Epstein-Barr Virus Independent Dysregulation of UBP43 Expression Alters Interferon-Stimulated Gene Expression in Burkitt Lymphoma

Epstein-Barr virus (EBV) persists as a life-long latent infection within memory B cells, but how EBV may circumvent the innate immune response within this virus reservoir is unclear. Recent studies suggest that the latency-associated non-coding RNAs of EBV may actually induce type I (antiviral) interferon production, raising the question of how EBV counters the negative consequences this is likely to have on viral persistence. We addressed this by examining the type I interferon response in Burkitt lymphoma (BL) cell lines, the only in vitro model of the restricted program of EBV latency-gene expression in persistently infected B cells in vivo. Importantly, we observed no effect of EBV on interferon alpha-induced signaling or evidence of type I interferon production, suggesting that EBV in this latent state is silent to the cell's innate antiviral surveillance. We did uncover, however, a defect in the negative feedback control of interferon signaling in a subpopulation of BL lines as was revealed by prolonged interferon-stimulated gene transcription consistent with sustained tyrosine phosphorylation on STAT1 and STAT2. This was due to inadequate induction of expression of the ubiquitin-specific protease UBP43, which removes the ubiquitin-like ISG15 polypeptide conjugated to proteins (ISGylation) in response to type I interferons. Results here are consistent with previous findings in genetically engineered Ubp43−/− murine cells that UBP43 down-regulates interferon signaling, independent of its ISG15 isopeptidase activity, by precluding the protein kinase JAK1 from the interferon receptor. This natural deficiency in UBP43 expression may therefore provide a useful model to further probe the biological roles of UBP43 and ISGylation

Clusters of Basic Amino Acids Contribute to RNA Binding and Nucleolar Localization of Ribosomal Protein L22

The ribosomal protein L22 is a component of the 60S eukaryotic ribosomal subunit. As an RNA-binding protein, it has been shown to interact with both cellular and viral RNAs including 28S rRNA and the Epstein-Barr virus encoded RNA, EBER-1. L22 is localized to the cell nucleus where it accumulates in nucleoli. Although previous studies demonstrated that a specific amino acid sequence is required for nucleolar localization, the RNA-binding domain has not been identified. Here, we investigated the hypothesis that the nucleolar accumulation of L22 is linked to its ability to bind RNA. To address this hypothesis, mutated L22 proteins were generated to assess the contribution of specific amino acids to RNA binding and protein localization. Using RNA-protein binding assays, we demonstrate that basic amino acids 80–93 are required for high affinity binding of 28S rRNA and EBER-1 by L22. Fluorescence localization studies using GFP-tagged mutated L22 proteins further reveal that basic amino acids 80–93 are critical for nucleolar accumulation and for incorporation into ribosomes. Our data support the growing consensus that the nucleolar accumulation of ribosomal proteins may not be mediated by a defined localization signal, but rather by specific interaction with established nucleolar components such as rRNA

Generation and characterization of wild-type and mutated L22 expression constructs.

<p>(A) Clusters of basic amino acids likely to be involved in RNA binding were identified within the L22 amino acid sequence and are shown underlined with the basic residues highlighted in bold font. The nine amino-terminal residues previously predicted to be the RNA-binding domain are also highlighted in bold <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005306#pone.0005306-ShuNu1" target="_blank">[29]</a>. (B) Location of mutations introduced into L22 coding sequence. Constructs expressing L22 lacking either nine amino-terminal or eight carboxy-terminal residues are designated Δ1–9 and Δ120–128, respectively. Point mutations generated in the basic amino acid clusters illustrated in (A) are shown relative to the wild-type sequence (shown directly below the line) and designated by arrows above and below the line. For m80 and m88, constructs with K to E mutations (and R to D) have been designated m80 and m88 while constructs with K to A mutations have been designated m80A and m88A. (C) N-terminal GFP-L22 fusion constructs, depicted in (B), were transiently transfected into 293T cells followed by analysis of protein lysates for protein expression by immunoblot using anti-GFP antibody. GAPDH served as a control for protein loading.</p

Mutation of residues 80–93 eliminates binding of L22 to multiple RNA substrates.

<p>The capacity of mutated L22 proteins to bind to EBER-1 (A) and 28S rRNA (B) was determined by specific capture of proteins on biotinylated RNAs immobilized on streptavidin magnetic beads. Protein lysates were generated from transiently transfected 293T cells and normalized for expression relative to wild-type GFP-L22. Total protein was incubated with 100 pmoles of biotinylated RNA and complexes were captured on streptavidin magnetic bead columns. Column flow-thru and eluate were subjected to SDS-PAGE and analyzed for GFP-L22 proteins by immunoblot using anti-GFP antibody. Protein lysate from cells transfected with GFP alone was used as a control for nonspecific binding to beads or RNA.</p

Mutation of residues 80–93 alters the subcellular localization of L22 and prevents incorporation into ribosomes.

<p>(A) The subcellular localization of mutated GFP-L22 proteins was analyzed by fluorescence microscopy following transient expression in 293T cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005306#pone-0005306-g005" target="_blank">Fig. 5A</a>. (B) Incorporation of m88 into ribosomes was analyzed by sucrose density gradient analysis of extracts generated from 293T cells engineered to stably express m88, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005306#pone-0005306-g005" target="_blank">Fig. 5B</a>. Migration of molecular weight standards (in kDa) is indicated to the left of the blots. Following detection of L22 (15 kDa) and m88 (43 kDa) with anti-L22 antibody, the blot was stripped of antibody and reprobed with anti-GFP antibody to confirm that the 43 kDa bands present in fractions 1–3 were in fact GFP-tagged L22.</p

GFP-L22 is localized to nucleoli and incorporated into ribosomes.

<p>(A) The subcellular localization of wild-type L22 fusion proteins and control proteins was analyzed by fluorescence microscopy following expression in 293T and HeLa cells. For transient expression, cells grown on coverslips were transfected with 2 µg of the indicated expression construct, fixed after 48 hrs and visualized using a Zeiss Axiovert inverted fluorescence microscope. HeLa-L22 cells stably express GFP-L22. BAP-L22 expression was visualized in transiently transfected HeLa cells following staining with Alexa Fluor 488-conjugated streptavidin. Fibrillarin served as an endogenous nucleolar marker and was detected in HeLa cells using anti-fibrillarin antibody and Alexa-conjugated secondary antibody. All coverslips were mounted in Vectashield plus DAPI. Bar equals 10 µm. (B) Nuclear (N) and cytoplasmic (C) fractions from untransfected and GFP-L22 transfected 293T cells were analyzed by immunblot using anti-L22 and anti-GAPDH antibodies. Extract from the indicated number of cells was analyzed. Following detection of L22, blots were stripped of antibody and reprobed for GAPDH which served as a control for cytoplasmic contamination of nuclear extracts. (C) Localization of endogenous L22 and GFP-L22 in HeLa-L22 cells was assessed by sucrose density gradient analysis. Ribosome-containing lysates were separated on a 10–50% w/v sucrose gradient and 0.5 ml fractions were collected from the top of the gradient. The protein and RNA content of each fraction was analyzed by western blot and agarose gel electrophoresis, respectively. Total RNA was visualized by ethidium bromide. Polysome profiles were recorded during fraction collection at 260 nm. The ribosomal subunit composition of each peak is indicated along with fraction numbers corresponding to the first and last fraction collected (1 and 12) as well as the start of collection of the 40S (fraction 6) and 60S (fraction 9) peaks.</p

Clusters of basic amino acids mediate L22 binding to EBER-1.

<p>(A) Binding of GFP-L22 to stem-loop III of EBER-1 (SL3) was tested in RNA EMSA experiments using increasing amounts of protein lysate (1, 5, and 20 µg) generated from 293T cells transfected with GFP-L22. 5 µg of control lysate expressing only GFP was used to assess nonspecific binding. Each 10 µl binding reaction contained 0.05 pmoles ³²P end-labeled SL3 RNA oligonucleotide. Reactions were electrophoresed on 8% native polyacrylamide gels and visualized by autoradiography. (B) L22 binds specifically to EBER-1. Binding specificity of L22 to SL3 was tested by antibody supershift and by competition with unlabeled oligonucleotides. 5 µg of GFP-L22 protein lysate was used in binding reactions. For antibody supershift experiments, 1 µl of anti-GFP or anti-polyhistidine (nonspecific control) antibody was added to binding reactions. In competition experiments, 10× and 100× unlabeled SL3 or mutated SL3 (mSL3) was added. (C) RNA binding capacity of GFP-L22 containing basic residue mutations or truncation of the amino-terminus (left panel) or with internal point mutations (right panel) was tested in RNA EMSA reactions, as described above. Amounts of each protein lysate used in binding reaction were determined by normalizing the level of expression of each mutated L22 construct to the level of GFP-L22 in 5 µg total protein lysate. Abbreviations used are: FP = free probe, NS = nonspecific, E = endogenous, GFP-L22 = all specific shifts generated with wild-type or mutated GFP-L22 proteins, SS = supershift.</p