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  • MUTATION  (4)
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  • 1
    Keywords: PEPTIDE ; Germany ; MICROSCOPY ; PATHWAY ; PATHWAYS ; PROTEIN ; PROTEINS ; RELEASE ; DOMAIN ; CONTRAST ; SEQUENCE ; PARTICLES ; virus ; MUTATION ; LINE ; inactivation ; OVEREXPRESSION ; MATRIX PROTEIN ; VIRION RELEASE ; electron microscopy ; MORPHOGENESIS ; ELECTRON-MICROSCOPY ; assembly ; AMINO-ACID ; interaction ; MUTANTS ; STRUCTURAL PROTEINS ; ENV ; VIRAL INFECTIVITY ; ENVELOPE GLYCOPROTEIN ; ROUS-SARCOMA VIRUS ; TSG101
    Abstract: Foamy viruses (FV) are unusual among retroviruses since they require both Gag and Env structural proteins for particle egress. Recently significant progress has been made towards the mechanistic understanding of the viral release process, in particular that of retroviruses, and the viral domains and cellular pathways involved. However little is currently known about domains of FV structural proteins and cellular proteins engaged in this process. By mutational analysis of sequence motifs in prototype FV (PFV) Gag, bearing homology to known late assembly (L) domains, a PSAP motif with L domain function that was functionally interchangeable by heterologous L domains was identified. In contrast the inactivation of a PPPI motif had no significant influence on PFV particle release, although mutant viral particles displayed reduced infectivity. Similarly mutation of an evolutionary conserved YXXL motif revealed no classical L-domain function but resulted in release of noninfectious viruslike particles. Biochemical and electron microscopy analysis demonstrated that these mutant particles incorporated all viral structural proteins but contained aberrantly capsid structures, suggesting a role in capsid assembly for this PFV Gag sequence motif. In line with the mutational analysis, overexpression of dominant negative (DN) mutants and wild-type TSG101 but not the DN mutant of AIP-1/ALIX reduced PFV particle release and infectivity. Furthermore, DN mutants of Vps4A, Vps4B, and CHMP3 inhibited PFV egress and infectivity. Taken together these results demonstrate that PFV, like other viruses, requires components of the vacuolar protein sorting (VPS) machinery for egress and enters the VPS pathway through interaction with TSG101
    Type of Publication: Journal article published
    PubMed ID: 15827161
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  • 2
    Keywords: Germany ; SITE ; GENE ; PROTEIN ; PROTEINS ; DOMAIN ; SEQUENCE ; ACID ; virus ; DELETION ; REVERSE-TRANSCRIPTASE ; MUTATION ; DELETIONS ; REGION ; MUTATIONS ; point mutation ; MATRIX PROTEIN ; MURINE LEUKEMIA-VIRUS ; RESIDUES ; assembly ; END ; AMINO-ACID ; MUTANTS ; D RETROVIRUSES ; ENV ; ENV LEADER PROTEIN ; ENVELOPE ; ENVELOPE PROTEIN ; POL PROTEINS ; REPLICATION STRATEGY ; VIRAL INFECTIVITY
    Abstract: Among the Retroviridae, foamy viruses (FVs) exhibit an unusual way of particle assembly and a highly specific incorporation of envelope protein into progeny virions. We have analyzed deletions and point mutants of the prototypic FV gag gene for capsid assembly and egress, envelope protein incorporation, infectivity, and ultrastructure. Deletions introduced at the 3' end of gag revealed the first 297 amino acids (aa) to be sufficient for specific Env incorporation and export of particulate material. Deletions introduced at the 5' end showed the region between as 6 and 200 to be dispensable for virus capsid assembly but required for the incorporation of Env and particle egress. Point mutations were introduced in the 5' region of gag to target residues conserved among FVs from different species. Alanine substitutions of residues in a region between as 40 and 60 resulted in severe alterations in particle morphology. Furthermore, at position 50, this region harbors the conserved arginine that is presumably at the center of a signal sequence directing FV Gag proteins to a cytoplasmic assembly site
    Type of Publication: Journal article published
    PubMed ID: 16160174
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  • 3
    Keywords: INHIBITOR ; Germany ; ENZYMES ; GENE ; EFFICIENCY ; DNA ; MECHANISM ; CONTRAST ; BIOLOGY ; MOLECULAR-BIOLOGY ; ACID ; virus ; MUTANT ; NO ; REVERSE-TRANSCRIPTASE ; DIFFERENCE ; ESCHERICHIA-COLI ; resistance ; MUTATION ; MUTATIONS ; REPLICATION ; sensitivity ; foamy virus ; INHIBITORS ; HIV-1 ; molecular biology ; molecular ; RECOMBINANT ; RE ; SUBSTRATE ; ENZYME ; MUTANTS ; ENGLAND ; Escherichia coli ; POLYMERIZATION ; MEDIATED EXCISION ; PRIMER-UNBLOCKING ; RNASE-H ; SELECTIVE EXCISION ; ZIDOVUDINE
    Abstract: Azidothymidine (AZT, zidovudine) is one of the few nucleoside inhibitors known to inhibit foamy virus replication. We have shown previously that up to four mutations in the reverse transcriptase gene of simian foamy virus from macaque (SFVmac) are necessary to confer high resistance against AZT. To characterize the mechanism of AZT resistance we expressed two recombinant reverse transcriptases of highly AZT-resistant SFVmac in Escherichia coli harboring three (K211I, S345T, E350K) or four mutations (K211I, I224T, S345T, E350K) in the reverse transcriptase gene. Our analyses show that the polymerization activity of these mutants is impaired. In contrast to the AZT-resistant reverse transcriptase of HIV-1, the AZT resistant enzymes of SFVmac reveal differences in their kinetic properties. The SFVmac enzymes exhibit lower specific activities on poly(rA)/oligo(dT) and higher K-M-values for polymerization but no change in K-D-values for DNA/DNA or RNA/DNA substrates. The AZT resistance of the mutant enzymes is based on the excision of the incorporated inhibitor in the presence of ATP. The additional amino acid change of the quadruple mutant appears to be important for regaining polymerization efficiency
    Type of Publication: Journal article published
    PubMed ID: 18096624
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  • 4
    Keywords: INHIBITOR ; CELL ; Germany ; SITE ; SITES ; GENE ; GENOME ; MECHANISM ; SEQUENCE ; culture ; virus ; ACQUISITION ; REVERSE-TRANSCRIPTASE ; resistance ; MUTATION ; MUTATIONS ; PURIFICATION ; PHENOTYPE ; REPLICATION ; SELECTION ; foamy virus ; POL ; ORDER ; molecular ; ENZYME ; POL PROTEINS ; REPLICATION STRATEGY ; USA ; NOR ; viral ; block ; reverse transcriptase ; NUCLEOTIDE ; viruses ; AZT resistance ; RIBONUCLEASE-H DOMAINS ; TYPE-1 RESISTANT ; ZIDOVUDINE AZT
    Abstract: Azidothymidine (AZT) is a reverse transcriptase (RT) inhibitor that efficiently blocks the replication of spumaretroviruses or foamy viruses (FVs). To more precisely elucidate the mechanism of action of the FV RT enzyme, we generated an AZT-resistant FV in cell culture. Biologically resistant virus was obtained for simian foamy virus from macaque (SFVmac), which was insensitive to AZT concentrations of 1 mM, but not for FVs derived from chimpanzees. Nucleotide sequencing revealed four non-silent mutations in the pol gene. Introduction of these mutations into an infectious molecular clone identified all changes to be required for the fully AZT-resistant phenotype of SFVmac. The alteration of individual sites showed that AZT resistance in SFVmac was likely acquired by consecutive acquisition of pol mutations in a defined order, because some alterations on their own did not result in an efficiently replicating virus, neither in the presence nor in the absence of AZT. The introduction of the mutations into the RT of the closely related prototypic FV (PFV) did not yield an AZT-resistant virus, instead they significantly impaired the viral fitness. (c) 2007 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17904181
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