Joachim Hauber

Functional dissection of the HIV-1 Rev trans-activator—Derivation of a trans-dominant repressor of Rev function

Human immunodeficiency virus type 1 (HIV-1) encodes a nuclear trans-activator, termed Rev, that is required for the expression of the viral structural proteins and, hence, for viral replication. The Rev protein acts posttranscriptionally to induce the sequence-specific nuclear export of unspliced HIV-1 mRNA species that are otherwise excluded from the cell cytoplasm. We have used site-directed mutagenesis to identify two distinct regions of the HIV-1 Rev protein that are required for in vivo biological activity. The larger and more N-terminal of these two regions includes, but extends beyond, an arginine-rich sequence element required for nuclear localization. Mutation of a second, more C-terminal Rev protein sequence element was found to yield defective Rev proteins that act as trans-dominant inhibitors of Rev function. These Rev mutants are shown to inhibit HIV-1 replication when expressed in transfected cells and may have potential application in the treatment of HIV-1 related disease.

Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells

This paper describes a novel eukaryotic reporter gene, secreted alkaline phosphatase (SEAP). In transient expression experiments using transfected mammalian cells, we demonstrate that SEAP yields results that are qualitatively and quantitatively similar, at both the mRNA and protein levels, to parallel results obtained using established reporter genes. However, SEAP offers significant advantages in terms of ease of assay and assay expense, and also has the potential for quantitative assay at levels as low as 0.2 pg/ml of culture medium. These attributes suggest that SEAP may have general utility in experiments which rely on the accurate measurement of reporter gene expression levels.

HIV-1 structural gene expression requires binding of the rev trans-activator to its RNA target sequence

Expression of human immunodeficiency virus type 1 structural proteins requires both the viral Rev trans-activator and its cis-acting RNA target sequence, the Rev response element (RRE). The RRE has been mapped to a conserved region of the HIV-1 env gene and is predicted to form a complex, highly stable RNA stem-loop structure. Site-directed mutagenesis was used to define a small subdomain of the RRE, termed stem-loop II, that is essential for biological activity. Gel retardation assays demonstrated that the Rev trans-activator is a sequence-specific RNA binding protein. The RRE stem-loop II subdomain was found to be both necessary and sufficient for the binding of Rev by the RRE. We propose that the HIV-1 Rev trans-activator belongs to a new class of sequence-specific RNA binding proteins characterized by the presence of an arginine-rich binding motif.

Mature Dendritic Cells Infected with Herpes Simplex Virus Type 1 Exhibit Inhibited T-Cell Stimulatory Capacity

ABSTRACT Mature dendritic cells (DC) are the most potent antigen-presenting cells within the entire immune system. Interference with the function of these cells therefore constitutes a very powerful mechanism for viruses to escape immune responses. Several members of theHerpesviridae family have provided examples of such escape strategies, including interference with antigen presentation and production of homologous cytokines. In this study we investigated the infection of mature DC with herpes simplex virus type 1 (HSV-1) and the way in which infection alters the phenotype and function of mature DC. Interestingly, the T-cell-stimulatory capacity of these DC was strongly impaired. Furthermore, we demonstrated that HSV-1 leads to the specific degradation of CD83, a cell surface molecule which is specifically upregulated during DC maturation. These data indicate that HSV-1 has developed yet another novel mechanism to escape immune responses.

Inhibition of HIV-1 replication in lymphocytes by mutants of the Rev cofactor eIF-5A.

Eukaryotic initiation factor 5A (eIF-5A) is a cellular cofactor required for the function of the human immunodeficiency virus type-1 (HIV-1) Rev trans-activator protein. The majority of a set of eIF-5A mutants did not support growth of yeast cells having an inactivated genomic copy of eIF-5A, indicating that the introduced mutation eliminated eIF-5A activity. Two nonfunctional mutants, eIF-5AM13 and eIF-5AM14, retained their binding capacity for the HIV-1 Rev response element: Rev complex. Both mutants were constitutively expressed in human T cells. When these T cells were infected with replication-competent HIV-1, virus replication was inhibited. The eIF-5AM13 and eIF-5AM14 proteins blocked Rev trans-activation and Rev-mediated nuclear export.

The adenovirus type 5 E1B-55K oncoprotein is a highly active shuttle protein and shuttling is independent of E4orf6, p53 and Mdm2

The E1B-55K and E4orf6 oncoproteins of adenovirus type 5 are involved in the export of viral mRNAs. Previously, it was suggested that a complex composed of E1B-55K and E4orf6 serves as a nucleocytoplasmic transporter for viral mRNAs in which the E4orf6 protein directs both nuclear import and export. We now demonstrate that the E1B-55K protein itself shuttles efficiently in the absence of E4orf6. In addition, E1B-55K trafficking was independent of the defined shuttle proteins Mdm2 or p53, which interacts with E1B-55K. The identified N-terminal E1B-55K leucine-rich nuclear-export signal (NES) conferred rapid nuclear export even in a heterologous system in contrast to the postulated E4orf6NES. Interestingly, although shuttling was blocked by inhibitors of the CRM1 mediated export pathway, E1B-55K inhibited neither the activity nor the trafficking of the retroviral shuttle proteins HIV-1 Rev and HTLV-1 Rex. In contrast, Rev or Rex blocked the nuclear export of E1B-55K, most likely by competing for essential export factors. Our results provide new insights into the regulation of the adenovirus mRNA export system and the processes of adenovirus mediated transformation.

Identification of cellular deoxyhypusine synthase as a novel target for antiretroviral therapy

The introduction of highly active antiretroviral therapy (HAART) has significantly decreased morbidity and mortality among patients infected with HIV-1. However, HIV-1 can acquire resistance against all currently available antiretroviral drugs targeting viral reverse transcriptase, protease, and gp41. Moreover, in a growing number of patients, the development of multidrug-resistant viruses compromises HAART efficacy and limits therapeutic options. Therefore, it is an ongoing task to develop new drugs and to identify new targets for antiretroviral therapy. Here, we identified the guanylhydrazone CNI-1493 as an efficient inhibitor of human deoxyhypusine synthase (DHS). By inhibiting DHS, this compound suppresses hypusine formation and, thereby, activation of eukaryotic initiation factor 5A (eIF-5A), a cellular cofactor of the HIV-1 Rev regulatory protein. We demonstrate that inhibition of DHS by CNI-1493 or RNA interference efficiently suppressed the retroviral replication cycle in cell culture and primary cells. We show that CNI-1493 inhibits replication of macrophage- and T cell-tropic laboratory strains, clinical isolates, and viral strains with high-level resistance to inhibitors of viral protease and reverse transcriptase. Moreover, no measurable drug-induced adverse effects on cell cycle transition, apoptosis, and general cytotoxicity were observed. Therefore, human DHS represents a novel and promising drug target for the development of advanced antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses.

Expression of CD83 Is Regulated by HuR via a Novel cis-Active Coding Region RNA Element

Dendritic cells are the most potent of the antigen-presenting cells and are characterized by surface expression of CD83. Here, we show that the coding region of CD83 mRNA contains a novel cis-acting structured RNA element that binds to HuR, a member of the ELAV family of AU-rich element RNA-binding proteins. Transient transfection of mammalian cells demonstrated that this CD83 mRNA-derived element acts as a post-transcriptional regulatory element in cells overexpressing HuR. Notably, binding of HuR to the CD83 post-transcriptional regulatory element did not affect mRNA stability. Using RNA interference, we show that HuR mediated efficient expression of CD83. In particular, HuR was required for cytoplasmic accumulation of CD83 transcripts. Likewise, inhibition of the CRM1 nuclear export pathway by leptomycin B or overexpression of a defective form of the nucleoporin Nup214/CAN diminished cytoplasmic CD83 mRNA levels. In summary, the data presented demonstrate that the HuR-CRM1 axis affects the nucleocytoplasmic translocation of CD83 mRNA under regular physiological conditions.

Eukaryotic Initiation Factor 5A Activity and HIV-1 Rev Function

Eukaryotic initiation factor 5A (eIF-5A) is the only cellular protein known to contain the unusual amino acid hypusine, a modification that appears to be required for cell proliferation. This hypusine-modified protein stimulates synthesis of methionyl-puromycin in an in vitro assay which mimics the formation of the first peptide bond during protein synthesis, although the exact role of eIF-5A in vivo is still unknown. The unexpected finding that eIF-5A is a cellular cofactor of the HIV-1 Rev trans-activator protein may, however, provide a novel opportunity to reveal precisely what function eIF-5A performs in eukaryotic cells. In this review article, we first present a brief description of HIV-1 Rev function, followed by an overview of the data that identified eIF-5A as a Rev cofactor and, finally, discuss novel findings with respect to cellular eIF-5A activities.

Multiple importins function as nuclear transport receptors for the Rev protein of human immunodeficiency virus type 1.

The Rev protein of human immunodeficiency virus type 1 is an RNA-binding protein that is required for nuclear export of unspliced and partially spliced viral mRNAs. Nuclear import of human immunodeficiency virus type 1 Rev has been suggested to depend on the classic nuclear transport receptor importin β, but not on the adapter protein importin α. We now show that, similar to importin α, Rev is able to dissociate RanGTP from recycling importin β, a reaction that leads to the formation of a novel import complex. Besides importin β, the transport receptors transportin, importin 5, and importin 7 specifically interact with Rev and promote its nuclear import in digitonin-permeabilized cells. A single arginine-rich nuclear localization sequence of Rev is required for interaction with all importins tested so far. In contrast to the importin β-binding domain of importin α, Rev interacts with an N-terminal fragment of importin β. Transportin contains two independent binding sites for Rev. Hence, the mode of interaction of importin β and transportin with Rev is clearly distinct from that with their classic import cargoes. Taken together, the viral protein takes advantage of multiple cellular transport pathways for its nuclear accumulation.