The Requirement for Cellular Transportin 3 (TNPO3 or TRN-SR2) during Infection Maps to Human Immunodeficiency Virus Type 1 Capsid and Not Integrase

Krishnan, Lavanya; Matreyek, Kenneth A.; Öztop, Ilker; Lee, Kyeongeun; Tipper, Christopher; Li, Xiang; Dar, Mohd Jamal; KewalRamani, Vineet N.; Engelman, Alan

doi:10.1128/jvi.01899-09

Cited by 167 publications

(244 citation statements)

References 54 publications

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“…Although biochemical analyses of intracellular HIV initially concluded that capsids were lost immediately postfusion (39,40), EM data revealed intact HIV capsids at or near nuclear pores (33) indicating that capsid uncoating does not necessarily precede viral trafficking to the NE. This is further supported by studies showing that premature uncoating upon retroviral restriction leads to abortive infection (41)(42)(43), and mutations in capsid proteins influence the requirement for nuclear pore components (35,(44)(45)(46). Recent work suggests that the trigger for uncoating is linked to the reverse transcription process (33,36).…”

Section: Stochastic Fluorescence Of Single Flash-labeled Tetracysteinesupporting

confidence: 49%

Superresolution imaging of HIV in infected cells with FlAsH-PALM

Lelek

Nunzio

Henriques

et al. 2012

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

143

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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Section: Stochastic Fluorescence Of Single Flash-labeled Tetracysteinesupporting

confidence: 49%

Superresolution imaging of HIV in infected cells with FlAsH-PALM

Lelek

Nunzio

Henriques

et al. 2012

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

143

135

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“…These studies, in which CA was artificially expressed as a discrete protein, do not, of course, exclude that core-associated CA derived from Gag cleavage during particle budding has important nuclear import functions in the postentry virion. Although the present work focused on the trafficking of de novo-expressed Gag, which is relevant to the assembly side of the life cycle, there is evidence that FIV and HIV-1 negotiate early events differently in some respects, including the use of nuclear entry pathways (32,34,54). Further comparative investigations of the roles of FIV versus HIV-1 Gag-derived virion structural proteins in preintegration trafficking steps may be informative.…”

Section: Discussionmentioning

confidence: 99%

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

Kemler

Saenz

Poeschla

2012

J Virol

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Lentiviral genomic RNAs are encapsidated by the viral Gag protein during virion assembly. The intracellular location of the initial Gag-RNA interaction is unknown. We previously observed feline immunodeficiency virus (FIV) Gag accumulating at the nuclear envelope during live-cell imaging, which suggested that trafficking of human immunodeficiency virus type 1 (HIV-1) and FIV Gag may differ. Here we analyzed the nucleocytoplasmic transport properties of both Gag proteins. We discovered that inhibition of the CRM1 nuclear export pathway with leptomycin B causes FIV Gag but not HIV-1 Gag to accumulate in the nucleus. Virtually all FIV Gag rapidly became intranuclear when the CRM1 export pathway was blocked, implying that most if not all FIV Gag normally undergoes nuclear cycling. In FIV-infected feline cells, some intranuclear Gag was detected in the steady state without leptomycin B treatment. When expressed individually, the FIV matrix (MA), capsid (CA), and nucleocapsid-p2 (NC-p2) domains were not capable of mediating leptomycin B-sensitive nuclear export of a fluorescent protein. In contrast, CA-NC-p2 did mediate nuclear export, with MA being dispensable. We conclude that HIV-1 and FIV Gag differ strikingly in a key intracellular trafficking property. FIV Gag is a nuclear shuttling protein that utilizes the CRM1 nuclear export pathway, while HIV-1 Gag is excluded from the nucleus. These findings expand the spectrum of lentiviral Gag behaviors and raise the possibility that FIV genome encapsidation may initiate in the nucleus.G ag is both necessary and sufficient for retrovirus particle formation and budding. The protein is translated on free polysomes and targeted to the plasma membrane, where virus particles are assembled (16). A central question is whether Gag and the viral genomic RNA (gRNA) associate at the plasma membrane or earlier during assembly and, if so, whether this occurs in the cytosol; in association with organelles, e.g., cotranslationally; or even in the nucleus, as was described previously for an alpharetrovirus (17). Human immunodeficiency virus type 1 (HIV-1) gRNAs become anchored at the plasma membrane before particle assembly is detectable, but it is not clear whether the Gag-gRNA complexes of this and other lentiviruses first form in the cytoplasm and then transit to the plasma membrane or the gRNA traffics there independently (26,27). Biochemical experiments supported the former scenario, with a monomer or low-order multimers forming on HIV-1 gRNAs and higher-order multimer formation depending on subsequent plasma membrane interactions (33).Gag is encoded by the full-length unspliced viral RNA. To circumvent the cellular checkpoint that prevents the export of intron-containing mRNA, lentiviruses express Rev, which functions as an adaptor between the cellular karyopherin CRM1/exportin-1 and a Rev response element (RRE) located in the 3= region of unspliced viral RNAs (9). However, the main cellular function of the CRM1 nuclear export pathway is the export of cellular proteins containing a...

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“…S6A). (P ut vs. 100nM AZT = 9.555e-05, P ut vs. 1uM = 4.485e-07, P ut vs. 10uM = 4.384e-08, P ut vs. 2uM nev = 0.0003, P ut vs. 10uM nev = 8.572e-13, P ut vs. 100nM ral = 3.466e-11, P ut vs. 500nM ral < 2.2e-16, P ut vs. 1uM ral < 2.2e-16, P ut vs. 100nM CX = 0.0004, P ut vs. 1uM CX = 3.046e-13, P ut vs. 10uM CX = 6.122e-10, KS test Transportin-SR2 (TRN-SR2, TNPO3) is a cellular import factor of SR-rich proteins that is involved in HIV-1 entry in the nuclei of infected cells (24)(25)(26)(27)(28)(29)(30)(31). In fact, after TRN-SR2 knockdown HIV-1 nuclear entry is inhibited and, consequently, the amount of integrated DNA is reduced.…”

Section: Resultsmentioning

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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The Requirement for Cellular Transportin 3 (TNPO3 or TRN-SR2) during Infection Maps to Human Immunodeficiency Virus Type 1 Capsid and Not Integrase

Cited by 167 publications

References 54 publications

Superresolution imaging of HIV in infected cells with FlAsH-PALM

Superresolution imaging of HIV in infected cells with FlAsH-PALM

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

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

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