Nuclear Localization of the Functional Bel 1 Transactivator but Not of the Gag Proteins of the Feline Foamy Virus

AbstractInteractions between host cells and foamy or spumaretroviruses are different from those of other known retroviruses. Previous work has suggested that the Gag and high-affinity DNA-binding Bel 1 transactivator of human foamy virus are localized in the nuclei of infected cells. Using two independent detection methods, we show here that the functionally active Bel 1 transactivator protein of feline foamy virus is of nuclear localization. In contrast to that reported for the human foamy virus Gag protein, the cat foamy virus Gag proteins exclusively localized in the cytoplasm close to perinuclear regions

A tudományos kommunikáció története a Journal des Scavans-tól az open access-ig

Background: The foamy viral genome encodes four central purine-rich elements localized in the integrase-coding region of pol. Previously, we have shown that the first two of these RNA elements (A and B) are required for protease dimerization and activation. The D element functions as internal polypurine tract during reverse transcription. Peters et al., described the third element (C) as essential for gag expression suggesting that it might serve as an RNA export element for the unspliced genomic transcript. Results: Here, we analysed env splicing and demonstrate that the described C element composed of three GAA repeats known to bind SR proteins regulates env splicing, thus balancing the amount of gag/pol mRNAs. Deletion of the C element effectively promotes a splice site switch from a newly identified env splice acceptor to the intrinsically strong downstream localised env 3′ splice acceptor permitting complete splicing of almost all LTR derived transcripts. We provide evidence that repression of this env splice acceptor is a prerequisite for gag expression. This repression is achieved by the C element, resulting in impaired branch point recognition and SF1/mBBP binding. Separating the branch point from the overlapping purine-rich C element, by insertion of only 20 nucleotides, liberated repression and fully restored splicing to the intrinsically strong env 3′ splice site. This indicated that the cis-acting element might repress splicing by blocking the recognition of essential splice site signals. Conclusions: The foamy viral purine-rich C element regulates splicing by suppressing the branch point recognition of the strongest env splice acceptor. It is essential for the formation of unspliced gag and singly spliced pol transcripts

Biochemical characterization of a multi-drug resistant HIV-1 subtype AG reverse transcriptase: antagonism of AZT discrimination and excision pathways and sensitivity to RNase H inhibitors

We analyzed a multi-drug resistant (MR) HIV-1 re- verse transcriptase (RT), subcloned from a patient- derived subtype CRF02 AG, harboring 45 amino acid exchanges, amongst them four thymidine analog mutations (TAMs) relevant for high-level AZT (azi- dothymidine) resistance by AZTMP excision (M41L, D67N, T215Y, K219E) as well as four substitutions of the AZTTP discrimination pathway (A62V, V75I, F116Y and Q151M). In addition, K65R, known to an- tagonize AZTMP excision in HIV-1 subtype B was present. Although MR-RT harbored the most signif- icant amino acid exchanges T215Y and Q151M of each pathway, it exclusively used AZTTP discrimi- nation, indicating that the two mechanisms are mu- tually exclusive and that the Q151M pathway is ob- viously preferred since it confers resistance to most nucleoside inhibitors. A derivative was created, ad- ditionally harboring the TAM K70R and the rever- sions M151Q as well as R65K since K65R antago- nizes excision. MR-R65K-K70R-M151Q was compe- tent of AZTMP excision, whereas other combinations thereof with only one or two exchanges still pro- moted discrimination. To tackle the multi-drug resis- tance problem, we tested if the MR-RTs could still be inhibited by RNase H inhibitors. All MR-RTs exhibited similar sensitivity toward RNase H inhibitors be- longing to different inhibitor classes, indicating the importance of developing RNase H inhibitors further as anti-HIV drugs

Regulation of foamy virus protease activity by viral RNA

No abstract available

Foamy Virus Biology and Its Application for Vector Development

Spuma- or foamy viruses (FV), endemic in most non-human primates, cats, cattle and horses, comprise a special type of retrovirus that has developed a replication strategy combining features of both retroviruses and hepadnaviruses. Unique features of FVs include an apparent apathogenicity in natural hosts as well as zoonotically infected humans, a reverse transcription of the packaged viral RNA genome late during viral replication resulting in an infectious DNA genome in released FV particles and a special particle release strategy depending capsid and glycoprotein coexpression and specific interaction between both components. In addition, particular features with respect to the integration profile into the host genomic DNA discriminate FV from orthoretroviruses. It appears that some inherent properties of FV vectors set them favorably apart from orthoretroviral vectors and ask for additional basic research on the viruses as well as on the application in Gene Therapy. This review will summarize the current knowledge of FV biology and the development as a gene transfer system

Low α2β1 Integrin Function Enhances the Proliferation of Fibroblasts from Patients with Idiopathic Pulmonary Fibrosis by Activation of the β-Catenin Pathway

Idiopathic pulmonary fibrosis (IPF) is a progressive and incurable fibroproliferative disorder characterized by unrelenting proliferation of fibroblasts and their deposition of collagen within alveoli, resulting in permanently scarred, nonfunctional airspaces. Normally, polymerized collagen suppresses fibroblast proliferation and serves as a physiological restraint to limit fibroproliferation after tissue injury. The IPF fibroblast, however, is a pathologically altered cell that has acquired the capacity to elude the proliferation-suppressive effects of polymerized collagen. The mechanism for this phenomenon remains incompletely understood. Here, we demonstrate that expression of α2β1 integrin, a major collagen receptor, is pathologically low in IPF fibroblasts interacting with polymerized collagen. Low integrin expression in IPF fibroblasts is associated with a failure to induce PP2A phosphatase activity, resulting in abnormally high levels of phosphorylated (inactive) GSK-3β and high levels of active β-catenin in the nucleus. Knockdown of β-catenin in IPF fibroblasts inhibits their ability to proliferate on collagen. Interdiction of α2β1 integrin in control fibroblasts reproduces the IPF phenotype and leads to the inability of these cells to activate PP2A, resulting in high levels of phosphorylated GSK-3β and active β-catenin and in enhanced proliferation on collagen. Our findings indicate that the IPF fibroblast phenotype is characterized by low α2β1 integrin expression, resulting in a failure of integrin to activate PP2A phosphatase, which permits inappropriate activation of the β-catenin pathway

Genotypic resistance testing in HIV by arrayed primer extension

The analysis of mutations that are associated with the occurrence of drug resistance is important for monitoring the antiretroviral therapy of patients infected with human immunodeficiency virus (HIV). Here, we describe the establishment and successful application of Arrayed Primer Extension (APEX) for genotypic resistance testing in HIV as a rapid and economical alternative to standard sequencing. The assay is based on an array of oligonucleotide primers that are immobilised via their 5′-ends. Upon hybridisation of template DNA, a primer extension reaction is performed in the presence of the four dideoxynucleotides, each labelled with a distinct fluorophore. The inserted label immediately indicates the sequence at the respective position. Any mutation changes the colour pattern. We designed a microarray for the analysis of 26 and 33 codons in the HIV protease and reverse transcriptase, respectively, which are of special interest with respect to drug resistance. The enormous genome variability of HIV represents a big challenge for genotypic resistance tests, which include a hybridisation step, both in terms of specificity and probe numbers. The use of degenerated oligonucleotides resulted in a significant reduction in the number of primers needed. For validation, DNA of 94 and 48 patients that exhibited resistance to inhibitors of HIV protease and reverse transcriptase, respectively, were analysed. The validation included HIV subtype B, prevalent in industrialised countries, as well as non-subtype B samples that are more common elsewhere