17 research outputs found
Antibody Guided Exploration of V3 Exposure on Subtype C HIV
The limited neutralizing activity of V3 antibodies is typically attributed to V3 masking. While relatively much effort has been devoted to exploring V3 accessibility on subtype B viruses, V3 exposure and mechanism(s) that might restrict V3 exposure on non-subtype B viruses have yet to be understood. I have focused on exploring the significance of the conserved V3 tip motifs GPGR and GPGQ of subtype B and nonsubtype B viruses for antibody recognition. Position 315 in representative subtype B and subtype C viruses was mutated to Gln and Arg respectively to assess the effect of the conserved Arg/Gln at position 315 on V3-specific neutralization. The Q315R subtype C viruses became sensitive to anti-V3 mAb B4e8 neutralization whereas the R315Q switched subtype B virus became resistant to V3 neutralization, even to V3 antibodies that do no contact the residue at position 315. These observations suggest that at least the tip of V3 is antibody accessible on the surface of some non-B viruses but it is the presence of Gln315 residue that modulates V3 antibody recognition. As such, engineering V3 tip antibodies to make high affinity interactions with a Gln315 residue could broaden V3 neutralization of non-subtype B viruses. To attempt to improve B4e8’s neutralizing activity, targeted mutagenesis of B4e8 complementarity determining region residues was done to generate antibody libraries using yeast and phage display. The libraries were subjected to multiple rounds of selection on subtype C gp120s, however B4e8 variants with enhanced affinity for subtype C gp120 were not recovered after the final rounds of selection. This suggests that the residues targeted here were not sufficient for enhancing B4e8 affinity for non-subtype B HIV
Characterizing the role of nuclear pore complexes in genome insertion of the yeast Ty1 retrotransposon
Nuclear pore complexes (NPCs) orchestrate cargo between the cytoplasm and nucleus and regulate chromatin organization. NPC proteins, or nucleoporins (Nups), are required for human immunodeficiency virus type 1 (HIV-1) gene expression and genomic integration of viral DNA. I utilize the Ty1 retrotransposon of Saccharomyces cerevisiae (S. cerevisiae) to study retroviral integration because retrotransposons are the progenitors of retroviruses and have conserved integrase (IN) enzymes. Ty1-IN targets Ty1 elements into the genome upstream of RNA polymerase (Pol) III-transcribed genes such as transfer RNA (tRNA) genes. Evidence that S. cerevisiae tRNA genes are recruited to NPCs prompted my investigation of a functional role for the NPC in Ty1 targeting into the genome. I find that Ty1 mobility is reduced in multiple Nup mutants that cannot be accounted for by defects in Ty1 gene expression, complementary DNA (cDNA) production or Ty1-IN nuclear entry. Instead, I find that Ty1 insertion upstream of tRNA genes is impaired. I also identify Nup mutants with wild type Ty1 mobility but impaired Ty1 targeting. The NPC nuclear basket, which interacts with chromatin, is required for both Ty1 expression and nucleosome targeting. Deletion of components of the NPC nuclear basket causes mis-targeting of Ty1 elements to the ends of chromosomes. The mis-targeting suggests that nuclear basket Nups are required directly or indirectly, perhaps as global architects or regulators of chromatin organization to orchestrate Ty1 targeting upstream of Pol III-transcribed genes.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat
Retrotransposon targeting to RNA polymerase III-transcribed genes
Retrotransposons are genetic elements that are similar in structure and life cycle to retroviruses by replicating via an RNA intermediate and inserting into a host genome. The Saccharomyces cerevisiae (S. cerevisiae) Ty1–5 elements are long terminal repeat (LTR) retrotransposons that are members of the Ty1-copia (Pseudoviridae) or Ty3-gypsy (Metaviridae) families. Four of the five S. cerevisiae Ty elements are inserted into the genome upstream of RNA Polymerase (Pol) III-transcribed genes such as transfer RNA (tRNA) genes. This particular genomic locus provides a safe environment for Ty element insertion without disruption of the host genome and is a targeting strategy used by retrotransposons that insert into compact genomes of hosts such as S. cerevisiae and the social amoeba Dictyostelium. The mechanism by which Ty1 targeting is achieved has been recently solved due to the discovery of an interaction between Ty1 Integrase (IN) and RNA Pol III subunits. We describe the methods used to identify the Ty1-IN interaction with Pol III and the Ty1 targeting consequences if the interaction is perturbed. The details of Ty1 targeting are just beginning to emerge and many unexplored areas remain including consideration of the 3-dimensional shape of genome. We present a variety of other retrotransposon families that insert adjacent to Pol III-transcribed genes and the mechanism by which the host machinery has been hijacked to accomplish this targeting strategy. Finally, we discuss why retrotransposons selected Pol III-transcribed genes as a target during evolution and how retrotransposons have shaped genome architecture.Land and Food Systems, Faculty ofMedicine, Faculty ofBiochemistry and Molecular Biology, Department ofReviewedFacult
A Role for the Budding Yeast Separase, Esp1, in Ty1 Element Retrotransposition
<div><p>Separase/Esp1 is a protease required at the onset of anaphase to cleave cohesin and thereby enable sister chromatid separation. Esp1 also promotes release of the Cdc14 phosphatase from the nucleolus to enable mitotic exit. To uncover other potential roles for separase, we performed two complementary genome-wide genetic interaction screens with a strain carrying the budding yeast <i>esp1-1</i> separase mutation. We identified 161 genes that when mutated aggravate <i>esp1-1</i> growth and 44 genes that upon increased dosage are detrimental to <i>esp1-1</i> viability. In addition to the expected cell cycle and sister chromatid segregation genes that were identified, 24% of the genes identified in the <i>esp1-1</i> genetic screens have a role in Ty1 element retrotransposition. Retrotransposons, like retroviruses, replicate through reverse transcription of an mRNA intermediate and the resultant cDNA product is integrated into the genome by a conserved transposon or retrovirus encoded integrase protein. We purified Esp1 from yeast and identified an interaction between Esp1 and Ty1 integrase using mass spectrometry that was subsequently confirmed by co-immunoprecipitation analysis. Ty1 transposon mobility and insertion upstream of the <i>SUF16</i> tRNA gene are both reduced in an <i>esp1-1</i> strain but increased in cohesin mutant strains. Securin/Pds1, which is required for efficient localization of Esp1 to the nucleus, is also required for efficient Ty1 transposition. We propose that Esp1 serves two roles to mediate Ty1 transposition – one to remove cohesin and the second to target Ty1-IN to chromatin.</p></div
Esp1 and Pds1 are required for Ty1 element insertion upstream of the <i>SUF16</i> locus.
<p>(A) Schematic of the <i>SUF16</i> genomic locus with PCR primers (arrows) designed to hybridize within the <i>SNR33</i> gene and the newly inserted Ty1 elements. (B) and (C) PCR analysis of yeast genomic DNA extracted from indicated strains grown in triplicate for 3 days at 20°C to induce transposition. Upper panel is the result of the PCR assay with primers shown in (A) whereas lower panel is a control PCR for the <i>CPR7</i> locus to demonstrate that yeast genomic DNA was present in each sample. Quantification of Ty1 insertion events is shown relative to wild type (WT) as described in the Materials and Methods.</p
Pds1 and Smc1 are not required for the Esp1-Ty1-IN interaction.
<p>(A) Quantitative PCR analysis of <i>PDS1</i> expression driven from the <i>TetO</i><sub><i>7</i></sub> promoter after 100μg/mL doxycycline addition. The relative quantity of <i>PDS1</i> transcript compared to a control (<i>TAF10</i>) is shown. (B) Esp1-GFP and untagged (No Tag) cells containing the <i>TetO</i><sub><i>7</i></sub>-<i>PDS1</i> allele and <i>pGAL1-Ty1-H3</i> were induced for 24 hours with 2% galactose, then 100μg/mL doxycycline was added (+) or not (-) for one hour. Esp1-GFP was purified with GFP-Trap beads, immunoblot analysis performed and probed with anti-GFP and anti-IN (8b11) antibodies. (C) Esp1-GFP and untagged (No Tag) cells containing the <i>TetO</i><sub><i>7</i></sub>—<i>SMC1</i> allele and <i>pGAL1-Ty1-H3</i> were induced for 24 hours with 2% galactose, then 100μg/mL doxycycline was added (+) or not (-) for six hours. Esp1-GFP was purified as in (B). The depletion of Smc1 was monitored with anti-Smc1 antibodies and the lysate was probed with anti-Pgk1 as a loading control.</p
All Esp1 domains contribute to Ty1 transposition.
<p>(A,B) The indicated strains, carrying the <i>pGTy1-H3-mhis3AI–URA3</i> plasmid, were patched and induced to undergo transposition on galactose media at the indicated temperatures. After transposition, plates were replica plated to 5-FOA to remove the <i>URA3</i> plasmid as described in the Materials and Methods. Patches are shown after the final step of replica plating. The SC (minimal complete) plates are a control for growth and the colonies present on the SC-HIS plates represent insertion of the <i>Ty1-HIS3</i> element into the genome. (C,D) Triplicate isolates of indicated <i>esp1</i> ts alleles, carrying a <i>pGAL-TyH3mHIS3AI-URA3</i> plasmid (pJBe376) were induced to undergo transposition at semi-restrictive temperature (30°C) by growth in 2% galactose for 24 hours (GAL). One isolate was grown for 24 hours in 2% glucose (D) as a control. <i>SUF16</i> PCR analysis of the extracted yeast genomic DNA is shown and <i>CPR7</i> is a control PCR. All bands in the three lanes for each GAL grown isolate were quantified and summed, then compared to the wild type strain. The relative values of the mutants compared to wild type (value of 1.0) is shown. The amino acid mutations of the <i>esp1</i> alleles have been previously been published: <i>esp1n122</i> (N90S, C511F), <i>esp1b120</i> (K782E, I951T, I1040T), <i>esp1c113</i> (F1327L H1391Y) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005109#pgen.1005109.ref026" target="_blank">26</a>].</p
Esp1 physically interacts with Ty1-IN.
<p>(A) Ty1 peptides identified in Esp1-Myc mass spectrometry versus untagged (mock) strain are color coded as follows: Gag/coat protein (blue), PR (yellow), IN (orange) and RT/RNAse H (green). (B) Immunoblot of whole cell lysate (Lysate) and GFP-Trap IP carried out from untagged wild type (No Tag), Esp1-GFP, Scc1-GFP, Pds1-GFP and Ndc80-GFP cells. Expression of a Ty1 element (<i>pGAL1-Ty1-H3</i>) was induced in all strains for 24 hours prior to cell lysis. Blots were probed with anti-GFP and anti-IN (8b11) antibodies. (C) Immunoblot of whole cell lysate (Lysate) and GFP-Trap IP carried out from Esp1-Myc or untagged wild type (No Tag) cells carrying a pGAL-GFP-LacZ-Ty1-IN plasmid (Ty1-IN) or pGAL-GFP-lacZ (vector) control. Cells were either grown in glucose (D) or galactose (GAL) for 24 hours to repress or induce GFP-LacZ-Ty1-IN expression, respectively. The asterisk marks a background band that is present in the lysate of the cells grown in glucose but is not detected in the GFP-Trap IP.</p
Ty1 cDNA levels are not affected in <i>esp1</i> and <i>pds1</i> mutants.
<p>(A) Southern blot analysis of <i>AflII</i> digested yeast genomic DNA isolated from the indicated mutants carrying the <i>pGTy1-H3-mhis3AI</i> plasmid after 24 hour induction with 2% galactose. One wild type (WT) sample was also grown in 2% glucose (GLU) as a control for <i>TY1</i> element expression. The blot was probed with a radiolabeled <i>HIS3</i> gene. Shown are the fragment sizes for Ty1 cDNA (∼2.4kb), the endogenous <i>HIS3</i> locus of the S288C deletion strain with 200bp deleted (<i>his3Δ1</i>, ∼6.5kb) and the <i>pGTy1-H3-mhis3AI</i> plasmid which is linearized (∼14kb). Below each lane is the quantitative ratio of Ty1 cDNA signal to <i>his3Δ1</i> signal. (B) Southern blot analysis of <i>PvuII</i> digested yeast genomic DNA isolated from the indicated mutants after 2 days growth at 20°C. Endogenous Ty1 elements and cDNA were detected with a radiolabelled <i>PvuII/SnaBI</i> Ty1 element fragment. Below each lane is the quantitative ratio of the Ty1 endogenous cDNA (∼2kb) to an endogenous Ty element (Ty1 control). <i>spt3Δ</i> serves as a negative control for Ty1 cDNA levels. (C) Endogenous Ty1-Gag processing in wild type (WT) versus <i>esp1-1</i> cells grown at 25°C or incubated for 6 hours at 30°C assessed by immunoblot. Blot was probed with anti-Gag antibody. p49 = unprocessed Gag; p45 = processed Gag. <i>spt3Δ</i> serves as a negative control for Gag expression.</p
<i>esp1-1</i> functional interaction map derived from the SDL screen.
<p>The 44 genes identified in the <i>esp1-1</i> SDL screen were subjected to Cytoscape analysis. All nodes are significantly enriched (p <0.05) GO Terms in the dataset and coloured nodes represent GO Terms that have been grouped into a significantly enriched category. Grey nodes are GO Terms that were not grouped into an enriched category. Edges define associations between groups and edge thickness indicates the level of significance within the network. Genes identified in the SDL screen that are associated with GO Terms are shown.</p