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DBL Revisited, a look back at the season of 2019
Posted on August 31, 2020 September 1, 2020 by Thomas McGivern
Today we have a post written by Ewan, a regular team member of One Turn Touchdowns UKTC team and a member of the DBL.
DBL 19 review: further thoughts on league play
Although this is my first blog for 1TTD, I've known Tom and Chris for a while having been part of their team at UKTC for the past 2 years. This article is my experience of the Dragon Bowl League 2019 (DBL 19) season and is a follow up piece to Tom's excellent overview of DBL 2018 [https://oneturntouchdown.com/2018/09/04/league-play-my-introduction-to-the-dbl/ ]. Hopefully this gives a slightly different perspective and highlights how the DBL has evolved. The first section is about my overall experience of playing in the DBL, whilst the second is an insight into the madness of Finals Day, when the two semi-finals and the final are played back to back in a glorious season finale! This post was originally meant to come out in the Spring, but then Coronavirus happened and it sat in draft until DBL 2020 kicked off and prompted me to get it finished.
But first a bit of background: I've been playing competitive blood bowl for about three years and I'm really enjoy this aspect of the game. Although I play several other board games, BB is definitely my favourite table-top game. Most of my competitive experience has been in regeneration tournaments (around a dozen of them, mostly finishing mid-table; NAF name lord_relictor), but I've also had the pleasure to play in the Dragon Bowl League the last two years (2018 and 2019). This is where I first met Tom and his Deadwood Rams, playing out two nail biting draws that could have gone either way. Although I've not played in other tabletop or online leagues, I'd definitely consider them in future.
Part 1 – The Glorious DBL
In 2019 the DBL expanded the league season to 60 players (yes, sixty!), which probably makes it one of the biggest table-top leagues in Britain, if not the world. This makes for some organisational challenges, but it also provides a large community keen to play BB in all its formats. To cater for this the DBL runs stunty and 7s leagues running in the off-season. It's also been a good place to organise teams and travelling groups for tournaments around the UK and overseas. They sent several players to the world cup in Austria and I was booked up to attend a tournament in Bilbao with around 10 others in July (stupid global pandemic). The DBL's "Underworld University" beginner/refresher league, which runs in parallel to the DBL, has also continued to grow and had around 12 players this year.
As far as abilities and competitiveness go, there's a good range of both in the DBL. There are several very serious and talented tournament players who've won NAF tournaments, but there are also plenty of people just playing for laughs. And even the good players often take lower tier teams to spice things up or to test themselves (Phil/Avecmontage took Halflings in the DBL and went on to win a competitive NAF event with them). Although there can be some tense games, it's definitely not WAAC and there's a big focus on the fluff and social sides. It was also pleasing to see almost all races were represented, with only the poor Pro Elves missing out (although let's not feel too sorry for them). This means there was huge variety in the racial match ups and very few mirror matches.
The Teams and Fluff
As it was last year, there's an incredible effort across the board on team painting, fluff/background and logos. My Orc team were the Brewgrog Punks, with a craft beer theme so all the players had brewery names (e.g. the troll was called Meantime and the goblin was Tiny Rebel). I knocked up the logo on a free website and (eventually) drafted some silly fluff to go with it. Every one of the 60 players made the effort to create a logo and bought into the story telling side of it. I think this really helps to build the community and the reasonably high effort 'barrier to entry' discourages flaky players.
The Organisers
The key to the DBL's success is the commitment and imagination of the Commissioners and the Governors. Two co-Commissioners (known as the Commishs) were in overall charge, with 6 Governors (Guvs) running each of the conferences. There were also two additional Guvs who do the merchandise and graphic design – both a key part of the overall experience. They organise the two draft nights, ensure all the matches are played on time, handle any rules enquiries and generally keep everyone motivated. This seems like a lot of effort, but it would be difficult to run an endeavour of this size without plenty of burden-sharing.
Some of the high quality logos
Each year the league has an overall theme, and this year it was "Revolution". On draft night each of the Guvs had a costume related to their favourite revolution and . I was drawn in the West Conference and my Guv (Alex) wore a top hat and handed out flat-caps to each of his players, representing the factory boss and his workers during the industrial revolution. We also had the Gillet Jaunes from protests in France and one of the Guvs was a very convincing Fidel Castro, complete with stick on beard and fake cigars! I think it's fair to say Revolution wasn't quite as memorable as the '80's' theme we had in 2018, but that's a high bar (especially when you factor in the brilliantly cheesy 80's sound track – think Wham, Footloose, Eye of the Tiger, Kenny Loggins…).
The League Format and Draft Night
In 2019 the Guvs added a 6th conference to accommodate the 10 additional players from last year (6 conferences of 10 players). This was called the "Exiles" conference, to go alongside the existing North, South, East, West and Central conferences. Each is split into two 5-person divisions: A and B and has its own colour and theme (Northern Hard Nuts, Southern Comforts etc). Each player then has a home and away game against each of the 4 opponents in their division. Although many league bowlers prefer playing different opponents in each game, I quite enjoyed the "return legs" against previous opponents, where scores can be settled and lessons identified can be put into practice. They stuck with a schedule of playing an average of 2 games every 3 weeks, which feels right to me.
As it was in 2018, qualification for the knock-outs was achieved by finishing in the top 32 of an overall league table for all 60 players, rather than your placing in the 5-person division. This means it's possible for 3 or even 4 people in your division to qualify, so it doesn't feel too cut-throat with other players in your division. It also encourages inter conference rivalry as you look at who has the most players near the top of the table.
The remaining 28 players went into the 'plate' tournament, known as the Cuckleberry Cup. This gives less experienced (or just unlucky!) coaches a chance to experience knock out games and make up for early season mistakes. Although it's not as serious or competitive as the Championship, it's still a high standard and included several experienced NAF coaches. The 2019 Cuck was won by Steve and his unpredictable Underworld team, led by their talismanic troll "Bumsmell".
Did you know, with the current global pandemic still causing disruption around the world, DBL 20 was very close to being the next casualty of Covid-19 but because of the unsurpising hard work of the commisioners and Guvs it has been able to kick off in a restriced and safe way for a limited number of coaches. It will be without a doubt a huge part of history but who will mak their mark on the pitch.
Draft nights
These were just as brilliant as last year. The first one is for the league draw in July and the 2nd is for the knock-outs draw in October. The organisers allocate players by drawing balls out of a bingo machine, which generates a huge amount of banter and excitement. Once the draw is done players can either play the opponent they've been drawn against or take part in games of Deathbowl. Several players come in fancy dress related to their team and everyone's in a party mood. Drinks flow in the club and we hit the town when it closes. Since the draws take place on a Monday I've learnt to book the Tuesday off work! The draft nights are always held at the club in 7 Dials, but league games are often played at other venues, with particular venues being Steam Engine in Waterloo and Rule Zero in Hackney Wick.
Draft night games and part of the draw
Every year there seems to be a random conference or division that sparks the imagination. In DBL 18 it was a division that had 4 elf teams out of 4, which led to some crazy, high scoring games, full of elfy bullshit! This year it was a division that included 4 stalwarts of the Edinboro Castle Blood Bowl League (ECBBL). The DBL has a friendly rivalry with the ECBBL, with several bowlers playing in both, so it was really funny to see most of them drawn together in a "division of death".
Another interesting twist the DBL has is the Conference Championship. After the league season, but before the knock-outs, the top player in each of the two divisions play off in a one off regen match for the conference title. Each of these 6 titles has its own name and lovingly painted miniature trophy. After a long league season these add a bit of spice to the intra-conference rivalry, with players in each division backing their representative to win the trophy.
The Exiles cup in progress and the West's "Magenta Fist"
The Merch and Rules Pack
An integral part of the DBL and its sense of camaraderie and community is the bespoke merchandise. In 2019 everyone got a pint glass, a coffee mug with their team's logo and some DBL dice – organised by the Merch Guv Oli. Each of the conference Guvs also arranged some conference specific dice and novelty clothing (related to the revolution theme). I was in the West conference and my excellent Guv Alex gave each of us a flat cap, whilst he wore a top hat (to represent the industrial revolution!). There was also additional merch for those that wanted it, including a red West t-shirts. Quite a few players wore these during their games and at NAF tournaments, which really helped build conference camaraderie.
West Conference merch, a Central coffee mug and some of the pin badges
The hard-copy Rules Pack is another amazing part of the DBL package. This is brought together by the Guvs before the season, particularly by the design Guv Sam. These are such beautiful documents, printed on high quality paper with gorgeous graphics linked to the theme, in this cse Revolution. They contain all the relevant league rules for the DBL and a handy reference doc for kick off tables and weather charts etc.
So how did I get on?
Well it's fair to say 2019 went much better for me than 2018. Playing with Orcs (I was keen to try bash after last season's dash with Skaven) I finished the regular season 4-4-0, topping my 5-person division and qualifying for the DBL round of 32 knock-outs (as one of the top 16 seeds). I had some great games against Dark Elves, Halflings, Nurgle and Bretonnians; none of which were easy. This also put me in the Conference Championship game, so before the knock-outs I competed against Sam's Khorne team for the West Conference trophy, the 'Magenta Fist'. This was a brutal affair with his Block, MB, Claw, Frenzy, Piling-On Blood Thirster tearing through my previously robust greenskins. It finished 2-0 to him and about 6-2 in Cas. Just as well it was regen!
After making generous use of the Magic Sponge, my team went into the round of 32 against Tom's Ogres. Although he's an excellent player, and very experienced with Ogres, it was a pretty good draw for me and I won 2-0. The last 16 was a grind fest against Lewis and his Khemri, but I sneaked it 1-0. The Quarter Final was a very different experience against Jerome's Vampires. He's a real maverick and almost pulled off a score on my drive after some sloppy caging. However, the Orc pain train got rolling and the numbers advantage saw me through. I won 3-0, but it was much closer that the score suggests. So against all expectations I was in the DBL finals day for the first time!
The DBL graphics are always top notch (thanks to Sam) and the DBL knock-outs and Cuckleberry draws are no exception
Part 2 – Finals Day
Finals day is something of an institution in the DBL. It's the culmination of a long season and one of the big three set piece events, alongside the two draft nights. The last couple of years it's been held in Loading Bar in Dalston, a boardgames pub that's happy for us to take up a large section of the pub and set up AV equipment for live streaming. As it's a fairly long event – with setup, the two semis, the final and the awards ceremony – it usually starts early afternoon and finishes well into the evening. Around 20 DBLers will attend during the day, some just come down for part of it whilst others stay for the full day and a few arrange to play friendly games of 7s or regular bowl in the margins. This year the UU final was also played their on the same day, so it was a real festival of Blood Bowl! My first finals day was in 2018, when I came down to watch the games and catch up with friends. That buzz and sense of occasion was really inspiring – definitely a motivator to play my best and qualify for future finals days.
It took a bit of organising and rescheduling to get all four semi-finalists there together, so despite the QFs finishing in early December, finals day didn't happen until February. Although it's definitely better than the alternatives (sub in replacement players or play the semis separately from the final), it's still a bit frustrating as the season lost a bit of momentum. Most players who'd already been knocked out had moved onto other boardgames or started in other BB leagues and competitions, so it look at bit of effort from the Guvs to get the rest of the DBL psyched up for it. Before the draw I wrote down and sent round the Punks' fluff. A few other players had done this earlier in the season and it really adds to the narrative.
DBL TV
Something that makes finals day really special is the live video and commentary on each game. The DBL is pretty good at reporting on other games, with a separate Whatsapp chat just for match updates, but having a full commentary throughout and live Youtube stream up takes it to the next level. We're lucky to have a few tech wizards in the club, so they come down early at set it up. One of the semis and the final get full video and commentary, whilst the other semi only gets text commentary via Whatsapp. I obviously wasn't able to see the video at the time, but it's still on Youtube (hyperlink: https://www.youtube.com/watch?v=SotbH9Bzq_Y&t=10962s&has_verified=1 ) and I have since watched it back. Quite strange watching yourself play and shouting at the screen saying "don't do that GFI, you're going to roll a 1", and "don't put the Goblin there you idiot!". The only previous time I'd been streamed before was with the 'Beer and Blood Bowl' guys, but that was at the bottom tables during the Europen in Cardiff, so I hadn't bothered to watch that one back!
The commentary team look less than impressed
The commentary team rotated through the day and each of the guys added some unique insight and humour. They covered the tactics, player skills, league record, history in the DBL, as well as numerous less serious comments and innuendo. If I have to pick a favourite it would be Phil G (king_ghidra), who brought wit and irreverence to his colour commentary. The video and commentary really helped bring the event to life for the folks who weren't able to make it to Loading Bar on the day. I also later found out that a few non-DBL friends of mine had even tuned in to watch the games and show support.
To keep me going through the day I'd brought along a banana, some cereal bars and a Red Bull. I've been to a few NAF tournaments and always have some snacks at hand in case games overrun and there isn't time for get food. I'd also thought about whether I wanted to drink alcohol during the day. Blood Bowl for me is closely associated with drinking and having a pint can be quite relaxing, but I definitely didn't want to get pissed. I ended up having 3 or 4 beers spread over several hours, which felt like a nice balance.
The Final Four (or five)
The four semi-finalists were myself, Gav (gcoleman76 – High Elves), Alex (cerumol – Wood Elves) and Fraser (Necromantic). Gav had finished top of the DBL league table having won 7 and drawn 1 of his 8 matches. Alex had won 7 of his and scored the most Tds (with Gav scoring the 2nd most!). So it's fair to say that many people were predicting an all-Elf final. Fraser also had a very tasty Necromantic team, which included an AG4 Werewolf (with 78 SPP after 11 games, the highest ever in the DBL).
But disaster struck and unfortunately Fraser was ill on the day so couldn't make it. The DBL organisers decided it should be one of the losing quarter finalists to fill his place. Only two of them were going to be at finals day and one of them was James the Commissioner, who stood aside due to his organising duties. So it was to be the former Commissioner Mark (overgone5), whose Brets had been narrowly knocked out in the QFs, who would take Fraser's place against Gav. Mark was also my compatriot from the West Conference, so we'd already played out two really tight draws in the regular season. So I was hoping we'd both make it to the final for some delicious narrative! Although it was a real shame that Fraser wasn't able to play, it was absolutely the right call. Trying to rearrange would at best have delayed the event, but could have led to it not happening at all.
The Semi-finals
I knew in advance I was playing Alex and his Woodies. He'd built up a really strong team and had thrashed another Orc player 5-0 in the knock-outs, but I was feeling reasonably relaxed going into this match. I was definitely the underdog and had already got further than expected, so as long as I played reasonably well there was no shame in going out now. The 'Maryland Marvels' had a lot of Dodge and was packed full of skilled up players, including an AG5 lineman and an a plus MV Catcher and Thrower. However, they were also missing the other dancer after he as MNG'ed in the QF game against Mark; so the remaining MV9 Strip Ball dancer would be priority target No1. We were drawn to play on the second table, without video streaming, but we had excellent text commentary on the Whatsapp chat from Tom (loynster from the TalkNuffle podcasts).
What followed was one of the most bizarre dicings I've ever been part of. Alex's first block was double skulls and that set the tone. By T4 he'd used all his rerolls. Of the first 20 D6s he rolled, I reckon 10 them were 1s (particularly galling for an elf!). Despite kicking to him, I got hold of the ball and slowly ground my way down the pitch. He mounted a stellar defence and by my T7 I was only just in range for the score with 2 GFIs. He just needed to dodge out and push me on 2D… but Nuffle had other ideas. He snake-eyed the dodge roll and reroll, then I made several more difficult rolls to score. 1-0 at HT. To make matters worse, I'd KO'ed the remaining dancer in T7, who then didn't come back second half. Second half I slowly ground my way done the pitch and tried to limit his chances. Eventually I ran it in for 2-0, giving him only 1 turn for a consolation score. He demonstrated his skill and experience to score the one-turner, but it wasn't enough.
Alex contemplates what he did to offend Nuffle, whilst Mark (wearing his West t-shirt) and Gav throw down on the main table
Throughout the game Alex was an absolute gent. Given the stakes involved, it would have been easy to get angry or miserable, but he accepted his rotten dice with good grace. It must have felt particularly tough given he narrow missed out on winning the DBL the year before, having been beaten by Paul's Dwarfs in the final. However, I have no doubt he'll be back to finals day again and the dice will hopefully be kinder.
Meanwhile on the "show table", Mark and Gav were playing out a tight game. The Brets had induced Dolfar Longstrde so both teams were moving it around quickly. I started watching at the beginning of overtime, after the first 16 turns ended 1-1. In the DBL extra-time is two 4-turn halves, with each side receiving once. Both teams scored on their drive, but Mark was forced to score his equaliser quickly, thereby giving Gav 2 turns to win it. His Strong Arm and Accurate thrower made the Long Bomb pass look easy, so their catcher could run it in to win 3-2.
The Final
Before the game there was a short break so I had time to nip out for a sandwich and some fresh air. Up until this point I'd been quite relaxed and was enjoying the occasion, but the realisation that I was now within touching distance of the cup had brought on the nerves. Making finals day had been an achievement in itself, so I wouldn't have been too disappointed if I'd played well and gone out to Alex in the semis. But it now I was in the final, and there was limited chance of me ever getting back there, the pressure had increased significantly. It was difficult to tell if Gav was also feeling the occasion but he looked fairly relaxed, probably down to his experience of playing at the top end of tournaments online and in table top.
The Punks prepare for the final, with skill rings from Magic Works – really handy when players have multiple skills
He had a 15 man squad and chose to buy a keg, so I had 250k in inducements. I thought about stars or a wizard, but ended up buying a 4th reroll with spare cash and then taking 4 dirty trick cards at 50k each. Most of them are useful and some are amazing at that price. One of the cards allowed me to get an extra player on the pitch, so when I won the toss I decided to receive and make the most of my drive. This decision had been complicated by the Sweltering Heat, but I thought it was worth the risk.
Tense moments and delivery of an emergency pint to calm the nerves!
The first half was a bit of a grind as I conservatively made my way up the pitch. Knowing how good and aggressive Gav was I was being quite risk averse. In the build up I'd decided it was better to miss out on the score than open up and give him a chance with AG4 and MV9 (+1MV catcher). This definitely led me to being too cautious and come turn 7 I'd had to move the cage sideways, leaving a gap, in order to get within 5 of the endzone. He duly dodged into the cage on a 5++ and sacked the ball carrier. Argh! I still had a chance to pick it up and run it in, but at this point the pressure definitely got to me. I failed to set up my blocks properly and had to made extra dice rolls. So when I eventually failed the pick-up in a TZ it was all over. HT 0-0.
At this point there were around a dozen people from the DBL gathered around the table and occasionally random punters would come over to see what was happening. I don't think I'd ever had more than a couple of neutrals watching me play so it felt a little bit claustrophobic, adding to the existing pressure of being in the final. The other new experience was the commentary team discussing my every move just 3 feet away. I couldn't really hear what they were saying into the mics, but knowing that everyone's analysing and judging your decisions was quite intimidating! I also remember being devastated when I failed to score: it definitely felt like that was my chance and I'd blown it.
At the start of the second half we rolled for sweltering heat. I got luckier than him, even though I'd had twelve on the pitch and only a squad of 13, so we both started a full team. He'd lost a couple to injury, but his deep squad and keg meant I still had a way to go to get the numbers advantage. He decided to score quickly and with the speed of his catchers and the super thrower, there wasn't much I could do to stop the 2-turner. His defence is really solid and he probably backed himself to turn me over. Although I had 7 turns to equalise, I wasn't hugely confident, especially given I'd previously failed to score after 8 turns with 12 players! It was looking even worse when he rolled a Blitz on the KO table!
He got two players to tag the Blitzer the under the ball and it was looking dicey. If I'd failed the pick up or rolled a double skulls at this point he would likely have gone 2-0 up and put it out of sight. However, at this point my Armour and Injury rolling picked up. He was already down to 10 players whilst I still had 11. In my first turn I managed to KO 2 elves so it was 11 v 8. I then made the big pick up roll and recovered into a reasonable cage.
Gav was using a dice tower for his rolls and I decided to drop my Injury dice into it – when they started coming up hot I continued the habit for fear of offending Nuffle. I'm not a superstitious person and I have a strong belief in probability and statistics, but given my fortunate run to the final I felt this was something I needed to continue! I still wasn't playing particularly well and needlessly gave him a couple of 1 dicers on the blodging ball carrier Blitzer (thankfully neither came off), as well as fluffing one of my Dirty Tricks (auto success on armour roll during a foul) by messing up my Goblin's positioning. However, by around turn 6 I was deep into his half and the casualties were starting to mount. I knew my luck was still in when I managed to argue the call to prevent my Goblin being sent off for a T15 foul (a moment that would later prove very significant). I was then able to escort it in and equalise on T16. FT 1-1.
Extra-time
Going into extra time it was 10 Orcs vs 9 Elves and thankfully the weather then reverted to normal. 4-turn halves probably slightly favour the faster teams, but my slight numerical advantage would help. I managed to move the ball downfield fairly quickly and was able to run it in on my T4, but he had a 1-turner attempt. I played the Dirty Trick 'Slippery Shoes' that makes GFIs fail on 1-3, but fortunately he double-skulled on one of the blocks, and then to rub salt in the wound I broke armour and Cas'ed the Blitzer!
In the second half of extra-time Gav repeated his 2-turner, charging down the wing and making the long throw to a catcher. He'd equalised to make it 2-2, but I still had 3 turns to win it. When DBL games are level after extra-time we go to 'penalties'. The players take it in turns to roll 2 block dice (repeated 5 times); if there's a Skull on either die you miss, if there isn't then you score. Player with the most scores after 5 rounds wins. However, you can use any team rerolls you have left at the end of the match to reroll the 2 dice. Before the match I'd probably have taken penalties, with the format favouring the less experienced coach. I also had a few rerolls more than him, so the odds in a shoot-out were probably in my favour. Therefore I didn't feel under huge pressure to score in those 3 turns. It was definitely more important to play it safe and avoid giving him any chances to steal the ball, especially having been Blitzed earlier in the game and knowing how aggressive he can be.
So I set up quite conservatively and made sure to get the ball safe. Assuming I could do this, I'd already decided to go for the throw team mate on the final turn. There was probably a higher probability play by running a couple of blitzers down the sidelines for the pass/hand-off, but I felt I needed maximum protection around the ball in the earlier turns. Come T4 the Goblin had the ball in a cage near halfway. All the Troll TTM rolls came off, but the Goblin scattered into the tackle zone of a well placed linemen; so a 5+ was needed to land. At this point the excitement in the crowd was building and with every successful roll the noise level went up a notch. So when the Goblin landed with a roll of a 5 all the spectators went crazy: screaming, taking photos and probably scaring other patrons of the pub! I still had to make the dodge to score, but when this came off the Goblin (who had earlier avoided an early bath by arguing the call) ran in for the winning TD.
Tiny Rebel makes his only useful contribution of the whole season!
After about 7 months, 14 games and numerous nail-biting moments, it was finally over. I'd won the cup! In terms of upsets and probability, I think it was probably akin to Leicester winning the Premier League or Greece winning the European Championship. Throughout the season it just felt like everything had gone my way, including avoiding injuries, rolling good skill ups, being drawn against lower tier teams, the draw opening up for me and a huge dose of luck in several matches (especially in the semi). Although my tactics and decision making has improved a bit over the past couple of years, I can't pretend it wasn't mostly down to Nuffle's benevolence (which means I'm due nothing but skulls next season).
Another great part of Finals Day is the post match awards. The Commissioner(s) do a quick speech to thank everyone for taking part and to the Guvs for helping organise it. They then hand out prizes to the four semi-finalists. Each player gets a framed print out of a Backbreaker Sport (the regular, light-hearted DBL 'newspaper' that records matches, player deaths etc) that featured their team. This is a really nice, personal touch, that again adds to the prestige of qualifying for finals day. On top of this Gav was also given a £20 gift voucher. I got a £40 voucher and the GW Ogres pitch. But most importantly, I also got the trophy, which was made by Mark, the ex-Commisioner. It's not fancy, but it has a real charm to it and having each winners name engraved on the side is a nice touch. At the end of the awards ceremony they also announced that I was going to be a Conference Guv for the following season, which for me was the perfect finish to a memorable day.
Although I'd really enjoyed my first season in the DBL in 2018, it had often felt like I was finding my feet and ending up kicking myself for making novice mistakes. This season I'd been a lot more on the ball (pardon the pun) and had walked away from all the games having had a good time and feeling like I'd played reasonably well. Winning the cup is something I'll never forget, but I would still enjoy playing in the DBL even if I had no chance of winning. To emphasise this, immediately after the final I decided to take Goblins in DBL 20 – in honour of the little fella who scored the winning touchdown!
In terms of the league Commissioners and Guvs, they did and exceptional job. This season was bigger and better than the last, with a record number of players and games. The only minor problem was the delay between the QFs and Finals Day, but in the grand scheme of things this is pretty trivial. The fluff, the organisational work, the merchandise, the commentary, the set pieces and the camaraderie were all absolutely brilliant. DBL 20 has just started (possibly a future article) and the number of players is down to 35 – hardly surprising given the global pandemic. However, it's great that it's still happening and I've no doubt it will still be a great experience and the numbers will bounce back in DBL 21.
And finally, a big thank you to all my opponents, conference colleagues, people who came to Finals Day, the Guvs, the Commissioners and everyone else in the DBL last year. I enjoyed every minute of it and look forward to playing in many more Dragon Bowl seasons.
Published by Thomas McGivern
View all posts by Thomas McGivern
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Welcome to Jack O'Connell Web, your premier source for the English actor Jack O'Connell. You may recognize Jack from the television series "Skins" or from the films "Starred Up", "Unbroken" and "Money Monster." The site aims to provide you with all the latest news, photos, media, and more on Jack and his career. Please take a look around the site and visit again soon!
The North Water (TV Mini-Series)
Jack as Patrick Sumner – Filming
The North Water tells the story of Patrick Sumner, a disgraced ex-army surgeon who signs up as ship's doctor on a whaling expedition to the Arctic. On board he meets Henry Drax, the harpooner, a brutish killer whose amorality has been shaped to fit the harshness of his world. Hoping to escape the horrors of his past, Sumner finds himself on an ill-fated journey with a murderous psychopath. In search of redemption, his story becomes a harsh struggle for survival in the Arctic wasteland. Jungleland (Movie)
Jack as Lion – Completed
A reluctant bare-knuckle boxer and his manager/brother must travel across the country for one last fight, but an unexpected travel companion exposes the cracks in their bond along the way. Little Fish (Movie)
Jack as Jude – Completed
A couple fights to hold their relationship together as a memory loss virus spreads and threatens to erase the history of their love and courtship.
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Archive for the 'Godless' Category
"Godless" New York Premiere
emily – November 22, 2017
Events Godless Photos
Jack's new Netflix series Godless is now avaliable for streaming! On Sunday (November 19), Jack attended the New York premiere of the series with Michelle Dockery and Jeff Daniels. The gallery has been updated with HQ photos from the premiere! I will try to find time soon to watch the show and will add screen captures and episode stills!
Public Appearances > 2017 > Nov 19 | "Godless" New York Premiere [+57]
"Godless" Official Trailer
emily – October 25, 2017
Godless Trailers Videos
The official trailer for Jack's upcoming Netflix series, Godless has been released. The series will release on November 22nd!
It is a fearful thing, to love what death can touch.
Godless follows the fates of Frank Griffin and his murderous gang… of Roy Goode, the member who turned against them… and of La Belle, the town full of women who must defend themselves when they get caught in the crossfire. Welcome to Godless country.
Jack O'Connell on 'Money Monster', Jodie Foster, and Playing Cowboy for Netflix's 'Godless'
emily – May 13, 2016
Godless Interviews Money Monster
COLLIDER – From director Jodie Foster, the real-time, high-stakes thriller Money Monster follows financial TV host Lee Gates (George Clooney) and his producer, Patty Fenn (Julia Roberts), as they are put in an extreme situation when a young man (Jack O'Connell) who has lost everything takes over their studio. During a tense stand-off that's broadcast to millions on live TV, Lee and Patty must unravel the mystery behind a conspiracy that led to the disappearance of $800 million of investor money.
At the film's press day, actor Jack O'Connell sat down with Collider at the Four Seasons Hotel in Los Angeles for this exclusive interview about what drew him to Money Monster, making the accent feel lived in, and whether his character was seeking redemption. He also talked about playing a cowboy in the upcoming Netflix series Godless, set in 1800s America. Be aware that there are some spoilers.
Collider: This is a wholly original adult thriller, it's smart and relevant, it relies much more on character development than action sequences and set pieces, it's opening in summer movie season and it's directed by a woman, which is very rare these days. When you got this script and read it, what was the draw for you?
JACK O'CONNELL: Just the reality of it all. What personally makes me feel very excited, as an actor, is when a film is brave and bold and it exposes wrongdoings, which are real. And it was a great opportunity to step into the shoes of someone that I could find quite instantly relevant. Being the same age, I'm sure if I wasn't acting, I'd probably be a lot worse off than Kyle is. That was quite instantaneous, the way I was able to adopt that, and that was very attractive. But also, to be in there with Julia [Roberts], George [Clooney] and Jodie [Foster], it gives you an instant gratification.
Doing an accent is a challenge, in and of itself, but in this film, you have a lot of dialogue. Does that make it even more challenging?
O'CONNELL: It can never be an impersonation. I can never be trying to imitate. I have to feel like I'm giving something that's lived in and natural. That's the main challenge. It's made a lot easier when all of the content is on the page 'cause then you have something very definite to work towards. Also, sometimes in a scene, you might go off page, so you have to give yourself enough room and prepare enough. That way, when you do find yourself in that scenario, you have something that you believe in. Otherwise, it's just too much to juggle. It requires preparation. I can't imagine taking on any role without trying to figure them things out first.
Jack O'Connell to Star in Topline Netflix Western 'Godless'
Godless News
THE HOLLYWOOD REPORTER – Jack O'Connell will lead the cast of the Steven Soderbergh and Scott Frank limited series.
Jack O'Connell is riding to Netflix.
The Unbroken star has been tapped to topline the streaming giant's Western drama Godless, The Hollywood Reporter has learned.
Godless, from Steven Soderbergh and Scott Frank, is set in 1884 and centers on Frank Griffin, a menacing outlaw who is terrorizing the West as he hunts down Roy Goode, his son-like partner-turned-mortal enemy. While Roy hides at Alice Fletcher's ranch, Frank's chase leads him to the quiet town of La Belle — that is mysteriously made up of entirely women.
O'Connell will star as Roy, the injured outlaw on the run from his former boss who was orphaned at a young age and was taken in by Griffin who ultimately breaks up a robbery and takes off with the loot, splitting from his father figure after he no longer can stomach his behavior.
For O'Connell, the role marks his first TV foray after his breakout film role as Olympic track star and World War II POW Louis Zamperini in Angelina Jolie's Unbroken. Before that, his TV credits included Skins and The Runaway. On the big screen, his feature follow-ups have included Money Monster and Tulip Fever. He is repped by CAA, Conway van Gelder Grant in the U.K. and Sloane Offer.
Billed as a limited series, Frank will pen the script, direct and executive produce alongside Soderbergh and Casey Silver.
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This morning, 10/18/2018, on Today with Meghan Kelly, former star of the Real Housewives of Orange County, Laurie Peterson sat down with Meghan to discuss her all-out war to prove her son's innocence who is charged with 3 counts of attempted murder. Her son, Josh Waring, has been incarcerated for over 2 years after being charged.
Peterson was one of the original cast members of RHOC and was on the show for 3 seasons. She chose to be a cast member when her son was only 14 and she was going thru a really bad divorce. At that time, Josh was using marijuana. However; it progressed to an addiction to heroin landing him in the courts on different charges. Due to the toll of the fighting on the show with other cast members and her son's addictions, Peterson chose to leave the show and work on her own family.
During her son's addiction problems, she was forced to ask her son to move out of their home. She tells Meghan that she always offered her son any financial help he would need to get counseling and to become clean but she would not enable him.
Peterson says they now have evidence to prove her son was framed before they go to trial. The victim first stated the gunshots came from another car but has since changed his story. There was no gunshot residue found on her son but there was old residue on the passenger side of the car where another passenger with a criminal history was riding. This friend of her son also has a long criminal history.
Waring was arrested in June 2016 after he allegedly shot a man in Costa Mesa, California, and then engaged in a car chase with police. Along with three counts of attempted murder, Waring faced charges of assault with a firearm, evading police while driving recklessly and resisting an offer and battery, among others, according to the Los Angeles Times.
"After keeping silent for nearly 2 years in regards to my son's arrest, I am going to begin laying out the facts of his case and how easily it is to be falsely accused!" Peterson tweeted.


text:
SpaceX will send a Japanese lander and an Emirati rover to the Moon
SpaceX's Falcon 9's take-off was supposed to take place from Cape Canaveral a few days ago. After an initial adjournment on 30 November to allow final scrutiny, the result led to a new postponement for an indefinite period.
The Falcon 9 rocket with the Hakuto-R M1 on board. Originally, the probe was supposed to board as a secondary passenger on a geostationary satellite, but eventually finds itself on its own. This allows it to have an orbit that brings it closer to the moon's south pole. © SpaceX
iSpace is a start-up that has survived the times. It was based on the work of a team that had responded for the XPrize challenge, whose goal was to send a rover to the Moon by 2018. No winner. Google withdraws from the challenge, which ends up disappearing, but iSpace continued to work on its lunar lander with its program Hakuto-R.
The Hakuto-R M1 probe when placed under the hood. © SpaceX
The first private mission to the Moon in history
iSpace's Hakuto-R program has two missions: a first in the coming days to test the lander's ability to land on lunar soil, and a second in 2024 with an additional rover. Like any future program mission, Hakuto-R M1 carries private and public payloads.
Full-size scale models of the Hakuto-R and Rashid rover on display at IAC in September 2022. On the extended legs, the lander is 2.3 m high and 2.6 m wide for a mass of 340 kilograms. The rover weighs 10 kilos. © Daniel Chretien, Futura
After launch, Hakuto-R M1's journey will last several months, during which the probe will gradually increase its altitude in Earth orbit until it injects itself into a translunar orbit. The probe will then remain in lunar orbit for two weeks before touching down in the Atlas Crater on the southeastern edge of the Frigoris Sea, a location that allows the probe to be illuminated by the Sun.
Hakuto-R mission 1 flight plan. © iSpace
> Read more about Futura
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UK wants to ban Chinese surveillance cameras on its sensitive sites
Microsoft Rewards – A 500 point card with featured Game Pass games


text:
Advances in Virology
Advances in Virology / 2017 / Article
AbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsConflicts of InterestAuthors' ContributionsAcknowledgmentsReferencesCopyright
Carolina de la Guardia, Mario Quijada, Ricardo Lleonart, "Phage-Displayed Peptides Selected to Bind Envelope Glycoprotein Show Antiviral Activity against Dengue Virus Serotype 2", Advances in Virology, vol. 2017, Article ID 1827341, 11 pages, 2017. https://doi.org/10.1155/2017/1827341
Phage-Displayed Peptides Selected to Bind Envelope Glycoprotein Show Antiviral Activity against Dengue Virus Serotype 2
Carolina de la Guardia ,1,2 Mario Quijada,1 and Ricardo Lleonart 1
1Center of Cellular and Molecular Biology of Diseases, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP), Building 219, Ciudad del Saber, Apartado 0843-01103, Panamá, Panama
2Department of Biotechnology, Acharya Nagarjuna University, Guntur, India
Academic Editor: Gary S. Hayward
Received25 May 2017
Accepted26 Jul 2017
Dengue virus is a growing public health threat that affects hundreds of million peoples every year and leave huge economic and social damage. The virus is transmitted by mosquitoes and the incidence of the disease is increasing, among other causes, due to the geographical expansion of the vector's range and the lack of effectiveness in public health interventions in most prevalent countries. So far, no highly effective vaccine or antiviral has been developed for this virus. Here we employed phage display technology to identify peptides able to block the DENV2. A random peptide library presented in M13 phages was screened with recombinant dengue envelope and its fragment domain III. After four rounds of panning, several binding peptides were identified, synthesized, and tested against the virus. Three peptides were able to block the infectivity of the virus while not being toxic to the target cells. Blind docking simulations were done to investigate the possible mode of binding, showing that all peptides appear to bind domain III of the protein and may be mostly stabilized by hydrophobic interactions. These results are relevant to the development of novel therapeutics against this important virus.
Dengue virus is a growing public health problem worldwide as about 390 million people get infected annually and almost 96 million people develop clinical manifestations of the disease [1]. Other authors estimate that about 3.9 billion people from 128 countries share the risk of infection with this virus [2]. Dengue virus can produce a wide spectrum of clinical presentations, from asymptomatic or mild manifestation to a more severe life threating manifestation, known as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Severe dengue can be deadly, especially in children, due to plasma leaking, respiratory distress, edema, severe bleeding, and organ impairment [3].
This virus is a Flavivirus that is transmitted by mosquitoes of the genus Aedes. The incidence of dengue infections has increased due to the spread of these mosquitoes into new regions [4]. DENV are present as 4 serotypes (DENV1, DENV2, DENV3, and DENV4) circulating in most endemic countries [5]. Dengue fever is considered the most rapidly growing mosquito-transmitted disease worldwide [6].
The dengue virion has an icosahedral symmetry, with diameter between 500 Å and 600 Å [7]. The viral genome consists of 11 Kbp single positive stranded RNA coding for a single polyprotein. The polyprotein is cleaved in the cytoplasm into several structural and nonstructural polypeptides [8]. The structural proteins include the capsid (C), premembrane (PrM)/membrane (M), and envelope glycoprotein (E), which are involved in the formation of the viral particle. The nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) are responsible for the viral replication, assembly, and immune response escape [8].
E glycoprotein is the most important molecule during the viral entry process as it appears to be responsible for receptor recognition and attachment to the cell surface, triggering the clathrin mediated endocytosis and the subsequent fusion of viral and cellular membranes. This protein can interact with diverse cellular molecules; therefore, it is an ideal target for the development of new antivirals [9–12]. E glycoprotein is formed by three domains, plus a membrane proximal stem and a transmembrane anchor [13–15]. Domain I is formed by eight β-strand barrels containing two insertion loops. Domain II contains hydrophobic sequences that are conserved among all flaviviruses. These hydrophobic sequences, also known as fusion peptides, are responsible for the insertion of the rearranged E trimer into the cellular membrane during fusion [9, 14–16]. Domain III (DIII) is an immunoglobulin-like carboxyterminal domain, responsible for the initial cellular receptor binding [17]. Additionally, the DIII is the main target for neutralizing antibodies [18].
Due to the complexities of the immune response and the pathology generated by this virus, particularly during subsequent infections, the development of vaccines has been slow and there is only one vaccine that has been registered, which is still in the early stages of testing in several territories [19, 20].
Currently, there is no approved antiviral for the treatment of dengue infection. It is known that the duration of the viremia is short in dengue patients. However, since high viremia has been related to a severe onset of the disease, the use of antivirals at early stages of the disease may block the progression of the disease and accelerate the recovery of patients. Here, we proposed the use of dengue virus envelope (E) glycoprotein as a target to search for peptides that could interfere with the first step of the infection process. We selected the DENV-2 E glycoprotein and the DIII as targets for the screening random peptides displayed on M13 bacteriophages.
Phage display technology has been used in past years to identify peptides with specific binding activities to a variety of targets. This technique has been very useful to identify mimotopes, novel antivirals [21], peptidomimetic drugs [22], and many other applications.
Here we show that the DENV2 E glycoprotein, as well as domain III (DIII), is useful target for the identification of phage-displayed peptides with potential as novel antivirals. Here we describe three peptides that inhibit the viral infectivity and are not cytotoxic to the permissive cells. Fully blind docking simulations with these peptides suggest binding sites at domain III of the envelope protein, stabilized by predominant hydrophobic interactions. These findings open new possibilities to optimize and refine the design of new peptidic inhibitors of infection by this virus.
2.1. Cells, Viruses, and Peptides
Vero cells (CCL-81, ATCC, USA) were used for dengue 2 virus propagation, plaque formation assays, and cytotoxicity assays. These cells were grown in MEM with 10% FCS and 50 μg/ml gentamicin, at 37°C and 5% CO2. Dengue virus, serotype 2, was a kind gift from Department of Research, Instituto Conmemorativo Gorgas de Estudios de la Salud (ICEGS), Panamá. Heptapeptide random library was obtained from New England Biolabs (USA). Synthetic peptides were obtained from Genscript (USA). Full DENV2 E glycoprotein (Cat. number Den-034) was obtained from ProSpec-Tany TechnoGene (Israel). Tissue culture, molecular biology grade, and general reagents were from Nunc and Sigma-Aldrich (USA).
2.2. Recombinant Protein Expression
Sequence coding for DENV2 E DIII (aa 289 to 405) was obtained from GenBank (complete genome NCBI Reference Sequence: NC_001474.2) and optimized for expression in Escherichia coli using an online tool (GeneOptimizer, Geneart). The optimized sequence was synthesized (IDT) and subcloned into NdeI-XhoI restriction sites of the expression plasmid pET-30b(+) (Novagen) using standard recombinant DNA techniques. This plasmid directs the inducible production of the DIII plus the amino acids Leu and Glu and a 6xHis tail at the C-terminal. This fragment of the E protein contains 125 amino acids and an approximate molecular weight of 14.2 kDa. The expression plasmid was verified by sequencing and transformed into E. coli strain BL21 (DE3) for expression. Single colonies were cultured in LB medium containing antibiotic at 37°C and 250 rpm and induced with 0.5 mM IPTG when OD600 reached 0.6. After 4 hours of induction cells were collected by centrifugation and kept at −20°C until purification. As expression of the recombinant protein was mostly at the insoluble fraction, further purification was done including inclusion body isolation, solubilization, and affinity purification in denaturing conditions, refolding, and dialysis.
2.3. Purification of Recombinant DIII
Frozen pellet from induced culture was suspended in cold lysis buffer (10 mM Tris-HCl pH 7.5, 5 mM benzamidine-HCl, 5 mM EDTA, 5 mM DTT, 0.3 mg/ml lysozyme, and 1 mM PMSF) and lysed by sonication on ice. The inclusion bodies were recovered by centrifugation, washed with 50 mM phosphate buffer, 5 mM EDTA, 200 mM NaCl, 0.5 M urea, and 1% Triton X-100, recovered as before, washed with 50 mM phosphate buffer, 1 mM EDTA, and 1 M NaCl, and solubilized in denaturing buffer (10 mM Tris-HCl pH 8.0, 100 mM NaH2PO4, 100 mM NaCl, and 6 M GuHCl). The solubilized inclusion bodies were purified using a Ni-NTA superflow cartridge (Qiagen). Following loading and washing with buffer C (6 M GuHCl, 100 mM NaH2PO4, and 100 mM HCl, pH 6.3), protein was eluted with buffer E (6 M GuHCl, 100 mM NaH2PO4, and 100 mM HCl, pH 4.5). Attempts to dialyze against PBS resulted in protein precipitation; therefore we tested several refolding conditions using a Protein Refolding kit (Pierce, USA). Finally, refolding was done by dilution into refolding buffer RB7 (1 mM GSH, 1 mM GSSH, 1 mM EDTA, 1.1 M GuHCl, 55 mM Tris-HCl, pH 8.0, 21 mM NaCl, and 0.88 mM KCl). The refolded protein was dialyzed against TBS, filter sterilized, and quantitated by BCA method.
2.4. In Vitro Virus Blocking Assay to Test Protein Refolding
Aliquots of refolded recombinant DIII, obtained from several refolding conditions, were first visually checked for aggregation. Those not showing aggregation were then tested in virus infection blocking experiments, to check for their ability to bind cellular receptors and inhibit these first steps in viral infection. Vero cells were plated in 96-well plates at 104 cells per well in complete medium (MEM, 10% FCS, and 50 μg/ml gentamicin). On the next day, cells were washed with PBS and incubated with the refolded protein (100 μl, 50 μg/ml) in maintenance medium (MEM, 1% FCS, and gentamicin) for 30 min at 37°C. Then cells were washed and incubated with DENV2 (MOI = 3, 50 μl), containing the refolded protein at the same concentration, for 1 h at 37°C in maintenance medium. Cells were again washed, replenished with maintenance medium, and incubated at 37°C, 5% CO2. After 5 days, cytopathic effect was quantified using chemiluminescent based ATP detection (CellTiter-Glo® cell viability assay, Promega).
2.5. Biopanning
Phage display was performed using a Ph.D.-7 Phage Display Peptide Library according to the manufacturer's instructions. Four rounds of panning were done using two targets, the full DENV2 envelope, or the recombinant DIII obtained as described above. For each panning step, several wells in ninety-six-well plates were coated overnight at 4°C and then blocked with 400 μl of 0.1 M NaHCO3 (pH 8.6) and 5 mg/ml BSA for 1 h at 4°C. After six washes with TBST (TBS, 0.1% Tween 20), 100 μl of phages in TBST, containing 1011 pfu were allowed to bind for 1 h at RT. Then wells were washed ten times with TBST and phages eluted with 0.2 M glycine-HCl pH 2.2, 1 mg/ml BSA. After neutralization with Tris pH 9.1, phages were titered and bulk amplified in E. coli for next round. In order to increase the stringency of the selection, successive rounds were done increasing the concentration of Tween-20 during washes and reducing the amount of target fixed to the solid phase. After the fourth round, eluted phages were plated, picked, and propagated for subsequent phage-ELISA and sequencing.
2.6. Phage-ELISA
DENV2 E, DIII, and BSA were used to coat 96-well microtiter plate (MaxiSorp, Nunc) overnight at 4°C. Plates were blocked with TBS-BSA, washed with TBST, and incubated with 1012 pfu of individual phages. After washing, HRP-conjugated anti-M13 monoclonal antibody (GE Healthcare, USA) was added, incubated, and washed. Color was developed by adding TMB substrate solution for 10 min and stopped with 1 N HCl. Plates were read at 405 nm. Phage clones with the best target-to-background signal ratio were selected for DNA sequencing. The DNA insert was determined by cycle sequencing using a 96-gIII primer as recommended by manufacturer. Sequence logos and residue coloring was done as implemented in JalView v.2.10.1 [23]. Coloring was following Zappo scheme, where residues are colored according to their physicochemical properties as follows: pink, aliphatic/hydrophobic; orange, aromatic; red, positive; green, negative; blue, hydrophilic; magenta, proline/glycine; and yellow, cysteine.
2.7. Viral Plaque Reduction Assay
Vero cells were seeded (2 × 105 cells per well) in 6-well plates and incubated overnight. The peptides were prepared at several dilutions and mixed with 100 pfu of DENV2, for a final volume of 150 μl. Mixtures were incubated at RT for 1 h and then added to cell monolayers and incubated 1 h at 37°C for adsorption. Then inoculum was removed and cells were washed and overlaid with maintenance medium with 1% methylcellulose and incubated for 5 days. Then medium was aspirated; cells were fixed with 10% formaldehyde and stained with 1% crystal violet. The viral plaques were visually counted and values from experimental groups compared to virus alone, nontreated controls.
2.8. Peptide Cytotoxicity Assay
Peptides were incubated with cells exactly as described in the viral plaque reduction assay (1 h for 37°C) and then washed away and cells refed with maintenance medium and incubated at 37°C for 24 h. Then cytotoxicity was estimated by measuring cellular ATP using luminescence as described by the manufacturer of the CellTiter-Glo® cell viability assay. Viability was estimated by comparing readings of peptide-treated cells with those of nontreated cells.
2.9. Peptide-Protein Docking Simulations
The proteins structures used for the peptide-docking analysis were either obtained from the database of protein structures (in the case of the DENV envelope protein, PDB 1OAN) or obtained in our lab by homology modeling (for DIII). Sequence corresponding to the expressed protein was used to obtain the structure by homology modeling using I-TASSER web server [24]. The best model predicted by I-TASSER presented good predictive value (-score = 0.77) that was structurally closely related to several viral envelope glycoproteins in the PDB database. To study the probable binding mode of the active peptides, we performed computational docking analysis using CABS-dock web server with the default parameters except for the increase of simulation cycles to 200 [25]. The best poses, ranked according to trajectory characteristics, were further analyzed to describe intermolecular interactions using LigPlot+ [26].
2.10. Statistical Analysis
Where indicated, medians were compared between the nontreated control and all other groups using Kruskal-Wallis with Dunn's multiple comparison test, as implemented in GraphPad Prism software.
3.1. Expression, Purification, and Refolding of Recombinant DENV2 Envelope Domain III
The dengue virus envelope is a class II virus fusion protein. This protein is a β strand rich, elongated molecule with three ectodomains, the centrally located DI, the apical DII which bears the fusion loop, and the Ig-like DIII, which is connected to the short, stem, and transmembrane C-terminus [14, 27]. DIII undergoes drastic repositioning during transition to the fusogenic conformation of the E protein, leading to rearrangement from dimeric to trimeric form [15, 28]. DENV E domain III forms an Ig-like β-barrel structure that is stabilized by one disulfide bridge.
The DIII is a key part of the envelope, as it is the receptor binding domain [13, 29]. It is also very antigenic, and antibodies to this domain are able to efficiently neutralize virus infection [18, 30]. Taking into account the important role of the envelope protein, as well as its domain III, we decided to search for peptides with binding activity to this protein, in an attempt to find novel molecules able to impair the infection process. For this purpose, we selected phage display as a robust methodology that allows the rapid screening and selection of millions of peptidic variants for binding capacity to a variety of targets. As the full envelope glycoprotein is a large protein that may present many possible nonproductive binding sites, we chose also to screen the random peptide library with domain III obtained in our lab.
The region coding for domain III of the envelope protein of DENV2 was obtained as a synthetic fragment from a commercial source after codon optimization for E. coli expression. This fragment was subcloned into the E. coli expression vector pET-30b under the control of a T7 promoter and fused to a 6xHis tail at the C-terminal. After IPTG induction, E. coli cultures showed high level of expression of the recombinant protein (Figure 1(a)), accounting for more than 50% of total protein, as judged from densitometric analysis of Coomassie stained SDS-PAGE gels (data not shown). As the recombinant protein was mainly present in insoluble aggregates, the purification protocol included an isolation and wash of inclusion bodies, followed by an affinity purification by affinity Ni-NTA chromatography under denaturing conditions. The affinity purification procedure implemented here allowed a high purity preparation of DIII (Figure 1(a)).
Cloning, expression in E. coli, and purification of codon optimized, dengue 2 envelope domain III. (a) SDS-PAGE showing several steps of protein expression and purification. 1, total lysate of cells before induction; 2, total lysate of cell after IPTG induction; 3, protein molecular weight standards; 4, solubilized inclusion bodies before loading into Ni-NTA column; 5, column flow-through; 6-7, two successive washes; 8-9, two successive elution. (b) Activity of purified and refolded recombinant domain III against DENV2 in Vero cells. Protein was refolded using several refolding conditions (RB1 to RB9) before dialysis and filtration for testing against the virus, in parallel with virus alone and ribavirin as a control. Data is presented as the mean and SD of two independent experiments. Condition RB1 was not used in this experiment as protein consistently precipitated.
Several attempts to refold this protein by dilution into PBS failed due to precipitation; therefore the refolding conditions for this protein were further explored using several experimental conditions. These tests included varying concentrations of refolding agents and additives, such as L-arginine, reduced and oxidized glutathione, and/or polyethylene glycol [31]. It has been shown that the dengue envelope DIII may act as a dominant negative inhibitor of the infection process [10, 32–35]; therefore we decided to use this criterion to assess the functionality of the refolded DIII. After the refolding tests, each protein preparation was dialyzed against TBS, filter sterilized, and tested in a DENV2 virus binding blocking assay in Vero cells. We found that recombinant protein refolded in buffer RB7 (1.1 M guanidine, 55 mM Tris-HCl, pH 8.2, 21 mM NaCl, 0.88 mM KCl, 1 mM GSH, and 1 mM EDTA) showed a significant inhibition of the infection (Figure 1(b)) while there were no signs of cytotoxicity (data not shown), suggesting that the protein was folded and functional. This procedure was then used at a higher scale to isolate the recombinant EDIII for the biopanning and further steps.
3.2. Selection of Phage-Displayed Envelope Binding Peptides
In order to select for peptidic binders to both targets, we used a random library of heptapeptides presented in the M13 phage pIII protein. These phages would contain 1 to 5 copies of the peptides per capsid, theoretically allowing for the selection of higher affinity binders. As baits for the panning procedure, two sources of proteins were used, (1) a recombinant, insect-expressed DENV2 envelope and (2) recombinant, E. coli expressed domain III. The panning procedure involved four rounds of phage selection on solid phase-bound protein and subsequent elution and amplification. During panning progression against both proteins, the amounts of eluted phages increase stepwise (Figure 2), suggesting an effective enrichment of particular phages. After final round, 24 phages were randomly selected from each panning scheme, propagated, and purified to test their binding ability to the respective bait by phage-ELISA.
Eluted phages were enriched in successive rounds or panning. During each panning, 1011 input phages were incubated with the target proteins (full DENV2 envelope or domain III) and eluted as described in Materials and Methods. The eluted phages were neutralized and titer was estimated. Data is presented as the absolute number of eluted phages at each step.
The analysis of eluted phages by ELISA showed that many appear to bind in a specific manner, indicated by absorbance values higher for the target as compared to those against the background (BSA) (Figure 3). Out of 24 randomly picked phages from each panning, 12 were confirmed for the full E glycoprotein and 21 were positive for the E domain III fragment. This behavior was shown by phages bearing the peptides STSFWIT, NERALTL, ELLASPW, SPSTHWK, LALAEIT, NLQIYAV, and SLSSVHD. Some other clones did not show good target-to-background signal ratio and were not used for sequencing or subsequent analyses. This result is not unexpected, as panning procedure may yield artifact binding phages which later do not bind well in the context of the ELISA.
In vitro binding of selected clones as shown by phage-ELISA. Absorbance values by ELISA when phages are incubated in the presence of solid phase-bound specific ligand (target) or unspecific ligand (bovine serum albumin, as background). The binding of phages was revealed by anti-M13 antibody. (a) Results of phage-ELISA using E domain III as target. (b) Results obtained when using full E glycoprotein as target.
The phage-ELISA positive phages, 33 in total, were submitted to DNA sequencing of the randomized region. DNA sequencing revealed that binding phages carried peptides with some common features, with similar consensus sequences for the envelope glycoprotein (SxSAxxx) and for the domain III protein (SxSxHTL) (Table 1, Figure 4). The corresponding peptides had abundance of negatively charged residues, intercalated with positively charged, hydrophilic, and aliphatic/hydrophobic residues (Figure 4). The resulting peptide sequences are consistent with one of the proteins (DIII) being a smaller portion of the other (E), as the consensus sequences share some resemblance, and the consensus for peptides selected against DIII appears to be more defined. Interestingly, several phages bearing the same peptide were selected with both proteins and appeared to be repeated in the final round elution (Table 1). The fact that some phages were observed several times is also congruent with a successful enrichment of specific binders during the panning steps.
Target: envelope Target: domain III
Peptide Frequency (%) Peptide Frequency (%)
DYPANKH 10 AAHYEHR 5
ELLASPW 20 FMXSHNG 5
LGSPMSN 20 HAMRAQP 5
NERALTL 20 HFWHLTP 5
SPSTHWK 10 LALAEIT 5
STSFWIT 10 LSNNNLR 10
YHKQIGP 10 MNPSKSL 5
NERALTL 15
NLQIYAV 5
SLSSVHD 5
SPSTHWK 15
STSFWIT 5
SYQSHYY 5
VSSTHLY 5
WNAKYTL 5
Peptide sequences at the randomized region and frequency of appearance of bearing phages.
Peptide sequences resulting from binder phages confirmed by phage-ELISA. (a) Peptides resulting from panning on full DENV2 envelope. (b) Peptides resulting from panning on recombinant DENV2 envelope domain III. Sequence logos and consensus sequence, as calculated using JalView, are depicted below each list of peptides. Coloring of residues and sequence logos as per Zappo scheme, where residues are colored according to their physicochemical properties as follows: pink, aliphatic/hydrophobic; orange, aromatic; red, positive; green, negative; blue, hydrophilic; magenta, proline/glycine; and yellow, cysteine.
Some of the binding phages shown here carried peptide sequences already reported at the biopanning data bank, a repository that, at the moment of writing this paper, had more than 23,700 sequences appearing in biopanning data [36]. The peptides SPSTHWK and WNAKYTL were also previously reported to bind crystalline Ni3B nanoparticles [37]. Also, the peptide NERALTL appeared twice in the database, with binding activity to epoxy covered surfaces [38] and to the fusion protein of the infectious salmon anemia virus [39]. Additionally, the peptide LSNNNLR was previously reported to bind poly(dimethylsiloxane) [40].
3.3. Activity of Selected Peptides and Possible Mode of Binding
Peptides from ELISA verified binding phages were synthesized containing the additional Gly-Gly-Gly-Ser spacer at the C-terminal and amidation of the carboxylate to block the negative charge, as suggested by the manufacturer to mimic the context of presentation in the M13 pIII protein. Then synthetic peptides were tested to check their ability to impair the infection of DENV2 in Vero cells. The synthetic peptides ELLASPW, SYQSHYY, and STSFWIT showed inhibitory activity against DENV2 infection in a plaque reduction test (Figure 5(b)). Some peptides that showed good in vitro binding to their target proteins did not show consistent inhibitory activity against the virus, that is, the peptide SPSTHWK, and were considered negative for viral inhibition (Figure 5(b)). Since the peptides were incubated only during the adsorption period (1 h at 37°C), it may be possible that the observed inhibition is due to interference in this initial step of the virus life cycle or due to virucidal activity. More experiments are required to elucidate the exact mechanism of action of these peptides. When the active peptides were incubated alone with Vero cells in the same conditions as those of the viral infection, no sign of cytotoxicity was observed (Figure 5(a)).
Biological activities of active peptides in Vero cells. (a) Cytotoxicity of active peptides. Cells were incubated with increasing concentrations of the peptide for 1 h and washed away and cells incubated in maintenance medium for 24. Then cytotoxicity was estimated from ATP levels using luminescence. The viability was estimated by comparing with nontreated cells. Data is presented as means and standard deviations of two independent experiments. Medians were compared between the nontreated control and all other groups using Kruskal-Wallis with Dunn's multiple comparison test. (b) DENV2 infectivity inhibition by peptides, as shown in a plaque reduction assay. The reduction in the number of plaques was estimated by comparison with virus-infected cells alone. The peptide SPSTHWK is presented as negative control since it showed no consistent inhibitory activity against the virus. Data is presented as means and standard deviations of two independent experiments.
The fact that some other peptides with good binding activity at the panning and ELISA tests did not inhibit the DENV2 infection is not surprising, as some of these small peptides might be binding to target regions that are not critical for their functions during binding and entry. It may be also possible that some of these peptides interact differently with their targets in their synthetic form compared with the case when they were presented in the context of the phage protein pIII.
In order to explore possible modes of binding of these peptides to their targets, we performed computational docking analysis using CABS-dock online server [25]. The algorithm pipeline followed by this software allows flexible, fully blind protein-peptide docking, finishing with all atom reconstruction and optimization of the best poses [41, 42]. The structures of the proteins used for the docking were either obtained from the database of protein structures (in the case of the DENV2 envelope protein, PDB 1OAN) or obtained in our lab by homology modeling (in the case of the DENV2 EDIII).
The docking simulations allowed prediction of possible poses for the binding of the active peptides to their target proteins. The best models predicted indicated a predominance of hydrophobic interactions over hydrogen bonds (Figure 6). Interestingly, the best pose predicted for the envelope glycoprotein and the corresponding peptide ELLASPW proposed a binding site also at the domain III of the protein (Figure 6). The suggested pose with the lowest interaction energy for the peptide ELLASPW on the envelope glycoprotein indicates hydrophobic interactions of the peptide with the protein residues I335, K334, L351, V354, F337, E338, V347, K344, R345, M340, Q386, and V382 and a hydrogen bond with E383. The suggested interactions of peptide STSFWIT with DIII include hydrophobic links with Y377, E403, L387, L389, K307, E311, K310, and F402 and hydrogen bonds with K388 and V308. Similarly, the best pose proposed for peptide SYQSHYY includes hydrophobic interactions with V382, M301, P336, E338, L294, K334, F337, I339, N355, M340, K295, V354, and R350 and hydrogen bond with K291.
Probable binding modes of active peptides on their targets, as explored by molecular docking simulation. (a) Most favorable pose of peptide ELLASPW on the DENV2 envelope. Inset shows more details of putative binding site. (b) Most favorable poses of peptides STSFWIT and SYQSHYY on domain III. Peptide STSFWIT occupies the upper binding site of the molecule in this diagram. Proteins are depicted in their hydrophobicity surfaces, while peptides are in stick model. (c) Interaction diagram for ELLASPW-envelope. (d) Interaction diagram for STSFWIT-domain III. (e) Interaction diagram for SYQSHYY-domain III. Interaction diagrams were done in LigPlot+. Intermolecular hydrogen bonds are indicated by green dashed lines, while hydrophobic interactions are represented by arcs and radiating spokes. Pose figures were prepared using UCSF Chimera.
Domain III of the dengue envelope has been shown to be involved in the initial steps of the binding of the virion to the receptor/attachment factors, particularly the amino acids in the sequence 380-IGVEPGQLKL-389 [13, 43, 44]. Interestingly, the best poses proposed by the simulation indicate some overlap with this region. The peptide STSFWIT appears to bind a region of DIII overlapping the 380–389 sequence, with three of its amino acids interacting with the peptide: L387, K388, and L389. The peptide ELLASPW does not appear to block or interfere with the receptor binding sequence, although two of its residues do participate in hydrophobic interactions with the peptide, E383 and Q386. The peptide SYQSHYY does not bind overlapping the receptor binding region, although one of its amino acids does interact with the peptide (V382). Interestingly, this last peptide also appears to bind very close to the sequence DKLQLK, interacting with the residues K291, Q293, and L294. This binding site may be relevant to the entry process as lysine 291 and lysine 295 were shown to be key for binding to the cellular GAGs [45]. Although best poses proposed here for the interactions between these peptides and their targets show favorable interaction energy profiles (data not shown), it should be also noted that, due to the complexities of modeling small peptides, there may be other valid binding sites. Further studies may be required to validate the proposed binding modes of the peptides and perform a rational, structure directed improvement of their biological activities.
The phage display technique has been used by several authors to search for interacting peptides, with the ultimate objective of developing new drugs against several viruses. Some examples include peptides described with activity against HIV [21, 46], Puumala virus [47], West Nile virus [48], Newcastle virus [49, 50], hepatitis C virus [51, 52], porcine reproductive and respiratory syndrome virus [53, 54], infectious bronchitis virus [55], hepatitis B virus [56], Mink enteritis virus [57], Japanese encephalitis virus [58], classical swine fever virus [59], and influenza virus [60].
Regarding peptidic inhibitors of dengue virus, three main strategies have been followed: (1) in silico designed peptides, (2) peptides mimicking the viral protein sequences, and (3) peptides obtained by panning against viral proteins. While the development of antiviral peptides against DENV has been dominated by reports using the first two approaches [61–64], a few attempts have been made to make use of the versatile phage display technology to find new antivirals. The peptides identified using random peptide libraries include those reported by Panya et al., 2014 [65], which target the envelope hydrophobic pocket, and those reported by Chew et al., 2015 [66], which target the full DENV2 virion. The peptides reported here do not share homology with the ones found by these authors, although results may not be fully comparable as different targets and libraries were used.
In conclusion, we have used a novel strategy to identify inhibitors of the DENV2 infectivity and we have found three peptides out of a random peptide library, which are able to specifically bind viral envelope protein and inhibit infectivity in vitro, without showing toxicity to the cells. The binding modes suggested by blind docking simulations indicate that the interactions between these active peptides and their targets may be stabilized by hydrophobic interactions, providing information relevant to the future improvement of new antivirals against this important virus.
The authors declare that there are no conflicts of interest.
Carolina de la Guardia participated in experimental work and drafting of the manuscript. Mario Quijada participated in the execution of the experimental work. Ricardo Lleonart participated in the design, experimental work, and manuscript preparation.
This work was partially supported by grants from Banco Interamericano de Desarrollo (Grant no. IND-JAL-02-DENGUE), Secretaría Nacional de Ciencia, Tecnología e Innovación de Panamá (SENACYT), and Sistema Nacional de Investigación de Panamá (SNI). Authors are also grateful to Alejandro Llanes for revision of the manuscript and to researchers from ICGES, Panamá, for donation of viral DENV2 strain.
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