Nucleolar Localization of GLTSCR2/PICT-1 Is Mediated by Multiple Unique Nucleolar Localization Sequences

The human glioma tumor suppressor candidate region 2 gene product, GLTSCR2, also called ‘protein interacting with carboxyl terminus 1’ (PICT-1), has been implicated in the regulation of two major tumor suppressor proteins, PTEN and p53, and reported to bind the membrane-cytoskeleton regulator of cell signaling, Merlin. PICT-1 is a nucleolar protein, conserved among eukaryotes, and its yeast homolog has been functionally associated with ribosomal RNA processing. By means of confocal microscopy of EGFP and myc-tagged PICT-1 fusion proteins, we delineate that the nucleolar localization of PICT-1 is mediated by two independent nucleolar localization sequences (NoLS). Unlike most NoLSs, these NoLSs are relatively long with flexible boundaries and contain arginine and leucine clusters. In addition, we show that PICT-1 exhibits a nucleolar distribution similar to proteins involved in ribosomal RNA processing, yet does not colocalize precisely with either UBF1 or Fibrillarin under normal or stressed conditions. Identification of the precise location of PICT-1 and the signals that mediate its nucleolar localization is an important step towards advancing our understanding of the demonstrated influence of this protein on cell fate and tumorigenesis

Latently KSHV-Infected Cells Promote Further Establishment of Latency upon Superinfection with KSHV

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a cancer-related virus which engages in two forms of infection: latent and lytic. Latent infection allows the virus to establish long-term persistent infection, whereas the lytic cycle is needed for the maintenance of the viral reservoir and for virus spread. By using recombinant KSHV viruses encoding mNeonGreen and mCherry fluorescent proteins, we show that various cell types that are latently-infected with KSHV can be superinfected, and that the new incoming viruses establish latent infection. Moreover, we show that latency establishment is enhanced in superinfected cells compared to primary infected ones. Further analysis revealed that cells that ectopically express the major latency protein of KSHV, LANA-1, prior to and during infection exhibit enhanced establishment of latency, but not cells expressing LANA-1 fragments. This observation supports the notion that the expression level of LANA-1 following infection determines the efficiency of latency establishment and avoids loss of viral genomes. These findings imply that a host can be infected with more than a single viral genome and that superinfection may support the maintenance of long-term latency

GLTSCR2/PICT-1, a Putative Tumor Suppressor Gene Product, Induces the Nucleolar Targeting of the Kaposi's Sarcoma-Associated Herpesvirus KS-Bcl-2 Protein▿

KS-Bcl-2, encoded by Kaposi's sarcoma-associated herpesvirus (KSHV), is a structural and functional homologue of the Bcl-2 family of apoptosis regulators. Like several other Bcl-2 family members, KS-Bcl-2 protects cells from apoptosis and autophagy. Using a yeast two-hybrid screen and coimmunoprecipitation assays, we identified a novel KS-Bcl-2-interacting protein, referred to as protein interacting with carboxyl terminus 1 (PICT-1), encoded by a candidate tumor suppressor gene, GLTSCR2. Confocal laser scanning microscopy revealed nucleolar localization of PICT-1, whereas KS-Bcl-2 was located mostly at the mitochondrial membranes with a small fraction in the nucleoli. Ectopic expression of PICT-1 resulted in a large increase in the nucleolar fraction of KS-Bcl-2, and only a minor fraction remained in the cytoplasm. Furthermore, knockdown of endogenous PICT-1 abolished the nucleolar localization of KS-Bcl-2. However, ectopically expressed PICT-1 did not alter the cellular distribution of human Bcl-2. Subsequent analysis mapped the crucial amino acid sequences of both KS-Bcl-2 and PICT-1 required for their interaction and for KS-Bcl-2 targeting to the nucleolus. Functional studies suggest a correlation between nucleolar targeting of KS-Bcl-2 by PICT-1 and reduction of the antiapoptotic activity of KS-Bcl-2. Thus, these studies demonstrate a cellular mechanism to sequester KS-Bcl-2 from the mitochondria and to downregulate its virally encoded antiapoptotic activity. Additional characterization of the interaction of KS-Bcl-2 and PICT-1 is likely to shed light on the functions of both proteins

Herpes Simplex Virus Type‑1 Attachment Inhibition by Functionalized Graphene Oxide

Graphene oxide and its derivatives have lately been the subject of increased attention in the field of bioscience and biotechnology. In this article, we report on the use of graphene oxide (GO) derivatives to inhibit herpes simplex virus type-1 (HSV-1) infections, mimicking the cell surface receptor heparan sulfate, and the GO derivatives compete with the latter in binding HSV-1. The inhibition does not affect cell-to-cell spreading. Media content has a significant effect on the inhibition properties of the nanomaterials. These have no cytotoxic effect, suggesting that this is a promising approach for the development of antiviral surfaces and for diagnostic purposes

Amino acids 387–478 contain a functional NoLS.

<p>(A) The sequences of myc-tagged PICT-1 deletion mutants are shown. Cellular localization as determined by confocal microscopy is indicated on the right. (B) Confocal microscopic analysis of fusion proteins. 24 hrs after transfection, cells were stained with anti-myc antibody and rhodamine-conjugated secondary antibody. Hoechst stained DNA appears blue. Corresponding differential interference contrast (DIC) images are also shown.</p

PICT-1 substitution mutants within the NoLS spanning amino-acids 347–395 retain the ability to be targeted to the nucleolus.

<p>(A) The sequences of myc-tagged full-length PICT-1 substitution mutants (M1–M6) are shown. Mutated residues are indicated by dotted lines. Cellular localization as determined by confocal microscopy is indicated on the right. (B) Confocal microscopic analysis of fusion proteins. 24 hrs after transfection, cells were stained with anti-myc antibody and rhodamine-conjugated secondary antibody. Hoechst stained DNA appears blue. Corresponding differential interference contrast (DIC) images are also shown.</p

Schematic representation of PICT-1 with its predicted monopartite (m) and bipartite (b) NLSs.

<p>Previously reported truncation mutants are shown with their cellular localization indicated on the right, ^a refers to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030825#pone.0030825-Kalt1" target="_blank">[19]</a> and ^b refers to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030825#pone.0030825-Yim1" target="_blank">[6]</a>. Shadowing denotes the putative nucleolar targeting sequence (aa 347–386) inferred from the localization of these mutants.</p

Amino acids 387–478 contain a functional NoLS that can target EGFP to the nucleolus.

<p>(A) PICT-1 sequences fused to EGFP are shown. Solid lines highlight LR/RL and KL/LK sequences. Cellular localization as determined by confocal microscopy is indicated on the right. (B) Confocal microscopic analysis of EGFP fusion proteins. Images were taken 24 hrs after transfection. Hoechst stained DNA appears blue. Corresponding differential interference contrast (DIC) images are also shown.</p

Cellular localization of endogenous PICT-1 upon stress induction.

<p>(A) Nucleolar segregation was induced by actinomycin D treatment (0.05 µg/ml) of HEK-293T for 4 hrs. Fibrillarin, UBF1 and PICT-1 were stained using Cy5 (orange), FITC (green) and Cy3 (red)-coupled secondary antibodies, respectively. Where indicated, merged images are shown. Nuclei were stained with Hoechst. Some PICT-1 is found in Fibrillarin containing nucleolar caps and to a greater extent in caps containing UBF1. (B) HEK-293T cells were treated with etoposide, cycloheximide, staurosporine or doxorubicin, for 24, 1, 24 and 2 hrs, respectively. Cells were then fixed and stained with anti PICT-1 and rhodamineconjugated secondary antibody.</p

The Portal Vertex of KSHV Promotes Docking of Capsids at the Nuclear Pores

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a cancer-related herpesvirus. Like other herpesviruses, the KSHV icosahedral capsid includes a portal vertex, composed of 12 protein subunits encoded by open reading frame (ORF) 43, which enables packaging and release of the viral genome into the nucleus through the nuclear pore complex (NPC). Capsid vertex-specific component (CVSC) tegument proteins, which directly mediate docking at the NPCs, are organized on the capsid vertices and are enriched on the portal vertex. Whether and how the portal vertex is selected for docking at the NPC is unknown. Here, we investigated the docking of incoming ORF43-null KSHV capsids at the NPCs, and describe a significantly lower fraction of capsids attached to the nuclear envelope compared to wild-type (WT) capsids. Like WT capsids, nuclear envelope-associated ORF43-null capsids co-localized with different nucleoporins (Nups) and did not detach upon salt treatment. Inhibition of nuclear export did not alter WT capsid docking. As ORF43-null capsids exhibit lower extent of association with the NPCs, we conclude that although not essential, the portal has a role in mediating the interaction of the CVSC proteins with Nups, and suggest a model whereby WT capsids can dock at the nuclear envelope through a non-portal penton vertex, resulting in an infection ‘dead end’