Transportin-SR2 imports HIV into the nucleus

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) and other lentiviruses have the capacity to infect nondividing cells like macrophages. This requires import of the preintegration complex (PIC) through the nuclear pore. Although many cellular and viral determinants have been proposed, the mechanism leading to nuclear import is not yet understood. RESULTS: Using yeast two-hybrid and pull-down, we identified and validated transportin-SR2 (TRN-SR2) as a bona fide binding partner of HIV-1 integrase. We confirmed the biological relevance of this interaction by RNAi. Depletion of TRN-SR2 interfered with the replication of HIV-1 and HIV-2 but not MoMLV in HeLaP4 cells. Knockdown of TRN-SR2 in primary macrophages likewise interfered with HIV-1 replication. Using Q-PCR, we pinpoint this block in replication to the early steps of the viral lifecycle. A reduction in 2-LTR formation suggests a block in PIC nuclear import upon siRNA-mediated knockdown. Different lines of evidence clearly proved that the late steps of viral replication are not affected. In an in vivo nuclear-import assay using labeled HIV-1 particles, the defect in nuclear import after depletion of TRN-SR2 was directly visualized. In comparison with control cell lines, the great majority of siRNA-treated cells did not contain any PIC in the nucleus. CONCLUSION: Our data clearly demonstrate that TRN-SR2 is the nuclear-import factor of HIV.     

Mechanisms of viral entry: sneaking in the front door

In this study, the filamentous green alga Zygogonium ericetorum (Zygnematales, Chlorophyta), collected at its natural habitat in the high alps, was investigated by light, scanning, and transmission electron microscopy. The field samples were separated into a moist fraction when wetted by splattering water of a nearby spring or a desiccated one when visually dried out. Light microscopy demonstrated a purple pigmentation of the sun-exposed upper layers, the central position of the nucleus, and the starch content in the pyrenoids. The smooth surface of the cells occasionally covered with fungal hyphae was shown by scanning electron microscopy. The cytoarchitecture of moist cells revealed many vacuoles and only a thin cytoplasmic area surrounding the two chloroplasts. The secondary cell walls of older cells were up to 4 µm thick. Organelle membranes as well as thylakoid membranes occasionally showed an inversion of contrast. In the chloroplasts, distinct areas with granular content surrounding the pyrenoids were detected. Within the cytoplasm, electron-dense particles with electron-translucent crystalloid structures were observed. When desiccated samples were investigated, the vacuoles and cytoplasmatic portions appeared destroyed, whereas nucleus and chloroplasts generally remained intact. The thylakoid membranes of desiccated samples showed lumen dilatations and numerous plastoglobules. Water-soluble extracts were separated by high-pressure liquid chromatography that revealed two major compounds with UV-absorbing capacities.     

Pathogen-endoplasmic-reticulum interactions: in through the out door

A key determinant for the survival of intracellular pathogens is their ability to subvert the cellular processes of the host to establish a compartment that allows replication. Although most microorganisms internalized by host cells are efficiently cleared following fusion with lysosomes, many pathogens have evolved mechanisms to escape this degradation. In this Review, we provide insight into the molecular processes that are targeted by pathogens that interact with the endoplasmic reticulum and thereby subvert the immune response, ensure their survival intracellularly and cause disease. We also discuss how the endoplasmic reticulum 'strikes back' and controls microbial growth.     

Structural basis for RanGTP independent entry of spliceosomal U snRNPs into the nucleus

The nuclear import of assembled spliceosomal subunits, the uridine-rich small nuclear ribonucleoprotein particles (U snRNPs), is mediated by a nuclear import receptor adaptor couple of importinβ (Impβ) and snurportin1 (SPN1). In contrast to any other characterized active nuclear import, the Impβ/SPN1/U snRNP complex does not require RanGTP for the terminal release from the nuclear basket of the nuclear pore complex (NPC). The crystal structure of Impβ (127-876) in complex with the Impβ-binding (IBB) domain of SPN1 (1-65) at 2.8-Å resolution reveals that Impβ adopts an open conformation, which is unique for a functional Impβ/cargo complex, and rather surprisingly, it resembles the conformation of the Impβ/RanGTP complex. As binding of RanGTP to Impβ usually triggers the release of import complexes from the NPC, we propose that by already mimicking a conformation similar to Impβ/RanGTP the independent dissociation of Impβ/SPN1 from the nuclear basket is energetically aided.     

Blocking HIV entry.

Targeting the fusogenic machinery of HIV with unnatural ligand libraries.     

Possible entry of fragments of a bacterial genome into the trophozoite nucleus of the dysentery amoeba

The use of F?lgen-like electron microscopy reaction has revealed particles in the cytoplasm and nucleus of the dysentery amoeba trophozoites which in their size and resistance to acidic hydrolysis and in their affinity for uranilacetate are similar to tightly packed elements of the bacterial genome. On this basis a conclusion was drawn that some fragments of genomes of phagocytic bacteria can be preserved in the amoebal cell. The community of microorganisms consisting of trophozoites of the dysentery amoeba and bacteria of the intestinal flora is supposed to be a suitable model for studying the possibility of penetration of transgenetic elements from procaryote to eucaryote.     

The Ty1 integrase protein can exploit the classical nuclear protein import machinery for entry into the nucleus

Like its retroviral relatives, the long terminal repeat retrotransposon Ty1 in the yeast Saccharomyces cerevisiae must traverse a permanently intact nuclear membrane for successful transposition and replication. For retrotransposition to occur, at least a subset of Ty1 proteins, including the Ty1 integrase, must enter the nucleus. Nuclear localization of integrase is dependent upon a C-terminal nuclear targeting sequence. However, the nuclear import machinery that recognizes this nuclear targeting signal has not been defined. We investigated the mechanism by which Ty1 integrase gains access to nuclear DNA as a model for how other retroelements, including retroviruses like HIV, may utilize cellular nuclear transport machinery to import their essential nuclear proteins. We show that Ty1 retrotransposition is significantly impaired in yeast mutants that alter the classical nuclear protein import pathway, including the Ran-GTPase, and the dimeric import receptor, importin-alpha/beta. Although Ty1 proteins are made and processed in these mutant cells, our studies reveal that an integrase reporter is not properly targeted to the nucleus in cells carrying mutations in the classical nuclear import machinery. Furthermore, we demonstrate that integrase coimmunoprecipitates with the importin-alpha transport receptor and directly binds to importin-alpha. Taken together, these data suggest Ty1 integrase can employ the classical nuclear protein transport machinery to enter the nucleus.     

HIV takes double hit before entry

In the absence of a vaccine or a cure, identification of novel HIV-1 inhibitors remains important. A paper in Retrovirology describes a rationally designed bi-specific protein that irreversibly damages the viral envelope glycoprotein complex via a two-punch mechanism. In contrast to traditional drugs that inhibit essential steps in the viral life cycle at the cell surface or in the infected cells, this inhibitor cripples free virus in the absence of cells.See research article: http://www.retrovirology.com/content/9/1/104     

pH-independent HIV entry into CD4-positive T cells via virus envelope fusion to the plasma membrane

CD4 functions as the cell-surface receptor for human immunodeficiency virus (HIV); however, the mechanism of virus entry into susceptible cells is unknown. To explore this question we used a human T lymphoblastic cell line (VB) expressing high levels of surface CD4. Neutralization of endosomal compartments (pH greater than 6.4) with lysosomotropic agents did not effectively inhibit HIV nucleocapsid entry into the cytoplasm, and virus treated at low pH (5.5) failed to induce rapid cell-to-cell fusion in uninfected cells. Electron microscopy of VB cells acutely exposed to HIV at neutral pH revealed direct fusion of the virus envelope with the plasma membrane within minutes at 4 degrees C. No endocytosed virions were visualized upon rewarming the HIV-exposed cells to 37 degrees C for as long as 60 min. These results indicate that HIV penetrates CD4-positive T cells via pH-independent membrane fusion.     

CD4 mimics targeting the mechanism of HIV entry

A structure–activity relationship study was conducted of several CD4 mimicking small molecules which block the interaction between HIV-1 gp120 and CD4. These CD4 mimics induce a conformational change in gp120, exposing its co-receptor-binding site. This induces a highly synergistic interaction in the use in combination with a co-receptor CXCR4 antagonist and reveals a pronounced effect on the dynamic supramolecular mechanism of HIV-1 entry.