High-Resolution In Vivo

Purpose. To investigate fundamental mechanisms of regimes of laser induced damage to the retina and the morphological changes associated with the damage response. Methods. Varying grades of photothermal, photochemical, and photomechanical retinal laser damage were produced in eyes of eight cynomolgus monkeys. An adaptive optics confocal scanning laser ophthalmoscope and spectral domain optical coherence tomographer were combined to simultaneously collect complementary in vivo images of retinal laser damage during and following exposure. Baseline color fundus photography was performed to complement high-resolution imaging. Monkeys were perfused with 10% buffered formalin and eyes were enucleated for histological analysis. Results. Laser energies for visible retinal damage in this study were consistent with previously reported damage thresholds. Lesions were identified in OCT images that were not visible in direct ophthalmoscopic examination or fundus photos. Unique diagnostic characteristics, specific to each damage regime, were identified and associated with shape and localization of lesions to specific retinal layers. Previously undocumented retinal healing response to blue continuous wave laser exposure was recorded through a novel experimental methodology. Conclusion. This study revealed increased sensitivity of lesion detection and improved specificity to the laser of origin utilizing high-resolution imaging when compared to traditional ophthalmic imaging techniques in the retina

HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors

Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent "burst-like" transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm

Working model for linkages between retroviral gRNA nuclear history and trafficking in the cytoplasm.

<p><b>(A)</b> In interphase cells, CRM1 regulates punctuated transitions of RRE-gRNAs from the nucleus to flood the cytoplasm in conjunction with Gag expression and the onset of virus particle assembly at the plasma membrane. 4xCTE-gRNAs (and M-PMV gRNAs) also leave the nucleus through the nuclear pore complex but instead are linked to microtubules that direct their trafficking to the microtubule organizing center (MTOC) and centrosome. <b>(B)</b> 4xCTE-gRNAs are also targeted to centrosomes during cell division. At the onset of mitosis, the nuclear membrane breaks down (phase 1) and 4xCTE-gRNAs are rapidly directed to duplicated centrosomes that form the poles of the mitotic spindle during metaphase (phase 2). Subsequently, CTE-gRNAs bound to centrosomes are partitioned to daughter cells and then released into the cytoplasm for Gag synthesis (phase 3).</p

The Rev/CRM1 or NXF1/NXT1 export modules are sufficient to trigger RNA burst export or MTOC targeting, respectively, and independently of an export element.

<p><b>(A)</b> Depiction of 24xMBL-bearing ΔEE HIV-1 gRNAs expressed with or without MS2-tagged fusion proteins in HeLa.MS2-YFP cells. <b>(B)</b> MS2-Rev expression led to ΔEE-gRNA evacuation from the nucleus (MS2-YFP signal in green), while MS2-NXF1 expression directed transcripts to the MTOC (yellow arrows, Pericentrin stain in red). MS2-YFP signal is also shown detected for ΔEE-gRNAs expressed with MS2-NXF1 at centrosomes (yellow arrows) in mitotic cells expressing mCherry-Tubulin (red). <b>(C)</b> Quantification of burst export and MTOC-targeting phenotypes for each condition as measured for Figs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005565#ppat.1005565.g001" target="_blank">1D</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005565#ppat.1005565.g004" target="_blank">4E</a>. <b>(D)</b> Depiction of 24xMBL-bearing ΔEE HIV-1 gRNAs expressed with or without Rev/RevM10-NXF1 fusion proteins. <b>(E)</b> As for (B), burst or MTOC-targeting for RRE-gRNAs expressed with Rev-NXF1 or RevM10-NXF1 fusion proteins, with or without NXT1 as indicated. <b>(F)</b> Quantification as for (C) for the experiments depicted in (D) and (E). All size bars represent 5 μm.</p

Full Length HIV-1 and M-PMV gRNA exhibit burst nuclear export and MTOC-targeting phenotypes, respectively.

<p><b>(A)</b> Representative images of full-length HIV-1 and M-PMV reporter viruses modified to carry the 24xMBL cassette and express Gag-CFP (blue) in HeLa.MS2-YFP cells at 24 hours post-transfection. Dotted line indicates nuclear/cytoplasmic boundary after gRNA (green) was evacuated from the nucleus. Black and yellow arrows indicate the location of the centrosomes based on Pericentrin (red) immunofluorescence. <b>(B)</b> MTOC-targeting for HIV-1 and M-PMV gRNAs as measured for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005565#ppat.1005565.g004" target="_blank">Fig 4E</a>. <b>(C)</b> M-PMV gRNAs accumulated at the MTOC in both human HeLa (top panels) and African green monkey Cos7 cells (bottom panels). <b>(D)</b> M-PMV gRNA clustering at the MTOC precedes Gag-CFP synthesis. Gag-CFP synchronized rise in mean fluorescence intensity (MFI) over time (lower panel, <i>n</i> = 10 cells) relative to the defined subcellular distribution of MS2-YFP for the same cells (top panel). All size bars represent 10 μm.</p

Differences to CRM1- and NXF1-dependent HIV-1 gRNA trafficking in the cytoplasm.

<p><b>(A)</b> Depiction of RRE (Rev/CRM1) or 4xCTE (NXF1/NXT1)-dependent surrogate gRNA transcripts. <b>(B)</b> VLP assembly assay as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005565#ppat.1005565.g001" target="_blank">Fig 1B</a> showing Gag-CFP expression and virus particle assembly for either RRE- or 4xCTE-gRNA transcripts expressed in the presence or absence of Rev-mCherry. <b>(C)</b> Whisker plot of VLP production times acquired from live cell movies encompassing a >35 hour acquisition time for Gag-CFP derived from either RRE- or 4xCTE-gRNA transcripts. <b>(D)</b> Select images from time lapse imaging over >24 hours showing MS2-YFP-tagged RRE-gRNAs moving from the nucleus to the cytoplasm over time in conjunction with the onset of Gag-CFP expression and virus particle production. White arrows indicate individual transcripts in the nucleus. Black arrows represent the transition of the MS2-YFP signal from the nucleus to the cytoplasm in a burst-like fashion. The dotted line indicates the nuclear/cytoplasmic boundary post-burst. Figure corresponds to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005565#ppat.1005565.s003" target="_blank">S1 Movie</a>. <b>(E)</b> Unlike RRE-gRNAs, MS2-YFP-tagged 4xCTE-gRNAs accumulate at a perinuclear location coincident with the onset of Gag-CFP expression (12.5 h). White arrows indicate individual transcripts in the nucleus and black arrows highlight 4xCTE-gRNA perinuclear accumulation over time. Figure corresponds to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005565#ppat.1005565.s004" target="_blank">S2 Movie</a>. Size bars represent 10 μm.</p

3-color system for studying HIV-1 gRNA trafficking.

<p><b>(A)</b> Depiction of surrogate gRNA transcripts bearing either the RRE (RRE-gRNA) or lacking an export element (ΔEE-gRNA). Transcripts retained the native viral leader sequence including the packaging signal and include the major splice donor (SD) and two splice acceptors (SAs). <b>(B)</b> Virion-like particle (VLP) assembly assay. The indicated transcripts were co-expressed in Hela.MS2-YFP cells with either mCherry alone as a control (lane 1) or Rev-mCherry (lanes 2–4). Cells and supernatants were harvested at 48 h post-transfection. Cell lysates were processed for SDS-PAGE and immunoblot detection of p24^Gag, mCherry, and HSP90 (loading control). VLPs were sedimented from supernatants by centrifugation through a sucrose cushion prior to SDS-PAGE and immunoblot detection of p24^Gag. <b>(C)</b> Representative images of HeLa.MS2-YFP cells for an experiment as for (B) showing Rev-mCherry expressed either alone (<i>i</i>.<i>-iii</i>), with ΔEE-gRNA (<i>iv</i>.<i>-vi</i>.) or RRE-gRNA transcripts (<i>vii</i>.<i>-ix</i>.). Cells were fixed at 24 h post-transfection and imaged using deconvolution fluorescence microscopy. Arrows in panel <i>v</i>. highlight nuclear punctae corresponding to nucleus-restricted ΔEE-gRNA transcripts. Arrows in panel <i>viii</i>. highlight instances of marked co-localization between MS2-YFP and Gag-CFP signals at punctae at the plasma membrane consistent with assembling virus particles. Size bar represents 10 μm. (<b>D)</b> Quantification of subcellular distribution phenotypes. HeLa.MS2-YFP cells were transfected as for B and C to express the indicated transcripts in the presence of either a mCherry control or Rev-mCherry. Cells were fixed at 36 h post-transfection and scored for instances wherein the fluorescent signal was predominantly nuclear (green bars), cytoplasmic (blue bars) or distributed equally between the nucleus and cytoplasm (red bars). Error bars represent the standard deviation of the mean for 3 independent transfections (<i>n</i> ≈ 300 cells for each condition). Panels on the right show representative phenotypes for the MS2-YFP (gRNA) channel. (<b>E)</b> Control experiment demonstrating similar subcellular distributions for Rev, gRNA, and Gag detected with or without fluorescent protein tags. Untagged Rev and Gag in the top panels were detected by indirect immunofluorescence using mouse anti-Rev or anti-Gag antisera, respectively, followed by secondary anti-mouse antibodies conjugated to AlexaFluor594. Untagged gRNAs were detected using digoxigenin-labeled RNA probes complementary to sequences within the <i>gag</i> reading frame as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005565#sec009" target="_blank">Materials and Methods</a>.</p

The RRE and CTE are both necessary and sufficient to trigger burst and MTOC-trafficking phenotypes, respectively.

<p><b>(A)</b> Depiction of heterologous, 24xMBL bearing ΔEE- and RRE-CFP transcripts and representative visual phenotypes in cells also expressing mCherry-Tubulin (red) as an MTOC marker. The MS2-YFP signal (green) was high within the nucleus for cells expressing ΔEE-CFP transcripts and RRE-CFP transcripts expressed in the absence of Rev. However, Rev triggered the relocalization of RRE-CFP transcripts to the cytoplasm. <b>(B)</b> MS2-YFP subcellular distribution for the indicated conditions as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005565#ppat.1005565.g001" target="_blank">Fig 1E</a>. <b>(C)</b> Depiction of heterologous, 24xMBL-bearing 1xCTE- and 4xCTE-CFP transcripts and representative visual phenotypes as for (A). <b>(D)</b> MTOC-targeting analysis as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005565#ppat.1005565.g004" target="_blank">Fig 4E</a> for the indicated conditions. (<b>E)</b> Selected frames from time-lapse imaging demonstrating CFP expression over time for all indicated model transcripts. Images are low magnification and show >100 cells per frame. <b>(F)</b> Quantification of CFP mean fluorescence intensity (MFI) for all conditions in (E) recorded at 24 hours. All size bars represent 10 μm.</p

CTE-gRNAs accumulate at centrosomes during mitosis and are subsequently partitioned to daughter cells.

<p><b>(A)</b> 4xCTE-gRNAs are partitioned to daughter cells via centrosomes during cell division. A representative HeLa.MS2-YFP cell expressing 4xCTE-gRNAs and tracked through metaphase, anaphase, and telophase. Yellow arrows indicate the enriched MS2-YFP signal (green) at the spindle poles (mCherry-tubulin in red) during metaphase (Hoechst DNA stain in blue). Figure corresponds to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005565#ppat.1005565.s007" target="_blank">S5 Movie</a>. <b>(B)</b> Unlike 4xCTE-gRNAs, RRE-gRNAs do not accumulate at the mitotic spindle. Imaging as for (A) but for cells co-expressing RRE-gRNAs and Rev. <b>(C,D)</b> 4xCTE-gRNAs (MS2-YFP; green) co-localize with the centrosome marker Pericentrin (C, white) and also NXF1 (D, white) at the poles of the spindle (shown using mCherry-tubulin, red). Pericentrin and NXF1 were detected by indirect immunofluorescence. For all panels, yellow arrows highlight 4xCTE-gRNA enrichment at the centrosome. Size bars represent 10 μm.</p

Cooperativity among Rev-associated nuclear export signals regulates HIV-1 gene expression and is a determinant of virus species tropism

Murine cells exhibit a profound block to HIV-1 virion production that was recently mapped to a species-specific structural attribute of the murine version of the chromosomal region maintenance 1 (mCRM1) nuclear export receptor and rescued by the expression of human CRM1 (hCRM1). In human cells, the HIV-1 Rev protein recruits hCRM1 to intron-containing viral mRNAs encoding the Rev response element (RRE), thereby facilitating viral late gene expression. Here we exploited murine 3T3 fibroblasts as a gain-of-function system to study hCRM1's species-specific role in regulating Rev's effector functions. We show that Rev is rapidly exported from the nucleus by mCRM1 despite only weak contributions to HIV-1's posttranscriptional stages. Indeed, Rev preferentially accumulates in the cytoplasm of murine 3T3 cells with or without hCRM1 expression, in contrast to human HeLa cells, where Rev exhibits striking en masse transitions between the nuclear and cytoplasmic compartments. Efforts to bias Rev's trafficking either into or out of the nucleus revealed that Rev encoding a second CRM1 binding domain (Rev-2xNES) or Rev-dependent viral gag-pol mRNAs bearing tandem RREs (GP-2xRRE), rescue virus particle production in murine cells even in the absence of hCRM1. Combined, these results suggest a model wherein Rev-associated nuclear export signals cooperate to regulate the number or quality of CRM1's interactions with viral Rev/RRE ribonucleoprotein complexes in the nucleus. This mechanism regulates CRM1-dependent viral gene expression and is a determinant of HIV-1's capacity to produce virions in nonhuman cell types. IMPORTANCE Cells derived from mice and other nonhuman species exhibit profound blocks to HIV-1 replication. Here we elucidate a block to HIV-1 gene expression attributable to the murine version of the CRM1 (mCRM1) nuclear export receptor. In human cells, hCRM1 regulates the nuclear export of viral intron-containing mRNAs through the activity of the viral Rev adapter protein that forms a multimeric complex on these mRNAs prior to recruiting hCRM1. We demonstrate that Rev-dependent gene expression is poor in murine cells despite the finding that, surprisingly, the bulk of Rev interacts efficiently with mCRM1 and is rapidly exported from the nucleus. Instead, we map the mCRM1 defect to the apparent inability of this factor to engage Rev multimers in the context of large viral Rev/RNA ribonucleoprotein complexes. These findings shed new light on HIV-1 gene regulation and could inform the development of novel antiviral strategies that target viral gene expression