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Although safety concerns preclude the use of live attenuated HIV vaccines in humans, they provide a useful system for identifying the elusive correlates of protective immunity in the SIV/macaque animal model. However, a number of pieces of evidence suggest that protection may result from prior occupancy of susceptible target cells by the vaccine virus rather than the immune response. To address this, we developed a Nef-deletion variant of an RT-SHIV whose active replication could be shut off by treatment with RT-inhibitors. Groups of macaques were inoculated with the ∆Nef-RT-SHIV and immune responses allowed to develop before antiretroviral treatment and subsequent challenge with wild-type SIVmac239. Vaccinated animals either resisted infection fully or significantly controlled the subsequent viremia. However, there was no difference between animals undergoing replication of the vaccine virus and those without. This strongly suggests that competition for available target cells does not play a role in protection., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The HERV-K(HML-2) family is the most recent addition to the collection of human endogenous retroviruses. It comprises proviruses that encode functional proteins that can assemble into replication defective particles carrying the envelope protein. Using a reconstituted HERV-K113 envelope sequence, we have analyzed its ability to mediate entry into a set of 33 cell lines from 10 species. Of these, 30 were permissive, demonstrating an amphotropism consistent with a broad expression of receptor protein(s). In an initial effort to identify a receptor for HERV-K(HML-2) we investigated whether transferrin receptor 1 and hyaluronidase 2, known cellular receptors of the closely related betaretroviruses mouse mammary tumor virus (MMTV) and Jaagsiekte sheep retrovirus (JSRV), could facilitate HERV-K(HML-2) entry. However, neither of these proteins could serve as a receptor for HERV-K(HML-2). Moreover, during attempts to further characterize the tropism of HERV-K(HML-2), we identified a cellular activity that inhibits infection at a post-entry, pre-integration step., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Preclinical evaluation in a small animal model would help the development of gene therapies and vaccines based on foamy virus vectors. The establishment of persistent, non-pathogenic infection with the prototype foamy virus in mice and rabbits has been described previously. To extend this spectrum of available animal models, hamsters were inoculated with infectious cell supernatant or bioballistically with a foamy virus plasmid. In addition, a novel foamy virus from a rhesus macaque was isolated and characterised genetically. Hamsters and mice were infected with this new SFVmac isolate to evaluate whether hamsters are also susceptible to infection. Both hamsters and mice developed humoral responses to either virus subtype. Virus integration and replication in different animal tissues were analysed by PCR and co-cultivation. The results strongly indicate establishment of a persistent infection in hamsters. These studies provide a further small animal model for studying FV-based vectors in addition to the established models., (© 2013 Elsevier Inc. All rights reserved.)

Plastic cell culture dishes that contain a thin bottom of highest optical quality including an imprinted finder grid (μ-Dish Grid-500) are optimally suited for routine correlative light and electron microscopy using chemical fixation. Such dishes allow high-resolution fluorescence and bright-field imaging using fixed and living cells and are compatible with standard protocols for scanning and transmission electron microscopy. Ease of use during cell culture and imaging, as well as a tight cover render the dishes particularly suitable for working with infectious organisms up to the highest biosafety level. Detailed protocols are provided and demonstrated by showing two examples: monitoring the production of virus-like particles of the Human Endogenous Retrovirus HERV-K(HML-2) by HeLa cells and investigation of Rab11-positive membrane-compartments of HeLa cells after infection with Chlamydia trachomatis., (© 2014 Elsevier Inc. All rights reserved.)

Chronic immune activation, triggered by plasmacytoid dendritic cell (PDC) interferon (IFN)-alpha production, plays an important role in HIV-1 pathogenesis. As the entry of HIV-1 seems to be important for the activation of PDC, we directly characterized the viral entry into these cells using immuno-electron microscopy, cellular fractionation, confocal imaging, and functional experiments. After attachment to PDC, viruses were taken up in an energy-dependent manner. The virions were located in compartments positive for caveolin; early endosomal antigen 1; Rab GTPases 5, 7 and 9; lysosomal-associated membrane protein 1. PDC harbored more virus in endocytic vesicles than CD4+ T cells (p<0.05). Blocking CD4 inhibited the uptake of virions into cytosolic and endosomal compartments. Dynasore, an inhibitor of dynamin-dependent endocytosis, not the fusion inhibitor T-20, reduced the HIV-1 induced IFN-alpha production. Altogether, our morphological and functional data support the role of endocytosis for the entry and IFN-alpha induction of HIV-1 in PDC., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Hantaan virus (HTNV), the prototype member of the Hantavirus genus in the family Bunyaviridae, causes hemorrhagic fever with renal syndrome (HFRS) in humans. Hemorrhage is due to endothelial barrier damage and a sharp decrease in platelet counts. The mechanisms underlying HTNV-associated acute thrombocytopenia have not been elucidated so far. Platelets are produced by mature megakaryocytes that develop during megakaryopoiesis. In this study, we show that HTNV targets megakaryocytic cells whereas rather non-pathogenic hantaviruses did not infect this cell type. After induction of differentiation megakaryocytic cells switched from low-level to high-level HTNV production without reduction in cell survival or alteration in differentiation. However, increased HTNV replication resulted in strong upregulation of HLA class I molecules although HTNV escaped type I interferon (IFN)-associated innate responses. Taken together, HTNV efficiently replicates in differentiating megakaryocytic cells resulting in upregulation of HLA class I molecules, the target structures for cytotoxic T cells (CTLs)., (Copyright 2010 Elsevier Inc. All rights reserved.)

The human endogenous retrovirus-K113 (HERV-K113) is the most complete HERV known to date. It contains open reading frames for all viral proteins. Depending on ethnicity, up to 30% of the human population carries the provirus on chromosome 19. To facilitate molecular and functional studies, we have cloned the HERV-K113 sequence into a small plasmid vector and characterized its functional properties. Here we show that based on a substantial LTR-promoter activity, full length messenger RNA and spliced env-, rec- and 1.5 kb (hel)-transcripts are produced. The envelope protein of HERV-K113 is synthesized as an 85 kDa precursor that is found partially processed. The accessory Rec protein is highly expressed and accumulates in the nucleus. Expression analysis revealed synthesis of the Gag precursor and the protease. However, the cloned HERV-K113 provirus is not replication competent. It carries inactivating mutations in the reverse transcriptase gene. These mutations can be reversed to reconstitute the active enzyme, but the reversion is not sufficient to reconstitute replication capacity of the virus.