Dynamic Fluorescent Imaging of Human Immunodeficiency Virus Type 1 Gag in Live Cells by Biarsenical Labeling

Rudner, Lynnie A.; Nydegger, Sascha; Coren, Lori V.; Nagashima, Kunio; Thali, Markus; Ott, David E.

doi:10.1128/jvi.79.7.4055-4065.2005

Cited by 116 publications

(115 citation statements)

References 72 publications

(113 reference statements)

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“…HIV from H9 cells buds at the plasma membrane, while HIV originates at deeply invaginated membrane structures that appear to be derived from the plasma membrane in MDMs (11,27,57). Their overall similarity in virion lipid composition is in agreement with the emerging evidence that both assembly sites are continuous with the plasma membrane (11,27,47,57). Microvesicles released from uninfected MDMs mostly share the lipid composition of HIV released from these cells but interestingly had very low levels of phosphoinositides, while HIV from the same cells contained these species (Fig.…”

Section: Lipid Profiles Of Hiv and Other Retrovirusessupporting

confidence: 71%

Retroviruses Human Immunodeficiency Virus and Murine Leukemia Virus Are Enriched in Phosphoinositides

Chan

Uchil

Jin

et al. 2008

J Virol

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251

366

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Retroviruses acquire a lipid envelope during budding from the membrane of their hosts. Therefore, the composition of this envelope can provide important information about the budding process and its location. Here, we present mass spectrometry analysis of the lipid content of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). The results of this comprehensive survey found that the overall lipid content of these viruses mostly matched that of the plasma membrane, which was considerably different from the total lipid content of the cells. However, several lipids are enriched in comparison to the composition of the plasma membrane: (i) cholesterol, ceramide, and GM3; and (ii) phosphoinositides, phosphorylated derivatives of phosphatidylinositol. Interestingly, microvesicles, which are similar in size to viruses and are also released from the cell periphery, lack phosphoinositides, suggesting a different budding mechanism/ location for these particles than for retroviruses. One phosphoinositide, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ], has been implicated in membrane binding by HIV Gag. Consistent with this observation, we found that PI(4,5)P 2 was enriched in HIV-1 and that depleting this molecule in cells reduced HIV-1 budding. Analysis of mutant virions mapped the enrichment of PI(4,5)P 2 to the matrix domain of HIV Gag. Overall, these results suggest that HIV-1 and other retroviruses bud from cholesterol-rich regions of the plasma membrane and exploit matrix/PI(4,5)P 2 interactions for particle release from cells.Retroviruses rely on their host for many essential parts of the viral replication cycle. Biochemical and antibody-based analyses of the replication cycle and proteins found in the virions have revealed many details of the molecular interactions between human immunodeficiency virus (HIV) and its host (20). In contrast, the role of lipids has been less well studied. With the increasing recognition that lipids play an important role in cellular signaling, it is no coincidence that lipid factors are slowly gaining prominence in our understanding of retroviral replication.Retroviruses, including HIV and murine leukemia virus (MLV), acquire their lipid coats by budding through host plasma membranes. Two important issues arise when considering the roles of lipids in retrovirus assembly and budding. First, the idea that HIV and other retroviruses bud from lipid rafts has gained widespread acceptance (39, 45). Lipid rafts are liquid ordered domains that exist within the liquid disordered phase of the bulk cell membrane. These dynamic lipid-protein assemblies are characterized by high levels of cholesterol, sphingolipids, saturated glycerophospholipids, and raft proteins. Because the half-lives for lipid rafts are extremely short (50), the assignment of HIV to lipid rafts is commonly established through the colocalization of HIV proteins with putative raft proteins and the preponderance of raft lipids, including cholesterol, sphingomyelin (SM), dihydrosphingomyelin (dhSM), ce...

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Section: Lipid Profiles Of Hiv and Other Retrovirusessupporting

confidence: 71%

Retroviruses Human Immunodeficiency Virus and Murine Leukemia Virus Are Enriched in Phosphoinositides

Chan

Uchil

Jin

et al. 2008

J Virol

Self Cite

251

366

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“…Although nonspecific interactions can be minimized by the addition of dithiols, the background labeling and the unspecific binding to hydrophobic sites in cellular components limits the potential of tetracysteine-biarsenical probes. Nevertheless, the FlAsH-tag has been used in a number of cell biology studies [36][37][38] and is the most established peptide tag to date.…”

Section: Non-enzymatic Peptide Tagsmentioning

confidence: 99%

Chemical tags: Applications in live cell fluorescence imaging

Wombacher

Cornish

2011

Journal of Biophotonics

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Technologies to visualize cellular structures and dynamics enable cell biologists to gain insight into complex biological processes. Currently, fluorescent proteins are used routinely to investigate the behavior of proteins in live cells. Chemical biology techniques for selective labeling of proteins with fluorescent labels have become an attractive alternative to fluorescent protein labeling. In the last ten years the progress in the development of chemical tagging methods have been substantial offering a broad palette of applications for live cell fluorescent microscopy. Several methods for protein labeling have been established, using protein tags, peptide tags and enzyme mediated tagging. This review focuses on the different strategies to achieve the attachment of fluorophores to proteins in live cells and cast light on the advantages and disadvantages of each individual method. Selected experiments in which chemical tags have been successfully applied to live cell imaging will be discussed and evaluated. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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“…HIV-1 virions labeled with fluorophores were pivotal in shedding light onto multiple aspects of the virus-host interplay during all steps of HIV-1 replication cycle (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Nevertheless, few optical approaches have been so far developed to visualize viral particles within the nuclear compartment (14,15), which limits our comprehension of the interaction between HIV-1 and the nuclear architecture.…”

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confidence: 99%

Single-Cell Imaging of HIV-1 Provirus (SCIP)

Primio

Quercioli

Allouch

et al. 2013

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

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Recent advances in fluorescence microscopy provided tools for the investigation and the analysis of the viral replication steps in the cellular context. In the HIV field, the current visualization systems successfully achieve the fluorescent labeling of the viral envelope and proteins, but not the genome. Here, we developed a system able to visualize the proviral DNA of HIV-1 through immunofluorescence detection of repair foci for DNA double-strand breaks specifically induced in the viral genome by the heterologous expression of the I-SceI endonuclease. The system for Single-Cell Imaging of HIV-1 Provirus, named SCIP, provides the possibility to individually track integrated-viral DNA within the nuclei of infected cells. In particular, SCIP allowed us to perform a topological analysis of integrated viral DNA revealing that HIV-1 preferentially integrates in the chromatin localized at the periphery of the nuclei.T echnical developments in imaging-based techniques have greatly improved our understanding of HIV-host cell interactions. HIV-1 virions labeled with fluorophores were pivotal in shedding light onto multiple aspects of the virus-host interplay during all steps of HIV-1 replication cycle (1-13). Nevertheless, few optical approaches have been so far developed to visualize viral particles within the nuclear compartment (14, 15), which limits our comprehension of the interaction between HIV-1 and the nuclear architecture. Moreover, the existing detection tools are based on the visualization of the viral protein complexes or envelope but not of the viral DNA with the only exception of the fluorescence in situ hybridization (FISH) technique. Even though FISH is a powerful technique, it is not very sensitive for HIV-1 detection and moreover disrupts the native architecture of the nuclear compartment as it requires harsh denaturation conditions. In addition, this technique does not allow the discrimination between integrated and nonintegrated viral DNA (16, 17). Here we describe a fluorescent approach to visualize HIV-1 DNA in the nuclear compartment of infected cells. We exploited a site-specific genome engineering technique that represents one of the most promising approaches to detect specific genome regions in modified organism (18) allowing for their spatial localization in the cell (19,20). This technique couples endogenous repair pathways, induced by rare cutting endonuclease, with immunofluorescence analysis. Rare cutting endonucleases, such as the yeast-homing endonuclease I-SceI, specifically cuts target sequences that cannot be found in the mammalian genome. By engineering DNA to contain the I-SceI cleavage site, it is thus possible to induce endogenous repair mechanism for double-strand breaks (DSBs) at specific genomic positions. DSB repair leads to the formation of distinct subnuclear structures that are generally referred to as "foci" (21). The first sensor of the DSB is the histone H2AX, which becomes massively phosphorylated at serine 139 (γ-H2AX). Foci of DNA repair are thus visible through immu...

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Dynamic Fluorescent Imaging of Human Immunodeficiency Virus Type 1 Gag in Live Cells by Biarsenical Labeling

Cited by 116 publications

References 72 publications

Retroviruses Human Immunodeficiency Virus and Murine Leukemia Virus Are Enriched in Phosphoinositides

Retroviruses Human Immunodeficiency Virus and Murine Leukemia Virus Are Enriched in Phosphoinositides

Chemical tags: Applications in live cell fluorescence imaging

Single-Cell Imaging of HIV-1 Provirus (SCIP)

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