Most retroviruses express all of their genes from a single primary transcript. In order to allow expression of more than one gene from this RNA, differential splicing is extensively used. Cellular quality control mechanisms retain and degrade unspliced or partially spliced RNAs in the nucleus. Two pathways have been described that explain how retroviruses circumvent this nuclear export inhibition. One involves a constitutive transport element in the viral RNA that interacts with the cellular mRNA transporter proteins NXF1 and NXT1 to facilitate nuclear export. The other pathway relies on the recognition of a viral RNA element by a virus-encoded protein that interacts with the karyopherin CRM1. In this report, we analyze the protein factors required for the nuclear export of unspliced foamy virus (FV) mRNA. We show that this export is CRM1 dependent. In contrast to other complex retroviruses, FVs do not encode an export-mediating protein. Crosslinking experiments indicated that the cellular protein HuR binds to the FV RNA. Inhibition studies showed that both ANP32A and ANP32B, which are known to bridge HuR and CRM1, are essential for FV RNA export. By using this export pathway, FVs solve a central problem of viral replication.The nuclear export of RNA molecules in eukaryotic cells is a tightly regulated process (18,59,63,70,71). Nuclear exit is usually allowed only for fully spliced cellular mRNAs, while intron-containing mRNAs are retained in the nucleus and subsequently degraded (17,18,59,63,70). This defines a specific problem in the replication of retroviruses (RVs), since they must export not only fully spliced but also unspliced or partially spliced mRNAs into the cytoplasm. For the export of the two latter RNA species, retroviruses have found ways to escape both the splicing machinery and the degradation of incompletely spliced mRNAs by making use of either of two strategies for nuclear export of mRNAs with intact splice donor (SD) and acceptor (SA) pairs.In complex retroviruses, such as lentiviruses, some betaretroviruses, and all deltaretroviruses, virus-encoded regulatory proteins (Rev, Rem, and Rex, respectively) bind to the unspliced or incompletely spliced viral mRNA on one hand and contact the karyopherin CRM1 on the other (1,29,33,48,49). Subsequently, this complex shuttles to the cytoplasm, where it delivers the RNA cargo in a regulated fashion that involves Ran in GTP-bound form. Normally CRM1 is used for nuclear export of ribosomal subunits, 5S rRNAs, cellular proteins containing a nuclear export signal (NES), and snRNAs (18,27,53,63). This pathway can also be hitchhiked by endogenous human retroviruses (12,47,74). The presence of regulatory proteins acting at the posttranscriptional level enables complex retroviruses to use a biphasic mode of gene expression ("early" versus "late" phase), resulting in a gain of complexity better known from DNA viruses (16).Alternatively, more simple retroviruses, such as the betaretrovirus Mason-Pfizer monkey virus (MPMV), can (42) contain a cis-acting constitu...

Fibrogenesis is usually initiated when regenerative processes have failed and/or chronic inflammation occurs. It is characterised by the activation of tissue fibroblasts and dysregulated synthesis of extracellular matrix proteins. FHL2 (four-and-a-half LIM domain protein 2) is a scaffolding protein that interacts with numerous cellular proteins, regulating signalling cascades and gene transcription. It is involved in tissue remodelling and tumour progression. Recent data suggest that FHL2 might support fibrogenesis by maintaining the transcriptional expression of alpha smooth muscle actin and the excessive synthesis and assembly of matrix proteins in activated fibroblasts. Here, we present evidence that FHL2 does not promote bleomycin-induced lung fibrosis, but rather suppresses this process by attenuating lung inflammation. Loss of FHL2 results in increased expression of the pro-inflammatory matrix protein tenascin C and downregulation of the macrophage activating C-type lectin receptor DC-SIGN. Consequently, FHL2 knockout mice developed a severe and long-lasting lung pathology following bleomycin administration due to enhanced expression of tenascin C and impaired activation of inflammation-resolving macrophages.

We recently described an inducible human TNF transgenic mouse line (ihTNFtg) that develops psoriasis-like arthritis after doxycycline stimulation and analysed the pathogenesis of arthritis in detail. Here, we show that the skin phenotype of these mice is characterized by hyperproliferation and aberrant activation of keratinocytes, induction of pro-inflammatory cytokines, and infiltration with Th1 and Treg lymphocytes, particularly with macrophage infiltration into lesional skin, thus pointing to a psoriasis-like phenotype. To reveal the contribution of T cells and macrophages to the development of TNF-mediated psoriasis, ihTNFtg mice were crossbred into RAG1 mice lacking mature T and B cells. Surprisingly, the psoriatic phenotype in the double mutants was not reduced; rather, it was enhanced. The skin showed significantly increased inflammation and in particular, increased infiltration by macrophages. Consequently, depletion of macrophages in RAG1 or wild-type mice led to decreased disease severity. On the contrary, depletion of Treg cells in wild-type mice increased both psoriasis and the number of infiltrating macrophages, while adoptive transfer of Foxp3-positive cells into RAG1 or wild-type mice decreased both the development of psoriasis and macrophage infiltration. Thus, we conclude that Treg lymphocytes inhibit the pro-inflammatory activity of macrophages, which are the major immune effector cells in hTNF-mediated psoriasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

ObjectiveTo generate doxycycline-inducible human tumor necrosis factor α (TNFα)–transgenic mice to overcome a major disadvantage of existing transgenic mice with constitutive expression of TNFα, which is the limitation in crossing them with various knockout or transgenic mice.MethodsA transgenic mouse line that expresses the human TNFα cytokine exclusively after doxycycline administration was generated and analyzed for the onset of diseases.ResultsDoxycycline-inducible human TNFα–transgenic mice developed an inflammatory arthritis– and psoriasis-like phenotype, with fore and hind paws being prominently affected. The formation of “sausage digits” with characteristic involvement of the distal interphalangeal joints and nail malformation was observed. Synovial hyperplasia, enthesitis, cartilage and bone alterations, formation of pannus tissue, and inflammation of the skin epidermis and nail matrix appeared as early as 1 week after the treatment of mice with doxycycline and became aggravated over time. The abrogation of human TNFα expression by the removal of doxycycline 6 weeks after beginning stimulation resulted in fast resolution of the most advanced macroscopic and histologic disorders, and 3–6 weeks later, only minimal signs of disease were visible.ConclusionUpon doxycycline administration, the doxycycline-inducible human TNFα–transgenic mouse displays the major features of inflammatory arthritis. It represents a unique animal model for studying the molecular mechanisms of arthritis, especially the early phases of disease genesis and tissue remodeling steps upon abrogation of TNFα expression. Furthermore, unlimited crossing of doxycycline-inducible human TNFα–transgenic mice with various knockout or transgenic mice opens new possibilities for unraveling the role of various signaling molecules acting in concert with TNFα.

Four-and-a-half LIM domain protein 2 (FHL2) is a multifunctional adaptor protein with fine-tuning adjustment properties. It acts as a regulator of signaling cascades but also as a cofactor of transcription and controls several anti-inflammatory immune responses. Recently, we described FHL2 as a novel regulator of influenza A virus propagation. We have shown that in vitro FHL2 restricts viral replication by accelerating the interferon regulatory factor 3-dependent transcription of the Ifnb1 gene. In this work, we unraveled an ambiguous role of FHL2 during influenza A virus infection in vivo. Although FHL2 restrained viral replication during the first 24 hours of infection, it significantly delayed viral clearance afterward. Comparison of lung immune status of wild-type and FHL2 knockout mice during influenza virus infection did not acknowledge significant differences in the innate host immune response but revealed an improved migration of dendritic cells from infected lungs into draining lymph nodes as well as increased levels of activated CD8 T lymphocytes accumulated in the lungs of FHL2 knockout mice.

Four and a half LIM domain protein 2 (Fhl2) is an intracellular adaptor molecule with a high protein-protein interaction capacity. It acts as a modulator of several signaling molecules in the cytosol and as a cofactor of transcription in the nucleus. Recent studies suggest the role of Fhl2 in tissue repair and the anti-inflammatory response. Herein, we show that Fhl2-deficient mice develop a more severe psoriatic arthritis disease under induction of the inducible human tumor necrosis factor (hTNF) transgene than wild-type mice. The disease was accompanied by increased infiltration of activated macrophages and T regulatory cells in skin and digit joints as well as by increased expression of matrix metalloproteases and bone-specific proteases. The more severe pathogenesis of psoriatic arthritis in Fhl2 knockout mice coincided with enhanced levels of soluble hTNF cytokine, but surprisingly not with transcription of the hTNF transgene. Studying the shedding of cell membrane-bound hTNF by Adam17, a known Fhl2 interacting protein, revealed an enhanced release of TNF in the absence of Fhl2. In summary, our results show that Fhl2 anticipates the emerging inflammation and specifically the development of psoriatic arthritis by impeding the Adam17-mediated release of TNF.

We have isolated a Chikungunya (Chik) virus from a patient who returned to Germany after a three-month visit to Mauritius in spring 2006. Upon return she developed a transient fever up to 40 degrees C. This was followed by myalgia and joint pain. IgG antibodies in serum to Chik virus were undetectable. Virus (Chik-Wü1) was isolated on Vero cells. We molecularly cloned the whole genome of Chik-Wü1 from viral RNA by RT-PCR. The complete sequence was determined and functional domains of the genome were assigned. Chik-Wü1 clearly belongs to the group of viruses analysed from the recent Indian Ocean outbreak. In order to develop tools useful for further characterization of Chik-Wü1, we bacterially expressed and purified the capsid (C) and envelope (E) proteins and established an immunoblot assay. Twenty-two of 30 serum samples from Chik virus-infected patients that scored positive in indirect immunofluorescence previously were also reactive in immunoblot analysis with recombinant C and E2 antigens.

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