A conserved structure within the HIV gag open reading frame that controls translation initiation directly recruits the 40S subunit and eIF3

Translation initiation on HIV genomic RNA relies on both cap and Internal Ribosome Entry Site (IRES) dependant mechanisms that are regulated throughout the cell cycle. During a unique phenomenon, the virus recruits initiation complexes through RNA structures located within Gag coding sequence, downstream of the initiation codon. We analyzed initiation complexes paused on the HIV-2 gag IRES and revealed that they contain all the canonical initiation factors except eIF4E and eIF1. We report that eIF3 and the small ribosomal subunit bind HIV RNA within gag open reading frame. We thus propose a novel two step model whereby the initial event is the formation of a ternary eIF3/40S/IRES complex. In a second step, dependent on most of the canonical initiation factors, the complex is rearranged to transfer the ribosome on the initiation codons. The absolute requirement of this large structure for HIV translation defines a new function for a coding region. Moreover, the level of information compaction within this viral genome reveals an additional level of evolutionary constraint on the coding sequence. The conservation of this IRES and its properties in rapidly evolving viruses suggest an important role in the virus life cycle and highlight an attractive new therapeutic target

The GTP- and Phospholipid-Binding Protein TTD14 Regulates Trafficking of the TRPL Ion Channel in Drosophila Photoreceptor Cells.

Recycling of signaling proteins is a common phenomenon in diverse signaling pathways. In photoreceptors of Drosophila, light absorption by rhodopsin triggers a phospholipase Cβ-mediated opening of the ion channels transient receptor potential (TRP) and TRP-like (TRPL) and generates the visual response. The signaling proteins are located in a plasma membrane compartment called rhabdomere. The major rhodopsin (Rh1) and TRP are predominantly localized in the rhabdomere in light and darkness. In contrast, TRPL translocates between the rhabdomeral plasma membrane in the dark and a storage compartment in the cell body in the light, from where it can be recycled to the plasma membrane upon subsequent dark adaptation. Here, we identified the gene mutated in trpl translocation defective 14 (ttd14), which is required for both TRPL internalization from the rhabdomere in the light and recycling of TRPL back to the rhabdomere in the dark. TTD14 is highly conserved in invertebrates and binds GTP in vitro. The ttd14 mutation alters a conserved proline residue (P75L) in the GTP-binding domain and abolishes binding to GTP. This indicates that GTP binding is essential for TTD14 function. TTD14 is a cytosolic protein and binds to PtdIns(3)P, a lipid enriched in early endosome membranes, and to phosphatidic acid. In contrast to TRPL, rhabdomeral localization of the membrane proteins Rh1 and TRP is not affected in the ttd14P75L mutant. The ttd14P75L mutation results in Rh1-independent photoreceptor degeneration and larval lethality suggesting that other processes are also affected by the ttd14P75L mutation. In conclusion, TTD14 is a novel regulator of TRPL trafficking, involved in internalization and subsequent sorting of TRPL into the recycling pathway that enables this ion channel to return to the plasma membrane

Proposed scheme of TRPL trafficking.

<p>Trafficking routes of TRPL in illuminated photoreceptors are illustrated with orange arrows while trafficking routes of TRPL in dark-kept photoreceptors are shown by blue arrows. De novo synthesis of TRPL is indicated by black arrows. Thick arrows and broken arrows indicate major and minor trafficking routes, respectively. Disturbed trafficking of TRPL in <i>ttd14</i> mutant photoreceptors is illustrated by red bars.</p

Characterization of the TRPL trafficking defect in the <i>ttd14</i>^<i>P75L</i> mutant.

<p>(A) Water immersion microscopy images of TRPL-eGFP fluorescence in white colored (<i>white</i>^<i>–</i>) eyes of wild type flies (wild type), <i>ttd14</i>^<i>P75L</i> mutant eye clones (<i>ttd14</i>^<i>P75L</i>), and flies expressing a <i>ttd14-myc</i> construct under the control of the Rh1 promoter in <i>ttd14</i>^<i>P75L</i> mutant eye clones (<i>ttd14</i>^<i>P75L</i><i>; Rh1</i>><i>ttd14-A-myc</i>). After eclosion, flies were kept in the dark for 1 day (upper row), 3 days (middle row) or 7 days (lower row) and subsequently exposed to orange light for 16 hours. After orange light illumination, flies were subjected to a second dark-adaptation for 24 hours. Localization of TRPL-eGFP in rhabdomeres or in the cell body appears, respectively, as distinct circular signal or as a diffuse signal with dark rhabdomeres. Scale bar: 10 μm. (B) Quantification of the relative fluorescence of TRPL-eGFP in rhabdomeres of the outer photoreceptor cells using water immersion images as shown in (<i>A</i>) (mean ± SD; n = 5). The relative rhabdomeral TRPL-eGFP fluorescence was determined as described in Material and Methods and the value obtained for wild type flies kept in darkness for 1 day was set to 100%. Statistically significant differences between wild type and <i>ttd14</i>^<i>P75L</i> as analyzed by an unpaired Student´s <i>t</i> test are indicated (***, p<0.001). (C) Localization of native TRPL on cross sections through ommatidia from wild type flies, <i>ttd14</i>^<i>P75L</i> mutant eye clones and <i>ttd14</i>^<i>P75L</i> mutant eye clones expressing a <i>Rh1</i>><i>ttd14-A-myc</i> construct (<i>ttd14</i>^<i>P75L</i><i>; Rh1</i>><i>ttd14-A-myc</i>). Flies were aged for 7 days in darkness and subsequently subjected to the same light-regime as in (A). Cross sections were probed with an anti-TRPL antibody (green, upper row) and Alexa Fluor 546-coupled phalloidin (red, middle row). Merged panels are shown in the lower row. Scale bar: 5 μm. (D) Immunoblot analysis of TRPL extracted from wild type heads or from heads with <i>ttd14</i>^<i>P75L</i> mutant eye clones (equivalent of 3 heads per lane). Freshly eclosed flies (1 day) or flies kept in the dark for 7 days were analyzed immediately (first black bars) or subjected to orange light illumination for 16 hours (white bars) followed by 24 hours of darkness (second black bars). The blots were probed with α-TRPL and α-Tubulin antibodies as indicated. The size of molecular weight markers in kilo Dalton is indicated at the left. (E) Quantification of the TRPL levels normalized to Tubulin. The TRPL level of 1 day old flies illuminated for 16 hours (second column) was set to 100%. Error bars show SEM (n = 5).</p

Amino acid sequence analysis of TTD14.

<p>(A) Scheme of the 475 amino acid long TTD14-A protein. The scheme illustrates a P-loop nucleoside triphosphate hydrolase domain (P-loop) spanning amino acids 65–167 and a CYTH domain (CYTH) containing amino acids 279–437 as predicted by InterPro (<a href="http://www.ebi.ac.uk/interpro" target="_blank">http://www.ebi.ac.uk/interpro</a>). (B) Amino acid sequence alignment of TTD14 homologs from <i>Drosophila melanogaster</i> (isoform A), <i>Musca domestica</i>, <i>Anopheles gambiae</i>, <i>Apis mellifera</i>, and <i>Caenorhabditis elegans</i>. * denotes amino acids identical in all sequences,: denotes conserved substitutions. The P-loop containing nucleoside triphosphate binding domain (green), a CYTH-like domain (orange), and the P75L point mutation (arrow) are indicated. (C) Electropherogram of a sequencing reaction from genomic DNA of a heterozygous <i>ttd14</i> mutant. The wild type and the mutant allele have, respectively, a CCT (encoding proline) or a CTT codon (encoding leucine) at amino acid position 75.</p

Aged <i>ttd14</i>^<i>P75L</i> mutant flies kept in a 12 hours light/ 12 hours dark cycle develop physiological defects.

<p>(A) Electroretinogram recordings from 7 day old wild type and <i>ttd</i>^<i>P75L</i> mutant flies, from 1 day old <i>ninaE</i>^<i>17</i> mutant, 14 day old wild type flies raised on vitamin A-deprived food, and 1 day old <i>trp</i>^<i>P343</i> mutant. All flies were raised and kept in the dark. Flies were stimulated with orange light (O) or blue light (<i>B</i>) for 5 seconds with dark intervals of 10 seconds between the stimuli. (B) ERG recordings from wild type flies, <i>ttd14</i>^<i>P75L</i> mutant flies and <i>ttd14</i>^<i>P75L</i><i>; Rh1</i>><i>ttd14-A-myc</i> flies (rescue) kept in a 12 hours light / 12 hours dark cycle for 1, 7, 14 and 21 days as well as after keeping the flies for 21 days in darkness (21 d dark, ERG traces at the right). Flies were stimulated with a 5 sec orange light pulse. (C) Quantification of the ERG amplitudes of flies kept in a 12 hours light / 12 hours dark cycle or in constant darkness as in <i>B</i>. Error bars represent SEM (n = 10). Statistically significant differences between wild type and <i>ttd14</i>^<i>P75L</i> as analyzed by an unpaired Student´s <i>t</i> test are indicated (***, p<0.001). (D) Immunoblot analysis assessing Rh1 in wild type heads and heads with <i>ttd14</i>^<i>P75L</i> mutant eye clones of flies kept in the dark for 14 days on either regular food (regular) or on a vitamin A-deprived diet (low vitA). The equivalent of 4 heads was loaded per lane. Tubulin was used as a loading control. Vitamin A deprivation resulted in a strong reduction of the Rh1 content. (E) Quantification of the ERG amplitude of 21 day old wild type and <i>ttd14</i>^<i>P75L</i> mutant flies kept in a 12 hours light / 12 hours dark cycle. Flies were raised and kept either on regular food (regular) or on a vitamin A-deprived diet (low vitA). Error bars represent SEM (n = 10). Vitamin A deprivation did not affect the ERG amplitude as analyzed by an unpaired Student´s <i>t</i> test and did not rescue the reduced ERG amplitude of <i>ttd14</i>^<i>P75L</i> mutant eyes.</p

Deficiency mapping of the <i>ttd14</i> locus.

<p>(A) Images on top show a wild type eye and a mosaic eye with white homocygous <i>ttd14</i>^<i>P75L</i> cell clones and red heterozygous cell clones. The panels show deep pseudopupils (red arrowhead) as revealed by TRPL-eGFP fluorescence in the eyes of wild type flies and <i>ttd14</i> mutant eye clones. Flies were raised in the dark for 1 day (upper row) or 7 days (lower row) and then subjected to 16 hours of orange light. Following orange light adaptation flies were again kept in darkness for 24 hours. (B) Using deletion strains from the Bloomington stock collection the lethality of <i>ttd14</i> was mapped to the region 55C8–55C9 on chromosome 2R. The gene region, the corresponding cytological bands, the position of genes in the mapped region as annotated in flybase (<a href="http://flybase.org/" target="_blank">http://flybase.org</a>), and the localization of deficiencies that complemented (black) or failed to complement (red) the lethality of <i>ttd14</i> are shown. (C) Three different mRNAs are predicted to be transcribed from the <i>ttd14</i> gene. In the transcript schemes, orange and gray boxes denote protein coding and non-coding exons, respectively. Black lines represent introns. <i>ttd14</i>-A and -B differ at the 3`end encoding proteins of 475 and 471 amino acids, respectively. <i>ttd14</i>-C has an additional exon and encodes a protein of 515 amino acids. The C to T mutation at position 751 and a P-element insertion (<i>CG30118</i>^{<i>KGO3769</i>}) are indicated by red arrowheads.</p

Photoreceptors of the <i>ttd14</i>^<i>P75L</i> mutant undergo light-enhanced, but Rh1-independent degeneration.

<p>(A) Transmission electron microscopy of tangential sections through eyes of wild type flies and homozygous mutant eye clones of <i>ttd14</i>^<i>P75L</i> mutant flies. Flies were assayed after eclosion (1 d), kept in the dark for 7 days followed by 16 hours orange light (7 d dark 16 h light), kept in the dark for 21 d, or subjected to a 12 hours light / 12 hours dark cycle for 21 d (21 d dark light). Flies were either raised on normal food (upper panels) or on vitamin A-deprived diet (low vitA; lower panels). 1–7 denotes rhabdomeres of photoreceptor cells R1 to R7. N, nucleus. Scale bar: 2.5 μm. Wild type flies display a normal morphology of the rhabdomeres at all conditions analyzed, except that vitamin A-deprived flies have smaller rhabdomeres due to the reduced amount of rhodopsin. Degeneration of inner and outer photoreceptor cells is obvious in <i>ttd14</i>^<i>P75L</i> mutants exposed to a light/dark cycle for 21 days. Interestingly, R7 cells show signs of cell death while R1-6 cells are not affected (arrowhead) in <i>ttd14</i>^<i>P75L</i> mutants kept in the dark for 21 days. (B) Time course of photoreceptor degeneration in the <i>ttd14</i>^<i>P75L</i> mutant raised on normal food or on vitamin A-deprived diet (low vitA). Fluorescent water immersion images of wild type flies and <i>ttd14</i>^<i>P75L</i> mutant eye clones, which express TRP-eGFP as a fluorescent marker for rhabdomeres in photoreceptor cells R1-6, were scored for the presence of rhabdomeres (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005578#sec012" target="_blank">Material and Methods</a>). 100% represents fully intact rhabdomeres. 3 ommatidia from five flies each were analyzed. Error bars denote SEM. Flies were kept in a 12 hours light / 12 hours dark cycle for the indicated number of days. Examples of original images used for this analysis are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005578#pgen.1005578.s003" target="_blank">S3 Fig</a>.</p

Rhabdomeral localization of Rh1 and TRP is not affected in the <i>ttd14</i>^<i>P75L</i> mutant.

<p>(A) Water immersion microscopy images of the Rh1-eGFP and TRP-eGFP fluorescence in the eyes of wild type flies (wild type) and <i>ttd14</i>^<i>P75L</i> mutant eye clones (<i>ttd14</i>^<i>P75L</i>). Flies were kept in constant darkness for 1 day (upper row), 7 days (middle row) or 28 days (lower row). Scale bar: 10 μm. (B) Analysis of Rh1-eGFP and TRP localization on cross sections through ommatidia of wild type flies (wild type) and <i>ttd14</i>^<i>P75L</i> mutant eye clones (<i>ttd14</i>^<i>P75L</i>), kept in darkness for 7 d. Rh1-eGFP localization was detected by its eGFP fluorescence (green, upper row), TRP localization was detected by an anti-TRP-antibody (green, upper row). The actin cytoskeleton of rhabdomeres was labeled with Alexa Fluor 546-coupled phalloidin (red, middle row). Overlay of red and green fluorescence appears yellow in the merged panels. Scale bar: 5 μm. (C) Immunoblot assessing Rh1 and TRP from heads of wild type flies (wild type, lanes 1–3), <i>ttd14</i>^<i>P75L</i> mutant flies (<i>ttd14</i>^<i>P75L</i>, lanes 4–6) and <i>ttd14</i>^<i>P75L</i><i>; Rh1</i>><i>ttd14-myc</i> flies (rescue, lane 7–9). Protein from 0.5 heads was loaded per lane. Flies were kept in darkness (lanes 1,4,7), exposed to white light (1800 lux) for 20 hours (lanes 2,5,8), or exposed to white light for 20 hours followed by a 6 hour recovery in the dark (lanes 3,6,9). The size of molecular weight markers in kilo Dalton is indicated at the left. (D) Quantification of the Rh1 levels normalized to TRP. Error bars show SEM (n = 4). Rh1 levels decrease after illumination due to lysosomal degradation of internalized Rh1. No significant differences were found between wild type, <i>ttd14</i>^<i>P75L</i> mutant and rescue flies at any light condition.</p