “second series” analogs were selected and tested to validate our SAR and structural models. Here, we report improvements to inhibitory activity ranging between ~2- and 60-fold, resulting in selective low micromolar dengue protease inhibitors. PMID:21185332

Flavonoid from Carica papaya inhibits NS2B-NS3 protease and prevents Dengue 2 viral assembly.

PubMed

Senthilvel, Padmanaban; Lavanya, Pandian; Kumar, Kalavathi Murugan; Swetha, Rayapadi; Anitha, Parimelzaghan; Bag, Susmita; Sarveswari, Sundaramoorthy; Vijayakumar, Vijayaparthasarathi; Ramaiah, Sudha; Anbarasu, Anand

2013-01-01

Dengue virus belongs to the virus family Flaviviridae. Dengue hemorrhagic disease caused by dengue virus is a public health problem worldwide. The viral non structural 2B and 3 (NS2B-NS3) protease complex is crucial for virus replication and hence, it is considered to be a good anti-viral target. Leaf extracts from Carica papaya is generally prescribed for patients with dengue fever, but there are no scientific evidences for its anti-dengue activity; hence we intended to investigate the anti-viral activity of compounds present in the leaves of Carica papaya against dengue 2 virus (DENV-2). We analysed the anti-dengue activities of the extracts from Carica papaya by using bioinformatics tools. Interestingly, we find the flavonoid quercetin with highest binding energy against NS2B-NS3 protease which is evident by the formation of six hydrogen bonds with the amino acid residues at the binding site of the receptor. Our results suggest that the flavonoids from Carica papaya have significant anti-dengue activities. ADME - Absorption, distribution, metabolism and excretion, BBB - Blood brain barrier, CYP - Cytochrome P450, DENV - - Dengue virus, DHF - Dengue hemorrhagic fever, DSS - Dengue shock syndrome, GCMS - - Gas chromatography- Mass spectrometry, MOLCAD - Molecular Computer Aided Design, NS - Non structural, PDB - Protein data bank, PMF - Potential Mean Force.

Hepatitis C Virus NS3/4A Protease Inhibitors: A Light at the End of the Tunnel

PubMed Central

Chatel-Chaix, Laurent; Baril, Martin; Lamarre, Daniel

2010-01-01

Hepatitis C virus (HCV) infection is a serious and growing threat to human health. The current treatment provides limited efficacy and is poorly tolerated, highlighting the urgent medical need for novel therapeutics. The membrane-targeted NS3 protein in complex with the NS4A comprises a serine protease domain (NS3/4A protease) that is essential for viral polyprotein maturation and contributes to the evasion of the host innate antiviral immunity by HCV. Therefore, the NS3/4A protease represents an attractive target for drug discovery, which is tied in with the challenge to develop selective small-molecule inhibitors. A rational drug design approach, based on the discovery of N-terminus product inhibition, led to the identification of potent and orally bioavailable NS3 inhibitors that target the highly conserved protease active site. This review summarizes the NS3 protease inhibitors currently challenged in clinical trials as one of the most promising antiviral drug class, and possibly among the first anti-HCV agents to be approved for the treatment of HCV infection. PMID:21994705

NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B-NS3 protease.

PubMed

Su, Xun-Cheng; Ozawa, Kiyoshi; Yagi, Hiromasa; Lim, Siew P; Wen, Daying; Ekonomiuk, Dariusz; Huang, Danzhi; Keller, Thomas H; Sonntag, Sebastian; Caflisch, Amedeo; Vasudevan, Subhash G; Otting, Gottfried

2009-08-01

The two-component NS2B-NS3 protease of West Nile virus is essential for its replication and presents an attractive target for drug development. Here, we describe protocols for the high-yield expression of stable isotope-labelled samples in vivo and in vitro. We also describe the use of NMR spectroscopy to determine the binding mode of new low molecular mass inhibitors of the West Nile virus NS2B-NS3 protease which were discovered using high-throughput in vitro screening. Binding to the substrate-binding sites S1 and S3 is confirmed by intermolecular NOEs and comparison with the binding mode of a previously identified low molecular mass inhibitor. Our results show that all these inhibitors act by occupying the substrate-binding site of the protease rather than by an allosteric mechanism. In addition, the NS2B polypeptide chain was found to be positioned near the substrate-binding site, as observed previously in crystal structures of the protease in complex with peptide inhibitors or bovine pancreatic trypsin inhibitor. This indicates that the new low molecular mass compounds, although inhibiting the protease, also promote the proteolytically active conformation of NS2B, which is very different from the crystal structure of the protein without inhibitor.

STD-NMR experiments identify a structural motif with novel second-site activity against West Nile virus NS2B-NS3 protease.

PubMed

Schöne, Tobias; Grimm, Lena Lisbeth; Sakai, Naoki; Zhang, Linlin; Hilgenfeld, Rolf; Peters, Thomas

2017-10-01

West Nile virus (WNV) belongs to the genus Flavivirus of the family Flaviviridae. This mosquito-borne virus that is highly pathogenic to humans has been evolving into a global threat during the past two decades. Despite many efforts, neither antiviral drugs nor vaccines are available. The viral protease NS2B-NS3 pro is essential for viral replication, and therefore it is considered a prime drug target. However, success in the development of specific NS2B-NS3 pro inhibitors had been moderate so far. In the search for new structural motifs with binding affinity for NS2B-NS3 pro , we have screened a fragment library, the Maybridge Ro5 library, employing saturation transfer difference (STD) NMR experiments as readout. About 30% of 429 fragments showed binding to NS2B-NS3 pro . Subsequent STD-NMR competition experiments using the known active site fragment A as reporter ligand yielded 14 competitively binding fragments, and 22 fragments not competing with A. In a fluorophore-based protease assay, all of these fragments showed inhibition in the micromolar range. Interestingly, 10 of these 22 fragments showed a notable increase of STD intensities in the presence of compound A suggesting cooperative binding. The most promising non-competitive inhibitors 1 and 2 (IC 50 ∼ 500 μM) share a structural motif that may guide the development of novel second-site (potentially allosteric) inhibitors of NS2B-NS3 pro . To identify the matching protein binding site, chemical shift perturbation studies employing 1 H, 15 N-TROSY-HSQC experiments with uniformly 2 H, 15 N-labeled protease were performed in the presence of 1, and in the concomitant absence or presence of A. The data suggest that 1 interacts with Met 52* of NS2B, identifying a secondary site adjacent to the binding site of A. Therefore, our study paves the way for the synthesis of novel bidentate NS2B-NS3 pro inhibitors. Copyright © 2017 Elsevier B.V. All rights reserved.

Exploring the Lead Compounds for Zika Virus NS2B-NS3 Protein: an e-Pharmacophore-Based Approach.

PubMed

Rohini, K; Agarwal, Pratika; Preethi, B; Shanthi, V; Ramanathan, K

2018-06-18

The rapid spread of the Zika virus and its association with the abnormal brain development constitute a global health emergency. With a continuing spread of the mosquito vector, the exposure is expected to accelerate in the coming years. Despite number of efforts, there is still no proper vaccine or medicine to combat this virus. Of note, the NS2B-NS3 protein is proven to be the potential target for the Zika virus therapeutics. Hence, e-pharmacophore-based drug design strategy was employed to identify potent inhibitors of NS2B-NS3 protein from ASINEX database consisting of 467,802 molecules. A 3D e-pharmacophore model was generated using PHASE module of Schrödinger Suite. The generated model consists of one hydrogen bond acceptor (A), two hydrogen bond donors (D), and two aromatic rings (R), ADDRR. The model was further evaluated for its ability to screen actives using enrichment analysis. Subsequently, high-throughput virtual screening protocol was employed, and the resultant hit molecules were also examined for its binding free energies and ADME properties using Prime MM-GBSA and Qikprop module of Schrodinger packages, respectively. Finally, the screened hit molecule was subjected to molecular dynamics simulation to examine its stability. Overall, the results from our analysis suggest that compound BAS 19192837 could be a potent inhibitor for the NS2B-NS3 protein of the Zika virus. It is also noteworthy to mention that our results are in good agreement with literature evidences. We hope that this result is of immense importance in designing potential drug molecules to combat the spread of Zika virus in the near future.

Structural basis of Zika virus helicase in recognizing its substrates.

PubMed

Tian, Hongliang; Ji, Xiaoyun; Yang, Xiaoyun; Zhang, Zhongxin; Lu, Zuokun; Yang, Kailin; Chen, Cheng; Zhao, Qi; Chi, Heng; Mu, Zhongyu; Xie, Wei; Wang, Zefang; Lou, Huiqiang; Yang, Haitao; Rao, Zihe

2016-08-01

The recent explosive outbreak of Zika virus (ZIKV) infection has been reported in South and Central America and the Caribbean. Neonatal microcephaly associated with ZIKV infection has already caused a public health emergency of international concern. No specific vaccines or drugs are currently available to treat ZIKV infection. The ZIKV helicase, which plays a pivotal role in viral RNA replication, is an attractive target for therapy. We determined the crystal structures of ZIKV helicase-ATP-Mn(2+) and ZIKV helicase-RNA. This is the first structure of any flavivirus helicase bound to ATP. Comparisons with related flavivirus helicases have shown that although the critical P-loop in the active site has variable conformations among different species, it adopts an identical mode to recognize ATP/Mn(2+). The structure of ZIKV helicase-RNA has revealed that upon RNA binding, rotations of the motor domains can cause significant conformational changes. Strikingly, although ZIKV and dengue virus (DENV) apo-helicases share conserved residues for RNA binding, their different manners of motor domain rotations result in distinct individual modes for RNA recognition. It suggests that flavivirus helicases could have evolved a conserved engine to convert chemical energy from nucleoside triphosphate to mechanical energy for RNA unwinding, but different motor domain rotations result in variable RNA recognition modes to adapt to individual viral replication.

Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains.

PubMed

Karow, Anne R; Theissen, Bettina; Klostermeier, Dagmar

2007-01-01

RNA helicases mediate structural rearrangements of RNA or RNA-protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis-Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication.

The HARP domain dictates the annealing helicase activity of HARP/SMARCAL1.

PubMed

Ghosal, Gargi; Yuan, Jingsong; Chen, Junjie

2011-06-01

Mutations in HepA-related protein (HARP, or SMARCAL1) cause Schimke immunoosseous dysplasia (SIOD). HARP has ATP-dependent annealing helicase activity, which helps to stabilize stalled replication forks and facilitate DNA repair during replication. Here, we show that the conserved tandem HARP (2HP) domain dictates this annealing helicase activity. Furthermore, chimeric proteins generated by fusing the 2HP domain of HARP with the SNF2 domain of BRG1 or HELLS show annealing helicase activity in vitro and, when targeted to replication forks, mimic the functions of HARP in vivo. We propose that the HARP domain endows HARP with this ATP-driven annealing helicase activity.

Synergistic interactions between the NS3(hel) and E proteins contribute to the virulence of dengue virus type 1.

PubMed

de Borba, Luana; Strottmann, Daisy M; de Noronha, Lucia; Mason, Peter W; Dos Santos, Claudia N Duarte

2012-01-01

Dengue includes a broad range of symptoms, ranging from fever to hemorrhagic fever and may occasionally have alternative clinical presentations. Many possible viral genetic determinants of the intrinsic virulence of dengue virus (DENV) in the host have been identified, but no conclusive evidence of a correlation between viral genotype and virus transmissibility and pathogenicity has been obtained. We used reverse genetics techniques to engineer DENV-1 viruses with subsets of mutations found in two different neuroadapted derivatives. The mutations were inserted into an infectious clone of DENV-1 not adapted to mice. The replication and viral production capacity of the recombinant viruses were assessed in vitro and in vivo. The results demonstrated that paired mutations in the envelope protein (E) and in the helicase domain of the NS3 (NS3(hel)) protein had a synergistic effect enhancing viral fitness in human and mosquito derived cell lines. E mutations alone generated no detectable virulence in the mouse model; however, the combination of these mutations with NS3(hel) mutations, which were mildly virulent on their own, resulted in a highly neurovirulent phenotype. The generation of recombinant viruses carrying specific E and NS3(hel) proteins mutations increased viral fitness both in vitro and in vivo by increasing RNA synthesis and viral load (these changes being positively correlated with central nervous system damage), the strength of the immune response and animal mortality. The introduction of only pairs of amino acid substitutions into the genome of a non-mouse adapted DENV-1 strain was sufficient to alter viral fitness substantially. Given current limitations to our understanding of the molecular basis of dengue neuropathogenesis, these results could contribute to the development of attenuated strains for use in vaccinations and provide insights into virus/host interactions and new information about the mechanisms of basic dengue biology.

Dynamic Nucleolar Targeting of Dengue Virus Polymerase NS5 in Response to Extracellular pH

PubMed Central

Fraser, Johanna E.; Rawlinson, Stephen M.; Heaton, Steven M.

2016-01-01

ABSTRACT The nucleolar subcompartment of the nucleus is increasingly recognized as an important target of RNA viruses. Here we document for the first time the ability of dengue virus (DENV) polymerase, nonstructural protein 5 (NS5), to accumulate within the nucleolus of infected cells and to target green fluorescent protein (GFP) to the nucleolus of live transfected cells. Intriguingly, NS5 exchange between the nucleus and nucleolus is dynamically modulated by extracellular pH, responding rapidly and reversibly to pH change, in contrast to GFP alone or other nucleolar and non-nucleolar targeted protein controls. The minimal pH-sensitive nucleolar targeting region (pHNTR), sufficient to target GFP to the nucleolus in a pH-sensitive fashion, was mapped to NS5 residues 1 to 244, with mutation of key hydrophobic residues, Leu-165, Leu-167, and Val-168, abolishing pHNTR function in NS5-transfected cells, and severely attenuating DENV growth in infected cells. This is the first report of a viral protein whose nucleolar targeting ability is rapidly modulated by extracellular stimuli, suggesting that DENV has the ability to detect and respond dynamically to the extracellular environment. IMPORTANCE Infections by dengue virus (DENV) threaten 40% of the world's population yet there is no approved vaccine or antiviral therapeutic to treat infections. Understanding the molecular details that govern effective viral replication is key for the development of novel antiviral strategies. Here, we describe for the first time dynamic trafficking of DENV nonstructural protein 5 (NS5) to the subnuclear compartment, the nucleolus. We demonstrate that NS5's targeting to the nucleolus occurs in response to acidic pH, identify the key amino acid residues within NS5 that are responsible, and demonstrate that their mutation severely impairs production of infectious DENV. Overall, this study identifies a unique subcellular trafficking event and suggests that DENV is able to detect and respond

West Nile Virus Temperature Sensitivity and Avian Virulence Are Modulated by NS1-2B Polymorphisms.

PubMed

Dietrich, Elizabeth A; Langevin, Stanley A; Huang, Claire Y-H; Maharaj, Payal D; Delorey, Mark J; Bowen, Richard A; Kinney, Richard M; Brault, Aaron C

2016-08-01

West Nile virus (WNV) replicates in a wide variety of avian species, which serve as reservoir and amplification hosts. WNV strains isolated in North America, such as the prototype strain NY99, elicit a highly pathogenic response in certain avian species, notably American crows (AMCRs; Corvus brachyrhynchos). In contrast, a closely related strain, KN3829, isolated in Kenya, exhibits a low viremic response with limited mortality in AMCRs. Previous work has associated the difference in pathogenicity primarily with a single amino acid mutation at position 249 in the helicase domain of the NS3 protein. The NY99 strain encodes a proline residue at this position, while KN3829 encodes a threonine. Introduction of an NS3-T249P mutation in the KN3829 genetic background significantly increased virulence and mortality; however, peak viremia and mortality were lower than those of NY99. In order to elucidate the viral genetic basis for phenotype variations exclusive of the NS3-249 polymorphism, chimeric NY99/KN3829 viruses were created. We show herein that differences in the NS1-2B region contribute to avian pathogenicity in a manner that is independent of and additive with the NS3-249 mutation. Additionally, NS1-2B residues were found to alter temperature sensitivity when grown in avian cells.

Replicative Functions of Minute Virus of Mice NS1 Protein Are Regulated In Vitro by Phosphorylation through Protein Kinase C

PubMed Central

Nüesch, Jürg P. F.; Dettwiler, Sabine; Corbau, Romuald; Rommelaere, Jean

1998-01-01

NS1, the major nonstructural protein of the parvovirus minute virus of mice, is a multifunctional phosphoprotein which is involved in cytotoxicity, transcriptional regulation, and initiation of viral DNA replication. For coordination of these various functions during virus propagation, NS1 has been proposed to be regulated by posttranslational modifications, in particular phosphorylation. Recent in vitro studies (J. P. F. Nüesch, R. Corbau, P. Tattersall, and J. Rommelaere, J. Virol. 72:8002–8012, 1998) provided evidence that distinct NS1 activities, notably the intrinsic helicase function, are modulated by the phosphorylation state of the protein. In order to study the dependence of the initiation of viral DNA replication on NS1 phosphorylation and to identify the protein kinases involved, we established an in vitro replication system that is devoid of endogenous protein kinases and is based on plasmid substrates containing the minimal left-end origins of replication. Cellular components necessary to drive NS1-dependent rolling-circle replication (RCR) were freed from endogenous serine/threonine protein kinases by affinity chromatography, and the eukaryotic DNA polymerases were replaced by the bacteriophage T4 DNA polymerase. While native NS1 (NS1P) supported RCR under these conditions, dephosphorylated NS1 (NS1O) was impaired. Using fractionated HeLa cell extracts, we identified two essential protein components which are able to phosphorylate NS1O, are enriched in protein kinase C (PKC), and, when present together, reactivate NS1O for replication. One of these components, containing atypical PKC, was sufficient to restore NS1O helicase activity. The requirement of NS1O reactivation for characteristic PKC cofactors such as Ca2+/phosphatidylserine or phorbol esters strongly suggests the involvement of this protein kinase family in regulation of NS1 replicative functions in vitro. PMID:9811734

Influenza A H3N2 subtype virus NS1 protein targets into the nucleus and binds primarily via its C-terminal NLS2/NoLS to nucleolin and fibrillarin

PubMed Central

2012-01-01

Background Influenza A virus non-structural protein 1 (NS1) is a virulence factor, which is targeted into the cell cytoplasm, nucleus and nucleolus. NS1 is a multi-functional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. Previously, we have shown that the NS1 protein of the H3N2 subtype influenza viruses possesses a C-terminal nuclear localization signal (NLS) that also functions as a nucleolar localization signal (NoLS) and targets the protein into the nucleolus. Results Here, we show that the NS1 protein of the human H3N2 virus subtype interacts in vitro primarily via its C-terminal NLS2/NoLS and to a minor extent via its N-terminal NLS1 with the nucleolar proteins, nucleolin and fibrillarin. Using chimeric green fluorescence protein (GFP)-NS1 fusion constructs, we show that the nucleolar retention of the NS1 protein is determined by its C-terminal NLS2/NoLS in vivo. Confocal laser microscopy analysis shows that the NS1 protein colocalizes with nucleolin in nucleoplasm and nucleolus and with B23 and fibrillarin in the nucleolus of influenza A/Udorn/72 virus-infected A549 cells. Since some viral proteins contain NoLSs, it is likely that viruses have evolved specific nucleolar functions. Conclusion NS1 protein of the human H3N2 virus interacts primarily via the C-terminal NLS2/NoLS and to a minor extent via the N-terminal NLS1 with the main nucleolar proteins, nucleolin, B23 and fibrillarin. PMID:22909121

Discovery and SAR studies of methionine-proline anilides as dengue virus NS2B-NS3 protease inhibitors.

PubMed

Zhou, Guo-Chun; Weng, Zhibing; Shao, Xiaoxia; Liu, Fang; Nie, Xin; Liu, Jinsong; Wang, Decai; Wang, Chunguang; Guo, Kai

2013-12-15

A series of methionine-proline dipeptide derivatives and their analogues were designed, synthesized and assayed against the serotype 2 dengue virus NS2B-NS3 protease, and methionine-proline anilides 1 and 2 were found to be the most active DENV 2 NS2B-NS3 competitive inhibitors with Ki values of 4.9 and 10.5 μM. The structure and activity relationship and the molecular docking revealed that L-proline, L-methionine and p-nitroaniline in 1 and 2 are the important characters in blocking the active site of NS2B-NS3 protease. Our current results suggest that the title dipeptidic scaffold represents a promising structural core to discover a new class of active NS2B-NS3 competitive inhibitors. Copyright © 2013 Elsevier Ltd. All rights reserved.

An In-Silico Investigation of Phytochemicals as Antiviral Agents Against Dengue Fever

PubMed Central

Powers, Chelsea N.; Setzer, William N.

2016-01-01

Abstract: A virtual screening analysis of our library of phytochemical structures with dengue virus protein targets has been carried out using a molecular docking approach. A total of 2194 plant-derived secondary metabolites have been docked. This molecule set comprised of 290 alkaloids (68 indole alkaloids, 153 isoquinoline alkaloids, 5 quinoline alkaloids, 13 piperidine alkaloids, 14 steroidal alkaloids, and 37 miscellaneous alkaloids), 678 terpenoids (47 monoterpenoids, 169 sesquiterpenoids, 265 diterpenoids, 81 steroids, and 96 triterpenoids), 20 aurones, 81 chalcones, 349 flavonoids, 120 isoflavonoids, 74 lignans, 58 stilbenoids, 169 miscellaneous polyphenolic compounds, 100 coumarins, 28 xanthones, 67 quinones, and 160 miscellaneous phytochemicals. Dengue virus protein targets examined included dengue virus protease (NS2B-NS3pro), helicase (NS3 helicase), methyltransferase (MTase), RNA-dependent RNA polymerase (RdRp), and the dengue virus envelope protein. Polyphenolic compounds, flavonoids, chalcones, and other phenolics were the most numerous of the strongly docking ligands for dengue virus protein targets. PMID:27151482

An In-Silico Investigation of Phytochemicals as Antiviral Agents Against Dengue Fever.

PubMed

Powers, Chelsea N; Setzer, William N

2016-01-01

A virtual screening analysis of our library of phytochemical structures with dengue virus protein targets has been carried out using a molecular docking approach. A total of 2194 plant-derived secondary metabolites have been docked. This molecule set comprised of 290 alkaloids (68 indole alkaloids, 153 isoquinoline alkaloids, 5 quinoline alkaloids, 13 piperidine alkaloids, 14 steroidal alkaloids, and 37 miscellaneous alkaloids), 678 terpenoids (47 monoterpenoids, 169 sesquiterpenoids, 265 diterpenoids, 81 steroids, and 96 triterpenoids), 20 aurones, 81 chalcones, 349 flavonoids, 120 isoflavonoids, 74 lignans, 58 stilbenoids, 169 miscellaneous polyphenolic compounds, 100 coumarins, 28 xanthones, 67 quinones, and 160 miscellaneous phytochemicals. Dengue virus protein targets examined included dengue virus protease (NS2B-NS3pro), helicase (NS3 helicase), methyltransferase (MTase), RNA-dependent RNA polymerase (RdRp), and the dengue virus envelope protein. Polyphenolic compounds, flavonoids, chalcones, and other phenolics were the most numerous of the strongly docking ligands for dengue virus protein targets.

The Changing Face of Hepatitis C: Recent Advances on HCV Inhibitors Targeting NS5A

PubMed

Rai, Diwakar; Wang, Liu; Jiang, Xuemei; Zhan, Peng; Jia, Haiyong; De Clercq, Erik; Liu, Xinyong

2015-05-05

Current treatment for HCV infections consists of approved direct acting antivirals (DAAs), viz. the protease inhibitors (boceprevir, telaprevir, and simeprevir), NS5B polymerase inhibitors (sofosbuvir) and NS5A inhibitor (ledipasvir) in combination with pegylated interferon α and ribavirin). These treatments have made a great improvement in the treatment of chronic HCV infections in recent years, but their adverse side effects, emergence of resistant mutants, high cost, and increased pill burden have limited their clinical use. Recently, with the increasing knowledge in understanding the HCV life cycle, more targets have been recognized. NS5A protein plays a critical role in assembly of infectious HCV particles and offering potential for HCV therapies. Therefore, discovery and development of novel DAAs targeting NS5A with novel mechanisms of action, is of great necessity to improve the quality of existing HCV treatments. In the present review, we discuss recent advances with NS5A inhibitors with potent anti-HCV activity, and the potential for the development of HCV NS5A inhibitors to combat HCV infections.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the DDX3 RNA helicase domain

PubMed Central

Rodamilans, Bernardo; Montoya, Guillermo

2007-01-01

DDX3 is a human RNA helicase that is involved in RNA processing and important human diseases. This enzyme belongs to the DEAD-box protein family, the members of which are characterized by the presence of nine conserved motifs including the Asp-Glu-Ala-Asp motif that defines the family. DDX3 has two distinct domains: an ATP-binding domain in the central region of the protein and a helicase domain in the carboxy-terminal region. The helicase domain of DDX3 was cloned and overexpressed in Escherichia coli. Crystallization experiments yielded crystals that were suitable for X-ray diffraction analysis. The final crystallization conditions were a reservoir solution consisting of 2 M ammonium sulfate, 0.1 M imidazole pH 6.4 plus 5 mM spermine tetrahydrochloride and a protein solution containing 10 mM HEPES, 500 mM ammonium sulfate pH 8.0. The crystals of the helicase domain belong to the monoclinic space group P21, with unit-cell parameters a = 43.85, b = 60.72, c = 88.39 Å, α = γ = 90, β = 101.02°, and contained three molecules per asymmetric unit. These crystals diffracted to a resolution limit of 2.2 Å using synchrotron radiation at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS). PMID:17401195

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the DDX3 RNA helicase domain.

PubMed

Rodamilans, Bernardo; Montoya, Guillermo

2007-04-01

DDX3 is a human RNA helicase that is involved in RNA processing and important human diseases. This enzyme belongs to the DEAD-box protein family, the members of which are characterized by the presence of nine conserved motifs including the Asp-Glu-Ala-Asp motif that defines the family. DDX3 has two distinct domains: an ATP-binding domain in the central region of the protein and a helicase domain in the carboxy-terminal region. The helicase domain of DDX3 was cloned and overexpressed in Escherichia coli. Crystallization experiments yielded crystals that were suitable for X-ray diffraction analysis. The final crystallization conditions were a reservoir solution consisting of 2 M ammonium sulfate, 0.1 M imidazole pH 6.4 plus 5 mM spermine tetrahydrochloride and a protein solution containing 10 mM HEPES, 500 mM ammonium sulfate pH 8.0. The crystals of the helicase domain belong to the monoclinic space group P2(1), with unit-cell parameters a = 43.85, b = 60.72, c = 88.39 A, alpha = gamma = 90, beta = 101.02 degrees , and contained three molecules per asymmetric unit. These crystals diffracted to a resolution limit of 2.2 A using synchrotron radiation at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS).

Pharmacophoric characteristics of dengue virus NS2B/NS3pro inhibitors: a systematic review of the most promising compounds.

PubMed

Leonel, Camyla Alves; Lima, William Gustavo; Dos Santos, Michelli; Ferraz, Ariane Coelho; Taranto, Alex Gutterres; de Magalhães, José Carlos; Dos Santos, Luciana Lara; Ferreira, Jaqueline Maria Siqueira

2018-03-01

Dengue virus (DENV) infection can lead to a wide range of clinical manifestations, including fatal hemorrhagic complications. There is a need to find effective pharmacotherapies to treat this disease due to the lack of specific immunotherapies and antiviral drugs. That said, the DENV NS2B/NS3pro protease complex is essential in both the viral multiplication cycle and in disease pathogenesis, and is considered a promising target for new antiviral therapies. Here, we performed a systematic review to evaluate the pharmacophoric characteristics of promising compounds against NS2B/NS3pro reported in the past 10 years. Online searches in the PUBMED/MEDLINE and SCOPUS databases resulted in 165 articles. Eight studies, which evaluated 3,384,268 molecules exhibiting protease inhibition activity, were included in this review. These studies evaluated anti-dengue activity in vitro and the IC 50 and EC 50 values were provided. Most compounds exhibited non-competitive inhibition. Cytotoxicity was evaluated in BHK-21, Vero, and LLC-MK2 cells, and the CC 50 values obtained ranged from < 1.0 to 780.5 µM. Several groups were associated with biological activity against dengue, including nitro, catechol, halogen and ammonium quaternaries. Thus, these groups seem to be potential pharmacophores that can be further investigated to treat dengue infections.

Quantitative Proteomic Analysis of the Influenza A Virus Nonstructural Proteins NS1 and NS2 during Natural Cell Infection Identifies PACT as an NS1 Target Protein and Antiviral Host Factor

PubMed Central

Tawaratsumida, Kazuki; Phan, Van; Hrincius, Eike R.; High, Anthony A.; Webby, Richard; Redecke, Vanessa

2014-01-01

ABSTRACT Influenza A virus (IAV) replication depends on the interaction of virus proteins with host factors. The viral nonstructural protein 1 (NS1) is essential in this process by targeting diverse cellular functions, including mRNA splicing and translation, cell survival, and immune defense, in particular the type I interferon (IFN-I) response. In order to identify host proteins targeted by NS1, we established a replication-competent recombinant IAV that expresses epitope-tagged forms of NS1 and NS2, which are encoded by the same gene segment, allowing purification of NS proteins during natural cell infection and analysis of interacting proteins by quantitative mass spectrometry. We identified known NS1- and NS2-interacting proteins but also uncharacterized proteins, including PACT, an important cofactor for the IFN-I response triggered by the viral RNA-sensor RIG-I. We show here that NS1 binds PACT during virus replication and blocks PACT/RIG-I-mediated activation of IFN-I, which represents a critical event for the host defense. Protein interaction and interference with IFN-I activation depended on the functional integrity of the highly conserved RNA binding domain of NS1. A mutant virus with deletion of NS1 induced high levels of IFN-I in control cells, as expected; in contrast, shRNA-mediated knockdown of PACT compromised IFN-I activation by the mutant virus, but not wild-type virus, a finding consistent with the interpretation that PACT (i) is essential for IAV recognition and (ii) is functionally compromised by NS1. Together, our data describe a novel approach to identify virus-host protein interactions and demonstrate that NS1 interferes with PACT, whose function is critical for robust IFN-I production. IMPORTANCE Influenza A virus (IAV) is an important human pathogen that is responsible for annual epidemics and occasional devastating pandemics. Viral replication and pathogenicity depends on the interference of viral factors with components of the host

Naringenin and quercetin--potential anti-HCV agents for NS2 protease targets.

PubMed

Lulu, S Sajitha; Thabitha, A; Vino, S; Priya, A Mohana; Rout, Madhusmita

2016-01-01

Nonstructural proteins of hepatitis C virus had drawn much attention for the scientific fraternity in drug discovery due to its important role in the disease. 3D structure of the protein was predicted using molecular modelling protocol. Docking studies of 10 medicinal plant compounds and three drugs available in the market (control) with NS2 protease were employed by using rigid docking approach of AutoDock 4.2. Among the molecules tested for docking study, naringenin and quercetin revealed minimum binding energy of - 7.97 and - 7.95 kcal/mol with NS2 protease. All the ligands were docked deeply within the binding pocket region of the protein. The docking study results showed that these compounds are potential inhibitors of the target; and also all these docked compounds have good inhibition constant, vdW+Hbond+desolv energy with best RMSD value.

Structural models for the design of novel antiviral agents against Greek Goat Encephalitis

PubMed Central

Papageorgiou, Louis; Loukatou, Styliani; Koumandou, Vassiliki Lila; Makałowski, Wojciech; Megalooikonomou, Vasileios

2014-01-01

The Greek Goat Encephalitis virus (GGE) belongs to the Flaviviridae family of the genus Flavivirus. The GGE virus constitutes an important pathogen of livestock that infects the goat’s central nervous system. The viral enzymes of GGE, helicase and RNA-dependent RNA polymerase (RdRP), are ideal targets for inhibitor design, since those enzymes are crucial for the virus’ survival, proliferation and transmission. In an effort to understand the molecular structure underlying the functions of those viral enzymes, the three dimensional structures of GGE NS3 helicase and NS5 RdRP have been modelled. The models were constructed in silico using conventional homology modelling techniques and the known 3D crystal structures of solved proteins from closely related species as templates. The established structural models of the GGE NS3 helicase and NS5 RdRP have been evaluated for their viability using a repertoire of in silico tools. The goal of this study is to present the 3D conformations of the GGE viral enzymes as reliable structural models that could provide the platform for the design of novel anti-GGE agents. PMID:25392762

Highly potent non-peptidic inhibitors of the HCV NS3/NS4A serine protease.

PubMed

Sperandio, David; Gangloff, Anthony R; Litvak, Joane; Goldsmith, Richard; Hataye, Jason M; Wang, Vivian R; Shelton, Emma J; Elrod, Kyle; Janc, James W; Clark, James M; Rice, Ken; Weinheimer, Steve; Yeung, Kap-Sun; Meanwell, Nicholas A; Hernandez, Dennis; Staab, Andrew J; Venables, Brian L; Spencer, Jeffrey R

2002-11-04

Screening of a diverse set of bisbenzimidazoles for inhibition of the hepatitis C virus (HCV) serine protease NS3/NS4A led to the identification of a potent Zn(2+)-dependent inhibitor (1). Optimization of this screening hit afforded a 10-fold more potent inhibitor (46) under Zn(2+) conditions (K(i)=27nM). This compound (46) binds also to NS3/NS4A in a Zn(2+) independent fashion (K(i)=1microM). The SAR of this class of compounds under Zn(2+) conditions is highly divergent compared to the SAR in the absence of Zn(2+), suggesting two distinct binding modes.

3D-QSAR pharmacophore-based virtual screening, molecular docking and molecular dynamics simulation toward identifying lead compounds for NS2B-NS3 protease inhibitors.

PubMed

Luo, Pei H; Zhang, Xuan R; Huang, Lan; Yuan, Lun; Zhou, Xang Z; Gao, X; Li, Ling S

2017-10-01

NS2B-NS3 protease has been identified to serve as lead drug design target due to its significant role in West Nile viral (WNV) and dengue virus (DENV) reproduction and replication. There are currently no approved chemotherapeutic drugs and effective vaccines to inhibit DENV and WNV infections. In this work, 3D-QSAR pharmacophore model has been developed to discover potential inhibitory candidates. Validation through Fischer's model and decoy test indicate that the developed 3D pharmacophore model is highly predictive for DENV inhibitors, which was then employed to screen ZINC chemical library to obtain reasonable hits. Following ADMET filtering, 15 hits were subjected to further filter through molecular docking and CoMFA modeling. Finally, top three hits were identified as lead compounds or potential inhibitory candidates with IC 50 values of ∼0.4637 µM and fitness of ∼57.73. It is implied from CoMFA modeling that substituents at the side site of benzotriazole such as a p-nitro group (e.g. biphenyl head) and a carbonyl (e.g. carboxylate function) at the side site of furan or amino group may improve bioactivity of ZINC85645245, respectively. Molecular dynamics simulations (MDS) were performed to discover new interactions and reinforce the binding modes from docking for the hits also. The QSAR and MDS results obtained from this work should be useful in determining structural requirements for inhibitor development as well as in designing more potential inhibitors for NS2B-NS3 protease.

West Nile Virus NS1 Antagonizes Interferon Beta Production by Targeting RIG-I and MDA5.

PubMed

Zhang, Hong-Lei; Ye, Han-Qing; Liu, Si-Qing; Deng, Cheng-Lin; Li, Xiao-Dan; Shi, Pei-Yong; Zhang, Bo

2017-09-15

West Nile virus (WNV) is a mosquito-borne flavivirus that causes epidemics of encephalitis and viscerotropic disease worldwide. This virus has spread rapidly and has posed a significant public health threat since the outbreak in New York City in 1999. The interferon (IFN)-mediated antiviral response represents an important component of virus-host interactions and plays an essential role in regulating viral replication. Previous studies have suggested that multifunctional nonstructural proteins encoded by flaviviruses antagonize the host IFN response via various means in order to establish efficient viral replication. In this study, we demonstrated that the nonstructural protein 1 (NS1) of WNV antagonizes IFN-β production, most likely through suppression of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) activation. In a dual-luciferase reporter assay, WNV NS1 significantly inhibited the activation of the IFN-β promoter after Sendai virus infection or poly(I·C) treatment. NS1 also suppressed the activation of the IFN-β promoter when it was stimulated by interferon regulatory factor 3 (IRF3)/5D or its upstream molecules in the RLR signaling pathway. Furthermore, NS1 blocked the phosphorylation and nuclear translocation of IRF3 upon stimulation by various inducers. Mechanistically, WNV NS1 targets RIG-I and melanoma differentiation-associated gene 5 (MDA5) by interacting with them and subsequently causing their degradation by the proteasome. Furthermore, WNV NS1 inhibits the K63-linked polyubiquitination of RIG-I, thereby inhibiting the activation of downstream sensors in the RLR signaling pathway. Taken together, our results reveal a novel mechanism by which WNV NS1 interferes with the host antiviral response. IMPORTANCE WNV Nile virus (WNV) has received increased attention since its introduction to the United States. However, the pathogenesis of this virus is poorly understood. This study demonstrated that the nonstructural protein 1 (NS1) of WNV

Helicases as molecular motors: An insight

NASA Astrophysics Data System (ADS)

Tuteja, Narendra; Tuteja, Renu

2006-12-01

Helicases are one of the smallest motors of biological system, which harness the chemical free energy of ATP hydrolysis to catalyze the opening of energetically stable duplex nucleic acids and thereby are involved in almost all aspect of nucleic acid metabolism including replication, repair, recombination, transcription, translation, and ribosome biogenesis. Basically, they break the hydrogen bonding between the duplex helix and translocate unidirectionally along the bound strand. Mostly all the helicases contain some conserved signature motifs, which act as an engine to power the unwinding. After the discovery of the first prokaryotic DNA helicase from Escherichia coli bacteria in 1976 and the first eukaryotic one from the lily plant in 1978, many more (>100) have been isolated. All the helicases share some common properties, including nucleic acid binding, NTP hydrolysis and unwinding of the duplex. Many helicases have been crystallized and their structures have revealed an underlying common structural fold for their function. The defects in helicases gene have also been reported to be responsible for variety of human genetic disorders, which can lead to cancer, premature aging or mental retardation. Recently, a new role of a helicase in abiotic stress signaling in plant has been discovered. Overall, helicases act as essential molecular tools for cellular machinery and help in maintaining the integrity of genome. Here an overview of helicases has been covered which includes history, biochemical assay, properties, classification, role in human disease and mechanism of unwinding and translocation.

Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication.

PubMed

Bozzacco, Leonia; Yi, Zhigang; Andreo, Ursula; Conklin, Claire R; Li, Melody M H; Rice, Charles M; MacDonald, Margaret R

2016-01-06

DNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesized that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that did not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected the NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3-to-NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of the NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication, and only revertant viruses with a restored wild-type arginine or lysine residue at the NS3/4A site were obtained. We conclude that DNAJC14 inhibition of RC formation upon DNAJC14 overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication. Flaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on endoplasmic reticulum (ER) membranes to produce a single polyprotein, which is

Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication

PubMed Central

Bozzacco, Leonia; Yi, Zhigang; Andreo, Ursula; Conklin, Claire R.; Li, Melody M. H.; Rice, Charles M.

2016-01-01

ABSTRACT DNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesized that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that did not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected the NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3-to-NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of the NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication, and only revertant viruses with a restored wild-type arginine or lysine residue at the NS3/4A site were obtained. We conclude that DNAJC14 inhibition of RC formation upon DNAJC14 overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication. IMPORTANCE Flaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on endoplasmic reticulum (ER) membranes to produce a single

Progress on New Hepatitis C Virus Targets: NS2 and NS5A

NASA Astrophysics Data System (ADS)

Marcotrigiano, Joseph

Hepatitis C virus (HCV) is a major global health problem, affecting about 170 million people worldwide. Chronic infection can lead to cirrhosis and liver cancer. The replication machine of HCV is a multi-subunit membrane associated complex, consisting of nonstructural proteins (NS2-5B), which replicate the viral RNA genome. The structures of NS5A and NS2 were recently determined. NS5A is an essential replicase component that also modulates numerous cellular processes ranging from innate immunity to cell growth and survival. The structure reveals a novel protein fold, a new zinc coordination motif, a disulfide bond and a dimer interface. Analysis of molecular surfaces suggests the location of the membrane interaction surface of NS5A, as well as hypothetical protein and RNA binding sites. NS2 is one of two virally encoded proteases that are required for processing the viral polyprotein into the mature nonstructural proteins. NS2 is a dimeric cysteine protease with two composite active sites. For each active site, the catalytic histidine and glutamate residues are contributed by one monomer and the nucleophilic cysteine by the other. The C-terminal residues remain coordinated in the two active sites, predicting an inactive post-cleavage form. The structure also reveals possible sites of membrane interaction, a rare cis-proline residue, and highly conserved dimer contacts. The novel features of both structures have changed the current view of HCV polyprotein replication and present new opportunities for antiviral drug design.

Characterisation of divergent flavivirus NS3 and NS5 protein sequences detected in Rhipicephalus microplus ticks from Brazil

PubMed Central

Maruyama, Sandra Regina; Castro-Jorge, Luiza Antunes; Ribeiro, José Marcos Chaves; Gardinassi, Luiz Gustavo; Garcia, Gustavo Rocha; Brandão, Lucinda Giampietro; Rodrigues, Aline Rezende; Okada, Marcos Ituo; Abrão, Emiliana Pereira; Ferreira, Beatriz Rossetti; da Fonseca, Benedito Antonio Lopes; de Miranda-Santos, Isabel Kinney Ferreira

2013-01-01

Transcripts similar to those that encode the nonstructural (NS) proteins NS3 and NS5 from flaviviruses were found in a salivary gland (SG) complementary DNA (cDNA) library from the cattle tick Rhipicephalus microplus. Tick extracts were cultured with cells to enable the isolation of viruses capable of replicating in cultured invertebrate and vertebrate cells. Deep sequencing of the viral RNA isolated from culture supernatants provided the complete coding sequences for the NS3 and NS5 proteins and their molecular characterisation confirmed similarity with the NS3 and NS5 sequences from other flaviviruses. Despite this similarity, phylogenetic analyses revealed that this potentially novel virus may be a highly divergent member of the genus Flavivirus. Interestingly, we detected the divergent NS3 and NS5 sequences in ticks collected from several dairy farms widely distributed throughout three regions of Brazil. This is the first report of flavivirus-like transcripts in R. microplus ticks. This novel virus is a potential arbovirus because it replicated in arthropod and mammalian cells; furthermore, it was detected in a cDNA library from tick SGs and therefore may be present in tick saliva. It is important to determine whether and by what means this potential virus is transmissible and to monitor the virus as a potential emerging tick-borne zoonotic pathogen. PMID:24626302

Virtual ligand screening of the National Cancer Institute (NCI) compound library leads to the allosteric inhibitory scaffolds of the West Nile Virus NS3 proteinase.

PubMed

Shiryaev, Sergey A; Cheltsov, Anton V; Gawlik, Katarzyna; Ratnikov, Boris I; Strongin, Alex Y

2011-02-01

Viruses of the genus Flavivirus are responsible for significant human disease and mortality. The N-terminal domain of the flaviviral nonstructural (NS)3 protein codes for the serine, chymotrypsin-fold proteinase (NS3pro). The presence of the nonstructural (NS)2B cofactor, which is encoded by the upstream gene in the flaviviral genome, is necessary for NS3pro to exhibit its proteolytic activity. The two-component NS2B-NS3pro functional activity is essential for the viral polyprotein processing and replication. Both the structure and the function of NS2B-NS3pro are conserved in the Flavivirus family. Because of its essential function in the posttranslational processing of the viral polyprotein precursor, NS2B-NS3pro is a promising target for anti-flavivirus drugs. To identify selective inhibitors with the reduced cross-reactivity and off-target effects, we focused our strategy on the allosteric inhibitors capable of targeting the NS2B-NS3pro interface rather than the NS3pro active site. Using virtual ligand screening of the diverse, ∼275,000-compound library and the catalytic domain of the two-component West Nile virus (WNV) NS2B-NS3pro as a receptor, we identified a limited subset of the novel inhibitory scaffolds. Several of the discovered compounds performed as allosteric inhibitors and exhibited a nanomolar range potency in the in vitro cleavage assays. The inhibitors were also potent in cell-based assays employing the sub-genomic, luciferase-tagged WNV and Dengue viral replicons. The selectivity of the inhibitors was confirmed using the in vitro cleavage assays with furin, a human serine proteinase, the substrate preferences of which are similar to those of WNV NS2B-NS3pro. Conceptually, the similar in silico drug discovery strategy may be readily employed for the identification of inhibitors of other flaviviruses.

Characterization and evaluation of apoptotic potential of double gene construct pVIVO.VP3.NS1.

PubMed

Saxena, Shikha; Desai, G S; Kumar, G Ravi; Sahoo, A P; Santra, Lakshman; Singh, Lakshya Veer

2015-05-01

Viral gene oncotherapy, targeted killing of cancer cells by viral genes, is an emerging non-infectious therapeutic cancer treatment modality. Chemo and radiotherapy in cancer treatment is limited due to their genotoxic side effects on healthy cells and need of functional p53, which is mutated in most of the cancers. VP3 (apoptin) of chicken infectious anaemia (CIA) and NS1 (Non structural protein 1) of Canine Parvovirus-2 (CPV-2) have been proven to have oncolytic potential in our laboratory. To evaluate oncolytic potential of VP3 and NS1 together these genes needed to be cloned in a bicistronic vector. In this study, both these genes were cloned and characterized for expression of their gene products and its apoptotic potential. The expression of VP3 and NS1 was studied by confocal microscopy and flowcytometry. Expression of VP3 and NS1 in pVIVO.VP3.NS1 transfected HeLa cells in comparison to mock transfected cells indicated that the double gene construct expresses both the products. This was further confirmed by flowcytometry where there was increase in cells expressing VP3 and NS1 in pVIVO.VP3.NS1 transfected group in comparison with the mock control group. The apoptotic inducing potential of this characterized pVIVO.VP3.NS1 was evaluated in human cervical cancer cell line (HeLa) by DNA fragmentation assay, TUNEL assay and Hoechst staning. This double construct was observed to induce apoptosis in HeLa cells.

Prokaryotic and eukaryotic DNA helicases. Essential molecular motor proteins for cellular machinery.

PubMed

Tuteja, Narendra; Tuteja, Renu

2004-05-01

DNA helicases are ubiquitous molecular motor proteins which harness the chemical free energy of ATP hydrolysis to catalyze the unwinding of energetically stable duplex DNA, and thus play important roles in nearly all aspects of nucleic acid metabolism, including replication, repair, recombination, and transcription. They break the hydrogen bonds between the duplex helix and move unidirectionally along the bound strand. All helicases are also translocases and DNA-dependent ATPases. Most contain conserved helicase motifs that act as an engine to power DNA unwinding. All DNA helicases share some common properties, including nucleic acid binding, NTP binding and hydrolysis, and unwinding of duplex DNA in the 3' to 5' or 5' to 3' direction. The minichromosome maintenance (Mcm) protein complex (Mcm4/6/7) provides a DNA-unwinding function at the origin of replication in all eukaryotes and may act as a licensing factor for DNA replication. The RecQ family of helicases is highly conserved from bacteria to humans and is required for the maintenance of genome integrity. They have also been implicated in a variety of human genetic disorders. Since the discovery of the first DNA helicase in Escherichia coli in 1976, and the first eukaryotic one in the lily in 1978, a large number of these enzymes have been isolated from both prokaryotic and eukaryotic systems, and the number is still growing. In this review we cover the historical background of DNA helicases, helicase assays, biochemical properties, prokaryotic and eukaryotic DNA helicases including Mcm proteins and the RecQ family of helicases. The properties of most of the known DNA helicases from prokaryotic and eukaryotic systems, including viruses and bacteriophages, are summarized in tables.

Structural view of the helicase reveals that Zika virus uses a conserved mechanism for unwinding RNA.

PubMed

Li, Lei; Wang, Jin; Jia, Zhihui; Shaw, Neil

2018-04-01

Recent studies suggest a link between infection by Zika virus (ZIKV) and the development of neurological complications. The lack of ZIKV-specific therapeutics has alarmed healthcare professionals worldwide. Here, crystal structures of apo and AMPPNP- and Mn 2+ -bound forms of the essential helicase of ZIKV refined to 1.78 and 1.3 Å resolution, respectively, are reported. The structures reveal a conserved trimodular topology of the helicase. ATP and Mn 2+ are tethered between two RecA-like domains by conserved hydrogen-bonding interactions. The binding of ligands induces the movement of backbone Cα and side-chain atoms. Numerous solvent molecules are observed in the vicinity of the AMPPNP, suggesting a role in catalysis. These high-resolution structures could be useful for the design of inhibitors targeting the helicase of ZIKV for the treatment of infections caused by ZIKV.

Purification and crystallization of dengue and West Nile virus NS2B-NS3 complexes.

PubMed

D'Arcy, Allan; Chaillet, Maxime; Schiering, Nikolaus; Villard, Frederic; Lim, Siew Pheng; Lefeuvre, Peggy; Erbel, Paul

2006-02-01

Both dengue and West Nile virus infections are an increasing risk to humans, not only in tropical and subtropical areas, but also in North America and parts of Europe. These viral infections are generally transmitted by mosquitoes, but may also be tick-borne. Infection usually results in mild flu-like symptoms, but can also cause encephalitis and fatalities. Approximately 2799 severe West Nile virus cases were reported this year in the United States, resulting in 102 fatalities. With this alarming increase in the number of West Nile virus infections in western countries and the fact that dengue virus already affects millions of people per year in tropical and subtropical climates, there is a real need for effective medicines. A possible therapeutic target to combat these viruses is the protease, which is essential for virus replication. In order to provide structural information to help to guide a lead identification and optimization program, crystallizations of the NS2B-NS3 protease complexes from both dengue and West Nile viruses have been initiated. Crystals that diffract to high resolution, suitable for three-dimensional structure determinations, have been obtained.

Identification of potential hit compounds for Dengue virus NS2B/NS3 protease inhibitors by combining virtual screening and binding free energy calculations.

PubMed

Wichapong, K; Nueangaudom, A; Pianwanit, S; Sippl, W; Kokpol, S

2013-09-01

Dengue virus (DV) infections are a serious public health problem and there is currently no vaccine or drug treatment. NS2B/NS3 protease, an essential enzyme for viral replication, is one of the promising targets in the search for drugs against DV. In this research work, virtual screening (VS) was carried out on four multi-conformational databases using several criteria. Firstly, molecular dynamics simulations of the NS2B/NS3 protease and four known inhibitors, which reveal an importance of both electrostatic and van der Waals interactions in stabilizing the ligand-enzyme interaction, were used to generate three different pharmacophore models (a structure-based, a static and a dynamic). Subsequently, these three models were employed for pharmacophore search in the VS. Secondly, compounds passing the first criterion were further reduced using the Lipinski's rule of five to keep only compounds with drug-like properties. Thirdly, molecular docking calculations were performed to remove compounds with unsuitable ligand-enzyme interactions. Finally, binding free energy of each compound was calculated. Compounds having better energy than the known inhibitors were selected and thus 20 potential hits were obtained.

Computational biology approach to uncover hepatitis C virus helicase operation.

PubMed

Flechsig, Holger

2014-04-07

Hepatitis C virus (HCV) helicase is a molecular motor that splits nucleic acid duplex structures during viral replication, therefore representing a promising target for antiviral treatment. Hence, a detailed understanding of the mechanism by which it operates would facilitate the development of efficient drug-assisted therapies aiming to inhibit helicase activity. Despite extensive investigations performed in the past, a thorough understanding of the activity of this important protein was lacking since the underlying internal conformational motions could not be resolved. Here we review investigations that have been previously performed by us for HCV helicase. Using methods of structure-based computational modelling it became possible to follow entire operation cycles of this motor protein in structurally resolved simulations and uncover the mechanism by which it moves along the nucleic acid and accomplishes strand separation. We also discuss observations from that study in the light of recent experimental studies that confirm our findings.

Altered RECQL5 expression in urothelial bladder carcinoma increases cellular proliferation and makes RECQL5 helicase activity a novel target for chemotherapy

PubMed Central

Patterson, Karl; Arya, Lovleen; Bottomley, Sarah; Morgan, Susan; Cox, Angela; Catto, James; Bryant, Helen E.

2016-01-01

RECQ helicases are a family of enzymes with both over lapping and unique functions. Functional autosomal recessive loss of three members of the family BLM, WRN and RECQL4, results in hereditary human syndromes characterized by cancer predisposition and premature aging, but despite the finding that RECQL5 deficient mice are cancer prone, no such link has been made to human RECQL5. Here we demonstrate that human urothelial carcinoma of the bladder (UCC) has increased expression of RECQL5 compared to normal bladder tissue and that increasing RECQL5 expression can drive proliferation of normal bladder cells and is associated with poor prognosis. Further, by expressing a helicase dead RECQL5 and by depleting bladder cancer cells of RECQL5 we show that inhibition of RECQL5 activity has potential as a new target for treatment of UCC. PMID:27764811

Regulation of MVM NS1 by protein kinase C: impact of mutagenesis at consensus phosphorylation sites on replicative functions and cytopathic effects.

PubMed

Corbau, R; Duverger, V; Rommelaere, J; Nüesch, J P

2000-12-05

Minute virus of mice NS1, an 83-kDa mainly nuclear phosphoprotein, is the only viral nonstructural protein required in all cell types and it is involved in multiple processes necessary for virus propagation. The diversity of functions assigned to NS1, together with the variation of its complex phosphorylation pattern during infection, suggested that the various activities of NS1 could be regulated by distinct phosphorylation events. So far, it has been demonstrated that NS1 replicative functions, in particular, DNA-unwinding activities, are regulated by protein kinase C (PKC), as exemplified by the modulation of NS1 helicase activity by PKClambda phosphorylation. In order to determine further impact of phosphorylation on NS1 functions, including the induction of cytopathic effects, a mutational approach was pursued in order to produce NS1 variants harboring amino acid substitutions at candidate PKC target residues. Besides the determination of two additional in vivo phosphorylation sites in NS1, this mutagenesis allowed the segregation of distinct NS1 functions from one another, generating NS1 variants with a distinct activity profile. Thus, we obtained NS1 mutants that were fully proficient for trans activation of the viral P38 promoter, while being impaired in their replicative functions. Moreover, the alterations of specific PKC phosphorylation sites gave rise to NS1 polypeptides that exerted reduced cytotoxicity, leading to sustained gene expression, while keeping functions necessary for progeny virus production, i.e., viral DNA replication and activation of the capsid gene promoter. These data suggested that in the course of a viral infection, NS1 may undergo a shift from productive to cytotoxic functions as a result of a phosphorylation-dependent regulation. Copyright 2000 Academic Press.

XPD Helicase: Shifting the Inchworm into Reverse

ERIC Educational Resources Information Center

Pugh, Robert A.

2009-01-01

Directional translocation by helicases results in duplex separation and displacement of bound proteins which allows for the DNA processing events associated with DNA repair, replication, recombination, and transcription. Unresolved questions regarding DNA helicases include: (1) how is directional translocation determined in SF2 helicases; (2) do…

Baseline Polymorphisms and Emergence of Drug Resistance in the NS3/4A Protease of Hepatitis C Virus Genotype 1 following Treatment with Faldaprevir and Pegylated Interferon Alpha 2a/Ribavirin in Phase 2 and Phase 3 Studies.

PubMed

Berger, K L; Scherer, J; Ranga, M; Sha, N; Stern, J O; Quinson, A-M; Kukolj, G

2015-10-01

Analysis of data pooled from multiple phase 2 (SILEN-C1 to 3) and phase 3 studies (STARTVerso1 to 4) of the hepatitis C virus (HCV) nonstructural protein 3/4A (NS3/4A) protease inhibitor faldaprevir plus pegylated interferon alpha/ribavirin (PR) provides a comprehensive evaluation of baseline and treatment-emergent NS3/4A amino acid variants among HCV genotype-1 (GT-1)-infected patients. Pooled analyses of GT-1a and GT-1b NS3 population-based pretreatment sequences (n = 3,124) showed that faldaprevir resistance-associated variants (RAVs) at NS3 R155 and D168 were rare (<1%). No single, noncanonical NS3 protease or NS4A cofactor baseline polymorphism was associated with a reduced sustained virologic response (SVR) to faldaprevir plus PR, including Q80K. The GT-1b NS3 helicase polymorphism T344I was associated with reduced SVR to faldaprevir plus PR (P < 0.0001) but was not faldaprevir specific, as reduced SVR was also observed with placebo plus PR. Among patients who did not achieve SVR and had available NS3 population sequences (n = 507 GT-1a; n = 349 GT-1b), 94% of GT-1a and 83% of GT-1b encoded faldaprevir treatment-emergent RAVs. The predominant GT-1a RAV was R155K (88%), whereas GT-1b encoded D168 substitutions (78%) in which D168V was predominant (67%). The novel GT-1b NS3 S61L substitution emerged in 7% of virologic failures as a covariant with D168V, most often among the faldaprevir breakthroughs; S61L in combination with D168V had a minimal impact on faldaprevir susceptibility compared with that for D168V alone (1.5-fold difference in vitro). The median time to loss of D168 RAVs among GT-1b-infected patients who did not have a sustained virologic response at 12 weeks posttreatment (non-SVR12) after virologic failure was 5 months, which was shorter than the 14 months for R155 RAVs among GT-1a-infected non-SVR12 patients, suggesting that D168V is less fit than R155K in the absence of faldaprevir selective pressure. Copyright © 2015, American Society for

DEAD-box RNA helicase DDX3X inhibits DENV replication via regulating type one interferon pathway.

PubMed

Li, Guanghao; Feng, Tingting; Pan, Wen; Shi, Xiaohong; Dai, Jianfeng

2015-01-02

Dengue virus (DENV) is a mosquito-borne virus that threatens approximately 2.5 billion people worldwide. Vaccines against DENV are currently unavailable. DEAD-box RNA helicases (DDXs) have been reported to participate in viral replication and host innate immune response. In the present study, we analyzed the role of 40 DDX proteins during DENV replication. Among these proteins, DDX3X showed antiviral effect against DENV infection. Viral replication significantly increased in DDX3X-silenced cells compared with the controls. The interferon (IFN)-β transcription level decreased during the early stage of DENV infection in DDX3X-silenced cells compared with that in the controls. DDX3X could stimulate IFN-β transcription through the IRF3 and the NFκB branches in DENV-infected cells. Our data imply that DDX3X, a member of DEAD-box RNA helicase, is necessary for IFN production and could inhibit DENV replication. Copyright © 2014 Elsevier Inc. All rights reserved.

RNA Helicases at work: binding and rearranging

PubMed Central

Jankowsky, Eckhard

2010-01-01

RNA helicases are ubiquitous, highly conserved enzymes that participate in nearly all aspects of RNA metabolism. These proteins bind or remodel RNA or RNA–protein complexes in an ATP-dependent fashion. How RNA helicases physically perform their cellular tasks has been a longstanding question, but in recent years, intriguing models have started to link structure, mechanism and biological function for some RNA helicases. This review outlines our current view on major structural and mechanistic themes of RNA helicase function, and on emerging physical models for cellular roles of these enzymes. PMID:20813532

Biochemical studies of the Saccharomyces cerevisiae Mph1 helicase on junction-containing DNA structures

PubMed Central

Kang, Young-Hoon; Munashingha, Palinda Ruvan; Lee, Chul-Hwan; Nguyen, Tuan Anh; Seo, Yeon-Soo

2012-01-01

Saccharomyces cerevisiae Mph1 is a 3–5′ DNA helicase, required for the maintenance of genome integrity. In order to understand the ATPase/helicase role of Mph1 in genome stability, we characterized its helicase activity with a variety of DNA substrates, focusing on its action on junction structures containing three or four DNA strands. Consistent with its 3′ to 5′ directionality, Mph1 displaced 3′-flap substrates in double-fixed or equilibrating flap substrates. Surprisingly, Mph1 displaced the 5′-flap strand more efficiently than the 3′ flap strand from double-flap substrates, which is not expected for a 3–5′ DNA helicase. For this to occur, Mph1 required a threshold size (>5 nt) of 5′ single-stranded DNA flap. Based on the unique substrate requirements of Mph1 defined in this study, we propose that the helicase/ATPase activity of Mph1 play roles in converting multiple-stranded DNA structures into structures cleavable by processing enzymes such as Fen1. We also found that the helicase activity of Mph1 was used to cause structural alterations required for restoration of replication forks stalled due to damaged template. The helicase properties of Mph1 reported here could explain how it resolves D-loop structure, and are in keeping with a model proposed for the error-free damage avoidance pathway. PMID:22090425

RK-33 Radiosensitizes Prostate Cancer Cells by Blocking the RNA Helicase DDX3

PubMed Central

Xie, Min; Vesuna, Farhad; Tantravedi, Saritha; Bol, Guus M.; Heerma van Voss, Marise R.; Nugent, Katriana; Malek, Reem; Gabrielson, Kathleen; van Diest, Paul J.; Tran, Phuoc T.; Raman, Venu

2017-01-01

Despite advances in diagnosis and treatment, prostate cancer is the most prevalent cancer in males and the second highest cause of cancer-related mortality. We identified an RNA helicase gene, DDX3 (DDX3X), which is overexpressed in prostate cancers, and whose expression is directly correlated with high Gleason scores. Knockdown of DDX3 in the aggressive prostate cancer cell lines DU145 and 22Rv1 resulted in significantly reduced clonogenicity. To target DDX3, we rationally designed a small molecule, RK-33, which docks into the ATP-binding domain of DDX3. Functional studies indicated that RK-33 preferentially bound to DDX3 and perturbed its activity. RK-33 treatment of prostate cancer cell lines DU145, 22Rv1, and LNCaP (which have high DDX3 levels) decreased proliferation and induced a G1 phase cell-cycle arrest. Conversely, the low DDX3–expressing cell line, PC3, exhibited few changes following RK-33 treatment. Importantly, combination studies using RK-33 and radiation exhibited synergistic effects both in vitro and in a xenograft model of prostate cancer demonstrating the role of RK-33 as a radiosensitizer. Taken together, these results indicate that blocking DDX3 by RK-33 in combination with radiation treatment is a viable option for treating locally advanced prostate cancer. PMID:27634756

NS6180, a new KCa3.1 channel inhibitor prevents T-cell activation and inflammation in a rat model of inflammatory bowel disease

PubMed Central

Strøbæk, D; Brown, DT; Jenkins, DP; Chen, Y-J; Coleman, N; Ando, Y; Chiu, P; Jørgensen, S; Demnitz, J; Wulff, H; Christophersen, P

2013-01-01

Background and Purpose The KCa3.1 channel is a potential target for therapy of immune disease. We identified a compound from a new chemical class of KCa3.1 inhibitors and assessed in vitro and in vivo inhibition of immune responses. Experimental Approach We characterized the benzothiazinone NS6180 (4-[[3-(trifluoromethyl)phenyl]methyl]-2H-1,4-benzothiazin-3(4H)-one) with respect to potency and molecular site of action on KCa3.1 channels, selectivity towards other targets, effects on T-cell activation as well as pharmacokinetics and inflammation control in colitis induced by 2,4-dinitrobenzene sulfonic acid, a rat model of inflammatory bowel disease (IBD). Key Results NS6180 inhibited cloned human KCa3.1 channels (IC50 = 9 nM) via T250 and V275, the same amino acid residues conferring sensitivity to triarylmethanes such as like TRAM-34. NS6180 inhibited endogenously expressed KCa3.1 channels in human, mouse and rat erythrocytes, with similar potencies (15–20 nM). NS6180 suppressed rat and mouse splenocyte proliferation at submicrolar concentrations and potently inhibited IL-2 and IFN-γ production, while exerting smaller effects on IL-4 and TNF-α and no effect on IL-17 production. Antibody staining showed KCa3.1 channels in healthy colon and strong up-regulation in association with infiltrating immune cells after induction of colitis. Despite poor plasma exposure, NS6180 (3 and 10 mg·kg−1 b.i.d.) dampened colon inflammation and improved body weight gain as effectively as the standard IBD drug sulfasalazine (300 mg·kg−1 q.d.). Conclusions and Implications NS6180 represents a novel class of KCa3.1 channel inhibitors which inhibited experimental colitis, suggesting KCa3.1 channels as targets for pharmacological control of intestinal inflammation. PMID:22891655

NS309 decreases rat detrusor smooth muscle membrane potential and phasic contractions by activating SK3 channels

PubMed Central

Parajuli, Shankar P; Hristov, Kiril L; Soder, Rupal P; Kellett, Whitney F; Petkov, Georgi V

2013-01-01

Background and Purpose Overactive bladder (OAB) is often associated with abnormally increased detrusor smooth muscle (DSM) contractions. We used NS309, a selective and potent opener of the small or intermediate conductance Ca2+-activated K+ (SK or IK, respectively) channels, to evaluate how SK/IK channel activation modulates DSM function. Experimental Approach We employed single-cell RT-PCR, immunocytochemistry, whole cell patch-clamp in freshly isolated rat DSM cells and isometric tension recordings of isolated DSM strips to explore how the pharmacological activation of SK/IK channels with NS309 modulates DSM function. Key Results We detected SK3 but not SK1, SK2 or IK channels expression at both mRNA and protein levels by RT-PCR and immunocytochemistry in DSM single cells. NS309 (10 μM) significantly increased the whole cell SK currents and hyperpolarized DSM cell resting membrane potential. The NS309 hyperpolarizing effect was blocked by apamin, a selective SK channel inhibitor. NS309 inhibited the spontaneous phasic contraction amplitude, force, frequency, duration and tone of isolated DSM strips in a concentration-dependent manner. The inhibitory effect of NS309 on spontaneous phasic contractions was blocked by apamin but not by TRAM-34, indicating no functional role of the IK channels in rat DSM. NS309 also significantly inhibited the pharmacologically and electrical field stimulation-induced DSM contractions. Conclusions and Implications Our data reveal that SK3 channel is the main SK/IK subtype in rat DSM. Pharmacological activation of SK3 channels with NS309 decreases rat DSM cell excitability and contractility, suggesting that SK3 channels might be potential therapeutic targets to control OAB associated with detrusor overactivity. PMID:23145946

AMP sensing by DEAD-box RNA helicases

PubMed Central

Putnam, Andrea A.; Jankowsky, Eckhard

2013-01-01

In eukaryotes, cellular levels of adenosine monophosphate (AMP) signal the metabolic state of the cell. AMP concentrations increase significantly upon metabolic stress, such as glucose deprivation in yeast. Here we show that several DEAD-box RNA helicases are sensitive to AMP, which is not produced during ATP hydrolysis by these enzymes. We find that AMP potently inhibits RNA binding and unwinding by the yeast DEAD-box helicases Ded1p, Mss116p, and eIF4A. However, the yeast DEAD-box helicases Sub2p and Dbp5p are not inhibited by AMP. Our observations identify a subset of DEAD-box helicases as enzymes with the capacity to directly link changes in AMP concentrations to RNA metabolism. PMID:23702290

The helicase domain of Polθ counteracts RPA to promote alt-NHEJ.

PubMed

Mateos-Gomez, Pedro A; Kent, Tatiana; Deng, Sarah K; McDevitt, Shane; Kashkina, Ekaterina; Hoang, Trung M; Pomerantz, Richard T; Sfeir, Agnel

2017-12-01

Mammalian polymerase theta (Polθ) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure-function analyses that reveal that, in addition to the polymerase domain, Polθ-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR-Cas9- mediated gene targeting by homologous recombination (HR). In vitro assays demonstrate that Polθ-helicase activity facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, inhibition of RPA1 enhances end joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Polθ and RPA, providing further insight into the regulation of repair-pathway choice in mammalian cells.

The effect of glycosylation on cytotoxicity of Ibaraki virus nonstructural protein NS3

PubMed Central

URATA, Maho; WATANABE, Rie; IWATA, Hiroyuki

2015-01-01

The cytotoxicity of Ibaraki virus nonstructural protein NS3 was confirmed, and the contribution of glycosylation to this activity was examined by using glycosylation mutants of NS3 generated by site-directed mutagenesis. The expression of NS3 resulted in leakage of lactate dehydrogenase to the culture supernatant, suggesting the cytotoxicity of this protein. The lack of glycosylation impaired the transport of NS3 to the plasma membrane and resulted in reduced cytotoxicity. Combined with the previous observation that NS3 glycosylation was specifically observed in mammalian cells (Urata et al., Virus Research 2014), it was suggested that the alteration of NS3 cytotoxicity through modulating glycosylation is one of the strategies to achieve host specific pathogenisity of Ibaraki virus between mammals and vector arthropods. PMID:26178820

Structural basis of Bloom syndrome (BS) causing mutations in the BLM helicase domain.

PubMed Central

Rong, S. B.; Väliaho, J.; Vihinen, M.

2000-01-01

BACKGROUND: Bloom syndrome (BS) is characterized by mutations within the BLM gene. The Bloom syndrome protein (BLM) has similarity to the RecQ subfamily of DNA helicases, which contain seven conserved helicase domains and share significant sequence and structural similarity with the Rep and PcrA DNA helicases. We modeled the three-dimensional structure of the BLM helicase domain to analyze the structural basis of BS-causing mutations. MATERIALS AND METHODS: The sequence alignment was performed for RecQ DNA helicases and Rep and PcrA helicases. The crystal structure of PcrA helicase (PDB entry 3PJR) was used as the template for modeling the BLM helicase domain. The model was used to infer the function of BLM and to analyze the effect of the mutations. RESULTS: The structural model with good stereochemistry of the BLM helicase domain contains two subdomains, 1A and 2A. The electrostatic potential of the model is highly negative over most of the surface, except for the cleft between subdomains 1A and 2A which is similar to the template protein. The ATP-binding site is located inside the model between subdomains 1A and 2A; whereas, the DNA-binding region is situated at the surface cleft, with positive potential between 1A and 2A. CONCLUSIONS: The three-dimensional structure of the BLM helicase domain was modeled and applied to interpret BS-causing mutations. The mutation I841T is likely to weaken DNA binding, while the mutations C891R, C901Y, and Q672R presumably disturb the ATP binding. In addition, other critical positions are discussed. PMID:10965492

AMP sensing by DEAD-box RNA helicases.

PubMed

Putnam, Andrea A; Jankowsky, Eckhard

2013-10-23

In eukaryotes, cellular levels of adenosine monophosphate (AMP) signal the metabolic state of the cell. AMP concentrations increase significantly upon metabolic stress, such as glucose deprivation in yeast. Here, we show that several DEAD-box RNA helicases are sensitive to AMP, which is not produced during ATP hydrolysis by these enzymes. We find that AMP potently inhibits RNA binding and unwinding by the yeast DEAD-box helicases Ded1p, Mss116p, and eIF4A. However, the yeast DEAD-box helicases Sub2p and Dbp5p are not inhibited by AMP. Our observations identify a subset of DEAD-box helicases as enzymes with the capacity to directly link changes in AMP concentrations to RNA metabolism. Copyright © 2013 Elsevier Ltd. All rights reserved.

PARP12 suppresses Zika virus infection through PARP-dependent degradation of NS1 and NS3 viral proteins.

PubMed

Li, Lili; Zhao, Hui; Liu, Ping; Li, Chunfeng; Quanquin, Natalie; Ji, Xue; Sun, Nina; Du, Peishuang; Qin, Cheng-Feng; Lu, Ning; Cheng, Genhong

2018-06-19

Zika virus infection stimulates a type I interferon (IFN) response in host cells, which suppresses viral replication. Type I IFNs exert antiviral effects by inducing the expression of hundreds of IFN-stimulated genes (ISGs). To screen for antiviral ISGs that restricted Zika virus replication, we individually knocked out 21 ISGs in A549 lung cancer cells and identified PARP12 as a strong inhibitor of Zika virus replication. Our findings suggest that PARP12 mediated the ADP-ribosylation of NS1 and NS3, nonstructural viral proteins that are involved in viral replication and modulating host defense responses. This modification of NS1 and NS3 triggered their proteasome-mediated degradation. These data increase our understanding of the antiviral activity of PARP12 and suggest a molecular basis for the potential development of therapeutics against Zika virus. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Key binding and susceptibility of NS3/4A serine protease inhibitors against hepatitis C virus.

PubMed

Meeprasert, Arthitaya; Hannongbua, Supot; Rungrotmongkol, Thanyada

2014-04-28

Hepatitis C virus (HCV) causes an infectious disease that manifests itself as liver inflammation, cirrhosis, and can lead to the development of liver cancer. Its NS3/4A serine protease is a potent target for drug design and development since it is responsible for cleavage of the scissile peptide bonds in the polyprotein important for the HCV life cycle. Herein, the ligand-target interactions and the binding free energy of the four current NS3/4A inhibitors (boceprevir, telaprevir, danoprevir, and BI201335) were investigated by all-atom molecular dynamics simulations with three different initial atomic velocities. The per-residue free energy decomposition suggests that the key residues involved in inhibitor binding were residues 41-43, 57, 81, 136-139, 155-159, and 168 in the NS3 domain. The van der Waals interactions yielded the main driving force for inhibitor binding at the protease active site for the cleavage reaction. In addition, the highest number of hydrogen bonds was formed at the reactive P1 site of the four studied inhibitors. Although the hydrogen bond patterns of these inhibitors were different, their P3 site was most likely to be recognized by the A157 backbone. Both molecular mechanic (MM)/Poisson-Boltzmann surface area and MM/generalized Born surface area approaches predicted the relative binding affinities of the four inhibitors in a somewhat similar trend to their experimentally derived biological activities.

Cleavage preference distinguishes the two-component NS2B-NS3 serine proteinases of Dengue and West Nile viruses.

PubMed

Shiryaev, Sergey A; Kozlov, Igor A; Ratnikov, Boris I; Smith, Jeffrey W; Lebl, Michal; Strongin, Alex Y

2007-02-01

Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1'-P2'-P3'-P4'-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1' or the P2' site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.

Preclinical Profile and Characterization of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir (BMS-650032)

PubMed Central

Sheaffer, Amy K.; Friborg, Jacques; Hernandez, Dennis; Falk, Paul; Zhai, Guangzhi; Levine, Steven; Chaniewski, Susan; Yu, Fei; Barry, Diana; Chen, Chaoqun; Lee, Min S.; Mosure, Kathy; Sun, Li-Qiang; Sinz, Michael; Meanwell, Nicholas A.; Colonno, Richard J.; Knipe, Jay; Scola, Paul

2012-01-01

Asunaprevir (ASV; BMS-650032) is a hepatitis C virus (HCV) NS3 protease inhibitor that has demonstrated efficacy in patients chronically infected with HCV genotype 1 when combined with alfa interferon and/or the NS5A replication complex inhibitor daclatasvir. ASV competitively binds to the NS3/4A protease complex, with Ki values of 0.4 and 0.24 nM against recombinant enzymes representing genotypes 1a (H77) and 1b (J4L6S), respectively. Selectivity was demonstrated by the absence of any significant activity against the closely related GB virus-B NS3 protease and a panel of human serine or cysteine proteases. In cell culture, ASV inhibited replication of HCV replicons representing genotypes 1 and 4, with 50% effective concentrations (EC50s) ranging from 1 to 4 nM, and had weaker activity against genotypes 2 and 3 (EC50, 67 to 1,162 nM). Selectivity was again demonstrated by the absence of activity (EC50, >12 μM) against a panel of other RNA viruses. ASV exhibited additive or synergistic activity in combination studies with alfa interferon, ribavirin, and/or inhibitors specifically targeting NS5A or NS5B. Plasma and tissue exposures in vivo in several animal species indicated that ASV displayed a hepatotropic disposition (liver-to-plasma ratios ranging from 40- to 359-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥110-fold above the inhibitor EC50s observed with HCV genotype-1 replicons. Based on these virologic and exposure properties, ASV holds promise for future utility in a combination with other anti-HCV agents in the treatment of HCV-infected patients. PMID:22869577

The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ziehr, Ben; Lenarcic, Erik; Cecil, Chad

Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compoundmore » that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics. - Highlights: • The host eIF4AIII RNA helicase is required for efficient HCMV replication. • Depleting eIF4AIII inhibited the nuclear export of HCMV mRNAs. • HCMV mRNAs did not require eIF4AIII to associate with polyribosomes. • The eIF4A family helicases may be new targets for host-directed antiviral drugs.« less

Sequence-dependent base pair stepping dynamics in XPD helicase unwinding

PubMed Central

Qi, Zhi; Pugh, Robert A; Spies, Maria; Chemla, Yann R

2013-01-01

Helicases couple the chemical energy of ATP hydrolysis to directional translocation along nucleic acids and transient duplex separation. Understanding helicase mechanism requires that the basic physicochemical process of base pair separation be understood. This necessitates monitoring helicase activity directly, at high spatio-temporal resolution. Using optical tweezers with single base pair (bp) resolution, we analyzed DNA unwinding by XPD helicase, a Superfamily 2 (SF2) DNA helicase involved in DNA repair and transcription initiation. We show that monomeric XPD unwinds duplex DNA in 1-bp steps, yet exhibits frequent backsteps and undergoes conformational transitions manifested in 5-bp backward and forward steps. Quantifying the sequence dependence of XPD stepping dynamics with near base pair resolution, we provide the strongest and most direct evidence thus far that forward, single-base pair stepping of a helicase utilizes the spontaneous opening of the duplex. The proposed unwinding mechanism may be a universal feature of DNA helicases that move along DNA phosphodiester backbones. DOI: http://dx.doi.org/10.7554/eLife.00334.001 PMID:23741615

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

PubMed Central

Bastia, Deepak; Srivastava, Pankaj; Zaman, Shamsu; Choudhury, Malay; Mohanty, Bidyut K.; Bacal, Julien; Langston, Lance D.; Pasero, Philippe; O’Donnell, Michael E.

2016-01-01

Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1–Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression. Here we show that mutations of Dbf4-dependent kinase (DDK) of Saccharomyces cerevisiae, but not other DNA replication factors, greatly reduced PFA at replication fork barriers in the spacer regions of the ribosomal DNA array. A key target of DDK is the mini chromosome maintenance (Mcm) 2–7 complex, which is known to require phosphorylation by DDK to form an active CMG [Cdc45 (cell division cycle gene 45), Mcm2–7, GINS (Go, Ichi, Ni, and San)] helicase. In vivo experiments showed that mutational inactivation of DDK caused release of Tof1 from the chromatin fractions. In vitro binding experiments confirmed that CMG and/or Mcm2–7 had to be phosphorylated for binding to phospho-Tof1–Csm3 but not to its dephosphorylated form. Suppressor mutations that bypass the requirement for Mcm2–7 phosphorylation by DDK restored PFA in the absence of the kinase. Retention of Tof1 in the chromatin fraction and PFA in vivo was promoted by the suppressor mcm5-bob1, which bypassed DDK requirement, indicating that under this condition a kinase other than DDK catalyzed the phosphorylation of Tof1. We propose that phosphorylation regulates the recruitment and retention of Tof1–Csm3 by the replisome and that this complex antagonizes the Rrm3 helicase, thereby promoting PFA, by preserving the integrity of the Fob1–Ter complex. PMID:27298353

OsSUV3 dual helicase functions in salinity stress tolerance by maintaining photosynthesis and antioxidant machinery in rice (Oryza sativa L. cv. IR64).

PubMed

Tuteja, Narendra; Sahoo, Ranjan Kumar; Garg, Bharti; Tuteja, Renu

2013-10-01

To overcome the salinity-induced loss of crop yield, a salinity-tolerant trait is required. The SUV3 helicase is involved in the regulation of RNA surveillance and turnover in mitochondria, but the helicase activity of plant SUV3 and its role in abiotic stress tolerance have not been reported so far. Here we report that the Oryza sativa (rice) SUV3 protein exhibits DNA and RNA helicase, and ATPase activities. Furthermore, we report that SUV3 is induced in rice seedlings in response to high levels of salt. Its expression, driven by a constitutive cauliflower mosaic virus 35S promoter in IR64 transgenic rice plants, confers salinity tolerance. The T1 and T2 sense transgenic lines showed tolerance to high salinity and fully matured without any loss in yields. The T2 transgenic lines also showed tolerance to drought stress. These results suggest that the introduced trait is functional and stable in transgenic rice plants. The rice SUV3 sense transgenic lines showed lesser lipid peroxidation, electrolyte leakage and H2 O2 production, along with higher activities of antioxidant enzymes under salinity stress, as compared with wild type, vector control and antisense transgenic lines. These results suggest the existence of an efficient antioxidant defence system to cope with salinity-induced oxidative damage. Overall, this study reports that plant SUV3 exhibits DNA and RNA helicase and ATPase activities, and provides direct evidence of its function in imparting salinity stress tolerance without yield loss. The possible mechanism could be that OsSUV3 helicase functions in salinity stress tolerance by improving photosynthesis and antioxidant machinery in transgenic rice. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Prevalence of polymorphisms with significant resistance to NS5A inhibitors in treatment-naive patients with hepatitis C virus genotypes 1a and 3a in Sweden.

PubMed

Lindström, Ida; Kjellin, Midori; Palanisamy, Navaneethan; Bondeson, Kåre; Wesslén, Lars; Lannergard, Anders; Lennerstrand, Johan

2015-08-01

The future treatment of hepatitis C virus (HCV) infection will be combinations of direct-acting antivirals (DAAs) that not only target multiple viral targets, but are also effective against different HCV genotypes. Of the many drug targets in HCV, one promising target is the non-structural 5A protein (NS5A), against which inhibitors, namely daclatasvir, ledipasvir and ombitasvir, have shown potent efficacy. However, since HCV is known to have very high sequence diversity, development of resistance is a problem against but not limited to NS5A inhibitors (i.e. resistance also found against NS3-protease and NS5B non-nucleoside inhibitors), when used in suboptimal combinations. Furthermore, it has been shown that natural resistance against DAAs is present in treatment-naïve patients and such baseline resistance will potentially complicate future treatment strategies. A pan-genotypic population-sequencing method with degenerated primers targeting the NS5A region was developed. We have investigated the prevalence of baseline resistant variants in 127 treatment-naïve patients of HCV genotypes 1a, 1b, 2b and 3a. The method could successfully sequence more than 95% of genotype 1a, 1b and 3a samples. Interpretation of fold resistance data against the NS5A inhibitors was done with the help of earlier published phenotypic data. Baseline resistance variants associated with high resistance (1000-50,000-fold) was found in three patients: Q30H or Y93N in genotype 1a patients and further Y93H in a genotype 3a patient. Using this method, baseline resistance can be examined and the data could have a potential role in selecting the optimal and cost-efficient treatment for the patient.

Synthesis and disulfide bond connectivity-activity studies of a kalata B1-inspired cyclopeptide against dengue NS2B-NS3 protease.

PubMed

Gao, Yaojun; Cui, Taian; Lam, Yulin

2010-02-01

Kalata B1 is a plant protein with remarkable thermal, chemical and enzymatic stability. Its potential applications could be centered on the possibility of using its cyclic structure and cystine knot motif as a scaffold for the design of stable pharmaceuticals. To discover potent dengue NS2B-NS3 protease inhibitors, we have prepared various kalata B1 analogues by varying the amino acid sequence. Mass spectrometric and biochemical investigations of these analogues revealed a cyclopeptide whose two fully oxidized forms are substrate-competitive inhibitors of the dengue viral NS2B-NS3 protease. Both oxidized forms showed potent inhibition with K(i) of 1.39+/-0.35 and 3.03+/-0.75 microM, respectively. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Nonstructural proteins nsP3 and nsP4 of Ross River and O'Nyong-nyong viruses: sequence and comparison with those of other alphaviruses.

PubMed

Strauss, E G; Levinson, R; Rice, C M; Dalrymple, J; Strauss, J H

1988-05-01

We have sequenced the nsP3 and nsP4 region of two alphaviruses, Ross River virus and O'Nyong-nyong virus, in order to examine these viruses for the presence or absence of an opal termination codon present between nsP3 and nsP4 in many alphaviruses. We found that Ross River virus possesses an in-phase opal termination codon between nsP3 and nsP4, whereas in O'Nyong-nyong virus this termination codon is replaced by an arginine codon. Previous studies have shown that two other alphaviruses, Sindbis virus and Middelburg virus, possess an opal termination codon separating nsP3 and nsP4 [E.G. Strauss, C.M. Rice, and J.H. Strauss (1983), Proc. Natl. Acad. Sci. USA 80, 5271-5275], whereas Semliki Forest virus possesses an arginine codon in lieu of the opal codon [K. Takkinen (1986), Nucleic Acids Res. 14, 5667-5682]. Thus, of the five alphaviruses examined to date, three possess the opal codon and two do not. Production of nsP4 requires readthrough of the opal codon in those alphaviruses that possess this termination codon and the function of the termination codon may be to regulate the amount of nsP4 produced. It is an open question then as to whether alphaviruses with no termination codon use other mechanisms to regulate the activity of this gene. The nsP4s of these five alphaviruses are highly conserved, sharing 71-76% amino acid sequence similarity, and all five contain the Gly-Asp-Asp motif found in many RNA virus replicases. The nsP3s are somewhat less conserved, sharing 52-73% amino acid sequence similarity throughout most of the protein, but each possesses a nonconserved C-terminal domain of 134 to 246 amino acids of unknown function.

Archaeal MCM has separable processivity, substrate choice and helicase domains

PubMed Central

Barry, Elizabeth R.; McGeoch, Adam T.; Kelman, Zvi; Bell, Stephen D.

2007-01-01

The mini-chromosome maintenance (MCM) complex is the principal candidate for the replicative helicase of archaea and eukaryotes. Here, we describe a functional dissection of the roles of the three principal structural modules of the homomultimeric MCM of the hyperthermophilic archaeon Sulfolobus solfataricus. Our results include the first analysis of the central AAA+ domain in isolation. This domain possesses ATPase and helicase activity, defining this as the minimal helicase domain. Reconstitution experiments show that the helicase activity of the AAA+ domain can be stimulated by addition of the isolated N-terminal half in trans. Addition of the N-terminus influences both the processivity of the helicase and the choice of substrate that can be melted by the ATPase domain. The degenerate helix-turn-helix domain at the C-terminus of MCM exerts a negative effect on the helicase activity of the complex. These results provide the first evidence for extensive regulatory inter-domain communication within the MCM complex. PMID:17259218

Helicase promotes replication re-initiation from an RNA transcript.

PubMed

Sun, Bo; Singh, Anupam; Sultana, Shemaila; Inman, James T; Patel, Smita S; Wang, Michelle D

2018-06-13

To ensure accurate DNA replication, a replisome must effectively overcome numerous obstacles on its DNA substrate. After encountering an obstacle, a progressing replisome often aborts DNA synthesis but continues to unwind. However, little is known about how DNA synthesis is resumed downstream of an obstacle. Here, we examine the consequences of a non-replicating replisome collision with a co-directional RNA polymerase (RNAP). Using single-molecule and ensemble methods, we find that T7 helicase interacts strongly with a non-replicating T7 DNA polymerase (DNAP) at a replication fork. As the helicase advances, the associated DNAP also moves forward. The presence of the DNAP increases both helicase's processivity and unwinding rate. We show that such a DNAP, together with its helicase, is indeed able to actively disrupt a stalled transcription elongation complex, and then initiates replication using the RNA transcript as a primer. These observations exhibit T7 helicase's novel role in replication re-initiation.

Human Pif1 helicase unwinds synthetic DNA structures resembling stalled DNA replication forks

PubMed Central

George, Tresa; Wen, Qin; Griffiths, Richard; Ganesh, Anil; Meuth, Mark; Sanders, Cyril M.

2009-01-01

Pif-1 proteins are 5′→3′ superfamily 1 (SF1) helicases that in yeast have roles in the maintenance of mitochondrial and nuclear genome stability. The functions and activities of the human enzyme (hPif1) are unclear, but here we describe its DNA binding and DNA remodeling activities. We demonstrate that hPif1 specifically recognizes and unwinds DNA structures resembling putative stalled replication forks. Notably, the enzyme requires both arms of the replication fork-like structure to initiate efficient unwinding of the putative leading replication strand of such substrates. This DNA structure-specific mode of initiation of unwinding is intrinsic to the conserved core helicase domain (hPifHD) that also possesses a strand annealing activity as has been demonstrated for the RecQ family of helicases. The result of hPif1 helicase action at stalled DNA replication forks would generate free 3′ ends and ssDNA that could potentially be used to assist replication restart in conjunction with its strand annealing activity. PMID:19700773

Identification of drug resistance and immune-driven variations in hepatitis C virus (HCV) NS3/4A, NS5A and NS5B regions reveals a new approach toward personalized medicine.

PubMed

Ikram, Aqsa; Obaid, Ayesha; Awan, Faryal Mehwish; Hanif, Rumeza; Naz, Anam; Paracha, Rehan Zafar; Ali, Amjad; Janjua, Hussnain Ahmed

2017-01-01

Cellular immune responses (T cell responses) during hepatitis C virus (HCV) infection are significant factors for determining the outcome of infection. HCV adapts to host immune responses by inducing mutations in its genome at specific sites that are important for HLA processing/presentation. Moreover, HCV also adapts to resist potential drugs that are used to restrict its replication, such as direct-acting antivirals (DAAs). Although DAAs have significantly reduced disease burden, resistance to these drugs is still a challenge for the treatment of HCV infection. Recently, drug resistance mutations (DRMs) observed in HCV proteins (NS3/4A, NS5A and NS5B) have heightened concern that the emergence of drug resistance may compromise the effectiveness of DAAs. Therefore, the NS3/4A, NS5A and NS5B drug resistance variations were investigated in this study, and their prevalence was examined in a large number of protein sequences from all HCV genotypes. Furthermore, potential CD4 + and CD8 + T cell epitopes were predicted and their overlap with genetic variations was explored. The findings revealed that many reported DRMs within NS3/4A, NS5A and NS5B are not drug-induced; rather, they are already present in HCV strains, as they were also detected in HCV-naïve patients. This study highlights several hot spots in which HLA and drug selective pressure overlap. Interestingly, these overlapping mutations were frequently observed among many HCV genotypes. This study implicates that knowledge of the host HLA type and HCV subtype/genotype can provide important information in defining personalized therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

SAD-3, a Putative Helicase Required for Meiotic Silencing by Unpaired DNA, Interacts with Other Components of the Silencing Machinery

PubMed Central

Hammond, Thomas M.; Xiao, Hua; Boone, Erin C.; Perdue, Tony D.; Pukkila, Patricia J.; Shiu, Patrick K. T.

2011-01-01

In Neurospora crassa, genes lacking a pairing partner during meiosis are suppressed by a process known as meiotic silencing by unpaired DNA (MSUD). To identify novel MSUD components, we have developed a high-throughput reverse-genetic screen for use with the N. crassa knockout library. Here we describe the screening method and the characterization of a gene (sad-3) subsequently discovered. SAD-3 is a putative helicase required for MSUD and sexual spore production. It exists in a complex with other known MSUD proteins in the perinuclear region, a center for meiotic silencing activity. Orthologs of SAD-3 include Schizosaccharomyces pombe Hrr1, a helicase required for RNAi-induced heterochromatin formation. Both SAD-3 and Hrr1 interact with an RNA-directed RNA polymerase and an Argonaute, suggesting that certain aspects of silencing complex formation may be conserved between the two fungal species. PMID:22384347

Further theoretical insight into the reaction mechanism of the hepatitis C NS3/NS4A serine protease

NASA Astrophysics Data System (ADS)

Martínez-González, José Ángel; Rodríguez, Alex; Puyuelo, María Pilar; González, Miguel; Martínez, Rodrigo

2015-01-01

The main reactions of the hepatitis C virus NS3/NS4A serine protease are studied using the second-order Møller-Plesset ab initio method and rather large basis sets to correct the previously reported AM1/CHARMM22 potential energy surfaces. The reaction efficiencies measured for the different substrates are explained in terms of the tetrahedral intermediate formation step (the rate-limiting process). The energies of the barrier and the corresponding intermediate are so close that the possibility of a concerted mechanism is open (especially for the NS5A/5B substrate). This is in contrast to the suggested general reaction mechanism of serine proteases, where a two-step mechanism is postulated.

Physical and functional interactions of Caenorhabditis elegans WRN-1 helicase with RPA-1.

PubMed

Hyun, Moonjung; Park, Sojin; Kim, Eunsun; Kim, Do-Hyung; Lee, Se-Jin; Koo, Hyeon-Sook; Seo, Yeon-Soo; Ahn, Byungchan

2012-02-21

The Caenorhabditis elegans Werner syndrome protein, WRN-1, a member of the RecQ helicase family, has a 3'-5' DNA helicase activity. Worms with defective wrn-1 exhibit premature aging phenotypes and an increased level of genome instability. In response to DNA damage, WRN-1 participates in the initial stages of checkpoint activation in concert with C. elegans replication protein A (RPA-1). WRN-1 helicase is stimulated by RPA-1 on long DNA duplex substrates. However, the mechanism by which RPA-1 stimulates DNA unwinding and the function of the WRN-1-RPA-1 interaction are not clearly understood. We have found that WRN-1 physically interacts with two RPA-1 subunits, CeRPA73 and CeRPA32; however, full-length WRN-1 helicase activity is stimulated by only the CeRPA73 subunit, while the WRN-1(162-1056) fragment that harbors the helicase activity requires both the CeRPA73 and CeRPA32 subunits for the stimulation. We also found that the CeRPA73(1-464) fragment can stimulate WRN-1 helicase activity and that residues 335-464 of CeRPA73 are important for physical interaction with WRN-1. Because CeRPA73 and the CeRPA73(1-464) fragment are able to bind single-stranded DNA (ssDNA), the stimulation of WRN-1 helicase by RPA-1 is most likely due to the ssDNA binding activity of CeRPA73 and the direct interaction of WRN-1 and CeRPA73.

Comparative Analysis of Disruption Tolerant Network Routing Simulations in the One and NS-3

DTIC Science & Technology

2017-12-01

real systems with less work compared to ns-2. In order to meet the design goals of ns-3, the entire code structure changed to a modular design . As a...NAVAL POSTGRADUATE SCHOOL MONTEREY, CALIFORNIA THESIS COMPARATIVE ANALYSIS OF DISRUPTION TOLERANT NETWORK ROUTING SIMULATIONS IN THE ONE AND NS-3...Thesis 03-23-2016 to 12-15-2017 4. TITLE AND SUBTITLE COMPARATIVE ANALYSIS OF DISRUPTION TOLERANT NETWORK ROUTING SIMULATIONS IN THE ONE AND NS-3 5

Getting it done at the ends: Pif1 family DNA helicases and telomeres

PubMed Central

Geronimo, Carly L.; Zakian, Virginia A.

2017-01-01

It is widely appreciated that the ends of linear DNA molecules cannot be fully replicated by the conventional replication apparatus. Less well known is that semi-conservative replication of telomeric DNA also presents problems for DNA replication. These problems likely arise from the atypical chromatin structure of telomeres, the GC-richness of telomeric DNA that makes it prone to forming DNA secondary structures, and from RNA-DNA hybrids, formed by transcripts of one or both DNA strands. Given the different aspects of telomeres that complicate their replication, it is not surprising that multiple DNA helicases promote replication of telomeric DNA. This review focuses on one such class of DNA helicases, the Pif1 family of 5′–3′ DNA helicases. In budding and fission yeasts, Pif1 family helicases impact both telomerase-mediated and semi-conservative replication of telomeric DNA as well as recombination-mediated telomere lengthening. PMID:27233114

Unraveling helicase mechanisms one molecule at a time

PubMed Central

Rasnik, Ivan; Myong, Sua; Ha, Taekjip

2006-01-01

Recent years have seen an increasing number of biological applications of single molecule techniques, evolving from a proof of principle type to the more sophisticated studies. Here we compare the capabilities and limitations of different single molecule techniques in studying the activities of helicases. Helicases share a common catalytic activity but present a high variability in kinetic and phenomenological behavior, making their studies ideal in exemplifying the use of the new single molecule techniques to answer biological questions. Unexpected phenomena have also been observed from individual molecules suggesting extended or alternative functionality of helicases in vivo. PMID:16935883

[RTEL1 (regulator of telomere elongation helicase 1), a DNA helicase essential for genome stability].

PubMed

Le Guen, Tangui; Jullien, Laurent; Schertzer, Mike; Lefebvre, Axelle; Kermasson, Laetitia; de Villartay, Jean-Pierre; Londoño-Vallejo, Arturo; Revy, Patrick

2013-12-01

RTEL1 (regulator of telomere length helicase 1) is a DNA helicase that has been identified more than 10 years ago. Many works since, mainly in the nematode Caenorhabditis elegans and the mouse, have highlighted its role in chromosomal stability, maintenance of telomere length, and DNA repair. Recently, four laboratories have characterized RTEL1 mutations in patients with dyskeratosis congenita (DC) and Hoyeraal-Hreidarsson (HH) syndrome, a rare and severe variant of DC. We here summarize the current knowledge on RTEL1 and discuss the possible other functions that RTEL1 could play. © 2013 médecine/sciences – Inserm.

The RNA Helicase DeaD Stimulates ExsA Translation To Promote Expression of the Pseudomonas aeruginosa Type III Secretion System

PubMed Central

Intile, Peter J.; Balzer, Grant J.; Wolfgang, Matthew C.

2015-01-01

ABSTRACT The Pseudomonas aeruginosa type III secretion system (T3SS) is a primary virulence factor important for phagocytic avoidance, disruption of host cell signaling, and host cell cytotoxicity. ExsA is the master regulator of T3SS transcription. The expression, synthesis, and activity of ExsA is tightly regulated by both intrinsic and extrinsic factors. Intrinsic regulation consists of the well-characterized ExsECDA partner-switching cascade, while extrinsic factors include global regulators that alter exsA transcription and/or translation. To identify novel extrinsic regulators of ExsA, we conducted a transposon mutagenesis screen in the absence of intrinsic control. Transposon disruptions within gene PA2840, which encodes a homolog of the Escherichia coli RNA-helicase DeaD, significantly reduced T3SS gene expression. Recent studies indicate that E. coli DeaD can promote translation by relieving inhibitory secondary structures within target mRNAs. We report here that PA2840, renamed DeaD, stimulates ExsA synthesis at the posttranscriptional level. Genetic experiments demonstrate that the activity of an exsA translational fusion is reduced in a deaD mutant. In addition, exsA expression in trans fails to restore T3SS gene expression in a deaD mutant. We hypothesized that DeaD relaxes mRNA secondary structure to promote exsA translation and found that altering the mRNA sequence of exsA or the native exsA Shine-Dalgarno sequence relieved the requirement for DeaD in vivo. Finally, we show that purified DeaD promotes ExsA synthesis using in vitro translation assays. Together, these data reveal a novel regulatory mechanism for P. aeruginosa DeaD and add to the complexity of global regulation of T3SS. IMPORTANCE Although members of the DEAD box family of RNA helicases are appreciated for their roles in mRNA degradation and ribosome biogenesis, an additional role in gene regulation is now emerging in bacteria. By relaxing secondary structures in mRNAs, DEAD box

Human RecQL4 helicase plays multifaceted roles in the genomic stability of normal and cancer cells.

PubMed

Mo, Dongliang; Zhao, Yongliang; Balajee, Adayabalam S

2018-01-28

Human RecQ helicases that share homology with E. coli RecQ helicase play critical roles in diverse biological activities such as DNA replication, transcription, recombination and repair. Mutations in three of the five human RecQ helicases (RecQ1, WRN, BLM, RecQL4 and RecQ5) result in autosomal recessive syndromes characterized by accelerated aging symptoms and cancer incidence. Mutational inactivation of Werner (WRN) and Bloom (BLM) genes results in Werner syndrome (WS) and Bloom syndrome (BS) respectively. However, mutations in RecQL4 result in three human disorders: (I) Rothmund-Thomson syndrome (RTS), (II) RAPADILINO and (III) Baller-Gerold syndrome (BGS). Cells from WS, BS and RTS are characterized by a unique chromosomal anomaly indicating that each of the RecQ helicases performs specialized function(s) in a non-redundant manner. Elucidating the biological functions of RecQ helicases will enable us to understand not only the aging process but also to determine the cause for age-associated human diseases. Recent biochemical and molecular studies have given new insights into the multifaceted roles of RecQL4 that range from genomic stability to carcinogenesis and beyond. This review summarizes some of the existing and emerging knowledge on diverse biological functions of RecQL4 and its significance as a potential molecular target for cancer therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Getting it done at the ends: Pif1 family DNA helicases and telomeres.

PubMed

Geronimo, Carly L; Zakian, Virginia A

2016-08-01

It is widely appreciated that the ends of linear DNA molecules cannot be fully replicated by the conventional replication apparatus. Less well known is that semi-conservative replication of telomeric DNA also presents problems for DNA replication. These problems likely arise from the atypical chromatin structure of telomeres, the GC-richness of telomeric DNA that makes it prone to forming DNA secondary structures, and from RNA-DNA hybrids, formed by transcripts of one or both DNA strands. Given the different aspects of telomeres that complicate their replication, it is not surprising that multiple DNA helicases promote replication of telomeric DNA. This review focuses on one such class of DNA helicases, the Pif1 family of 5'-3' DNA helicases. In budding and fission yeasts, Pif1 family helicases impact both telomerase-mediated and semi-conservative replication of telomeric DNA as well as recombination-mediated telomere lengthening. Copyright © 2016. Published by Elsevier B.V.

Concerted action of the MutLβ heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion

PubMed Central

Duroc, Yann; Kumar, Rajeev; Ranjha, Lepakshi; Adam, Céline; Guérois, Raphaël; Md Muntaz, Khan; Marsolier-Kergoat, Marie-Claude; Dingli, Florent; Laureau, Raphaëlle; Loew, Damarys; Llorente, Bertrand; Charbonnier, Jean-Baptiste; Cejka, Petr; Borde, Valérie

2017-01-01

Gene conversions resulting from meiotic recombination are critical in shaping genome diversification and evolution. How the extent of gene conversions is regulated is unknown. Here we show that the budding yeast mismatch repair related MutLβ complex, Mlh1-Mlh2, specifically interacts with the conserved meiotic Mer3 helicase, which recruits it to recombination hotspots, independently of mismatch recognition. This recruitment is essential to limit gene conversion tract lengths genome-wide, without affecting crossover formation. Contrary to expectations, Mer3 helicase activity, proposed to extend the displacement loop (D-loop) recombination intermediate, does not influence the length of gene conversion events, revealing non-catalytical roles of Mer3. In addition, both purified Mer3 and MutLβ preferentially recognize D-loops, providing a mechanism for limiting gene conversion in vivo. These findings show that MutLβ is an integral part of a new regulatory step of meiotic recombination, which has implications to prevent rapid allele fixation and hotspot erosion in populations. DOI: http://dx.doi.org/10.7554/eLife.21900.001 PMID:28051769

The eukaryotic translation initiation factor 3 subunit E binds to classical swine fever virus NS5A and facilitates viral replication.

PubMed

Liu, Xiaofeng; Wang, Xiaoyu; Wang, Qian; Luo, Mingyang; Guo, Huancheng; Gong, Wenjie; Tu, Changchun; Sun, Jinfu

2018-02-01

Classical swine fever virus (CSFV) NS5A protein is a multifunctional protein, playing critical roles in viral RNA replication, translation and assembly. To further explore its functions in viral replication, interaction of NS5A with host factors was assayed using a his-tag "pull down" assay coupled with shotgun LC-MS/MS. Host protein translation initiation factor 3 subunit E was identified as a binding partner of NS5A, and confirmed by co-immunoprecipitation and co-localization analysis. Overexpression of eIF3E markedly enhanced CSFV genomic replication, viral protein expression and production of progeny virus, and downregulation of eIF3E by siRNA significantly decreased viral proliferation in PK-15 cells. Luciferase reporter assay showed an enhancement of translational activity of the internal ribosome entry site of CSFV by eIF3E and a decrease in cellular translation by NS5A. These data indicate that eIF3E plays an important role in CSFV replication, thereby identifying it as a potential target for inhibition of the virus. Copyright © 2017 Elsevier Inc. All rights reserved.

DNA unwinding by Escherichia coli DNA helicase I (TraI) provides evidence for a processive monomeric molecular motor.

PubMed

Sikora, Bartek; Eoff, Robert L; Matson, Steven W; Raney, Kevin D

2006-11-24

The F plasmid TraI protein (DNA helicase I) plays an essential role in conjugative DNA transfer as both a transesterase and a helicase. Previous work has shown that the 192-kDa TraI protein is a highly processive helicase, catalytically separating >850 bp under steady-state conditions. In this report, we examine the kinetic mechanism describing DNA unwinding of TraI. The kinetic step size of TraI was measured under both single turnover and pre-steady-state conditions. The resulting kinetic step-size estimate was approximately 6-8 bp step(-1). TraI can separate double-stranded DNA at a rate of approximately 1100 bp s(-1), similar to the measured unwinding rate of the RecBCD helicase, and appears to dissociate very slowly from the 3' terminus following translocation and strand-separation events. Analyses of pre-steady-state burst amplitudes indicate that TraI can function as a monomer, similar to the bacteriophage T4 helicase, Dda. However, unlike Dda, TraI is a highly processive monomeric helicase, making it unique among the DNA helicases characterized thus far.

ARCPHdb: A comprehensive protein database for SF1 and SF2 helicase from archaea.

PubMed

Moukhtar, Mirna; Chaar, Wafi; Abdel-Razzak, Ziad; Khalil, Mohamad; Taha, Samir; Chamieh, Hala

2017-01-01

Superfamily 1 and Superfamily 2 helicases, two of the largest helicase protein families, play vital roles in many biological processes including replication, transcription and translation. Study of helicase proteins in the model microorganisms of archaea have largely contributed to the understanding of their function, architecture and assembly. Based on a large phylogenomics approach, we have identified and classified all SF1 and SF2 protein families in ninety five sequenced archaea genomes. Here we developed an online webserver linked to a specialized protein database named ARCPHdb to provide access for SF1 and SF2 helicase families from archaea. ARCPHdb was implemented using MySQL relational database. Web interfaces were developed using Netbeans. Data were stored according to UniProt accession numbers, NCBI Ref Seq ID, PDB IDs and Entrez Databases. A user-friendly interactive web interface has been developed to browse, search and download archaeal helicase protein sequences, their available 3D structure models, and related documentation available in the literature provided by ARCPHdb. The database provides direct links to matching external databases. The ARCPHdb is the first online database to compile all protein information on SF1 and SF2 helicase from archaea in one platform. This database provides essential resource information for all researchers interested in the field. Copyright © 2016 Elsevier Ltd. All rights reserved.

Close encounters for the first time: Helicase interactions with DNA damage.

PubMed

Khan, Irfan; Sommers, Joshua A; Brosh, Robert M

2015-09-01

DNA helicases are molecular motors that harness the energy of nucleoside triphosphate hydrolysis to unwinding structured DNA molecules that must be resolved during cellular replication, DNA repair, recombination, and transcription. In vivo, DNA helicases are expected to encounter a wide spectrum of covalent DNA modifications to the sugar phosphate backbone or the nitrogenous bases; these modifications can be induced by endogenous biochemical processes or exposure to environmental agents. The frequency of lesion abundance can vary depending on the lesion type. Certain adducts such as oxidative base modifications can be quite numerous, and their effects can be helix-distorting or subtle perturbations to DNA structure. Helicase encounters with specific DNA lesions and more novel forms of DNA damage will be discussed. We will also review the battery of assays that have been used to characterize helicase-catalyzed unwinding of damaged DNA substrates. Characterization of the effects of specific DNA adducts on unwinding by various DNA repair and replication helicases has proven to be insightful for understanding mechanistic and biological aspects of helicase function in cellular DNA metabolism. Published by Elsevier B.V.

Helicase-inactivating mutations as a basis for dominant negative phenotypes

PubMed Central

Wu, Yuliang

2010-01-01

There is ample evidence from studies of both unicellular and multicellular organisms that helicase-inactivating mutations lead to cellular dysfunction and disease phenotypes. In this review, we will discuss the mechanisms underlying the basis for abnormal phenotypes linked to mutations in genes encoding DNA helicases. Recent evidence demonstrates that a clinically relevant patient missense mutation in Fanconi Anemia Complementation Group J exerts detrimental effects on the biochemical activities of the FANC J helicase, and these molecular defects are responsible for aberrant genomic stability and a poor DNA damage response. The ability of FANC J to use the energy from AT P hydrolysis to produce the force required to unwind duplex or G-quadruplex DNA structures or destabilize protein bound to DNA is required for its DNA repair functions in vivo. Strikingly, helicase-inactivating mutations can exert a spectrum of dominant negative phenotypes, indicating that expression of the mutant helicase protein potentially interferes with normal DNA metabolism and has an effect on basic cellular processes such as DNA replication, the DNA damage response and protein trafficking. This review emphasizes that future studies of clinically relevant mutations in helicase genes will be important to understand the molecular pathologies of the associated diseases and their impact on heterozygote carriers. PMID:20980836

Switch on the engine: how the eukaryotic replicative helicase MCM2-7 becomes activated.

PubMed

Tognetti, Silvia; Riera, Alberto; Speck, Christian

2015-03-01

A crucial step during eukaryotic initiation of DNA replication is the correct loading and activation of the replicative DNA helicase, which ensures that each replication origin fires only once. Unregulated DNA helicase loading and activation, as it occurs in cancer, can cause severe DNA damage and genomic instability. The essential mini-chromosome maintenance proteins 2-7 (MCM2-7) represent the core of the eukaryotic replicative helicase that is loaded at DNA replication origins during G1-phase of the cell cycle. The MCM2-7 helicase activity, however, is only triggered during S-phase once the holo-helicase Cdc45-MCM2-7-GINS (CMG) has been formed. A large number of factors and several kinases interact and contribute to CMG formation and helicase activation, though the exact mechanisms remain unclear. Crucially, upon DNA damage, this reaction is temporarily halted to ensure genome integrity. Here, we review the current understanding of helicase activation; we focus on protein interactions during CMG formation, discuss structural changes during helicase activation, and outline similarities and differences of the prokaryotic and eukaryotic helicase activation process.

Structure of a helicase–helicase loader complex reveals insights into the mechanism of bacterial primosome assembly

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Bin; Eliason, William K.; Steitz, Thomas A.

2013-09-19

During the assembly of the bacterial loader-dependent primosome, helicase loader proteins bind to the hexameric helicase ring, deliver it onto the oriC DNA and then dissociate from the complex. Here, to provide a better understanding of this key process, we report the crystal structure of the ~570-kDa prepriming complex between the Bacillus subtilis loader protein and the Bacillus stearothermophilus helicase, as well as the helicase-binding domain of primase with a molar ratio of 6:6:3 at 7.5 Å resolution. The overall architecture of the complex exhibits a three-layered ring conformation. Moreover, the structure combined with the proposed model suggests that themore » shift from the ‘open-ring’ to the ‘open-spiral’ and then the ‘closed-spiral’ state of the helicase ring due to the binding of single-stranded DNA may be the cause of the loader release.« less

Saccharomyces cerevisiae Sen1 Helicase Domain Exhibits 5'- to 3'-Helicase Activity with a Preference for Translocation on DNA Rather than RNA.

PubMed

Martin-Tumasz, Stephen; Brow, David A

2015-09-18

In the yeast Saccharomyces cerevisiae, the essential nuclear helicase Sen1 is required for efficient termination of transcription of short noncoding RNA genes by RNA polymerase II. However, the mechanism by which Sen1 promotes transcription termination is not known. Prior biochemical studies on the Sen1 homolog from Schizosaccharomyces pombe showed that it can bind and unwind both DNA and RNA, but the S. pombe protein is not essential and has not been demonstrated to function in transcription. Furthermore, Sen1 from either yeast has not previously been expressed as a recombinant protein, due to its large molecular mass (252 kDa in S. cerevisiae). Here, we report the purification and characterization of the 89-kDa S. cerevisiae Sen1 helicase domain (Sen1-HD) produced in Escherichia coli. Sen1-HD binds single-stranded RNA and DNA with similar affinity in the absence of ATP, but it binds RNA more stably than DNA in the presence of ATP, apparently due to a slower translocation rate on RNA. Translocation occurs in the 5' to 3' direction, as for the S. pombe protein. When purified from E. coli at a moderate salt concentration, Sen1-HD was associated with short RNAs that are enriched for the trinucleotide repeat (CAN)4. We propose that Sen1 binds to RNAs and prevents their stable pairing with DNA, consistent with in vivo studies by others showing increased R-loop (RNA/DNA hybrid) formation when Sen1 activity is impaired by mutations. Our results are consistent with a model in which Sen1 promotes transcription termination by resolving R-loops. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Rational Drug Discovery of HCV Helicase Inhibitor: Improved Docking Accuracy with Multiple Seeding in AutoDock Vina and In Situ Minimization.

PubMed

Lim, See K; Othman, Rozana; Yusof, Rohana; Heh, Choon H

2017-01-01

Hepatitis C is a significant cause for end-stage liver diseases and liver transplantation which affects approximately 3% of the global populations. Despite the current several direct antiviral agents in the treatment of Hepatitis C, the standard treatment for HCV infection is accompanied by several drawbacks, such as adverse side effects, high pricing of medications and the rapid emerging rate of resistant HCV variants. To discover potential inhibitors for HCV helicase through an optimized in silico approach. In this study, a homology model (HCV Genotype 3 helicase) was used as the target and screened through a benzopyran-based virtual library. Multiple-seedings of AutoDock Vina and in situ minimization were to account for the non-deterministic nature of AutoDock Vina search algorithm and binding site flexibility, respectively. ADME/T and interaction analyses were also done on the top hits via FAFDRUG3 web server and Discovery Studio 4.5. This study involved the development of an improved flow for virtual screening via implemention of multiple-seeding screening approach and in situ minimization. With the new docking protocol, the redocked standards have shown better RMSD value in reference to their native conformations. Ten benzopyran-like compounds with satisfactory physicochemical properties were discovered to be potential inhibitors of HCV helicase. ZINC38649350 was identified as the most potential inhibitor. Ten potential HCV helicase inhibitors were discovered via a new docking optimization protocol with better docking accuracy. These findings could contribute to the discovery of novel HCV antivirals and serve as an alternative approach of in silico rational drug discovery. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

BLM helicase facilitates telomere replication during leading strand synthesis of telomeres

PubMed Central

Kosiyatrakul, Settapong T.

2015-01-01

Based on its in vitro unwinding activity on G-quadruplex (G4) DNA, the Bloom syndrome–associated helicase BLM is proposed to participate in telomere replication by aiding fork progression through G-rich telomeric DNA. Single molecule analysis of replicated DNA (SMARD) was used to determine the contribution of BLM helicase to telomere replication. In BLM-deficient cells, replication forks initiating from origins within the telomere, which copy the G-rich strand by leading strand synthesis, moved slower through the telomere compared with the adjacent subtelomere. Fork progression through the telomere was further slowed in the presence of a G4 stabilizer. Using a G4-specific antibody, we found that deficiency of BLM, or another G4-unwinding helicase, the Werner syndrome-associated helicase WRN, resulted in increased G4 structures in cells. Importantly, deficiency of either helicase led to greater increases in G4 DNA detected in the telomere compared with G4 seen genome-wide. Collectively, our findings are consistent with BLM helicase facilitating telomere replication by resolving G4 structures formed during copying of the G-rich strand by leading strand synthesis. PMID:26195664

Putative SF2 helicases of the early-branching eukaryote Giardia lamblia are involved in antigenic variation and parasite differentiation into cysts

PubMed Central

2012-01-01

Background Regulation of surface antigenic variation in Giardia lamblia is controlled post-transcriptionally by an RNA-interference (RNAi) pathway that includes a Dicer-like bidentate RNase III (gDicer). This enzyme, however, lacks the RNA helicase domain present in Dicer enzymes from higher eukaryotes. The participation of several RNA helicases in practically all organisms in which RNAi was studied suggests that RNA helicases are potentially involved in antigenic variation, as well as during Giardia differentiation into cysts. Results An extensive in silico analysis of the Giardia genome identified 32 putative Super Family 2 RNA helicases that contain almost all the conserved RNA helicase motifs. Phylogenetic studies and sequence analysis separated them into 22 DEAD-box, 6 DEAH-box and 4 Ski2p-box RNA helicases, some of which are homologs of well-characterized helicases from higher organisms. No Giardia putative helicase was found to have significant homology to the RNA helicase domain of Dicer enzymes. Additionally a series of up- and down-regulated putative RNA helicases were found during encystation and antigenic variation by qPCR experiments. Finally, we were able to recognize 14 additional putative helicases from three different families (RecQ family, Swi2/Snf2 and Rad3 family) that could be considered DNA helicases. Conclusions This is the first comprehensive analysis of the Super Family 2 helicases from the human intestinal parasite G. lamblia. The relative and variable expression of particular RNA helicases during both antigenic variation and encystation agrees with the proposed participation of these enzymes during both adaptive processes. The putatives RNA and DNA helicases identified in this early-branching eukaryote provide initial information regarding the biological role of these enzymes in cell adaptation and differentiation. PMID:23190735

CD4+ T-cell recovery with suppressive ART-induced rapid sequence evolution in hepatitis C virus envelope but not NS3.

PubMed

Liu, Lin; Nardo, David; Li, Eric; Wang, Gary P

2016-03-13

CD4 T-cell depletion from HIV infection leads to a global decline in anti-hepatitis C virus (HCV) envelope neutralizing antibody (nAb) response, which may play a role in accelerating liver fibrosis. An increase in anti-HCV nAb titers has been reported during antiretroviral therapy (ART) but its impact on HCV remains poorly understood. The objective of this study is to determine the effects of ART on long-term HCV evolution. We examined HCV quasispecies structure and long-term evolution in HIV/HCV coinfected patients with ART-induced CD4 T-cell recovery, and compared with patients with CD4 T-cell depletion from delayed ART. We applied a single-variant sequencing (SVS) method to construct authentic viral quasispecies and compared sequence evolution in HCV envelope, the primary target for humoral immune responses, and NS3, a target for cellular immunity, between the two cohorts. The SVS method corrected biases known to skew the proportions of viral variants, revealing authentic HCV quasispeices structures. We observed higher rates of HCV envelope sequence evolution in patients with ART-induced CD4 T-cell recovery, compared with patients with CD4 T-cell depletion from delayed ART (P = 0.03). Evolutionary rates for NS3 were considerably lower than the rates for envelope (P < 0.01), with no significant difference observed between the two groups. ART-induced CD4 T-cell recovery results in rapid sequence evolution in HCV envelope, but not in NS3. These results suggest that suppressive ART disproportionally enhances HCV-specific humoral responses more than cellular responses, resulting in rapid sequence evolution in HCV envelope but not NS3.

Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus.

PubMed

Mutso, Margit; Morro, Ainhoa Moliner; Smedberg, Cecilia; Kasvandik, Sergo; Aquilimeba, Muriel; Teppor, Mona; Tarve, Liisi; Lulla, Aleksei; Lulla, Valeria; Saul, Sirle; Thaa, Bastian; McInerney, Gerald M; Merits, Andres; Varjak, Margus

2018-04-27

Infection by Chikungunya virus (CHIKV) of the Old World alphaviruses (family Togaviridae) in humans can cause arthritis and arthralgia. The virus encodes four non-structural proteins (nsP) (nsP1, nsp2, nsP3 and nsP4) that act as subunits of the virus replicase. These proteins also interact with numerous host proteins and some crucial interactions are mediated by the unstructured C-terminal hypervariable domain (HVD) of nsP3. In this study, a human cell line expressing EGFP tagged with CHIKV nsP3 HVD was established. Using quantitative proteomics, it was found that CHIKV nsP3 HVD can bind cytoskeletal proteins, including CD2AP, SH3KBP1, CAPZA1, CAPZA2 and CAPZB. The interaction with CD2AP was found to be most evident; its binding site was mapped to the second SH3 ligand-like element in nsP3 HVD. Further assessment indicated that CD2AP can bind to nsP3 HVDs of many different New and Old World alphaviruses. Mutation of the short binding element hampered the ability of the virus to establish infection. The mutation also abolished ability of CD2AP to co-localise with nsP3 and replication complexes of CHIKV; the same was observed for Semliki Forest virus (SFV) harbouring a similar mutation. Similar to CD2AP, its homolog SH3KBP1 also bound the identified motif in CHIKV and SFV nsP3.

The organization and contribution of helicases to RNA splicing.

PubMed

De, Inessa; Schmitzová, Jana; Pena, Vladimir

2016-01-01

Splicing is an essential step of gene expression. It occurs in two consecutive chemical reactions catalyzed by a large protein-RNA complex named the spliceosome. Assembled on the pre-mRNA substrate from five small nuclear proteins, the spliceosome acts as a protein-controlled ribozyme to catalyze the two reactions and finally dissociates into its components, which are re-used for a new round of splicing. Upon following this cyclic pathway, the spliceosome undergoes numerous intermediate stages that differ in composition as well as in their internal RNA-RNA and RNA-protein contacts. The driving forces and control mechanisms of these remodeling processes are provided by specific molecular motors called RNA helicases. While eight spliceosomal helicases are present in all organisms, higher eukaryotes contain five additional ones potentially required to drive a more intricate splicing pathway and link it to an RNA metabolism of increasing complexity. Spliceosomal helicases exhibit a notable structural diversity in their accessory domains and overall architecture, in accordance with the diversity of their task-specific functions. This review summarizes structure-function knowledge about all spliceosomal helicases, including the latter five, which traditionally are treated separately from the conserved ones. The implications of the structural characteristics of helicases for their functions, as well as for their structural communication within the multi-subunits environment of the spliceosome, are pointed out. © 2016 Wiley Periodicals, Inc.

Measuring Helicase Inhibition of the DEAD-box Protein Dbp2 by Yra1

PubMed Central

Ma, Wai Kit; Tran, Elizabeth J.

2016-01-01

Despite the highly conserved helicase core, individual DEAD-box proteins are specialized in diverse RNA metabolic processes. One mechanism that determines DEAD-box protein specificity is enzymatic regulation by other protein cofactors. In this chapter, we describe a protocol for purifying the Saccharomyces cerevisiae DEAD-box RNA helicase Dbp2 and RNA-binding protein Yra1 and subsequent analysis of helicase regulation. The experiments described here can be adapted to RNA helicase and purified co-factor. PMID:25579587

RNA helicase proteins as chaperones and remodelers

PubMed Central

Jarmoskaite, Inga; Russell, Rick

2014-01-01

Superfamily 2 helicase proteins are ubiquitous in RNA biology and have an extraordinarily broad set of functional roles. Central among these roles are to promote rearrangements of structured RNAs and to remodel RNA-protein complexes (RNPs), allowing formation of native RNA structure or progression through a functional cycle of structures. While all superfamily 2 helicases share a conserved helicase core, they are divided evolutionarily into several families, and it is principally proteins from three families, the DEAD-box, DEAH/RHA and Ski2-like families, that function to manipulate structured RNAs and RNPs. Strikingly, there are emerging differences in the mechanisms of these proteins, both between families and within the largest family (DEAD-box), and these differences appear to be tuned to their RNA or RNP substrates and their specific roles. This review outlines basic mechanistic features of the three families and surveys individual proteins and the current understanding of their biological substrates and mechanisms. PMID:24635478

microRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3' untranslated region: function in replication and influence of RNA secondary structure.

PubMed

Gerresheim, Gesche K; Dünnes, Nadia; Nieder-Röhrmann, Anika; Shalamova, Lyudmila A; Fricke, Markus; Hofacker, Ivo; Höner Zu Siederdissen, Christian; Marz, Manja; Niepmann, Michael

2017-02-01

We have analyzed the binding of the liver-specific microRNA-122 (miR-122) to three conserved target sites of hepatitis C virus (HCV) RNA, two in the non-structural protein 5B (NS5B) coding region and one in the 3' untranslated region (3'UTR). miR-122 binding efficiency strongly depends on target site accessibility under conditions when the range of flanking sequences available for the formation of local RNA secondary structures changes. Our results indicate that the particular sequence feature that contributes most to the correlation between target site accessibility and binding strength varies between different target sites. This suggests that the dynamics of miRNA/Ago2 binding not only depends on the target site itself but also on flanking sequence context to a considerable extent, in particular in a small viral genome in which strong selection constraints act on coding sequence and overlapping cis-signals and model the accessibility of cis-signals. In full-length genomes, single and combination mutations in the miR-122 target sites reveal that site 5B.2 is positively involved in regulating overall genome replication efficiency, whereas mutation of site 5B.3 showed a weaker effect. Mutation of the 3'UTR site and double or triple mutants showed no significant overall effect on genome replication, whereas in a translation reporter RNA, the 3'UTR target site inhibits translation directed by the HCV 5'UTR. Thus, the miR-122 target sites in the 3'-region of the HCV genome are involved in a complex interplay in regulating different steps of the HCV replication cycle.

Virtual screening of commercial cyclic peptides as NS2B-NS3 protease inhibitor of dengue virus serotype 2 through molecular docking simulation

NASA Astrophysics Data System (ADS)

Nasution, M. A. F.; Aini, R. N.; Tambunan, U. S. F.

2017-04-01

A disease caused by dengue virus infection has become one of the major health problems in the world, particularly in Asia, Africa, and South America. This disease has become endemic in more than 100 countries, and approximately 100 million cases occur each year with 2.5 billion people or 40% of the world population at risk of having this virus infection. Therefore, we need an antiviral drug that can inhibit the activity of the enzymes that involved in the virus replication in the body. Lately, the peptide-based drug design has been developed and proved to have interesting pharmacological properties. This study uses commercially cyclic peptides that have already marketed. The purpose of this study is to screen the commercial cyclic peptides that can be used as an inhibitor of the NS2B-NS3 protease of dengue virus serotype 2 (DENV-2) through molecular docking simulations. Inhibition of NS3 protease enzyme can lead to enzymatic inhibition activity so the formed polyprotein from the translation of RNA cannot be cut into pieces and remain in the long strand form. Consequently, proteins that are vital for the sustainability of dengue virus replication cannot be formed. This research resulted in [alpha]-ANF (1-28), rat, Brain Natriuretic Peptide, porcine, Atrial Natriuretic Factor (3-28) (human) and Atrial Natriuretic Peptide (126-150) (rat) as the best drug candidate for inhibiting the NS2B-NS3 protease of DENV-2.

Recombinant dengue 2 virus NS3 protein conserves structural antigenic and immunological properties relevant for dengue vaccine design.

PubMed

Ramírez, Rosa; Falcón, Rosabel; Izquierdo, Alienys; García, Angélica; Alvarez, Mayling; Pérez, Ana Beatriz; Soto, Yudira; Muné, Mayra; da Silva, Emiliana Mandarano; Ortega, Oney; Mohana-Borges, Ronaldo; Guzmán, María G

2014-10-01

The NS3 protein is a multifunctional non-structural protein of flaviviruses implicated in the polyprotein processing. The predominance of cytotoxic T cell lymphocytes epitopes on the NS3 protein suggests a protective role of this protein in limiting virus replication. In this work, we studied the antigenicity and immunogenicity of a recombinant NS3 protein of the Dengue virus 2. The full-length NS3 gene was cloned and expressed as a His-tagged fusion protein in Escherichia coli. The pNS3 protein was purified by two chromatography steps. The recombinant NS3 protein was recognized by anti-protease NS3 polyclonal antibody and anti-DENV2 HMAF by Western Blot. This purified protein was able to stimulate the secretion of high levels of gamma interferon and low levels of interleukin-10 and tumor necrosis factor-α in mice splenocytes, suggesting a predominantly Th-1-type T cell response. Immunized BALB/c mice with the purified NS3 protein showed a strong induction of anti-NS3 IgG antibodies, essentially IgG2b, as determined by ELISA. Immunized mice sera with recombinant NS3 protein showed specific recognition of native dengue protein by Western blotting and immunofluorescence techniques. The successfully purified recombinant protein was able to preserv the structural and antigenic determinants of the native dengue protein. The antigenicity shown by the recombinant NS3 protein suggests its possible inclusion into future DENV vaccine preparations.

Broadening CD4+ and CD8+ T Cell Responses against Hepatitis C Virus by Vaccination with NS3 Overlapping Peptide Panels in Cross-Priming Liposomes

PubMed Central

Filskov, Jonathan; Mikkelsen, Marianne; Hansen, Paul R.; Christensen, Jan P.; Thomsen, Allan R.; Andersen, Peter; Agger, Else Marie

2017-01-01

ABSTRACT Despite the introduction of effective drugs to treat patients with chronic hepatitis C virus (HCV) infection, a vaccine would be the only means to substantially reduce the worldwide disease burden. An incomplete understanding of how HCV interacts with its human host and evades immune surveillance has hampered vaccine development. It is generally accepted that in infected individuals, a narrow repertoire of exhausted T cells is a hallmark of persistent infection, whereas broad, vigorous CD4+ and CD8+ T cell responses are associated with control of acute hepatitis C. We employed a vaccine approach based on a mixture of peptides (pepmix) spanning the entire sequence of HCV nonstructural protein 3 (NS3) in cross-priming cationic liposomes (CAF09) to facilitate a versatile presentation of all possible T cell epitopes, regardless of the HLA background of the vaccine recipient. Here, we demonstrate that vaccination of mice with NS3 pepmix broadens the repertoire of epitope-specific T cells compared to the corresponding recombinant protein (rNS3). Moreover, vaccination with rNS3 induced only CD4+ T cells, whereas the NS3 pepmix induced a far more vigorous CD4+ T cell response and was as potent a CD8+ T cell inducer as an adenovirus-vectored vaccine expressing NS3. Importantly, the cellular responses are dominated by multifunctional T cells, such as gamma interferon-positive (IFN-γ+) tumor necrosis factor alpha-positive (TNF-α+) coproducers, and displayed cytotoxic capacity in mice. In conclusion, we present a novel vaccine approach against HCV, inducing a broadened T cell response targeting both immunodominant and potential subdominant epitopes, which may be key elements to counter T cell exhaustion and prevent chronicity. IMPORTANCE With at least 700,000 annual deaths, development of a vaccine against hepatitis C virus (HCV) has high priority, but the tremendous ability of the virus to dodge the human immune system poses great challenges. Furthermore, many

DEAD-box Helicases as Integrators of RNA, Nucleotide and Protein Binding

PubMed Central

Putnam, Andrea A.

2013-01-01

DEAD-box helicases perform diverse cellular functions in virtually all steps of RNA metabolism from Bacteria to Humans. Although DEAD-box helicases share a highly conserved core domain, the enzymes catalyze a wide range of biochemical reactions. In addition to the well established RNA unwinding and corresponding ATPase activities, DEAD-box helicases promote duplex formation and displace proteins from RNA. They can also function as assembly platforms for larger ribonucleoprotein complexes, and as metabolite sensors. This review aims to provide a perspective on the diverse biochemical features of DEAD-box helicases and connections to structural information. We discuss these data in the context of a model that views the enzymes as integrators of RNA, nucleotide, and protein binding. PMID:23416748

Antiviral Activity and Resistance Analysis of NS3/4A Protease Inhibitor Grazoprevir and NS5A Inhibitor Elbasvir in Hepatitis C Virus GT4 Replicons.

PubMed

Asante-Appiah, Ernest; Curry, Stephanie; McMonagle, Patricia; Ingravallo, Paul; Chase, Robert; Nickle, David; Qiu, Ping; Howe, Anita; Lahser, Frederick C

2017-07-01

Although genotype 4 (GT4)-infected patients represent a minor overall percentage of the global hepatitis C virus (HCV)-infected population, the high prevalence of the genotype in specific geographic regions coupled with substantial sequence diversity makes it an important genotype to study for antiviral drug discovery and development. We evaluated two direct-acting antiviral agents-grazoprevir, an HCV NS3/4A protease inhibitor, and elbasvir, an HCV NS5A inhibitor-in GT4 replicons prior to clinical studies in this genotype. Following a bioinformatics analysis of available GT4 sequences, a set of replicons bearing representative GT4 clinical isolates was generated. For grazoprevir, the 50% effective concentration (EC 50 ) against the replicon bearing the reference GT4a (ED43) NS3 protease and NS4A was 0.7 nM. The median EC 50 for grazoprevir against chimeric replicons encoding NS3/4A sequences from GT4 clinical isolates was 0.2 nM (range, 0.11 to 0.33 nM; n = 5). The difficulty in establishing replicons bearing NS3/4A resistance-associated substitutions was substantially overcome with the identification of a G162R adaptive substitution in NS3. Single NS3 substitutions D168A/V identified from de novo resistance selection studies reduced grazoprevir antiviral activity by 137- and 47-fold, respectively, in the background of the G162R replicon. For elbasvir, the EC 50 against the replicon bearing the reference full-length GT4a (ED43) NS5A gene was 0.0002 nM. The median EC 50 for elbasvir against chimeric replicons bearing clinical isolates from GT4 was 0.0007 nM (range, 0.0002 to 34 nM; n = 14). De novo resistance selection studies in GT4 demonstrated a high propensity to suppress the emergence of amino acid substitutions that confer high-potency reductions to elbasvir. Phenotypic characterization of the NS5A amino acid substitutions identified (L30F, L30S, M31V, and Y93H) indicated that they conferred 15-, 4-, 2.5-, and 7.5-fold potency losses, respectively, to elbasvir

Pre-steady-state DNA unwinding by bacteriophage T4 Dda helicase reveals a monomeric molecular motor.

PubMed

Nanduri, Bindu; Byrd, Alicia K; Eoff, Robert L; Tackett, Alan J; Raney, Kevin D

2002-11-12

Helicases are molecular motor enzymes that unwind and translocate nucleic acids. One of the central questions regarding helicase activity is whether the process of coupling ATP hydrolysis to DNA unwinding requires an oligomeric form of the enzyme. We have applied a pre-steady-state kinetics approach to address this question with the bacteriophage T4 Dda helicase. If a helicase can function as a monomer, then the burst amplitude in the pre-steady state might be similar to the concentration of enzyme, whereas if the helicase required oligomerization, then the amplitude would be significantly less than the enzyme concentration. DNA unwinding of an oligonucleotide substrate was conducted by using a Kintek rapid quench-flow instrument. The substrate consisted of 12 bp adjacent to 12 nucleotides of single-stranded DNA. Dda (4 nM) was incubated with substrate (16 nM) in buffer, and the unwinding reaction was initiated by the addition of ATP (5 mM) and Mg(2+) (10 mM). The reaction was stopped by the addition of 400 mM EDTA. Product formation exhibited biphasic kinetics, and the data were fit to the equation for a single exponential followed by a steady state. The amplitude of the first phase was 3.5 +/- 0.2 nM, consistent with a monomeric helicase. The burst amplitude of product formation was measured over a range of enzyme and substrate concentrations and remained consistent with a functional monomer. Thus, Dda can rapidly unwind oligonucleotide substrates as a monomer, indicating that the functional molecular motor component of a helicase can reside within a single polypeptide.

The interaction of RNA helicase DDX3 with HIV-1 Rev-CRM1-RanGTP complex during the HIV replication cycle

DOE PAGES

Mahboobi, Seyed Hanif; Javanpour, Alex A.; Mofrad, Mohammad R. K.

2015-02-27

Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV’s reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNAmore » transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP.« less

The interaction of RNA helicase DDX3 with HIV-1 Rev-CRM1-RanGTP complex during the HIV replication cycle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mahboobi, Seyed Hanif; Javanpour, Alex A.; Mofrad, Mohammad R. K.

Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV’s reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNAmore » transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP.« less

Structure-based discovery of clinically approved drugs as Zika virus NS2B-NS3 protease inhibitors that potently inhibit Zika virus infection in vitro and in vivo.

PubMed

Yuan, Shuofeng; Chan, Jasper Fuk-Woo; den-Haan, Helena; Chik, Kenn Ka-Heng; Zhang, Anna Jinxia; Chan, Chris Chung-Sing; Poon, Vincent Kwok-Man; Yip, Cyril Chik-Yan; Mak, Winger Wing-Nga; Zhu, Zheng; Zou, Zijiao; Tee, Kah-Meng; Cai, Jian-Piao; Chan, Kwok-Hung; de la Peña, Jorge; Pérez-Sánchez, Horacio; Cerón-Carrasco, José Pedro; Yuen, Kwok-Yung

2017-09-01

Zika virus (ZIKV) infection may be associated with severe complications in fetuses and adults, but treatment options are limited. We performed an in silico structure-based screening of a large chemical library to identify potential ZIKV NS2B-NS3 protease inhibitors. Clinically approved drugs belonging to different drug classes were selected among the 100 primary hit compounds with the highest predicted binding affinities to ZIKV NS2B-NS3-protease for validation studies. ZIKV NS2B-NS3 protease inhibitory activity was validated in most of the selected drugs and in vitro anti-ZIKV activity was identified in two of them (novobiocin and lopinavir-ritonavir). Molecular docking and molecular dynamics simulations predicted that novobiocin bound to ZIKV NS2B-NS3-protease with high stability. Dexamethasone-immunosuppressed mice with disseminated ZIKV infection and novobiocin treatment had significantly (P < 0.05) higher survival rate (100% vs 0%), lower mean blood and tissue viral loads, and less severe histopathological changes than untreated controls. This structure-based drug discovery platform should facilitate the identification of additional enzyme inhibitors of ZIKV. Copyright © 2017 Elsevier B.V. All rights reserved.

Single-molecule fluorescence reveals the unwinding stepping mechanism of replicative helicase.

PubMed

Syed, Salman; Pandey, Manjula; Patel, Smita S; Ha, Taekjip

2014-03-27

Bacteriophage T7 gp4 serves as a model protein for replicative helicases that couples deoxythymidine triphosphate (dTTP) hydrolysis to directional movement and DNA strand separation. We employed single-molecule fluorescence resonance energy transfer methods to resolve steps during DNA unwinding by T7 helicase. We confirm that the unwinding rate of T7 helicase decreases with increasing base pair stability. For duplexes containing >35% guanine-cytosine (GC) base pairs, we observed stochastic pauses every 2-3 bp during unwinding. The dwells on each pause were distributed nonexponentially, consistent with two or three rounds of dTTP hydrolysis before each unwinding step. Moreover, we observed backward movements of the enzyme on GC-rich DNAs at low dTTP concentrations. Our data suggest a coupling ratio of 1:1 between base pairs unwound and dTTP hydrolysis, and they further support the concept that nucleic acid motors can have a hierarchy of different-sized steps or can accumulate elastic energy before transitioning to a subsequent phase. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Introgression of Chromosome 3Ns from Psathyrostachys huashanica into Wheat Specifying Resistance to Stripe Rust

PubMed Central

Kang, Houyang; Wang, Yi; Fedak, George; Cao, Wenguang; Zhang, Haiqin; Fan, Xing; Sha, Lina; Xu, Lili; Zheng, Youliang; Zhou, Yonghong

2011-01-01

Wheat stripe rust is a destructive disease in the cool and humid wheat-growing areas of the world. Finding diverse sources of stripe rust resistance is critical for increasing genetic diversity of resistance for wheat breeding programs. Stripe rust resistance was identified in the alien species Psathyrostachys huashanica, and a wheat- P. huashanica amphiploid line (PHW-SA) with stripe rust resistance was reported previously. In this study, a P. huashanica 3Ns monosomic addition line (PW11) with superior resistance to stripe rust was developed, which was derived from the cross between PHW-SA and wheat J-11. We evaluated the alien introgressions PW11-2, PW11-5 and PW11-8 which were derived from line PW11 for reaction to new Pst race CYR32, and used molecular and cytogenetic tools to characterize these lines. The introgressions were remarkably resistant to CYR32, suggesting that the resistance to stripe rust of the introgressions thus was controlled by gene(s) located on P. huashanica chromosome 3Ns. All derived lines were cytologically stable in term of meiotic chromosome behavior. Two 3Ns chromosomes of P. huashanica were detected in the disomic addition line PW11-2. Chromosomes 1B of substitution line PW11-5 had been replaced by a pair of P. huashanica 3Ns chromosomes. In PW11-8, a small terminal segment from P. huashanica chromosome arm 3NsS was translocated to the terminal region of wheat chromosomes 3BL. Thus, this translocated chromosome is designated T3BL-3NsS. These conclusions were further confirmed by SSR analyses. Two 3Ns-specific markers Xgwm181 and Xgwm161 will be useful to rapidly identify and trace the translocated fragments. These introgressions, which had significant characteristics of resistance to stripe rust, could be utilized as novel germplasms for wheat breeding. PMID:21760909

A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase.

PubMed

Levin, Mikhail K; Gurjar, Madhura; Patel, Smita S

2005-05-01

Helicases translocate along their nucleic acid substrates using the energy of ATP hydrolysis and by changing conformations of their nucleic acid-binding sites. Our goal is to characterize the conformational changes of hepatitis C virus (HCV) helicase at different stages of ATPase cycle and to determine how they lead to translocation. We have reported that ATP binding reduces HCV helicase affinity for nucleic acid. Now we identify the stage of the ATPase cycle responsible for translocation and unwinding. We show that a rapid directional movement occurs upon helicase binding to DNA in the absence of ATP, resulting in opening of several base pairs. We propose that HCV helicase translocates as a Brownian motor with a simple two-stroke cycle. The directional movement step is fueled by single-stranded DNA binding energy while ATP binding allows for a brief period of random movement that prepares the helicase for the next cycle.

Structural and biochemical basis for the difference in the helicase activity of two different constructs of SARS-CoV helicase.

PubMed

Adedeji, A O; Singh, K; Sarafianos, S G

2012-12-22

The non—structural protein 13 (nsp13) of Severe Acute Respiratory Syndrome Coronavirus (SARS—CoV) is a helicase that separates double—stranded RNA or DNA with a 5'—3' polarity, using the energy of nucleotide hydrolysis. We have previously determined the minimal mechanism of helicase function by nsp13 where we demonstrated that the enzyme unwinds nucleic acid in discrete steps of 9.3 base—pairs each with a catalytic rate of 30 steps per second. In that study we used different constructs of nsp13 (GST and H6 constructs). GST—nsp13 showed much more efficient nucleic acid unwinding than the H6—tagged counterpart. At 0.1 second, more than 50% of the ATP is hydrolyzed by GST—nsp13 compared to less than 5% ATP hydrolysis by H6—nsp13. Interestingly, the two constructs have the same binding affinity for nucleic acids. We, therefore propose that the difference in the catalytic efficiency of these two constructs is due to the interference of ATP binding by the histidine tag at the amino—terminus of nsp13.

Zirconium catalyzed synthesis of 2-arylidene Indan-1,3-diones and evaluation of their inhibitory activity against NS2B-NS3 WNV protease.

PubMed

Oliveira, Ana Flávia C da S; de Souza, Ana Paula M; de Oliveira, André S; da Silva, Milene L; de Oliveira, Fabrício M; Santos, Edjon G; da Silva, Ítalo Esposti P; Ferreira, Rafaela S; Villela, Filipe S; Martins, Felipe T; Leal, Daniel H S; Vaz, Boniek G; Teixeira, Róbson R; de Paula, Sergio O

2018-04-10

A simple and efficient Knoevenagel procedure for the synthesis of 2-arylidene indan-1,3-diones is herein reported. These compounds were prepared via ZrOCl2·8H2O catalyzed reactions of indan-1,3-dione with several aromatic aldehydes and using water as the solvent. The 2-arylidene indan-1,3-diones were obtained with 53%-95% yield within 10-45 min. The synthesized compounds were evaluated as inhibitors of the NS2B-NS3 protease of West Nile Virus (WNV). It was found that hydroxylated derivatives impaired enzyme activity with varying degrees of effectiveness. The most active hydroxylated derivatives, namely 2-(4-hydroxybenzylidene)-1H-indene-1,3(2H)-dione (14) and 2-(3,4-dihydroxybenzylidene)-1H-indene-1,3(2H)-dione (17), were characterized as noncompetitive enzymes inhibitors, with IC 50 values of 11 μmol L -1 and 3 μmol L -1 , respectively. Docking and electrostatic potential surfaces investigations provided insight on the possible binding mode of the most active compounds within an allosteric site. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

In-silico identification and evaluation of plant flavonoids as dengue NS2B/NS3 protease inhibitors using molecular docking and simulation approach.

PubMed

Qamar, Muhammad Tahirul; Ashfaq, Usman Ali; Tusleem, Kishver; Mumtaz, Arooj; Tariq, Quratulain; Goheer, Alina; Ahmed, Bilal

2017-11-01

Dengue infection is prevailing among the people not only from the developing countries but also from the developed countries due to its high morbidity rate around the globe. Hence, due to the unavailability of any suitable vaccine for rigorous dengue virus (DENV), the only mode of its treatment is prevention. The circumstances require an urgent development of efficient and practical treatment to deal with these serotypes. The severe effects and cost of synthetic vaccines simulated researchers to find anti-viral agents from medicinal plants. Flavonoids present in medicinal plants, holds anti-viral activity and can be used as vaccine against viruses. Therefore, present study was planned to find anti-viral potential of 2500 flavonoids inhibitors against the DENVNS2B/NS3 protease through computational screening which can hinder the viral replication within the host cell. By using molecular docking, it was revealed that flavonoids showed strong and stable bonding in the binding pocket of DENV NS2B/NS3 protease and had strong interactions with catalytic triad. Drug capability and anti-dengue potential of the flavonoids was also evaluated by using different bioinformatics tools. Some flavonoids effectively blocked the catalytic triad of DENV NS2B/NS3 protease and also passed through drug ability evaluation. It can be concluded from this study that these flavonoids could act as potential inhibitors to stop the replication of DENV and there is a need to study the action of these molecules in-vitro to confirm their action and other properties.

A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease

PubMed Central

Liu, Binbin; Zhang, Jing; Koetzner, Cheri A.; Jones, Susan A.; Lin, Qishan

2017-01-01

The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC) to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2) in vitro, with IC50 values of 1.8 μM, 11.4 μM, and 4.8 μM, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV), West Nile virus (WNV), and Yellow fever virus (YFV) on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 μM on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and mutagenesis

Characterization of biochemical properties of Bacillus subtilis RecQ helicase.

PubMed

Qin, Wei; Liu, Na-Nv; Wang, Lijun; Zhou, Min; Ren, Hua; Bugnard, Elisabeth; Liu, Jie-Lin; Zhang, Lin-Hu; Vendôme, Jeremie; Hu, Jin-Shan; Xi, Xu Guang

2014-12-01

RecQ family helicases function as safeguards of the genome. Unlike Escherichia coli, the Gram-positive Bacillus subtilis bacterium possesses two RecQ-like homologues, RecQ[Bs] and RecS, which are required for the repair of DNA double-strand breaks. RecQ[Bs] also binds to the forked DNA to ensure a smooth progression of the cell cycle. Here we present the first biochemical analysis of recombinant RecQ[Bs]. RecQ[Bs] binds weakly to single-stranded DNA (ssDNA) and blunt-ended double-stranded DNA (dsDNA) but strongly to forked dsDNA. The protein exhibits a DNA-stimulated ATPase activity and ATP- and Mg(2+)-dependent DNA helicase activity with a 3' → 5' polarity. Molecular modeling shows that RecQ[Bs] shares high sequence and structure similarity with E. coli RecQ. Surprisingly, RecQ[Bs] resembles the truncated Saccharomyces cerevisiae Sgs1 and human RecQ helicases more than RecQ[Ec] with regard to its enzymatic activities. Specifically, RecQ[Bs] unwinds forked dsDNA and DNA duplexes with a 3'-overhang but is inactive on blunt-ended dsDNA and 5'-overhung duplexes. Interestingly, RecQ[Bs] unwinds blunt-ended DNA with structural features, including nicks, gaps, 5'-flaps, Kappa joints, synthetic replication forks, and Holliday junctions. We discuss these findings in the context of RecQ[Bs]'s possible functions in preserving genomic stability. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The DEAD-box helicase eIF4A: paradigm or the odd one out?

PubMed

Andreou, Alexandra Z; Klostermeier, Dagmar

2013-01-01

DEAD-box helicases catalyze the ATP-dependent unwinding of RNA duplexes. They share a helicase core formed by two RecA-like domains that carries a set of conserved motifs contributing to ATP binding and hydrolysis, RNA binding and duplex unwinding. The translation initiation factor eIF4A is the founding member of the DEAD-box protein family, and one of the few examples of DEAD-box proteins that consist of a helicase core only. It is an RNA-stimulated ATPase and a non-processive helicase that unwinds short RNA duplexes. In the catalytic cycle, a series of conformational changes couples the nucleotide cycle to RNA unwinding. eIF4A has been considered a paradigm for DEAD-box proteins, and studies of its function have revealed the governing principles underlying the DEAD-box helicase mechanism. However, as an isolated helicase core, eIF4A is rather the exception, not the rule. Most helicase modules in other DEAD-box proteins are modified, some by insertions into the RecA-like domains, and the majority by N- and C-terminal appendages. While the basic catalytic function resides within the helicase core, its modulation by insertions, additional domains or a network of interaction partners generates the diversity of DEAD-box protein functions in the cell. This review summarizes the current knowledge on eIF4A and its regulation, and discusses to what extent eIF4A serves as a model DEAD-box protein.

DDX3 DEAD-box RNA helicase plays a central role in mitochondrial protein quality control in Leishmania

PubMed Central

Padmanabhan, Prasad Kottayil; Zghidi-Abouzid, Ouafa; Samant, Mukesh; Dumas, Carole; Aguiar, Bruno Guedes; Estaquier, Jerome; Papadopoulou, Barbara

2016-01-01

DDX3 is a highly conserved member of ATP-dependent DEAD-box RNA helicases with multiple functions in RNA metabolism and cellular signaling. Here, we describe a novel function for DDX3 in regulating the mitochondrial stress response in the parasitic protozoan Leishmania. We show that genetic inactivation of DDX3 leads to the accumulation of mitochondrial reactive oxygen species (ROS) associated with a defect in hydrogen peroxide detoxification. Upon stress, ROS production is greatly enhanced, causing mitochondrial membrane potential loss, mitochondrial fragmentation, and cell death. Importantly, this phenotype is exacerbated upon oxidative stress in parasites forced to use the mitochondrial oxidative respiratory machinery. Furthermore, we show that in the absence of DDX3, levels of major components of the unfolded protein response as well as of polyubiquitinated proteins increase in the parasite, particularly in the mitochondrion, as an indicator of mitochondrial protein damage. Consistent with these findings, immunoprecipitation and mass-spectrometry studies revealed potential interactions of DDX3 with key components of the cellular stress response, particularly the antioxidant response, the unfolded protein response, and the AAA-ATPase p97/VCP/Cdc48, which is essential in mitochondrial protein quality control by driving proteosomal degradation of polyubiquitinated proteins. Complementation studies using DDX3 deletion mutants lacking conserved motifs within the helicase core support that binding of DDX3 to ATP is essential for DDX3's function in mitochondrial proteostasis. As a result of the inability of DDX3-depleted Leishmania to recover from ROS damage and to survive various stresses in the host macrophage, parasite intracellular development was impaired. Collectively, these observations support a central role for the Leishmania DDX3 homolog in preventing ROS-mediated damage and in maintaining mitochondrial protein quality control. PMID:27735940

Helicase Dependent Isothermal Amplification of DNA and RNA using Self-Avoiding Molecular Recognition Systems

PubMed Central

Yang, Zunyi; McLendon, Chris; Hutter, Daniel; Bradley, Kevin M.; Hoshika, Shuichi; Frye, Carole; Benner, Steven A.

2015-01-01

Assays that target DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low resource environments, requiring equipment and power that may not be available in these environments. However, isothermal procedures that avoid thermal cycling are often confounded by primer dimers, off-target priming, and other artifacts. Here, we show how a “self avoiding molecular recognition system” (SAMRS) eliminates these artifacts to give clean amplicons in a helicase-dependent isothermal amplification (SAMRS-HDA). We also show that incorporating SAMRS into the 3′-ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS-HDA can be twofold multiplexed, something difficult to achieve with HDA using standard primers. This shows that SAMRS-HDA is a more versatile approach than standard HDA with a broader applicability for xNA-targeted diagnostics and research. PMID:25953623

Crystal structure of full-length Zika virus NS5 protein reveals a conformation similar to Japanese encephalitis virus NS5

DOE Office of Scientific and Technical Information (OSTI.GOV)

Upadhyay, Anup K.; Cyr, Matthew; Longenecker, Kenton

The rapid spread of the recentZika virus(ZIKV) epidemic across various countries in the American continent poses a major health hazard for the unborn fetuses of pregnant women. To date, there is no effective medical intervention. The nonstructural protein 5 ofZika virus(ZIKV-NS5) is critical for ZIKV replication through the 5'-RNA capping and RNA polymerase activities present in its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent RNA polymerase (RdRp) domains, respectively. The crystal structure of the full-length ZIKV-NS5 protein has been determined at 3.05 Å resolution from a crystal belonging to space groupP2 12 12 and containing two protein molecules in the asymmetricmore » unit. The structure is similar to that reported for the NS5 protein fromJapanese encephalitis virusand suggests opportunities for structure-based drug design targeting either its MTase or RdRp domain.« less

Structure and sequence based functional annotation of Zika virus NS2b protein: Computational insights.

PubMed

Aguilera-Pesantes, Daniel; Méndez, Miguel A

2017-10-28

While Zika virus (ZIKV) outbreaks are a growing concern for global health, a deep understanding about the virus is lacking. Here we report a contribution to the basic science on the virus- a detailed computational analysis of the non structural protein NS2b. This protein acts as a cofactor for the NS3 protease (NS3Pro) domain that is important on the viral life cycle, and is an interesting target for drug development. We found that ZIKV NS2b cofactor is highly similar to other virus within the Flavivirus genus, especially to West Nile Virus, suggesting that it is completely necessary for the protease complex activity. Furthermore, the ZIKV NS2b has an important role to the function and stability of the ZIKV NS3 protease domain even when presents a low conservation score. In addition, ZIKV NS2b is mostly rigid, which could imply a non dynamic nature in substrate recognition. Finally, by performing a computational alanine scanning mutagenesis, we found that residues Gly 52 and Asp 83 in the NS2b could be important in substrate recognition. Copyright © 2017 Elsevier Inc. All rights reserved.

Accessory replicative helicases and the replication of protein-bound DNA.

PubMed

Brüning, Jan-Gert; Howard, Jamieson L; McGlynn, Peter

2014-12-12

Complete, accurate duplication of the genetic material is a prerequisite for successful cell division. Achieving this accuracy is challenging since there are many barriers to replication forks that may cause failure to complete genome duplication or result in possibly catastrophic corruption of the genetic code. One of the most important types of replicative barriers are proteins bound to the template DNA, especially transcription complexes. Removal of these barriers demands energy input not only to separate the DNA strands but also to disrupt multiple bonds between the protein and DNA. Replicative helicases that unwind the template DNA for polymerases at the fork can displace proteins bound to the template. However, even occasional failures in protein displacement by the replicative helicase could spell disaster. In such circumstances, failure to restart replication could result in incomplete genome duplication. Avoiding incomplete genome duplication via the repair and restart of blocked replication forks also challenges viability since the involvement of recombination enzymes is associated with the risk of genome rearrangements. Organisms have therefore evolved accessory replicative helicases that aid replication fork movement along protein-bound DNA. These helicases reduce the dangers associated with replication blockage by protein-DNA complexes, aiding clearance of blocks and resumption of replication by the same replisome thus circumventing the need for replication repair and restart. This review summarises recent work in bacteria and eukaryotes that has begun to delineate features of accessory replicative helicases and their importance in genome stability. Copyright © 2014. Published by Elsevier Ltd.

Multiple Sensing Application on Wireless Sensor Network Simulation using NS3

NASA Astrophysics Data System (ADS)

Kurniawan, I. F.; Bisma, R.

2018-01-01

Hardware enhancement provides opportunity to install various sensor device on single monitoring node which then enables users to acquire multiple data simultaneously. Constructing multiple sensing application in NS3 is a challenging task since numbers of aspects such as wireless communication, packet transmission pattern, and energy model must be taken into account. Despite of numerous types of monitoring data available, this study only considers two types such as periodic, and event-based data. Periodical data will generate monitoring data follows configured interval, while event-based transmit data when certain determined condition is met. Therefore, this study attempts to cover mentioned aspects in NS3. Several simulations are performed with different number of nodes on arbitrary communication scheme.

Eukaryotic Replicative Helicase Subunit Interaction with DNA and Its Role in DNA Replication

PubMed Central

Martinez, Matthew P.; Wacker, Amanda L.; Bruck, Irina; Kaplan, Daniel L.

2017-01-01

The replicative helicase unwinds parental double-stranded DNA at a replication fork to provide single-stranded DNA templates for the replicative polymerases. In eukaryotes, the replicative helicase is composed of the Cdc45 protein, the heterohexameric ring-shaped Mcm2-7 complex, and the tetrameric GINS complex (CMG). The CMG proteins bind directly to DNA, as demonstrated by experiments with purified proteins. The mechanism and function of these DNA-protein interactions are presently being investigated, and a number of important discoveries relating to how the helicase proteins interact with DNA have been reported recently. While some of the protein-DNA interactions directly relate to the unwinding function of the enzyme complex, other protein-DNA interactions may be important for minichromosome maintenance (MCM) loading, origin melting or replication stress. This review describes our current understanding of how the eukaryotic replicative helicase subunits interact with DNA structures in vitro, and proposed models for the in vivo functions of replicative helicase-DNA interactions are also described. PMID:28383499

Bromovirus RNA Replication Compartment Formation Requires Concerted Action of 1a's Self-Interacting RNA Capping and Helicase Domains

PubMed Central

Diaz, Arturo; Gallei, Andreas

2012-01-01

All positive-strand RNA viruses replicate their genomes in association with rearranged intracellular membranes such as single- or double-membrane vesicles. Brome mosaic virus (BMV) RNA synthesis occurs in vesicular endoplasmic reticulum (ER) membrane invaginations, each induced by many copies of viral replication protein 1a, which has N-terminal RNA capping and C-terminal helicase domains. Although the capping domain is responsible for 1a membrane association and ER targeting, neither this domain nor the helicase domain was sufficient to induce replication vesicle formation. Moreover, despite their potential for mutual interaction, the capping and helicase domains showed no complementation when coexpressed in trans. Cross-linking showed that the capping and helicase domains each form trimers and larger multimers in vivo, and the capping domain formed extended, stacked, hexagonal lattices in vivo. Furthermore, coexpressing the capping domain blocked the ability of full-length 1a to form replication vesicles and replicate RNA and recruited full-length 1a into mixed hexagonal lattices with the capping domain. Thus, BMV replication vesicle formation and RNA replication depend on the direct linkage and concerted action of 1a's self-interacting capping and helicase domains. In particular, the capping domain's strong dominant-negative effects showed that the ability of full-length 1a to form replication vesicles was highly sensitive to disruption by non-productively titrating lattice-forming self-interactions of the capping domain. These and other findings shed light on the roles and interactions of 1a domains in replication compartment formation and support prior results suggesting that 1a induces replication vesicles by forming a capsid-like interior shell. PMID:22090102

Chloroplast- or Mitochondria-Targeted DEAD-Box RNA Helicases Play Essential Roles in Organellar RNA Metabolism and Abiotic Stress Responses

PubMed Central

Nawaz, Ghazala; Kang, Hunseung

2017-01-01

The yields and productivity of crops are greatly diminished by various abiotic stresses, including drought, cold, heat, and high salinity. Chloroplasts and mitochondria are cellular organelles that can sense diverse environmental stimuli and alter gene expression to cope with adverse environmental stresses. Organellar gene expression is mainly regulated at posttranscriptional levels, including RNA processing, intron splicing, RNA editing, RNA turnover, and translational control, during which a variety of nucleus-encoded RNA-binding proteins (RBPs) are targeted to chloroplasts or mitochondria where they play essential roles in organellar RNA metabolism. DEAD-box RNA helicases (RHs) are enzymes that can alter RNA structures and affect RNA metabolism in all living organisms. Although a number of DEAD-box RHs have been found to play important roles in RNA metabolism in the nucleus and cytoplasm, our understanding on the roles of DEAD-box RHs in the regulation of RNA metabolism in chloroplasts and mitochondria is only at the beginning. Considering that organellar RNA metabolism and gene expression are tightly regulated by anterograde signaling from the nucleus, it is imperative to determine the functions of nucleus-encoded organellar RBPs. In this review, we summarize the emerging roles of nucleus-encoded chloroplast- or mitochondria-targeted DEAD-box RHs in organellar RNA metabolism and plant response to diverse abiotic stresses. PMID:28596782

The helicase HAGE expressed by malignant melanoma-initiating cells is required for tumor cell proliferation in vivo.

PubMed

Linley, Adam J; Mathieu, Morgan G; Miles, Amanda K; Rees, Robert C; McArdle, Stephanie E B; Regad, Tarik

2012-04-20

Malignant melanoma-initiating cells (MMIC) are a subpopulation of cells responsible for melanoma tumor growth and progression. They are defined by the expression of the ATP-binding cassette (ABC) subfamily B member 5 (ABCB5). Here, we identified a critical role for the DEAD-box helicase antigen (HAGE) in ABCB5+ MMIC-dependent tumorigenesis and show that HAGE-specific inactivation inhibits melanoma tumor growth mediated by this tumor-initiating population. Knockdown of HAGE led to a significant decrease in RAS protein expression with a concomitant decrease in activation of the AKT and ERK signaling pathways implicated to play an important role in melanoma progression. To confirm that the reduction in NRAS (Neuroblastoma RAS) expression was dependent on the HAGE helicase activity, we showed that NRAS, effectively silenced by siRNA, could be rescued by reintroduction of HAGE in cells lacking HAGE. Furthermore, we provide a mechanism by which HAGE promotes NRAS unwinding in vitro. We also observed using tumor transplantation in Non-obese diabetic/severe combined immunodeficiency mice that the HAGE knockdown in a ABCB5+ melanoma cell line displayed a significant decrease in tumor growth and compared with the control. Our results suggest that the helicase HAGE is required for ABCB5+ MMIC-dependent tumor growth through promoting RAS protein expression and that cancer therapies targeting HAGE helicase may have broad applications for treating malignant melanoma and potentially other cancer types.

The Helicase HAGE Expressed by Malignant Melanoma-Initiating Cells Is Required for Tumor Cell Proliferation in Vivo*

PubMed Central

Linley, Adam J.; Mathieu, Morgan G.; Miles, Amanda K.; Rees, Robert C.; McArdle, Stephanie E. B.; Regad, Tarik

2012-01-01

Malignant melanoma-initiating cells (MMIC) are a subpopulation of cells responsible for melanoma tumor growth and progression. They are defined by the expression of the ATP-binding cassette (ABC) subfamily B member 5 (ABCB5). Here, we identified a critical role for the DEAD-box helicase antigen (HAGE) in ABCB5+ MMIC-dependent tumorigenesis and show that HAGE-specific inactivation inhibits melanoma tumor growth mediated by this tumor-initiating population. Knockdown of HAGE led to a significant decrease in RAS protein expression with a concomitant decrease in activation of the AKT and ERK signaling pathways implicated to play an important role in melanoma progression. To confirm that the reduction in NRAS (Neuroblastoma RAS) expression was dependent on the HAGE helicase activity, we showed that NRAS, effectively silenced by siRNA, could be rescued by reintroduction of HAGE in cells lacking HAGE. Furthermore, we provide a mechanism by which HAGE promotes NRAS unwinding in vitro. We also observed using tumor transplantation in Non-obese diabetic/severe combined immunodeficiency mice that the HAGE knockdown in a ABCB5+ melanoma cell line displayed a significant decrease in tumor growth and compared with the control. Our results suggest that the helicase HAGE is required for ABCB5+ MMIC-dependent tumor growth through promoting RAS protein expression and that cancer therapies targeting HAGE helicase may have broad applications for treating malignant melanoma and potentially other cancer types. PMID:22393060

ClpXP protease targets long-lived DNA translocation states of a helicase-like motor to cause restriction alleviation

PubMed Central

Simons, Michelle; Diffin, Fiona M.; Szczelkun, Mark D.

2014-01-01

We investigated how Escherichia coli ClpXP targets the helicase-nuclease (HsdR) subunit of the bacterial Type I restriction–modification enzyme EcoKI during restriction alleviation (RA). RA is a temporary reduction in endonuclease activity that occurs when Type I enzymes bind unmodified recognition sites on the host genome. These conditions arise upon acquisition of a new system by a naïve host, upon generation of new sites by genome rearrangement/mutation or during homologous recombination between hemimethylated DNA. Using recombinant DNA and proteins in vitro, we demonstrate that ClpXP targets EcoKI HsdR during dsDNA translocation on circular DNA but not on linear DNA. Protein roadblocks did not activate HsdR proteolysis. We suggest that DNA translocation lifetime, which is elevated on circular DNA relative to linear DNA, is important to RA. To identify the ClpX degradation tag (degron) in HsdR, we used bioinformatics and biochemical assays to design N- and C-terminal mutations that were analysed in vitro and in vivo. None of the mutants produced a phenotype consistent with loss of the degron, suggesting an as-yet-unidentified recognition pathway. We note that an EcoKI nuclease mutant still produces cell death in a clpx− strain, consistent with DNA damage induced by unregulated motor activity. PMID:25260590

Distinct functions of human RecQ helicases during DNA replication.

PubMed

Urban, Vaclav; Dobrovolna, Jana; Janscak, Pavel

2017-06-01

DNA replication is the most vulnerable process of DNA metabolism in proliferating cells and therefore it is tightly controlled and coordinated with processes that maintain genomic stability. Human RecQ helicases are among the most important factors involved in the maintenance of replication fork integrity, especially under conditions of replication stress. RecQ helicases promote recovery of replication forks being stalled due to different replication roadblocks of either exogenous or endogenous source. They prevent generation of aberrant replication fork structures and replication fork collapse, and are involved in proper checkpoint signaling. The essential role of human RecQ helicases in the genome maintenance during DNA replication is underlined by association of defects in their function with cancer predisposition. Copyright © 2016 Elsevier B.V. All rights reserved.

The crystal structure of Zika virus NS5 reveals conserved drug targets.

PubMed

Duan, Wenqian; Song, Hao; Wang, Haiyuan; Chai, Yan; Su, Chao; Qi, Jianxun; Shi, Yi; Gao, George F

2017-04-03

Zika virus (ZIKV) has emerged as major health concern, as ZIKV infection has been shown to be associated with microcephaly, severe neurological disease and possibly male sterility. As the largest protein component within the ZIKV replication complex, NS5 plays key roles in the life cycle and survival of the virus through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent RNA polymerase (RdRp) domains. Here, we present the crystal structures of ZIKV NS5 MTase in complex with an RNA cap analogue ( m7 GpppA) and the free NS5 RdRp. We have identified the conserved features of ZIKV NS5 MTase and RdRp structures that could lead to development of current antiviral inhibitors being used against flaviviruses, including dengue virus and West Nile virus, to treat ZIKV infection. These results should inform and accelerate the structure-based design of antiviral compounds against ZIKV. © 2017 The Authors.

GINS complex protein Sld5 recruits SIK1 to activate MCM helicase during DNA replication.

PubMed

Joshi, Kiranmai; Shah, Varun Jayeshkumar; Maddika, Subbareddy

2016-12-01

In eukaryotes, proper loading and activation of MCM helicase at chromosomal origins plays a central role in DNA replication. Activation of MCM helicase requires its association with CDC45-GINS complex, but the mechanism of how this complex activates MCM helicase is poorly understood. Here we identified SIK1 (salt-inducible kinase 1), an AMPK related protein kinase, as a molecular link that connects GINS complex with MCM helicase activity. We demonstrated that Sld5 a component of GINS complex interacts with SIK1 and recruits it to the sites of DNA replication at the onset of S phase. Depletion of SIK1 leads to defective DNA replication. Further, we showed that SIK1 phosphorylates MCM2 at five conserved residues at its N-terminus, which is essential for the activation of MCM helicase. Collectively, our results suggest SIK1 as a novel integral component of CMG replicative helicase during eukaryotic DNA replication. Copyright © 2016 Elsevier Inc. All rights reserved.

MCM Paradox: Abundance of Eukaryotic Replicative Helicases and Genomic Integrity.

PubMed

Das, Mitali; Singh, Sunita; Pradhan, Satyajit; Narayan, Gopeshwar

2014-01-01

As a crucial component of DNA replication licensing system, minichromosome maintenance (MCM) 2-7 complex acts as the eukaryotic DNA replicative helicase. The six related MCM proteins form a heterohexamer and bind with ORC, CDC6, and Cdt1 to form the prereplication complex. Although the MCMs are well known as replicative helicases, their overabundance and distribution patterns on chromatin present a paradox called the "MCM paradox." Several approaches had been taken to solve the MCM paradox and describe the purpose of excess MCMs distributed beyond the replication origins. Alternative functions of these MCMs rather than a helicase had also been proposed. This review focuses on several models and concepts generated to solve the MCM paradox coinciding with their helicase function and provides insight into the concept that excess MCMs are meant for licensing dormant origins as a backup during replication stress. Finally, we extend our view towards the effect of alteration of MCM level. Though an excess MCM constituent is needed for normal cells to withstand stress, there must be a delineation of the threshold level in normal and malignant cells. This review also outlooks the future prospects to better understand the MCM biology.

The RNA helicase RHAU (DHX36) suppresses expression of the transcription factor PITX1.

PubMed

Booy, Evan P; Howard, Ryan; Marushchak, Oksana; Ariyo, Emmanuel O; Meier, Markus; Novakowski, Stefanie K; Deo, Soumya R; Dzananovic, Edis; Stetefeld, Jörg; McKenna, Sean A

2014-03-01

RNA Helicase associated with AU-rich element (RHAU) (DHX36) is a DEAH (Aspartic acid, Glumatic Acid, Alanine, Histidine)-box RNA helicase that can bind and unwind G4-quadruplexes in DNA and RNA. To detect novel RNA targets of RHAU, we performed an RNA co-immunoprecipitation screen and identified the PITX1 messenger RNA (mRNA) as specifically and highly enriched. PITX1 is a homeobox transcription factor with roles in both development and cancer. Primary sequence analysis identified three probable quadruplexes within the 3'-untranslated region of the PITX1 mRNA. Each of these sequences, when isolated, forms stable quadruplex structures that interact with RHAU. We provide evidence that these quadruplexes exist in the endogenous mRNA; however, we discovered that RHAU is tethered to the mRNA via an alternative non-quadruplex-forming region. RHAU knockdown by small interfering RNA results in significant increases in PITX1 protein levels with only marginal changes in mRNA, suggesting a role for RHAU in translational regulation. Involvement of components of the microRNA machinery is supported by similar and non-additive increases in PITX1 protein expression on Dicer and combined RHAU/Dicer knockdown. We also demonstrate a requirement of argonaute-2, a key RNA-induced silencing complex component, to mediate RHAU-dependent changes in PITX1 protein levels. These results demonstrate a novel role for RHAU in microRNA-mediated translational regulation at a quadruplex-containing 3'-untranslated region.

The Flavivirus Protease As a Target for Drug Discovery

PubMed Central

Brecher, Matthew; Zhang, Jing; Li, Hongmin

2014-01-01

Many flaviviruses are significant human pathogens causing considerable disease burdens, including encephalitis and hemorrhagic fever, in the regions in which they are endemic. A paucity of treatments for flaviviral infections has driven interest in drug development targeting proteins essential to flavivirus replication, such as the viral protease. During viral replication, the flavivirus genome is translated as a single polyprotein precursor, which must be cleaved into individual proteins by a complex of the viral protease, NS3, and its cofactor, NS2B. Because this cleavage is an obligate step of the viral life-cycle, the flavivirus protease is an attractive target for antiviral drug development. In this review, we will survey recent drug development studies targeting the NS3 active site, as well as studies targeting an NS2B/NS3 interaction site determined from flavivirus protease crystal structures. PMID:24242363

The flavivirus protease as a target for drug discovery.

PubMed

Brecher, Matthew; Zhang, Jing; Li, Hongmin

2013-12-01

Many flaviviruses are significant human pathogens causing considerable disease burdens, including encephalitis and hemorrhagic fever, in the regions in which they are endemic. A paucity of treatments for flaviviral infections has driven interest in drug development targeting proteins essential to flavivirus replication, such as the viral protease. During viral replication, the flavivirus genome is translated as a single polyprotein precursor, which must be cleaved into individual proteins by a complex of the viral protease, NS3, and its cofactor, NS2B. Because this cleavage is an obligate step of the viral life-cycle, the flavivirus protease is an attractive target for antiviral drug development. In this review, we will survey recent drug development studies targeting the NS3 active site, as well as studies targeting an NS2B/NS3 interaction site determined from flavivirus protease crystal structures.

Natural prevalence of resistance-associated variants in hepatitis C virus NS5A in genotype 3a-infected people who inject drugs in Germany.

PubMed

Walker, Andreas; Siemann, Holger; Groten, Svenja; Ross, R Stefan; Scherbaum, Norbert; Timm, Jörg

2015-09-01

People who inject drugs (PWID) are the most important risk group for incident Hepatitis C virus (HCV) infection. In PWID in Europe HCV genotype 3a is highly prevalent. Unfortunately, many of the recently developed directly acting antiviral drugs against HCV (DAAs) are suboptimal for treatment of this genotype. Detection of resistance-associated variants (RAV) in genotype 3a may help to optimize treatment decisions, however, robust protocols for amplification and sequencing of HCV NS5A as an important target for treatment of genotype 3a are currently lacking. The aim of this study was to establish a protocol for sequencing of HCV NS5A in genotype 3a and to determine the frequency of RAVs in treatment-naïve PWID living in Germany. The full NS5A region was amplified and sequenced from 110 HCV genotype 3a infected PWID using an in-house PCR protocol. With the established protocol the complete NS5A region was successfully amplified and sequenced from 110 out of 112 (98.2%) genotype 3a infected PWID. Phylogenetic analysis of sequences from PWID together with unrelated genotype 3a sequences from a public database showed a scattered distribution without geographic clustering. Viral polymorphisms A30K and Y93H known to confer resistance in a GT3a replication model were present in 8 subjects (7.2%). A protocol for amplification of nearly all GT3a samples was successfully established. Substitutions conferring resistance to NS5A inhibitors were detected in a few treatment-naive PWID. Copyright © 2015 Elsevier B.V. All rights reserved.

Ctf4 Is a Hub in the Eukaryotic Replisome that Links Multiple CIP-Box Proteins to the CMG Helicase.

PubMed

Villa, Fabrizio; Simon, Aline C; Ortiz Bazan, Maria Angeles; Kilkenny, Mairi L; Wirthensohn, David; Wightman, Mel; Matak-Vinkovíc, Dijana; Pellegrini, Luca; Labib, Karim

2016-08-04

Replisome assembly at eukaryotic replication forks connects the DNA helicase to DNA polymerases and many other factors. The helicase binds the leading-strand polymerase directly, but is connected to the Pol α lagging-strand polymerase by the trimeric adaptor Ctf4. Here, we identify new Ctf4 partners in addition to Pol α and helicase, all of which contain a "Ctf4-interacting-peptide" or CIP-box. Crystallographic analysis classifies CIP-boxes into two related groups that target different sites on Ctf4. Mutations in the CIP-box motifs of the Dna2 nuclease or the rDNA-associated protein Tof2 do not perturb DNA synthesis genome-wide, but instead lead to a dramatic shortening of chromosome 12 that contains the large array of rDNA repeats. Our data reveal unexpected complexity of Ctf4 function, as a hub that connects multiple accessory factors to the replisome. Most strikingly, Ctf4-dependent recruitment of CIP-box proteins couples other processes to DNA synthesis, including rDNA copy-number regulation. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

HCV NS3 protease enhances liver fibrosis via binding to and activating TGF-β type I receptor

NASA Astrophysics Data System (ADS)

Sakata, Kotaro; Hara, Mitsuko; Terada, Takaho; Watanabe, Noriyuki; Takaya, Daisuke; Yaguchi, So-Ichi; Matsumoto, Takehisa; Matsuura, Tomokazu; Shirouzu, Mikako; Yokoyama, Shigeyuki; Yamaguchi, Tokio; Miyazawa, Keiji; Aizaki, Hideki; Suzuki, Tetsuro; Wakita, Takaji; Imoto, Masaya; Kojima, Soichi

2013-11-01

Viruses sometimes mimic host proteins and hijack the host cell machinery. Hepatitis C virus (HCV) causes liver fibrosis, a process largely mediated by the overexpression of transforming growth factor (TGF)-β and collagen, although the precise underlying mechanism is unknown. Here, we report that HCV non-structural protein 3 (NS3) protease affects the antigenicity and bioactivity of TGF-β2 in (CAGA)9-Luc CCL64 cells and in human hepatic cell lines via binding to TGF-β type I receptor (TβRI). Tumor necrosis factor (TNF)-α facilitates this mechanism by increasing the colocalization of TβRI with NS3 protease on the surface of HCV-infected cells. An anti-NS3 antibody against computationally predicted binding sites for TβRI blocked the TGF-β mimetic activities of NS3 in vitro and attenuated liver fibrosis in HCV-infected chimeric mice. These data suggest that HCV NS3 protease mimics TGF-β2 and functions, at least in part, via directly binding to and activating TβRI, thereby enhancing liver fibrosis.

Staphylococcal SCCmec elements encode an active MCM-like helicase and thus may be replicative

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mir-Sanchis, Ignacio; Roman, Christina A.; Misiura, Agnieszka

2016-08-29

Methicillin-resistant Staphylococcus aureus (MRSA) is a public-health threat worldwide. Although the mobile genomic island responsible for this phenotype, staphylococcal cassette chromosome (SCC), has been thought to be nonreplicative, we predicted DNA-replication-related functions for some of the conserved proteins encoded by SCC. We show that one of these, Cch, is homologous to the self-loading initiator helicases of an unrelated family of genomic islands, that it is an active 3'-to-5' helicase and that the adjacent ORF encodes a single-stranded DNA–binding protein. Our 2.9-Å crystal structure of intact Cch shows that it forms a hexameric ring. Cch, like the archaeal and eukaryotic MCM-familymore » replicative helicases, belongs to the pre–sensor II insert clade of AAA+ ATPases. Additionally, we found that SCC elements are part of a broader family of mobile elements, all of which encode a replication initiator upstream of their recombinases. Replication after excision would enhance the efficiency of horizontal gene transfer.« less

A chloroplast-targeted cabbage DEAD-box RNA helicase BrRH22 confers abiotic stress tolerance to transgenic Arabidopsis plants by affecting translation of chloroplast transcripts.

PubMed

Nawaz, Ghazala; Lee, Kwanuk; Park, Su Jung; Kim, Yeon-Ok; Kang, Hunseung

2018-06-01

Although the roles of many DEAD-box RNA helicases (RHs) have been determined in the nucleus as well as in cytoplasm during stress responses, the importance of chloroplast-targeted DEAD-box RHs in stress response remains largely unknown. In this study, we determined the function of BrRH22, a chloroplast-targeted DEAD-box RH in cabbage (Brassica rapa), in abiotic stress responses. The expression of BrRH22 was markedly increased by drought, heat, salt, or cold stress and by ABA treatment, but was largely decreased by UV stress. Expression of BrRH22 in Arabidopsis enhanced germination and plantlet growth under high salinity or drought stress. BrRH22-expressing plants displayed a higher cotyledon greening and better plantlet growth upon ABA treatment due to decreases in the levels of ABI3, ABI4, and ABI5. Further, BrRH22 affected translation of several chloroplast transcripts under stress. Notably, BrRH22 had RNA chaperone function. These results altogether suggest that chloroplast-transported BrRH22 contributes positively to the response of transgenic Arabidopsis to abiotic stress by affecting translation of chloroplast genes via its RNA chaperone activity. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.

PubMed

Hochstrasser, Megan L; Taylor, David W; Bhat, Prashant; Guegler, Chantal K; Sternberg, Samuel H; Nogales, Eva; Doudna, Jennifer A

2014-05-06

In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

Naturally occurring mutations associated with resistance to HCV NS5B polymerase and NS3 protease inhibitors in treatment-naïve patients with chronic hepatitis C.

PubMed

Costantino, Angela; Spada, Enea; Equestre, Michele; Bruni, Roberto; Tritarelli, Elena; Coppola, Nicola; Sagnelli, Caterina; Sagnelli, Evangelista; Ciccaglione, Anna Rita

2015-11-14

The detection of baseline resistance mutations to new direct-acting antivirals (DAAs) in HCV chronically infected treatment-naïve patients could be important for their management and outcome prevision. In this study, we investigated the presence of mutations, which have been previously reported to be associated with resistance to DAAs in HCV polymerase (NS5B) and HCV protease (NS3) regions, in sera of treatment-naïve patients. HCV RNA from 152 naïve patients (84 % Italian and 16 % immigrants from various countries) infected with different HCV genotypes (21,1a; 21, 1b; 2, 2a; 60, 2c; 22, 3a; 25, 4d and 1, 4k) was evaluated for sequence analysis. Amplification and sequencing of fragments in the NS5B (nt 8256-8640) and NS3 (nt 3420-3960) regions of HCV genome were carried out for 152 and 28 patients, respectively. The polymorphism C316N/H in NS5B region, associated with resistance to sofosbuvir, was detected in 9 of the 21 (43 %) analysed sequences from genotype 1b-infected patients. Naturally occurring mutations V36L, and M175L in the NS3 protease region were observed in 100 % of patients infected with subtype 2c and 4. A relevant proportion of treatment naïve genotype 1b infected patients evaluated in this study harboured N316 polymorphism and might poorly respond to sofosbuvir treatment. As sofosbuvir has been approved for treatment of HCV chronic infection in USA and Europe including Italy, pre-treatment testing for N316 polymorphism on genotype 1b naïve patients should be considered for this drug.

Structure of the eukaryotic replicative CMG helicase suggests a pumpjack motion for translocation

DOE PAGES

Yuan, Zuanning; Bai, Lin; Sun, Jingchuan; ...

2016-02-08

The CMG helicase is composed of Cdc45, Mcm2–7 and GINS. Here we report the structure of the Saccharomyces cerevisiae CMG, determined by cryo-EM at a resolution of 3.7–4.8 Å. The structure reveals that GINS and Cdc45 scaffold the N tier of the helicase while enabling motion of the AAA+ C tier. CMG exists in two alternating conformations, compact and extended, thus suggesting that the helicase moves like an inchworm. The N-terminal regions of Mcm2–7, braced by Cdc45–GINS, form a rigid platform upon which the AAA+ C domains make longitudinal motions, nodding up and down like an oil-rig pumpjack attached tomore » a stable platform. The Mcm ring is remodeled in CMG relative to the inactive Mcm2–7 double hexamer. In addition, the Mcm5 winged-helix domain is inserted into the central channel, thus blocking entry of double-stranded DNA and supporting a steric-exclusion DNA-unwinding model.« less

TES/Aura L2 Ozone (O3) Nadir V6 (TL2O3NS)

Atmospheric Science Data Center

2018-01-22

TES/Aura L2 Ozone (O3) Nadir (TL2O3NS) News:  TES News Join ... Project Title:  TES Discipline:  Tropospheric Composition Version:  V6 Level:  L2 Platform:  TES/Aura L2 Ozone Spatial Coverage:  5.3 x 8.5 km nadir ...

Molecular Mechanism by Which a Potent Hepatitis C Virus NS3-NS4A Protease Inhibitor Overcomes Emergence of Resistance

PubMed Central

O'Meara, Jeff A.; Lemke, Christopher T.; Godbout, Cédrickx; Kukolj, George; Lagacé, Lisette; Moreau, Benoît; Thibeault, Diane; White, Peter W.; Llinàs-Brunet, Montse

2013-01-01

Although optimizing the resistance profile of an inhibitor can be challenging, it is potentially important for improving the long term effectiveness of antiviral therapy. This work describes our rational approach toward the identification of a macrocyclic acylsulfonamide that is a potent inhibitor of the NS3-NS4A proteases of all hepatitis C virus genotypes and of a panel of genotype 1-resistant variants. The enhanced potency of this compound versus variants D168V and R155K facilitated x-ray determination of the inhibitor-variant complexes. In turn, these structural studies revealed a complex molecular basis of resistance and rationalized how such compounds are able to circumvent these mechanisms. PMID:23271737

MCM Paradox: Abundance of Eukaryotic Replicative Helicases and Genomic Integrity

PubMed Central

Das, Mitali; Singh, Sunita; Pradhan, Satyajit

2014-01-01

As a crucial component of DNA replication licensing system, minichromosome maintenance (MCM) 2–7 complex acts as the eukaryotic DNA replicative helicase. The six related MCM proteins form a heterohexamer and bind with ORC, CDC6, and Cdt1 to form the prereplication complex. Although the MCMs are well known as replicative helicases, their overabundance and distribution patterns on chromatin present a paradox called the “MCM paradox.” Several approaches had been taken to solve the MCM paradox and describe the purpose of excess MCMs distributed beyond the replication origins. Alternative functions of these MCMs rather than a helicase had also been proposed. This review focuses on several models and concepts generated to solve the MCM paradox coinciding with their helicase function and provides insight into the concept that excess MCMs are meant for licensing dormant origins as a backup during replication stress. Finally, we extend our view towards the effect of alteration of MCM level. Though an excess MCM constituent is needed for normal cells to withstand stress, there must be a delineation of the threshold level in normal and malignant cells. This review also outlooks the future prospects to better understand the MCM biology. PMID:25386362

Dna2 nuclease-helicase structure, mechanism and regulation by Rpa.

PubMed

Zhou, Chun; Pourmal, Sergei; Pavletich, Nikola P

2015-11-02

The Dna2 nuclease-helicase maintains genomic integrity by processing DNA double-strand breaks, Okazaki fragments and stalled replication forks. Dna2 requires ssDNA ends, and is dependent on the ssDNA-binding protein Rpa, which controls cleavage polarity. Here we present the 2.3 Å structure of intact mouse Dna2 bound to a 15-nucleotide ssDNA. The nuclease active site is embedded in a long, narrow tunnel through which the DNA has to thread. The helicase domain is required for DNA binding but not threading. We also present the structure of a flexibly-tethered Dna2-Rpa interaction that recruits Dna2 to Rpa-coated DNA. We establish that a second Dna2-Rpa interaction is mutually exclusive with Rpa-DNA interactions and mediates the displacement of Rpa from ssDNA. This interaction occurs at the nuclease tunnel entrance and the 5' end of the Rpa-DNA complex. Hence, it only displaces Rpa from the 5' but not 3' end, explaining how Rpa regulates cleavage polarity.

To peep into Pif1 helicase: multifaceted all the way from genome stability to repair-associated DNA synthesis.

PubMed

Chung, Woo-Hyun

2014-02-01

Pif1 DNA helicase is the prototypical member of a 5' to 3' helicase superfamily conserved from bacteria to humans. In Saccharomyces cerevisiae, Pif1 and its homologue Rrm3, localize in both mitochondria and nucleus playing multiple roles in the maintenance of genomic homeostasis. They display relatively weak processivities in vitro, but have largely non-overlapping functions on common genomic loci such as mitochondrial DNA, telomeric ends, and many replication forks especially at hard-to-replicate regions including ribosomal DNA and G-quadruplex structures. Recently, emerging evidence shows that Pif1, but not Rrm3, has a significant new role in repair-associated DNA synthesis with Polδ during homologous recombination stimulating D-loop migration for conservative DNA replication. Comparative genetic and biochemical studies on the structure and function of Pif1 family helicases across different biological systems are further needed to elucidate both diversity and specificity of their mechanisms of action that contribute to genome stability.

Srs2 overexpression reveals a helicase-independent role at replication forks that requires diverse cell functions

PubMed Central

León Ortiz, Ana María; Reid, Robert J. D.; Dittmar, John C.; Rothstein, Rodney; Nicolas, Alain

2011-01-01

Srs2 is a 3’ to 5’ DNA helicase that regulates many aspects of DNA metabolism in Saccharomyces cerevisiae. It is best known for its ability to counteract homologous recombination by dismantling Rad51 filaments, but is also involved in checkpoint activation, adaptation and recovery, and in resolution of late recombination intermediates. To further address its biological roles and uncover new genetic interactions, we examined the consequences of overexpressing SRS2 as well as two helicase-dead mutants, srs2-K41A and srs2-K41R, in the collection of 4827 yeast haploid deletion mutants. We identified 274 genes affecting a large variety of cellular functions that are required for cell growth when SRS2 or its mutants are overexpressed. Further analysis of these interactions reveals that Srs2 acts independently of its helicase function at replication forks likely through its recruitment by the sumoylated PCNA replication clamp. This helicase-independent function is responsible for the negative interactions with DNA metabolism genes and for the toxicity of SRS2 overexpression in many of the diverse cellular pathways revealed in our screens. PMID:21459050

DNA binding polarity, dimerization, and ATPase ring remodeling in the CMG helicase of the eukaryotic replisome

PubMed Central

Costa, Alessandro; Renault, Ludovic; Swuec, Paolo; Petojevic, Tatjana; Pesavento, James J; Ilves, Ivar; MacLellan-Gibson, Kirsty; Fleck, Roland A; Botchan, Michael R; Berger, James M

2014-01-01

The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3′ single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational and assembly states as it matures into a functional helicase. DOI: http://dx.doi.org/10.7554/eLife.03273.001 PMID:25117490

TES/Aura L2 Ammonia (NH3) Nadir V6 (TL2NH3NS)

Atmospheric Science Data Center

2018-01-22

TES/Aura L2 Ammonia (NH3) Nadir (TL2NH3NS) News:  TES News ... Level:  L2 Platform:  TES/Aura L2 Ammonia Spatial Coverage:  5.3 x 8.5 km nadir ... Contact ASDC User Services Parameters:  Ammonia Legacy:  Retired data product , click here for ...

Structural Insights into the Regulation of Foreign Genes in Salmonella by the Hha/H-NS Complex*

PubMed Central

Ali, Sabrina S.; Whitney, John C.; Stevenson, James; Robinson, Howard; Howell, P. Lynne; Navarre, William Wiley

2013-01-01

The bacterial nucleoid-associated proteins Hha and H-NS jointly repress horizontally acquired genes in Salmonella, including essential virulence loci encoded within Salmonella pathogenicity islands. Hha is known to interact with the N-terminal dimerization domain of H-NS; however, the manner in which this interaction enhances transcriptional silencing is not understood. To further understand this process, we solved the x-ray crystal structure of Hha in complex with the N-terminal dimerization domain of H-NS (H-NS(1–46)) to 3.2 Å resolution. Two monomers of Hha bind to symmetrical sites on either side of the H-NS(1–46) dimer. Disruption of the Hha/H-NS interaction by the H-NS site-specific mutation I11A results in increased expression of the Hha/H-NS co-regulated gene hilA without affecting the expression levels of proV, a target gene repressed by H-NS in an Hha-independent fashion. Examination of the structure revealed a cluster of conserved basic amino acids that protrude from the surface of Hha on the opposite side of the Hha/H-NS(1–46) interface. Hha mutants with a diminished positively charged surface maintain the ability to interact with H-NS but can no longer regulate hilA. Increased expression of the hilA locus did not correspond to significant depletion of H-NS at the promoter region in chromatin immunoprecipitation assays. However, in vitro, we find Hha improves H-NS binding to target DNA fragments. Taken together, our results show for the first time how Hha and H-NS interact to direct transcriptional repression and reveal that a positively charged surface of Hha enhances the silencing activity of H-NS nucleoprotein filaments. PMID:23515315

Combined 3D-QSAR, molecular docking, molecular dynamics simulation, and binding free energy calculation studies on the 5-hydroxy-2H-pyridazin-3-one derivatives as HCV NS5B polymerase inhibitors.

PubMed

Yu, Haijing; Fang, Yu; Lu, Xia; Liu, Yongjuan; Zhang, Huabei

2014-01-01

The NS5B RNA-dependent RNA polymerase (RdRP) is a promising therapeutic target for developing novel anti-hepatitis C virus (HCV) drugs. In this work, a combined molecular modeling study was performed on a series of 193 5-hydroxy-2H-pyridazin-3-one derivatives as inhibitors of HCV NS5B Polymerase. The best 3D-QSAR models, including CoMFA and CoMSIA, are based on receptor (or docking). Furthermore, a 40-ns molecular dynamics (MD) simulation and binding free energy calculations using docked structures of NS5B with ten compounds, which have diverse structures and pIC50 values, were employed to determine the detailed binding process and to compare the binding modes of the inhibitors with different activities. On one side, the stability and rationality of molecular docking and 3D-QSAR results were validated by MD simulation. The binding free energies calculated by the MM-PBSA method gave a good correlation with the experimental biological activity. On the other side, by analyzing some differences between the molecular docking and the MD simulation results, we can find that the MD simulation could also remedy the defects of molecular docking. The analyses of the combined molecular modeling results have identified that Tyr448, Ser556, and Asp318 are the key amino acid residues in the NS5B binding pocket. The results from this study can provide some insights into the development of novel potent NS5B inhibitors. © 2013 John Wiley & Sons A/S.

Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity

PubMed Central

Allonso, Diego; Andrade, Iamara S.; Conde, Jonas N.; Coelho, Diego R.; Rocha, Daniele C. P.; da Silva, Manuela L.; Ventura, Gustavo T.

2015-01-01

ABSTRACT Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. IMPORTANCE Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the

Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity.

PubMed

Allonso, Diego; Andrade, Iamara S; Conde, Jonas N; Coelho, Diego R; Rocha, Daniele C P; da Silva, Manuela L; Ventura, Gustavo T; Silva, Emiliana M; Mohana-Borges, Ronaldo

2015-12-01

Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the intracellular and the

Dilatonic parallelizable NS-NS backgrounds

NASA Astrophysics Data System (ADS)

Kawano, Teruhiko; Yamaguchi, Satoshi

2003-08-01

We complete the classification of parallelizable NS-NS backgrounds in type II supergravity by adding the dilatonic case to the result of Figueroa-O'Farrill on the non-dilatonic case. We also study the supersymmetry of these parallelizable backgrounds. It is shown that all the dilatonic parallelizable backgrounds have sixteen supersymmetries.

Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116

NASA Astrophysics Data System (ADS)

Zhang, Yuan; Palla, Mirkó; Sun, Andrew; Liao, Jung-Chi

2013-09-01

DEAD-box RNA helicases are ATP-dependent proteins implicated in nearly all aspects of RNA metabolism. The yeast DEAD-box helicase Mss116 is unique in its functions of splicing group I and group II introns and activating mRNA translation, but the structural understanding of why it performs these unique functions remains unclear. Here we used sequence analysis and molecular dynamics simulation to identify residues in the flexible linker specific for yeast Mss116, potentially associated with its unique functions. We first identified residues that are 100% conserved in Mss116 of different species of the Saccharomycetaceae family. The amino acids of these conserved residues were then compared with the amino acids of the corresponding residue positions of other RNA helicases to identify residues that have distinct amino acids from other DEAD-box proteins. Four residues in the flexible linker, i.e. N334, E335, P336 and H339, are conserved and Mss116-specific. Molecular dynamics simulation was conducted for the wild-type Mss116 structure and mutant models to examine mutational effects of the linker on the conformational equilibrium. Relatively short MD simulation runs (within 20 ns) were enough for us to observe mutational effects, suggesting serious structural perturbations by these mutations. The mutation of E335 depletes the interactions between E335 and K95 in domain 1. The interactions between N334/P336 and N496/I497 of domain 2 are also abolished by mutation. Our results suggest that tight interactions between the Mss116-specific flexible linker and the two RecA-like domains may be mechanically required to crimp RNA for the unique RNA processes of yeast Mss116.

Molecular Docking Based Screening of Plant Flavonoids as Dengue NS1 Inhibitors

PubMed Central

Qamar, Muhammad Tahir ul; Mumtaz, Arooj; Naseem, Rabbia; Ali, Amna; Fatima, Tabeer; Jabbar, Tehreem; Ahmad, Zubair; Ashfaq, Usman Ali

2014-01-01

Dengue infection has turned into a serious health concern globally due to its high morbidity rate and a high possibility of increase in its mortality rate on the account of unavailability of any proper treatment for severe dengue infection. The situation demands an urgent development of efficient and practicable treatment to deal with Dengue virus (DENV). Flavonoids, a class of phytochemicals present in medicinal plants, possess anti-viral activity and can be strong drug candidates against viruses. NS1 glycoprotein of Dengue virus is involved in its RNA replication and can be a strong target for screening of drugs against this virus. Current study focuses on the identification of flavonoids which can block Asn-130 glycosylation site of Dengue virus NS1 to inhibit viral replication as glycosylation of NS1 is required for its biological functioning. Molecular docking approach was used in this study and the results revealed that flavonoids have strong potential interactions with active site of NS1. Six flavonoids (Deoxycalyxin A; 3,5,7,3',4'-pentahydroxyflavonol-3-O-beta-D-galactopyranoside; (3R)-3',8-Dihydroxyvestitol; Sanggenon O; Epigallocatechin gallate; Chamaejasmin) blocked the Asn-130 glycosylation site of NS1 and could be able to inhibit the viral replication. It can be concluded from this study that these flavonoids could serve as antiviral drugs for dengue infections. Further in-vitro analyses are required to confirm their efficacy and to evaluate their drug potency. PMID:25187688

The RNA helicase DDX17 controls the transcriptional activity of REST and the expression of proneural microRNAs in neuronal differentiation.

PubMed

Lambert, Marie-Pierre; Terrone, Sophie; Giraud, Guillaume; Benoit-Pilven, Clara; Cluet, David; Combaret, Valérie; Mortreux, Franck; Auboeuf, Didier; Bourgeois, Cyril F

2018-06-21

The Repressor Element 1-silencing transcription factor (REST) represses a number of neuronal genes in non-neuronal cells or in undifferentiated neural progenitors. Here, we report that the DEAD box RNA helicase DDX17 controls important REST-related processes that are critical during the early phases of neuronal differentiation. First, DDX17 associates with REST, promotes its binding to the promoter of a subset of REST-targeted genes and co-regulates REST transcriptional repression activity. During neuronal differentiation, we observed a downregulation of DDX17 along with that of the REST complex that contributes to the activation of neuronal genes. Second, DDX17 and its paralog DDX5 regulate the expression of several proneural microRNAs that are known to target the REST complex during neurogenesis, including miR-26a/b that are also direct regulators of DDX17 expression. In this context, we propose a new mechanism by which RNA helicases can control the biogenesis of intronic miRNAs. We show that the processing of the miR-26a2 precursor is dependent on RNA helicases, owing to an intronic regulatory region that negatively impacts on both miRNA processing and splicing of its host intron. Our work places DDX17 in the heart of a pathway involving REST and miRNAs that allows neuronal gene repression.

The Kinetic Mechanism for DNA Unwinding by Multiple Molecules of Dda Helicase Aligned on DNA†

PubMed Central

Eoff, Robert L.; Raney, Kevin D.

2010-01-01

Helicases catalyze the separation of double-stranded nucleic acids to form single-stranded intermediates. Using transient state kinetic methods we have determined the kinetic properties of DNA unwinding under conditions that favor a monomeric form of the Dda helicase as well as conditions that allow multiple molecules to function on the same substrate. Multiple helicase molecules can align like a train on the DNA track. The number of base pairs unwound in a single binding event for Dda is increased from ~19 bp for the monomeric form to ~64 bp when as many as four Dda molecules are aligned on the same substrate, while the kinetic step-size (3.2 ± 0.7 bp) and unwinding rate (242 ± 25 bp s−1) appear to be independent of the number of Dda molecules present on a given substrate. The data support a model in which the helicase molecules bound to the same substrate move along the DNA track independently during DNA unwinding. The observed increase in processivity arises from the increased probability that at least one of the helicases will completely unwind the DNA prior to dissociation. These results are in contrast to previous reports in which multiple Dda molecules on the same track greatly enhanced the rate and amplitude for displacement of protein blocks on the track. Therefore, only when the progress of the lead molecule in the train is impeded by some type of block, such as a protein bound to DNA, do the trailing molecules interact with the lead molecule in order to overcome the block. The fact that trailing helicase molecules have little impact on the lead molecule in the train during routine DNA unwinding suggests that the trailing molecules are moving at similar rates as the lead molecule. This result implicates a step in the translocation mechanism as contributing greatly to the overall rate-limiting step for unwinding of duplex DNA. PMID:20408588

EM structure of a helicase-loader complex depicting a 6:2 binding sub-stoichiometry from Geobacillus kaustophilus HTA426

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Yen-Chen; Naveen, Vankadari; Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan

During DNA replication, bacterial helicase is recruited as a complex in association with loader proteins to unwind the parental duplex. Previous structural studies have reported saturated 6:6 helicase-loader complexes with different conformations. However, structural information on the sub-stoichiometric conformations of these previously-documented helicase-loader complexes remains elusive. Here, with the aid of single particle electron-microscopy (EM) image reconstruction, we present the Geobacillus kaustophilus HTA426 helicase-loader (DnaC-DnaI) complex with a 6:2 binding stoichiometry in the presence of ATPγS. In the 19 Å resolution EM map, the undistorted and unopened helicase ring holds a robust loader density above the C-terminal RecA-like domain. Meanwhile, themore » path of the central DNA binding channel appears to be obstructed by the reconstructed loader density, implying its potential role as a checkpoint conformation to prevent the loading of immature complex onto DNA. Our data also reveals that the bound nucleotides and the consequently induced conformational changes in the helicase hexamer are essential for active association with loader proteins. These observations provide fundamental insights into the formation of the helicase-loader complex in bacteria that regulates the DNA replication process. - Highlights: • Helicase-loader complex structure with 6:2 sub-stoichiometry is resolved by EM. • Helicase hexamer in 6:2 sub-stoichiometry is constricted and un-opened. • 6:2 binding ratio of helicase-loader complex could act as a DNA loading checkpoint. • Nucleotides stabilize helicase-loader complex at low protein concentrations.« less

Systems Biology Reveals NS4B-Cyclophilin A Interaction: A New Target to Inhibit YFV Replication.

PubMed

Vidotto, Alessandra; Morais, Ana T S; Ribeiro, Milene R; Pacca, Carolina C; Terzian, Ana C B; Gil, Laura H V G; Mohana-Borges, Ronaldo; Gallay, Philippe; Nogueira, Mauricio L

2017-04-07

Yellow fever virus (YFV) replication is highly dependent on host cell factors. YFV NS4B is reported to be involved in viral replication and immune evasion. Here interactions between NS4B and human proteins were determined using a GST pull-down assay and analyzed using 1-DE and LC-MS/MS. We present a total of 207 proteins confirmed using Scaffold 3 Software. Cyclophilin A (CypA), a protein that has been shown to be necessary for the positive regulation of flavivirus replication, was identified as a possible NS4B partner. 59 proteins were found to be significantly increased when compared with a negative control, and CypA exhibited the greatest difference, with a 22-fold change. Fisher's exact test was significant for 58 proteins, and the p value of CypA was the most significant (0.000000019). The Ingenuity Systems software identified 16 pathways, and this analysis indicated sirolimus, an mTOR pathway inhibitor, as a potential inhibitor of CypA. Immunofluorescence and viral plaque assays showed a significant reduction in YFV replication using sirolimus and cyclosporine A (CsA) as inhibitors. Furthermore, YFV replication was strongly inhibited in cells treated with both inhibitors using reporter BHK-21-rep-YFV17D-LucNeoIres cells. Taken together, these data suggest that CypA-NS4B interaction regulates YFV replication. Finally, we present the first evidence that YFV inhibition may depend on NS4B-CypA interaction.

Demonstration of helicase activity in the nonstructural protein, NSs, of the negative-sense RNA virus, groundnut bud necrosis virus.

PubMed

Bhushan, Lokesh; Abraham, Ambily; Choudhury, Nirupam Roy; Rana, Vipin Singh; Mukherjee, Sunil Kumar; Savithri, Handanahal Subbarao

2015-04-01

The nonstructural protein NSs, encoded by the S RNA of groundnut bud necrosis virus (GBNV) (genus Tospovirus, family Bunyaviridae) has earlier been shown to possess nucleic-acid-stimulated NTPase and 5' α phosphatase activity. ATP hydrolysis is an essential function of a true helicase. Therefore, NSs was tested for DNA helicase activity. The results demonstrated that GBNV NSs possesses bidirectional DNA helicase activity. An alanine mutation in the Walker A motif (K189A rNSs) decreased DNA helicase activity substantially, whereas a mutation in the Walker B motif resulted in a marginal decrease in this activity. The parallel loss of the helicase and ATPase activity in the K189A mutant confirms that NSs acts as a non-canonical DNA helicase. Furthermore, both the wild-type and K189A NSs could function as RNA silencing suppressors, demonstrating that the suppressor activity of NSs is independent of its helicase or ATPase activity. This is the first report of a true helicase from a negative-sense RNA virus.

Molecular biology of Hel308 helicase in archaea.

PubMed

Woodman, Isabel L; Bolt, Edward L

2009-02-01

Hel308 is an SF2 (superfamily 2) helicase with clear homologues in metazoans and archaea, but not in fungi or bacteria. Evidence from biochemistry and genetics implicates Hel308 in remodelling compromised replication forks. In the last 4 years, significant advances have been made in understanding the biochemistry of archaeal Hel308, most recently through atomic structures from cren- and eury-archaea. These are good templates for SF2 helicase function more generally, highlighting co-ordinated actions of accessory domains around RecA folds. We review the emerging molecular biology of Hel308, drawing together ideas of how it may contribute to genome stability through the control of recombination, with reference to paradigms developed in bacteria.

A mechanical mechanism for translocation of ring-shaped helicases on DNA and its demonstration in a macroscopic simulation system

NASA Astrophysics Data System (ADS)

Chou, Y. C.

2018-04-01

The asymmetry in the two-layered ring structure of helicases and the random thermal fluctuations of the helicase and DNA molecules are considered as the bases for the generation of the force required for translocation of the ring-shaped helicase on DNA. The helicase comprises a channel at its center with two unequal ends, through which strands of DNA can pass. The random collisions between the portion of the DNA strand in the central channel and the wall of the channel generate an impulsive force toward the small end. This impulsive force is the starting point for the helicase to translocate along the DNA with the small end in front. Such a physical mechanism may serve as a complementary for the chemomechanical mechanism of the translocation of helicase on DNA. When the helicase arrives at the junction of ssDNA and dsDNA (a fork), the collision between the helicase and the closest base pair may produce a sufficient impulsive force to break the weak hydrogen bond of the base pair. Thus, the helicase may advance and repeat the process of unwinding the dsDNA strand. This mechanism was tested in a macroscopic simulation system where the helicase was simulated using a truncated-cone structure and DNA was simulated with bead chains. Many features of translocation and unwinding such as translocation on ssDNA and dsDNA, unwinding of dsDNA, rewinding, strand switching, and Holliday junction resolution were reproduced.

A host YB-1 ribonucleoprotein complex is hijacked by hepatitis C virus for the control of NS3-dependent particle production.

PubMed

Chatel-Chaix, Laurent; Germain, Marie-Anne; Motorina, Alena; Bonneil, Éric; Thibault, Pierre; Baril, Martin; Lamarre, Daniel

2013-11-01

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production.

A Host YB-1 Ribonucleoprotein Complex Is Hijacked by Hepatitis C Virus for the Control of NS3-Dependent Particle Production

PubMed Central

Chatel-Chaix, Laurent; Germain, Marie-Anne; Motorina, Alena; Bonneil, Éric; Thibault, Pierre; Baril, Martin

2013-01-01

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production. PMID:23986595

Characterization of the Caenorhabditis elegans HIM-6/BLM helicase: unwinding recombination intermediates.

PubMed

Jung, Hana; Lee, Jin A; Choi, Seoyoon; Lee, Hyunwoo; Ahn, Byungchan

2014-01-01

Mutations in three human RecQ genes are implicated in heritable human syndromes. Mutations in BLM, a RecQ gene, cause Bloom syndrome (BS), which is characterized by short stature, cancer predisposition, and sensitivity to sunlight. BLM is a RecQ DNA helicase that, with interacting proteins, is able to dissolve various DNA structures including double Holliday junctions. A BLM ortholog, him-6, has been identified in Caenorhabditis elegans, but little is known about its enzymatic activities or its in vivo roles. By purifying recombinant HIM-6 and performing biochemical assays, we determined that the HIM-6 has DNA-dependent ATPase activity HIM-6 and helicase activity that proceeds in the 3'-5' direction and needs at least five 3' overhanging nucleotides. HIM-6 is also able to unwind DNA structures including D-loops and Holliday junctions. Worms with him-6 mutations were defective in recovering the cell cycle arrest after HU treatment. These activities strongly support in vivo roles for HIM-6 in processing recombination intermediates.

Prokaryotic Expression of Hepatitis C Virus-NS3 Protein and Preparation of a Monoclonal Antibody.

PubMed

Xi, Yun; Zhang, Yuming; Fang, Jianmin; Whittaker, Kelly; Luo, Shuhong; Huang, Ruo-Pan

2017-12-01

Hepatitis C virus (HCV) is a significant health threat that has been extensively investigated worldwide. Improving the sensitivity and specificity of laboratory tests for screening and early diagnosis of HCV in a relevant population is an effective measure to control the spread of HCV. To build a more reliable diagnostic method for HCV, we expressed gene fragments of HCV-NS3 linked to a carrier, pET28a, and then transformed this vector into Escherichia. coli. The produced recombinant NS3 protein with a molecular weight of 38 kDa, which was purified through Ni-chelating affinity chromatography, was used to immunize BALB/C mice, which generated a serum antibody titer of 1:160,000 against the immunogen. Three positive monoclonal isolates (2A5, 2A6, and 5B12) were screened and established. Western blot and enzyme-linked immunosorbent assay (ELISA) results of these monoclonal cells show that each could specifically recognize the recombinant protein. Antibodies 2A5 and 2A6 were developed into an ELISA sandwich antibody pair for the recombinant protein. The detection sensitivity of our developed ELISA was 1.6 ng/mL, with a linear range of 2.5-80 ng/mL (R 2  = 0.998). Serum NS3 ELISA results show that the average value in the healthy group, liver disease group, and hepatitis C group was 3.71, 7.28, and 13.11 ng/mL, respectively. The positive rates of HCV-NS3 protein in the liver disease group and hepatitis C group was 17.2% and 41.7%, respectively. Detection of HCV-NS3 antigen can be used as an auxiliary test for anti-HCV antibody detection, thus reducing leakage detection and providing a reliable basis for clinical practice.

Bis-aptazyme sensors for hepatitis C virus replicase and helicase without blank signal

PubMed Central

Cho, Suhyung; Kim, Ji-Eun; Lee, Bo-Rahm; Kim, June-Hyung; Kim, Byung-Gee

2005-01-01

The fusion molecule (i.e. aptazyme) of aptamer and hammerhead ribozyme was developed as in situ sensor. Previously, the hammerhead ribozyme conjugated with aptamer through its stem II module showed a significant blank signal by self-cleavage. To reduce or remove its self-cleavage activity in the absence of target molecule, rational designs were attempted by reducing the binding affinity of the aptazyme to its RNA substrate, while maintaining the ribonuclease activity of the aptazyme. Interestingly, the bis-aptazymes which comprise the two aptamer-binding sites at both stem I and stem III of the hammerhead ribozyme showed very low blank signals, and their ratios of reaction rate constants, i.e. signal to noise ratios, were several tens to hundred times higher than those of the stem II-conjugated bis-aptazymes. The reduction in the blank signals seems to be caused by a higher dissociation constant between the main strand of the bis-aptazyme and its substrate arising from multi-point base-pairing of the bis-aptazymes. The bis-aptazymes for HCV replicase and helicase showed high selectivity against other proteins, and a linear relationship existed between their ribozyme activities and the target concentrations. In addition, a bis-aptazyme of dual functions was designed by inserting both aptamers for HCV replicase and helicase into the stem I and stem III of hammerhead ribozyme, respectively, and it also showed greater sensitivity and specificity for both proteins without blank signal. PMID:16314308

The rem Mutations in the ATP-Binding Groove of the Rad3/XPD Helicase Lead to Xeroderma pigmentosum-Cockayne Syndrome-Like Phenotypes

PubMed Central

Montelone, Beth A.; Aguilera, Andrés

2014-01-01

The eukaryotic TFIIH complex is involved in Nucleotide Excision Repair and transcription initiation. We analyzed three yeast mutations of the Rad3/XPD helicase of TFIIH known as rem (recombination and mutation phenotypes). We found that, in these mutants, incomplete NER reactions lead to replication fork breaking and the subsequent engagement of the homologous recombination machinery to restore them. Nevertheless, the penetrance varies among mutants, giving rise to a phenotype gradient. Interestingly, the mutations analyzed reside at the ATP-binding groove of Rad3 and in vivo experiments reveal a gain of DNA affinity upon damage of the mutant Rad3 proteins. Since mutations at the ATP-binding groove of XPD in humans are present in the Xeroderma pigmentosum-Cockayne Syndrome (XP-CS), we recreated rem mutations in human cells, and found that these are XP-CS-like. We propose that the balance between the loss of helicase activity and the gain of DNA affinity controls the capacity of TFIIH to open DNA during NER, and its persistence at both DNA lesions and promoters. This conditions NER efficiency and transcription resumption after damage, which in human cells would explain the XP-CS phenotype, opening new perspectives to understand the molecular basis of the role of XPD in human disease. PMID:25500814

The rem mutations in the ATP-binding groove of the Rad3/XPD helicase lead to Xeroderma pigmentosum-Cockayne syndrome-like phenotypes.

PubMed

Herrera-Moyano, Emilia; Moriel-Carretero, María; Montelone, Beth A; Aguilera, Andrés

2014-12-01

The eukaryotic TFIIH complex is involved in Nucleotide Excision Repair and transcription initiation. We analyzed three yeast mutations of the Rad3/XPD helicase of TFIIH known as rem (recombination and mutation phenotypes). We found that, in these mutants, incomplete NER reactions lead to replication fork breaking and the subsequent engagement of the homologous recombination machinery to restore them. Nevertheless, the penetrance varies among mutants, giving rise to a phenotype gradient. Interestingly, the mutations analyzed reside at the ATP-binding groove of Rad3 and in vivo experiments reveal a gain of DNA affinity upon damage of the mutant Rad3 proteins. Since mutations at the ATP-binding groove of XPD in humans are present in the Xeroderma pigmentosum-Cockayne Syndrome (XP-CS), we recreated rem mutations in human cells, and found that these are XP-CS-like. We propose that the balance between the loss of helicase activity and the gain of DNA affinity controls the capacity of TFIIH to open DNA during NER, and its persistence at both DNA lesions and promoters. This conditions NER efficiency and transcription resumption after damage, which in human cells would explain the XP-CS phenotype, opening new perspectives to understand the molecular basis of the role of XPD in human disease.

Dna2 nuclease-helicase structure, mechanism and regulation by Rpa

PubMed Central

Zhou, Chun; Pourmal, Sergei; Pavletich, Nikola P

2015-01-01

The Dna2 nuclease-helicase maintains genomic integrity by processing DNA double-strand breaks, Okazaki fragments and stalled replication forks. Dna2 requires ssDNA ends, and is dependent on the ssDNA-binding protein Rpa, which controls cleavage polarity. Here we present the 2.3 Å structure of intact mouse Dna2 bound to a 15-nucleotide ssDNA. The nuclease active site is embedded in a long, narrow tunnel through which the DNA has to thread. The helicase domain is required for DNA binding but not threading. We also present the structure of a flexibly-tethered Dna2-Rpa interaction that recruits Dna2 to Rpa-coated DNA. We establish that a second Dna2-Rpa interaction is mutually exclusive with Rpa-DNA interactions and mediates the displacement of Rpa from ssDNA. This interaction occurs at the nuclease tunnel entrance and the 5’ end of the Rpa-DNA complex. Hence, it only displaces Rpa from the 5’ but not 3’ end, explaining how Rpa regulates cleavage polarity. DOI: http://dx.doi.org/10.7554/eLife.09832.001 PMID:26491943

Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication.

PubMed Central

Xu, J; Mendez, E; Caron, P R; Lin, C; Murcko, M A; Collett, M S; Rice, C M

1997-01-01

Members of the Flaviviridae encode a serine proteinase termed NS3 that is responsible for processing at several sites in the viral polyproteins. In this report, we show that the NS3 proteinase of the pestivirus bovine viral diarrhea virus (BVDV) (NADL strain) is required for processing at nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B but not for cleavage at the junction between NS2 and NS3. Cleavage sites of the proteinase were determined by amino-terminal sequence analysis of the NS4A, NS4B, NS5A, and NS5B proteins. A conserved leucine residue is found at the P1 position of all four cleavage sites, followed by either serine (3/4A, 4B/5A, and 5A/5B sites) or alanine (4A/4B site) at the P1' position. Consistent with this cleavage site preference, a structural model of the pestivirus NS3 proteinase predicts a highly hydrophobic P1 specificity pocket. trans-Processing experiments implicate the 64-residue NS4A protein as an NS3 proteinase cofactor required for cleavage at the 4B/5A and 5A/5B sites. Finally, using a full-length functional BVDV cDNA clone, we demonstrate that a catalytically active NS3 serine proteinase is essential for pestivirus replication. PMID:9188600

Targeting DDX3 with a small molecule inhibitor for lung cancer therapy

PubMed Central

Bol, Guus M; Vesuna, Farhad; Xie, Min; Zeng, Jing; Aziz, Khaled; Gandhi, Nishant; Levine, Anne; Irving, Ashley; Korz, Dorian; Tantravedi, Saritha; Heerma van Voss, Marise R; Gabrielson, Kathleen; Bordt, Evan A; Polster, Brian M; Cope, Leslie; van der Groep, Petra; Kondaskar, Atul; Rudek, Michelle A; Hosmane, Ramachandra S; van der Wall, Elsken; van Diest, Paul J; Tran, Phuoc T; Raman, Venu

2015-01-01

Lung cancer is the most common malignancy worldwide and is a focus for developing targeted therapies due to its refractory nature to current treatment. We identified a RNA helicase, DDX3, which is overexpressed in many cancer types including lung cancer and is associated with lower survival in lung cancer patients. We designed a first-in-class small molecule inhibitor, RK-33, which binds to DDX3 and abrogates its activity. Inhibition of DDX3 by RK-33 caused G1 cell cycle arrest, induced apoptosis, and promoted radiation sensitization in DDX3-overexpressing cells. Importantly, RK-33 in combination with radiation induced tumor regression in multiple mouse models of lung cancer. Mechanistically, loss of DDX3 function either by shRNA or by RK-33 impaired Wnt signaling through disruption of the DDX3–β-catenin axis and inhibited non-homologous end joining—the major DNA repair pathway in mammalian somatic cells. Overall, inhibition of DDX3 by RK-33 promotes tumor regression, thus providing a compelling argument to develop DDX3 inhibitors for lung cancer therapy. PMID:25820276

Conserved mechanism for coordinating replication fork helicase assembly with phosphorylation of the helicase

PubMed Central

Bruck, Irina; Kaplan, Daniel L.

2015-01-01

Dbf4-dependent kinase (DDK) phosphorylates minichromosome maintenance 2 (Mcm2) during S phase in yeast, and Sld3 recruits cell division cycle 45 (Cdc45) to minichromosome maintenance 2-7 (Mcm2-7). We show here DDK-phosphoryled Mcm2 preferentially interacts with Cdc45 in vivo, and that Sld3 stimulates DDK phosphorylation of Mcm2 by 11-fold. We identified a mutation of the replication initiation factor Sld3, Sld3-m16, that is specifically defective in stimulating DDK phosphorylation of Mcm2. Wild-type expression levels of sld3-m16 result in severe growth and DNA replication defects. Cells expressing sld3-m16 exhibit no detectable Mcm2 phosphorylation in vivo, reduced replication protein A-ChIP signal at an origin, and diminished Go, Ichi, Ni, and San association with Mcm2-7. Treslin, the human homolog of Sld3, stimulates human DDK phosphorylation of human Mcm2 by 15-fold. DDK phosphorylation of human Mcm2 decreases the affinity of Mcm5 for Mcm2, suggesting a potential mechanism for helicase ring opening. These data suggest a conserved mechanism for replication initiation: Sld3/Treslin coordinates Cdc45 recruitment to Mcm2-7 with DDK phosphorylation of Mcm2 during S phase. PMID:26305950

Conserved mechanism for coordinating replication fork helicase assembly with phosphorylation of the helicase.

PubMed

Bruck, Irina; Kaplan, Daniel L

2015-09-08

Dbf4-dependent kinase (DDK) phosphorylates minichromosome maintenance 2 (Mcm2) during S phase in yeast, and Sld3 recruits cell division cycle 45 (Cdc45) to minichromosome maintenance 2-7 (Mcm2-7). We show here DDK-phosphoryled Mcm2 preferentially interacts with Cdc45 in vivo, and that Sld3 stimulates DDK phosphorylation of Mcm2 by 11-fold. We identified a mutation of the replication initiation factor Sld3, Sld3-m16, that is specifically defective in stimulating DDK phosphorylation of Mcm2. Wild-type expression levels of sld3-m16 result in severe growth and DNA replication defects. Cells expressing sld3-m16 exhibit no detectable Mcm2 phosphorylation in vivo, reduced replication protein A-ChIP signal at an origin, and diminished Go, Ichi, Ni, and San association with Mcm2-7. Treslin, the human homolog of Sld3, stimulates human DDK phosphorylation of human Mcm2 by 15-fold. DDK phosphorylation of human Mcm2 decreases the affinity of Mcm5 for Mcm2, suggesting a potential mechanism for helicase ring opening. These data suggest a conserved mechanism for replication initiation: Sld3/Treslin coordinates Cdc45 recruitment to Mcm2-7 with DDK phosphorylation of Mcm2 during S phase.

NS1-binding protein abrogates the elevation of cell viability by the influenza A virus NS1 protein in association with CRKL

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miyazaki, Masaya; Nishihara, Hiroshi, E-mail: hnishihara@med.hokudai.ac.jp; Hasegawa, Hideki

Highlights: •NS1 induced excessive phosphorylation of ERK and elevated cell viability. •NS1-BP expression and CRKL knockdown abolished survival effect of NS1. •NS1-BP and NS1 formed the complex through the interaction with CRKL-SH3(N). -- Abstract: The influenza A virus non-structural protein 1 (NS1) is a multifunctional virulence factor consisting of an RNA binding domain and several Src-homology (SH) 2 and SH3 binding motifs, which promotes virus replication in the host cell and helps to evade antiviral immunity. NS1 modulates general host cell physiology in association with various cellular molecules including NS1-binding protein (NS1-BP) and signaling adapter protein CRK-like (CRKL), while themore » physiological role of NS1-BP during influenza A virus infection especially in association with NS1 remains unclear. In this study, we analyzed the intracellular association of NS1-BP, NS1 and CRKL to elucidate the physiological roles of these molecules in the host cell. In HEK293T cells, enforced expression of NS1 of A/Beijing (H1N1) and A/Indonesia (H5N1) significantly induced excessive phosphorylation of ERK and elevated cell viability, while the over-expression of NS1-BP and the abrogation of CRKL using siRNA abolished such survival effect of NS1. The pull-down assay using GST-fusion CRKL revealed the formation of intracellular complexes of NS1-BP, NS1 and CRKL. In addition, we identified that the N-terminus SH3 domain of CRKL was essential for binding to NS1-BP using GST-fusion CRKL-truncate mutants. This is the first report to elucidate the novel function of NS1-BP collaborating with viral protein NS1 in modulation of host cell physiology. In addition, an alternative role of adaptor protein CRKL in association with NS1 and NS1-BP during influenza A virus infection is demonstrated.« less

XPD Helicase Structures And Activities: Insights Into the Cancer And Aging Phenotypes From XPD Mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fan, L.; Fuss, J.O.; Cheng, Q.J.

2009-05-18

Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicasemore » activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.« less

XPD Helicase Structures and Activities: Insights into the Cancer and Aging Phenotypes from XPD Mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tainer, John; Fan, Li; Fuss, Jill O.

2008-06-02

Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicasemore » activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.« less

Helicase-Dependent Isothermal Amplification of DNA and RNA by Using Self-Avoiding Molecular Recognition Systems.

PubMed

Yang, Zunyi; McLendon, Chris; Hutter, Daniel; Bradley, Kevin M; Hoshika, Shuichi; Frye, Carole B; Benner, Steven A

2015-06-15

Assays that detect DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low-resource environments, and requires equipment and power that might not be available in these environments. Isothermal procedures, which avoid thermal cycling, are often confounded by primer dimers, off-target priming, and other artifacts. Here, we show how a "self avoiding molecular recognition system" (SAMRS) eliminates these artifacts and gives clean amplicons in a helicase-dependent isothermal amplification (SAMRS-HDA). We also show that incorporating SAMRS into the 3'-ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS-HDA can be twofold multiplexed, difficult to achieve with HDA using standard primers. Thus, SAMRS-HDA is a more versatile approach than standard HDA, with a broader applicability for xNA-targeted diagnostics and research. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases

PubMed Central

Pinto, Cosimo; Kasaciunaite, Kristina; Seidel, Ralf; Cejka, Petr

2016-01-01

Human DNA2 (hDNA2) contains both a helicase and a nuclease domain within the same polypeptide. The nuclease of hDNA2 is involved in a variety of DNA metabolic processes. Little is known about the role of the hDNA2 helicase. Using bulk and single-molecule approaches, we show that hDNA2 is a processive helicase capable of unwinding kilobases of dsDNA in length. The nuclease activity prevents the engagement of the helicase by competing for the same substrate, hence prominent DNA unwinding by hDNA2 alone can only be observed using the nuclease-deficient variant. We show that the helicase of hDNA2 functionally integrates with BLM or WRN helicases to promote dsDNA degradation by forming a heterodimeric molecular machine. This collectively suggests that the hDNA2 motor promotes the enzyme's capacity to degrade dsDNA in conjunction with BLM or WRN and thus promote the repair of broken DNA. DOI: http://dx.doi.org/10.7554/eLife.18574.001 PMID:27612385

Probing the ATP-induced conformational flexibility of the PcrA helicase protein using molecular dynamics simulation.

PubMed

Mhashal, Anil R; Choudhury, Chandan Kumar; Roy, Sudip

2016-03-01

Helicases are enzymes that unwind double-stranded DNA (dsDNA) into its single-stranded components. It is important to understand the binding and unbinding of ATP from the active sites of helicases, as this knowledge can be used to elucidate the functionality of helicases during the unwinding of dsDNA. In this work, we investigated the unbinding of ATP and its effect on the active-site residues of the helicase PcrA using molecular dynamic simulations. To mimic the unbinding process of ATP from the active site of the helicase, we simulated the application of an external force that pulls ATP from the active site and computed the free-energy change during this process. We estimated an energy cost of ~85 kJ/mol for the transformation of the helicase from the ATP-bound state (1QHH) to the ATP-free state (1PJR). Unbinding led to conformational changes in the residues of the protein at the active site. Some of the residues at the ATP-binding site were significantly reoriented when the ATP was pulled. We observed a clear competition between reorientation of the residues and energy stabilization by hydrogen bonds between the ATP and active-site residues. We also checked the flexibility of the PcrA protein using a principal component analysis of domain motion. We found that the ATP-free state of the helicase is more flexible than the ATP-bound state.

Hepatitis C Virus NS3/4A Protease Inhibitors Incorporating Flexible P2 Quinoxalines Target Drug Resistant Viral Variants.

PubMed

Matthew, Ashley N; Zephyr, Jacqueto; Hill, Caitlin J; Jahangir, Muhammad; Newton, Alicia; Petropoulos, Christos J; Huang, Wei; Kurt-Yilmaz, Nese; Schiffer, Celia A; Ali, Akbar

2017-07-13

A substrate envelope-guided design strategy is reported for improving the resistance profile of HCV NS3/4A protease inhibitors. Analogues of 5172-mcP1P3 were designed by incorporating diverse quinoxalines at the P2 position that predominantly interact with the invariant catalytic triad of the protease. Exploration of structure-activity relationships showed that inhibitors with small hydrophobic substituents at the 3-position of P2 quinoxaline maintain better potency against drug resistant variants, likely due to reduced interactions with residues in the S2 subsite. In contrast, inhibitors with larger groups at this position were highly susceptible to mutations at Arg155, Ala156, and Asp168. Excitingly, several inhibitors exhibited exceptional potency profiles with EC 50 values ≤5 nM against major drug resistant HCV variants. These findings support that inhibitors designed to interact with evolutionarily constrained regions of the protease, while avoiding interactions with residues not essential for substrate recognition, are less likely to be susceptible to drug resistance.

RNA Helicase Associated with AU-rich Element (RHAU/DHX36) Interacts with the 3′-Tail of the Long Non-coding RNA BC200 (BCYRN1)*

PubMed Central

Booy, Evan P.; McRae, Ewan K. S.; Howard, Ryan; Deo, Soumya R.; Ariyo, Emmanuel O.; Dzananovic, Edis; Meier, Markus; Stetefeld, Jörg; McKenna, Sean A.

2016-01-01

RNA helicase associated with AU-rich element (RHAU) is an ATP-dependent RNA helicase that demonstrates high affinity for quadruplex structures in DNA and RNA. To elucidate the significance of these quadruplex-RHAU interactions, we have performed RNA co-immunoprecipitation screens to identify novel RNAs bound to RHAU and characterize their function. In the course of this study, we have identified the non-coding RNA BC200 (BCYRN1) as specifically enriched upon RHAU immunoprecipitation. Although BC200 does not adopt a quadruplex structure and does not bind the quadruplex-interacting motif of RHAU, it has direct affinity for RHAU in vitro. Specifically designed BC200 truncations and RNase footprinting assays demonstrate that RHAU binds to an adenosine-rich region near the 3′-end of the RNA. RHAU truncations support binding that is dependent upon a region within the C terminus and is specific to RHAU isoform 1. Tests performed to assess whether BC200 interferes with RHAU helicase activity have demonstrated the ability of BC200 to act as an acceptor of unwound quadruplexes via a cytosine-rich region near the 3′-end of the RNA. Furthermore, an interaction between BC200 and the quadruplex-containing telomerase RNA was confirmed by pull-down assays of the endogenous RNAs. This leads to the possibility that RHAU may direct BC200 to bind and exert regulatory functions at quadruplex-containing RNA or DNA sequences. PMID:26740632

Evolution of Bacterial Global Modulators: Role of a Novel H-NS Paralogue in the Enteroaggregative Escherichia coli Strain 042

PubMed Central

2018-01-01

ABSTRACT Bacterial genomes sometimes contain genes that code for homologues of global regulators, the function of which is unclear. In members of the family Enterobacteriaceae, cells express the global regulator H-NS and its paralogue StpA. In Escherichia coli, out of providing a molecular backup for H-NS, the role of StpA is poorly characterized. The enteroaggregative E. coli strain 042 carries, in addition to the hns and stpA genes, a third gene encoding an hns paralogue (hns2). We present in this paper information about its biological function. Transcriptomic analysis has shown that the H-NS2 protein targets a subset of the genes targeted by H-NS. Genes targeted by H-NS2 correspond mainly with horizontally transferred (HGT) genes and are also targeted by the Hha protein, a fine-tuner of H-NS activity. Compared with H-NS, H-NS2 expression levels are lower. In addition, H-NS2 expression exhibits specific features: it is sensitive to the growth temperature and to the nature of the culture medium. This novel H-NS paralogue is widespread within the Enterobacteriaceae. IMPORTANCE Global regulators such as H-NS play key relevant roles enabling bacterial cells to adapt to a changing environment. H-NS modulates both core and horizontally transferred (HGT) genes, but the mechanism by which H-NS can differentially regulate these genes remains to be elucidated. There are several instances of bacterial cells carrying genes that encode homologues of the global regulators. The question is what the roles of these proteins are. We noticed that the enteroaggregative E. coli strain 042 carries a new hitherto uncharacterized copy of the hns gene. We decided to investigate why this pathogenic E. coli strain requires an extra H-NS paralogue, termed H-NS2. In our work, we show that H-NS2 displays specific expression and regulatory properties. H-NS2 targets a subset of H-NS-specific genes and may help to differentially modulate core and HGT genes by the H-NS cellular pool. PMID

Evolution of Bacterial Global Modulators: Role of a Novel H-NS Paralogue in the Enteroaggregative Escherichia coli Strain 042.

PubMed

Prieto, A; Bernabeu, M; Aznar, S; Ruiz-Cruz, S; Bravo, A; Queiroz, M H; Juárez, A

2018-01-01

Bacterial genomes sometimes contain genes that code for homologues of global regulators, the function of which is unclear. In members of the family Enterobacteriaceae , cells express the global regulator H-NS and its paralogue StpA. In Escherichia coli , out of providing a molecular backup for H-NS, the role of StpA is poorly characterized. The enteroaggregative E. coli strain 042 carries, in addition to the hns and stpA genes, a third gene encoding an hns paralogue ( hns2 ). We present in this paper information about its biological function. Transcriptomic analysis has shown that the H-NS2 protein targets a subset of the genes targeted by H-NS. Genes targeted by H-NS2 correspond mainly with horizontally transferred (HGT) genes and are also targeted by the Hha protein, a fine-tuner of H-NS activity. Compared with H-NS, H-NS2 expression levels are lower. In addition, H-NS2 expression exhibits specific features: it is sensitive to the growth temperature and to the nature of the culture medium. This novel H-NS paralogue is widespread within the Enterobacteriaceae . IMPORTANCE Global regulators such as H-NS play key relevant roles enabling bacterial cells to adapt to a changing environment. H-NS modulates both core and horizontally transferred (HGT) genes, but the mechanism by which H-NS can differentially regulate these genes remains to be elucidated. There are several instances of bacterial cells carrying genes that encode homologues of the global regulators. The question is what the roles of these proteins are. We noticed that the enteroaggregative E. coli strain 042 carries a new hitherto uncharacterized copy of the hns gene. We decided to investigate why this pathogenic E. coli strain requires an extra H-NS paralogue, termed H-NS2. In our work, we show that H-NS2 displays specific expression and regulatory properties. H-NS2 targets a subset of H-NS-specific genes and may help to differentially modulate core and HGT genes by the H-NS cellular pool.

ATP-dependent RecG Helicase Is Required for the Transcriptional Regulator OxyR Function in Pseudomonas species*

PubMed Central

Yeom, Jinki; Lee, Yunho; Park, Woojun

2012-01-01

The oxyR gene appears to reside in an operon with the recG helicase gene in many bacteria, including pathogenic Pseudomonas aeruginosa and Pseudomonas putida. Analysis of P. putida transcriptomes shows that many OxyR-controlled genes are regulated by the ATP-dependent RecG helicase and that RecG alone modulates the expression of many genes. We found that purified RecG binds to the promoters of many OxyR-controlled genes and that expression of these genes was not induced under conditions of oxidative stress in recG mutants of P. aeruginosa, P. putida, and Escherichia coli. In vitro data revealed that promoters containing palindromic sequences are essential for RecG binding and that single-strand binding proteins and ATP are also needed for RecG to promote transcription, whereas a magnesium ion has the opposite effect. The OxyR tetramer preferentially binds to promoters after RecG has generated linear DNA in the presence of ATP; otherwise, the OxyR dimer has higher affinity. This study provides new insights into the mechanism of bacterial transcription by demonstrating that RecG might be required for the induction of the OxyR regulon by unwinding palindromic DNA for transcription. This work describes a novel bacterial transcriptional function by RecG helicase with OxyR and may provide new targets for controlling Pseudomonas species pathogen. PMID:22621928

Action of CMG with strand-specific DNA blocks supports an internal unwinding mode for the eukaryotic replicative helicase

PubMed Central

Langston, Lance; O’Donnell, Mike

2017-01-01

Replicative helicases are ring-shaped hexamers that encircle DNA for duplex unwinding. The currently accepted view of hexameric helicase function is by steric exclusion, where the helicase encircles one DNA strand and excludes the other, acting as a wedge with an external DNA unwinding point during translocation. Accordingly, strand-specific blocks only affect these helicases when placed on the tracking strand, not the excluded strand. We examined the effect of blocks on the eukaryotic CMG and, contrary to expectations, blocks on either strand inhibit CMG unwinding. A recent cryoEM structure of yeast CMG shows that duplex DNA enters the helicase and unwinding occurs in the central channel. The results of this report inform important aspects of the structure, and we propose that CMG functions by a modified steric exclusion process in which both strands enter the helicase and the duplex unwinding point is internal, followed by exclusion of the non-tracking strand. DOI: http://dx.doi.org/10.7554/eLife.23449.001 PMID:28346143

Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids.

PubMed

Kivlehan, Francine; Mavré, François; Talini, Luc; Limoges, Benoît; Marchal, Damien

2011-09-21

We described an electrochemical method to monitor in real-time the isothermal helicase-dependent amplification of nucleic acids. The principle of detection is simple and well-adapted to the development of portable, easy-to-use and inexpensive nucleic acids detection technologies. It consists of monitoring a decrease in the electrochemical current response of a reporter DNA intercalating redox probe during the isothermal DNA amplification. The method offers the possibility to quantitatively analyze target nucleic acids in less than one hour at a single constant temperature, and to perform at the end of the isothermal amplification a DNA melt curve analysis for differentiating between specific and non-specific amplifications. To illustrate the potentialities of this approach for the development of a simple, robust and low-cost instrument with high throughput capability, the method was validated with an electrochemical system capable of monitoring up to 48 real-time isothermal HDA reactions simultaneously in a disposable microplate consisting of 48-electrochemical microwells. Results obtained with this approach are comparable to that obtained with a well-established but more sophisticated and expensive fluorescence-based method. This makes for a promising alternative detection method not only for real-time isothermal helicase-dependent amplification of nucleic acid, but also for other isothermal DNA amplification strategies.

[Determination of drug resistance mutations of NS3 inhibitors in chronic hepatitis C patients infected with genotype 1].

PubMed

Şanlıdağ, Tamer; Sayan, Murat; Akçalı, Sinem; Kasap, Elmas; Buran, Tahir; Arıkan, Ayşe

2017-04-01

Direct-acting antiviral agents (DAA) such as NS3 protease inhibitors is the first class of drugs used for chronic hepatitis C (CHC) treatment. NS3 inhibitors (PI) with low genetic barrier have been approved to be used in the CHC genotype 1 infections, and in the treatment of compensated liver disease including cirrhosis together with pegile interferon and ribavirin. Consequently, the development of drug resistance during DAA treatment of CHC is a major problem. NS3 resistant variants can be detected before treatment as they can occurnaturally. The aim of this study was to investigate new and old generation NS3 inhibitors resistance mutations before DAA treatment in hepatitis C virus (HCV) that were isolated from CHC. The present study was conducted in 2015 and included 97 naive DAA patients infected with HCV genotype 1, who were diagnosed in Manisa and Kocaeli cities of Turkey. Magnetic particle based HCV RNA extraction and than RNA detection and quantification were performed using commercial real-time PCR assay QIASypmhony + Rotorgene Q/ArtusHCV QS-RGQ and COBAS Ampliprep/COBAS TaqMan HCV Tests. HCV NS3 viral protease genome region was amplified with PCR and mutation analysis was performed by Sanger dideoxy sequencing technique of NS3 protease codons (codon 32-185). HCV NS3 protease inhibitors; asunaprevir, boceprevir, faldaprevir, grazoprevir, pariteprevir, simeprevir and telaprevir were analysed for resistant mutations by Geno2pheno-HCV resistance tool. HCV was genotyped in all patients and 88 patients (n= 88/97, 91%) had genotype 1. Eight (n= 8/97, 8.2%) and 80 (n= 80/97, 82.4%) HCC patients were subgenotyped as 1a and 1b, respectively. Many aminoacid substitutions and resistance mutations were determined in 39/88 (44%) patients in the study group. Q80L, S122C/N, S138W were defined as potential substitutions (6/88 patients; 7%); R109K, R117C, S122G, I132V, I170V, N174S were described as potential resistance (34/88 patients; 39%); V36L, T54S, V55A, Q80H were

ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method.

PubMed

Yang, Mu; Wang, Ganggang

2016-09-15

The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication. In this work, a non-radioactive method for measuring ATPase activity of DnaBBst helicase was described. The working parameters and conditions were optimized. Furthermore, this method was applied to investigate effects of DnaG primase, ssDNA and helicase loader protein (DnaI) on ATPase activity of DnaBBst. Our results showed this method was sensitive and efficient. Moreover, it is suitable for the investigation of functional interaction between DnaB and related factors. Copyright © 2016 Elsevier Inc. All rights reserved.

DDX3X RNA helicase affects breast cancer cell cycle progression by regulating expression of KLF4.

PubMed

Cannizzaro, Ester; Bannister, Andrew John; Han, Namshik; Alendar, Andrej; Kouzarides, Tony

2018-05-21

DDX3X is a multifunctional RNA helicase with documented roles in different cancer types. Here, we demonstrate that DDX3X plays an oncogenic role in breast cancer cells by modulating the cell cycle. Depletion of DDX3X in MCF7 cells slows cell proliferation by inducing a G1 phase arrest. Notably, DDX3X inhibits expression of Kruppel-like factor 4 (KLF4), a transcription factor and cell cycle repressor. Moreover, DDX3X directly interacts with KLF4 mRNA and regulates its splicing. We show that DDX3X-mediated repression of KLF4 promotes expression of S-phase inducing genes in MCF7 breast cancer cells. These findings provide evidence for a novel function of DDX3X in regulating expression and downstream functions of KLF4, a master negative regulator of the cell cycle. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Discovery of Dengue Virus NS4B Inhibitors

PubMed Central

Wang, Qing-Yin; Dong, Hongping; Zou, Bin; Karuna, Ratna; Wan, Kah Fei; Zou, Jing; Susila, Agatha; Yip, Andy; Shan, Chao; Yeo, Kim Long; Xu, Haoying; Ding, Mei; Chan, Wai Ling; Gu, Feng; Seah, Peck Gee; Liu, Wei; Lakshminarayana, Suresh B.; Kang, CongBao; Lescar, Julien; Blasco, Francesca; Smith, Paul W.

2015-01-01

ABSTRACT The four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogens in humans. No clinically approved vaccine or antiviral is currently available for DENV. Here we report a spiropyrazolopyridone compound that potently inhibits DENV both in vitro and in vivo. The inhibitor was identified through screening of a 1.8-million-compound library by using a DENV-2 replicon assay. The compound selectively inhibits DENV-2 and -3 (50% effective concentration [EC50], 10 to 80 nM) but not DENV-1 and -4 (EC50, >20 μM). Resistance analysis showed that a mutation at amino acid 63 of DENV-2 NS4B (a nonenzymatic transmembrane protein and a component of the viral replication complex) could confer resistance to compound inhibition. Genetic studies demonstrate that variations at amino acid 63 of viral NS4B are responsible for the selective inhibition of DENV-2 and -3. Medicinal chemistry improved the physicochemical properties of the initial “hit” (compound 1), leading to compound 14a, which has good in vivo pharmacokinetics. Treatment of DENV-2-infected AG129 mice with compound 14a suppressed viremia, even when the treatment started after viral infection. The results have proven the concept that inhibitors of NS4B could potentially be developed for clinical treatment of DENV infection. Compound 14a represents a potential preclinical candidate for treatment of DENV-2- and -3-infected patients. IMPORTANCE Dengue virus (DENV) threatens up to 2.5 billion people and is now spreading in many regions in the world where it was not previously endemic. While there are several promising vaccine candidates in clinical trials, approved vaccines or antivirals are not yet available. Here we describe the identification and characterization of a spiropyrazolopyridone as a novel inhibitor of DENV by targeting the viral NS4B protein. The compound potently inhibits two of the four serotypes of DENV (DENV-2 and -3) both in vitro and in vivo. Our

Infection of Common Marmosets with GB Virus B Chimeric Virus Encoding the Major Nonstructural Proteins NS2 to NS4A of Hepatitis C Virus

PubMed Central

Zhu, Shaomei; Liu, Bochao; Xu, Yuxia; Sun, Yachun; Wang, Yilin; Wang, Yuanzhan; Shuai, Lifang; Chen, Zixuan; Allain, Jean-Pierre

2016-01-01

for HCV infection, but ethical considerations have restricted their utility in biomedical research. GB virus B (GBV-B) is a flavivirus related to HCV. It can infect common marmosets, a New World small primate, and induces viral hepatitis similar to HCV infection in humans. To minimize differences between GBV-B and HCV, we generated HCV NS2 to -4A/GBV-B chimeric viruses and established a chimera-infected marmoset model. HCV NS2 to -4A chimera-infected marmosets provide a small-animal model for evaluating novel antiviral drugs targeting HCV NS3-NS4A protease and T-cell-based HCV vaccines. PMID:27384651

Infection of Common Marmosets with GB Virus B Chimeric Virus Encoding the Major Nonstructural Proteins NS2 to NS4A of Hepatitis C Virus.

PubMed

Zhu, Shaomei; Li, Tingting; Liu, Bochao; Xu, Yuxia; Sun, Yachun; Wang, Yilin; Wang, Yuanzhan; Shuai, Lifang; Chen, Zixuan; Allain, Jean-Pierre; Li, Chengyao

2016-09-15

, but ethical considerations have restricted their utility in biomedical research. GB virus B (GBV-B) is a flavivirus related to HCV. It can infect common marmosets, a New World small primate, and induces viral hepatitis similar to HCV infection in humans. To minimize differences between GBV-B and HCV, we generated HCV NS2 to -4A/GBV-B chimeric viruses and established a chimera-infected marmoset model. HCV NS2 to -4A chimera-infected marmosets provide a small-animal model for evaluating novel antiviral drugs targeting HCV NS3-NS4A protease and T-cell-based HCV vaccines. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Engineered Toxins “Zymoxins” Are Activated by the HCV NS3 Protease by Removal of an Inhibitory Protein Domain

PubMed Central

Shapira, Assaf; Gal-Tanamy, Meital; Nahary, Limor; Litvak-Greenfeld, Dana; Zemel, Romy; Tur-Kaspa, Ran; Benhar, Itai

2011-01-01

The synthesis of inactive enzyme precursors, also known as “zymogens,” serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated “zymogenized” chimeric toxins (which we denote “zymoxins”). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the “zymoxin” approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected. PMID:21264238

DEAD-box RNA helicase DDX23 modulates glioma malignancy via elevating miR-21 biogenesis.

PubMed

Yin, Jinlong; Park, Gunwoo; Lee, Jeong Eun; Choi, Eun Young; Park, Ju Young; Kim, Tae-Hoon; Park, Nayun; Jin, Xiong; Jung, Ji-Eun; Shin, Daye; Hong, Jun Hee; Kim, Hyunggee; Yoo, Heon; Lee, Seung-Hoon; Kim, Youn-Jae; Park, Jong Bae; Kim, Jong Heon

2015-09-01

Upregulation of microRNA-21 (miR-21) is known to be strongly associated with the proliferation, invasion, and radio-resistance of glioma cells. However, the regulatory mechanism that governs the biogenesis of miR-21 in glioma is still unclear. Here, we demonstrate that the DEAD-box RNA helicase, DDX23, promotes miR-21 biogenesis at the post-transcriptional level. The expression of DDX23 was enhanced in glioma tissues compared to normal brain, and expression level of DDX23 was highly associated with poor survival of glioma patients. Specific knockdown of DDX23 expression suppressed glioma cell proliferation and invasion in vitro and in vivo, which is similar to the function of miR-21. We found that DDX23 increased the level of miR-21 by promoting primary-to-precursor processing of miR-21 through an interaction with the Drosha microprocessor. Mutagenesis experiments critically demonstrated that the helicase activity of DDX23 was essential for the processing (cropping) of miR-21, and we further found that ivermectin, a RNA helicase inhibitor, decreased miR-21 levels by potentially inhibiting DDX23 activity and blocked invasion and cell proliferation. Moreover, treatment of ivermectin decreased glioma growth in mouse xenografts. Taken together, these results suggest that DDX23 plays an essential role in glioma progression, and might thus be a potential novel target for the therapeutic treatment of glioma. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation.

PubMed

Deegan, Tom D; Yeeles, Joseph Tp; Diffley, John Fx

2016-05-02

The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45-MCM-GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK-dependent manner. Sld3 binds specifically to DDK-phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho-MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK-independent replication. Thus, Sld3 is an essential "reader" of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Interference of transcription across H-NS binding sites and repression by H-NS.

PubMed

Rangarajan, Aathmaja Anandhi; Schnetz, Karin

2018-05-01

Nucleoid-associated protein H-NS represses transcription by forming extended DNA-H-NS complexes. Repression by H-NS operates mostly at the level of transcription initiation. Less is known about how DNA-H-NS complexes interfere with transcription elongation. In vitro H-NS has been shown to enhance RNA polymerase pausing and to promote Rho-dependent termination, while in vivo inhibition of Rho resulted in a decrease of the genome occupancy by H-NS. Here we show that transcription directed across H-NS binding regions relieves H-NS (and H-NS/StpA) mediated repression of promoters in these regions. Further, we observed a correlation of transcription across the H-NS-bound region and de-repression. The data suggest that the transcribing RNA polymerase is able to remodel the H-NS complex and/or dislodge H-NS from the DNA and thus relieve repression. Such an interference of transcription and H-NS mediated repression may imply that poorly transcribed AT-rich loci are prone to be repressed by H-NS, while efficiently transcribed loci escape repression. © 2018 John Wiley & Sons Ltd.

Helicase-Dependent RNA Decay Illuminated by a Cryo-EM Structure of a Human Nuclear RNA Exosome-MTR4 Complex.

PubMed

Weick, Eva-Maria; Puno, M Rhyan; Januszyk, Kurt; Zinder, John C; DiMattia, Michael A; Lima, Christopher D

2018-06-14

The ribonucleolytic RNA exosome interacts with RNA helicases to degrade RNA. To understand how the 3' to 5' Mtr4 helicase engages RNA and the nuclear exosome, we reconstituted 14-subunit Mtr4-containing RNA exosomes from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human and show that they unwind structured substrates to promote degradation. We loaded a human exosome with an optimized DNA-RNA chimera that stalls MTR4 during unwinding and determined its structure to an overall resolution of 3.45 Å by cryoelectron microscopy (cryo-EM). The structure reveals an RNA-engaged helicase atop the non-catalytic core, with RNA captured within the central channel and DIS3 exoribonuclease active site. MPP6 tethers MTR4 to the exosome through contacts to the RecA domains of MTR4. EXOSC10 remains bound to the core, but its catalytic module and cofactor C1D are displaced by RNA-engaged MTR4. Competition for the exosome core may ensure that RNA is committed to degradation by DIS3 when engaged by MTR4. Copyright © 2018 Elsevier Inc. All rights reserved.

Characterization of the Caenorhabditis elegans HIM-6/BLM Helicase: Unwinding Recombination Intermediates

PubMed Central

Choi, Seoyoon; Lee, Hyunwoo; Ahn, Byungchan

2014-01-01

Mutations in three human RecQ genes are implicated in heritable human syndromes. Mutations in BLM, a RecQ gene, cause Bloom syndrome (BS), which is characterized by short stature, cancer predisposition, and sensitivity to sunlight. BLM is a RecQ DNA helicase that, with interacting proteins, is able to dissolve various DNA structures including double Holliday junctions. A BLM ortholog, him-6, has been identified in Caenorhabditis elegans, but little is known about its enzymatic activities or its in vivo roles. By purifying recombinant HIM-6 and performing biochemical assays, we determined that the HIM-6 has DNA-dependent ATPase activity HIM-6 and helicase activity that proceeds in the 3'-5' direction and needs at least five 3' overhanging nucleotides. HIM-6 is also able to unwind DNA structures including D-loops and Holliday junctions. Worms with him-6 mutations were defective in recovering the cell cycle arrest after HU treatment. These activities strongly support in vivo roles for HIM-6 in processing recombination intermediates. PMID:25036527

A Novel Benzodiazepine Compound Inhibits Yellow Fever Virus Infection by Specifically Targeting NS4B Protein.

PubMed

Guo, Fang; Wu, Shuo; Julander, Justin; Ma, Julia; Zhang, Xuexiang; Kulp, John; Cuconati, Andrea; Block, Timothy M; Du, Yanming; Guo, Ju-Tao; Chang, Jinhong

2016-09-21

-risk regions. It has been estimated that up to 1.7 million YFV infections occur in Africa each year, resulting in 29,000 to 60,000 death. Thus far, there is no specific antiviral treatment for yellow fever. To cope with this medical challenge, we identified a benzodiazepine compound that selectively inhibits YFV by targeting the viral NS4B protein. To our knowledge, this is the first report demonstrating in vivo safety and antiviral efficacy of an YFV NS4B inhibitor in an animal model. We have thus reached a critical milestone toward the development of specific antiviral therapeutics for clinical management of yellow fever. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A Novel Benzodiazepine Compound Inhibits Yellow Fever Virus Infection by Specifically Targeting NS4B Protein

PubMed Central

Guo, Fang; Wu, Shuo; Julander, Justin; Ma, Julia; Zhang, Xuexiang; Kulp, John; Cuconati, Andrea; Block, Timothy M.; Du, Yanming; Guo, Ju-Tao

2016-01-01

rate fails to prevent outbreaks in at-risk regions. It has been estimated that up to 1.7 million YFV infections occur in Africa each year, resulting in 29,000 to 60,000 deaths. Thus far, there is no specific antiviral treatment for yellow fever. To cope with this medical challenge, we identified a benzodiazepine compound that selectively inhibits YFV by targeting the viral NS4B protein. To our knowledge, this is the first report demonstrating in vivo safety and antiviral efficacy of a YFV NS4B inhibitor in an animal model. We have thus reached a critical milestone toward the development of specific antiviral therapeutics for clinical management of yellow fever. PMID:27654301

Homozygosity for the WRN Helicase-Inactivating Variant, R834C, does not confer a Werner syndrome clinical phenotype

PubMed Central

Kamath-Loeb, Ashwini S.; Zavala-van Rankin, Diego G.; Flores-Morales, Jeny; Emond, Mary J.; Sidorova, Julia M.; Carnevale, Alessandra; Cárdenas-Cortés, Maria del Carmen; Norwood, Thomas H.; Monnat, Raymond J.; Loeb, Lawrence A.; Mercado-Celis, Gabriela E.

2017-01-01

Loss-of-function mutations in the WRN helicase gene cause Werner syndrome- a progeroid syndrome with an elevated risk of cancer and other age-associated diseases. Large numbers of single nucleotide polymorphisms have been identified in WRN. We report here the organismal, cellular, and molecular phenotypes of variant rs3087425 (c. 2500C > T) that results in an arginine to cysteine substitution at residue 834 (R834C) and up to 90% reduction of WRN helicase activity. This variant is present at a high (5%) frequency in Mexico, where we identified 153 heterozygous and three homozygous individuals among 3,130 genotyped subjects. Family studies of probands identified ten additional TT homozygotes. Biochemical analysis of WRN protein purified from TT lymphoblast cell lines confirmed that the R834C substitution strongly and selectively reduces WRN helicase, but not exonuclease activity. Replication track analyses showed reduced replication fork progression in some homozygous cells following DNA replication stress. Among the thirteen TT homozygotes, we identified a previously unreported and statistically significant gender bias in favor of males (p = 0.0016), but none of the clinical findings associated with Werner syndrome. Our results indicate that WRN helicase activity alone is not rate-limiting for the development of clinical WS. PMID:28276523

Role of the phosphatidylinositol-3-kinase/Akt/target of rapamycin pathway during ambidensovirus infection of insect cells.

PubMed

Salasc, F; Mutuel, D; Debaisieux, S; Perrin, A; Dupressoir, T; Grenet, A-S Gosselin; Ogliastro, M

2016-01-01

The phosphatidylinositol-3-kinase (PI3K)/Akt/target of rapamycin (TOR) signalling pathway controls cell growth and survival, and is targeted by a number of viruses at different phases of their infection cycle to control translation. Whether and how insect viruses interact with this pathway remain poorly addressed. Here, we investigated the role of PI3K/Akt/TOR signalling during lethal infection of insect cells with an insect parvovirus. Using Junonia coenia densovirus (JcDV; lepidopteran ambidensovirus 1) and susceptible insect cells as experimental models, we first described JcDV cytopathology, and showed that viral infection affects cell size, cell proliferation and survival. We deciphered the role of PI3K/Akt/TOR signalling in the course of infection and found that non-structural (NS) protein expression correlates with the inhibition of TOR and the shutdown of cellular synthesis, concomitant with the burst of viral protein expression. Together, these results suggest that NS proteins control the cellular translational machinery to favour the translation of viral mRNAs at the expense of cellular mRNAs. As a consequence of TOR inhibition, cell autophagy is activated. These results highlight new functions for NS proteins in the course of multiplication of an insect parvovirus.

Replacement of the respiratory syncytial virus nonstructural proteins NS1 and NS2 by the V protein of parainfluenza virus 5

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tran, Kim C.; He, Biao; Teng, Michael N.

2007-11-10

Paramyxoviruses have been shown to produce proteins that inhibit interferon production and signaling. For human respiratory syncytial virus (RSV), the nonstructural NS1 and NS2 proteins have been shown to have interferon antagonist activity through an unknown mechanism. To understand further the functions of NS1 and NS2, we generated recombinant RSV in which both NS1 and NS2 were replaced by the PIV5 V protein, which has well-characterized IFN antagonist activities ({delta}NS1/2-V). Expression of V was able to partially inhibit IFN responses in {delta}NS1/2-V-infected cells. In addition, the replication kinetics of {delta}NS1/2-V were intermediate between {delta}NS1/2 and wild-type (rA2) in A549 cells.more » However, expression of V did not affect the ability of {delta}NS1/2-V to activate IRF3 nuclear translocation and IFN{beta} transcription. These data indicate that V was able to replace some of the IFN inhibitory functions of the RSV NS1 and NS2 proteins, but also that NS1 and NS2 have functions in viral replication beyond IFN antagonism.« less

The annealing helicase and branch migration activities of Drosophila HARP.

PubMed

Kassavetis, George A; Kadonaga, James T

2014-01-01

HARP (SMARCAL1, MARCAL1) is an annealing helicase that functions in the repair and restart of damaged DNA replication forks through its DNA branch migration and replication fork regression activities. HARP is conserved among metazoans. HARP from invertebrates differs by the absence of one of the two HARP-specific domain repeats found in vertebrates. The annealing helicase and branch migration activity of invertebrate HARP has not been documented. We found that HARP from Drosophila melanogaster retains the annealing helicase activity of human HARP, the ability to disrupt D-loops and to branch migrate Holliday junctions, but fails to regress model DNA replication fork structures. A comparison of human and Drosophila HARP on additional substrates revealed that both HARPs are competent in branch migrating a bidirectional replication bubble composed of either DNA:DNA or RNA:DNA hybrid. Human, but not Drosophila, HARP is also capable of regressing a replication fork structure containing a highly stable poly rG:dC hybrid. Persistent RNA:DNA hybrids in vivo can lead to replication fork arrest and genome instability. The ability of HARP to strand transfer hybrids may signify a hybrid removal function for this enzyme, in vivo.

The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

PubMed Central

2013-01-01

Background Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication. Methods To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference. Results In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L

The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication.

PubMed

Morais, Ana Ts; Terzian, Ana Cb; Duarte, Danilo Vb; Bronzoni, Roberta Vm; Madrid, Maria Cfs; Gavioli, Arieli F; Gil, Laura Hvg; Oliveira, Amanda G; Zanelli, Cleslei F; Valentini, Sandro R; Rahal, Paula; Nogueira, Mauricio L

2013-06-22

Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication. To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference. In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L with YFV NS5 may play a role

Targeting DDX3 with a small molecule inhibitor for lung cancer therapy.

PubMed

Bol, Guus M; Vesuna, Farhad; Xie, Min; Zeng, Jing; Aziz, Khaled; Gandhi, Nishant; Levine, Anne; Irving, Ashley; Korz, Dorian; Tantravedi, Saritha; Heerma van Voss, Marise R; Gabrielson, Kathleen; Bordt, Evan A; Polster, Brian M; Cope, Leslie; van der Groep, Petra; Kondaskar, Atul; Rudek, Michelle A; Hosmane, Ramachandra S; van der Wall, Elsken; van Diest, Paul J; Tran, Phuoc T; Raman, Venu

2015-05-01

Lung cancer is the most common malignancy worldwide and is a focus for developing targeted therapies due to its refractory nature to current treatment. We identified a RNA helicase, DDX3, which is overexpressed in many cancer types including lung cancer and is associated with lower survival in lung cancer patients. We designed a first-in-class small molecule inhibitor, RK-33, which binds to DDX3 and abrogates its activity. Inhibition of DDX3 by RK-33 caused G1 cell cycle arrest, induced apoptosis, and promoted radiation sensitization in DDX3-overexpressing cells. Importantly, RK-33 in combination with radiation induced tumor regression in multiple mouse models of lung cancer. Mechanistically, loss of DDX3 function either by shRNA or by RK-33 impaired Wnt signaling through disruption of the DDX3-β-catenin axis and inhibited non-homologous end joining-the major DNA repair pathway in mammalian somatic cells. Overall, inhibition of DDX3 by RK-33 promotes tumor regression, thus providing a compelling argument to develop DDX3 inhibitors for lung cancer therapy. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

Virtual Screening and Molecular Dynamics Simulations from a Bank of Molecules of the Amazon Region Against Functional NS3-4A Protease-Helicase Enzyme of Hepatitis C Virus.

PubMed

Pinheiro, Alan Sena; Duarte, Jaqueline Bianca Carvalho; Alves, Cláudio Nahum; de Molfetta, Fábio Alberto

2015-07-01

Hepatitis C virus (HCV) infection is a disease that affects approximately 3% of the global population and requires new therapeutic agents without the inconvenience associated with current anti-HCV treatment. This paper reports on a study of a virtual screening and a molecular dynamics simulation of compounds derived from natural products from the Amazon region that are potentially effective against the NS3-4A enzyme of HCV, which plays an important role in the replication process of this virus. According to the results of the molecular docking calculations and subsequent consensual analysis, the best scored compounds showed interactions between hydrogen and residues of the catalytic triad as well as interactions with residues that guide ligands to the active site of the enzyme. They also showed stability in the molecular dynamics simulation, as the structures preserved important interactions at the active site of the enzyme. The root mean square deviation (RMSD) values were stabilized at the end of the simulation time. Such compounds are considered promising as novel therapies against HCV.

Helicase Stepping Investigated with One-Nucleotide Resolution Fluorescence Resonance Energy Transfer

NASA Astrophysics Data System (ADS)

Lin, Wenxia; Ma, Jianbing; Nong, Daguan; Xu, Chunhua; Zhang, Bo; Li, Jinghua; Jia, Qi; Dou, Shuoxing; Ye, Fangfu; Xi, Xuguang; Lu, Ying; Li, Ming

2017-09-01

Single-molecule Förster resonance energy transfer is widely applied to study helicases by detecting distance changes between a pair of dyes anchored to overhangs of a forked DNA. However, it has been lacking single-base pair (1-bp) resolution required for revealing stepping kinetics of helicases. We designed a nanotensioner in which a short DNA is bent to exert force on the overhangs, just as in optical or magnetic tweezers. The strategy improved the resolution of Förster resonance energy transfer to 0.5 bp, high enough to uncover differences in DNA unwinding by yeast Pif1 and E. coli RecQ whose unwinding behaviors cannot be differentiated by currently practiced methods. We found that Pif1 exhibits 1-bp-stepping kinetics, while RecQ breaks 1 bp at a time but sequesters the nascent nucleotides and releases them randomly. The high-resolution data allowed us to propose a three-parameter model to quantitatively interpret the apparently different unwinding behaviors of the two helicases which belong to two superfamilies.

Structural characterization of β-catenin and RX-5902 binding to phospho-p68 RNA helicase by molecular dynamics simulation.

PubMed

Ali, Waqar; Shafique, Shagufta; Rashid, Sajid

2018-05-02

Emerging implications of probable ATP-dependent RNA helicase p68 in tumorigenesis and progression makes it a discerning target for cancer therapy. Recently it has been reported that tyrosyl-phosphorylation of p68 promotes β-catenin nuclear translocation and cancer metastasis through elevating the epithelial-mesenchymal transition. Despite recent advances, the structural characterization of this interaction, mode of action and induced conformational changes remain elusive. Here, through comparative structure analysis and molecular dynamics simulation assays, we explored comparative binding pattern of phospho-p68 against β-catenin. Conversely, due to the promising therapeutic potential of p68 in blocking the invasiveness and metastasis of cancer cells, we investigated the binding of heterocyclic N-substituted piperazine derivative-RX-5902 that inhibits the binding of phospho-p68 and β-catenin. Evidently, transactivation and C-terminal helicase domains of phospho-p68 exhibited dramatic conformational alterations to assist β-catenin and RX-5902 binding. As compared to unbound phospho-p68 (56.1 Å), the residual distances between transactivation domain-Ser79 and C-terminal helicase domain-Gln555 were reduced to 34.1 Å and 31 Å upon binding to β-catenin and RX-5902, respectively. In contrast, helicase ATP-binding domain remained conformationally stable throughout simulations. Clearly, the comparative docking-for-functional analysis of phospho-p68 against RX-5902 and β-catenin uncovered a spectrum of structural linkages associated with the molecular basis of β-catenin-dependent ATPase activity. Thus the outcomes of this study may provide a platform for the rational design of specific and potent inhibitors against phospho-p68 with a special emphasis on anticancer activity. Copyright © 2018 Elsevier Ltd. All rights reserved.

Transcriptome-wide analysis of DEAD-box RNA helicase gene family in an Antarctic psychrophilic alga Chlamydomonas sp. ICE-L.

PubMed

Liu, Chenlin; Huang, Xiaohang

2015-09-01

DEAD-box RNA helicase family proteins have been identified in almost all living organisms. Some of them play a crucial role in adaptation to environmental changes and stress response, especially in the low-temperature acclimation in different kinds of organisms. Compared with the full swing study in plants and bacteria, the characters and functions of DEAD-box family proteins had not been surveyed in algae. To identify genes critical for freezing acclimation in algae, we screened DEAD-box RNA helicase genes from the transcriptome sequences of a psychrophilic microalga Chlamydomonas sp. ICE-L which was isolated from Antarctic sea ice. Totally 39 DEAD-box RNA helicase genes had been identified. Most of the DEAD-box RNA helicase have 1:1 homologous relationships in Chlamydomonas reinhardtii and Chlamydomonas sp. ICE-L with several exceptions. The homologous proteins in ICE-L to the helicases critical for cold or freezing tolerance in Arabidopsis thaliana had been identified based on phylogenetic comparison studies. The response of these helicase genes is not always identical in the Chlamydomonas sp. ICE-L and Arabidopsis under the same low-temperature treatment. The expression of several DEAD-box RNA helicase genes including CiRH5, CiRH25, CiRH28, and CiRH55 were significantly up-regulated under freezing treatment of ICE-L and their function in freezing acclimation of ICE-L deserved further investigation.

Structural rearrangements in the mitochondrial genome of Drosophila melanogaster induced by elevated levels of the replicative DNA helicase

PubMed Central

Ciesielski, Grzegorz L; Nadalutti, Cristina A; Oliveira, Marcos T; Griffith, Jack D; Kaguni, Laurie S

2018-01-01

Abstract Pathological conditions impairing functions of mitochondria often lead to compensatory upregulation of the mitochondrial DNA (mtDNA) replisome machinery, and the replicative DNA helicase appears to be a key factor in regulating mtDNA copy number. Moreover, mtDNA helicase mutations have been associated with structural rearrangements of the mitochondrial genome. To evaluate the effects of elevated levels of the mtDNA helicase on the integrity and replication of the mitochondrial genome, we overexpressed the helicase in Drosophila melanogaster Schneider cells and analyzed the mtDNA by two-dimensional neutral agarose gel electrophoresis and electron microscopy. We found that elevation of mtDNA helicase levels increases the quantity of replication intermediates and alleviates pausing at the replication slow zones. Though we did not observe a concomitant alteration in mtDNA copy number, we observed deletions specific to the segment of repeated elements in the immediate vicinity of the origin of replication, and an accumulation of species characteristic of replication fork stalling. We also found elevated levels of RNA that are retained in the replication intermediates. Together, our results suggest that upregulation of mtDNA helicase promotes the process of mtDNA replication but also results in genome destabilization. PMID:29432582

The NS1 polypeptide of the murine parvovirus minute virus of mice binds to DNA sequences containing the motif [ACCA]2-3.

PubMed Central

Cotmore, S F; Christensen, J; Nüesch, J P; Tattersall, P

1995-01-01

A DNA fragment containing the minute virus of mice 3' replication origin was specifically coprecipitated in immune complexes containing the virally coded NS1, but not the NS2, polypeptide. Antibodies directed against the amino- or carboxy-terminal regions of NS1 precipitated the NS1-origin complexes, but antibodies directed against NS1 amino acids 284 to 459 blocked complex formation. Using affinity-purified histidine-tagged NS1 preparations, we have shown that the specific protein-DNA interaction is of moderate affinity, being stable in 0.1 M salt but rapidly lost at higher salt concentrations. In contrast, generalized (or nonspecific) DNA binding by NS1 could be demonstrated only in low salt. Addition of ATP or gamma S-ATP enhanced specific DNA binding by wild-type NS1 severalfold, but binding was lost under conditions which favored ATP hydrolysis. NS1 molecules with mutations in a critical lysine residue (amino acid 405) in the consensus ATP-binding site bound to the origin, but this binding could not be enhanced by ATP addition. DNase I protection assays carried out with wild-type NS1 in the presence of gamma S-ATP gave footprints which extended over 43 nucleotides on both DNA strands, from the middle of the origin bubble sequence to a position some 14 bp beyond the nick site. The DNA-binding site for NS1 was mapped to a 22-bp fragment from the middle of the 3' replication origin which contains the sequence ACCAACCA. This conforms to a reiterated motif (ACCA)2-3, which occurs, in more or less degenerate form, at many sites throughout the minute virus of mice genome (J. W. Bodner, Virus Genes 2:167-182, 1989). Insertion of a single copy of the sequence (ACCA)3 was shown to be sufficient to confer NS1 binding on an otherwise unrecognized plasmid fragment. The functions of NS1 in the viral life cycle are reevaluated in the light of this result. PMID:7853501

The NS1 polypeptide of the murine parvovirus minute virus of mice binds to DNA sequences containing the motif [ACCA]2-3.

PubMed

Cotmore, S F; Christensen, J; Nüesch, J P; Tattersall, P

1995-03-01

A DNA fragment containing the minute virus of mice 3' replication origin was specifically coprecipitated in immune complexes containing the virally coded NS1, but not the NS2, polypeptide. Antibodies directed against the amino- or carboxy-terminal regions of NS1 precipitated the NS1-origin complexes, but antibodies directed against NS1 amino acids 284 to 459 blocked complex formation. Using affinity-purified histidine-tagged NS1 preparations, we have shown that the specific protein-DNA interaction is of moderate affinity, being stable in 0.1 M salt but rapidly lost at higher salt concentrations. In contrast, generalized (or nonspecific) DNA binding by NS1 could be demonstrated only in low salt. Addition of ATP or gamma S-ATP enhanced specific DNA binding by wild-type NS1 severalfold, but binding was lost under conditions which favored ATP hydrolysis. NS1 molecules with mutations in a critical lysine residue (amino acid 405) in the consensus ATP-binding site bound to the origin, but this binding could not be enhanced by ATP addition. DNase I protection assays carried out with wild-type NS1 in the presence of gamma S-ATP gave footprints which extended over 43 nucleotides on both DNA strands, from the middle of the origin bubble sequence to a position some 14 bp beyond the nick site. The DNA-binding site for NS1 was mapped to a 22-bp fragment from the middle of the 3' replication origin which contains the sequence ACCAACCA. This conforms to a reiterated motif (ACCA)2-3, which occurs, in more or less degenerate form, at many sites throughout the minute virus of mice genome (J. W. Bodner, Virus Genes 2:167-182, 1989). Insertion of a single copy of the sequence (ACCA)3 was shown to be sufficient to confer NS1 binding on an otherwise unrecognized plasmid fragment. The functions of NS1 in the viral life cycle are reevaluated in the light of this result.

Modular magnetic tweezers for single-molecule characterizations of helicases.

PubMed

Kemmerich, Felix E; Kasaciunaite, Kristina; Seidel, Ralf

2016-10-01

Magnetic tweezers provide a versatile toolkit supporting the mechanistic investigation of helicases. In the present article, we show that custom magnetic tweezers setups are straightforward to construct and can easily be extended to provide adaptable platforms, capable of addressing a multitude of enquiries regarding the functions of these fascinating molecular machines. We first address the fundamental components of a basic magnetic tweezers scheme and review some previous results to demonstrate the versatility of this instrument. We then elaborate on several extensions to the basic magnetic tweezers scheme, and demonstrate their applications with data from ongoing research. As our methodological overview illustrates, magnetic tweezers are an extremely useful tool for the characterization of helicases and a custom built instrument can be specifically tailored to suit the experimenter's needs. Copyright © 2016 Elsevier Inc. All rights reserved.

DNA binding and unwinding by Hel308 helicase requires dual functions of a winged helix domain.

PubMed

Northall, Sarah J; Buckley, Ryan; Jones, Nathan; Penedo, J Carlos; Soultanas, Panos; Bolt, Edward L

2017-09-01

Hel308 helicases promote genome stability linked to DNA replication in archaea, and have homologues in metazoans. In the crystal structure of archaeal Hel308 bound to a tailed DNA duplex, core helicase domains encircle single-stranded DNA (ssDNA) in a "ratchet" for directional translocation. A winged helix domain (WHD) is also present, but its function is mysterious. We investigated the WHD in full-length Hel308, identifying that mutations in a solvent exposed α-helix resulted in reduced DNA binding and unwinding activities. When isolated from the rest of Hel308, the WHD protein alone bound to duplex DNA but not ssDNA, and DNA binding by WHD protein was abolished by the same mutations as were analyzed in full-length Hel308. Isolated WHD from a human Hel308 homologue (HelQ) also bound to duplex DNA. By disrupting the interface between the Hel308 WHD and a RecA-like domain, a topology typical of Ski2 helicases, we show that this is crucial for ATPase and helicase activities. The data suggest a model in which the WHD promotes activity of Hel308 directly, through binding to duplex DNA that is distinct from ssDNA binding by core helicase, and indirectly through interaction with the RecA-like domain. We propose how the WHD may contribute to ssDNA translocation, resulting in DNA helicase activity or in removal of other DNA bound proteins by "reeling" ssDNA. Copyright © 2017 Elsevier B.V. All rights reserved.

Amino acids 16-275 of minute virus of mice NS1 include a domain that specifically binds (ACCA)2-3-containing DNA.

PubMed

Mouw, M; Pintel, D J

1998-11-10

GST-NS1 purified from Escherichia coli and insect cells binds double-strand DNA in an (ACCA)2-3-dependent fashion under similar ionic conditions, independent of the presence of anti-NS1 antisera or exogenously supplied ATP and interacts with single-strand DNA and RNA in a sequence-independent manner. An amino-terminal domain (amino acids 1-275) of NS1 [GST-NS1(1-275)], representing 41% of the full-length NS1 molecule, includes a domain that binds double-strand DNA in a sequence-specific manner at levels comparable to full-length GST-NS1, as well as single-strand DNA and RNA in a sequence-independent manner. The deletion of 15 additional amino-terminal amino acids yielded a molecule [GST-NS1(1-275)] that maintained (ACCA)2-3-specific double-strand DNA binding; however, this molecule was more sensitive to increasing ionic conditions than full-length GST-NS1 and GST-NS1(1-275) and could not be demonstrated to bind single-strand nucleic acids. A quantitative filter binding assay showed that E. coli- and baculovirus-expressed GST-NS1 and E. coli GST-NS1(1-275) specifically bound double-strand DNA with similar equilibrium kinetics [as measured by their apparent equilibrium DNA binding constants (KD)], whereas GST-NS1(16-275) bound 4- to 8-fold less well. Copyright 1998 Academic Press.

Sus scrofa miR-204 and miR-4331 Negatively Regulate Swine H1N1/2009 Influenza A Virus Replication by Targeting Viral HA and NS, Respectively.

PubMed

Zhang, Shishuo; Wang, Ruifang; Su, Huijuan; Wang, Biaoxiong; Sizhu, Suolang; Lei, Zhixin; Jin, Meilin; Chen, Huanchun; Cao, Jiyue; Zhou, Hongbo

2017-04-03

The prevalence of swine pandemic H1N1/2009 influenza A virus (SIV-H1N1/2009) in pigs has the potential to generate novel reassortant viruses, posing a great threat to human health. Cellular microRNAs (miRNAs) have been proven as promising small molecules for regulating influenza A virus replication by directly targeting viral genomic RNA. In this study, we predicted potential Sus scrofa (ssc-, swine) miRNAs targeting the genomic RNA of SIV-H1N1/2009 by RegRNA 2.0, and identified ssc-miR-204 and ssc-miR-4331 to target viral HA and NS respectively through dual-luciferase reporter assays. The messenger RNA (mRNA) levels of viral HA and NS were significantly suppressed when newborn pig trachea (NPTr) cells respectively overexpressed ssc-miR-204 and ssc-miR-4331 and were infected with SIV-H1N1/2009, whereas the suppression effect could be restored when respectively decreasing endogenous ssc-miR-204 and ssc-miR-4331 with inhibitors. Because of the importance of viral HA and NS in the life cycle of influenza A virus, ssc-miR-204 and ssc-miR-4331 exhibited an inhibition effect on SIV-H1N1/2009 replication. The antiviral effect was sequence-specific of SIV-H1N1/2009, for the target sites in HA and NS of H5N1 or H9N2 influenza A virus were not conserved. Furthermore, SIV-H1N1/2009 infection reversely downregulated the expression of ssc-miR-204 and ssc-miR-4331, which might facilitate the virus replication in the host. In summary, this work will provide us some important clues for controlling the prevalence of SIV-H1N1/2009 in pig populations.

3D-QSAR and molecular docking studies on designing inhibitors of the hepatitis C virus NS5B polymerase

NASA Astrophysics Data System (ADS)

Li, Wenlian; Si, Hongzong; Li, Yang; Ge, Cuizhu; Song, Fucheng; Ma, Xiuting; Duan, Yunbo; Zhai, Honglin

2016-08-01

Viral hepatitis C infection is one of the main causes of the hepatitis after blood transfusion and hepatitis C virus (HCV) infection is a global health threat. The HCV NS5B polymerase, an RNA dependent RNA polymerase (RdRp) and an essential role in the replication of the virus, has no functional equivalent in mammalian cells. So the research and development of efficient NS5B polymerase inhibitors provides a great strategy for antiviral therapy against HCV. A combined three-dimensional quantitative structure-activity relationship (QSAR) modeling was accomplished to profoundly understand the structure-activity correlation of a train of indole-based inhibitors of the HCV NS5B polymerase to against HCV. A comparative molecular similarity indices analysis (COMSIA) model as the foundation of the maximum common substructure alignment was developed. The optimum model exhibited statistically significant results: the cross-validated correlation coefficient q2 was 0.627 and non-cross-validated r2 value was 0.943. In addition, the results of internal validations of bootstrapping and Y-randomization confirmed the rationality and good predictive ability of the model, as well as external validation (the external predictive correlation coefficient rext2 = 0.629). The information obtained from the COMSIA contour maps enables the interpretation of their structure-activity relationship. Furthermore, the molecular docking study of the compounds for 3TYV as the protein target revealed important interactions between active compounds and amino acids, and several new potential inhibitors with higher activity predicted were designed basis on our analyses and supported by the simulation of molecular docking. Meanwhile, the OSIRIS Property Explorer was introduced to help select more satisfactory compounds. The satisfactory results from this study may lay a reliable theoretical base for drug development of hepatitis C virus NS5B polymerase inhibitors.

DNA unwinding by ring-shaped T4 helicase gp41 is hindered by tension on the occluded strand.

PubMed

Ribeck, Noah; Saleh, Omar A

2013-01-01

The replicative helicase for bacteriophage T4 is gp41, which is a ring-shaped hexameric motor protein that achieves unwinding of dsDNA by translocating along one strand of ssDNA while forcing the opposite strand to the outside of the ring. While much study has been dedicated to the mechanism of binding and translocation along the ssDNA strand encircled by ring-shaped helicases, relatively little is known about the nature of the interaction with the opposite, 'occluded' strand. Here, we investigate the interplay between the bacteriophage T4 helicase gp41 and the ss/dsDNA fork by measuring, at the single-molecule level, DNA unwinding events on stretched DNA tethers in multiple geometries. We find that gp41 activity is significantly dependent on the geometry and tension of the occluded strand, suggesting an interaction between gp41 and the occluded strand that stimulates the helicase. However, the geometry dependence of gp41 activity is the opposite of that found previously for the E. coli hexameric helicase DnaB. Namely, tension applied between the occluded strand and dsDNA stem inhibits unwinding activity by gp41, while tension pulling apart the two ssDNA tails does not hinder its activity. This implies a distinct variation in helicase-occluded strand interactions among superfamily IV helicases, and we propose a speculative model for this interaction that is consistent with both the data presented here on gp41 and the data that had been previously reported for DnaB.

Mcm3 replicative helicase mutation impairs neuroblast proliferation and memory in Drosophila.

PubMed

Blumröder, R; Glunz, A; Dunkelberger, B S; Serway, C N; Berger, C; Mentzel, B; de Belle, J S; Raabe, T

2016-09-01

In the developing Drosophila brain, a small number of neural progenitor cells (neuroblasts) generate in a co-ordinated manner a high variety of neuronal cells by integration of temporal, spatial and cell-intrinsic information. In this study, we performed the molecular and phenotypic characterization of a structural brain mutant called small mushroom bodies (smu), which was isolated in a screen for mutants with altered brain structure. Focusing on the mushroom body neuroblast lineages we show that failure of neuroblasts to generate the normal number of mushroom body neurons (Kenyon cells) is the major cause of the smu phenotype. In particular, the premature loss of mushroom body neuroblasts caused a pronounced effect on the number of late-born Kenyon cells. Neuroblasts showed no obvious defects in processes controlling asymmetric cell division, but generated less ganglion mother cells. Cloning of smu uncovered a single amino acid substitution in an evolutionarily conserved protein interaction domain of the Minichromosome maintenance 3 (Mcm3) protein. Mcm3 is part of the multimeric Cdc45/Mcm/GINS (CMG) complex, which functions as a helicase during DNA replication. We propose that at least in the case of mushroom body neuroblasts, timely replication is not only required for continuous proliferation but also for their survival. The absence of Kenyon cells in smu reduced learning and early phases of conditioned olfactory memory. Corresponding to the absence of late-born Kenyon cells projecting to α'/β' and α/β lobes, smu is profoundly defective in later phases of persistent memory. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Tumor Necrosis Factor Receptor-Associated Factor 5 Interacts with the NS3 Protein and Promotes Classical Swine Fever Virus Replication.

PubMed

Lv, Huifang; Dong, Wang; Guo, Kangkang; Jin, Mingxing; Li, Xiaomeng; Li, Cunfa; Zhang, Yanming

2018-06-05

Classical swine fever, caused by classical swine fever virus (CSFV), is a highly contagious and high-mortality viral disease, causing huge economic losses in the swine industry worldwide. CSFV non-structural protein 3 (NS3), a multifunctional protein, plays crucial roles in viral replication. However, how NS3 exactly exerts these functions is currently unknown. Here, we identified tumor necrosis factor receptor-associated factor 5 (TRAF5) as a novel binding partner of the NS3 protein via yeast two-hybrid, co-immunoprecipitation and glutathione S -transferase pull-down assays. Furthermore, we observed that TRAF5 promoted CSFV replication in porcine alveolar macrophages (PAMs). Additionally, CSFV infection or NS3 expression upregulated TRAF5 expression, implying that CSFV may exploit TRAF5 via NS3 for better growth. Moreover, CSFV infection and TRAF5 expression activated p38 mitogen activated protein kinase (MAPK) activity, and inhibition of p38 MAPK activation by the SB203580 inhibitor suppressed CSFV replication. Notably, TRAF5 overexpression did not promote CSFV replication following inhibition of p38 MAPK activation. Our findings reveal that TRAF5 promotes CSFV replication via p38 MAPK activation. This work provides a novel insight into the role of TRAF5 in CSFV replication capacity.

Dengue and Zika viruses subvert reticulophagy by NS2B3-mediated cleavage of FAM134B.

PubMed

Lennemann, Nicholas J; Coyne, Carolyn B

2017-02-01

The endoplasmic reticulum (ER) is exploited by several diverse viruses during their infectious life cycles. Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), utilize the ER as a source of membranes to establish their replication organelles and to facilitate their assembly and eventual maturation along the secretory pathway. To maintain normal homeostasis, host cells have evolved highly efficient processes to dynamically regulate the ER, such as through reticulophagy, a selective form of autophagy that leads to ER degradation. Here, we identify the ER-localized reticulophagy receptor FAM134B as a host cell restriction factor for both DENV and ZIKV. We show that RNAi-mediated depletion of FAM134B significantly enhances both DENV and ZIKV replication at an early stage of the viral life cycle. Consistent with its role as an antiviral host factor, we found that several flaviviruses including DENV, ZIKV, and West Nile virus (WNV), utilize their NS3 virally-encoded proteases to directly cleave FAM134B at a single site within its reticulon homology domain (RHD). Mechanistically, we show that NS3-mediated cleavage of FAM134B blocks the formation of ER and viral protein-enriched autophagosomes, suggesting that the cleavage of FAM134B serves to specifically suppress the reticulophagy pathway. These findings thus point to an important role for FAM134B and reticulophagy in the regulation of flavivirus infection and suggest that these viruses specifically target these pathways to promote viral replication.

RTEL1: functions of a disease-associated helicase.

PubMed

Vannier, Jean-Baptiste; Sarek, Grzegorz; Boulton, Simon J

2014-07-01

DNA secondary structures that arise during DNA replication, repair, and recombination (3R) must be processed correctly to prevent genetic instability. Regulator of telomere length 1 (RTEL1) is an essential DNA helicase that disassembles a variety of DNA secondary structures to facilitate 3R processes and to maintain telomere integrity. The past few years have witnessed the emergence of RTEL1 variants that confer increased susceptibility to high-grade glioma, astrocytomas, and glioblastomas. Mutations in RTEL1 have also been implicated in Hoyeraal-Hreidarsson syndrome, a severe form of the bone-marrow failure and cancer predisposition disorder, dyskeratosis congenita. We review these recent findings and highlight its crucial link between DNA secondary-structure metabolism and human disease. Copyright © 2014 Elsevier Ltd. All rights reserved.

Species-Specific Inhibition of RIG-I Ubiquitination and IFN Induction by the Influenza A Virus NS1 Protein

PubMed Central

Rajsbaum, Ricardo; Albrecht, Randy A.; Wang, May K.; Maharaj, Natalya P.; Versteeg, Gijs A.; Nistal-Villán, Estanislao; García-Sastre, Adolfo; Gack, Michaela U.

2012-01-01

Influenza A viruses can adapt to new host species, leading to the emergence of novel pathogenic strains. There is evidence that highly pathogenic viruses encode for non-structural 1 (NS1) proteins that are more efficient in suppressing the host immune response. The NS1 protein inhibits type-I interferon (IFN) production partly by blocking the TRIM25 ubiquitin E3 ligase-mediated Lys63-linked ubiquitination of the viral RNA sensor RIG-I, required for its optimal downstream signaling. In order to understand possible mechanisms of viral adaptation and host tropism, we examined the ability of NS1 encoded by human (Cal04), avian (HK156), swine (SwTx98) and mouse-adapted (PR8) influenza viruses to interact with TRIM25 orthologues from mammalian and avian species. Using co-immunoprecipitation assays we show that human TRIM25 binds to all tested NS1 proteins, whereas the chicken TRIM25 ortholog binds preferentially to the NS1 from the avian virus. Strikingly, none of the NS1 proteins were able to bind mouse TRIM25. Since NS1 can inhibit IFN production in mouse, we tested the impact of TRIM25 and NS1 on RIG-I ubiquitination in mouse cells. While NS1 efficiently suppressed human TRIM25-dependent ubiquitination of RIG-I 2CARD, NS1 inhibited the ubiquitination of full-length mouse RIG-I in a mouse TRIM25-independent manner. Therefore, we tested if the ubiquitin E3 ligase Riplet, which has also been shown to ubiquitinate RIG-I, interacts with NS1. We found that NS1 binds mouse Riplet and inhibits its activity to induce IFN-β in murine cells. Furthermore, NS1 proteins of human but not swine or avian viruses were able to interact with human Riplet, thereby suppressing RIG-I ubiquitination. In conclusion, our results indicate that influenza NS1 protein targets TRIM25 and Riplet ubiquitin E3 ligases in a species-specific manner for the inhibition of RIG-I ubiquitination and antiviral IFN production. PMID:23209422

Species-specific inhibition of RIG-I ubiquitination and IFN induction by the influenza A virus NS1 protein.

PubMed

Rajsbaum, Ricardo; Albrecht, Randy A; Wang, May K; Maharaj, Natalya P; Versteeg, Gijs A; Nistal-Villán, Estanislao; García-Sastre, Adolfo; Gack, Michaela U

2012-01-01

Influenza A viruses can adapt to new host species, leading to the emergence of novel pathogenic strains. There is evidence that highly pathogenic viruses encode for non-structural 1 (NS1) proteins that are more efficient in suppressing the host immune response. The NS1 protein inhibits type-I interferon (IFN) production partly by blocking the TRIM25 ubiquitin E3 ligase-mediated Lys63-linked ubiquitination of the viral RNA sensor RIG-I, required for its optimal downstream signaling. In order to understand possible mechanisms of viral adaptation and host tropism, we examined the ability of NS1 encoded by human (Cal04), avian (HK156), swine (SwTx98) and mouse-adapted (PR8) influenza viruses to interact with TRIM25 orthologues from mammalian and avian species. Using co-immunoprecipitation assays we show that human TRIM25 binds to all tested NS1 proteins, whereas the chicken TRIM25 ortholog binds preferentially to the NS1 from the avian virus. Strikingly, none of the NS1 proteins were able to bind mouse TRIM25. Since NS1 can inhibit IFN production in mouse, we tested the impact of TRIM25 and NS1 on RIG-I ubiquitination in mouse cells. While NS1 efficiently suppressed human TRIM25-dependent ubiquitination of RIG-I 2CARD, NS1 inhibited the ubiquitination of full-length mouse RIG-I in a mouse TRIM25-independent manner. Therefore, we tested if the ubiquitin E3 ligase Riplet, which has also been shown to ubiquitinate RIG-I, interacts with NS1. We found that NS1 binds mouse Riplet and inhibits its activity to induce IFN-β in murine cells. Furthermore, NS1 proteins of human but not swine or avian viruses were able to interact with human Riplet, thereby suppressing RIG-I ubiquitination. In conclusion, our results indicate that influenza NS1 protein targets TRIM25 and Riplet ubiquitin E3 ligases in a species-specific manner for the inhibition of RIG-I ubiquitination and antiviral IFN production.

Could GRB170817A be really correlated to an NS-NS merging?

NASA Astrophysics Data System (ADS)

Fargion, D.; Khlopov, M. Yu.; Oliva, P.

The exciting development of gravitational wave (GW) astronomy in the correlation of LIGO and VIRGO detection of GW signals makes possible to expect registration of effects of not only binary black hole (BH) coalescence but also binary neutron star (NS) merging accompanied by electromagnetic (gamma ray burst; GRB) signal. Here we consider the possibility that an NS, merging in an NS-NS or NS-BH system might be (soon) observed in correlation with any LIGO-VIRGO GWs detection. We analyze as an example the recent case of the short GRB170817A observed by Fermi and integral. The associated optical transient (OT) source in NGC4993 implies a rare near source, a consequent averaged large rate of such events (almost) compatible with expected NS-NS merging rate. However the expected beamed GRB (or short GRB) may be mostly aligned to a different direction than ours. Therefore, even soft GRB photons, spread more than hard ones, might be hardly able to shower to us. Nevertheless, a prompt spiraling electron turbine jet in largest magnetic fields, at the base of the NS-NS collapse, might shine by its tangential synchrotron radiation in spread way with its skimming photons shining in large open disk. The consequent solid angle for such soft disk gamma radiation may be large enough to be nevertheless often observed.

18F-labeled norepinephrine transporter tracer [18F]NS12137: radiosynthesis and preclinical evaluation.

PubMed

Kirjavainen, Anna K; Forsback, Sarita; López-Picón, Francisco R; Marjamäki, Päivi; Takkinen, Jatta; Haaparanta-Solin, Merja; Peters, Dan; Solin, Olof

2018-01-01

Several psychiatric and neurodegenerative diseases are associated with malfunction of brain norepinephrine transporter (NET). However, current clinical evaluations of NET function are limited by the lack of sufficiently sensitive methods of detection. To this end, we have synthesized exo-3-[(6-[ 18 F]fluoro-2-pyridyl)oxy]-8-azabicyclo[3.2.1]-octane ([ 18 F]NS12137) as a radiotracer for positron emission tomography (PET) and have demonstrated that it is highly specific for in vivo detection of NET-rich regions of rat brain tissue. We applied two methods of electrophilic, aromatic radiofluorination of the precursor molecule, exo-3-[(6-trimethylstannyl-2-pyridyl)oxy]-8-azabicyclo-[3.2.1]octane-8-carboxylate: (1) direct labeling with [ 18 F]F 2 , and (2) labeling with [ 18 F]Selectfluor, a derivative of [ 18 F]F 2 , using post-target produced [ 18 F]F 2 . The time-dependent distribution of [ 18 F]NS12137 in brain tissue of healthy, adult Sprague-Dawley rats was determined by ex vivo autoradiography. The specificity of [ 18 F]NS12137 binding was demonstrated on the basis of competitive binding by nisoxetine, a known NET antagonist of high specificity. [ 18 F]NS12137 was successfully synthesized with radiochemical yields of 3.9% ± 0.3% when labeled with [ 18 F]F 2 and 10.2% ± 2.7% when labeled with [ 18 F]Selectfluor. The molar activity of radiotracer was 8.8 ± 0.7 GBq/μmol with [ 18 F]F 2 labeling and 6.9 ± 0.4 GBq/μmol with [ 18 F]Selectfluor labeling at the end of synthesis of [ 18 F]NS12137. Uptake of [ 18 F]NS12137 in NET-rich areas in rat brain was demonstrated with the locus coeruleus (LCoe) having the highest regional uptake. Prior treatment of rats with nisoxetine showed no detectable [ 18 F]NS12137 in the LCoe. Analyses of whole brain samples for radiometabolites showed only the parent compound [ 18 F]NS12137. Uptake of 18 F-radioactivity in bone increased with time. The two electrophilic 18 F-labeling methods proved to be suitable for synthesis of [ 18 F]NS

Downregulation of viral RNA translation by hepatitis C virus non-structural protein NS5A requires the poly(U/UC) sequence in the 3' UTR.

PubMed

Hoffman, Brett; Li, Zhubing; Liu, Qiang

2015-08-01

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is essential for viral replication; however, its effect on HCV RNA translation remains controversial partially due to the use of reporters lacking the 3' UTR, where NS5A binds to the poly(U/UC) sequence. We investigated the role of NS5A in HCV translation using a monocistronic RNA containing a Renilla luciferase gene flanked by the HCV UTRs. We found that NS5A downregulated viral RNA translation in a dose-dependent manner. This downregulation required both the 5' and 3' UTRs of HCV because substitution of either sequence with the 5' and 3' UTRs of enterovirus 71 or a cap structure at the 5' end eliminated the effects of NS5A on translation. Translation of the HCV genomic RNA was also downregulated by NS5A. The inhibition of HCV translation by NS5A required the poly(U/UC) sequence in the 3' UTR as NS5A did not affect translation when it was deleted. In addition, we showed that, whilst the amphipathic α-helix of NS5A has no effect on viral translation, the three domains of NS5A can inhibit translation independently, also dependent on the presence of the poly(U/UC) sequence in the 3' UTR. These results suggested that NS5A downregulated HCV RNA translation through a mechanism involving the poly(U/UC) sequence in the 3' UTR.

The C-terminal domain of the Bloom syndrome DNA helicase is essential for genomic stability

PubMed Central

Yankiwski, Victor; Noonan, James P; Neff, Norma F

2001-01-01

Background Bloom syndrome is a rare cancer-prone disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. Bloom syndrome cells have a distinctive high frequency of sister chromatid exchange and quadriradial formation. BLM, the protein altered in BS, is a member of the RecQ DNA helicase family, whose members share an average of 40% identity in the helicase domain and have divergent N-terminal and C-terminal flanking regions of variable lengths. The BLM DNA helicase has been shown to localize to the ND10 (nuclear domain 10) or PML (promyelocytic leukemia) nuclear bodies, where it associates with TOPIIIα, and to the nucleolus. Results This report demonstrates that the N-terminal domain of BLM is responsible for localization of the protein to the nuclear bodies, while the C-terminal domain directs the protein to the nucleolus. Deletions of the N-terminal domain of BLM have little effect on sister chromatid exchange frequency and chromosome stability as compared to helicase and C-terminal mutations which can increase SCE frequency and chromosome abnormalities. Conclusion The helicase activity and the C-terminal domain of BLM are critical for maintaining genomic stability as measured by the sister chromatid exchange assay. The localization of BLM into the nucleolus by the C-terminal domain appears to be more important to genomic stability than localization in the nuclear bodies. PMID:11472631

Flaviviral NS4b, chameleon and jack-in-the-box roles in viral replication and pathogenesis, and a molecular target for antiviral intervention.

PubMed

Zmurko, Joanna; Neyts, Johan; Dallmeier, Kai

2015-07-01

Dengue virus and other flaviviruses such as the yellow fever, West Nile, and Japanese encephalitis viruses are emerging vector-borne human pathogens that affect annually more than 100 million individuals and that may cause debilitating and potentially fatal hemorrhagic and encephalitic diseases. Currently, there are no specific antiviral drugs for the treatment of flavivirus-associated disease. A better understanding of the flavivirus-host interactions during the different events of the flaviviral life cycle may be essential when developing novel antiviral strategies. The flaviviral non-structural protein 4b (NS4b) appears to play an important role in flaviviral replication by facilitating the formation of the viral replication complexes and in counteracting innate immune responses such as the following: (i) type I IFN signaling; (ii) RNA interference; (iii) formation of stress granules; and (iv) the unfolded protein response. Intriguingly, NS4b has recently been shown to constitute an excellent target for the selective inhibition of flavivirus replication. We here review the current knowledge on NS4b. © 2015 The Authors. Reviews in Medical Virology published by John Wiley & Sons Ltd.

Infectious Bovine Viral Diarrhea Virus (Strain NADL) RNA from Stable cDNA Clones: a Cellular Insert Determines NS3 Production and Viral Cytopathogenicity

PubMed Central

Mendez, Ernesto; Ruggli, Nicolas; Collett, Marc S.; Rice, Charles M.

1998-01-01

Bovine viral diarrhea virus (BVDV), strain NADL, was originally isolated from an animal with fatal mucosal disease. This isolate is cytopathic in cell culture and produces two forms of NS3-containing proteins: uncleaved NS2-3 and mature NS3. For BVDV NADL, the production of NS3, a characteristic of cytopathic BVDV strains, is believed to be a consequence of an in-frame insertion of a 270-nucleotide cellular mRNA sequence (called cIns) in the NS2 coding region. In this study, we constructed a stable full-length cDNA copy of BVDV NADL in a low-copy-number plasmid vector. As assayed by transfection of MDBK cells, uncapped RNAs transcribed from this template were highly infectious (>105 PFU/μg). The recovered virus was similar in plaque morphology, growth properties, polyprotein processing, and cytopathogenicity to the BVDV NADL parent. Deletion of cIns abolished processing at the NS2/NS3 site and produced a virus that was no longer cytopathic for MDBK cells. This deletion did not affect the efficiency of infectious virus production or viral protein production, but it reduced the level of virus-specific RNA synthesis and accumulation. Thus, cIns not only modulates NS3 production but also upregulates RNA replication relative to an isogenic noncytopathic derivative lacking the insert. These results raise the possibility of a linkage between enhanced BVDV NADL RNA replication and virus-induced cytopathogenicity. PMID:9573238

Staufen2 functions in Staufen1-mediated mRNA decay by binding to itself and its paralog and promoting UPF1 helicase but not ATPase activity

PubMed Central

Park, Eonyoung; Gleghorn, Michael L.; Maquat, Lynne E.

2013-01-01

Staufen (STAU)1-mediated mRNA decay (SMD) is a posttranscriptional regulatory mechanism in mammals that degrades mRNAs harboring a STAU1-binding site (SBS) in their 3′-untranslated regions (3′ UTRs). We show that SMD involves not only STAU1 but also its paralog STAU2. STAU2, like STAU1, is a double-stranded RNA-binding protein that interacts directly with the ATP-dependent RNA helicase up-frameshift 1 (UPF1) to reduce the half-life of SMD targets that form an SBS by either intramolecular or intermolecular base-pairing. Compared with STAU1, STAU2 binds ∼10-fold more UPF1 and ∼two- to fivefold more of those SBS-containing mRNAs that were tested, and it comparably promotes UPF1 helicase activity, which is critical for SMD. STAU1- or STAU2-mediated augmentation of UPF1 helicase activity is not accompanied by enhanced ATP hydrolysis but does depend on ATP binding and a basal level of UPF1 ATPase activity. Studies of STAU2 demonstrate it changes the conformation of RNA-bound UPF1. These findings, and evidence for STAU1−STAU1, STAU2−STAU2, and STAU1−STAU2 formation in vitro and in cells, are consistent with results from tethering assays: the decrease in mRNA abundance brought about by tethering siRNA-resistant STAU2 or STAU1 to an mRNA 3′ UTR is inhibited by downregulating the abundance of cellular STAU2, STAU1, or UPF1. It follows that the efficiency of SMD in different cell types reflects the cumulative abundance of STAU1 and STAU2. We propose that STAU paralogs contribute to SMD by “greasing the wheels” of RNA-bound UPF1 so as to enhance its unwinding capacity per molecule of ATP hydrolyzed. PMID:23263869

Staufen2 functions in Staufen1-mediated mRNA decay by binding to itself and its paralog and promoting UPF1 helicase but not ATPase activity.

PubMed

Park, Eonyoung; Gleghorn, Michael L; Maquat, Lynne E

2013-01-08

Staufen (STAU)1-mediated mRNA decay (SMD) is a posttranscriptional regulatory mechanism in mammals that degrades mRNAs harboring a STAU1-binding site (SBS) in their 3'-untranslated regions (3' UTRs). We show that SMD involves not only STAU1 but also its paralog STAU2. STAU2, like STAU1, is a double-stranded RNA-binding protein that interacts directly with the ATP-dependent RNA helicase up-frameshift 1 (UPF1) to reduce the half-life of SMD targets that form an SBS by either intramolecular or intermolecular base-pairing. Compared with STAU1, STAU2 binds ~10-fold more UPF1 and ~two- to fivefold more of those SBS-containing mRNAs that were tested, and it comparably promotes UPF1 helicase activity, which is critical for SMD. STAU1- or STAU2-mediated augmentation of UPF1 helicase activity is not accompanied by enhanced ATP hydrolysis but does depend on ATP binding and a basal level of UPF1 ATPase activity. Studies of STAU2 demonstrate it changes the conformation of RNA-bound UPF1. These findings, and evidence for STAU1-STAU1, STAU2-STAU2, and STAU1-STAU2 formation in vitro and in cells, are consistent with results from tethering assays: the decrease in mRNA abundance brought about by tethering siRNA-resistant STAU2 or STAU1 to an mRNA 3' UTR is inhibited by downregulating the abundance of cellular STAU2, STAU1, or UPF1. It follows that the efficiency of SMD in different cell types reflects the cumulative abundance of STAU1 and STAU2. We propose that STAU paralogs contribute to SMD by "greasing the wheels" of RNA-bound UPF1 so as to enhance its unwinding capacity per molecule of ATP hydrolyzed.

Uncommon and Emissive {[Au2(C3H6NS2)2][Au(C3H6NS2)2]2(PF6)2} Mixed Au+ and Au3+ Pseudotetranuclear Crystalline Compound: Synthesis, Structural Characterization, and Optical Properties.

PubMed

Langaro, Ana P; Souza, Ana K R; Morassuti, Claudio Y; Lima, Sandro M; Casagrande, Gleison A; Deflon, Victor M; Nunes, Luiz A O; Da Cunha Andrade, Luis H

2016-11-23

An uncommon emissive pseudotetranuclear compound, {[Au 2 (C 3 H 6 NS 2 ) 2 ][Au(C 3 H 6 NS 2 ) 2 ] 2 (PF 6 ) 2 }, was synthesized and characterized in terms of its structure and optical properties. The synthesis produced a crystalline compound composed of four gold atoms with two different oxidation states (Au + and Au 3+ ) in the same crystalline structure. The title complex belonged to a triclinic crystalline system involving the centrosymmetric P1̅ space group. X-ray diffractometry and vibrational spectroscopy (infrared, Raman, and SERS) were used for structural characterization of the new crystal. The vibrational spectroscopy techniques supported the X-ray diffraction results and confirmed the presence of bonds including Au-Au and Au-S. Optical characterization performed using UV-vis spectroscopy showed that under ultraviolet excitation, the emissive crystalline complex presented characteristic broad luminescent bands centered at 420 and 670 nm.

Recombinant Nonstructural 3 Protein, rNS3, of Hepatitis C Virus Along With Recombinant GP96 Induce IL-12, TNFα and α5integrin Expression in Antigen Presenting Cells

PubMed Central

Hajizadeh, Mohammad Reza; Mokarram, Pooneh; Kamali sarvestani, Eskandar; Bolhassani, Azam; Mostafavi Pour, Zohreh

2013-01-01

Background Hepatitis C virus (HCV) infection is the main cause of chronic liver disease and to date there has been no vaccine development to prevent this infection. Among non-structural HCV proteins, NS3 protein is an excellent goal for a therapeutic vaccine, due to its large size and less variation in conserved regions. The immunogenic properties of heat shock proteins (HSPs) for instance GP96 have prompted investigations into their function as strong adjuvant to improve innate and adaptive immunity. Objectives The aim of this study was to examine additive effects of recombinant GP96 (rGP96) fragments accompanied by rNS3 on expression levels of α5integrin and pro-inflammatory cytokines, IL-12 and TNFα, in Antigen Presenting Cells (APCs). Materials and Methods Recombinant viral proteins (rNS3 and rRGD-NS3), N-terminal and C-terminal fragments of GP96 were produced and purified from E. coli in order to treat the cells; mouse spleen Dendritic Cells (DCs) and THP-1 macrophages. Results Our results showed that rNT-GP96 alone significantly increases the expression level of IL-12, TNFα and α5integrin in THP-1 macrophages and DCs, while IL-12 and TNFα expression levels were unaffected by either rNS3 or rRGD-NS3. Interestingly, the co-addition of these recombinant proteins with rNT-GP96 increased IL-12, TNFα and α5integrin expression. Pearson Correlation showed a direct association between α5integrin with IL-12 and TNF-α expression. Conclusions we have highlighted the role of rNS3 plus rNT-GP96 mediated by α5integrin in producing IL-12 and TNFα. It can be suggested that rNT-GP96 could enhance immunity characteristic of rNS3 protein via production of pro-inflammatory cytokines. PMID:24032046

Severe acute respiratory syndrome coronavirus replication inhibitor that interferes with the nucleic acid unwinding of the viral helicase.

PubMed

Adedeji, Adeyemi O; Singh, Kamalendra; Calcaterra, Nicholas E; DeDiego, Marta L; Enjuanes, Luis; Weiss, Susan; Sarafianos, Stefan G

2012-09-01

Severe acute respiratory syndrome (SARS) is a highly contagious disease, caused by SARS coronavirus (SARS-CoV), for which there are no approved treatments. We report the discovery of a potent inhibitor of SARS-CoV that blocks replication by inhibiting the unwinding activity of the SARS-CoV helicase (nsp13). We used a Förster resonance energy transfer (FRET)-based helicase assay to screen the Maybridge Hitfinder chemical library. We identified and validated a compound (SSYA10-001) that specifically blocks the double-stranded RNA (dsRNA) and dsDNA unwinding activities of nsp13, with 50% inhibitory concentrations (IC(50)s) of 5.70 and 5.30 μM, respectively. This compound also has inhibitory activity (50% effective concentration [EC(50)] = 8.95 μM) in a SARS-CoV replicon assay, with low cytotoxicity (50% cytotoxic concentration [CC(50)] = >250 μM), suggesting that the helicase plays a still unidentified critical role in the SARS-CoV life cycle. Enzyme kinetic studies on the mechanism of nsp13 inhibition revealed that SSYA10-001 acts as a noncompetitive inhibitor of nsp13 with respect to nucleic acid and ATP substrates. Moreover, SSYA10-001 does not affect ATP hydrolysis or nsp13 binding to the nucleic acid substrate. SSYA10-001 did not inhibit hepatitis C virus (HCV) helicase, other bacterial and viral RNA-dependent RNA polymerases, or reverse transcriptase. These results suggest that SSYA10-001 specifically blocks nsp13 through a novel mechanism and is less likely to interfere with the functions of cellular enzymes that process nucleic acids or ATP. Hence, it is possible that SSYA10-001 inhibits unwinding by nsp13 by affecting conformational changes during the course of the reaction or translocation on the nucleic acid. SSYA10-001 will be a valuable tool for studying the specific role of nsp13 in the SARS-CoV life cycle, which could be a model for other nidoviruses and also a candidate for further development as a SARS antiviral target.

The Arabidopsis At1g30680 gene encodes a homologue to the phage T7 gp4 protein that has both DNA primase and DNA helicase activities.

PubMed

Diray-Arce, Joann; Liu, Bin; Cupp, John D; Hunt, Travis; Nielsen, Brent L

2013-03-04

The Arabidopsis thaliana genome encodes a homologue of the full-length bacteriophage T7 gp4 protein, which is also homologous to the eukaryotic Twinkle protein. While the phage protein has both DNA primase and DNA helicase activities, in animal cells Twinkle is localized to mitochondria and has only DNA helicase activity due to sequence changes in the DNA primase domain. However, Arabidopsis and other plant Twinkle homologues retain sequence homology for both functional domains of the phage protein. The Arabidopsis Twinkle homologue has been shown by others to be dual targeted to mitochondria and chloroplasts. To determine the functional activity of the Arabidopsis protein we obtained the gene for the full-length Arabidopsis protein and expressed it in bacteria. The purified protein was shown to have both DNA primase and DNA helicase activities. Western blot and qRT-PCR analysis indicated that the Arabidopsis gene is expressed most abundantly in young leaves and shoot apex tissue, as expected if this protein plays a role in organelle DNA replication. This expression is closely correlated with the expression of organelle-localized DNA polymerase in the same tissues. Homologues from other plant species show close similarity by phylogenetic analysis. The results presented here indicate that the Arabidopsis phage T7 gp4/Twinkle homologue has both DNA primase and DNA helicase activities and may provide these functions for organelle DNA replication.

Interplay of cis- and trans-regulatory mechanisms in the spliceosomal RNA helicase Brr2.

PubMed

Absmeier, Eva; Becke, Christian; Wollenhaupt, Jan; Santos, Karine F; Wahl, Markus C

2017-01-02

RNA helicase Brr2 is implicated in multiple phases of pre-mRNA splicing and thus requires tight regulation. Brr2 can be auto-inhibited via a large N-terminal region folding back onto its helicase core and auto-activated by a catalytically inactive C-terminal helicase cassette. Furthermore, it can be regulated in trans by the Jab1 domain of the Prp8 protein, which can inhibit Brr2 by intermittently inserting a C-terminal tail in the enzyme's RNA-binding tunnel or activate the helicase after removal of this tail. Presently it is unclear, whether these regulatory mechanisms functionally interact and to which extent they are evolutionarily conserved. Here, we report crystal structures of Saccharomyces cerevisiae and Chaetomium thermophilum Brr2-Jab1 complexes, demonstrating that Jab1-based inhibition of Brr2 presumably takes effect in all eukaryotes but is implemented via organism-specific molecular contacts. Moreover, the structures show that Brr2 auto-inhibition can act in concert with Jab1-mediated inhibition, and suggest that the N-terminal region influences how the Jab1 C-terminal tail interacts at the RNA-binding tunnel. Systematic RNA binding and unwinding studies revealed that the N-terminal region and the Jab1 C-terminal tail specifically interfere with accommodation of double-stranded and single-stranded regions of an RNA substrate, respectively, mutually reinforcing each other. Additionally, such analyses show that regulation based on the N-terminal region requires the presence of the inactive C-terminal helicase cassette. Together, our results outline an intricate system of regulatory mechanisms, which control Brr2 activities during snRNP assembly and splicing.

Evolution of the DEAD box helicase family in chicken: chickens have no DHX9 ortholog.

PubMed

Sato, Haruko; Oshiumi, Hiroyuki; Takaki, Hiromi; Hikono, Hirokazu; Seya, Tsukasa

2015-10-01

Viral RNA represents a pattern molecule that can be recognized by RNA sensors in innate immunity. Humans and mice possess cytoplasmic DNA/RNA sensors for detecting viral replication. There are a number of DEAD (Asp-Glu-Ala-Asp; DExD/H) box-type helicases in mammals, among which retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated protein 5 (MDA50) are indispensable for RNA sensing; however, they are functionally supported by a number of sensors that directly bind viral RNA or replicative RNA intermediates to convey signals to RIG-I and MDA5. Some DEAD box helicase members recognize DNA irrespective of the origin. These sensors transmit IFN-inducing signals through adaptors, including mitochondrial antiviral signaling. Viral double-stranded RNAs are reportedly sensed by the helicases DDX1, DDX21, DHX36, DHX9, DDX3, DDX41, LGP2 and DDX60, in addition to RIG-I and MDA5, and induce type I IFNs, thereby blocking viral replication. Humans and mice have all nucleic acid sensors listed here. In the RNA sensing system in chicken, it was found in the present study that most DEAD box helicases are conserved; however, DHX9 is genetically deficient in addition to reported RIG-I. Based on the current genome databases, similar DHX9 deficiency was observed in ducks and several other bird species. Because chicken, but not duck, was found to be deficient in RIG-I, the RNA-sensing system of chicken lacks RIG-I and DHX9 and is thus more fragile than that of duck or mammal. DHX9 may generally compensate for the function of RIG-I and deficiency of DHX9 possibly participates in exacerbations of viral infection such as influenza in chickens. © 2015 The Societies and Wiley Publishing Asia Pty Ltd.

DNA secondary structure of the released strand stimulates WRN helicase action on forked duplexes without coordinate action of WRN exonuclease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahn, Byungchan, E-mail: bbccahn@mail.ulsan.ac.kr; Bohr, Vilhelm A.

2011-08-12

Highlights: {yields} In this study, we investigated the effect of a DNA secondary structure on the two WRN activities. {yields} We found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. {yields} These results imply that WRN helicase and exonuclease activities can act independently. -- Abstract: Werner syndrome (WS) is an autosomal recessive premature aging disorder characterized by aging-related phenotypes and genomic instability. WS is caused by mutations in a gene encoding a nuclear protein, Werner syndrome protein (WRN), a member of the RecQ helicase family, that interestingly possessesmore » both helicase and exonuclease activities. Previous studies have shown that the two activities act in concert on a single substrate. We investigated the effect of a DNA secondary structure on the two WRN activities and found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. These results imply that WRN helicase and exonuclease activities can act independently, and we propose that the uncoordinated action may be relevant to the in vivo activity of WRN.« less

Tick-Borne Encephalitis Virus Nonstructural Protein NS5 Induces RANTES Expression Dependent on the RNA-Dependent RNA Polymerase Activity.

PubMed

Zheng, Zifeng; Yang, Jieyu; Jiang, Xuan; Liu, Yalan; Zhang, Xiaowei; Li, Mei; Zhang, Mudan; Fu, Ming; Hu, Kai; Wang, Hanzhong; Luo, Min-Hua; Gong, Peng; Hu, Qinxue

2018-05-14

Tick-borne encephalitis virus (TBEV) is one of the flaviviruses that targets the CNS and causes encephalitis in humans. The mechanism of TBEV that causes CNS destruction remains unclear. It has been reported that RANTES-mediated migration of human blood monocytes and T lymphocytes is specifically induced in the brain of mice infected with TBEV, which causes ensuing neuroinflammation and may contribute to brain destruction. However, the viral components responsible for RANTES induction and the underlying mechanisms remain to be fully addressed. In this study, we demonstrate that the NS5, but not other viral proteins of TBEV, induces RANTES production in human glioblastoma cell lines and primary astrocytes. TBEV NS5 appears to activate the IFN regulatory factor 3 (IRF-3) signaling pathway in a manner dependent on RIG-I/MDA5, which leads to the nuclear translocation of IRF-3 to bind with RANTES promoter. Further studies reveal that the activity of RNA-dependent RNA polymerase (RdRP) but not the RNA cap methyltransferase is critical for TBEV NS5-induced RANTES expression, and this is likely due to RdRP-mediated synthesis of dsRNA. Additional data indicate that the residues at K359, D361, and D664 of TBEV NS5 are critical for RdRP activity and RANTES induction. Of note, NS5s from other flaviviruses, including Japanese encephalitis virus, West Nile virus, Zika virus, and dengue virus, can also induce RANTES expression, suggesting the significance of NS5-induced RANTES expression in flavivirus pathogenesis. Our findings provide a foundation for further understanding how flaviviruses cause neuroinflammation and a potential viral target for intervention. Copyright © 2018 by The American Association of Immunologists, Inc.

CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication.

PubMed

Langston, Lance D; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E; Finkelstein, Jeff; Yao, Nina Y; Indiani, Chiara; O'Donnell, Mike E

2014-10-28

DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG-Pol ε complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.

CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication

PubMed Central

Langston, Lance D.; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E.; Finkelstein, Jeff; Yao, Nina Y.; Indiani, Chiara; O’Donnell, Mike E.

2014-01-01

DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG–Pol ε complex and showed that it is a functional polymerase–helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes. PMID:25313033

Activation of BRCA1/BRCA2-Associated Helicase BACH1 Is Required for Timely Progression through S Phase▿

PubMed Central

Kumaraswamy, Easwari; Shiekhattar, Ramin

2007-01-01

BACH1 (also known as FANCJ and BRIP1) is a DNA helicase that directly interacts with the C-terminal BRCT repeat of the breast cancer susceptibility protein BRCA1. Previous biochemical and functional analyses have suggested a role for the BACH1 homolog in Caenorhabditis elegans during DNA replication. Here, we report the association of BACH1 with a distinct BRCA1/BRCA2-containing complex during the S phase of the cell cycle. Depletion of BACH1 or BRCA1 using small interfering RNAs results in delayed entry into the S phase of the cell cycle. Such timely progression through S phase requires the helicase activity of BACH1. Importantly, cells expressing a dominant negative mutation in BACH1 that results in a defective helicase displayed increased activation of DNA damage checkpoints and genomic instability. BACH1 helicase is silenced during the G1 phase of the cell cycle and is activated through a dephosphorylation event as cells enter S phase. These results point to a critical role for BACH1 helicase activity not only in the timely progression through the S phase but also in maintaining genomic stability. PMID:17664283

The structure of SV40 large T hexameric helicase in complex with AT-rich origin DNA

PubMed Central

Gai, Dahai; Wang, Damian; Li, Shu-Xing; Chen, Xiaojiang S

2016-01-01

DNA replication is a fundamental biological process. The initial step in eukaryotic DNA replication is the assembly of the pre-initiation complex, including the formation of two head-to-head hexameric helicases around the replication origin. How these hexameric helicases interact with their origin dsDNA remains unknown. Here, we report the co-crystal structure of the SV40 Large-T Antigen (LT) hexameric helicase bound to its origin dsDNA. The structure shows that the six subunits form a near-planar ring that interacts with the origin, so that each subunit makes unique contacts with the DNA. The origin dsDNA inside the narrower AAA+ domain channel shows partial melting due to the compression of the two phosphate backbones, forcing Watson-Crick base-pairs within the duplex to flip outward. This structure provides the first snapshot of a hexameric helicase binding to origin dsDNA, and suggests a possible mechanism of origin melting by LT during SV40 replication in eukaryotic cells. DOI: http://dx.doi.org/10.7554/eLife.18129.001 PMID:27921994

Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2.

PubMed

Tossavainen, Helena; Aitio, Olli; Hellman, Maarit; Saksela, Kalle; Permi, Perttu

2016-07-29

We show that a peptide from Chikungunya virus nsP3 protein spanning residues 1728-1744 binds the amphiphysin-2 (BIN1) Src homology-3 (SH3) domain with an unusually high affinity (Kd 24 nm). Our NMR solution complex structure together with isothermal titration calorimetry data on several related viral and cellular peptide ligands reveal that this exceptional affinity originates from interactions between multiple basic residues in the target peptide and the extensive negatively charged binding surface of amphiphysin-2 SH3. Remarkably, these arginines show no fixed conformation in the complex structure, indicating that a transient or fluctuating polyelectrostatic interaction accounts for this affinity. Thus, via optimization of such dynamic electrostatic forces, viral peptides have evolved a superior binding affinity for amphiphysin-2 SH3 compared with typical cellular ligands, such as dynamin, thereby enabling hijacking of amphiphysin-2 SH3-regulated host cell processes by these viruses. Moreover, our data show that the previously described consensus sequence PXRPXR for amphiphysin SH3 ligands is inaccurate and instead define it as an extended Class II binding motif PXXPXRpXR, where additional positive charges between the two constant arginine residues can give rise to extraordinary high SH3 binding affinity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Interactive Roles of DNA Helicases and Translocases with the Single-Stranded DNA Binding Protein RPA in Nucleic Acid Metabolism.

PubMed

Awate, Sanket; Brosh, Robert M

2017-06-08

Helicases and translocases use the energy of nucleoside triphosphate binding and hydrolysis to unwind/resolve structured nucleic acids or move along a single-stranded or double-stranded polynucleotide chain, respectively. These molecular motors facilitate a variety of transactions including replication, DNA repair, recombination, and transcription. A key partner of eukaryotic DNA helicases/translocases is the single-stranded DNA binding protein Replication Protein A (RPA). Biochemical, genetic, and cell biological assays have demonstrated that RPA interacts with these human molecular motors physically and functionally, and their association is enriched in cells undergoing replication stress. The roles of DNA helicases/translocases are orchestrated with RPA in pathways of nucleic acid metabolism. RPA stimulates helicase-catalyzed DNA unwinding, enlists translocases to sites of action, and modulates their activities in DNA repair, fork remodeling, checkpoint activation, and telomere maintenance. The dynamic interplay between DNA helicases/translocases and RPA is just beginning to be understood at the molecular and cellular levels, and there is still much to be learned, which may inform potential therapeutic strategies.

Interactive Roles of DNA Helicases and Translocases with the Single-Stranded DNA Binding Protein RPA in Nucleic Acid Metabolism

PubMed Central

Awate, Sanket; Brosh, Robert M.

2017-01-01

Helicases and translocases use the energy of nucleoside triphosphate binding and hydrolysis to unwind/resolve structured nucleic acids or move along a single-stranded or double-stranded polynucleotide chain, respectively. These molecular motors facilitate a variety of transactions including replication, DNA repair, recombination, and transcription. A key partner of eukaryotic DNA helicases/translocases is the single-stranded DNA binding protein Replication Protein A (RPA). Biochemical, genetic, and cell biological assays have demonstrated that RPA interacts with these human molecular motors physically and functionally, and their association is enriched in cells undergoing replication stress. The roles of DNA helicases/translocases are orchestrated with RPA in pathways of nucleic acid metabolism. RPA stimulates helicase-catalyzed DNA unwinding, enlists translocases to sites of action, and modulates their activities in DNA repair, fork remodeling, checkpoint activation, and telomere maintenance. The dynamic interplay between DNA helicases/translocases and RPA is just beginning to be understood at the molecular and cellular levels, and there is still much to be learned, which may inform potential therapeutic strategies. PMID:28594346

The putative RNA helicase Dbp6p functionally interacts with Rpl3p, Nop8p and the novel trans-acting Factor Rsa3p during biogenesis of 60S ribosomal subunits in Saccharomyces cerevisiae.

PubMed Central

de la Cruz, Jesús; Lacombe, Thierry; Deloche, Olivier; Linder, Patrick; Kressler, Dieter

2004-01-01

Ribosome biogenesis requires at least 18 putative ATP-dependent RNA helicases in Saccharomyces cerevisiae. To explore the functional environment of one of these putative RNA helicases, Dbp6p, we have performed a synthetic lethal screen with dbp6 alleles. We have previously characterized the nonessential Rsa1p, whose null allele is synthetically lethal with dbp6 alleles. Here, we report on the characterization of the four remaining synthetic lethal mutants, which reveals that Dbp6p also functionally interacts with Rpl3p, Nop8p, and the so-far-uncharacterized Rsa3p (ribosome assembly 3). The nonessential Rsa3p is a predominantly nucleolar protein required for optimal biogenesis of 60S ribosomal subunits. Both Dbp6p and Rsa3p are associated with complexes that most likely correspond to early pre-60S ribosomal particles. Moreover, Rsa3p is co-immunoprecipitated with protA-tagged Dbp6p under low salt conditions. In addition, we have established a synthetic interaction network among factors involved in different aspects of 60S-ribosomal-subunit biogenesis. This extensive genetic analysis reveals that the rsa3 null mutant displays some specificity by being synthetically lethal with dbp6 alleles and by showing some synthetic enhancement with the nop8-101 and the rsa1 null allele. PMID:15126390

Allosteric ligands for the pharmacologically important Flavivirus target (NS5) from ZINC database based on pharmacophoric points, free energy calculations and dynamics correlation.

PubMed

Khan, Abbas; Saleem, Shoaib; Idrees, Muhammad; Ali, Syed Shujait; Junaid, Muhammad; Chandra Kaushik, Aman; Wei, Dong-Qing

2018-04-11

Dengue virus belongs to a group of human pathogens, which causes different diseases, dengue hemorrhagic fever and dengue shock syndrome in humans. It possesses RNA as a genetic material and is replicated with the aid of NS5 protein. RNA-dependent RNA polymerase (RdRp) is an important domain of NS5 in the replication of that virus. The catalytic process activity of RdRp is making it an important target for antiviral chemical therapy. To date, No FDA drug has been approved and marketed for the treatment of diseases caused by Dengue virus. So, there is a dire need to advance an area of active antiviral inhibitors that is safe, less expensive and widely available. An experimentally validated complex of Dengue NS5 and compound 27 (6LS) were used as pharmacophoric input and hits were identified. We also used Molecular dynamics (MD) simulations alongside free energy and dynamics of the internal residues of the apo and holo systems to understand the binding mechanism. Our analysis resulted that the three inhibitors (ZINC72070002, ZINC6551486, and ZINC39588257) greatly affected the interior dynamics and residual signaling to dysfunction the replicative role of NS5. The interaction of these inhibitors caused the loss of the correlated motion of NS5 near to the N terminus and helped the stability of the binding complex. This investigation provided a methodological route to discover allosteric inhibitors against the epidemics of this Flaviviruses. Allosteric inhibitors are important and major assets in considering the development of the competitive and robust antiviral agents such as against Dengue viral infection. Copyright © 2018 Elsevier Inc. All rights reserved.

A single-chip 32-channel analog beamformer with 4-ns delay resolution and 768-ns maximum delay range for ultrasound medical imaging with a linear array transducer.

PubMed

Um, Ji-Yong; Kim, Yoon-Jee; Cho, Seong-Eun; Chae, Min-Kyun; Kim, Byungsub; Sim, Jae-Yoon; Park, Hong-June

2015-02-01

A single-chip 32-channel analog beamformer is proposed. It achieves a delay resolution of 4 ns and a maximum delay range of 768 ns. It has a focal-point based architecture, which consists of 7 sub-analog beamformers (sub-ABF). Each sub-ABF performs a RX focusing operation for a single focal point. Seven sub-ABFs perform a time-interleaving operation to achieve the maximum delay range of 768 ns. Phase interpolators are used in sub-ABFs to generate sampling clocks with the delay resolution of 4 ns from a low frequency system clock of 5 MHz. Each sub-ABF samples 32 echo signals at different times into sampling capacitors, which work as analog memory cells. The sampled 32 echo signals of each sub-ABF are originated from one target focal point at one instance. They are summed at one instance in a sub-ABF to perform the RX focusing for the target focal point. The proposed ABF chip has been fabricated in a 0.13- μ m CMOS process with an active area of 16 mm (2). The total power consumption is 287 mW. In measurement, the digital echo signals from a commercial ultrasound medical imaging machine were applied to the fabricated chip through commercial DAC chips. Due to the speed limitation of the DAC chips, the delay resolution was relaxed to 10 ns for the real-time measurement. A linear array transducer with no steering operation is used in this work.

Tripeptide inhibitors of dengue and West Nile virus NS2B-NS3 protease.

PubMed

Schüller, Andreas; Yin, Zheng; Brian Chia, C S; Doan, Danny N P; Kim, Hyeong-Kyu; Shang, Luqing; Loh, Teck Peng; Hill, Jeffery; Vasudevan, Subhash G

2011-10-01

A series of tripeptide aldehyde inhibitors were synthesized and their inhibitory effect against dengue virus type 2 (DENV2) and West Nile virus (WNV) NS3 protease was evaluated side by side with the aim to discover potent flaviviral protease inhibitors and to examine differences in specificity of the two proteases. The synthesized inhibitors feature a varied N-terminal cap group and side chain modifications of a P2-lysine residue. In general a much stronger inhibitory effect of the tripeptide inhibitors was observed toward WNV protease. The inhibitory concentrations against DENV2 protease were in the micromolar range while they were submicromolar against WNV. The data suggest that a P2-arginine shifts the specificity toward DENV2 protease while WNV protease favors a lysine in the P2 position. Peptides with an extended P2-lysine failed to inhibit DENV2 protease suggesting a size-constrained S2 pocket. Our results generally encourage the investigation of di- and tripeptide aldehydes as inhibitors of DENV and WNV protease. Copyright © 2011 Elsevier B.V. All rights reserved.

Differential involvement of the related DNA helicases Pif1p and Rrm3p in mtDNA point mutagenesis and stability.

PubMed

O'Rourke, Thomas W; Doudican, Nicole A; Zhang, Hong; Eaton, Jana S; Doetsch, Paul W; Shadel, Gerald S

2005-07-18

With the exception of base excision repair, conserved pathways and mechanisms that maintain mitochondrial genome stability have remained largely undelineated. In the budding yeast, Saccharomyces cerevisiae, Pif1p is a unique DNA helicase that is localized both to the nucleus and mitochondria, where it is involved in maintaining DNA integrity. We previously elucidated a role for Pif1p in oxidative mtDNA damage resistance that appears to be distinct from its postulated function in mtDNA recombination. Strains lacking Pif1p (pif1Delta) exhibit an increased rate of formation of petite mutants (an indicator of mtDNA instability) and elevated mtDNA point mutagenesis. Here we show that deletion of the RRM3 gene, which encodes a DNA helicase closely related to Pif1p, significantly rescues the petite-induction phenotype of a pif1Delta strain. However, suppression of this phenotype was not accompanied by a corresponding decrease in mtDNA point mutagenesis. Instead, deletion of RRM3 alone resulted in an increase in mtDNA point mutagenesis that was synergistic with that caused by a pif1Delta mutation. In addition, we found that over-expression of RNR1, encoding a large subunit of ribonucleotide reductase (RNR), rescued the petite-induction phenotype of a pif1Delta mutation to a similar extent as deletion of RRM3. This, coupled to our finding that the Rad53p protein kinase is phosphorylated in the rrm3Delta pif1Delta double-mutant strain, leads us to conclude that one mechanism whereby deletion of RRM3 influences mtDNA stability is by modulating mitochondrial deoxynucleoside triphosphate pools. We propose that this is accomplished by signaling through the conserved Mec1/Rad53, S-phase checkpoint pathway to induce the expression and activity of RNR. Altogether, our results define a novel role for Rrm3p in mitochondrial function and indicate that Pif1p and Rrm3p influence a common process (or processes) involved in mtDNA replication, repair, or stability.

TFaNS-Tone Fan Noise Design/Prediction System: Users' Manual TFaNS Version 1.5

NASA Technical Reports Server (NTRS)

Topol, David A.; Huff, Dennis L. (Technical Monitor)

2003-01-01

TFaNS is the Tone Fan Noise Design/Prediction System developed by Pratt & Whitney under contract to NASA Glenn. The purpose of this system is to predict tone noise emanating from a fan stage including the effects of reflection and transmission by the rotor and stator and by the duct inlet and nozzle. The first version of this design system was developed under a previous NASA contract. Several improvements have been made to TFaNS. This users' manual shows how to run this new system. TFaNS consists of the codes that compute the acoustic properties (reflection and transmission coefficients) of the various elements and writes them to files, CUP3D Fan Noise Coupling Code that reads these files, solves the coupling problem, and outputs the desired noise predictions, and AWAKEN CFD/Measured Wake Postprocessor which reformats CFD wake predictions and/or measured wake data so they can be used by the system. This report provides information on code input and file structure essential for potential users of TFaNS.

Quantification of NS1 dengue biomarker in serum via optomagnetic nanocluster detection

NASA Astrophysics Data System (ADS)

Antunes, Paula; Watterson, Daniel; Parmvi, Mattias; Burger, Robert; Boisen, Anja; Young, Paul; Cooper, Matthew A.; Hansen, Mikkel F.; Ranzoni, Andrea; Donolato, Marco

2015-11-01

Dengue is a tropical vector-borne disease without cure or vaccine that progressively spreads into regions with temperate climates. Diagnostic tools amenable to resource-limited settings would be highly valuable for epidemiologic control and containment during outbreaks. Here, we present a novel low-cost automated biosensing platform for detection of dengue fever biomarker NS1 and demonstrate it on NS1 spiked in human serum. Magnetic nanoparticles (MNPs) are coated with high-affinity monoclonal antibodies against NS1 via bio-orthogonal Cu-free ‘click’ chemistry on an anti-fouling surface molecular architecture. The presence of the target antigen NS1 triggers MNP agglutination and the formation of nanoclusters with rapid kinetics enhanced by external magnetic actuation. The amount and size of the nanoclusters correlate with the target concentration and can be quantified using an optomagnetic readout method. The resulting automated dengue fever assay takes just 8 minutes, requires 6 μL of serum sample and shows a limit of detection of 25 ng/mL with an upper detection range of 20000 ng/mL. The technology holds a great potential to be applied to NS1 detection in patient samples. As the assay is implemented on a low-cost microfluidic disc the platform is suited for further expansion to multiplexed detection of a wide panel of biomarkers.

Transmembrane Domains of NS2B Contribute to both Viral RNA Replication and Particle Formation in Japanese Encephalitis Virus.

PubMed

Li, Xiao-Dan; Deng, Cheng-Lin; Ye, Han-Qing; Zhang, Hong-Lei; Zhang, Qiu-Yan; Chen, Dong-Dong; Zhang, Pan-Tao; Shi, Pei-Yong; Yuan, Zhi-Ming; Zhang, Bo

2016-06-15

Flavivirus nonstructural protein 2B (NS2B) is a transmembrane protein that functions as a cofactor for viral NS3 protease. The cytoplasmic region (amino acids 51 to 95) alone of NS2B is sufficient for NS3 protease activity, whereas the role of transmembrane domains (TMDs) remains obscure. Here, we demonstrate for the first time that flavivirus NS2B plays a critical role in virion assembly. Using Japanese encephalitis virus (JEV) as a model, we performed a systematic mutagenesis at the flavivirus conserved residues within the TMDs of NS2B. As expected, some mutations severely attenuated (L38A and R101A) or completely destroyed (G12L) viral RNA synthesis. Interestingly, two mutations (G37L and P112A) reduced viral RNA synthesis and blocked virion assembly. None of the mutations affected NS2B-NS3 protease activity. Because mutations G37L and P112A affected virion assembly, we selected revertant viruses for these two mutants. For mutant G37L, replacement with G37F, G37H, G37T, or G37S restored virion assembly. For mutant P112A, insertion of K at position K127 (leading to K127KK) of NS2B rescued virion assembly. A biomolecular fluorescent complementation (BiFC) analysis demonstrated that (i) mutation P112A selectively weakened NS2B-NS2A interaction and (ii) the adaptive mutation K127KK restored NS2B-NS2A interaction. Collectively, our results demonstrate that, in addition to being a cofactor for NS3 protease, flavivirus NS2B also functions in viral RNA replication, as well as virion assembly. Many flaviviruses are important human pathogens. Understanding the molecular mechanisms of the viral infection cycle is essential for vaccine and antiviral development. In this study, we demonstrate that the TMDs of JEV NS2B participate in both viral RNA replication and virion assembly. A viral genetic study and a BiFC assay demonstrated that interaction between NS2B and NS2A may participate in modulating viral assembly in the flavivirus life cycle. Compensatory-mutation analysis

Chemical genetics-based development of small molecules targeting hepatitis C virus.

PubMed

Jin, Guanghai; Lee, Jisu; Lee, Kyeong

2017-09-01

Hepatitis C virus (HCV) infection is a major worldwide problem that has emerged as one of the most significant diseases affecting humans. There are currently no vaccines or efficient therapies without side effects, despite today's advanced medical technology. Currently, the common therapy for most patients (i.e. genotype 1) is combination of HCV-specific direct-acting antivirals (DAAs). Up to 2011, the standard of care (SOC) was a combination of peg-IFNα with ribavirin (RBV). After approval of NS3/4A protease inhibitor, SOC was peg-IFNα and RBV with either the first-generation DAAs boceprevir or telaprevir. In the past several years, various novel small molecules have been discovered and some of them (i.e., HCV polymerase, protease, helicase and entry inhibitors) have undergone clinical trials. Between 2013 and 2016, the second-generation DAA drugs simeprevir, asunaprevir, daclatasvir, dasabuvir, sofosbuvir, and elbasvir were approved, as well as the combinational drugs Harvoni ® , Zepatier ® , Technivie ® , and Epclusa ® . A number of reviews have been recently published describing the structure-activity relationship (SAR) in the development of HCV inhibitors and outlining current therapeutic approaches to hepatitis C infection. Target identification involves studying a drug's mechanism of action (MOA), and a variety of target identification methods have been developed in the past few years. Chemical biology has emerged as a powerful tool for studying biological processes using small molecules. The use of chemical genetic methods is a valuable strategy for studying the molecular mechanisms of the viral lifecycle and screening for anti-viral agents. Two general screening approaches have been employed: forward and reverse chemical genetics. This review reveals information on the small molecules in HCV drug discovery by using chemical genetics for targeting the HCV protein and describes successful examples of targets identified with these methods.

Molecular characterisation of RIG-I-like helicases in the black flying fox, Pteropus alecto.

PubMed

Cowled, Christopher; Baker, Michelle L; Zhou, Peng; Tachedjian, Mary; Wang, Lin-Fa

2012-04-01

The RIG-I like helicases, RIG-I, mda5 and LGP2 are an evolutionarily conserved family of cytosolic pattern recognition receptors important in the recognition of viral RNA, and responsible for the innate induction of interferons and proinflammatory cytokines upon viral infection. Bats are natural reservoir hosts to a variety of RNA viruses that cause significant morbidity and mortality in other species; however the mechanisms responsible for the control of viral replication in bats are not understood. This report describes the molecular cloning and expression analysis of RIG-I, mda5 and LGP2 genes in the fruit bat Pteropus alecto, and is the first description of RIG-I like helicases from any species of bat. Our results demonstrate that P. alecto RIG-I, mda5 and LGP2 have similar primary structures and tissue expression patterns to their counterparts in humans and other mammals. Stimulation of bat kidney cells with synthetic dsRNA (poly I:C) induced high levels of interferon β and rapid upregulation of all three helicases. These findings reveal that the cytoplasmic virus sensing machinery is present and intact in P. alecto. This study provides the foundation for further investigations into the interactions between bat RIG-I-like helicases and viruses to elucidate the mechanisms responsible for the asymptomatic nature of viral infections in bats. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

Biochemical Characterization of a Mycobacteriophage Derived DnaB Ortholog Reveals New Insight into the Evolutionary Origin of DnaB Helicases

PubMed Central

Bhowmik, Priyanka; Das Gupta, Sujoy K.

2015-01-01

The bacterial replicative helicases known as DnaB are considered to be members of the RecA superfamily. All members of this superfamily, including DnaB, have a conserved C- terminal domain, known as the RecA core. We unearthed a series of mycobacteriophage encoded proteins in which the RecA core domain alone was present. These proteins were phylogenetically related to each other and formed a distinct clade within the RecA superfamily. A mycobacteriophage encoded protein, Wildcat Gp80 that roots deep in the DnaB family, was found to possess a core domain having significant sequence homology (Expect value < 10-5) with members of this novel cluster. This indicated that Wildcat Gp80, and by extrapolation, other members of the DnaB helicase family, may have evolved from a single domain RecA core polypeptide belonging to this novel group. Biochemical investigations confirmed that Wildcat Gp80 was a helicase. Surprisingly, our investigations also revealed that a thioredoxin tagged truncated version of the protein in which the N-terminal sequences were removed was fully capable of supporting helicase activity, although its ATP dependence properties were different. DnaB helicase activity is thus, primarily a function of the RecA core although additional N-terminal sequences may be necessary for fine tuning its activity and stability. Based on sequence comparison and biochemical studies we propose that DnaB helicases may have evolved from single domain RecA core proteins having helicase activities of their own, through the incorporation of additional N-terminal sequences. PMID:26237048

Flaviviral NS4b, chameleon and jack‐in‐the‐box roles in viral replication and pathogenesis, and a molecular target for antiviral intervention

PubMed Central

Zmurko, Joanna; Dallmeier, Kai

2015-01-01

Summary Dengue virus and other flaviviruses such as the yellow fever, West Nile, and Japanese encephalitis viruses are emerging vector‐borne human pathogens that affect annually more than 100 million individuals and that may cause debilitating and potentially fatal hemorrhagic and encephalitic diseases. Currently, there are no specific antiviral drugs for the treatment of flavivirus‐associated disease. A better understanding of the flavivirus–host interactions during the different events of the flaviviral life cycle may be essential when developing novel antiviral strategies. The flaviviral non‐structural protein 4b (NS4b) appears to play an important role in flaviviral replication by facilitating the formation of the viral replication complexes and in counteracting innate immune responses such as the following: (i) type I IFN signaling; (ii) RNA interference; (iii) formation of stress granules; and (iv) the unfolded protein response. Intriguingly, NS4b has recently been shown to constitute an excellent target for the selective inhibition of flavivirus replication. We here review the current knowledge on NS4b. © 2015 The Authors. Reviews in Medical Virology published by John Wiley & Sons Ltd. PMID:25828437

RNA helicase-like protein as an early regulator of transcription factors for plant chilling and freezing tolerance

PubMed Central

Gong, Zhizhong; Lee, Hojoung; Xiong, Liming; Jagendorf, André; Stevenson, Becky; Zhu, Jian-Kang

2002-01-01

Susceptibility to chilling injury prevents the cultivation of many important crops and limits the extended storage of horticultural commodities. Although freezing tolerance is acquired through cold-induced gene expression changes mediated in part by the CBF family of transcriptional activators, whether plant chilling resistance or sensitivity involves the CBF genes is not known. We report here that an Arabidopsis thaliana mutant impaired in the cold-regulated expression of CBF genes and their downstream target genes is sensitive to chilling stress. Expression of CBF3 under a strong constitutive promoter restores chilling resistance to the mutant plants. The mutated gene was cloned and found to encode a nuclear localized RNA helicase. Our results identify a regulator of CBF genes, and demonstrate the importance of gene regulation and the CBF transcriptional activators in plant chilling resistance. PMID:12165572

Rationalizing meat consumption. The 4Ns.

PubMed

Piazza, Jared; Ruby, Matthew B; Loughnan, Steve; Luong, Mischel; Kulik, Juliana; Watkins, Hanne M; Seigerman, Mirra

2015-08-01

Recent theorizing suggests that the 4Ns - that is, the belief that eating meat is natural, normal, necessary, and nice - are common rationalizations people use to defend their choice of eating meat. However, such theorizing has yet to be subjected to empirical testing. Six studies were conducted on the 4Ns. Studies 1a and 1b demonstrated that the 4N classification captures the vast majority (83%-91%) of justifications people naturally offer in defense of eating meat. In Study 2, individuals who endorsed the 4Ns tended also to objectify (dementalize) animals and included fewer animals in their circle of moral concern, and this was true independent of social dominance orientation. Subsequent studies (Studies 3-5) showed that individuals who endorsed the 4Ns tend not to be motivated by ethical concerns when making food choices, are less involved in animal-welfare advocacy, less driven to restrict animal products from their diet, less proud of their animal-product decisions, tend to endorse Speciesist attitudes, tend to consume meat and animal products more frequently, and are highly committed to eating meat. Furthermore, omnivores who strongly endorsed the 4Ns tended to experience less guilt about their animal-product decisions, highlighting the guilt-alleviating function of the 4Ns. Copyright © 2015 Elsevier Ltd. All rights reserved.

Archaeal orthologs of Cdc45 and GINS form a stable complex that stimulates the helicase activity of MCM.

PubMed

Xu, Yuli; Gristwood, Tamzin; Hodgson, Ben; Trinidad, Jonathan C; Albers, Sonja-Verena; Bell, Stephen D

2016-11-22

The regulated recruitment of Cdc45 and GINS is key to activating the eukaryotic MCM(2-7) replicative helicase. We demonstrate that the homohexameric archaeal MCM helicase associates with orthologs of GINS and Cdc45 in vivo and in vitro. Association of these factors with MCM robustly stimulates the MCM helicase activity. In contrast to the situation in eukaryotes, archaeal Cdc45 and GINS form an extremely stable complex before binding MCM. Further, the archaeal GINS•Cdc45 complex contains two copies of Cdc45. Our analyses give insight into the function and evolution of the conserved core of the archaeal/eukaryotic replisome.

The Helicase Activity of Hyperthermophilic Archaeal MCM is Enhanced at High Temperatures by Lysine Methylation.

PubMed

Xia, Yisui; Niu, Yanling; Cui, Jiamin; Fu, Yang; Chen, Xiaojiang S; Lou, Huiqiang; Cao, Qinhong

2015-01-01

Lysine methylation and methyltransferases are widespread in the third domain of life, archaea. Nevertheless, the effects of methylation on archaeal proteins wait to be defined. Here, we report that recombinant sisMCM, an archaeal homolog of Mcm2-7 eukaryotic replicative helicase, is methylated by aKMT4 in vitro. Mono-methylation of these lysine residues occurs coincidently in the endogenous sisMCM protein purified from the hyperthermophilic Sulfolobus islandicus cells as indicated by mass spectra. The helicase activity of mini-chromosome maintenance (MCM) is stimulated by methylation, particularly at temperatures over 70°C. The methylated MCM shows optimal DNA unwinding activity after heat-treatment between 76 and 82°C, which correlates well with the typical growth temperatures of hyperthermophilic Sulfolobus. After methylation, the half life of MCM helicase is dramatically extended at 80°C. The methylated sites are located on the accessible protein surface, which might modulate the intra- and inter- molecular interactions through changing the hydrophobicity and surface charge. Furthermore, the methylation-mimic mutants of MCM show heat resistance helicase activity comparable to the methylated MCM. These data provide the biochemical evidence that posttranslational modifications such as methylation may enhance kinetic stability of proteins under the elevated growth temperatures of hyperthermophilic archaea.

The Helicase Activity of Hyperthermophilic Archaeal MCM is Enhanced at High Temperatures by Lysine Methylation

PubMed Central

Xia, Yisui; Niu, Yanling; Cui, Jiamin; Fu, Yang; Chen, Xiaojiang S.; Lou, Huiqiang; Cao, Qinhong

2015-01-01

Lysine methylation and methyltransferases are widespread in the third domain of life, archaea. Nevertheless, the effects of methylation on archaeal proteins wait to be defined. Here, we report that recombinant sisMCM, an archaeal homolog of Mcm2-7 eukaryotic replicative helicase, is methylated by aKMT4 in vitro. Mono-methylation of these lysine residues occurs coincidently in the endogenous sisMCM protein purified from the hyperthermophilic Sulfolobus islandicus cells as indicated by mass spectra. The helicase activity of mini-chromosome maintenance (MCM) is stimulated by methylation, particularly at temperatures over 70°C. The methylated MCM shows optimal DNA unwinding activity after heat-treatment between 76 and 82°C, which correlates well with the typical growth temperatures of hyperthermophilic Sulfolobus. After methylation, the half life of MCM helicase is dramatically extended at 80°C. The methylated sites are located on the accessible protein surface, which might modulate the intra- and inter- molecular interactions through changing the hydrophobicity and surface charge. Furthermore, the methylation-mimic mutants of MCM show heat resistance helicase activity comparable to the methylated MCM. These data provide the biochemical evidence that posttranslational modifications such as methylation may enhance kinetic stability of proteins under the elevated growth temperatures of hyperthermophilic archaea. PMID:26617586

RADH, a gene of Saccharomyces cerevisiae encoding a putative DNA helicase involved in DNA repair. Characteristics of radH mutants and sequence of the gene.

PubMed

Aboussekhra, A; Chanet, R; Zgaga, Z; Cassier-Chauvat, C; Heude, M; Fabre, F

1989-09-25

A new type of radiation-sensitive mutant of S. cerevisiae is described. The recessive radH mutation sensitizes to the lethal effect of UV radiations haploids in the G1 but not in the G2 mitotic phase. Homozygous diploids are as sensitive as G1 haploids. The UV-induced mutagenesis is depressed, while the induction of gene conversion is increased. The mutation is believed to channel the repair of lesions engaged in the mutagenic pathway into a recombination process, successful if the events involve sister-chromatids but lethal if they involve homologous chromosomes. The sequence of the RADH gene reveals that it may code for a DNA helicase, with a Mr of 134 kDa. All the consensus domains of known DNA helicases are present. Besides these consensus regions, strong homologies with the Rep and UvrD helicases of E. coli were found. The RadH putative helicase appears to belong to the set of proteins involved in the error-prone repair mechanism, at least for UV-induced lesions, and could act in coordination with the Rev3 error-prone DNA polymerase.

Mechanism of Archaeal MCM Helicase Recruitment to DNA Replication Origins

PubMed Central

Samson, Rachel Y.; Abeyrathne, Priyanka D.; Bell, Stephen D.

2015-01-01

Summary Cellular DNA replication origins direct the recruitment of replicative helicases via the action of initiator proteins belonging to the AAA+ superfamily of ATPases. Archaea have a simplified subset of the eukaryotic DNA replication machinery proteins and possess initiators that appear ancestral to both eukaryotic Orc1 and Cdc6. We have reconstituted origin-dependent recruitment of the homohexameric archaeal MCM in vitro with purified recombinant proteins. Using this system, we reveal that archaeal Orc1-1 fulfills both Orc1 and Cdc6 functions by binding to a replication origin and directly recruiting MCM helicase. We identify the interaction interface between these proteins and reveal how ATP binding by Orc1-1 modulates recruitment of MCM. Additionally, we provide evidence that an open-ring form of the archaeal MCM homohexamer is loaded at origins. PMID:26725007

Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid-State NMR Spectroscopy.

PubMed

Wiegand, Thomas; Cadalbert, Riccardo; Gardiennet, Carole; Timmins, Joanna; Terradot, Laurent; Böckmann, Anja; Meier, Beat H

2016-11-02

DnaB helicases are bacterial, ATP-driven enzymes that unwind double-stranded DNA during DNA replication. Herein, we study the sequential binding of the "non-hydrolysable" ATP analogue AMP-PNP and of single-stranded (ss) DNA to the dodecameric DnaB helicase from Helicobacter pylori using solid-state NMR. Phosphorus cross-polarization experiments monitor the binding of AMP-PNP and DNA to the helicase. 13 C chemical-shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding. The helicase switches upon AMP-PNP addition into a conformation apt for ssDNA binding, and AMP-PNP is hydrolyzed and released upon binding of ssDNA. Our study sheds light on the conformational changes which are triggered by the interaction with AMP-PNP and are needed for ssDNA binding of H. pylori DnaB in vitro. They also demonstrate the level of detail solid-state NMR can provide for the characterization of protein-DNA interactions and the interplay with ATP or its analogues. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The DEAD box helicase RDE-12 promotes amplification of RNAi in cytoplasmic foci in C. elegans

PubMed Central

Yang, Huan; Vallandingham, Jim; Shiu, Philip; Li, Hua; Hunter, Craig P.; Mak, Ho Yi

2014-01-01

Summary RNA interference (RNAi) is a potent mechanism for down-regulating gene expression. Conserved RNAi pathway components are found in animals, plants, fungi and other eukaryotes [1–3]. In C. elegans, the RNAi response is greatly amplified by the synthesis of abundant secondary siRNAs [4–6]. Exogenous double stranded RNA is processed by Dicer and RDE-1/Argonaute into primary siRNA that guides target mRNA recognition. The RDE-10/RDE-11 complex and the RNA dependent RNA polymerase RRF-1 then engage the target mRNA for secondary siRNA synthesis [7, 8]. However, the molecular link between primary siRNA production and secondary siRNA synthesis remains largely unknown. Furthermore, it is unclear if the sub-cellular sites for target mRNA recognition and degradation coincide with sites where siRNA synthesis and amplification occur. In the C. elegans germline, cytoplasmic P granules at the nuclear pores and perinuclear Mutator foci contribute to target mRNA surveillance and siRNA amplification, respectively [9–11]. We report that RDE-12, a conserved FG domain containing DEAD-box helicase, localizes in P-granules and cytoplasmic foci that are enriched in RSD-6 but are excluded from the Mutator foci. Our results suggest that RDE-12 promotes secondary siRNA synthesis by orchestrating the recruitment of RDE-10 and RRF-1 to primary siRNA targeted mRNA in distinct cytoplasmic compartments. PMID:24684930