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Arthropod-borne flaviviruses, such as Zika virus (ZIKV), Usutu virus (USUV), and West Nile virus (WNV), are a growing cause of human illness and death around the world. Presently, no licensed antivirals to control them are available and, therefore, search for broad-spectrum antivirals, including host-directed compounds, is essential. The PI3K/Akt pathway controls essential cellular functions involved in cell metabolism and proliferation. Moreover, Akt has been found to participate in modulating replication in different viruses including the flaviviruses. In this work we studied the interaction of flavivirus NS5 polymerases with the cellular kinase Akt. In vitro NS5 phosphorylation experiments with Akt showed that flavivirus NS5 polymerases are phosphorylated and co-immunoprecipitate by Akt. Polymerase activity assays of Ala- and Glu-generated mutants for the Akt-phosphorylated residues also indicate that Glu mutants of ZIKV and USUV NS5s present a reduced primer-extension activity that was not observed in WNV mutants. Furthermore, treatment with Akt inhibitors (MK-2206, honokiol and ipatasertib) reduced USUV and ZIKV titers in cell culture but, except for honokiol, not WNV. All these findings suggest an important role for Akt in flavivirus replication although with specific differences among viruses and encourage further investigations to examine the PI3K/Akt/mTOR pathway as an antiviral potential target.

According to separation studies it has been evident that a tobacco RNA-replicase after TMV-infection consists of two parts. The larger part is host-directed and will be combined with a virus borne small protein which considerably enhances the RNA-replicase activity and is therefore named replicase enhancer, Ree. This compound was found at HPLC-separations of TMV-coat proteins, and was detected using polymerase assay with a radioactive nucleotide involved. Molecular weight has been determined by mass spectrometry: with FT ICR MS to get the size – 6 023.3 - and with MALDI TOF MS to obtain a sequence of the 54 amino acids involved. Presumably Ree is fixed to the TMV-RNA at infection. The enzyme seems to produce minus-strands of the virus RNA.

According to separation studies it has been evident that a tobacco RNA-replicase after TMV-infection consists of two parts. The larger part is host-directed and will be combined with a virus borne small protein which considerably enhances the RNA-replicase activity and is therefore named replicase enhancer, Ree. This compound was found at HPLC-separations of TMV-coat proteins, and was detected using polymerase assay with a radioactive nucleotide involved. Molecular weight has been determined by mass spectrometry: with FT ICR MS to get the size – 6 023.3 - and with MALDI TOF MS to obtain a sequence of the 54 amino acids involved. Presumably Ree is fixed to the TMV-RNA at infection. The enzyme seems to produce minus-strands of the virus RNA.

According to separation studies it has been evident that a tobacco RNA-replicase after TMV-infection consists of two parts. The larger part is host-directed and will be combined with a virus borne small protein which considerably enhances the RNA-replicase activity and is therefore named replicase enhancer, Ree. This compound was found at HPLC-separations of TMV-coat proteins, and was detected using polymerase assay with a radioactive nucleotide involved. Molecular weight has been determined by mass spectrometry: with FT ICR MS to get the size – 6 023.3 - and with MALDI TOF MS to obtain a sequence of the 54 amino acids involved. Presumably Ree is fixed to the TMV-RNA at infection. The enzyme seems to produce minus-strands of the virus RNA.

According to separation studies it has been evident that a tobacco RNA-replicase after TMV-infection consists of two parts. The larger part is host-directed and will be combined with a virus borne small protein which considerably enhances the RNA-replicase activity and is therefore named replicase enhancer, Ree. This compound was found at HPLC-separations of TMV-coat proteins, and was detected using polymerase assay with a radioactive nucleotide involved. Molecular weight has been determined by mass spectrometry: with FT ICR MS to get the size – 6 023.3 - and with MALDI TOF MS to obtain a sequence of the 54 amino acids involved. Presumably Ree is fixed to the TMV-RNA at infection. The enzyme seems to produce minus-strands of the virus RNA.

According to separation studies it has been evident that a tobacco RNA-replicase after TMV-infection consists of two parts. The larger part is host-directed and will be combined with a virus borne small protein which considerably enhances the RNA-replicase activity and is therefore named replicase enhancer, Ree. This compound was found at HPLC-separations of TMV-coat proteins, and was detected using polymerase assay with a radioactive nucleotide involved. Molecular weight has been determined by mass spectrometry: with FT ICR MS to get the size – 6 023.3 - and with MALDI TOF MS to obtain a sequence of the 54 amino acids involved. Presumably Ree is fixed to the TMV-RNA at infection. The enzyme seems to produce minus-strands of the virus RNA.

Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules., authorCount :35

Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules.

Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules. © 2007 Elsevier B.V. All rights reserved.

Tobacco Mosaic Virus and Arabidopsis thaliana serve as ideal model systems to study the molecular aspects of virus - host interactions. Using this system, an interaction between the helicase domain within TMV replicase protein and an auxin responsive protein, IAA26 was identified. IAA26 is a member of the Aux/IAA family of transcription factors that function as repressors in signaling pathways controlled by the phytohormone auxin. Characterization of the interaction was carried out utilizing a helicase mutant defective in its interaction with IAA26 and by creating transgenic plants silenced for IAA26 expression. These studies suggest that the interaction was not essential for either viral replication or movement but promoted the development of disease symptoms. Cellular co-localization studies revealed that in TMV infected tissue, the nuclear localization and stability of IAA26 was compromised and the protein was relocalized to distinct cytoplasmic vesicles in association with the viral replicase. In keeping with its role as a transcription factor, the alterations in IAA26 function should lead to misregulation of downstream auxin responsive genes and this is supported by the fact that ~ 30% of the Arabidopsis genes displaying transcriptional alterations to TMV could be linked to the auxin signaling pathway. Aux/IAA family members share significant sequence and functional homology, and an additional interaction screen identified two more Arabidopsis Aux/IAA proteins, IAA27 and IAA18 and a putative tomato Aux/IAA protein, LeIAA26 that could interact with TMV helicase. The nuclear localization of these three proteins was disrupted by TMV and alterations in LeIAA26 levels induced virus infection-like symptoms in tomato. Additionally, transgenic plants over-expressing a proteolysis resistant mutant of IAA26 showed abnormal developmental phenotype, the severity of which was abrogated during TMV infection which blocked nuclear accumulation of the protein. Taken together, t

Potato leafroll virus (PLRV) is a major menace for the potato production all over the world. PLRV is transmitted by aphids, and until now, the only strategy available to control this pest has been to use large amounts of insecticides. Transgenic approaches involving the expression of viral replicases are being developed to provide protection for plants against viral diseases. The purpose of this study was to compare the protection afforded by the differential expression of PLRV replicase transgene in potato plants cv. Desirée. Plants were genetically modified to express the complete sense PLRV replicase gene. Two constructions were used, one containing the constitutive 35SCaMV promoter and the other the phloem-specific RolA promoter from Agrobacterium rhizogenes. Transgenic plants were infected with PLRV in vitro, using infested aphids. In plants in which 35SCaMV controlled the expression of the PLRV replicase gene, signs of infection were initially detected, although most plants later developed a recovery phenotype showing undetectable virus levels 40 days after infection. In turn, those plants with the RolA promoter displayed an initial resistance that was later overcome. Different molecular mechanisms are likely to participate in the response to PLRV infection of these two types of transgenic plants.

A putative zinc finger (ZF) domain in the Equine arteritis virus (EAV) nsp 1 protein was described recently to be required for viral transcription. The nsp 1 ZF (50 aa) was expressed on the surface of M13KE gIII phage, fused to the N terminus of the phage pIII protein. To evaluate the functionality of the ZF domain, a binding assay was developed, based on the use of immobilized Ni2+ ions (Ni-NTA). Phages displaying ZF bound significantly better to Ni-NTA than did phages displaying negative-control peptides, which also contained metal-coordinating residues. Also, binding of ZF-displaying phages could be inhibited by an anti-nsp 1 serum, or by mutation of residues predicted to be important for zinc coordination. Finally, binding was abolished by low concentrations (0.1%) Tween 20, and rescued by including Zn2+, Ni2+ or Cu2+, but not Mg2+, in the binding buffer, suggesting that formation of secondary structure was involved in binding of the ZF to Ni-NTA. These findings provide the first experimental evidence that the putative nsp 1 ZF domain can coordinate divalent metal ions, and that this property is associated with the secondary structure of the domain. The Ni-NTA binding assay developed in the present study may have general applications in the study of other ZF domains.