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. Author manuscript; available in PMC: 2014 Feb 1.
Published in final edited form as: Virus Res. 2012 Oct 2;171(2):304–318. doi: 10.1016/j.virusres.2012.09.011

NC-Mediated Nucleolar Localization of Retroviral Gag Proteins

Timothy L Lochmann 1, Darrin V Bann 1, Eileen P Ryan 2, Andrea R Beyer 2, Annie Mao 3, Alan Cochrane 3, Leslie J Parent 1,2,✉
PMCID: PMC3578147  NIHMSID: NIHMS418906  PMID: 23036987

Abstract

The assembly and release of retrovirus particles from the cell membrane is directed by the Gag polyprotein. The Gag protein of Rous sarcoma virus (RSV) traffics through the nucleus prior to plasma membrane localization. We previously reported that nuclear localization of RSV Gag is linked to efficient packaging of viral genomic RNA, however the intranuclear activities of RSV Gag are not well understood. To gain insight into the properties of the RSV Gag protein within the nucleus, we examined the subnuclear localization and dynamic trafficking of RSV Gag. Restriction of RSV Gag to the nucleus by mutating its nuclear export signal (NES) in the p10 domain or interfering with CRM1-mediated nuclear export of Gag by leptomycin B (LMB) treatment led to the accumulation of Gag in nucleoli and discrete nucleoplasmic foci. Retention of RSV Gag in nucleoli was reduced with cis-expression of the 5′ untranslated RU5 region of the viral RNA genome, suggesting the psi (ψ packaging signal may alter the subnuclear localization of Gag. Fluorescence recovery after photobleaching (FRAP) demonstrated that the nucleolar fraction of Gag was highly mobile, indicating that the there was rapid exchange with Gag proteins in the nucleoplasm. RSV Gag is targeted to nucleoli by a nucleolar localization signal (NoLS) in the NC domain, and similarly, the human immunodeficiency virus type 1 (HIV-1) NC protein also contains an NoLS consisting of basic residues. Interestingly, co-expression of HIV-1 NC or Rev with HIV-1 Gag resulted in accumulation of Gag in nucleoli. Moreover, a subpopulation of HIV-1 Gag was detected in the nucleoli of HeLa cells stably expressing the entire HIV-1 genome in a Rev-dependent fashion. These findings suggest that the RSV and HIV-1 Gag proteins undergo nucleolar trafficking in the setting of viral infection.

Introduction

Nucleoli are distinct subnuclear compartments historically known for their central role in ribosome biogenesis (Boisvert et al., 2007; Cisterna and Biggiogera, 2010). The nucleolus contains three regions based on their appearance by electron microscopy: the fibrillar center (FC), where rRNA transcription occurs; the dense fibrillar component (DFC) surrounding the FC, where rRNAs are processed and ribosomal proteins are localized; and the outermost layer, the granular component (GC), which is the site of further rRNA processing and ribosomal subunit assembly. Accordingly, the nucleolus contains many factors involved in RNA metabolism and ribonucleoprotein complex formation. In addition, proteomic analyses identified more than 700 proteins in nucleoli (Andersen et al., 2005; Andersen et al., 2002), including factors involved in DNA replication and repair, pre-mRNA processing, stress responses, posttranslational modification (sumoylation and ubiquitination), apoptosis, and cell-cycle control (Boisvert et al., 2007). Through these studies, it has become apparent that the nucleolus is a key regulator of critical cellular processes in addition to ribosomal subunit assembly.

Over 100 viral proteins encoded by greater than 50 distinct viruses have been reported to localize to nucleoli, prompting the nucleolus to be coined the “gateway to viral infection” (Hiscox, 2002; Matthews et al., 2011). Some viruses disrupt nucleolar architecture [e.g., herpes simplex virus type 1; (Bertrand and Pearson, 2008; Lopez et al., 2008; Lymberopoulos and Pearson, 2007; Xing et al., 2010)], recruit nucleolar proteins to virus replication centers [e.g., adenovirus (Hindley et al., 2007; Lawrence et al., 2006; Okuwaki et al., 2001)], or re-localize nucleolar proteins to impair cellular metabolism [e.g., poliovirus (Waggoner and Sarnow, 1998)]. Other viruses, such as poliovirus, arterivirus, and human immunodeficiency virus type 1 (HIV-1), disrupt nontraditional nucleolar functions and deregulate the cell cycle to enhance virus replication (Miyazaki et al., 1996; Waggoner and Sarnow, 1998; Yoo et al., 2003).

Many viral proteins that localize to nucleoli are capsid and nucleocapsid proteins that bind to viral genomes and direct virus particle assembly (Matthews et al., 2011). Most of these proteins bind RNA, so nucleolar localization has been assumed to be a consequence of the high concentration of RNA in nucleoli. However, the amount of mRNA in the nucleoplasm is greater than within the nucleolus (Amano, 1967), and most proteins that exhibit nucleolar localization are actively retained due to interactions with cellular proteins that reside there (Westman et al., 2010). The nucleolar localization of viral proteins has been functionally linked to viral ribonucloprotein complex formation, viral RNA export, genome packaging, genome replication, capsid assembly, and pathogenesis. In the case of the influenza A virus NP protein, nucleolar localization is required for the replication and packaging of the influenza NP ribonucloprotein complex (Ozawa et al., 2007). For herpesvirus saimiri, nucleolar trafficking of ORF57 is required for the nuclear export of intronless viral RNA (Boyne and Whitehouse, 2006). Deletion of the nucleolar retention signal (NoRS) in ORF57 abrogates nuclear export of viral RNA, whereas addition of a heterologous NoLS restores RNA export. These examples demonstrate that both RNA and DNA viruses commandeer nucleoli as sites of viral protein-RNA interaction that facilitate RNA export and packaging.

Among retroviruses, both viral proteins and unspliced viral RNAs interact with nucleoli and nucleolar proteins. The nucleolus plays a key role in the function of the HIV-1 Rev protein, which mediates the nuclear export of unspliced viral RNA (Cochrane et al., 1990; Perkins et al., 1989). The nucleolus is the site of Rev dimerization, which is required for its activity (Daelemans et al., 2004). Rev localizes to the nucleolus through its interaction with the cellular protein B23. When unspliced HIV-1 RNA containing the Rev response element (RRE) RNA binds to Rev, B23 is displaced, and Rev moves into the nucleoplasm (Fankhauser et al., 1991). Further evidence that the nucleolus plays an important role in HIV-1 biology is provided by the finding that linking a ribozyme to a small nucleolar RNA interferes with HIV-1 replication, suggesting that the ribozyme cleaves HIV-1 RNA as it traffics through the nucleolus (Michienzi et al., 2000). Moreover, the nucleolar protein nucleolin enhances HIV-1 assembly, but only when an RNA containing the psi packaging sequence is expressed (Ueno et al., 2004). In the case of human T cell lymphotropic virus type I (HTLV-I), the Rex, p21, pX, p30, p30II, and HBZ-SP1 proteins localize to the nucleolus, as does the unspliced viral RNA (Bartoe et al., 2000; Ghorbel et al., 2006; Hivin et al., 2007; Kubota et al., 1996; Kubota et al., 1989; Nosaka et al., 1989; Siomi et al., 1988). Taken together, these studies suggest that nucleoli may serve as a site of interaction between retroviral proteins and unspliced viral RNA.

Previously, we demonstrated a link between nuclear trafficking of the avian oncoretrovirus Rous sarcoma virus (RSV) Gag protein and genomic RNA (gRNA) packaging (Garbitt-Hirst et al., 2009; Scheifele et al., 2002). Viral mutants bearing Gag proteins that bypass the nucleus package gRNA less efficiently than the wild-type virus. Insertion of a heterologous nuclear localization sequence (NLS) restores both nuclear trafficking and gRNA packaging (Garbitt-Hirst et al., 2009). In addition to RSV Gag-RNA binding, Gag-Gag interactions occur in the nucleus in an RNA-dependent manner (Kenney et al., 2008). Here we report that a subset of RSV Gag proteins in the nucleus localize to nucleoli when the NES in p10 is mutated or the CRM1 nuclear export pathway in inhibited with LMB. The nucleolar population of RSV Gag is highly mobile, trafficking rapidly between the nucleolus and the nucleoplasm, with biophysical properties similar to endogenous nucleolar proteins. Nucleolar retention of RSV Gag was reduced in the presence of the ψ packaging signal, suggesting that the intranuclear Gag-ψ RNA interaction influences RSV Gag localization. Additionally, HIV-1 Gag was detected within nucleoli upon co-expression of either HIV-1 NC or HIV-1 Rev and also in the context of an integrated provirus in which Gag expression was Rev-dependent. These findings provide evidence that the Gag polyproteins of HIV-1 and RSV traffic through the nucleolus during the virus replication cycle.

Materials and Methods

Expression vectors and cells

The proviral vectors derived from RSV Prague C, pRC.V8 and pRC.L219A, and the plasmids pGag-GFP/CFP, p219A.Gag-GFP/YFP, pNES-A.Gag-GFP/YFP, pYFP-RSV.NC have been described (Butterfield-Gerson et al., 2006; Craven et al., 1995; Kenney et al., 2008; Parent et al., 2000; Scheifele et al., 2007). Rev-independent pHIV.Gag-CFP was created using a HindIII-BamHI digest from pHIV.Gag-GFP (a kind gift from Marilyn Resh, Memorial Sloan-Kettering Cancer Center, New York) (Hermida-Matsumoto and Resh, 2000). To construct pKoz.L219A.Gag-YFP, a primer containing the 12 nucleotides of the Kozak sequence immediately upstream of the Gag initiation codon was used for PCR amplification with an antisense primer in the CA coding region. The resulting fragment was digested with NheI-ScaI and inserted into the pL219A.Gag-YFP vector. pRU5.L219A.Gag-YFP was constructed in a similiar fashion, with the upstream PCR primer beginning at nucleotide 1 of the RSV 5′ untranslated region. pYFP-HIV.NC was created using PCR amplification of the HXB2 HIV-1 NC coding region followed by insertion into the pYFP-N1 vector using HindIII-BamHI. The plasmid pMLV.NC-YFP was constructed by PCR amplification of the MLV NC coding region of MLV Gag from pRR88 (Gorelick et al., 1988) and inserted into pYFP-N1 using HindIII-BamHI. The NC domain of MMTV Gag (1486–1767, sequence accession number AF228552 for MMTV(C3H) provirus) was PCR amplified, digested with BglII, treated with Klenow, digested with BamHI and ligated into pEGFP.N2 to produce pMMTV.NC-GFP. The plasmids pFibrillarin-YFP and pB23-CFP were made from pFibrillarin-GFP and pB23-GFP (gifts from Mark Olson, University of Mississippi Medical Center) (Dundr et al., 2000) by fragment exchange of the fluorophore coding regions using BamHI-NotI. The sequences of PCR primers are available upon request. All plasmids were sequenced through the coding regions and were found to be faithful copies of the corresponding genes.

Cell lines used in these experiments were quail fibroblasts (QT6), chicken fibroblasts (DF1), mouse fibroblasts (3T3), mouse mammary cells (NMuMG), and human cervical cancer cells (HeLa) (Azad et al., 1993; Ben-Av and Liscovitch, 1989; Himly et al., 1998; Salmivirta et al., 1992). Transfections were performed using the calcium phosphate method for QT6 cells (Fujiwara et al., 1988), FuGene (Roche) for DF-1 cells, or Lipofectamine 2000 (Invitrogen) for HeLa, 3T3, and NMuMG cells. The HeLa HIVΔmls rtTA cell line contains an integrated HIV-1 provirus whose expression is dependent upon addition of doxycycline to the culture media, as described previously (Wong et al., 2011). The HeLa HIV.Gag-GFP rtTA cell line was generated by replacing the PR and RT portions of Pol with GFP in frame with Gag to express a Gag-GFP fusion protein. As with the HeLa HIVΔmls rtTA cell line, expression of the integrated HIV provirus is dependent upon the presence of doxycycline in the media. In both cell lines, expression of Gag is completely dependent on Rev expression (Wong et al., 2011).

Immunofluorescence and live cell imaging using laser scanning confocal microscopy

Coverslips were seeded with QT6 or HeLa cells that were infected or transfected, fixed, permeabilized using a 3:1 methanol:acetone mixture at −20°C (Scheifele et al., 2002), blocked with 5% goat serum, incubated with polyclonal anti-RSV (Craven et al., 1995), washed, stained with either a Cy5 (AbCam) or FITC (Sigma) conjugated anti-rabbit IgG secondary antibodies, and mounted with SlowFade (Invitrogen). Cells were either imaged live or were fixed in 4% paraformaldehyde with no detectable differences in subcellular localization of the fluorophore-tagged proteins. For live cell imaging experiments, cells were seeded onto 35-mm glass-bottomed dishes (MatTek Corporation), transfected as above, and imaged by confocal microscopy at 14 to 24 h post-transfection. Imaging was performed using a Leica AOBS SP2 confocal microscope using sequential scanning under conditions in which there was no crosstalk between fluorophores. Sequential scanning settings were used to differentiate the emission spectra of: CFP (excitation at 458 nm, emission at 465–490 nm, 50% laser power) and YFP (excitation 514 nm, emission 530–600 nm, 10% laser power); CFP and Cy5 (excitation 633 nm, emission 645–750 nm, 100% laser power); GFP (excitation 488 nm, emission 495–540 nm, 35% laser power) and Texas Red (excitation 543, emission 600–700, 50% laser power); or FITC (excitation 488, emission 501–552, 30% laser power) and Texas Red (excitation 543, emission 600–654, 50% laser power). Sequential single channel images using FITC (excitation at 488 nm, emission at 500–535 nm, 25% laser power) emission spectra were also captured as indicated in the Figure Legends. Differential interphase contrast (DIC) images were obtained using transmitted light.

For analysis of HIV-1 Gag or HIV-1 Gag-GFP localization, provirus expression was induced in HeLa cells by addition of doxycycline (2 μg/ml) to media for 24 h. Cells were subsequently fixed by addition of 4% paraformaldehyde in PBS for 30 min. then permeabiilzed by treating with 1% Triton X-100 in PBS for 5 min. For HIV-1 Gag-GFP analysis, cells were blocked with 3% BSA in PBS and nucleoli were stained with rabbit anti-B23 (a kind gift from L. Frappier, University of Toronto) and Texas Red anti-rabbit antibody (Jackson Immunoresearch). For HIV-1 Gag imaging, cells were blocked with 3% BSA in PBS then incubated with mouse anti-p24(CA) antibody (183, a kind gift from M. Tremblay, Laval University) and rabbit anti-hEBP2 (a kind gift from L. Frappier, University of Toronto) followed by staining with Texas Red anti-rabbit antibody (Jackson Immunoresearch). To quantify the amount of HIV-1 Gag in nucleoli, single optical slices through the largest diameter of the nucleus were chosen for analysis. Nucleoli were identified by staining for B23, and the amount of Gag or Gag-GFP signal present in the nucleoli compared to the entire cell was calculated using the “measure” tool in ImageJ (Rasband, 1997–2012).

Fluorescence Recovery after Photobleaching (FRAP) analysis

FRAP was performed on live cells transfected with either pL219A.Gag-YFP or pGag-YFP. All assays were performed at a scan speed of 800Hz with an acquisition time of 0.8 seconds. Five pre-bleach images were acquired using the YFP channel settings at 10% laser power. The YFP signal in nucleoli (L219A.Gag-YFP) or at the membrane (Gag-YFP) was specifically photobleached using the 514nm laser at 100% power over four time-points. Recovery was monitored for approximately 120 seconds at 10% laser power. As a control for bleaching and acquisition fluorescence loss, fluorescence of the entire cell was monitored during the experiment. The background from both the region of interest and the total cell fluorescence was subtracted. Correction for photobleaching and normalization of the data was performed as previously described (Phair and Misteli, 2000). Briefly, the relative fluorescence intensity was calculated by IRel = T0It/TtI0, where T0 is the total cell intensity before bleaching, Tt is the total cell intensity at time-point t, I0 is the intensity of the region of interest before bleaching, and It is the intensity of the region of interest at time-point t. The mobile fraction of each region was determined using Fm = IE − I0/II − I0, where IE is the fluorescence in the bleached region after full recovery, I0 is the fluorescence immediately after bleaching, and II is the pre-bleach fluorescence. Statistical analysis was performed using Prism (Graphpad Software) with a two-tailed Student’s t test performed to compare mobile fractions in the nucleolus and at the plasma membrane. FRAP analysis was performed using separate regions of interest from at least 8 individual cells and the data were analyzed using the Leica SP2 software package.

Fluorescence Resonance Energy Transfer (FRET) measurements

Acceptor photobleaching FRET was performed on live cells at 14 to 24 h post-transfection as previously described (Kenney et al., 2008). Pre-bleach images of CFP (excitation at 458nm, emission at 460–496nm, 50% laser power) and YFP (excitation at 514nm, emission at 538–600nm, 10% laser power) were acquired. Using the 514nm laser at 100% power, YFP was specifically photobleached until fluorescence intensity was reduced to 10% of pre-bleach levels. Post-bleach images were acquired for both the CFP and YFP channels. FRET efficiency was calculated using the formula FRETEff = donorPost − donorPre/donorPost, when donorPost is greater than donorPre. FRET analysis was performed on at least two different sets of transfected cells using a minimum of 10 different cells per transfection. Nucleolar FRET was performed by bleaching the entire nucleus through a single optical slice of the nuclear plane. After photobleaching, individual nucleoli in each cell were selected as regions of interest for FRET measurement and data were analyzed using the Leica software package accompanying the confocal microscope.

Results

Subnuclear localization of the nuclear-restricted RSV Gag polyprotein

Our previous studies revealed that RSV Gag-Gag interactions occur within the nucleus (Kenney et al., 2008). Restriction of Gag to the nucleus by treating cells with LMB to inhibit nuclear export through the CRM1 pathway or by mutating critical hydrophobic residues in the Gag p10 NES (Fig. 1A) results in Gag accumulation within discrete subnuclear foci in a subset of cells. The formation of Gag intranuclear foci was dependent on the NC domain and could not be complemented by an RNA-independent protein-protein interaction domain. Further examination of those cells containing Gag nuclear puncta revealed that Gag proteins were frequently observed in structures that resembled nucleoli. To formally test this possibility, we used fluorescent confocal microscopy to image cells expressing wild-type Gag-YFP and treated with LMB, or cells expressing Gag mutants impaired in nuclear export (Koz.L219A.Gag-YFP or Koz.NES-A.Gag-YFP) (Fig. 1B). DIC images were acquired simultaneously to identify nucleoli (white arrows), as described previously (Li et al., 1996; Speil and Kubitscheck, 2010; Zhang et al., 2009). The number of cells in which Gag formed intranuclear foci varied among different experiments, ranging from 20–70% of transfected cells. Within the subset of cells containing intranuclear Gag foci, punctate spots were present in the nucleoplasm in ~30% of cells (left panels). In the remaining cells (~70%), Gag accumulated within nucleoli (right panels).

FIG. 1.

FIG. 1

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Subnuclear localization of the nuclear-restricted RSV Gag Protein. (A) Schematic diagram of the RSV Gag nuclear export sequence (NES) mutants used in this study with amino acids delineated; MA, matrix; CA, capsid; NC, nucleocapsid; (B) Images show either punctate or nucleolar localization of either NES mutant or wildtype RSV Gag treated with leptomycin B (LMB). Both YFP and DIC images are shown with white arrows pointing to nucleoli. Differential interference contrast (DIC) images were adjusted for brightness and contrast to improve clarity using CorelDraw.

Evidence for transient nucleolar localization of the wild-type RSV Gag protein

Based on our previous findings that the wild-type and NES-mutant (L219A.Gag; Fig. 1A) Gag proteins interact within the nucleus (Kenney et al., 2008), we tested whether Gag-Gag interactions also occurred in the nucleolus. If so, then the NES-mutant Gag protein might act as a “trap” for the wild-type Gag protein, resulting in accumulation of the wild-type protein within nucleoli. We co-expressed wild-type RSV.Gag-CFP with L219A.Gag-YFP and found that wild-type Gag was indeed retained in nucleoli (Fig. 2A). By contrast, RSV.Gag-CFP did not relocalize to nucleoli with co-expression of fibrillarin-YFP, another nucleolar-localized protein, demonstrating specificity of the effect (Fig. 2B). These data suggest that wild-type Gag transiently traffics through the nucleolus, although nucleolar localization only becomes evident with co-expression of an interacting nucleolar protein, in this case L219A.Gag (Fig. 2A), or with LMB treatment (Fig. 1).

FIG. 2.

FIG. 2

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Nucleolar localization of the wild-type RSV Gag protein under different conditions. Cells co-expressing wild-type RSV Gag-CFP and L219A.Gag-YFP (A) or RSV Gag-CFP and Fibrillarin-YFP (B) were imaged by confocal microscopy. RSV Gag-CFP was partially relocalized to nucleoli (white arrowheads) when co-expressed with L219A.Gag-YFP, but not Fibrillarin-YFP. Transfected QT6 cells expressing the wild-type proviral construct pRC.V8 treated with LMB (C) or the pRC.L219A mutant provirus (D) were immunostained with anti-RSV antibody to detect the Gag protein, with arrowheads indicating nucleoli. (E) Immunofluorescence of DF1 cells chronically infected with wild-type RSV (RC.V8) and treated with LMB, with nucleoli indicated by white arrowheads. For each panel, cells were cotransfected with fibrillarin-CFP or imaged with DIC (data not shown) to identify nucleoli.

Next, we examined whether the RSV Gag protein could be detected in nucleoli when expressed within the context of a replication-competent proviral vector or during authentic virus infection. To test these possibilities, cells were co-transfected with the proviral vector pRC.V8 and fibrillarin-CFP (as a nucleolar marker), treated with LMB, fixed, and immunostained with an anti-RSV antibody to detect Gag (Craven et al., 1995; Scheifele et al., 2002; Weldon et al., 1990). RSV Gag was found within nucleoli in a subset of LMB-treated cells expressing the wild-type provirus (Fig. 2C, white arrowheads). Additionally, in cells expressing a proviral construct bearing the Gag.L219A NES mutant (RC.L219A), we observed a subpopulation of Gag within nucleoli (Fig. 2D). Finally, in cells infected with RSV and treated with LMB, wild-type Gag was present within nucleoli in approximately 5–10% of cells (Fig. 2E). In general, fewer cells exhibited a nucleolar subpopulation of Gag in cells treated with LMB compared to cells expressing RC.Gag.L219A. We presume that this difference is due to the short period of LMB treatment (1.5 hours), which provides only a limited amount of time for free Gag proteins to enter the nucleus and accumulate in nucleoli. By contrast, cells transfected with pRC.Gag.L219A express the mutant protein for ~18 hours prior to imaging, allowing a much longer time for Gag proteins to be retained in nucleoli.

Rapid exchange of RSV Gag between the nucleolus and nucleoplasm

Most nucleolar proteins in the cell move rapidly between the nucleolus and nucleoplasm depending on the availability of binding factors that alter their subnuclear distribution (Boulon et al., 2004; Emmott et al., 2008; Emmott and Hiscox, 2009; Ernoult-Lange et al., 2009; Leung and Lamond, 2003; Pradet-Balade et al., 2011; Verheggen and Bertrand, 2012; You et al., 2008). To determine whether RSV Gag behaves in a similar fashion as cellular nucleolar shuttling proteins, fluorescence recovery after photobleaching (FRAP) was performed by photobleaching an individual nucleolus containing fluorescence signal derived from RSV L219A.Gag-GFP and measuring the rate of fluorescence recovery (Fig. 3A, top). Five pre-bleach images were taken with 0.8 seconds between each frame (a representative frame is shown in the left panel). A single nucleolus (center panel, solid arrowhead) was precisely photobleached using the 514nm laser at 100% intensity for four frames, and fluorescence recovery within the nucleolar region was measured from 0–122.613 s (top center and right panels). The recovery of fluorescence in the bleached nucleolus was clearly visible by the end of the time-course (t=122.613 s, top right panel, solid arrowhead). As expected, the unbleached nucleolus (unfilled arrowhead) demonstrated a slight reduction in fluorescence over time, which was due to photobleaching during image acquisition (compare top left and right panels). To control for this nonspecific bleaching during imaging, the same amount of signal loss was subtracted from both nucleoli during the quantitative analysis of the data (Phair and Misteli, 2000).

FIG. 3.

FIG. 3

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Mobility of RSV Gag proteins in nucleoli assessed by fluorescence recovery after photobleaching (FRAP) analysis. (A) Images of QT6 cells expressing RSV L219A.Gag-GFP (top) demonstrate fluorescence in the nucleolus (indicated by arrowheads) prior to photobleaching (Pre-Bleach). The nucleolus indicated by the filled arrowhead was specifically bleached with high-intensity laser (100% laser power at 514 nm wavelength) to reduce YFP fluorescence to ~40% of baseline levels (center image, t=0s; time 0 seconds). Post-bleach images were acquired for a total of 122.613 seconds, with recovery of fluorescence shown in the top right panel (t=122.613s). The open arrowhead indicates a nucleolus that was not subjected to photobleaching, but its fluorescence was monitored to correct for fluorescence lost from image acquisition. FRAP analysis was also performed in cells expressing RSV Gag-YFP, with the region of interest at the plasma membrane indicated by a white open rectangle (bottom). The experiment was performed in the same fashion as the images in the top panels, and loss of fluorescence from image acquisition was monitored. (B) Quantitative analysis of relative fluorescence (y-axis) in the nucleolus (solid line) and at the plasma membrane (dashed line) over time was performed as described in Materials and Methods. The point of maximum photobleaching was set as time 0 (x-axis). A representative experiment is shown. (C) Average mobile fractions (Fm) of L219A.Gag-YFP in the nucleolus and Gag-YFP at the plasma membrane were calculated from 8 individual cells over 2–3 separate transfections. Error bars represent the standard error of the mean.

Overall, a decrease in nucleolar fluorescence to ~40% of the initial level was observed in the bleached nucleolus, followed by recovery of fluorescence to the plateau level by 25 s (Fig. 3B). The half-time of recovery for RSV L219A.Gag-GFP was calculated to be 18.4 s, which is in the same range or faster than recovery times reported for other viral and cellular proteins that transit between the nucleolus and nucleoplasm via active mechanisms (Sansam et al., 2003; You et al., 2008). The rapid recovery of fluorescence of the bleached nucleolus indicated that L219A.Gag-GFP cycled rapidly from an unbleached region of the nucleoplasm into the nucleolus.

By contrast, when cells expressing RSV.Gag-YFP were bleached in the region of interest corresponding to the plasma membrane (Fig. 3A, lower set of panels, white rectangles), there was little to no recovery of fluorescence even at the end of the 122.613 s imaging period. Thus, as expected, the fraction of RSV Gag-YFP that was bound to the plasma membrane had very little mobility (Fig. 3B) because these fluorescent membrane puncta represent nascent virus-like particles (Gomez and Hope, 2006; Larson et al., 2005).

The fluorescence recovery of the nucleolar fraction of RSV L219A.Gag-YFP was measured and compared to the plasma membrane fraction of RSV Gag-YFP for 8 individual cells and the average mobile fraction was determined (Fig. 3C). The fraction of L219A.Gag-YFP in nucleoli was highly mobile (72.1%), whereas Gag-YFP at the membrane was much less so (19.6%) (p ≤ 0.001). Therefore, L219A.Gag-YFP within nucleoli appears to be a highly dynamic protein that moves between nucleoli and the nucleoplasm, possibly facilitated by an interaction with a protein or RNA partner in the nucleolus.

Expression of RSV 5′ untranslated region (UTR) alters the nucleolar accumulation of Gag

Based on data demonstrating that complexes of HIV-1 Rev and the cis-acting Rev-response element in unspliced viral RNA traffic through the nucleolus [reviewed in (Arizala and Rossi, 2011)], we tested the idea that the 5′ noncoding region of RSV RNA, which contains the ψ packaging region selectively bound by the NC domain of Gag, might alter the subnuclear distribution of Gag. To pursue this line of investigation, we designed two L219A.Gag-YFP vectors bearing different 5′ noncoding sequences upstream of the Gag initiation codon (Fig. 4A). pRU5.L219A.Gag-YFP contains the entire 5′ untranslated region (the R and U5 sequences) from the RS.V8 proviral clone (Fig. 4A, top) whereas pKoz.L219A.Gag-YFP contains the RSV Kozak sequence and includes only the 12 nucleotides immediately preceding the translational start site for Gag (Fig. 4A, bottom).

FIG. 4.

FIG. 4

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Dependency of RSV Gag nucleolar accumulation on upstream untranslated RNA sequences. (A) Schematic diagram of L219A.Gag-YFP expression constructs. pRU5.L219A.Gag-YFP contains the entire 5′UTR of RSV (nt 1–379) upstream of the Gag translation initiation site (nt 380). pKoz.L219A.Gag-YFP contains only the 12 nucleotides of the 5′UTR (nt 368–379) immediately upstream of the Gag start site. Thin black lines represent RNA sequences present prior to the start of Gag translation. Thick lines represent the translated proteins, L219A.Gag (dark gray) or YFP (green). The ψ-sequence (Aψ) (Aronoff et al., 1993) is represented by the dashed bracket (nt 126–395). Nucleotides numbers are derived from GenBank accession number J02342; beginning of R region in LTR is nucleotide 1. (B) QT6 cells co-expressing Koz.L219A.Gag-YFP and fibrillarin-CFP (as a nucleolar marker) were imaged using confocal microscopy. Cells displaying Gag nuclear foci were analyzed for nucleolar accumulation. Both nucleolar (top) and nucleaoplasmic foci (bottom) phenotypes are shown. The percentage of each phenotype represented in the cell population is indicated to the right of each row of images. (C) QT6 cells co-expressing RU5.L219A.Gag-YFP and fibrillarin-CFP were analyzed as in part A. (D) Cells co-expressing either Koz.L219A.Gag-YFP or RU5.L219A.Gag-YFP and B23-YFP were imaged using confocal microscopy to demonstrate regions of co-localization (yellow).

QT6 cells were transfected with either pKoz.L219A.Gag-YFP or pRU5.L219A.Gag-YFP and confocal microscopy images were acquired (Fig. 4B, C, D). The nucleolar protein fibrillarin-CFP served as a marker for the nucleolar DFC and GC regions, whereas the B23-CFP fusion protein was expressed to highlight the GC region (Lam et al., 2005). Interestingly, Gag was localized primarily to the GC, just outside of fibrillarin-CFP, and within distinct foci that overlapped partially with the B23 signal (Fig. 4C, D). Because the CG contains specialized sites where non-ribosome related activities take place (Politz et al., 2002; Politz et al., 2005), higher resolution examination of the subnucleolar localization of RSV Gag is warranted in future studies.

Transfected cells that contained nuclear RSV Gag foci were scored for the presence of nucleolar Gag-YFP fluorescence signal, with approximately 25 individual cells analyzed for each construct. In cells expressing Koz.L219A.Gag-YFP (lacking the ψ sequence), the protein was localized to nucleoli in 70% of cells (Fig. 4B). By contrast, in cells expressing RU5.L219A.Gag-YFP containing the entire 5′ noncoding sequence, including ψ, Gag was nucleolar-localized in only 46% of cells, with the majority of cells containing nucleoplasmic foci (Fig. 4C). These results indicate that the RNA sequence present in the 5′UTR altered the subnuclear localization of RSV Gag, decreasing its retention in nucleoli. One plausible explanation for this result is that like HIV-1 Rev, RSV Gag may associate with a factor that retains it in the nucleolus; when the ψ RNA packaging element is present, the viral RNA may bind to Gag with higher affinity and displace Gag from its nucleolar binding partner. Alternatively, it is also possible that the decrease in nucleolar localization of RU5.L219A.Gag occurs because RSV Gag binds the ψ sequence in the nucleoplasm, retaining the viral ribonucleoprotein complex outside the nucleolus. Further experimentation will be needed to distinguish between these possibilities. Nonetheless, we can conclude that expressing L219A.Gag along with the upstream RNA sequences containing ψ results in reduced nucleolar localization of Gag.

The NC domain of Gag contains nucleolar localization properties

The mechanism by which proteins localize to nucleoli typically requires the presence of an intrinsic nucleolar localization signal (NoLS) and may result from the protein binding to a resident component of the nucleolus (nucleic acid or protein) that retains the protein in the nucleolus [reviewed in (Emmott and Hiscox, 2009; Pederson and Tsai, 2009; Scott et al., 2010)]. Most NoLSs contain basic residues, although a true NoLS consensus sequence has not been derived (Scott et al., 2010). A recently developed computational tool for predicting NoLSs (Scott et al., 2010) identified two putative NoLSs in the NC domain of RSV Gag: one between residues 28–51 and the other between residues 54–73 (Fig. 5A, black boxes). This prediction agrees with our previous report showing that the RSV NC domain alone is capable of localizing to the nucleus and accumulating in nucleoli when fused to GFP (Butterfield-Gerson et al., 2006).

FIG. 5.

FIG. 5

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Nucleolar localization signals in retroviral NC proteins. (A) Amino acid sequence comparison of NC proteins used in the cell expression studies in panel C. Cys-His boxes are shaded gray and the predicted NoLSs are outlined with black boxes with a consensus sequence shown at the bottom. (B) QT6 cells expressing RSV Gag.ΔNC-YFP and fibrillarin-YFP were imaged in the absence (−) or presence (+) of LMB. (C) Nucleolar localization of YFP or GFP fused to NC proteins derived from RSV, HIV, MMTV, and MLV Gag. Top panels show localization of various NC proteins in QT6 cells. Bottom panels show localization in different cell types, including HeLa (HIV NC), NMuMG (MMTV NC), or 3T3 (MLV NC) cells.

To determine whether the NC sequence was required for nucleolar localization of Gag, we examined the distribution of a mutant bearing a deletion of the NC domain (Fig. 5B). GagΔNC-YFP was co-expressed with fibrillarin-CFP in cells with or without LMB treatment, and its subcellular localization was analyzed by confocal microscopy. As previously reported, GagΔNC-YFP was diffuse throughout the cytoplasm in untreated cells (-LMB images) (Kenney et al., 2008). With LMB treatment, there was no RSV GagΔNC-YFP signal observed in nucleoli (Fig. 5B, +LMB images). These data indicate that the presence of the NC domain is necessary for nucleolar localization of Gag, suggesting that an NoLS motif in NC is needed to retain RSV Gag in nucleoli.

In addition to RSV, the NC proteins of HIV and murine leukemia virus (MLV) have been reported to accumulate within nucleoli (Butterfield-Gerson et al., 2006; Gallay et al., 1995; Risco et al., 1995b; Zhang and Crumpacker, 2002). Pedicted NoLSs in each protein were identified as indicated in Fig. 5A (outlined by black boxes) (Scott et al., 2010), although none of these putative NoLSs had been experimentally verified. Moreover, it was not known whether nucleolar accumulation of NC would be cell-type specific, which would suggest that the NC protein of each retrovirus would need to be expressed in a particular host to interact with a species-specific binding partner. To address this question, the subcellular localization of RSV, HIV-1, MLV or mouse mammary tumor virus (MMTV) NC proteins fused to variants of GFP were examined by confocal microscopy. The NC protein of each retrovirus localized to nucleoli in avian (QT6) cells and in cells derived from species naturally infected by each retrovirus (Fig. 5C). The only significant cell-type difference was observed for MLV.YFP-NC, which was present in low amounts within nucleoli in QT6 cells but appeared diffuse throughout the nucleus (nucleoplasm and nucleoli) in mouse (3T3) cells. These observations indicate that nucleolar localization of the NC protein is common among retroviruses and suggests that if NC is retained in nucleoli by a binding partner, such as a nucleolar-localized RNA or protein, it is apparently conserved in chicken, mouse, and human cells.

Identification of the NoLS in the RSV NC protein

To test empirically whether the NoLS of RSV NC predicted by computational methods possessed nucleolar localization activity, we mapped the regions of RSV NC that were sufficient for nucleolar localization. The NC protein was separated into five regions divided by the Cys-His boxes (CH1 and CH2): the N-terminal (NT, aa 1–19) region, the linker (L, aa 35–46) region, and the C-terminal (CT, aa 61–92) region (Fig. 6A). Although YFP-NC is theoretically below the size threshold for passive diffusion into the nucleus, the protein was strongly localized to the nucleolus, indicating that it contains an NoLS. An internal deletion mutant removing both Cys-His motifs (YFP-NC.ΔCH1/ΔCH2) retained nucleolar localization in QT6 cells (Fig. 6B), demonstrating that the Cys-His sequences were dispensable for nucleolar localization.

FIG. 6.

FIG. 6

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Subcellular localization of RSV NC mutants. (A) Schematic diagrams of YFP-RSV.NC wild-type (YFP.RSV.NC, top) and mutant amino acid sequences (below) are shown with the N-terminal (NT), linker (L), C-terminal (CT) regions and the Cys-His box 1 (CH1) and 2 (CH2) indicated. Altered residues are depicted in bold and residues identical to WT are represented by dots. (B) Localization of YFP alone (top left) or NC mutants containing three (NC, ΔCH1/ΔCH2), one (L), or two (NT/L, L/CT) basic regions as analyzed by confocal microscopy of transfected QT6 cells. (C) Localization of NC mutants bearing the indicated amino acid substitutions were visualized via confocal microscopy in QT6 cells.

To determine whether any of the individual basic regions (NT, L, or CT) were sufficient to direct nucleolar localization, each was expressed as a fusion protein with YFP (Fig. 6B). Both YFP-NT and YFP-CT appeared diffuse throughout the nucleus and cytoplasm, like YFP alone, indicating that they did not possess any specific targeting signals (data not shown). YFP-L appeared to be concentrated in the nucleus, but was present equally in the nucleolus and nucleoplasm. This observation suggested that the L region possessed nucleolar localization activity, but the protein was not as concentrated within nucleoli as full-length NC. Thus, it was possible that the NoLS in RSV NC was a bipartite signal. Indeed, we found that when two basic regions were fused (with the intervening Cys-His motif removed; YFP-NT/L), the protein was present in the nucleolus and nucleoplasm. By contrast, YFP-L/CT was highly concentrated within nucleoli, suggesting the NoLS likely mapped to the L and CT regions of RSV NC, as predicted (Fig. 5A) (Scott et al., 2010).

To identify the specific residues within the L and CT regions involved in nucleolar localization, site-directed mutagenesis was used to target specific basic amino acids (Fig. 6A and C). Within the L region, changing residues 36–39 (KKRK) to alanines (RSV YFP-NC.M1) resulted in a localization pattern similar to YFP alone (compare to YFP in Fig. 6B), indicating a loss of nuclear and nucleolar localization. This result suggested that the KKRK sequence contained a nuclear localization signal (NLS) as well as an NoLS. Alteration of the other basic residues in the L region caused the YFP-NC.M2 protein to become more diffuse throughout the nucleus; however, fluorescence was still detectable within nucleoli. Within the CT region, mutating amino acids 61–63 (RKR) to alanine residues (YFP-NC.M3) or residues R61 and R63 to nonbasic residues (SKL) reduced but did not eliminate retention in nucleoli, and nuclear localization was unaltered. YFP-NC.M4, which mutated residues 70R and 73K to alanines was localized primarily within nucleoli, indicating that these basic residues were not required. However, combining the alanine substitutions in M3 and M4 led to nuclear localization with loss of nucleolar localization. To test whether basic residues within the NT contributed to nucleolar localization, even though they were not identified by the NoLS predictor, we tested YFP-NC.M5 (residues R5-to-A and R7-to-A) and YFP-NC.M6 (residues R16-to-A and R18-to-A), and both proteins remained nucleolar.

In concusion, residues KKRK at 36–39 in the L region contained an NLS and NoLS. Additional NoLS activity was located within amino acids RKR (61–63) and 70R/73K in the CT region. Thus, the NoLS predicted by the algorithm (Scott et al., 2010) for RSV NC were validated empirically.

Mapping the nucleolar localization signal in the HIV-1 NC protein

Analogous to RSV NC, the HIV-1 NC protein encodes basic regions flanking the Cys-His motifs. Additionally, there is a PRKK sequence (residues 31–34) in HIV-1 NC strikingly similar to the NLS we identified in RSV NC. To determine whether these basic residues in HIV-1 NC mediated nuclear or nucleolar localization, we performed mutational analysis. The HIV-1 NC protein was divided into five regions (NT, CH1, L, CH2, and CT) (Fig. 7A). Unlike RSV NC, the HIV-1 NC protein contains basic amino acid clusters only within the NT and L regions. Deletion of the Cys-His boxes did not disrupt nucleolar localization, indicating that the Cys-His motifs were not required (Fig. 7B). We next examined the localization of each region flanking the Cys-His boxes. YFP-CT was diffuse throughout the nucleus and cytoplasm, indistinguishable from YFP. By contrast, the YFP-NT and YFP-L, when expressed separately, were located throughout the nucleoplasm and nucleoli. This observation suggests that sequences within the NT region and/or the L region contributed to the nucleolar localization of HIV-1 NC.

FIG. 7.

FIG. 7

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Subcellular localization of HIV-1 NC mutants in HeLa cells. (A) Schematic diagram of YFP-HIV.NC is shown with the WT sequence of the amino acids immediately flanking CH1 (top) and mutant sequences below, indicated in bold. (B) Localization of HIV-1 NC mutants was analyzed for each mutant using confocal microscopy.

We next mutated the basic clusters located within the NT and L regions using site-directed mutagenesis. The M1 and M2 mutant NC proteins were present in nucleoli, although there was also an increase in the cytoplasmic signal, perhaps reflecting inefficient nuclear entry or misfolding (Fig. 7B). The double mutant M1/M2 did not localize to nucleoli, but was found within the nucleoplasm and cytoplasm, similar to YFP alone. These results suggest that HIV-1 NC contains two independent NoLS’s (R10/K11 and R32/K33/K34). Although each basic cluster alone was sufficient for nucleolar localization, both sequences were required for proper localization of NC.

Nucleolar localization of a subpopulation of the HIV-1 Gag protein

Because the HIV-1 NC protein localizes to the nucleolus and Gag proteins interact through RNA-mediated NC-NC associations, we tested the possibility that HIV-1 NC and Gag might interact in the nucleolus. However, based on recent reports suggesting that HIV-1 Gag does not undergo nuclear trafficking (Baluyot et al., 2012; Grewe et al., 2012), we did not expect to see a change in the localization of HIV-1 Gag with co-expression of HIV-1 NC or any other nucleolar protein. To our surprise, co-expression of a Rev-independent (RI) allele of HIV.Gag-CFP [RI.HIV.Gag-CFP; (Zhou et al., 1994)] with the nucleolar-localized proteins HIV.YFP-NC or HIV.Rev-YFP resulted in nucleolar localization of a subpopulation of RI.HIV.Gag-CFP in HeLa cells (Fig. 8A). This effect on HIV-1 Gag localization was specific for NC and Rev, as no Gag was seen in nucleoli with co-expression of fibrillarin-YFP.

FIG. 8.

FIG. 8

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Interaction of HIV-1 Gag with NC and Rev in nucleoli. (A) Confocal images showing Rev-independent (RI) HIV-1 Gag-CFP co-transfected with either HIV.NC-YFP, HIV.Rev-YFP or fibrillarin-YFP. Nucleolar fluorescence of HIV-1 Gag-CFP was seen in cells co-transfected with NC or Rev but not fibrillarin. (B) FRET was performed as described in Materials and Methods. HeLa cells were co-transfected with RI.HIV.Gag-CFP and YFP-HIV.NC, HIV.Rev-YFP, or Fibrillarin-YFP. YFP fluorescence was selectively photobleached using the 514nm laser aftery selecting the entire nucleus (dashed white circle) as the region of interest. Fluorescence of the nucleoli was measured before and after bleaching. An increase in the fluorescence of the RI.HIV.Gag-CFP nucleolar signal can be seen by comparing the intensity of the signals in the Gag pre-bleach and post-bleach images in cells co-expressing Gag with NC and Rev but not fibrillarin. (C) FRET efficiency was calculated (in percentage) for RI.HIV.Gag-CFP paired with YFP-HIV.NC, Rev-YFP, or fibrillarin-YFP using the formula described in Materials and Methods. The mean for each value +/− standard error of the mean was plotted. (D) Diagram of the HIV-1 Gag-GFP rtTA proviral expression vector used to create a stable HeLa cell line expressing Gag-GFP under the control of a doxycycline-inducible promoter. (E) Confocal images of HeLa cells expressing HIV-1 GagGFP rtTA or HIVΔ mls rtTA (F) were obtained after induction of Gag expression by adding doxycycline to the culture media. Images in the middle panels were adjusted to increase the overall intensity to bring out the fluorescence of Gag in nucleoli. Gag localization was determined by either direct detection of GFP (E) or by staining with anti-HIV CA antibody (F) and nucleoli were detected using anti-B23 (E) or anti-EBP2 antibodies (F).

To determine whether there was evidence for a direct interaction between RI.HIV.Gag-CFP and NC or Rev, acceptor photobleaching FRET analysis was performed (Fig. 8B). The entire nucleus was selected as the region of interest (denoted by a dashed circle shown), and the YFP-specific laser was used to bleach the HIV.NC-YFP, HIV.Rev-YFP and fibrillarin-YFP signals in the nucleus. Post-bleach analysis was performed on individual nucleoli to measure the change in CFP donor fluorescence for RI.HIV.Gag-CFP. The post-bleach images for RI.HIV.Gag-CFP showed an increase in CFP signal when HIV.YFP-NC or HIV.Rev-YFP were photobleached, but no change was seen for fibrillarin-YFP. Quantitative analysis of 10 cells indicated that RI.HIV.Gag-CFP had a significantly higher FRET efficiency when co-expressed with either NC (7.8%) or Rev (16.4%) (Fig. 8C). By contrast, FRET efficiency of Gag co-expressed with fibrillarin (3.0%) was similar to the background FRET between free CFP and YFP (2.6%) (Fig. 8C). These data demonstrate evidence for a direct protein-protein interaction between Rev-independent HIV-1 Gag with NC and Rev within nucleoli.

The RI.HIV.Gag-CFP construct used in the preceding experiments contains several mutations that eliminate the need for the Rev protein to facilitate nuclear export of the unspliced mRNA that encodes gag and enhances Gag protein expression (Schwartz et al., 1992). Next, we asked whether nucleolar localization of HIV-1 Gag was limited to this Rev-independent construct. To this end, we examined the localization of HIV-1 Gag in HeLa cells containing an integrated HIV-1 GagGFP rtTA proviral construct (Fig. 8D). In this cell line, Gag-GFP expression is dependent on doxycycline (dox), and in the absence of dox no Gag-GFP was detected (data not shown) (Wong et al., 2011). However, incubating the cells with dox induced strong expression of HIV-1 Gag-GFP, and immunostaining for the nucleolar protein B23 revealed that a sub-population of Gag colocalized with B23 in nucleoli (Fig. 8E). Subsequent analysis of single confocal slices from 30 individual cells revealed that on average 2.76±0.12% of the HIV-1 Gag-GFP signal localized to nucleoli. In agreement with previous reports, LMB treatment had no effect on the nuclear accumulation of HIV-1 Gag-GFP (data not shown) (Baluyot et al., 2012; Grewe et al., 2012; Kemler et al., 2012).

It was very unlikely that a Gag cleavage product such as NC-GFP was responsible for the GFP signal in nucleoli because the HIV.Gag-GFP rtTA provirus lacks the gene encoding the HIV-1 protease (Fig. 8D), therefore any processing of Gag-GFP would have to occur through the activity of a cellular protease. Furthermore, analysis of the GFP signal in cell lysates by SDS-PAGE and Western blotting revealed that full-length Gag-GFP was by far the major GFP-containing protein present (data not shown). However, to ensure that the nucleolar fluorescence signal observed was derived from the Gag protein rather than from NC-GFP, we examined the localization of HeLa cells stably expressing untagged HIV-1 Gag from the HIVΔ mls rtTA provirus under control of the dox-inducible promoter (Fig. 8F) (Wong et al., 2011). HeLa cells induced with dox and immunostained using an antibody against HIV-1 CA (p24) revealed evidence for HIV-1 Gag in nucleoli, as confirmed by co-staining with an antibody against a host nucleolar protein (EBP2). The Gag fluorescence signal was more evident when the image intensity was increased (center panel). Together, these observations suggest that a small population of HIV-1 Gag localized to nucleoli when expressed in the context of a Rev-dependent proviral vector.

Discussion

Retroviral Gag protein trafficking during assembly is an important, yet incompletely understood process. The RSV Gag protein transiently traffics through the nucleus in a cRM1-dependent manner. When RSV Gag is highly concentrated in the nucleus, either by treating cells with LMB or by mutating hydrophobic residues in the p10 NES, Gag proteins form discrete subnuclear foci (Kenney et al., 2008; Scheifele et al., 2002). Here we report that RSV Gag also accumulates within nucleoli in a subset of cells in which nuclear export is inhibited.

The formation of RSV Gag-containing nucleolar and nucleoplasmic foci depend on the presence of the NC domain, suggesting a requirement for RNA binding. Although it is possible that Gag accumulates in nucleoli due to nonspecific RNA binding, this explanation is unlikely given that only 10% of the RNA in the nucleus is found within nucleoli (Amano, 1967). Furthermore, RSV Gag localizes primarily to the nucleolar GC, which contains regions that contain very little rRNA and instead are protein-rich (Politz et al., 2002; Politz et al., 2005). Our biophysical analysis of RSV Gag nucleolar foci using FRAP demonstrated that the Gag proteins in nucleoli are highly dynamic, with a substantial portion of the protein (~75%, Fig. 3) moving continuously between nucleoli and the nucleoplasm. This property is characteristic of nucleolar proteins, which rapidly shuttle between the nucleolus and nucleoplasm (Chen and Huang, 2001) and is in contradistinction to other proteins found in subnuclear foci with very long recovery times that are stably associated with the nuclear matrix (Ernoult-Lange et al., 2009).

Based on our results, we conclude that under normal conditions RSV Gag cycles very rapidly through the nucleolus as part of its intranuclear trafficking pathway. It is likely that CRM1 plays a role in the bidirectional movement of RSV Gag between the nucleoplasm and nucleolus, as CRM1 has been implicated in determining the composition of ribonucloprotein complexes and directing their transport between the nucleolus and other intranuclear sites (Boulon et al., 2004; Ernoult-Lange et al., 2009; Pradet-Balade et al., 2011; Verheggen and Bertrand, 2012). Thus, CRM1 may perform similar functions in RSV infection, defining the composition of Gag nucleoprotein complexes (Gudleski et al., 2010); promoting subnuclear targeting of the complexes; and mediating their export through the nuclear pore.

At this point, it is not clear whether RSV Gag binds to a cellular protein or RNA that retains it in the nucleolus. It is possible that nucleoli serve as a storage depot for Gag proteins when they are highly concentrated in the nucleus, owing to an interaction with a host protein or RNA that has a substantial dwell-time in the nucleolus. Likely candidates for host RNAs that bind RSV Gag in nucleoli include tRNA-Trp, 5S rRNA, 7SL, and U6, which are selectively packaged into RSV virions (Dahlberg et al., 1974; Faras et al., 1973; Giles et al., 2004). These small noncoding RNAs are all transcripts made by RNA Polymerase III (pol III), they are transcribed within or adjacent to nucleoli (Matera et al., 1995), and at some stage in their biogenesis, these RNAs are localized to the nucleolus. Interestingly, murine leukemia virus (MLV) and HIV-1 virions contain an enriched population of pol III transcripts (Onafuwa-Nuga et al., 2005; Onafuwa-Nuga et al., 2006). Intriguingly, some of the RNAs packaged in MLV, e.g., mY1 and mY3, appear to be incorporated very early after their synthesis at perinucleolar sites (Garcia et al., 2009). These finding are particularly intriguing in light of a previous report that a subpopulation of the MLV Gag protein localized to the nucleus (Nash et al., 1993). Our current results demonstrating that a subset of RSV and HIV-1 Gag proteins localize to the nucleolus under specific conditions raises the possibility that small amount of these proteins traffic through the nucleolus transiently and encounter pol III transcripts early after their synthesis. The question of whether these noncoding RNAs play a role in subcellular targeting of the viral nucleoprotein complex as suggested by others (Garcia et al., 2009) or they are encapsidated due to a bystander effect has yet to be answered.

During HIV-1 and MLV infection, the NC protein localizes to nucleoli during the early stages of infection (Gallay et al., 1995; Risco et al., 1995a; Zhang and Crumpacker, 2002), suggesting that nucleolar localizing properties of the NC domain may serve two distinct roles, one during assembly (as part of Gag) and the other after cleavage from the Gag precursor during association of the reverse-transcription complex with the nuclear compartment. Such a role for RSV NC has not yet been demonstrated, and because available antibodies lack specificity for NC itself, we were not able to confirm the presence of NC in the nucleus or nucleolus early in infection (data not shown). However, now that we have identified the residues in both proteins that are required for nucleolar localization, studies with viral constructs bearing these mutant sequences RSV and HIV-1 NC have the potential to address interesting questions about the potential role of nucleolar targeting in both early and late events. These experiments will undoubtedly be very complicated because of the difficulty in separating the roles of basic residues in NC in nucleolar targeting, nuclear entry, RNA binding, Gag-Gag interactions, RNA chaperone functions, annealing of the tRNA primer to the genome, and steps in reverse transcription.

Several recent reports indicate that HIV-1 Gag does not undergo CRM1-mediated nuclear export like RSV does, although the published studies could not exclude whether there was a small fraction of HIV-1 Gag present in the nucleus in LMB-treated or untreated cells (Baluyot et al., 2012; Grewe et al., 2012; Kemler et al., 2012). Nonetheless, we were surprised to observe nucleolar localization of HIV-1 Gag when co-expressed with either HIV-1 NC or Rev. Further experiments will need to be performed to determine whether HIV-1 Gag and Rev interact in nucleoli by protein-protein contacts or through an RNA intermediate. Furthermore, we found that the Rev-dependent Gag-GFP fusion protein and the untagged Gag protein expressed from two different integrated proviral constructs also localized to nucleoli. It is possible that our detection of HIV-1 Gag in nucleoli was facilitated by the robust Rev-dependent expression of Gag-GFP using the inducible system. Together, these findings suggest that nucleolar trafficking of HIV-1 Gag may occur during viral infection.

Although the biological relevance of our findings remains to be determined, a number reports have indicated that the nucleolus plays an important role in HIV-1 replication. Rev was recently reported to enhance gRNA packaging during viral assembly (Blissenbach et al., 2010). Moreover, HIV-1 gRNA itself has nucleolar localization properties, suggesting that genome packaging could be linked to the nucleolus through an interaction with Rev (Michienzi et al., 2000). Co-expression of HIV-1 Gag, gRNA, and the nucleolar protein nucleolin promote efficient assembly of HIV-1 virus-like particles (Ueno et al., 2004). Together with our findings that a subpopulation of HIV-1 Gag is present in the nucleolus under steady state conditions, it is possible that the nucleolus could play a contributing role to HIV-1 gRNA packaging and virus assembly.

In addition to our data presented in the current study that RSV and HIV-1 Gag undergo nucleolar localization, it was recent reported that the Gag protein of feline immunodeficiency virus (FIV) accumulates in the nucleus and nucleolus in a CRM1-dependent fashion (Kemler et al., 2012). These studies raise the intriguing possibility that RSV, HIV-1, and FIV Gag may have conserved roles in the nucleolus. Whether nucleolar trafficking of retroviral Gag proteins is related to the packaging of nuclear-localized small cellular RNAs or gRNAs is a question that warrants further exploration. At this point, however, there does appear to be compelling evidence that nuclear/nucleolar trafficking of Gag is a more general feature of retroviruses than previously appreciated.

  • The NC domains of the RSV and HIV Gag proteins contain nucleolar localization signals

  • RSV Gag can accumulate in nucleoli when CRM1-mediated nuclear export is inhibited

  • Nuclear RSV Gag proteins are mobile, cycling rapidly between nucleoli and nucleoplasm

  • A subpopulation of Rev-dependent HIV-1 Gag localizes to nucleoli

Acknowledgments

This research was supported by NIH grants R01 CA76534 to L.J.P., T32CA60395 to T.L.L. (Richard C. Courtney PI), F30 CA165774 to D.V.B, and a Canadian Institutes of Health Research grant to A.C. We are grateful for support from the Department of Medicine to T.L.L. This work was funded, in part, from the Pennsylvania Department of Health using Tobacco CURE funds (to A.R.B. and L.J.P) and the Department specifically disclaims responsibility for any analyses, interpretations or conclusions. We thank Marilyn Resh and Mark Olson for generously providing DNA constructs used in this study. We acknowledge the importance of insightful discussions with our colleagues David J. Spector and Sergei Grigoryev and the technical contributions of the Confocal Imaging Core and DNA Sequencing Core at the Penn State College of Medicine.

Footnotes

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References

  1. Amano M. Metabolism of RNA in the liver cells of the rat. I. Isolation and chemical composition of nucleus, nucleolus, chromatin, nuclear sap and cytoplasm. Exp Cell Res. 1967;46(1):169–179. doi: 10.1016/0014-4827(67)90418-1. [DOI] [PubMed] [Google Scholar]
  2. Andersen JS, Lam YW, Leung AK, Ong SE, Lyon CE, Lamond AI, Mann M. Nucleolar proteome dynamics. Nature. 2005;433(7021):77–83. doi: 10.1038/nature03207. [DOI] [PubMed] [Google Scholar]
  3. Andersen JS, Lyon CE, Fox AH, Leung AK, Lam YW, Steen H, Mann M, Lamond AI. Directed proteomic analysis of the human nucleolus. Curr Biol. 2002;12(1):1–11. doi: 10.1016/s0960-9822(01)00650-9. [DOI] [PubMed] [Google Scholar]
  4. Arizala J, Rossi JJ. Role of the Nucleolus in HIV Infection and Therapy. In: Olson MOJ, editor. The Nucleolus. Springer; New York, NY: 2011. pp. 381–402. [Google Scholar]
  5. Aronoff R, Hajjar AM, Linial ML. Avian retroviral RNA encapsidation: reexamination of functional 5′ RNA sequences and the role of nucleocapsid Cys-His motifs. J Virol. 1993;67(1):178–188. doi: 10.1128/jvi.67.1.178-188.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Azad RF, Driver VB, Tanaka K, Crooke RM, Anderson KP. Antiviral activity of a phosphorothioate oligonucleotide complementary to RNA of the human cytomegalovirus major immediate-early region. Antimicrob Agents Chemother. 1993;37(9):1945–1954. doi: 10.1128/aac.37.9.1945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Baluyot MF, Grosse SA, Lyddon TD, Janaka SK, Johnson MC. CRM1-dependent trafficking of retroviral Gag proteins revisited. J Virol. 2012;86(8):4696–4700. doi: 10.1128/JVI.07199-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bartoe JT, Albrecht B, Collins ND, Robek MD, Ratner L, Green PL, Lairmore MD. Functional role of pX open reading frame II of human T-lymphotropic virus type 1 in maintenance of viral loads in vivo. J Virol. 2000;74(3):1094–1100. doi: 10.1128/jvi.74.3.1094-1100.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ben-Av P, Liscovitch M. Phospholipase D activation by the mitogens platelet-derived growth factor and 12-O-tetradecanoylphorbol 13-acetate in NIH-3T3 cells. FEBS Lett. 1989;259(1):64–66. doi: 10.1016/0014-5793(89)81495-4. [DOI] [PubMed] [Google Scholar]
  10. Bertrand L, Pearson A. The conserved N-terminal domain of herpes simplex virus 1 UL24 protein is sufficient to induce the spatial redistribution of nucleolin. J Gen Virol. 2008;89(Pt 5):1142–1151. doi: 10.1099/vir.0.83573-0. [DOI] [PubMed] [Google Scholar]
  11. Blissenbach M, Grewe B, Hoffmann B, Brandt S, Uberla K. Nuclear RNA export and packaging functions of HIV-1 Rev revisited. J Virol. 2010;84(13):6598–6604. doi: 10.1128/JVI.02264-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Boisvert FM, van Koningsbruggen S, Navascues J, Lamond AI. The multifunctional nucleolus. Nat Rev Mol Cell Biol. 2007;8(7):574–585. doi: 10.1038/nrm2184. [DOI] [PubMed] [Google Scholar]
  13. Boulon S, Verheggen C, Jady BE, Girard C, Pescia C, Paul C, Ospina JK, Kiss T, Matera AG, Bordonne R, Bertrand E. PHAX and CRM1 are required sequentially to transport U3 snoRNA to nucleoli. Molecular Cell. 2004;16(5):777–787. doi: 10.1016/j.molcel.2004.11.013. [DOI] [PubMed] [Google Scholar]
  14. Boyne JR, Whitehouse A. Nucleolar trafficking is essential for nuclear export of intronless herpesvirus mRNA. Proc Natl Acad Sci U S A. 2006;103(41):15190–15195. doi: 10.1073/pnas.0604890103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Butterfield-Gerson KL, Scheifele LZ, Ryan EP, Hopper AK, Parent LJ. Importin-beta family members mediate alpharetrovirus gag nuclear entry via interactions with matrix and nucleocapsid. J Virol. 2006;80(4):1798–1806. doi: 10.1128/JVI.80.4.1798-1806.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Chen D, Huang S. Nucleolar components involved in ribosome biogenesis cycle between the nucleolus and nucleoplasm in interphase cells. J Cell Biol. 2001;153(1):169–176. doi: 10.1083/jcb.153.1.169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Cisterna B, Biggiogera M. Ribosome biogenesis: from structure to dynamics. Int Rev Cell Mol Biol. 2010;284:67–111. doi: 10.1016/S1937-6448(10)84002-X. [DOI] [PubMed] [Google Scholar]
  18. Cochrane AW, Perkins A, Rosen CA. Identification of sequences important in the nucleolar localization of human immunodeficiency virus Rev: relevance of nucleolar localization to function. J Virol. 1990;64(2):881–885. doi: 10.1128/jvi.64.2.881-885.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Craven RC, Leure-duPree AE, Weldon RA, Jr, Wills JW. Genetic analysis of the major homology region of the Rous sarcoma virus Gag protein. J Virol. 1995;69(7):4213–4227. doi: 10.1128/jvi.69.7.4213-4227.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Daelemans D, Costes SV, Cho EH, Erwin-Cohen RA, Lockett S, Pavlakis GN. In vivo HIV-1 Rev multimerization in the nucleolus and cytoplasm identified by fluorescence resonance energy transfer. J Biol Chem. 2004;279(48):50167–50175. doi: 10.1074/jbc.M407713200. [DOI] [PubMed] [Google Scholar]
  21. Dahlberg JE, Sawyer RC, Taylor JM, Faras AJ, Levinson WE, Goodman HM, Bishop JM. Transcription of DNA from the 70S RNA of Rous sarcoma virus. I. Identification of a specific 4S RNA which serves as primer. J Virol. 1974;13(5):1126–1133. doi: 10.1128/jvi.13.5.1126-1133.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Dundr M, Misteli T, Olson MO. The dynamics of postmitotic reassembly of the nucleolus. J Cell Biol. 2000;150(3):433–446. doi: 10.1083/jcb.150.3.433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Emmott E, Dove BK, Howell G, Chappell LA, Reed ML, Boyne JR, You JH, Brooks G, Whitehouse A, Hiscox JA. Viral nucleolar localisation signals determine dynamic trafficking within the nucleolus. Virology. 2008;380(2):191–202. doi: 10.1016/j.virol.2008.05.032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Emmott E, Hiscox JA. Nucleolar targeting: the hub of the matter. EMBO Rep. 2009;10(3):231–238. doi: 10.1038/embor.2009.14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ernoult-Lange M, Wilczynska A, Harper M, Aigueperse C, Dautry F, Kress M, Weil D. Nucleocytoplasmic traffic of CPEB1 and accumulation in Crm1 nucleolar bodies. Mol Biol Cell. 2009;20(1):176–181. doi: 10.1091/mbc.E08-09-0904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Fankhauser C, Izaurralde E, Adachi Y, Wingfield P, Laemmli UK. Specific complex of human immunodeficiency virus type 1 rev and nucleolar B23 proteins: dissociation by the Rev response element. Mol Cell Biol. 1991;11(5):2567–2575. doi: 10.1128/mcb.11.5.2567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Faras AJ, Garapin AC, Levinson WE, Bishop JM, Goodman HM. Characterization of the low-molecular-weight RNAs associated with the 70S RNA of Rous sarcoma virus. J Virol. 1973;12(2):334–342. doi: 10.1128/jvi.12.2.334-342.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Fujiwara T, Oda K, Yokota S, Takatsuki A, Ikehara Y. Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the endoplasmic reticulum. J Biol Chem. 1988;263(34):18545–18552. [PubMed] [Google Scholar]
  29. Gallay P, Swingler S, Song J, Bushman F, Trono D. HIV nuclear import is governed by the phosphotyrosine-mediated binding of matrix to the core domain of integrase. Cell. 1995;83 (4):569–576. doi: 10.1016/0092-8674(95)90097-7. [DOI] [PubMed] [Google Scholar]
  30. Garbitt-Hirst R, Kenney SP, Parent LJ. Genetic evidence for a connection between Rous sarcoma virus gag nuclear trafficking and genomic RNA packaging. J Virol. 2009;83(13):6790–6797. doi: 10.1128/JVI.00101-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Garcia EL, Onafuwa-Nuga A, Sim S, King SR, Wolin SL, Telesnitsky A. Packaging of host mY RNAs by murine leukemia virus may occur early in Y RNA biogenesis. J Virol. 2009;83(23):12526–12534. doi: 10.1128/JVI.01219-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ghorbel S, Sinha-Datta U, Dundr M, Brown M, Franchini G, Nicot C. Human T-cell leukemia virus type I p30 nuclear/nucleolar retention is mediated through interactions with RNA and a constituent of the 60 S ribosomal subunit. J Biol Chem. 2006;281(48):37150–37158. doi: 10.1074/jbc.M603981200. [DOI] [PubMed] [Google Scholar]
  33. Giles KE, Caputi M, Beemon KL. Packaging and reverse transcription of snRNAs by retroviruses may generate pseudogenes. RNA. 2004;10(2):299–307. doi: 10.1261/rna.2150604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Gomez CY, Hope TJ. Mobility of human immunodeficiency virus type 1 Pr55Gag in living cells. J Virol. 2006;80(17):8796–8806. doi: 10.1128/JVI.02159-05. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Gorelick RJ, Henderson LE, Hanser JP, Rein A. Point mutants of Moloney murine leukemia virus that fail to package viral RNA: evidence for specific RNA recognition by a “zinc finger-like” protein sequence. Proc Natl Acad Sci U S A. 1988;85(22):8420–8424. doi: 10.1073/pnas.85.22.8420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Grewe B, Hoffmann B, Ohs I, Blissenbach M, Brandt S, Tippler B, Grunwald T, Uberla K. Cytoplasmic utilization of human immunodeficiency virus type 1 genomic RNA is not dependent on a nuclear interaction with gag. J Virol. 2012;86(6):2990–3002. doi: 10.1128/JVI.06874-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Gudleski N, Flanagan JM, Ryan EP, Bewley MC, Parent LJ. Directionality of nucleocytoplasmic transport of the retroviral gag protein depends on sequential binding of karyopherins and viral RNA. Proc Natl Acad Sci U S A. 2010;107(20):9358–9363. doi: 10.1073/pnas.1000304107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Hermida-Matsumoto L, Resh MD. Localization of human immunodeficiency virus type 1 Gag and Env at the plasma membrane by confocal imaging. J Virol. 2000;74(18):8670–8679. doi: 10.1128/jvi.74.18.8670-8679.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Himly M, Foster DN, Bottoli I, Iacovoni JS, Vogt PK. The DF-1 chicken fibroblast cell line: transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses. Virology. 1998;248(2):295–304. doi: 10.1006/viro.1998.9290. [DOI] [PubMed] [Google Scholar]
  40. Hindley CE, Davidson AD, Matthews DA. Relationship between adenovirus DNA replication proteins and nucleolar proteins B23.1 and B23.2. J Gen Virol. 2007;88(Pt 12):3244–3248. doi: 10.1099/vir.0.83196-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Hiscox JA. The nucleolus--a gateway to viral infection? Arch. Virol. 2002;147(6):1077–1089. doi: 10.1007/s00705-001-0792-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Hivin P, Basbous J, Raymond F, Henaff D, Arpin-Andre C, Robert-Hebmann V, Barbeau B, Mesnard JM. The HBZ-SP1 isoform of human T-cell leukemia virus type I represses JunB activity by sequestration into nuclear bodies. Retrovirology. 2007;4:14. doi: 10.1186/1742-4690-4-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Kemler I, Saenz D, Poeschla E. Feline Immunodeficiency Virus Gag is a Nuclear Shuttling Protein. J Virol. 2012 doi: 10.1128/JVI.00692-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Kenney SP, Lochmann TL, Schmid CL, Parent LJ. Intermolecular interactions between retroviral Gag proteins in the nucleus. J Virol. 2008;82(2):683–691. doi: 10.1128/JVI.02049-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Kubota S, Hatanaka M, Pomerantz RJ. Nucleo-cytoplasmic redistribution of the HTLV-I Rex protein: alterations by coexpression of the HTLV-I p21x protein. Virology. 1996;220(2):502–507. doi: 10.1006/viro.1996.0339. [DOI] [PubMed] [Google Scholar]
  46. Kubota S, Siomi H, Satoh T, Endo S, Maki M, Hatanaka M. Functional similarity of HIV-I rev and HTLV-I rex proteins: identification of a new nucleolar-targeting signal in rev protein. Biochem Biophys Res Commun. 1989;162(3):963–970. doi: 10.1016/0006-291x(89)90767-5. [DOI] [PubMed] [Google Scholar]
  47. Lam YW, Trinkle-Mulcahy L, Lamond AI. The nucleolus. J Cell Sci. 2005;118(Pt 7):1335–1337. doi: 10.1242/jcs.01736. [DOI] [PubMed] [Google Scholar]
  48. Larson DR, Johnson MC, Webb WW, Vogt VM. Visualization of retrovirus budding with correlated light and electron microscopy. Proc Natl Acad Sci U S A. 2005;102(43):15453–15458. doi: 10.1073/pnas.0504812102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Lawrence FJ, McStay B, Matthews DA. Nucleolar protein upstream binding factor is sequestered into adenovirus DNA replication centres during infection without affecting RNA polymerase I location or ablating rRNA synthesis. J Cell Sci. 2006;119(Pt 12):2621–2631. doi: 10.1242/jcs.02982. [DOI] [PubMed] [Google Scholar]
  50. Leung AK, Lamond AI. The dynamics of the nucleolus. Crit Rev Eukaryot Gene Expr. 2003;13(1):39–54. doi: 10.1615/critreveukaryotgeneexpr.v13.i1.40. [DOI] [PubMed] [Google Scholar]
  51. Li YP, Busch RK, Valdez BC, Busch H. C23 interacts with B23, a putative nucleolar-localization-signal-binding protein. Eur J Biochem. 1996;237(1):153–158. doi: 10.1111/j.1432-1033.1996.0153n.x. [DOI] [PubMed] [Google Scholar]
  52. Lopez MR, Schlegel EF, Wintersteller S, Blaho JA. The major tegument structural protein VP22 targets areas of dispersed nucleolin and marginalized chromatin during productive herpes simplex virus 1 infection. Virus Res. 2008;136(1–2):175–188. doi: 10.1016/j.virusres.2008.05.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Lymberopoulos MH, Pearson A. Involvement of UL24 in herpes-simplex-virus-1-induced dispersal of nucleolin. Virology. 2007;363(2):397–409. doi: 10.1016/j.virol.2007.01.028. [DOI] [PubMed] [Google Scholar]
  54. Matera AG, Frey MR, Margelot K, Wolin SL. A perinucleolar compartment contains several RNA polymerase III transcripts as well as the polypyrimidine tract-binding protein, hnRNP I. J Cell Biol. 1995;129(5):1181–1193. doi: 10.1083/jcb.129.5.1181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Matthews D, Emmott E, Hiscox J. Viruses and the Nucleolus. The Nucleolus. 2011:321–345. [Google Scholar]
  56. Michienzi A, Cagnon L, Bahner I, Rossi JJ. Ribozyme-mediated inhibition of HIV 1 suggests nucleolar trafficking of HIV-1 RNA. Proc Natl Acad Sci U S A. 2000;97(16):8955–8960. doi: 10.1073/pnas.97.16.8955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Miyazaki Y, Nosaka T, Hatanaka M. The post-transcriptional regulator Rev of HIV: implications for its interaction with the nucleolar protein B23. Biochimie. 1996;78(11–12):1081–1086. doi: 10.1016/s0300-9084(97)86733-8. [DOI] [PubMed] [Google Scholar]
  58. Nash MA, Meyer MK, Decker GL, Arlinghaus RB. A subset of Pr65gag is nucleus associated in murine leukemia virus-infected cells. J Virol. 1993;67(3):1350–1356. doi: 10.1128/jvi.67.3.1350-1356.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Nosaka T, Siomi H, Adachi Y, Ishibashi M, Kubota S, Maki M, Hatanaka M. Nucleolar targeting signal of human T-cell leukemia virus type I rex-encoded protein is essential for cytoplasmic accumulation of unspliced viral mRNA. Proc Natl Acad Sci U S A. 1989;86(24):9798–9802. doi: 10.1073/pnas.86.24.9798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Okuwaki M, Iwamatsu A, Tsujimoto M, Nagata K. Identification of nucleophosmin/B23, an acidic nucleolar protein, as a stimulatory factor for in vitro replication of adenovirus DNA complexed with viral basic core proteins. J Mol Biol. 2001;311(1):41–55. doi: 10.1006/jmbi.2001.4812. [DOI] [PubMed] [Google Scholar]
  61. Onafuwa-Nuga AA, King SR, Telesnitsky A. Nonrandom packaging of host RNAs in moloney murine leukemia virus. J Virol. 2005;79(21):13528–13537. doi: 10.1128/JVI.79.21.13528-13537.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Onafuwa-Nuga AA, Telesnitsky A, King SR. 7SL RNA, but not the 54-kd signal recognition particle protein, is an abundant component of both infectious HIV-1 and minimal virus-like particles. RNA. 2006;12(4):542–546. doi: 10.1261/rna.2306306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Ozawa M, Fujii K, Muramoto Y, Yamada S, Yamayoshi S, Takada A, Goto H, Horimoto T, Kawaoka Y. Contributions of two nuclear localization signals of influenza A virus nucleoprotein to viral replication. J Virol. 2007;81(1):30–41. doi: 10.1128/JVI.01434-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Parent LJ, Cairns TM, Albert JA, Wilson CB, Wills JW, Craven RC. RNA dimerization defect in a Rous sarcoma virus matrix mutant. J Virol. 2000;74(1):164–172. doi: 10.1128/jvi.74.1.164-172.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Pederson T, Tsai RY. In search of nonribosomal nucleolar protein function and regulation. J Cell Biol. 2009;184(6):771–776. doi: 10.1083/jcb.200812014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Perkins A, Cochrane AW, Ruben SM, Rosen CA. Structural and functional characterization of the human immunodeficiency virus rev protein. J Acquir Immune Defic Syndr. 1989;2(3):256–263. [PubMed] [Google Scholar]
  67. Phair RD, Misteli T. High mobility of proteins in the mammalian cell nucleus. Nature. 2000;404(6778):604–609. doi: 10.1038/35007077. [DOI] [PubMed] [Google Scholar]
  68. Politz JC, Lewandowski LB, Pederson T. Signal recognition particle RNA localization within the nucleolus differs from the classical sites of ribosome synthesis. J Cell Biol. 2002;159(3):411–418. doi: 10.1083/jcb.200208037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Politz JC, Polena I, Trask I, Bazett-Jones DP, Pederson T. A nonribosomal landscape in the nucleolus revealed by the stem cell protein nucleostemin. Mol Biol Cell. 2005;16(7):3401–3410. doi: 10.1091/mbc.E05-02-0106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Pradet-Balade, Girard C, Boulon S, Paul C, Azzag K, Bordonne R, Bertrand E, Verheggen C. CRM1 controls the composition of nucleoplasmic pre-snoRNA complexes to licence them for nucleolar transport. EMBO Journal. 2011;30(11):2205–2218. doi: 10.1038/emboj.2011.128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Rasband WS. ImageJ. National Institutes of Health; Bethesda, Maryland : 1997–2012. http://imageJ.nih.gov/ij/ [Google Scholar]
  72. Risco C, Menendez-Arias L, Copeland TD, Pinto da Silva P, Oroszlan S. Intracellular transport of the murine leukemia virus during acute infection of NIH 3T3 cells: nuclear import of nucleocapsid protein and integrase. J Cell Sci. 1995a;108 (Pt 9):3039–3050. doi: 10.1242/jcs.108.9.3039. [DOI] [PubMed] [Google Scholar]
  73. Risco C, Menendez-Arias L, Copeland TD, Pinto da SP, Oroszlan S. Intracellular transport of the murine leukemia virus during acute infection of NIH 3T3 cells: nuclear import of nucleocapsid protein and integrase. J Cell Sci. 1995b;108(Pt 9):3039–3050. doi: 10.1242/jcs.108.9.3039. [DOI] [PubMed] [Google Scholar]
  74. Salmivirta M, Heino J, Jalkanen M. Basic fibroblast growth factor-syndecan complex at cell surface or immobilized to matrix promotes cell growth. J Biol Chem. 1992;267(25):17606–17610. [PubMed] [Google Scholar]
  75. Sansam CL, Wells KS, Emeson RB. Modulation of RNA editing by functional nucleolar sequestration of ADAR2. Proc Natl Acad Sci U S A. 2003;100(24):14018–14023. doi: 10.1073/pnas.2336131100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Scheifele LZ, Garbitt RA, Rhoads JD, Parent LJ. Nuclear entry and CRM1-dependent nuclear export of the Rous sarcoma virus Gag polyprotein. Proc Natl Acad Sci U S A. 2002;99(6):3944–3949. doi: 10.1073/pnas.062652199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Scheifele LZ, Kenney SP, Cairns TM, Craven RC, Parent LJ. Overlapping roles of the Rous sarcoma virus Gag p10 domain in nuclear export and virion core morphology. J Virol. 2007;81(19):10718–10728. doi: 10.1128/JVI.01061-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Schwartz S, Campbell M, Nasioulas G, Harrison J, Felber BK, Pavlakis GN. Mutational inactivation of an inhibitory sequence in human immunodeficiency virus type 1 results in Rev-independent gag expression. J Virol. 1992;66(12):7176–7182. doi: 10.1128/jvi.66.12.7176-7182.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. Scott MS, Boisvert FM, McDowall MD, Lamond AI, Barton GJ. Characterization and prediction of protein nucleolar localization sequences. Nucleic Acids Res. 2010;38(21):7388–7399. doi: 10.1093/nar/gkq653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Siomi H, Shida H, Nam SH, Nosaka T, Maki M, Hatanaka M. Sequence requirements for nucleolar localization of human T cell leukemia virus type I pX protein, which regulates viral RNA processing. Cell. 1988;55(2):197–209. doi: 10.1016/0092-8674(88)90043-8. [DOI] [PubMed] [Google Scholar]
  81. Speil J, Kubitscheck U. Single ovalbumin molecules exploring nucleoplasm and nucleoli of living cell nuclei. Biochim Biophys Acta. 2010;1803(3):396–404. doi: 10.1016/j.bbamcr.2009.10.010. [DOI] [PubMed] [Google Scholar]
  82. Ueno T, Tokunaga K, Sawa H, Maeda M, Chiba J, Kojima A, Hasegawa H, Shoya Y, Sata T, Kurata T, Takahashi H. Nucleolin and the packaging signal, psi, promote the budding of human immunodeficiency virus type-1 (HIV-1) Microbiol Immunol. 2004;48(2):111–118. doi: 10.1111/j.1348-0421.2004.tb03496.x. [DOI] [PubMed] [Google Scholar]
  83. Verheggen C, Bertrand E. CRM1 plays a nuclear role in transporting snoRNPs to nucleoli in higher eukaryotes. Nucleus. 2012;3(2) doi: 10.4161/nucl.19266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  84. Waggoner S, Sarnow P. Viral ribonucleoprotein complex formation and nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells. J Virol. 1998;72(8):6699–6709. doi: 10.1128/jvi.72.8.6699-6709.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  85. Weldon RA, Jr, Erdie CR, Oliver MG, Wills JW. Incorporation of chimeric gag protein into retroviral particles. J Virol. 1990;64(9):4169–4179. doi: 10.1128/jvi.64.9.4169-4179.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  86. Westman BJ, Verheggen C, Hutten S, Lam YW, Bertrand E, Lamond AI. A proteomic screen for nucleolar SUMO targets shows SUMOylation modulates the function of Nop5/Nop58. Molecular cell. 2010;39(4):618–631. doi: 10.1016/j.molcel.2010.07.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  87. Wong R, Balachandran A, Mao AY, Dobson W, Gray-Owen S, Cochrane A. Differential effect of CLK SR Kinases on HIV-1 gene expression: potential novel targets for therapy. Retrovirology. 2011;8:47. doi: 10.1186/1742-4690-8-47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  88. Xing J, Wu F, Pan W, Zheng C. Molecular anatomy of subcellular localization of HSV-1 tegument protein US11 in living cells. Virus Res. 2010;153(1):71–81. doi: 10.1016/j.virusres.2010.07.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  89. Yoo D, Wootton SK, Li G, Song C, Rowland RR. Colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar RNA-associated protein fibrillarin. J Virol. 2003;77(22):12173–12183. doi: 10.1128/JVI.77.22.12173-12183.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  90. You JH, Howell G, Pattnaik AK, Osorio FA, Hiscox JA. A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein based on live cell imaging. Virology. 2008;378(1):34–47. doi: 10.1016/j.virol.2008.04.037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  91. Zhang J, Crumpacker CS. Human immunodeficiency virus type 1 nucleocapsid protein nuclear localization mediates early viral mRNA expression. J Virol. 2002;76(20):10444–10454. doi: 10.1128/JVI.76.20.10444-10454.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  92. Zhang J, Yang Y, Wu J. B23 interacts with PES1 and is involved in nucleolar localization of PES1. Acta Biochim Biophys Sin (Shanghai) 2009;41(12):991–997. doi: 10.1093/abbs/gmp096. [DOI] [PubMed] [Google Scholar]
  93. Zhou W, Parent LJ, Wills JW, Resh MD. Identification of a membrane-binding domain within the amino-terminal region of human immunodeficiency virus type 1 Gag protein which interacts with acidic phospholipids. J Virol. 1994;68(4):2556–2569. doi: 10.1128/jvi.68.4.2556-2569.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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