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Hey! I'm Julia. Welcome to my blog. Here's every post I've ever written. If you want somewhere to start, try my favorites (the ones with stars next to them, below). Enjoy!
|A few sketches
|Benchmarking correctly is hard (and techniques for doing it better)
|One way to make containers network: BGP
|How do HTTP requests get sent to the right place?
|Linux debugging tools I love
|PolyConf 2016
|Why do we use the Linux kernel's TCP stack?
|Using ltrace to debug a memory leak
|Should you be scared of Unix signals?
|How to spy on a Ruby program
|A useful new strace feature
|Ideas for making better conference talks & conferences
|Learning to like design documents
|Three ways to solve hard programming problems
|"advice" for aspiring tech bloggers
|Notes from my PyData Berlin keynote
|A few reasons to be skeptical of machine learning
|Investigating Erlang by reading its system calls
|homu + highfive: awesome bots that make open source projects easier
|A second try at using Rust
|A workshop on strace & tcpdump
|What are SSL ciphers & session keys?
|The Etsy site performance report is amazing
|How does knowledge get locked up in people's heads?
|CDNs aren't just for caching
|you can take the derivative of a regular expression?!
|Some links on Java garbage collection
|Java garbage collection can be really slow
|Looking inside machine learning black boxes
|Some of my favorite blogs
|I conquered thread pools! For today, at least.
|Thread pools! How do I use them?
|I/O matters.
|How do you do capacity planning?
|tcpdump is amazing
|How does perf work? (in which we read the Linux kernel source)
|Women-only spaces are a hack
|Surprises in Ruby HTTP libraries
|What is "the stack"?
|perf top: an awesome way to spy on CPU usage
|How to measure your CPU time: clock_gettime!
|Why I love log files
|Have high expectations for your computers
|TIL: clock skew exists
|How CPU load averages work (and using them to triage webserver performance!)
|Fast integer sets with Roaring Bitmaps (and, making friends with your modern CPU)
|Sendfile (a system call for web developers to know about!)
|Guessing Linux kernel registers
|Calling C from Rust
|A few notes on my CUSEC talk
|Why I ❤ Rust
|Talking about stock options = awesome
|Java isn't slow
|Winning the bias-variance tradeoff
|Surviving meetings while remote
|Things you should know about stock options before negotiating an offer
|2015: Year in review
|How to trick a neural network into thinking a panda is a vulture
|Women in Machine Learning 2015 (fun!!!)
|Why Ruby's Timeout is dangerous (and Thread.raise is terrifying)
|How I got better at debugging
|Why you should understand (a little) about TCP
|Docker is amazing
|PAPERS ARE AMAZING: Profiling threaded programs with Coz
|A millisecond isn't fast (and how we made it 100x faster)
|Is machine learning safe to use?
|An argument for hiring junior developers
|Some easy statistics: Bootstrap confidence intervals
|AdaCamp Montreal 2015
|A zine about strace
|Learning at open source sprints (no preparation required)
|A few spy tools for your operating system (other than strace!)
|Seeing system calls with perf instead of strace
|Senior engineering & fantasy heroes
|Nancy Drew and the Case of the Slow Program
|You can choose who submits talks to your conference
|1:1 topic ideas
|How the locate command works (and let's write a faster version in one minute!)
|How gzip uses Huffman coding
|How I learned to program in 10 years
|A/A testing
|Why a C++ programmer might say "I could never write Python"
|Data Day Texas 2015
|On reading the source code, not the docs
|Programming doesn't belong to men (it belongs to me)
|Fear makes you a worse programmer
|Reproducing awesomeness
|Diving into concurrency: trying out mutexes and atomics
|Spying on Hadoop with strace
|LD_PRELOAD is super fun. And easy!
|PyData NYC (I gave a machine learning talk! yay!)
|Harm reduction for developers
|Fun with machine learning: logistic regression!
|Fun with machine learning: does your model actually work?
|What women in technology really think (150 of them, at least)
|Working remote, 8 months in (seeing humans is important!)
|How to set up a blog in 5 minutes
|How does SQLite work? Part 2: btrees! (or: disk seeks are slow don't do them!)
|How does SQLite work? Part 1: pages!
|Strange Loop 2014
|You can be a kernel hacker!
|How is a binary executable organized? Let's explore it!
|What happens if you write a TCP stack in Python?
|Pair programming is amazing! Except... when it's not.
|Open sourced talks!
|Ruby Rogues podcast: systems programming tricks!
|Fun with stats: How big of a sample size do I need?
|How I did Hacker School: ignoring things I understand and doing the impossible
|!!Con talks are up
|Machine learning isn't Kaggle competitions
|Asking questions is a superpower
|Working remote, 3 months in
|Should my conference do anonymous review?
|Anonymous talk review is amazing.
|Diving into HDFS
|I can spy on my CPU cycles with perf!
|Computers are *fast*!
|Stopping to think
|Don't feel guilty about not contributing to open source
|Debug your programs like they're closed source!
|♥ PyCon
|Becoming a better developer (it's not just writing more programs)
|"Ask if you have questions" isn't enough
|Reports from remote-land: remote pairing works great!
|Recovering files using /proc (and spying, too!)
|My Rust OS will never be finished (and it's a success!)
|Writing an OS in Rust in tiny steps (Steps 1-5)
|Debugging shared library problems with strace
|Hacker School's Secret Strategy for Being Super Productive (or: Help.)
|Pair programming
|More practical uses for strace!
|Using strace to avoid reading Ruby code
|A resume workshop idea
|A bookmarklet to flee from Mailman archives
|Spying on ssh with strace
|"How was Hacker School?"
|A few blogs I really like
|Three steps to learning GDB
|Sounding confident in interviews
|CUSEC = fun
|What my technical interviews have looked like
|You should give talks. They will be awesome. Here's why!
|4 paths to being a kernel hacker
|Some things my kernel can't do
|Sketches for a graph layout engine
|Questions I'm asking in interviews
|Coding strategies
|When is "guys" gender neutral? I did a survey!
|Understanding how killall works using strace
|A pandas cookbook
|Day 46: Never Graduate
|Day 45: I now have Rust code for executing executables!
|Day 44: qemu + gdb = so great
|Day 43: SOMETHING IS ERASING MY PROGRAM WHILE IT'S RUNNING (oh wait oops)
|Day 42: How to run a simple ELF executable, from scratch (I don't know)
|Day 41: Linkers are upsetting
|Day 40: 12 things I learned today about linkers.
|Day 40: Linkers are amazing.
|Day 39: Writing malloc wrong, for fun
|Day 38: After 6 days, I have problems that I can't understand at all
|Day 37: After 5 days, my OS doesn't crash when I press a key
|Day 36: On programming without malloc
|Day 35: Types in Rust, for beginners
|How to call Rust from assembly, and vice versa
|Videos from PyData NYC are up!
|Writing a kernel using rustboot & rust-core
|What happens when you run 'Hello, world'
|Day 34b: Writing curl using my TCP stack
|Day 34: The tiniest operating system
|Day 33: How to make music with ClojureScript
|Day 32: Magical Orchestra!!!
|Day 31: Binary trees with core.logic!
|Day 30: Making music in Clojure with Overtone. Clojure bugs with laziness! Fun!
|Day 29: Trying out Emacs!
|Day 28: Made a git workflow visualization webapp!
|Day 27: Automatically testing changes in state! Visualizing my Git workflow! Floats!
|Women in Technology workshop at PyData NYC
|Day 26: Trying to describe the TCP state machine in a readable way. Failing.
|Day 25: ACK all the things
|Day 24: Unit testing this TCP library
|Day 23: Started writing a TCP stack in Python
|NYC Python talk
|Day 22: Got some TCP packets back!
|Day 21: Trying to TCP
|Day 20: Traceroute in 15 lines of code using Scapy
|Day 19: A few reasons why networking is hard
|Day 18: ARP cache poisoning (or: In ur connection, sniffing ur packets)
|Day 17: How to write a buffer overflow exploit
|Do Rails programmers use node.js? Visualizing correlations in command usage
|Day 16: gzip + poetry = awesome
|Day 15: How a .gz file is structured, redux
|Day 14: When it's hard to write tests, that's when I should be testing
|Day 13: Off by one errors
|Day 12: Why Julia likes Julia
|Day 11: How does gzip work?
|Day 10: Goals. Goals? Maybe I need to be *reading* more code?
|Day 9: Bytecode is made of bytes! CPython isn't scary!
|Day 8: Julia writes Julia! And remembers that open source is hard.
|Day 7: An echo server in Clojure
|Day 6: I wrote a rootkit!
|Day 5: I wrote a kernel module!!!
|Where to find bike sharing systems' data feeds
|Day 4: Processes vs threads, and kernel modules!
|Day 3: What does the Linux kernel even do?
|Day 2: netcat fun!
|Day 1: What does a shell even do?
|Hacker School Day 1: Messing around with the stack in C
|Hacker School Day -4: unit testing in C. checkmk!
|Julia Serano's 'Excluded'
|PyData Boston 2013
|Open Data Exchange 2013
|Transit maps: 1941 vs 2011
|Graphing bike path data with IPython Notebook and pandas
|Trying out Octopress
|Montreal All-Girl Hack Night #2: now with cookies
|Montreal All-Girl Hack Night #1: AMAZING


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Assistant Professor , Dance Concentration
Salem State College
Direct Phone: (978) ***-****
Email: m***@***.edu
352 Lafayette Street
Salem, Massachusetts 01970
United States
Salem State College is a comprehensive, fully accredited, four-year institution. Approximately 9,200 undergraduate and graduate students representing 27 states and 52 nations matriculated during the 2003-04 academic year. Salem State is the fifth largest ... more
Assistant Professor
Massachusetts Dance Education Organization
The Colleges
Point Park University
dance and arts management
American University
master classes
Massachusetts Association for Health , Physical Education , Recreation and Dance
master classes
Tufts University
master classes
American College Dance Festival
master classes
Green Street Studios
Trident Gallery - representing contemporary artists on historic Cape Ann - 189 Main Street - Gloucester - Massachusetts
tridentgallery.com [cached]
Three showcases the distinct choreographic voices of Caitlin Corbett, Meghan McLyman, and James Morrow in the intimate setting of Trident Gallery.
Mentors for the Summer 2012 session ...
greenstreetstudios.org [cached]
Mentors for the Summer 2012 session are Heidi Henderson, Meghan McLyman (EA Alumni), and Micki Taylor-Pinney.
www.fallingflightproject.com [cached]
Accumulation Dance: Boston Contemporary Dance Ensemble
www.accumulationdance.org [cached]
Social networking gone haywire and much more | NORTH SHORE ART THROB
www.nsartthrob.com [cached]
Greg McLyman
OST Inc
Linda McLyman
Progress Associates
Ted McLyman
Apexx Behavioral Solutions Group LLC
Mischa McLyman
Peter McLyman
Country Club of Vermont

subset: commoncrawl


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Update custom web part
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We have a custom webpart for Sharepoint 2010. We would like to add to additional features to the existing code.
1. Make the hyperlink url open in the modal dialog window
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We have a custom webpart to display news that works great. We would just like these two enhancements and our developer is new to Sharepoint development. We will provide you with the code once a bid has been accepted.
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Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.
Hepatitis C virus (HCV; a single-stranded, positive-sense RNA virus that is classified in the family Flaviviridae) is the main cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (5, 26, 45). More than 170 million people worldwide are chronically infected with HCV (41). The approximately 9.6-kb HCV genome encodes a unique open reading frame that is translated into a polyprotein (5, 54). The polyprotein is cleaved cotranslationally into at least 10 proteins by viral proteases and cellular signalases (6, 10).
The HCV core protein represents the first 1 to 191 amino acids (aa) of the polyprotein and is followed by two glycoproteins, E1 and E2 (6). The core protein plays a central role in the packaging of viral RNA (25, 40); modulates various cellular processes, including signal transduction pathways, transcriptional control, cell cycle progression, apoptosis, lipid metabolism, and the immune response (9, 40); and has transforming potential in certain cells (43). Mice transgenic for the HCV core gene develop steatosis (32) and later hepatocellular carcinoma (31). These findings suggest that HCV core protein plays a crucial role in hepatocarcinogenesis.
Two major forms of the HCV core protein, p21 (mature form) and p23 (immature form), can be generated in cultured cells (60). Cellular signal peptidase cleaves at the junction of the core/E1, releasing the immature form of the core protein from the polypeptide (12, 46). Signal peptide peptidase cleaves just before the signal sequence, liberating the mature form of the HCV core protein at the cytoplasmic face of the endoplasmic reticulum (29). Several different sites have been proposed as potential cleavage sites of signal peptide peptidase, such as Leu-179 (15, 29), Phe-177 (36, 37), Leu-182 (15), and Ser-173 (46). Further processing of the HCV core protein yields a 17-kDa product with a C terminus at around amino acid 152. A truncated form of the core protein, p17, was found in transfected cells (42, 52) and liver tissues from humans with hepatocellular carcinoma (59). The majority of this protein translocates to the nucleus. The C terminus of the core protein is important for regulating the stability of the protein (20, 52).
We previously showed that the C-terminally truncated forms of the core protein are degraded through the ubiquitin-proteasome pathway (52). We found that the mature form of the core protein, p21, also links to a few ubiquitin moieties, suggesting that the ubiquitin-proteasome pathway involves proteolysis of heterologous species of the core protein (52). Overexpression of PA28γ (a REG family proteasome activator also known as REGγ or Ki antigen) enhances the proteasomal degradation of the HCV core protein (30). A recent study has shown that PA28γ is involved in the degradation of the steroid receptor coactivator 3 (SRC-3) in an ATP- and ubiquitin-independent manner (27). It is still unclear what E3 ubiquitin ligase is responsible for ubiquitylation of the HCV core protein.
E6AP was initially identified as the cellular factor that stimulates ubiquitin-mediated degradation of the tumor suppressor p53 in conjunction with the E6 protein of cancer-associated human papillomavirus types 16 and 18 (14, 48). The E6-E6AP complex functions as a E3 ubiquitin ligase in the ubiquitylation of p53 (49). E6AP is the prototype of a family of ubiquitin ligases called HECT domain ubiquitin ligases, all of which contain a domain homologous to the E6AP carboxyl terminus (13). Interestingly, E6AP is not involved in the regulation of p53 ubiquitylation in the absence of E6 (55). Several potential E6-independent substrates for E6AP have been identified, such as hHR23A, Blk, and Mcm7 (23, 24, 35). E6AP is also a candidate gene for Angelman syndrome, which is a severe neurological disorder characterized by mental retardation (21).
This study aimed to identify endogenous ubiquitin-proteasome pathway proteins that are associated with HCV core protein. Tandem affinity purification and mass spectrometry analysis identified E6AP as an HCV core-binding protein. Here we present evidence that E6AP associates with HCV core protein in vitro and in vivo and is involved in ubiquitylation and degradation of HCV core protein. We propose that an E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of HCV core protein.
Human embryonic kidney 293T cells, human hepatoblastoma HepG2 cells, and human hepatoma Huh-7 cells were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 50 IU/ml penicillin, 50 μg/ml streptomycin (Invitrogen), and 10% (vol/vol) fetal bovine serum (JRH Biosciences) at 37°C in a 5% CO2 incubator. 293T cells and HepG2 cells were transfected with plasmid DNA using FuGene 6 transfection reagents (Roche). Huh-7 cells were transfected with plasmid DNA using TransIT LT1 transfection reagents (Mirus).
MEF tag cassette (containing myc tag, the tobacco etch virus protease cleavage site, and FLAG tag) (16) was fused to the N terminus of the cDNA encoding core protein of HCV NIHJ1 (genotype 1b) (1). To express MEF-tagged core protein in mammalian cells, the genome coding for HCV core protein (amino acids 1 to 191) was amplified by PCR using pBR HCV NIHJ1 as a template. Sense oligonucleotide containing a Kozak consensus translation initiation codon and antisense oligonucleotide containing an in-frame translation stop codon were synthesized by PCR. The amplified PCR product was purified, digested with EcoRI and EcoRV, and then inserted into the EcoRI-EcoRV site of pcDNA3-MEF. FLAG-tagged HCV core expression plasmids based upon pCAGGS (34) were described previously (30). To express E6AP and the active-site cysteine-to-alanine mutant of E6AP in mammalian cells, pCMV4-HA-E6AP isoform II and pCMV4-HA-E6AP C-A were utilized (19). The C-A mutation was introduced at the site of E6AP C843. To express E6AP and E6AP C-A under the CAG promoter, the E6AP fragment and the E6AP C-A fragment were amplified by PCR, purified, digested with SmaI and NotI, and blunt ended using a DNA blunting kit (Takara). These PCR fragments were subcloned into pCAGGS.
To make a fusion protein consisting of glutathione S-transferase (GST) fused to the N terminus of E6AP in Escherichia coli, the E6AP fragment was amplified by PCR and the resultant product was cloned into the SmaI-NotI site of pGEX4T-1 vector (Amersham Biosciences). To express a series of E6AP truncation mutants as GST fusion proteins, each fragment was amplified by PCR and cloned into the SmaI-NotI site of pGEX4T-1. To purify GST core protein efficiently by two-step affinity purification, we fused hexahistidine (His) tag to the C terminus of GST fusion proteins. To bacterially express HCV core (aa 1 to 173) protein as a fusion protein containing N-terminal GST tag and C-terminal His tag, core fragment was amplified by PCR and the resultant product was cloned into the EcoRI-NotI site of pGEX4T-1 vector. The resultant plasmid was designated pGEX GST-C173HT. To express GST core (1-152)-His and GST-His in E. coli, pGEX core (1-152)-His and pGEX-His were constructed similarly. The resultant plasmids were designated pGEX GST-C152HT and pGEX GST-HT, respectively.
To generate recombinant baculoviruses expressing GST-E6AP, GST-E6AP fragment was excised from pGEX E6AP by digestion with SmaI and Tth111I and ligated into the SmaI-Tth111I site of pVL1392 (Invitrogen). To express GST-E6AP C-A, pVLGST-E6AP C-A was constructed similarly. To generate recombinant baculovirus expressing HCV core (aa 1 to 173) protein as a fusion protein containing N-terminal GST tag and C-terminal His tag, GST-C173HT fragment was amplified by PCR using pGEX GST-C173HT as a template, digested with BglII-XbaI, and subcloned into the BglII-XbaI site of pVL1392. To generate recombinant baculoviruses expressing GST-C152HT and GST-HT, cDNA fragments corresponding to GST-C152HT and GST-HT were amplified by PCR and subcloned into pVL1392, respectively. The resultant plasmids were designated pVLGST-C173HT, pVLGST-C152HT, and pVLGST-HT. To generate recombinant baculovirus expressing MEF-tagged E6AP, cDNA fragment encoding MEF-E6AP was subcloned into pVL1392. To express HCV core protein in the TNT-coupled wheat germ lysate system (Promega), HCV core cDNA was inserted in the EcoRI site of pCMVTNT (Promega). The primer sequences used in this study are available from the authors upon request. The sequences of the inserts were extensively verified using an ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems). Recombinant baculoviruses were recovered using a BaculoGold transfection kit (Pharmingen) according to the manufacturer's instructions.
The mouse monoclonal antibodies (MAbs) used in this study were anti-hemagglutinin (anti-HA) MAb (12CA5; Roche), anti-FLAG (M2) MAb (Sigma), anti-c-myc MAb (9E10; Santa Cruz), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) MAb (Chemicon), anti-GST MAb (Santa Cruz), anti-ubiquitin MAb (Chemicon), anti-E6AP MAb (E6AP-330) (Sigma), anticore MAb (B2; Anogen), and another anti-core MAb (2H9) (56). Polyclonal antibodies (PAbs) used in this study were anti-HA rabbit PAb (Y-11; Santa Cruz), anti-FLAG rabbit PAb (F7425; Sigma), anti-E6AP rabbit PAb (H-182; Santa Cruz), anti-DDX3 rabbit PAb (47), anti-PA28γ rabbit PAb (Affiniti), and anti-GST goat PAb (Amersham). Anticore rabbit PAb (TS1) was raised against the recombinant GST core protein.
293T cells were transfected with the plasmid expressing MEF core by the calcium phosphate precipitation method (4). After the cells were lysed, the expressed MEF core and its binding proteins were recovered following the procedure described previously (16). 293T cells transfected with pcDNA3-MEF core in four 10-cm dishes were lysed in 2 ml of lysis buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% (wt/vol) glycerol, 100 mM NaF, 1 mM Na3VO4, 1% (wt/vol) Triton X-100, 5 μM ZnCl2, 2 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 1 μg/ml leupeptin. The lysate was centrifuged at 100,000 × g for 20 min at 4°C. The supernatant was passed through a 5-μm filter, incubated with 100 μl of Sepharose beads for 60 min at 4°C, and then passed through a 0.65-μm filter. The filtered supernatant was mixed with 100 μl of anti-myc-conjugated Sepharose beads for the first immunoprecipitation. After incubation for 90 min at 4°C, the beads were washed five times with 1 ml of TNTG buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% [wt/vol] glycerol, and 1% [wt/vol] Triton X-100), twice with 1 ml of buffer A (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1% [wt/vol] Triton X-100), and finally once with 1 ml of TNT buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% [wt/vol] Triton X-100). The washed beads were incubated with 10 U of tobacco etch virus protease (Invitrogen) in TNT buffer (100 μl) to release bound protein complexes from the beads. After incubation for 60 min at room temperature, the supernatant was pooled and the beads were washed twice with 70 μl of buffer A. The resulting supernatants were combined and incubated with 12 μl of FLAG-Sepharose beads for the second immunoprecipitation. After incubation for 60 min at room temperature, the beads were washed three times with 240 μl of buffer A, and proteins bound to the immobilized HCV core protein on the FLAG beads were dissociated by incubation with 80 μg/ml FLAG peptide (NH2-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-COOH) (Sigma).
Proteins were separated by 9% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by silver staining. The stained bands were excised and digested in the gel with lysylendoprotease-C (Lys-C), and the resulting peptide mixtures were analyzed using a direct nanoflow liquid chromatography-tandem mass spectrometry (MS/MS) system (33), equipped with an electrospray interface reversed-phase column, a nanoflow gradient device, a high-resolution Q-time of flight hybrid mass spectrometer (Q-TOF2; Micromass), and an automated data analysis system. All the MS/MS spectra were searched against the nonredundant protein sequence database maintained at the National Center for Biotechnology Information using the Mascot program (Matrixscience) to identify proteins. The MS/MS signal assignments were also confirmed manually.
E. coli BL21(DE3) cells were transformed with plasmids expressing GST fusion protein or His-tagged protein and grown at 37°C. Expression of the fusion protein was induced by 1 mM isopropyl-β-d-thiogalactopyranoside at 37°C for 4 h. Bacteria were harvested, suspended in lysis buffer (phosphate-buffered saline [PBS] containing 1% Triton X-100), and sonicated on ice.
Hi5 cells were infected with recombinant baculoviruses to produce GST-C173HT, GST-C152HT, GST-HT, MEF-E6AP, and His-tagged mouse E1 (17). GST and GST fusion proteins were purified on glutathione-Sepharose beads (Amersham Bioscience) according to the manufacturer's protocols. His-tagged proteins were purified on nickel-nitrilotriacetic acid beads (QIAGEN) according to the manufacturer's protocols. MEF-E6AP and MEF-E6AP C-A were purified on anti-FLAG M2 agarose beads (Sigma) according to the manufacturer's protocols.
Immunoblot analysis was performed essentially as described previously (11). The membrane was visualized with SuperSignal West Pico chemiluminescent substrate (Pierce).
To map the E6AP binding site on HCV core protein, 2.5 μg of purified recombinant GST-E6AP expressed in Hi5 cells was mixed with 1,000 μg of 293T cell lysates transfected with a series of FLAG-tagged HCV core deletion mutants as indicated. The protein concentration of the cells was determined using the bicinchoninic acid protein assay kit (Pierce). The mixtures were immunoprecipitated with anti-FLAG M2 agarose beads (Sigma), and proteins bound to the immobilized HCV core protein on anti-FLAG beads were dissociated with FLAG peptide (Sigma). The eluates were analyzed by immunoblotting with anti-GST PAb. To map the HCV core-binding site on E6AP, GST pull-down assays were performed as described previously (51).
In vivo ubiquitylation assays were performed essentially as described previously (57). FLAG-core was immunoprecipitated with anti-FLAG beads. Immunoprecipitates were analyzed by immunoblotting, using either anti-HA PAb or anticore PAb (TS1) to detect ubiquitylated core proteins.
For in vitro ubiquitylation of HCV core protein, purified GST-C173HT and GST-C152HT were used as substrates. Purified GST-HT was used as a negative control. Assays were done in 40-μl volumes containing 20 mM Tris-HCl, pH 7.6, 50 mM NaCl, 5 mM ATP, 10 mM MgCl2, 8 μg of bovine ubiquitin (Sigma), 0.1 mM dithiothreitol, 200 ng mouse E1, 200 ng E2 (UbcH7), and 0.5 μg each of MEF-E6AP or MEF-E6AP C-A. The reaction mixtures were incubated at 37°C for 120 min followed by purification with glutathione-Sepharose beads and immunoblotting with the indicated antibodies.
293T cells or Huh-7 cells at 3 × 105 cells in a six-well plate were transfected with 40 pmol of either E6AP-specific short interfering RNA (siRNA; Sigma) or scramble negative-control siRNA duplexes (Sigma) using HiPerFect transfection reagent (QIAGEN) following the manufacturer's instructions. The siRNA target sequences were as follows: E6AP (sense), 5′-GGGUCUACACCAGAUUGCUTT-3′; scramble negative control (sense), 5′-UUGCGGGUCUAAUCACCGATT-3′.
To examine the half-life of HCV core protein, transfected 293T cells were treated with 50 μg/ml cycloheximide (CHX) at 44 h posttransfection. The cells at zero time points were harvested immediately after treatment with CHX. Cells from subsequent time points were incubated in medium containing CHX at 37°C for 3, 6, and 9 h as indicated.
Infectious HCV JFH1 was produced in Huh-7.5.1 cells (61) as described previously (56). Culture supernatant containing infectious HCV JFH1 was collected and passed through a 0.22-μm filter. Naïve Huh-7 cells were seeded 24 h before infection at a density of 1 × 106 in a 10-cm dish. The cells were incubated with 2.5 ml of the inoculum (6.5 × 103 50% tissue culture infectious dose [TCID50]/ml) for 3 h, washed three times with PBS, and supplemented with fresh complete Dulbecco's modified Eagle's medium. Then the cells were transfected with 6 μg each of pCAGGS, pCAG-HA-E6AP, or pCAG-HA-E6AP C-A by using TransIT LT1 (Mirus). The cells were trypsinized and replated in six-well plates at 1 day postinfection. The culture medium was changed every 2 days. The culture supernatants and the cells were collected at days 3 and 7 postinfection.
We quantitated HCV core protein in cell lysate using the HCV core antigen enzyme-linked immunosorbent assay (ELISA) (Ortho-Clinical Diagnostics). Total RNA was extracted from cells using TRIzol reagent (Invitrogen). To quantitate HCV RNAs, real-time reverse transcription-PCR was performed as described previously (53).
The TCID50 was calculated essentially based on the method described previously (28). Virus titration was performed by seeding Huh-7 cells in 96-well plates at 1 × 104 cells/well. Samples were serially diluted fivefold in complete growth medium and used to infect the seeded cells (six wells per dilution). Following 3 days of incubation, the cells were immunostained for core with anticore MAb (2H9). Wells that expressed at least one core-expressing cell were counted as positive, and the TCID50 was calculated.
Cells on collagen-coated coverslips were washed with PBS, fixed with 4% paraformaldehyde for 30 min at 4°C, and permeabilized with PBS containing 0.2% Triton X-100. Cells were preincubated with BlockAce (Dainippon Pharmaceuticals), incubated with specific antibodies as primary antibodies, washed, and incubated with rhodamine-conjugated goat anti-rabbit immunoglobulin G (ICN Pharmaceuticals, Inc.) and Qdot 565-conjugated goat anti-mouse immunoglobulin G (Quantumdot) as secondary antibody. Then the cells were washed with PBS, counterstained with DAPI (4′,6′-diamidino-2-phenylindole) solution (Sigma) for 3 min, mounted on glass slides, and examined with a BZ-8000 microscope (Keyence).
Naïve Huh-7 cells at 106 cells/10-cm dish were inoculated with 2.5 ml of the inoculum including infectious HCV JFH1 (6.5 × 103 TCID50/ml) and cultured. The cells were replated in a six-well plate at 3 × 105 cells/well at day 11 postinfection and transfected with 40 pmol of E6AP siRNA or control siRNA. The culture medium was changed at 24 h after transfection. The cells were harvested at day 2 after transfection, and the intracellular core protein levels were quantitated using the HCV core antigen ELISA. The culture supernatants were collected at day 2 after transfection and assayed for TCID50 determinations.
To identify the molecular machinery for HCV core ubiquitylation, we searched for endogenous ubiquitin-proteasome pathway proteins that associated with HCV core protein. HCV core-binding proteins (i.e., MEF core and its binding proteins, recovered from lysed cells) were purified by a tandem affinity purification procedure using a tandem tag (known as MEF tag) (16). Ten proteins were reproducibly detected (Fig. (Fig.1A,1A, lane 2), but none were recovered from lysed control cells transfected with empty vector alone (Fig. (Fig.1A,1A, lane 1).
To identify the proteins, silver-stained bands were excised from the gel, digested by Lys-C, and analyzed using a direct nanoflow liquid chromatography-MS/MS system. Nine proteins were identified: two known HCV core-binding proteins, human DEAD box protein DDX3 (38) and proteasome activator PA28γ (30), and seven potential HCV core-binding proteins. E6AP was identified (Fig. (Fig.1A,1A, lane 2) on the basis of five independent MS/MS spectra (Table (Table1).1). Immunoblot analyses confirmed the proteomic identification of E6AP, DDX3, PA28γ, and MEF-core (Fig. 1B to E).
The E6AP binding domain for HCV core protein was investigated. Figure Figure2A2A is a schematic representation of E6AP and known motifs in E6AP. A series of deletion mutants of E6AP as GST fusion proteins were expressed in E. coli. GST pull-down assays found that the carboxyl-terminal deletion mutant E6AP (1-517), but not E6AP (1-418) (Fig. (Fig.2C,2C, lanes C and D), and the amino-terminal deletion mutant E6AP (418-875), but not E6AP (517-875) (Fig. (Fig.2C,2C, lanes J and K), were able to bind to the core protein. The signal was absent when unprogrammed wheat germ extracts (the negative control) were used as a source of proteins (data not shown). GST pull-down assays (Fig. (Fig.2B)2B) found that the region from aa 418 to aa 517 is important for binding to the HCV core protein. An assay of the ability of GST-E6AP (418-517) to bind to the HCV core protein was confirmatory (Fig. (Fig.2C,2C, lane N) and led to the conclusion that the HCV core-binding domain of E6AP was aa 418 to aa 517.
By use of a panel of HCV core deletion mutants (Fig. (Fig.3A),3A), GST-E6AP was found to coimmunoprecipitate with all of the FLAG-core proteins (Fig. (Fig.3A,3A, lanes A to H) except FLAG-core (72-191) or FLAG-core (92-191) (Fig. (Fig.3A,3A, lanes I and J). No association of control GST protein with any FLAG-core proteins was observed (data not shown). These data suggest that the aa-58-to-aa-71 segment of the HCV core binds to E6AP. The ability of GST-core (58-71) to associate with purified MEF-E6AP confirmed that the core (aa 58-71) was the site for E6AP binding on the HCV core protein (Fig. (Fig.3B3B).
One of the features of HECT domain ubiquitin ligases is direct association with their substrates (50). Thus, we hypothesized that E6AP would function as an E3 ubiquitin ligase for the HCV core protein. We assessed the effects of E6AP on the HCV core protein in 293T cells. FLAG-core (1-191) together with HA-tagged wild-type E6AP, catalytically inactive mutant E6AP, E6AP C-A (19), or WWP1 (another HECT domain ubiquitin ligase) (22) was introduced into 293T cells, and the levels of the core protein were examined by immunoblotting. The steady-state levels of the core protein decreased with an increase in the amount of E6AP plasmids (Fig. 4A and B). However, neither E6AP C-A mutant nor WWP1 decreased the steady-state levels of the core protein, suggesting that E6AP enhances degradation of the core protein.
To verify the critical need for endogenous E6AP in the core degradation, expression of E6AP was knocked down by siRNA and the expression of the core protein and E6AP was assayed by immunoblotting. Transfection of the E6AP-specific siRNA duplex reduced the protein level of E6AP by 90% at 48 h posttransfection (Fig. (Fig.4C,4C, middle panel). Immunoblotting revealed a 4.1-fold increase in the level of the core protein in the cells transfected with E6AP siRNA (Fig. (Fig.4C,4C, top panel), suggesting that endogenous E6AP plays a role in the proteolysis of the HCV core protein.
Then we examined whether E6AP reduces the steady-state levels of the core protein in hepatic cells as well as in 293T cells. Exogenous expression of E6AP resulted in reduction of the core protein in human hepatoblastoma HepG2 cells (Fig. (Fig.4D).4D). Treatment of the cells with the proteasome inhibitor MG132 increased the core protein level, suggesting that the core protein was degraded through the ubiquitin-proteasome pathway. These results indicate that E6AP enhances proteasomal degradation of the HCV core protein in both hepatic cells and nonhepatic cells.
To determine whether the E6AP-induced reduction of the core protein is due to an increase in the rate of core degradation, we performed kinetic analysis using the protein synthesis inhibitor CHX. HCV core protein together with wild-type E6AP or inactive mutant E6AP C-A was expressed in 293T cells. At 44 h after transfection, cells were treated with either 50 μg/ml CHX alone or 50 μg/ml CHX plus 25 μM MG132 to inhibit proteasome function. Cells were collected at 0, 3, 6, and 9 h following treatment and analyzed by immunoblotting (Fig. (Fig.5A).5A). Overexpression of E6AP resulted in rapid degradation of the core protein, whereas inactive mutant E6AP C-A increased the half-life of the core protein (Fig. (Fig.5B),5B), suggesting that the inactive E6AP inhibited degradation of the core protein in a dominant-negative manner, which is in agreement with previous studies (19, 55). Treatment of the cells with MG132 inhibited the degradation of the core protein (Fig. (Fig.5B).5B). Reverse transcription-PCR to determine mRNA levels of the HCV core gene and GAPDH gene found that neither wild-type E6AP nor inactive E6AP changed mRNA levels of the HCV core gene and GAPDH gene (data not shown). These results indicate that E6AP enhances proteasomal degradation of the core protein.
To determine whether the full-length HCV core protein expressed in hepatic cells is degraded through an E6AP-dependent pathway, human hepatoma Huh-7 cells were transfected with pCAG HCV core (1-191) along with either E6AP or E6AP C-A. To rule out the effects of N-terminal FLAG tag on the core degradation, HCV core protein was expressed as untagged protein. Expression of wild-type E6AP resulted in reduction of the core protein (Fig. (Fig.6).6). On the other hand, HCV core protein was not decreased after transfection of inactive E6AP, indicating that the full-length core protein expressed in Huh-7 cells is also degraded through an E6AP-dependent pathway.
To determine whether E6AP can induce ubiquitylation of HCV core protein in cells, we performed in vivo ubiquitylation assays. 293T cells were cotransfected with FLAG-core (1-191) and either E6AP or empty plasmid, together with a plasmid encoding HA-tagged ubiquitin to facilitate detection of ubiquitylated core protein. Cell lysates were immunoprecipitated with anti-FLAG MAb and immunoblotted with anti-HA PAb to detect ubiquitylated core protein (Fig. (Fig.7A).7A). Only a little ubiquitin signal was observed on the core protein in the absence of cotransfected E6AP (Fig. (Fig.7A,7A, lane 3). In contrast, coexpression of E6AP led to readily detectable ubiquitylated forms of the core protein as a ladder and a smear of higher-molecular-weight bands (Fig. (Fig.7A,7A, compare lane 3 with lane 4). Immunoblot analysis with anticore PAb confirmed that FLAG-core proteins were immunoprecipitated (Fig. (Fig.7B,7B, lanes 2 to 4, short exposure) and that higher-molecular-weight bands conjugated with HA-ubiquitin were indeed ubiquitylated forms of the core protein (Fig. (Fig.7B,7B, lanes 3 and 4, long exposure).
To rule out the possibility that E6AP contributes to core protein degradation by inducing degradation of inhibitors of core turnover, we determined whether E6AP functions directly as a ubiquitin ligase by testing the ability of purified MEF-E6AP to mediate in vitro ubiquitylation of the purified recombinant HCV core protein. HCV core protein was expressed as a fusion protein containing N-terminal GST tag and C-terminal His tag and purified as described in Materials and Methods. GST-C173HT (aa 1-173) and GST-C152HT (aa 1-152) (see Materials and Methods) were used to determine whether the mature core protein and the C-terminally truncated core protein are targeted for ubiquitylation in vitro. The validity of this assay was established by demonstrating that E6AP but not E6AP C-A induced ATP-dependent ubiquitylation of GST-core protein. When in vitro ubiquitylation reactions were carried out either in the absence of MEF-E6AP or in the presence of MEF-E6AP C-A, no ubiquitylation signal was detected (Fig. (Fig.8A,8A, lanes 4 and 5). However, inclusion of purified MEF-E6AP in the reaction mixture resulted in marked ubiquitylation of GST-C173HT (Fig. (Fig.8A,8A, lane 6), while no ubiquitylation was observed in the absence of ATP (Fig. (Fig.8A,8A, lane 7). No signal was detected when GST-HT was used as a substrate (Fig. (Fig.8A,8A, lane 8). The higher-molecular-weight species of GST-core proteins were reactive with both anti-ubiquitin MAb (Fig. (Fig.8B,8B, right panel, lanes 2 and 4) and anti-GST MAb (Fig. (Fig.8B,8B, left panel, lanes 2 and 4). Both GST-C152HT and GST-C173HT were polyubiquitylated by E6AP in vitro (Fig. (Fig.8B),8B), indicating that both the C-terminally truncated core and the mature core are polyubiquitylated by E6AP in vitro. These results revealed that E6AP directly mediated ubiquitylation of HCV core proteins in an ATP-dependent manner.
We used a recently developed system for the production of infectious HCV particles using the HCV JFH1 strain (28, 56, 61) to examine whether E6AP can promote degradation of HCV core protein expressed from infectious HCV. E6AP-dependent core degradation was assessed in Huh-7 cells inoculated with the culture supernatant containing HCV JFH1. Levels of HCV core protein were detectable at day 3 postinfection and increased with time. Immunofluorescence staining for the core protein indicated that the percentage of HCV core-positive cells in the Huh-7 cells was almost 100 at day 7 postinfection. Transfection efficiency was 50 to 60% as measured with GFP-expressing plasmid. At day 7 postinfection, exogenous expression of E6AP reduced the intracellular core protein level by about 60% compared to the empty plasmid-transfected control cells (Fig. (Fig.9A).9A). Inactive E6AP had little effect on the core protein levels. Total protein levels in the cells (Fig. (Fig.9B)9B) and intracellular HCV RNA levels (Fig. (Fig.9C)9C) did not change after transfection of wild-type E6AP or inactive E6AP. The immunofluorescence study revealed that HCV core protein was variably detected and the intensity of core staining was reduced in the cells staining positive for wild-type E6AP compared with neighboring cells staining negative for E6AP (Fig. (Fig.9E).9E). Using inactive E6AP revealed colocalization of the core protein and E6AP in the perinuclear region (Fig. (Fig.9F)9F) of HCV-infected cells. These results suggest that E6AP enhanced degradation of HCV core protein expressed from infectious HCV. Then we titrated HCV infectivity in the culture supernatant at day 7 postinfection by limiting dilution assays. Exogenous expression of E6AP reduced the supernatant infectivity titer, whereas inactive E6AP had no effect on its infectivity titer (Fig. (Fig.9D),9D), suggesting that the E6AP-dependent ubiquitin proteasome pathway affects the production of HCV particles through downregulation of the core protein.
Finally, to further validate the role of E6AP in HCV production, expression of endogenous E6AP was knocked down by siRNA and the HCV infectivity titers released from HCV JFH1-infected cells were examined. Knockdown of E6AP by siRNA led to an increase in intracellular core protein levels (Fig. 10A) and supernatant HCV infectivity titers (Fig. 10B). Taken together, our results suggest that E6AP mediates ubiquitylation and degradation of HCV core protein in HCV-infected cells, thereby affecting the production of HCV particles.
HCV core protein is a major component of viral nucleocapsid, plays a central role in viral assembly (25, 40), and contributes to viral pathogenesis and hepatocarcinogenesis (9). Therefore, it is important to clarify the molecular mechanisms that govern the cellular stability of this viral protein. We have previously reported that processing at the C-terminal hydrophobic domain of the core protein leads to efficient polyubiquitylation of the core protein (52). In this study, we identified E6AP as an HCV core-binding protein and showed that HCV core protein interacts with E6AP in vivo and in vitro, that E6AP enhances ubiquitylation and degradation of the mature core protein as well as the C-terminally truncated core protein, and that HCV core protein expressed from infectious HCV is degraded via E6AP-dependent proteolysis. HCV core protein and E6AP were found to colocalize in the cytoplasm, especially in the perinuclear region. Moreover, exogenous expression of E6AP reduces intracellular core protein levels and supernatant HCV infectivity titers in HCV-infected Huh-7 cells. Knockdown of endogenous E6AP by siRNA increases intracellular core protein levels and supernatant infectivity titers in HCV-infected cells. These findings suggest that E6AP mediates ubiquitylation and degradation of HCV core protein, thereby affecting the production of HCV particles.
HCV core protein interacts with E6AP through the region of the core protein between aa 58 and aa 71. These 14 amino acids are highly conserved, with the first nine amino acids (PRGRRQPIP) present in the core protein of all the HCV genotypes (3). This result suggests that E6AP-dependent degradation of HCV core protein is common to all HCV genotypes and plays an important role in the HCV life cycle or viral pathogenesis. Our data indicated that HCV core proteins of genotypes 1b and 2a are subjected to proteolysis through an E6AP-mediated degradation pathway. We are currently examining whether E6AP promotes degradation of HCV core proteins of other genotypes.
Studies in addition to ours have reported that other HCV proteins, such as NS5B (8), the unglycosylated cytosolic form of E2 (39), NS2 (7), and F protein (58), are degraded through the ubiquitin-proteasome pathway. These studies suggest that the ubiquitin-proteasome pathway plays a role in the HCV life cycle or viral pathogenesis. To our knowledge, the present study is the first to demonstrate that the ubiquitin-proteasome pathway affects the HCV life cycle.
PA28γ was found to interact with HCV core protein in hepatocytes and promote proteasomal degradation of HCV core protein (30). PA28γ, however, has been shown to function in a ubiquitin-independent, ATP-independent, and 20S proteasome-dependent pathway (27). There have been reports that several cellular factors, such as p53 (2), p73 (2), and RPN4 (18), are degraded through two alternative pathways, the ubiquitin-dependent 26S proteasome-dependent pathway and the ubiquitin-independent 20S proteasome-dependent pathway. Here we provide evidence that E6AP mediates ubiquitylation of HCV core protein. Still unclear is whether the PA28γ-dependent pathway requires polyubiquitylation of HCV core protein. HCV core protein is predominantly localized in the cytoplasm, especially at the endoplasmic reticulum membrane, on the surface of lipid droplets, and on mitochondria and mitochondrion-associated membranes (51). In HCV JFH1-infected cells, HCV core was found to localize in the cytoplasm and frequently to accumulate in the perinuclear region and the lipid droplets (44). Our results indicated that E6AP colocalized with HCV core protein especially in the perinuclear region. PA28γ was found to colocalize with HCV core protein in the nucleus. Functional differences may exist between the E6AP-dependent pathway and the PA28γ-dependent pathway in the stability control of HCV core protein. The functional role of the E6AP-dependent pathway and the PA28γ-dependent pathway remains to be elucidated.
The HCV core-binding region of E6AP was mapped to the region between aa 418 and aa 517. The multicopy maintenance protein 7, Mcm7, interacts with E6AP through a short motif, termed the L2G box (aa 412 to 414), that lies within the E6 binding site of E6AP (23). Our data indicated that the E6 binding region containing the L2G motif is not required for interaction between HCV core protein and E6AP (Fig. (Fig.2C,2C, lane M).
We propose here that E6AP may affect the production of HCV particles through controlling the amounts of HCV core protein. This mechanism may contribute to persistent infection. The E6AP binding domain of the core protein resides in the RNA-binding domain and binding domains for many host factors (40). These factors may affect the binding between E6AP and HCV core protein, resulting in control of E6AP-dependent core degradation. Another possibility is that HCV core protein may affect the normal function of E6AP, thereby contributing to pathogenesis. It will be intriguing to investigate whether HCV core protein has any effect on E6AP-dependent degradation of host factors. The other intriguing possibility is that HCV core-E6AP complex may function as an E3 ligase-like E6-E6AP complex to target host factors for proteasomal degradation and contribute to viral pathogenesis.
In conclusion, we have demonstrated that E6AP interacts with HCV core protein in vitro and in vivo and mediates ubiquitin-dependent degradation of the core protein, leading to downregulation of HCV particles. We propose that the E6AP-mediated ubiquitin-proteasome pathway may play a role in affecting the production of HCV particles through controlling the amounts of viral nucleocapsid protein. Identification of the specific E3 ubiquitin ligase may contribute to gaining a better understanding of the biology of the HCV life cycle as well as molecular details of the ubiquitin-dependent degradation of HCV core protein.
We thank D. Bohmann (EMBL) for providing pMT123, K. Miyazono (University of Tokyo) for pcDEF3-6Myc-WWP1, and K. Iwai (Osaka City University) for recombinant baculovirus carrying His 6-mouse E1. Huh-7.5.1 cells and Huh-7 cells were kindly provided by F. V. Chisari (Scripps Research Institute). We also thank P. Zhou (Weill Medical College of Cornell University), S. I. Wells (Cincinnati Children's Hospital Medical Center), and A. W. Hudson (Medical College of Wisconsin) for critical readings of the manuscript; M. Matsuda, S. Yoshizaki, M. Ikeda, and M. Sasaki for technical assistance; Y. Sugiyama and S. Senzui for plasmid construction; and T. Mizoguchi for secretarial work.
This work was supported in part by a grant for Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation; by grants-in-aid from the Ministry of Health, Labor and Welfare; by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology; and by the program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), Japan. T.I. was supported in part by a grant from Novartis Foundation (Japan) for the Promotion of Science and by the Tokyo Metropolitan University President's Fund, Special Emphasis Research Project of Japan.
Published ahead of print on 15 November 2006.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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The Sebastian - Vail
The Sebastian - Vail
Hotel Guest Rating: 9.3/10
16 Vail Road
Vail, CO 81657
Welcome to the The Sebastian - Vail
The pool, on-site dining options, ski valet and modern in-room amenities get high marks from Priceline guests at the Sebastian - Vail.
Hotel Features
The five-story Sebastian offers 107 resort-style rooms featuring free Wi-Fi, dark furnishings, iPod docks, flat-panel HDTVs, DVD players, mini-fridges and coffeemakers. Some rooms also offer balconies and full kitchens. The heated, outdoor pool is open year-round and provides mountain views, outdoor dining and multiple hot tubs. There are also a business center and a stocked library available on-site. Dining options include an organic restaurant, a trendy lounge, a cafe, a beignet spot and 24-hour room service. Guests can keep fit with a workout in the gym and stay zen with a body treatment in the on-site spa. Private ski valet service and valet parking are available.
Hotel Location
The Sebastian is situated off I-70 surrounded by Vail's exciting dining, shopping and popular snow-capped sites. Guests are within a 10-minute stroll of Dobson Ice Arena and a five-minute walk of Colorado Ski Museum. Eagle County Airport is 35 miles west.
Guest Favorites
Priceline guests appreciate the convenience to restaurants and shops, the "welcoming environment" and the "spacious, well-appointed" and "modern rooms" at The Sebastian - Vail.
The Sebastian - Vail Amenities
- 24-hour front desk
- 24-hour security
- Accessible facilities
- Accessible parking
- Air conditioning
- Air conditioning individually controlled in room
- Alarm clock
- AM/FM radio
- Baby sitting
- Bell staff/porter
- Business center
- Cable television
- Catering services
- Children welcome
- Color television
- Concierge desk
- Conference facilities
- Connecting rooms
- Elevators
- Family plan
- Free high speed internet connection
- Full service housekeeping
- Hairdryer
- Handicap rooms
- Heated pool
- High speed internet access for laptop in public areas
- Hotspots
- Housekeeping - daily
- Interior corridors
- Iron
- Ironing board
- Jacuzzi
- Lounges/bars
- Massage services
- Meeting rooms
- Non-smoking
- Non-smoking rooms (generic)
- On-site business center
- Outdoor pool
- Parking fee managed by hotel
- Pay per view movies on TV
- Pets allowed
- Photocopy center
- Pool
- Public address system
- Remote control television
- Restaurant
- Room service
- Room service - limited hours
- Security
- Spa
- Storage space
- Technical concierge
- Telephone
- Transportation services - local area
- Transportation services - local office
- Turndown service
- Valet parking
- Voice mail
- Wakeup service
- Wireless internet connection in public areas
Hotel Information
Upon check-in photo identification and credit card is required. All special requests are subject to availability upon check-in. Special requests cannot be guaranteed and may incur additional charges. Guests are required to show a photo identification and credit card upon check-in. Please note that all Special Requests are subject to availability and additional charges may apply.
Hotel Guest Reviews of the The Sebastian - VailOur hotel ratings and reviews are from real hotel guests. Priceline.com ensures that its reviews are guests-only by proactively sending hotel surveys to customers after they've completed their hotel stays. In the post-stay surveys, priceline.com hotel customers are asked a number of questions about their stay and the hotel. On a scale of 1-10 with 10 being the best, the survey asks guests to rate their overall experience and specifically rate the hotel's cleanliness, staff and location. Priceline.com does not allow site visitors to post unsolicited hotel reviews.
July 12, 2016
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July 10, 2016
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November 11, 2015
Family Traveling with Young Children
August 30, 2015
Family Traveling with Young Children
I love this resort! Great location and amazing staff. at the bar is the best bartender we have ever had. I will return to this resort and I cant imagine that anything changes. Take our advice, go to Hotel Employee at the bar and ask him to make you a custom drink. This was our favorite thing about the resort. The concierges was also great. They were able to tell us wonderful hiking and dinner recommendations. LOVE VAIL ...
August 26, 2015
Family Traveling with Teens
August 19, 2015
July 28, 2015
July 27, 2015
June 16, 2015
Solo Traveler
This guest did not leave comments about this hotel.
June 9, 2015
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Friday Nov 07, 2014
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What is Modern Customer Service?
By Tony Berk on Jul 10, 2014
Make it easy for customers to engage
Barry Lieberman is Chief Customer Care Officer at Q-See, a global leader in providing advanced surveillance solutions for homes and small businesses. In response to our topic question, Barry shared: "Modern customer service to me is offering service to the customer where they want to be serviced". Barry suggests being proactive and deliver consistent, outstanding service on every channel. [Read More]
Monday Aug 19, 2013
Become a part of your customer's life at CRM Evolution 2013
By Tuula Fai on Aug 19, 2013
The first day of the CRM Evolution 2013 Conference and Exhibition began with a dynamic keynote by Jeanne Bliss, Founder of CustomerBliss.com. She talked about organizations "becoming a part of the story of their customers' lives" through:
Getting Real—walking a mile in their customers' shoes
Example: USAA has new hires eat army rations, wear flak jackets and boots and go through the same insurance application processes
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Example: WestJet empowers its frontline staff to give 'honored guests' the best experience possible
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Example: Southwest sends apology letters offering discount coupons to fly again.
Oracle's very own Matt Kresch, Principal Product Strategy Director, spoke about the Modern Contact Center. To be 'modern,' your service organization must be able to carry on a seamless conversation with the customer across channels. A unified agent desktop—that brings all the customer information and engagement tools together on one screen—makes this possible.
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For policymakers anxious about changing patterns of relationships, the royal engagement offers some reassurance.
Defining the Modern British Family
Shelley Budgeon is a senior lecturer in sociology and Sociology Programme Director at the University of Birmingham.
November 17, 2010
Certainly the history of the relationship between Prince William and Kate Middleton is a departure from royal norms. They met in college, dated a long time and they will enter marriage knowing each other and themselves better for it. Both will be nearing 30 when they marry — Diana was only 19.
That William is able to choose to marry someone from a middle class background has been granted much significance. The factors in their relationship are quite consistent with relationship norms in Britain now. Over the past 40 years, the age at first marriage has been steadily increasing and personal satisfaction is more greatly emphasized, with far more diversity in how people choose to organize their intimate relationships. It may be that royal marriages in Britain, like personal relationships amongst the populace, are becoming "detraditionalized."
However, from another perspective William and Kate are out of step with key social trends. As a social institution, marriage rates are in decline as are birth rates. Births outside of wedlock are increasing and the numbers of women choosing to remain childless are rising. The transformation of gender relations has made work/life balance an ongoing challenge for many couples. Divorce rates and single parenthood are up.
When set against this backdrop, it's hard to define their choice as "normal." Moreover, definitions of the family in Britain remain contested. Indeed these trends are often cited as evidence of social decline. The rhetoric of "strong marriages equal a strong society" has resurfaced as a prominent feature of the Conservative Party's analysis of the "broken society."
For politicians and policy makers who are anxious about changing patterns of relationships, the symbolic importance that is being granted to the royal engagement will offer some reassurance that individuals are still willing to enter into "proper commitments."


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Lawmakers could make Mardi Gras throws tax exempt
Published: Wednesday, May 11, 2005 at 11:54 a.m.
Last Modified: Wednesday, May 11, 2005 at 12:10 p.m.
BATON ROUGE -- Hey mister, throw me something tax free!
It might be a tad bit formal for a Mardi Gras parade, but if a Crescent City lawmaker has his druthers this session, the phrase would apply to future carnivals.
Rep. Peppi Bruneau, R-New Orleans, is pushing legislation that would allow Mardi Gras krewes to sell specialty items to their memberships -- as long as the official logo is emblazoned on the items -- without charging them state and local taxes.
That means everything from doubloons and plush toys to costumes and bead bags would be tax-free for float-riders if the items are krewe-specific.
The House Ways and Means Committee passed House Bill 90 Tuesday with little objection. And there are already plans to add other parading organizations, such those associated with St. Patrick's Day.
Earl Millet, the New Orleans regional director for the state Department of Revenue, said there is already such an exemption in existence, but it is rarely enforced and not officially in statute.
As such, the proposed change would have little or no impact on the revenue streams of state and local governments.
News of the bill passing its first legislative hurdle Monday was met with cheers by revelers in the Terrebonne-Lafourche area.
S.P. LaRussa, a Houma real-estate broker and a founding member and parade captain of the Krewe of Hercules, said the 22-year-old parade keeps getting more expensive each year and the tax proposal should help slow things down for coming Carnivals.
"We have 150 riders that pay a $350 annual due," LaRussa said. "We can't keep raising that. The only way to make money for the parade is to sell things to your members with your logo on them. Honestly, I think everything we do should be tax-free."
The bill specifies that only nonprofit organizations would be able to take advantage of the tax-free offer, and only after they receive a yearly certification from the state Department of Revenue.
Jerry Jones, president of Krewe of Shaka in Thibodaux, said the tax proposal would give his krewe an opportunity to shift savings over into another area that is often overlooked -- charity.
For 11 years, Jones said, his krewe has been sponsoring underprivileged children with an annual scholarship and hosting "adopt-a-child" programs that aid local children through a school year.
"We can take that money we're saving and forward it to helping more kids," Jones said.
The only opposition to the bill came from Rep. Rick Farrar, D-Pineville, who said he voted against the legislation for symbolic reasons.
"I'm trying to get a tax credit for our churches," Farrar said. "I can't get that passed here, but we can give one to Mardi Gras?"
Bruneau, who managed to brag on his New Orleans krewe during the committee hearing without divulging its identity, which is a well-kept secret for some Big Easy revelers, said Mardi Gras is a privately-funded venture and the tax break would have little impact on state and local coffers.
Bruneau said he would consider amending his own bill in coming weeks to add other parades.
The legislation moves to the House floor for debate.
Jeremy Alford can be reached at firstname.lastname@example.com.
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"When her church volunteer faces deportation to Sierra Leone, Vicar Stella offers to help the only way she can. One white lie, one fake wedding, a good cause. Soon the church is overrun with bogus brides and a day of reckoning dawns. All Saints is a rollicking comedy about right and wrong; good and evil, and unexpected consequences.
"Based on a true story and inspired by the exuberant churches and communities of Peckham, Furious Theatre's All Saints arrives at the King's Head straight from its sold-out run at The Last Refuge.
"Nicola Baldwin's first play Confetti won the George Devine and Time Out awards and her most recent play Blackshirts won the 2013 East 15/Stagetime competition on IdeasTap... Join Furious at All Saints for a month of Sundays (and Mondays) celebrating the triumph of love in an imperfect world." – from the King's Head website
All Saints has been very popular with audiences and quite well reviewed by critics:
"Helen Sheals's lively production underscores the play's humour and gets good performances" – Michael Billington in the Guardian
"Baldwin … composed a skilful script for All Saints; it's intelligent, witty and grounds some superb characters. The actors, director and designers have developed it into a fabulously funny production, building on great character work with subtlety and panache." – from What's Peen Seen
"I also left with a big grin and a warm sense of satisfaction at a story that captured and celebrated the meaning of community and, from the standing ovation by those behind me, I wasn't alone." – from One Stop Arts
"This is an extremely funny piece which, in the second half, also achieves unexpected emotional depth. It's helped by a whole ensemble of stellar performances without a weak link among them... a superbly entertaining evening – even the cast seemed surprised by how many times they were called back by applause that refused to die down. Highly recommended." – from Remotegoat
Following an initial phase when I purchased group tickets for those who prepaid, this option is now closed. Please purchase your ticket direct from the theatre.
I would be grateful if all attendees would contribute £1 each on arrival, to help defray the approximately £100 that it costs per year to maintain a group on Meetup.
I plan to arrange dinner or drinks after the play (drinks at 69 Colebrook Row or the King's Head – or dinner at Pasha or Le Mercury), depending on preference – please let me know if you would like to stay on.
All in all I prefer the Monday if people are happy with that. Since no one so far has indicated they have a preference, I'll go with Monday - unless people raise any objections during the course of today. I'm happy to buy tickets for anyone who wants to sit together in a group(s). All seats are the same price, £16 - unless you're eligible for a concession price of £12 (please see http://www.kingsheadtheatre.com/faq.html for details). If you're eligible and want me to buy a group ticket for you, please let me know by private message and I'll organise it.
I'll make the ticket payment option open from this evening (immediately after scheduling the date) for 36 hours (I'll buy the tickets at 10 am on Thursday).
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A. Baluch, A. Humar, D. Eurich, A. Egli, A. Liacini, K. Hoschler, P. Campbell, N. Berka, S. Urschel, L. Wilson and D. Kumar Randomized Controlled Trial of High-Dose Intradermal Versus Standard-Dose Intramuscular Influenza Vaccine in Organ Transplant Recipients American Journal of Transplantation 13
Version of Record online: 13 FEB 2013 | DOI: 10.1111/ajt.12149
This trial shows that high-dose intradermal vaccine has similar immunogenicity to standard intramuscular vaccine, and that neither is associated with alloantibody upregulation.
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