UIS2: A Unique Phosphatase Required for the Development of <i>Plasmodium</i> Liver Stages

<div><p><i>Plasmodium</i> salivary sporozoites are the infectious form of the malaria parasite and are dormant inside salivary glands of <i>Anopheles</i> mosquitoes. During dormancy, protein translation is inhibited by the kinase UIS1 that phosphorylates serine 59 in the eukaryotic initiation factor 2α (eIF2α). De-phosphorylation of eIF2α-P is required for the transformation of sporozoites into the liver stage. In mammalian cells, the de-phosphorylation of eIF2α-P is mediated by the protein phosphatase 1 (PP1). Using a series of genetically knockout parasites we showed that in malaria sporozoites, contrary to mammalian cells, the eIF2α-P phosphatase is a member of the PP2C/PPM phosphatase family termed UIS2. We found that eIF2α was highly phosphorylated in <i>uis2</i> conditional knockout sporozoites. These mutant sporozoites maintained the crescent shape after delivery into mammalian host and lost their infectivity. Both <i>uis1</i> and <i>uis2</i> were highly transcribed in the salivary gland sporozoites but <i>uis2</i> expression was inhibited by the Pumilio protein Puf2. The repression of <i>uis2</i> expression was alleviated when sporozoites developed into liver stage. While most eukaryotic phosphatases interact transiently with their substrates, UIS2 stably bound to phosphorylated eIF2α, raising the possibility that high-throughput searches may identify chemicals that disrupt this interaction and prevent malaria infection.</p></div

The N-terminus of UIS2 binds eIF2α-P.

<p>(A) PfeIF2α-P interacts with endogenous UIS2. In these experiments we used the codon-optimized eIF2α of <i>P</i>. <i>falciparum</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref007" target="_blank">7</a>] that shares 89% identity with its <i>P</i>. <i>berghei</i> ortholog. The immobilized GST-PfeIF2α was incubated with the lysates of <i>P</i>. <i>berghei</i> blood stage parasites in the presence or absence of Sal (50 μM) or GA (70 μM). The bound proteins were detected by immunoblot using antibodies against PP1, UIS2, phosphorylated eIF2α, and total eIF2α. Levels of PbeIF2α-P and total PbeIF2α were quantified by densitometry analysis. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.s010" target="_blank">S8 Fig</a>: The mouse anti-PbPP1 antibody recognized the 35 kDa endogenous PP1 from the parasite lysates. (B) PfeIF2α<i>S59D</i> mutant protein interaction with UIS2. The immobilized GST-PfeIF2α wt, <i>S59A</i>, or <i>S59D</i> were incubated with the lysates of <i>P</i>. <i>berghei</i> blood stage parasites, with or without Sal, and UIS2 was detected by immunoblot. (C) Schematic representation of the PbUIS2 coding sequence. UIS2 contains a putative conserved phosphatase domain (PD, 535–1054 amino acids) and <i>Plasmodium</i> specific sequences at N- and C- terminus. (D) The N-terminus of PbUIS2 bound PfeIF2α-P. The PbUIS2 N-ter, PD, and C-ter were fused to GST-tag at their N-terminus and His-tag at their C-terminus, respectively. After 2-step affinity purification, the <i>E</i>. <i>coli</i> expressed fusion proteins were immobilized on glutathione sepharose 4B. After incubation with purified recombinant PfeIF2α-P, the sepharose was washed three times with high-salt NETN buffer (300 mM NaCl, 20 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, and 0.5% (v/v) Nonidet P-40). The retained proteins were detected by SDS-PAGE followed by coomassie brilliant blue staining. Lane 1,3,5: GST and His-tagged PbUIS2 N-ter, PD, and C-ter, respectively. Lane 2,4,6: proteins retained on the glutathione sepharose 4B after pull down assays with PfeIF2α-P. Lane 7, PfeIF2α-P control. The pulled down 38 kDa protein in lane 2 was analyzed by mass spectrometry and identified as PfeIF2α-P. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.s011" target="_blank">S9 Fig</a>. (E) Co-immunoprecipitation (IP) of endogenous complex between UIS2 and eIF2α-P. Whole cell extracts from <i>P</i>. <i>berghei</i> blood stage parasites were subjected to immunoprecipitation with anti-eIF2α-P or anti-UIS2 antibodies followed by immunoblot analysis.</p

Regulation of UIS2 expression by Puf2 in Ssp.

<p>(A) The <i>uis2</i> mRNA levels of wt Ssp, <i>puf2(-)</i> Ssp, and wt liver stages were measured by Real-time PCR. Each value is the mean ± SD of two independent experiments. (B) Immunoblot depicts UIS2 protein levels in wt Ssp, <i>puf2(-)</i> Ssp, and wt liver stages using mouse anti-UIS2 antibodies. CSP was used as internal control (n = 3). (C) mRNA levels of <i>Puf</i> proteins at different stages of parasite development. <i>puf1</i> and <i>puf2</i> mRNA levels were analyzed by real-time PCR using cDNAs from different stages of <i>P</i>. <i>berghei</i> development. mRNA levels were normalized to the control mRNA, arginyl-tRNA synthetase (PB000094.03.0). MG: midgut; SG: salivary gland; Liver: liver stages; BS: blood stages; Gtc: gametocytes. Each value is the mean ± SD of two independent experiments. (D) The <i>uis2</i> mRNA binding to Puf2 was analyzed by RNA gel shift assays. Lane 1: The reaction contains only the biotinylated <i>uis2</i>-RNA probe (negative control). Lane 2: The band shift of the <i>uis2</i>-RNA probe was detected by the addition of recombinant <i>P</i>. <i>berghei</i> Puf2 protein. Lane 3: The labeled probe is outcompeted by the addition of a 200-fold excess of un-labeled <i>uis2</i>-RNA. Lane 4 and 5: The labeled probe was not outcompeted by the addition of a 200-fold excess of non-specific unlabeled <i>Pbpp1</i> or <i>Pbuis1</i> probe.</p

Phenotypes of <i>uis2 cKO parasites</i>.

<p>(A) mRNA levels of <i>uis2</i> in the <i>P</i>. <i>bergthei</i> life cycle. <i>Pbuis2</i> (<i>PBANKA_132800</i>) mRNA levels were analyzed by real-time PCR using cDNAs from different stages of <i>P</i>. <i>berghei</i>. mRNA level was normalized to arginyl-tRNA synthetase (<i>PbArgRS</i>, PB000094.03.0). MG: midgut; SG: salivary gland; Liver: liver stages; BS: asexual blood stages; Gtc: gametocytes. Each value is the mean ± SD of two independent experiments. (B) Phosphorylation level of eIF2α was higher in <i>uis2</i> cKO Ssp. Levels of UIS2, PbeIF2α-P and total PbeIF2α from 5X10⁵ Ssp are shown by immunoblots. CSP was used as a control. Values of densitometry analysis are shown below the bands. This experiment was repeated three times and similar results were obtained. (C) The mutant sporozoites invaded HepG2 cells as effectively as wt parasites. Wild-type TRAP/FlpL(-) and <i>uis2</i> cKO Ssp were added to HepG2 cells and fixed 1 h post-infection. The parasites inside and outside the HepG2 cells were quantified by the hepatocyte invasion assay. Each value is the mean ± SD of two independent experiments. <i>(</i><b><i>D</i></b><i>)</i> The mutant parasites maintained the crescent shape in contrast to the round shape of the wt parasites in HepG2 cells. Wt TRAP/FlpL(-) and <i>uis2</i> cKO Ssp were added to HepG2 cells and were detected 48 h post-infection by immunofluorescence using antibodies against the liver stage antigen UIS4. Bars, 10 μm. (E) The development of <i>uis2</i> cKO sporozoites was blocked inside HepG2 cells. <i>P</i>. <i>berghei</i> Ssp infectivity of HepG2 cells was evaluated 48 h post-infection by counting exo-erythrocytic stage (EEF) numbers and measuring18S rRNA level. The left panel shows the mean of EEF numbers ± SD of two independent experiments. The right panel shows liver-stage parasite burden measured by real-time RT-PCR. (F) The development of <i>uis2</i> cKO sporozoites was blocked in the mouse liver. C57BL/6 mice (five per group) were injected intravenously with 1×10⁴ wt TRAP/FlpL(-) or with the same number of <i>uis2</i> cKO Ssp. Liver-stage parasite burden was measured 42 hours post-infection by real-time RT-PCR. Each value is the mean ± SD of two independent experiments.</p

Characterization of <i>Plasmodium pp1</i> (<i>PBANKA_102830</i>).

<p><i>(A) pp1</i> mRNA levels during the <i>P</i>. <i>berghei</i> life cycle. <i>Pbpp1</i> mRNA levels were analyzed by real-time PCR using cDNAs from the different stages of <i>P</i>. <i>berghei</i>. The <i>arginyl-tRNA synthetase</i> (<i>PbArgRS</i>, <i>PB000094</i>.<i>03</i>.<i>0</i>) was used as internal control. Each value is the mean ± SD of two independent experiments. MG: midgut; SG: salivary gland; Liver: liver stages; BS: asexual blood stages; Gtc: gametocyte. <i>(B) Pbpp1</i> mRNA levels in <i>Pbpp1</i> cKO Ssp were quantified by qPCR. P = 0.041. P value was calculated by t test. Shown are mean ± SD of two independent experiments. <i>(C)</i> Immunoblot analysis of <i>Pbpp1</i> cKO and wt TRAP/FlpL(-) Ssp using anti-PbPP1 serum. CSP was used as control. <i>(D)</i> The liver stage development of <i>Pbpp1</i> cKO Ssp in HepG2 cells was indistinguishable from the wild type TRAP/FlpL(-) Ssp. Hepatic parasites were stained with anti-PbHSP70 and anti-PbPP1 antibodies at 6h, 24h, 36h, 48h, and 55h post-infection. Bar, 10 μm. <i>(E)</i> eIF2α phosphorylation level in <i>Pbpp1</i> cKO sporozoites was indistinguishable from wild type. Five hundred thousand wt TRAP/FlpL(-) or <i>Pbpp1</i> cKO sporozoites were dissected from mosquito salivary glands. Levels of PbeIF2α-P and total PbeIF2α were quantified by densitometry analysis of immunoblots performed with antibodies against anti-eIF2α-P and anti-total eIF2α [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref015" target="_blank">15</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref016" target="_blank">16</a>]. Values are shown below the bands. Results were similar in two independent experiments.</p

Model for UIS2 function in the <i>Plasmodium</i>.

<p>The eIF2α kinase eIK2 (also called UIS1) and Pumilio protein Puf2 are highly transcribed and translated in the mosquito salivary gland [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref003" target="_blank">3</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref005" target="_blank">5</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref025" target="_blank">25</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref026" target="_blank">26</a>]. The highly transcribed eIF2α phosphatase <i>uis2</i> mRNA binds to Puf2 and the translation of phosphatase UIS2 is likely repressed. The eIF2α of Ssp is highly phosphorylated since the eIF2α kinase eIK2 activity is dominant, leading to translation inhibition and latency of sporozoites in the mosquito salivary glands. When sporozoites are injected into the mammalian host, the repression of UIS2 translation is probably alleviated, the eIF2α phosphatase UIS2 activity is dominant, eIF2α is dephosphorylated, and Ssp transform into the liver stages. Knockout of <i>puf2</i> or <i>eIK2</i> contributes to the dominance of UIS2 and the mutant sporozoites inside of mosquito salivary glands prematurely transform into liver stages [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref004" target="_blank">4</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref023" target="_blank">23</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref028" target="_blank">28</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref029" target="_blank">29</a>].</p

PbeIF2α-P phosphatase activity of UIS2.

<p>(A) PbUIS2 phosphatase domain (PD) dephosphorylated eIF2α-P i<i>n vitro</i>. PfeIF2α-P[γ-³²P] was incubated with GST-PbUIS2PD in the presence of 5mM MnCl₂. (B) Characterization of PbUIS2 as a PP2C/PPM phosphatase. PbUIS2PD phosphatase activity was measured by malachite green colorimetric assay in the presence of EDTA, okadaic acid (OA), or CdCl₂. E: GST-PbUIS2PD; S: eIF2α-P. The inorganic phosphate was detected with malachite green by measuring the absorbance at 630 nm. Each value is the mean ± SD of three independent experiments.</p

hnRNP K is required for optimal M2 protein production and influenza A virus replication.

<p>A549 cells were transfected with non-targeting or hnRNP K siRNAs for 48 h prior to infection. siRNA transfected cells were infected with A/WSN/33 at (A) MOI 2 or (B) MOI 0.001. Cells were harvested at the indicated hours post-infection, and viral protein accumulation was assessed by immunoblot analysis. Each protein band in (A) and (B) was quantified by ImageJ and normalized to α-tubulin levels. (C) Control or hnRNP K siRNA transfected cells were infected with A/WSN/33 at MOI 0.001. At 36 hours post-infection, cell supernatants were collected and subjected to viral titer analysis (n = 3) or (D) ATP level analysis to determine cell viability (n = 3, representative experiment). Error bars denote mean + SEM. *p<0.05.</p

NS1-BP interacts with RNA-binding proteins, RNA polymerase II, and influenza A virus polymerase.

<p>(A) Top panel, HeLa cell lysates were immunoprecipitated with control IgG or NS1-BP antibodies. Interacting proteins were resolved by SDS-PAGE and identified by mass spectrometry. Bottom panel, A549 cells were treated with non-targeting or NS1-BP siRNAs (1, 2, and 3) for 48 h. Cell extracts were subjected to immunoblot analysis, which shows NS1-BP knockdown. β-actin served as loading control. (B) Immunoprecipitation was performed with control IgG or anti-NS1-BP antibodies, in the absence or presence of RNase A. Western blots were then performed with the depicted antibodies, selected based on the proteins identified in A. (C) A549 cells were mock-infected or infected with influenza virus at MOI 5 for 5 h. Cells were lysed and subjected to size exclusion chromatography. The fractions were concentrated by TCA precipitation and analyzed by western blot with the indicated antibodies. (D) A549 cells were mock-infected or infected with influenza virus (A/WSN/33) at MOI 2 for 16 h. Cell lysates were immunoprecipitated with control IgG or anti-NS1-BP antibodies. Western blots were performed with the depicted antibodies.</p

Cellular RNA Binding Proteins NS1-BP and hnRNP K Regulate Influenza A Virus RNA Splicing

<div><p>Influenza A virus is a major human pathogen with a genome comprised of eight single-strand, negative-sense, RNA segments. Two viral RNA segments, NS1 and M, undergo alternative splicing and yield several proteins including NS1, NS2, M1 and M2 proteins. However, the mechanisms or players involved in splicing of these viral RNA segments have not been fully studied. Here, by investigating the interacting partners and function of the cellular protein NS1-binding protein (NS1-BP), we revealed novel players in the splicing of the M1 segment. Using a proteomics approach, we identified a complex of RNA binding proteins containing NS1-BP and heterogeneous nuclear ribonucleoproteins (hnRNPs), among which are hnRNPs involved in host pre-mRNA splicing. We found that low levels of NS1-BP specifically impaired proper alternative splicing of the viral M1 mRNA segment to yield the M2 mRNA without affecting splicing of mRNA₃, M4, or the NS mRNA segments. Further biochemical analysis by formaldehyde and UV cross-linking demonstrated that NS1-BP did not interact directly with viral M1 mRNA but its interacting partners, hnRNPs A1, K, L, and M, directly bound M1 mRNA. Among these hnRNPs, we identified hnRNP K as a major mediator of M1 mRNA splicing. The M1 mRNA segment generates the matrix protein M1 and the M2 ion channel, which are essential proteins involved in viral trafficking, release into the cytoplasm, and budding. Thus, reduction of NS1-BP and/or hnRNP K levels altered M2/M1 mRNA and protein ratios, decreasing M2 levels and inhibiting virus replication. Thus, NS1-BP-hnRNPK complex is a key mediator of influenza A virus gene expression.</p></div