Construction and Characterization of a Fluorescently Labeled Infectious Human Immunodeficiency Virus Type 1 Derivative

Müller, Bárbara; Daecke, Jessica; Fackler, Oliver T.; Dittmar, Matthias T.; Zentgraf, Hanswalter; Kräusslich, Hans‐Georg

doi:10.1128/jvi.78.19.10803-10813.2004

Cited by 205 publications

(231 citation statements)

References 49 publications

(32 reference statements)

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“…CA is not tolerant to large insertions, probably because of its "cage"-like structure. The constructs MA.GFP, GFP.p12, NC.GFP, and IN.GFP may serve as tools for virus trafficking studies (38)(39)(40)(41)(42)(43)(44)(45)(46).…”

Section: Discussionmentioning

confidence: 99%

Protein transduction from retroviral Gag precursors

Voelkel¹,

Galla²,

Maetzig³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

125

118

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Retroviral particles assemble a few thousand units of the Gag polyproteins. Proteolytic cleavage mediated by the retroviral protease forms the bioactive retroviral protein subunits before cell entry. We hypothesized that this process could be exploited for targeted, transient, and dose-controlled transduction of nonretroviral proteins into cultured cells. We demonstrate that gammaretroviral particles tolerate the incorporation of foreign protein at several positions of their Gag or Gag-Pol precursors. Receptor-mediated and thus potentially cell-specific uptake of engineered particles occurred within minutes after cell contact. Dose and kinetics of nonretroviral protein delivery were dependent upon the location within the polyprotein precursor. Proteins containing nuclear localization signals were incorporated into retroviral particles, and the proteins of interest were released from the precursor by the retroviral protease, recognizing engineered target sites. In contrast to integration-defective lentiviral vectors, protein transduction by retroviral polyprotein precursors was completely transient, as protein transducing retrovirus-like particles could be produced that did not transduce genes into target cells. Alternatively, bifunctional protein-delivering particle preparations were generated that maintained their ability to serve as vectors for retroviral transgenes. We show the potential of this approach for targeted genome engineering of induced pluripotent stem cells by delivering the site-specific DNA recombinase, Flp. Protein transduction of Flp after proteolytic release from the matrix position of Gag allowed excision of a lentivirally transduced cassette that concomitantly expresses the canonical reprogramming transcription factors (Oct4, Klf4, Sox2, c-Myc) and a fluorescent marker gene, thus generating induced pluripotent stem cells that are free of lentivirally transduced reprogramming genes.Flp recombinase | murine leukemia virus | pluripotent cells | protein transfer

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Section: Discussionmentioning

confidence: 99%

Protein transduction from retroviral Gag precursors

Voelkel¹,

Galla²,

Maetzig³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

125

118

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“…For a detailed investigation of HIV-cell interactions by SVT, we have generated and characterized entry-competent single-as well as double-fluorescently labeled HIV-1 derivatives (Lampe et al 2007;Müller et al 2004). The dual-labeled virus like particles (VLPs) carry two differently colored fluorescent labels at two viral proteins and were designed to distinguish between complete virions and subviral particles resulting from membrane fusion.…”

Section: Introductionmentioning

confidence: 99%

HIV-1–cellular interactions analyzed by single virus tracing

et al. 2008

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Single virus tracing (SVT) allows the direct investigation of the entry pathway of viruses into living cells. Using fluorescently labeled virus-like particles (VLPs) and SVT, we have studied the interaction between human immunodeficiency virus type 1 (HIV-1) and the plasma membrane of living cells. From the trajectories of freely diffusing VLPs in solution, we established that the particle preparation was homogeneous and the particles had a hydrodynamic radius of 86 ± 5 nm, consistent with the size of single HI viruses. The VLPs that come in contact with the cell surface either become immobilized or rapidly dissociate from the cell surface. The fraction of virions that become immobilized on the plasma membrane correlates with the surface heparan sulfate linked proteoglycans (HSPG) concentration of the cell line tested. The particles that are not immobilized make an average of 1.5 contacts with the cell surface before diffusing away. For most cell lines investigated, the contact duration follows an exponential distribution with a lifetime between 20 and 50 ms depending on the cell type.

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“…This has proven problematic for a number of studies that imaged pathogen invasion of host cells. For example, GFP-labeling interferes with the delivery of bacterial effector proteins into host cells via type III secretion (13), and fluorescent protein fusions of the HIV-1 gag or pol can impair the production of infectious virions unless the labeled protein or wild-type Gag-Pol are provided in trans (14)(15)(16)). …”

mentioning

confidence: 99%

Superresolution imaging of HIV in infected cells with FlAsH-PALM

Lelek

Nunzio

Henriques

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

140

135

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Imaging protein assemblies at molecular resolution without affecting biological function is a long-standing goal. The diffractionlimited resolution of conventional light microscopy (∼200-300 nm) has been overcome by recent superresolution (SR) methods including techniques based on accurate localization of molecules exhibiting stochastic fluorescence; however, SR methods still suffer important restrictions inherent to the protein labeling strategies. Antibody labels are encumbered by variable specificity, limited commercial availability and affinity, and are mostly restricted to fixed cells. Fluorescent protein fusions, though compatible with live cell imaging, substantially increase protein size and can interfere with their biological activity. We demonstrate SR imaging of proteins tagged with small tetracysteine motifs and the fluorescein arsenical helix binder (FlAsH-PALM). We applied FlAsH-PALM to image the integrase enzyme (IN) of HIV in fixed and living cells under experimental conditions that fully preserved HIV infectivity. The obtained resolution (∼30 nm) allowed us to characterize the distribution of IN within virions and intracellular complexes and to distinguish different HIV structural populations based on their morphology. We could thus discriminate ∼100 nm long mature conical cores from immature Gag shells and observe that in infected cells cytoplasmic (but not nuclear) IN complexes display a morphology similar to the conical capsid. Together with the presence of capsid proteins, our data suggest that cytoplasmic IN is largely present in intact capsids and that these can be found deep within the cytoplasm. FlAsH-PALM opens the door to in vivo SR studies of microbial complexes within host cells and may help achieve truly molecular resolution.

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Construction and Characterization of a Fluorescently Labeled Infectious Human Immunodeficiency Virus Type 1 Derivative

Cited by 205 publications

References 49 publications

Protein transduction from retroviral Gag precursors

Protein transduction from retroviral Gag precursors

HIV-1–cellular interactions analyzed by single virus tracing

Superresolution imaging of HIV in infected cells with FlAsH-PALM

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