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1.
Virology ; 506: 28-33, 2017 06.
Article in English | MEDLINE | ID: mdl-28314126

ABSTRACT

Foamy viruses are non-pathogenic retroviruses and represent a tool for vector development. For gene therapy applications and for analyses of viral protein composition infectious particles need to be purified, which has been difficult for foamy viruses in the past. Here, we describe a novel, simple, and fast purification method for prototype foamy viruses with high purity using size exclusion and affinity chromatography. More than 99,9% of the contaminating proteins were removed. The purified viruses were used to determine the amount of the incorporated Pol protein relative to Gag. The determined Gag to Pol PR-RT ratio of 30:1 confirmed previous studies suggesting FV virions encapsidate fewer number of Pol molecules than orthoretroviruses.


Subject(s)
Chromatography, Affinity/methods , Chromatography, Gel/methods , Spumavirus/growth & development , Virion/growth & development , Gene Expression Regulation, Viral , Gene Products, pol/genetics , Gene Products, pol/metabolism , Humans , Retroviridae Infections/virology , Spumavirus/genetics , Spumavirus/isolation & purification , Spumavirus/physiology , Virion/genetics , Virion/isolation & purification , Virion/physiology , Virus Assembly , Virus Cultivation
2.
Antimicrob Agents Chemother ; 59(2): 1100-9, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25487800

ABSTRACT

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.


Subject(s)
Dengue Virus/drug effects , Protease Inhibitors/pharmacology , Serine Endopeptidases/metabolism , Viral Nonstructural Proteins/metabolism , Antiviral Agents/pharmacology , Cell Line , Dengue Virus/enzymology , HIV-1/drug effects , Humans , Molecular Docking Simulation , Virus Replication/drug effects
3.
Virology ; 454-455: 145-56, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24725941

ABSTRACT

In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2׳ and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies. The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation.


Subject(s)
Aspartic Acid Endopeptidases/metabolism , RNA-Directed DNA Polymerase/metabolism , Ribonuclease H/metabolism , Spumavirus/enzymology , Aspartic Acid Endopeptidases/genetics , Protein Structure, Tertiary , RNA-Directed DNA Polymerase/genetics , Ribonuclease H/genetics
4.
J Virol ; 87(13): 7774-6, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23616664

ABSTRACT

In contrast to orthoretroviruses, processing of foamy viral p71 Gag is limited to a single cleavage site. Nevertheless, Gag maturation is essential for infectivity, but deletion of p3 results in a modest drop in infectivity. Here, we show that Gag processing of p71 to p68 and p3 is essential for full-length cDNA synthesis, while inactivation of Gag cleavage results in cDNAs containing only the RU5 region; cDNAs encompassing the U3 region were almost undetectable.


Subject(s)
Aspartic Acid Endopeptidases/metabolism , DNA, Complementary/biosynthesis , Gene Products, gag/metabolism , Protein Processing, Post-Translational/physiology , RNA-Directed DNA Polymerase/metabolism , Spumavirus/metabolism , Blotting, Western , Gene Products, gag/genetics , Mutagenesis, Site-Directed , Protein Processing, Post-Translational/genetics , Spumavirus/genetics , Spumavirus/pathogenicity
5.
Retrovirology ; 9: 41, 2012 May 10.
Article in English | MEDLINE | ID: mdl-22574974

ABSTRACT

BACKGROUND: Recently, contradictory results on foamy virus protease activity were published. While our own results indicated that protease activity is regulated by the viral RNA, others suggested that the integrase is involved in the regulation of the protease. RESULTS: To solve this discrepancy we performed additional experiments showing that the protease-reverse transcriptase (PR-RT) exhibits protease activity in vitro and in vivo, which is independent of the integrase domain. In contrast, Pol incorporation, and therefore PR activity in the viral context, is dependent on the integrase domain. To further analyse the regulation of the protease, we incorporated Pol in viruses by expressing a GagPol fusion protein, which supported near wild-type like infectivity. A GagPR-RT fusion, lacking the integrase domain, also resulted in wild-type like Gag processing, indicating that the integrase is dispensable for viral Gag maturation. Furthermore, we demonstrate with a trans-complementation assays that the PR in the context of the PR-RT protein supports in trans both, viral maturation and infectivity. CONCLUSION: We provide evidence that the FV integrase is required for Pol encapsidation and that the FV PR activity is integrase independent. We show that an active PR can be encapsidated in trans as a GagPR-RT fusion protein.


Subject(s)
Aspartic Acid Endopeptidases/chemistry , Fusion Proteins, gag-pol/chemistry , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Viral , Integrases/chemistry , RNA, Viral/chemistry , Simian foamy virus/enzymology , Enzyme Activation , Fusion Proteins, gag-pol/genetics , Genetic Complementation Test , HEK293 Cells , Humans , Plasmids/chemistry , Plasmids/genetics , Protein Structure, Tertiary , Proteolysis , RNA, Viral/genetics , RNA-Directed DNA Polymerase/chemistry , Simian foamy virus/chemistry , Simian foamy virus/genetics , Transfection , Viral Proteins/chemistry , Viral Proteins/genetics
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