TY  - JOUR
AB  - OBJECTIVE: Macroglial cells like astrocytes are key targets for the formation of HIV-1 reservoirs in the brain. The 'shock-and-kill' HIV-1 cure strategy proposes eradication of reservoirs by clinical treatment with latency reversing agents (LRAs). However, virus activation may endanger the brain, due to limited cell turnover, viral neurotoxicity and poor penetration of antiretroviral drugs. Since the brain is not accessible to clinical sampling, we established an experimental model to investigate the LRA effects on HIV-1 latency in macroglial reservoirs. DESIGN: Human neural stem cells (HNSC.100) were used to generate a system that models HIV-1 transcriptional latency in proliferating progenitor, as well as differentiated macroglial cell populations and latency-modulating effects of LRAs and compounds targeting HIV-1 transcription were analysed. METHODS: HNSCs were infected with pseudotyped Env-defective HIV-1 viruses. HIV-1 DNA and RNA levels were quantified by qPCR. Expression of latent GFP-reporter viruses was analysed by confocal microscopy and flow cytometry. NF-κB signalling was investigated by confocal microscopy and chromatin immunoprecipitation. RESULTS: Two of the eight well known LRAs (tumour necrosis factor-alpha, suberoylanilide hydroxamic acid) reactivated HIV-1 in latently infected HNSCs. Tumour necrosis factor-alpha reactivated HIV-1 in progenitor and differentiated populations, whereas suberoylanilide hydroxamic acid was more potent in progenitors. Pre-treatment with inhibitors of key HIV-1 transcription factors (NF-κB, Cdk9) suppressed HIV-1 reactivation. CONCLUSION: We conclude that latent HIV-1 in macroglial reservoirs can be activated by selected LRAs. Identification of small molecules that suppress HIV-1 reactivation supports functional cure strategies. We propose using the HNSC model to develop novel strategies to enforce provirus quiescence in the brain.
AU  - Schneider, M.
AU  - Tigges, B.
AU  - Meggendorfer, M.
AU  - Helfer, M.
AU  - Ziegenhain, C.
AU  - Brack-Werner, R.
C1  - 45096
C2  - 37239
SP  - 1147-1159
TI  - A new model for post-integration latency in macroglial cells to study HIV-1 reservoirs of the brain.
JO  - Aids
VL  - 29
IS  - 10
PY  - 2015
SN  - 0269-9370
ER  - 

TY  - JOUR
AB  - OBJECTIVE:: The identification of still unrevealed mechanisms affecting the anti-HIV CD8 T-cell response in HIV-1 infection. DESIGN:: Starting from the observation that anti-Tat immunization is associated with improved CD8 T-cell immunity, we developed both in-vitro and ex-vivo assays to characterize the effects of extra-cellular Tat on the adaptive CD8 T-cell response. METHODS:: The effects of Tat on CD8 T-cell activation were assayed using CD8 T-cell clones specific for either cellular (MART-1) or viral (HIV-1 Nef) antigens, and HIV-1 Gag-specific CD8 T cells from HIV-1 patients. RESULTS:: The interaction between CD8 T lymphocytes and immobilized Tat, but not its soluble form, inhibits peptide-specific CD8 T-lymphocyte activation. The inhibition does not depend on Tat trans-activation activity, but on the interaction of the Tat RGD domain with α5β1 and αvβ3 integrins. Impaired CD8 T-cell activation was also observed in cocultures of CD8 T cells with HIV-1-infected cells. Anti-Tat Abs abrogate the inhibitory effect, consistently with the evidence that extracellular Tat accumulates on the cell membrane of virus-producing cells. The Tat-induced inhibition of cell activation associates with increased apoptosis of CD8 T cells. Finally, the inhibition of cell activation also takes place in Gag-specific CD8 T lymphocytes from HIV-1-infected patients. CONCLUSION:: Our results support the idea that CD8 T-cell apoptosis induced by surface-bound extracellular Tat can contribute to the dysregulation of the CD8 T-cell adaptive response against HIV as well as other pathogens present in AIDS patients.
AU  - Chiozzini, C.*
AU  - Collacchi, B.*
AU  - Nappi, F.*
AU  - Bauer, T.
AU  - Arenaccio, C.*
AU  - Tripiciano, A.*
AU  - Longo, O.*
AU  - Ensoli, F.*
AU  - Cafaro, A.*
AU  - Ensoli, B.*
AU  - Federico, M.*
C1  - 32553
C2  - 35232
CY  - Philadelphia
SP  - 2189-2200
TI  - Surface-bound Tat inhibits antigen-specific CD8+ T-cell activation in an integrin-dependent manner.
JO  - Aids
VL  - 28
IS  - 15
PB  - Lippincott Williams & Wilkins
PY  - 2014
SN  - 0269-9370
ER  - 

TY  - JOUR
AB  - OBJECTIVE: In human astrocytes, restriction of HIV replication involves inhibition of HIV Rev activity. We previously identified a Rev-interacting human protein fragment (16.4.1) that can reduce Rev activity. The 16.4.1 sequence is contained in a group of highly similar host cell proteins, which we call the Risp family. Here we investigate whether the Risp family is connected to HIV replication in astrocytes. METHODS: Cell/tissue lysates were analyzed for Risp expression by western blot with various anti-Risp antibodies. The interaction of astrocytic Risp members with Rev was investigated by affinity chromatography. Astrocytes were transfected with expression plasmids containing cDNAs encoding full-length Risp or the isolated 16.4.1 region for Risp overexpression or with siRNAs designed for Risp knock-down. Rev activity was investigated with a Rev-reporter assay. RNA levels were quantified by real-time RT-PCR, HIV Gag levels by p24ELISA. RESULTS: Expression of the Risp family was demonstrated in human brain tissues and astrocytes. Astrocytes were shown to produce Risp family members that interact with Rev. Production of HIV Gag proteins and Rev-dependent RNAs in persistently infected astrocytes increased upon Risp knock-down and decreased upon Risp overexpression. Risp knock-down increased Rev activity and raised proportions of Rev proteins in the nucleus of astrocytes. CONCLUSION: Our results link the Risp family to restriction of HIV production and inhibition of Rev activity in astrocytes. We conclude that the Risp family represents a novel family of host factors that can control HIV replication and may be important for the containment of HIV infection in brain reservoirs.
AU  - Vincendeau, M.
AU  - Kramer, S.
AU  - Hadian, K.
AU  - Rothenaigner, I.
AU  - Bell, J.*
AU  - Hauck, S.M.
AU  - Bickel, C.
AU  - Nagel, D.
AU  - Kremmer, E.
AU  - Werner, T.*
AU  - Leib-Mösch, C.
AU  - Brack-Werner, R.
C1  - 5948
C2  - 27636
SP  - 2433-2442
TI  - Control of HIV replication in astrocytes by a family of highly conserved host proteins with a common Rev-interacting domain (Risp).
JO  - Aids
VL  - 24
IS  - 6
PB  - Lippincott Williams&Wilkins
PY  - 2010
SN  - 0269-9370
ER  - 

TY  - JOUR
AB  - HIV can reside in the brain for many years. While astrocytes are known to tolerate long-term HIV infection, the potential of other neural cell types to harbour HIV is unclear. To investigate whether HIV can persist in neural progenitor cell populations. A multipotent human neural stem cell line (HNSC.100) was used to compare HIV infection in neural progenitor and astrocyte cell populations. Expression of cellular genes/proteins was analysed by real-time reverse transcriptase PCR, Western blot, immunocytochemistry and flow cytometry. Morphological properties of cells were measured by quantitative fluorescent image analysis. Virus release by cells exposed to HIV-1IIIB was monitored by enzyme-linked immunosorbent assay for Gag. Proviral copy numbers were determined by real-time PCR and early HIV transcripts by reverse transcriptase PCR. Rev activity was determined with a fluorescent-based reporter assay. Progenitor populations differed from astrocyte populations by showing much lower glial fibrillary acidic protein (GFAP) production, higher cell-surface expression of the CXCR4 chemokine receptor, higher Rev activity and distinct cell morphologies. HIV-exposed progenitor cultures released moderate amounts of virus for over 2 months and continued to display cell-associated HIV markers (proviral DNA, early HIV transcripts) during the entire observation period (115 days). Differentiation of HIV-infected progenitor cells to astrocytes was associated with transient activation of virus production. Long-term HIV infection of progenitor populations led to upregulation of GFAP and changes in cell morphology. These studies suggest that neural progenitor populations can contribute to the reservoir for HIV in the brain and undergo changes as a consequence of HIV persistence.
AU  - Rothenaigner, I.
AU  - Kramer, S.
AU  - Ziegler, M.
AU  - Wolff, H.
AU  - Kleinschmidt, A.
AU  - Brack-Werner, R.
C1  - 887
C2  - 24819
SP  - 2271-2281
TI  - Long-term HIV-1 infection of neural progenitor populations.
JO  - Aids
VL  - 21
IS  - 17
PB  - Lippincott Williams & Wilkins
PY  - 2007
SN  - 0269-9370
ER  - 

TY  - JOUR
AU  - Sutter, G.
AU  - Haas, J.*
C1  - 21755
C2  - 19953
SP  - 139-145
TI  - Novel vaccine delivery systems : Solutions to HIV vaccine dilemmas ?.
JO  - Aids
VL  - 15
PY  - 2001
SN  - 0269-9370
ER  - 

TY  - JOUR
AB  - OBJECTIVES:
      	
      		The primary objective of this study was to expand the safety and immunogenicity database of recombinant gp160 as a therapeutic vaccine in the treatment of HIV-infection. Preliminary efficacy data was also sought.
      	
      		DESIGN:
      	
      		This trial was a randomized, double-blind, placebo-controlled study. Two-hundred and eight volunteers, 96 therapy-naive with CD4 cell count >500x10(6)/l (group A) and 112 with CD4 cell count of 200-500x10(6)/l (group B, 51 out of 112 on treatment with one or two nucleoside analogues), received monthly injections of rgp160 IIIB vaccine or placebo for the first 6 months of the study; booster immunizations with rgp160 MN or placebo were given at times 15, 18, and 21 months.
      	
      		METHODS:
      	
      		Safety and immunogenicity data were obtained and measurements of CD4 cell count, plasma viral RNA, and proviral DNA were performed. Clinical outcome was recorded for the 24 months of study.
      	
      		RESULTS:
      	
      		The vaccine was safe and well tolerated. Despite the induction of new rgp160-specific lymphoproliferative responses and the presence of positive delayed type hypersensitivity skin tests to rgp160 at the end of the 24 month study, no effect on the natural history of HIV infection was detected. Within 24 months, AIDS-defining illnesses had occurred in 19 of the vaccinated volunteers and in 18 of the placebo recipients. Persons with higher plasma viral RNA levels and higher proviral DNA had a more rapid decline in CD4 cell count when compared to persons with lower values. Vaccine did not alter viral RNA or proviral DNA levels.
      	
      		CONCLUSION:
      	
      		There was no clinical benefit to therapeutic immunizations with rgp160, despite the induction of new lymphoproliferative responses.
      
      
      	 
AU  - Goebel, F.-D.*
AU  - Mannhalter, J.W.*
AU  - Belshe, R.B.*
AU  - Eibl, M.M.*
AU  - de Gruttola, V.*
AU  - Griffiths, P.D.*
AU  - Erfle, V.
AU  - Kunschak, M.*
AU  - Engl, W.*
C1  - 21246
C2  - 19357
SP  - 1461-1468
TI  - Recombinant gp160 as a therapeutic vaccine for HIV-infection: results of a large randomized, controlled trial.
JO  - Aids
VL  - 13
IS  - 12
PY  - 1999
SN  - 0269-9370
ER  - 

TY  - JOUR
AB  - Objective: To study expression of HIV-1 in human glial cell lines. Design: Chronically HIV-1-infected glial cell lines were established to evade potential artefacts resulting from unphysiological viral entry (i.e., transfection). These cell lines were used to study viral expression and regulation. Methods: Chronically infected glial cell lines were established by terminal dilution cloning of human glioma cells exposed to HIV-1. Virus production and expression were assayed by measuring reverse transcriptase activity, p24-antigen levels and syncytia-inducing capacity in C8166 target cells (extracellular), or by indirect immunoperoxidase staining, immunoblot analysis, and p24- and Nef-antigen-capture enzyme-linked immunosorbent assays (intracellular). HIV-long terminal repeat (LTR)-dependent expression of the chloramphenicol acetyltransferase reporter gene was determined in transient transfection assays. Results: Culture supernatant from chronically HIV-1-infected glial cells contained only low levels of virus compared with chronically HIV-infected fibroblasts and T-lymphoma cells. Detailed study of HIV-antigen expression in representative glial cell line TH4-7-5 indicated the presence of all major structural proteins, albeit at low levels, and of Vif, Tat, Rev and Nef. Intracellular levels of Nef exceeded p24-antigen levels by approximately 10-fold. Virus was recovered from TH4-7-5 cells by cocultivation with blood-derived target cells, indicating that low-level virus production is not due to defective provirus. Prominent negative regulatory element (NRE)-mediated suppression of exogenous HIV-LTR activity was observed in TH4-7-5 cells and was unequalled by chronically HIV-producing fibroblast cells or by uninfected fibroblast and glial cells. Conclusions: Our results suggest that restricted virus production by chronically infected glial cells involves LTR-mediated regulation of virus expression.
AU  - Brack-Werner, R.
AU  - Kleinschmidt, A.M.
AU  - Ludvigsen, A.
AU  - Mellert, W.
AU  - Neumann, M.C.
AU  - Herrmann, R.
AU  - Khim, M.C.L.*
AU  - Burny, A.*
AU  - Müller-Lantzsch, N.*
AU  - Stavrou, D.K.*
AU  - Erfle, V.F.
C1  - 40614
C2  - 13022
SP  - 273-285
TI  - Infection of human brain cells by HIV-1: Restricted virus production in chronically infected human glial cell lines.
JO  - Aids
VL  - 6
IS  - 3
PY  - 1992
SN  - 0269-9370
ER  - 

TY  - JOUR
AB  - Objectives: The characterization and localization of HIV-1 Nef highly expressed in permanently infected astrocytes (TH4-7-5) as a model for latent infection of human brain cells. Design: Immunochemical methods are an appropriate tool to investigate expression and localization of cellular proteins. Methods: Nef expression was analysed by Western blot and immunoperoxidase staining using a panel of monoclonal and polyclonal antibodies. Cellular localization studies were performed by indirect immunofluorescence and subcellular fractionation of TH4-7-5 cells. Myristoylation of Nef was investigated by immunoprecipitation of [3H]myristic acid-labelled cell extract. TH4-7-5 nef gene was cloned and amplified by polymerase chain reaction and the nef nucleotide sequence analysed. Results: Reactivities of various Nef-specific antibodies with Nef antigen in TH4-7-5 cells were demonstrated by Western blot analysis. Immunofluorescence revealed cytoplasmic perinuclear staining of Nef with most antibodies. However, one monoclonal antibody against amino acids 168-175 of Nef showed intense homogeneous nuclear staining in TH4-7-5 cells. Reactivity of this Nef antibody was blocked with recombinant Nef derived from TH4-7-5 cells. After subcellular fractionation, Nef was detected in nuclear, membrane and cytosolic fractions of TH4-7-5 cells. No myristoylated Nef antigen was detectable, perhaps because of a serine residue at position 2 of the TH4-7-5 nef gene instead of the glycine residue required for myristoylation. Conclusions: Chronically HIV-1-infected astrocytoma cells with restricted virus production express different antigenic forms of Nef, which can be distinguished by their subcellular localization. Variant subcellular targeting of Nef suggests the existence of multiple activities of Nef within HIV-infected cells.
AU  - Kohleisen, B.
AU  - Neumann, M.C.
AU  - Herrmann, R.
AU  - Brack-Werner, R.
AU  - Krohn, K.J.E.
AU  - Ovod, V.V.
AU  - Ranki, A.M.
AU  - Erfle, V.F.
C1  - 40507
C2  - 34541
SP  - 1427-1436
TI  - Cellular localization of Nef expressed in persistently HIV-1-infected low-producer astrocytes.
JO  - Aids
VL  - 6
IS  - 12
PY  - 1992
SN  - 0269-9370
ER  - 

TY  - JOUR
AU  - Marquart, K.H.
AU  - Engst, R.H.
AU  - Oehlschlaegel, G.
C1  - 40821
C2  - 34552
SP  - 346-348
TI  - An 8-year history of Kaposi's sarcoma in an HIV-negative bisexual man [8].
JO  - Aids
VL  - 5
IS  - 3
PY  - 1991
SN  - 0269-9370
ER  - 

TY  - JOUR
AB  - The persistent infection of human glial cells with HIV-1 is characterized by prominent expression of the Nef protein. In order to evaluate the possible role of Nef in the development of HIV-1-associated neurological disorders, we compared Nef with known neuroactive proteins. We found that HIV Nef shares sequence and structural features with scorpion peptides known to interact with K+ channels. Sequence similarity encompasses two distinct regions of scorpion peptides. Based on crystallography data, both regions in scorpion peptides cooperate in forming a common domain stabilized by ion pairs between charged amino-acid residues. Recombinant Nef protein, as well as a synthetic part of a scorpion channel active peptide (M10), reversibly increased the total K+ current of chick dorsal root ganglions in patch-clamp experiments without killing the cells. These results indicate that a region conserved in HIV Nef and scorpion peptides concurs in both structure and electrophysiological activity and suggest that Nef, like scorpion peptides, may affect neuronal cell function.
AU  - Werner, T.
AU  - Ferroni, S.*
AU  - Særmark, T.*
AU  - Brack-Werner, R.
AU  - Banati, R.B.*
AU  - Mager, R.*
AU  - Steinaa, L.*
AU  - Kreutzberg, G.W.*
AU  - Erfle, V.F.
C1  - 40835
C2  - 34556
SP  - 1301-1308
TI  - HIV-1 Nef protein exhibits structural and functional similarity to scorpion peptides interacting with K+ channels.
JO  - Aids
VL  - 5
IS  - 11
PY  - 1991
SN  - 0269-9370
ER  - 

TY  - JOUR
AU  - Jager, H.C.
AU  - Postma, M.J.
AU  - Leidl, R.
AU  - Majnoni d'Intignano, B.
AU  - Baert, A.E.
C1  - 18501
C2  - 11650
SP  - 1166-1167
TI  - AIDS Impact Scenarios: Questions for the Years to come.
JO  - Aids
VL  - 4
PY  - 1990
SN  - 0269-9370
ER  - 

TY  - JOUR
AB  - Primary human skin- and lung-derived fibroblast cell cultures and continuous human osteoblast-like and fibroblast-like cell lines were infected with different strains of HIV-1. Infection was measured at the single-cell level using the immunoperoxidase staining method to detect viral proteins. No cytopathic effects were observed in HIV-1-infected cell cultures. One continuous cell line (LC5), derived from embryonic lung, was readily infectable with HIV-1 and showed continuous production of infectious virus. Infection of LC5 cells could be blocked with anti-CD4 monoclonal antibodies. These findings indicate that fibroblasts of skin and lung, and osteogenic cells may be considered as potential target cells for HIV-1, thereby possibly contributing to the establishment of local HIV reservoirs.
AU  - Mellert, W.*
AU  - Kleinschmidt, A.M.*
AU  - Schmidt, J.*
AU  - Festl, H.*
AU  - Emler, S.*
AU  - Roth, W.K.*
AU  - Erfle, V.F.
C1  - 42261
C2  - 34618
SP  - 527-535
TI  - Infection of human fibroblasts and osteoblast-like cells with HIV-1.
JO  - Aids
VL  - 4
IS  - 6
PY  - 1990
SN  - 0269-9370
ER  -