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Jackson Hewitt Tax Service
This business is no longer available.
This information has been provided for your convenience and may no longer be valid.
5927 Ridge Ave
(215) 877-4181
Open Map
Since 2010 the company has been providing Tax Return Preparation Services.
by dandb on September 17, 2015 from
Editorial Reviews
National tax preparation franchise takes the ""I"" out of IRS during tax season with traditional paper and e-file services.:
In Short
Each of the over 6,000 Jackson Hewitt office locations across the country is a veritable one-stop shop for tax filing. With services for state and federal filings, the New...
by by Erin Chambers at Citysearch on July 29, 2008 from
Information from the business
Jackson Hewitt® is an industry leader providing full-service tax return preparation. We prepare millions of federal, state and local individual income tax returns each year. Jackson Hewitt operates approximately 6,500 locations throughout the U.S., including locations in Walmart and Sears stores.
Our Tax Pros understand the tax code, and are up-to-date on all the tax law changes. We'll sit down and discuss your personal situation in complete confidentiality to make sure nothing is overlooked.
Jackson Hewitt provides:
- Federal and state tax preparation
- Help with government paperwork...
by Citysearch on March 19, 2014 from
Jackson Hewitt Tax Service Inc. is an industry-leading provider of full service individual federal and state income tax preparation.
by foursquare on May 02, 2014 from
Full-service income tax preparation for local, state, and federal returns
by citysquares on April 22, 2014 from
Privacy & Terms


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Love this spring's gypsyish fashions, but wary of looking like a sloppy hippie? Meet designer Catherine Malandrino, of the SoHo shop Catherine, who always mixes ethnic finds with bright basics: "You have to bring south-of-France colors to the Hamptons, otherwise it's so . . . boring," she says. To add a bit of pizzazz to our essentials, we tagged along on a shopping trip.
At the Chelsea Market, we make a beeline for Imports From Marrakesh. From a cauldron full of leather slippers, Malandrino picks a gold pair ($50). "So sophisticated with khaki pants." Next comes a lime-green-and-brown scarf with sequined tassels ("Perfect for the beach.") With the gold slippers? "No, no! Flip-flops or Manolo Blahniks -- always combine ethnic with modern," she explains.
Taking the scarf, we head on to Little India's Royal Sari House, a trove of colorful silks. "I'm looking for green -- bright green," she presses the saleswoman. But she also loves a pink and gold tunic ($250, with pants): "Fantastic with capri pants, no?" (The pink pants will go with a white top.) She's not nearly so concerned with the traditional way of wearing things ("A great strapless wrap dress," she proclaims of a large shawl, $200). In the end, she picks up another green silk scarf ($35): "You can do a lot with this."
At York Novelty Import, bright strands of beads ($3-$12 each) blanket the walls. "When you're tan, you have to wear lots of natural, colorful beads," she insists. But not prissily: "Lots and lots together, like in Africa." Mixing and matching, Malandrino reflects on her choices: "I'm very colorful."
Imports From Marrakesh, 88 Tenth Ave., near 15th St. (675-9700); Royal Sari House, 264 Fifth Ave., at 29th St. (679-0732); York Novelty Import, 10 W. 37th St. (594-7040).
Picks of the Week
ABH Design's pashmina shawls (very, very impressive Mother's Day gifts) are on sale for much less than you'd pay elsewhere: from $160 for light embroidered styles to $240 for mid-weight solids to $395 for oversize, heavily embroidered shawls. 153 E. 61st St. (688-3764); A.E., M.C., V.; Mon.-Fri. 10-5:30; 4/19-5/10.
Patrick Robinson's cool downtown clothes for women are $350 and more at uptown department stores. At the showroom sale, they're half off: from $150 for cashmere sweaters to $350 for fluid jersey dresses. Sizes 2-14. 84 Wooster St., second floor (431-1301); checks accepted; Mon.-Fri. 10-6, 4/19-4/23.
Renée DuMarr women's casual and career clothes are on sale at the showroom for 70 percent off retail: from $50 for simple dresses to $200 for tweed suits. Sizes 4-14. 530 Seventh Ave., near 38th St., seventh floor (768-3734); A.E., checks accepted; Fri.-Sat. 11-4; 4/23-4/24.
Carla Westcott and Heidi Weisel's eveningwear is ideal for dressy summer weddings. At Barneys, gowns start around $1,000; at the showroom sale, samples and stock are $100-$300. Sizes 4-14. 530 Seventh Ave., near 39th St., twentieth floor (768-9794); checks accepted; Fri. 8-7; 4/23.
Save a Bunch
Brighten your home (or a friend's dinner party) with budget weekend pickings from 800-FLOWERS. Stems are usually from $1 for carnations to $2.99 for yellow lilies to $4.99 for long-stemmed roses, but on Friday afternoons from 4 till 8, all are two for one. Mix your own, or snap up a ready-made bouquet ($12.99 for tulips) -- either way, they'll match it for free. 380 Second Ave., near 22nd St. (780-0120); 576 Third Ave., at 38th St. (490-0990).

subset: commoncrawl


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NASA GISS Director James Hansen Plays Climate Science Victimization Card.
Claims of human caused global warming began with a deception and the pattern continues. It appears a new response of climate scientists caught in malfeasance has evolved as the original deception fails. It's called self-victimization that Wikipedia defines as;
"the fabrication of victimhood for a variety of reasons such to justify abuse of others, to manipulate others, a coping strategy or attention seeking."
James Hansen, 71 years old and director of the NASA Goddard Institute of Space Studies (GISS), produced a recent example. Going to Scotland for another speech, he played the victimization card now familiar to IPCC affiliated and Climatic Research Unit (CRU) people, like Director Phil Jones who claimed he considered suicide after the emails were leaked. Hansen played the card in October 2011 when he said,
"Climate sceptics are winning the argument with the public over global warming."
His latest comments are an expansion and suggest political tunnel vision rather than scientific awareness. Here are his comments (H) with my commentary (B).
(H) "There is remarkable inconsistency between the scientific story and public story,"
(B) True, but it's a result of IPCC strategy designed to distort the difference. They produce a Science Report, which virtually nobody reads or can understand, months after they release the grossly distorted Summary for Policymakers (SPM).
(H) "The science has become stronger and stronger over the past five years while the public perception has gone in completely the other direction. That is not an accident."
(B) Again true, but not because the public are stupid or misled by the skeptics as Hansen arrogantly infers. There was a campaign to block the normal scientific method whereby the Anthropogenic Global Warming (AGW) hypothesis is subjected to attempts by scientists to disprove it. Now, despite the blocking, evidence and failed predictions prove the hypothesis is wrong. As said, "…if your prediction is wrong then your hypothesis is wrong. Period."
(H) "There is a very concerted effort by people who would prefer to see business to continue as usual. They have been winning the public debate with the help of tremendous resources."
(B) Totally untrue, but characteristic of self-victimization. "Manipulators often play the victim role ("poor me") by portraying themselves as victims of circumstances or someone else"s behavior in order to gain pity or sympathy or to evoke compassion."
Hansen is saying failure is not their fault: they're victims of a small unethical self-serving group backed by equally unethical self-serving agencies. Oil and energy companies are the favorite target, but it's laughable when compared with money spent by government agencies promoting the IPCC version of global warming. As Joanne Nova notes,
"Thousands of scientists have been funded to find a connection between human carbon emissions and the climate. Hardly any have been funded to find the opposite. Throw 30 billion dollars at one question and how could bright, dedicated people not find 800 pages worth of connections, links, predictions, projections and scenarios? (What's amazing is what they haven't found: empirical evidence.)"
There is "empirical evidence" but it's identified by skeptics despite efforts by government funded agencies and environmental groups to block, deny, and discredit with personal attacks.
Hansen was central to the deception about global warming when he took it from behind the scenes of the UN bureaucratic machinations into the political, public, and mainstream media spotlight. His June 1988 appearance before a US Senate Committee at which he claimed he was certain that human CO2 was causing global warming, was an orchestrated event. The maestros of the deception were proud of what they did. It parallels Hansen's actions and reflects the end justifies the means mentality of politically driven climate science.
Director's of the drama were Al Gore and Timothy Wirth. In a 2007 PBS Frontline documentary Wirth said,
We knew there was this scientist at NASA, you know, who had really identified the human impact before anybody else had done so and was very certain about it. So we called him up and asked him if he would testify.
Then they took actions that explain why people distrust and despise most politicians.
We called the Weather Bureau and found out what historically was the hottest day of the summer. Well, it was June 6th or June 9th or whatever it was. So we scheduled the hearing that day, and bingo, it was the hottest day on record in Washington, or close to it.
The interviewer asked Wirth;
Did you also alter the temperature in the hearing room that day?
Wirth replied;
What we did is that we went in the night before and opened all the windows, I will admit, right, so that the air conditioning wasn't working inside the room.
Since that first deliberate deception Hansen has tried to confirm his claim that;
"the global warming is now large enough that we can ascribe, with a high degree of confidence, a cause-and-effect relationship to the greenhouse effect."
He has failed to prove the claim or even counteract proof that it's incorrect. His efforts include arrests for public protests; giving testimony at a trial in England on behalf of Greenpeace protesters; tampering with data; claiming, falsely, he was "muzzled" by the Bush White House; and claiming climate change was morally comparable to slavery. His political activism recently prompted 50 fellow NASA scientists and engineers to sign a protest. It's a disgrace he hasn't been challenged for behaviour unacceptable for a bureaucrat and possibly in violation of the Hatch Act.
Facts contradicting the hypothesis increase, predictions are consistently wrong, corruption of the process are revealed almost daily yet Hansen blames a massive conspiracy driven by non-existent funding. He infers the public are being bamboozled, but the polls demonstrate they are not as stupid as Hansen apparently believes. It appears his self-deception matches his self-victimization.


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Amber Teething Necklace - 3 Lines Multicolour
0 reviews | write a review
New Design Multicolour Baby Teething Necklace is made from 3 twisted and knotted threads of amber beads and has screw clasp. Real Amber Necklace will help relieve drooling, gumming, pain and sleepless nights for you baby, because Amber absorbs pain and is a natural analgesic. Help your baby through this terrible time with 100% Baltic Amber Necklace!
Necklaces size: about 6 grams, 13 inches long.
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For years I have been buying both running and walking shoes, but always end up with pain along the outside edge of my right foot (I'm heavy, under-pronate and have a high arch). Just last week, I walked into the New Balance store wearing a brand new pair of 993's, and was still in pain while researching the idea of purchasing the new 2002 model running shoes. Yesterday, I received my new shoes from Amazon and put them on, and went walking. NO PAIN! The cushioning is like a pillow under my feet! The fit is wonderful and the craftsmanship is superior (as you would expect for a $249.00 pair of running shoes), and the shoe looks like a work of art. In my case, the money I save by not needing to purchase custom made orthotics (which was my next step), pays for the price of the shoes. Money well spent for my situation. I am getting ready to purchase a second pair so I can rotate them daily. If your in the market for a neutral cushioned running shoe, this model might be worth considering.
Great shoes. I\'m in Iraq right now and they keep my feet cool and provide excellent traction in the dirt and on the rocks. Very comfortable to run in. I will be replacing them with another pair of the same shoes when the time comes.
complete foot comfort !!!!!!!!!!!!!! complete


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Rins earns new two-year Suzuki deal
Alex Rins signed a two-year contract renewal with Suzuki Ecstar ahead of the French Grand Prix.
After an injury-blighted first year in MotoGP, Rins claimed his first podium at this season's second race in Argentina.
The 22-year-old was delighted to sign on with Suzuki for the 2019 and 2020 campaigns and will be aiming to finish for just the second time this year in Le Mans this weekend.
"I'm very happy about this contract renewal," said Rins. "No matter the results, whether good or bad, the mood in the box has always been positive.
Great news ahead of the #FrenchGP; @Rins42 stays with us for two more years! #SUZUKing@MotoGP#BreakingNewspic.twitter.com/FHnXS9pPu9-- Team SUZUKI ECSTAR (@suzukimotogp) May 17, 2018
"The project the team have set up for me is fantastic, I can fully trust the capabilities of the company and the team when it comes to development and improvement, and I'm sure we can continue to aim for big results.
"Now we only have to keep on working hard to see just how far we can go."


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One of the leads on the Epigeneium nakaharaei has bloomed. I am looking forward to two more flowers this year from the remaining leads.
Epigeneium nakaharaei grows in Taiwan in the central mountain range. It is cool to warm growing, fragrant and blooms in the fall and winter. It needs bright light. The genus Epigeneium (Epig) contains 35 species in Southeast Asia and the Philippines. They do well mounted in warm, humid and shady conditions.
This plant is mounted on a 5-inch hardwood stick. There are 19 pseudobulbs with three leads. Each of the leads has enough pseudobulbs to be a nice division. Besides that, there is a section of older pseudobulbs.
This needs to be re-mounted. I will take one of the leads and move it to the center of the stick. I will sell the other two divisions on eBay next week.


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Here's Brent Sutter on Dion Phaneuf's hit to Kyle Okposo last night:
"There has been some disrespect brought into the game from player to player. That's something I've said for 10 years... In today's game it's considered a clean check. And yet it's a very dangerous check. When a player is vulnerable like that, you're putting yourself in a position to injure another player."
Wait... no... that was Brent Sutter on Doug Weight's check to his son Brandon Sutter. Well then let's see what Brent Sutter actually said about Dion Phaneuf's hit to Kyle Okposo last night:
"That's part of Dion's game. When he sees that open ice, Dion has to use it. It's one part of his game that makes him a unique player. You never like to see anybody get hurt obviously, and it's too bad that happened, and hopefully we've been told, he's going to be okay. But Dion still can't pass up that hit."
Huh. I guess the main difference is that the Weight check was one-on-one while Phaneuf hit a guy that had already been engaged by two other players. Thus, Phaneuf's hit deserves no criticism. Or maybe it's that the preseason is a higher stakes environment than the regular season since Phaneuf is probably fighting for his job with guys like Anton Stralman. Maybe I'll just give Sutter the last word:
"I probably got knocked out 10 to 11 times in my career and played through it. I'm not saying that's a good thing. That's why I'm so screwed up today. You can write that, too."
Friday, September 18, 2009
Brent Sutter and Cognitive Dissonance
Posted by Scott Reynolds at 9/18/2009 02:23:00 PM
Labels: B Sutter, Calgary Flames, Hockey, K Okposo, New York Islanders
1 comment:
- JLikens said...
That's hilarious.
Nice find.
- September 21, 2009 at 11:49 AM
Subscribe to: Post Comments (Atom)


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4sight pushes Crufts brand with BLE promo
Do you think you have a particularly intelligent dog?
Has your canine perfected a special trick for example? Or do you just think their talent is being particularly cute (like the one pictured) or just plain barking mad?
Whatever your mutt has mastered, 4sight Licensing Solutions is asking visitors to Brand Licensing Europe next week to bring along a picture to its stand (DO60). Up for grabs are three sets of tickets for a family of four to Crufts in Birmingham or Discover Dogs in London.
The ultimate Best in Show will also win a bottle of champagne. And have their picture posted on Licensing.biz.
The competition is part of 4sight's plans to push the Crufts and Kennel Club brands, which it will be showcasing at BLE.
It begins today and will run through until October 8th. Pictures can also be emailed to email@example.com, where you can also enquire over terms and conditions.


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All Pointe Care
Since 2000, it has been our privilege to support Connecticut's families and children. Our highly trained, well-educated and experienced team of support staff, therapists, counselors, nurses and others are ready to face the many varied challenges of life.We have earned a reputation of being trustworthy and reliable – and that is the most important element of our business. That is why we:
- conduct thorough background checks and reference verification for every single employee
- provide comprehensive training and professional development
- keep our management personally involved in every case or assignment we accept
This website gives you detailed information of our services and activities. We welcome your phone call to set up a personalized consultation.
Steve & Nancy LaPointe


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Contact: Marsha McCabe, 541-594-3091
Washington - Spending by national park visitors in 2010 added $31 billion to the national economy and supported more than 258,000 jobs across the U.S., an increase of $689 million and 11,500 jobs over 2009.
The figures are based on $12 billion of direct spending by 281 million visitors in 394 national parks and nearby communities and are included in an annual, peer-reviewed, visitor spending analysis conducted by Dr. Daniel Stynes of Michigan State University for the National Park Service.
"National parks are best known for their incredible beauty and the preservation of America's historical legacy but they are also the significant, clean, green fuel of economic engines for local communities and our entire nation," said National Park Service Director Jonathan B. Jarvis.
"The investment American taxpayers make in their national parks continues to offer a huge return, not only in priceless family experiences but in real jobs and economic growth in our gateway communities that ripples to suppliers across the nation," Jarvis added.
Vicki Snitzler, Superintendent at Oregon Caves National Monument, and Craig Ackerman, Superintendent at Crater Lake National Park, said the numbers show these two southern Oregon attractions reflect the same trend as the national results. Snitzler added, "We are proud to be members of the Southern Oregon community and of our contributions to the local economy." Oregon Caves generates about $6.5 million and 62 jobs through direct and indirect spending while Crater Lake generates over $34 million and 540 jobs. Most of the spending/jobs are related to lodging, food, and beverage service (52 percent), followed by other retail (29 percent), entertainment/amusements (10 percent), gas and local transportation (7 percent) and groceries (2 percent). Ackerman said that the two sites have been working closely with the Southern Oregon Visitors Association and Travel Oregon to increase the number of visitors to the region and to lengthen the stays of those who travel here. Ackerman noted that "both parks have added new visitor attractions or amenities, such as the Trolley Tours and new trails at Crater Lake and new, off-trail caving experiences at the Monument."
The entire National Park Service report which includes information on visitor spending by park and by state, is online at https://www.nature.nps.gov/socialscience/products.cfm#MGM , click Economic Benefits to Local Communities from National Park Visitation and Payroll, 2010 . For more state-by-state, information on national parks and how the National Park Service is working with communities go to www.nps.gov/STATE , for example: https://www.nps.gov/oregon .


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Top London Budget Hotels Brands
The tourists who come to visit the capital city of England have the intention of seeing as many of the tourists attractions of this city as possible – and, in order to do that, they need to save the money and not spend it on the luxurious hotels (that money can buy the ticket, to, sat, the London Eye) but to book a room at one of the more affordable hotels. And there is a great number of such hotels in London – there is definitely cheaper than the expensive hotels. The list of the Top London Budget Hotel Brands will try to answer the dilemma about which chains of the cheaper hotels are the best? Some of them are older, some newer, but these five brands are guaranteed to deliver nice accommodation to their guests, providing them with all the necessary things, like the access to Internet or the machines which make coffee or tea. These places, of course, don't have that luxurious look and feel like The Ritz or The Lanesborough – but that is not important to those who just want to explore one of the biggest cities of Europe. The Top London Budget Hotel Brands are:
There are over 50 Travelodge hotels in the capital city of the United Kingdom, as this chain of affordable hotels is rapidly expanding throughout the country. Travelodge has over 30,000 bedrooms in the country, placing it on the second place of budget hotels sector, the first one being Premier Inn. The guests can book these rooms at the stunning price of just 30 pounds. The rooms are separated into three categories – there are the family ones, the rooms for the disabled people and the double rooms, each of them being equipped with free coffee and tea, as well as with the en-suite bathrooms. Wi-Fi can be found at all of the Travelodge hotels, but it requires a fee. New Travelodge buildings have the new design for the rooms while the older buildings have been refurbished and modernized with new color schemes and the addition of a flat TV screen. Some of the locations of London's Travelodge hotels are Marylebone, Covent Garden, Southwark and others.
Tune Hotels
This chain of cheaper hotels originates from Malaysia, and it has just a few hotels in the United Kingdom. But fear not – these places are already known to be really good choices who are searching for a combination of a lower price and a pleasant accommodation. There are five Tune Hotels in London – in Westminster, Canary Wharf, King's Cross, Paddington, and on the Liverpool Street. Besides these hotels in London, the other ones can be found in Edinburgh, in Newcastle, and in Liverpool. The most well-known of them would probably be the one in Westminster. It offers bright, clean rooms which are equipped with flat-screen TVs, en-suite bathrooms, and access to the Internet in the form of Wi-Fi. There is even the air-conditioning – always a nice addition in the budget range of hotels. Breakfast, unfortunately, requires a small surcharge, but the customers are still giving praise the other perks of Tune Hotels, especially to their location, as they are always close to some of the major attractions and to transport stations.
Holiday Inn Express
Holiday Inn Express is a chain of both mid-price and low-cost hotels which was established in 1991, in the United States, with the first hotel in Europe being opened five years later – in 1996 (in Scotland). There are over 2,300 Holiday Inn Express hotels in the world, and they are offering a good accommodation for a small amount of money. There is a number of these hotels surrounding London, as there are some of them in the center of it, like the ones on the Old Street and at Victoria and Earl's Court. The best known of them is probably HIE London City, which is close to the Old Street station and represents a good base for those about to explore the city. This hotel is a nice example of the Holiday Inn Express hotels – there are some really comfortable rooms with the en-suite bathrooms, satellite television and there is even a tasty breakfast in the morning. Holiday Inn Express is certainly among the Top Budget Hotel Brands in London.
Premier Inn
Premier Inn is most certainly one of Top London's Budget Hotel Brands. It is the biggest hotel brand in the entire country, as it has a stunning number of 700 hotels which house over 50,000 rooms. Premier Inn was established in 1987 to compete with Travelodge, and it has more locations than the rival now, these locations being in the centers of the cities, near the airports and in the suburbs. Premier Inn hotels guarantee coziness and safety, and it's pretty hard to go wrong by booking a room at one of their hotels. Every room is clean and has all the commodities, like the comfortable beds, friendly staff and, of course, access to the Internet (Wi-Fi). One of the best things about the Premier Inn chain of hotels is that every hotel has a restaurant, making the guests able to eat tasty meals after waking up in the morning. The capital city of England has plenty of Premier Inn hotels, all of them easy to find.
Ibis is an international hotel company which was founded in 1974, in France – in 2014, there was the opening of the 1000th Ibis hotel in Indonesia. A large number of these hotels is based in the home country (France), with the rest of them distributed across Europe and other continents. Some of those hotels can be found in London, and booking a room at one of them is a good choice – Ibis hotels are known for the spotless rooms, featuring comfortable beds and the so-called power showers. Guests are especially praising the friendly staff which is the hallmark of Ibis, as well as the emphasis that this chain puts on the economy – a simple yet functional accommodation for a small sum of money (it's should be also noted that Ibis hotels offer an array of small extras, like bottled water or newspapers). Some of the London locations of Ibis hotels are Kensington, Blackfriars, the one in the financial district (official name is City of London), and others.


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Deadline - Lionsgate is getting serious about the second installment of The Hunger Games. The mini-major is courting Oscar-winning Slumdog Millionaire scribe Simon Beaufoy to write Catching Fire, the second installment of the three book series that tracks the life and death adventures of Katniss Everdeen (Jennifer Lawrence).
I'm just hoping for a good first movie so that they will have a sequel. From what I understand it's never a sure thing until after they see what the first one makes.
Let's hope the first one doesn't disappoint. I guess Lionsgate doesn't think it will.


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Marisano James and Dr. Shaffer at Quail Ridge during the Feb 16, 2008 Recruitment Weekend field trip.
How does the admission process work?
Admission to the Population Biology Graduate Group is basically a three-part process.
Part 1: In the first part of the admissions process, the Admissions Committee reviews all parts of the written application materials (transcripts, GRE scores, statement of purpose, personal history statement, and letters of recommendation) to identify a pool of candidates with outstanding academic credentials and research experience. The Admissions Committee creates a list of potentially admissible students. Thus, the major responsibility for the first part of the admission process lies with the PBGG program.
Part 2: The second part of the admissions process is no less critical than the first, and is different because the major responsibility for its execution lies with YOU, the prospective graduate student (although we will certainly try to be as helpful as we possibly can).
The second part of the admission process is to find ideal 'matches' between prospective students and PBGG faculty who can serve as their First Year Mentors (and often subsequently as their major professors). In some cases, students may choose to be co-advised by two faculty. Finding the right match involves first and foremost identifying faculty who share research interests with the prospective student, but also who have space in their laboratory group and can contribute to the financial support of the prospective student.
It is essential that prospective students contact faculty whose laboratories are conducting research in the areas that the student wishes to pursue to introduce themselves and inquire about faculty willingness to accept a new student. This process of communicating with potential faculty sponsors often begins well before the written application is completed. (There is little time between the application deadline of January 2 and the date when invitations go out for the recruitment visit; thus, it is helpful to correspond with potential faculty sponsors relatively early in the admissions 'season' -- students often correspond with faculty between September and December. Please see the descriptions of faculty research programs under the section "People...Faculty".)
Part 3: Having been deemed admissible by the Admissions Committee and successfully identified one or more potential faculty advisors (who have expressed to PBGG staff their serious interest in having the prospective student join their lab), the prospective student will be invited to campus during the recruitment weekend. This in-person visit is an essential final step in determining if the hoped-for match between prospective student and sponsoring faculty member (the First Year Mentor) exists. Offers of admission to the Population Biology Graduate Group are generally sent out approximately one week following the recruitment weekend visit.
We wish you every success.
Drs. Begun and Yang


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Android phones are getting cheaper these days.There are many brands selling android phones under rs 5000 in India.
Don't react fast on buying an android phones under rs 5000, look for quality as well.I checked some android phones online within the range of Rs 5000/-.
I must say there are only few good brands selling phones at this price with android OS.While researching online I found many brands such as Micromax, Karbonn, Spice, Iball, Lava, Spice, Lemon, Intex and Alcatel.
These are the brands selling android phones under rs 5000 in India.Check for best specification phone that suits your need as you know, you wont get that things which you will find high end phones.
So I would only say look for two things in any android phone.
1. RAM (Don't expect more RAM in budget phones.)
2. Processor
Without discussing any further, lets look at Best Android phones under rs 5000 in India
Best Android phones under rs 5000 in India
1. Micromax Ninja 3 A57
If you are in budget of 5000 rupess than this is the best phone for you.It has 1 GHz Qualcomm Processor with 3 mp camera.
3.5″ TFT Capacitive Touchscreen which comes with Android v2.3 (Gingerbread) OS.
This phone is very powerful after rooting (Warning : Rooting process is only for professional's who knows how to do it,Search online for more information.)
Price: Rs 4850
2. Karbonn A5
This is another good phone at this price.It has same specification as Micromax Ninja 3 A57 except camera.It has 2 MP camera.
Price: Rs 5000
3. Karbonn A1+
This has good specs as seen in Micromax Ninja 3 A57. It comes with 1 GHz Processor, 3.5″ Capacitive Touchscreen and 3 MP HD Primary Camera.
Better phone than Karbonn A5.
Price: 4290
4. Micromax Smarty A25
This is an entry level android phone with all basic features.Good thing is that it has Android V2.3.6 Gingerbread, Dual Sim, WiFi,etc
Price: Rs 3390
This was the current list of budget android phones under rs 5000 in India, Also look for android phones under Rs 10000


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- Research
- Open Access
TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm
© De laco et al; licensee BioMed Central Ltd. 2013
- Received: 16 January 2013
- Accepted: 11 February 2013
- Published: 15 February 2013
Despite intensive investigation the mechanism by which HIV-1 reaches the host cell nucleus is unknown. TNPO3, a karyopherin mediating nuclear entry of SR-proteins, was shown to be required for HIV-1 infectivity. Some investigators have reported that TNPO3 promotes HIV-1 nuclear import, as would be expected for a karyopherin. Yet, an equal number of investigators have failed to obtain evidence that supports this model. Here, a series of experiments were performed to better elucidate the mechanism by which TNPO3 promotes HIV-1 infectivity.
To examine the role of TNPO3 in HIV-1 replication, the 2-LTR circles that are commonly used as a marker for HIV-1 nuclear entry were cloned after infection of TNPO3 knockdown cells. Potential explanation for the discrepancy in the literature concerning the effect of TNPO3 was provided by sequencing hundreds of these clones: a significant fraction resulted from autointegration into sites near the LTRs and therefore were not bona fide 2-LTR circles. In response to this finding, new techniques were developed to monitor HIV-1 cDNA, including qPCR reactions that distinguish 2-LTR circles from autointegrants, as well as massive parallel sequencing of HIV-1 cDNA. With these assays, TNPO3 knockdown was found to reduce the levels of 2-LTR circles. This finding was puzzling, though, since previous work has shown that the HIV-1 determinant for TNPO3-dependence is capsid (CA), an HIV-1 protein that forms a mega-dalton protein lattice in the cytoplasm. TNPO3 imports cellular splicing factors via their SR-domain. Attention was therefore directed towards CPSF6, an SR-protein that binds HIV-1 CA and inhibits HIV-1 nuclear import when the C-terminal SR-domain is deleted. The effect of 27 HIV-1 capsid mutants on sensitivity to TNPO3 knockdown was then found to correlate strongly with sensitivity to inhibition by a C-terminal deletion mutant of CPSF6 (R2 = 0.883, p < 0.0001). TNPO3 knockdown was then shown to cause CPSF6 to accumulate in the cytoplasm. Mislocalization of CPSF6 to the cytoplasm, whether by TNPO3 knockdown, deletion of the CPSF6 nuclear localization signal, or by fusion of CPSF6 to a nuclear export signal, resulted in inhibition of HIV-1 replication. Additionally, targeting CPSF6 to the nucleus by fusion to a heterologous nuclear localization signal rescued HIV-1 from the inhibitory effects of TNPO3 knockdown. Finally, mislocalization of CPSF6 to the cytoplasm was associated with abnormal stabilization of the HIV-1 CA core.
TNPO3 promotes HIV-1 infectivity indirectly, by shifting the CA-binding protein CPSF6 to the nucleus, thus preventing the excessive HIV-1 CA stability that would otherwise result from cytoplasmic accumulation of CPSF6.
- Capsid
- Nuclear transport
Early events in the human immunodeficiency virus type 1 (HIV-1) replication cycle commence when HIV-1 binds and fuses to a susceptible host cell. With fusion, the conical HIV-1 virion core is released into the cytosol. The virion core measures 119 × 60 nm on average [1] and bears a proteinaceous coat of more than 1,000 capsid monomers [2]. The viral genomic RNA within the coat serves as template for the viral reverse transcriptase (RT), in a process that links disassembly of the virion core to viral cDNA synthesis [3]. The resulting pre-integration complex (PIC) then gains access to the nucleus, presumably through the nuclear pore complex, where the viral integrase (IN) ligates the viral cDNA to host cell chromosomal DNA.
Exactly how the viral genome reaches the nucleus of the host cell is not clear. Correlations between biochemistry and function are rendered difficult by the fact that only a minority of retrovirion particles within any given preparation succeed in transducing susceptible target cells [4]. Genetic approaches have been fruitful in that several cellular factors have been identified that influence early HIV-1 replication steps occurring after reverse transcription [5–10]. One such factor, Transportin-3 (TNPO3), is a karyopherin that imports SR-rich splicing factors into the nucleus [11].
TNPO3 clearly plays an important, though controversial role in HIV-1 replication [5, 6, 8, 9, 12–16]. Some studies suggest that TNPO3 promotes nuclear import of the PIC [6, 9, 14, 17]. Others indicate that it has a role in a step after the viral PIC reaches the nucleus [8, 12, 13, 15, 16]. TNPO3 has been reported to interact with both IN and CA [6, 13, 16, 18], but the relevance for HIV-1 replication of the interaction with IN was not confirmed [15, 19]. Evidence supporting the functional significance of interaction with CA is stronger than that for interaction with IN. MLV does not require TNPO3 for transduction; chimeras in which HIV-1 CA and IN are swapped with the MLV counterparts reveal a central role for CA in TNPO3 function and fail to demonstrate a role for IN [19]. Additionally, nearly 30 HIV-1 CA mutants have been identified that alter HIV-1 dependence on TNPO3 [7, 8, 13, 19]. How TNPO3 would promote HIV-1 infectivity via effects on CA is not obvious. Shah et al. suggests that TNPO3 acts directly on the process of CA core uncoating [20].
Cleavage and polyadenylation specific factor 6 (CPSF6) is a 68-kD subunit of the mammalian cleavage factor I (CF Im), a component of the mRNA cleavage/polyadenylation machinery [21]. CPSF6 possesses an N-terminal RNA recognition motif (RRM), a central proline-rich domain, and a C-terminal domain enriched in arginine/serine, arginine/glutamate and arginine/aspartate repeats, similar to the RS-domain of SR splicing factors [22]. A CPSF6 C-terminal deletion mutant lacking the RS-like domain (CPSF6-358) was isolated in an expression screen for cDNAs that inhibit HIV-1 replication [7]. CPSF6-358 binds HIV-1 CA and viral restriction activity depends on this interaction [7, 23]. Closer examination of CPSF6 revealed a number of functional links with TNPO3. The CPSF6 RS-like domain is required for localization to the nuclear compartment [7, 24]. Interestingly, TNPO3 imports cargo proteins by interacting with the RS-domain of SR-proteins [25]. CPSF6-358 binds specifically an HIV-1 CA pocket where amino acids important in HIV-1 dependency to TNPO3 are located [7, 8, 23]. HIV-1, HIV-2 and SIV macaque (SIVmac), but not MLV and FIV, are inhibited by CPSF6-358 expression and TNPO3 depletion [6, 7, 19].
Here we set out to clarify the mechanism by which TNPO3 promotes HIV-1 infectivity. We began by pinpointing the step in the HIV-1 replication cycle that is blocked by TNPO3 knockdown and then sought evidence for functional links between HIV-1, TNPO3, and CPSF6.
TNPO3 depletion blocks HIV-1 replication in a step before the virus enters the nucleus
Several groups reported experiments designed to determine at which step HIV-1 replication is blocked when TNPO3 is depleted [5, 6, 8, 9, 12–17]. All observed a block after completion of reverse transcription but results differed with respect to the effects of TNPO3 knockdown on the formation of 2-LTR circles, a marker for nuclear import of the retrovirus replication machinery [26]. Some studies showed that TNPO3 acts to promote integration, without effects on the levels of 2-LTR circles [8, 12, 13, 15, 16]. Other studies reported that the levels of 2-LTR circles were decreased, indicating that TNPO3 is required before nuclear import [6, 9, 14, 17].
Then, the levels of 2-LTR circles were assessed 24 hrs post-infection with the VSV G-pseudotyped HIV-1NL4-3. The PCR primers were commonly used sequences that flank the junction of the ligated, viral cDNA termini, but do not overlap with the junction (Figure 1D) [8, 26]. Despite an 11-fold decrease in the efficiency of transduction of TNPO3 KD cells by WT virus, as compared with the TNPO3-independent A105T CA mutant virus, the replication of which is TNPO3-independent (Figure 1B), no significant reduction in the amount of 2-LTR circles was observed (Figure 1E).
PCR products from the reactions designed to detect 2-LTR circles were ligated into a plasmid and at least 85 clones from each condition were sequenced and evaluated, as previously described [27, 28]. 2-LTR circles with consensus sequence were observed, as well as point mutations, insertion of PBS or PPT sequences at the junctions, and deletions at the termini of the viral cDNA (Additional file 1: Table S1). Autointegration resulting from opposite-strand joining was also detected [29, 30]; these circular viral cDNAs contain 2-LTRs but are distinguished from 2-LTR circles that result from end-joining by the lack of the terminal dinucleotides that are removed by IN when the viral cDNA is inserted into various regions of the viral genome.
Since the PCR reaction that most labs use to amplify 2-LTR circles also amplified significant quantities of autointegrants, the effect of TNPO3 KD on the signal in this reaction necessitated adjustment for the quantity of autointegrants. The relative amount of 2-LTR circles produced by WT virus, divided by the amount of the opposite-strand autointegrants, was decreased roughly 10-fold by TNPO3 KD (Figure 1F). No significant change in this adjusted ratio was observed with the TNPO3-independent A105T virus. To determine if this change in ratio was due to an absolute decrease in 2-LTR circles or to an increase in autointegrants, TNPO3 KD and control cells were transduced in the presence of raltegravir, a drug that blocks integration. Under this condition the level of 2-LTR circles was reduced 4-fold in the absence of TNPO3 (Figure 1G). Autointegration was assessed independently using a PCR method developed by Yan et al. [31]; when WT or A105T capsid mutant viruses were used to transduce either control or TNPO3 KD cells, the levels of autointegrants were very similar (Figure 1H). Autointegration was not detected when WT virus was used to infect raltegravir-treated cells (data not shown). These experiments indicate that, in the absence of the confounding variable of autointegration, TNPO3 KD was associated with an absolute decrease in the level of 2-LTR circles.
To confirm a block prior to the formation of 2-LTR circles, control and TNPO3 KD cells were treated with raltegravir, in order to block viral integration. These cells were then challenged with HIV-1NL4-3-based vectors encoding the green fluorescence protein (GFP) reporter gene and carrying WT or A105T capsid mutation (Figure 2C). Expression of the reporter gene from the unintegrated viral DNA was detectable 48 hrs after infection, though the mean fluorescence intensity was much weaker than the signal in the absence of raltegravir, and it disappeared if cells were cultured for more than 7 days (data not shown). This signal required de novo cDNA synthesis in the target cells since GFP was not detected if reverse transcription was blocked by AZT treatment of the target cells, or by mutation of the catalytic site of RT (D185K/D186L) in the challenging vector (data not shown). GFP expression from WT virus was strongly reduced when TNPO3 was depleted, while the A105T CA mutant virus was independent. These results clearly show that TNPO3 KD blocks HIV-1 in a step before 2-LTR circles are formed.
Finally, the effect of TNPO3 depletion on 2-LTR circles and autointegration was assessed by high-throughput sequencing (Figure 2D). The complete high throughput sequencing dataset has been submitted to the Sequence Read Archive (SRA), http://www.ncbi.nlm.nih.gov/sra, under accession number SRA056122. TNPO3 KD and control cells were infected with WT and A105T viruses for 24 hours. Low-molecular weight DNA was extracted from the infected cells, primed with random hexamers, and amplified by PCR. The reads were first aligned to human DNA, and the unmapped reads were extracted and remapped to the HIV-1 genome. Then, only reads encompassing a junction were considered (Additional file 1: Table S2). That is, reads having two mapping points on the HIV-1 genome, such as LTR/LTR or LTR/other location, were considered. Finally, junctions were classified according to position and whether the LTR terminal CA was maintained (circles) or cleaved (autointegrants). High-throughput sequencing of the PCR products detected ~105 reads with definitive LTR sequences for each condition, of which, 91% were autointegrants. (Figure 2D). The level of autointegration was similar in WT and A105T CA mutant virus infecting either control or TNPO3 KD cells. 2-LTR circles were significantly reduced when WT virus infected TNPO3 KD cells, while A105T CA mutant virus was totally independent. These results show unequivocally that TNPO3 depletion inhibits HIV-1 replication in a step prior to 2-LTR circle formation.
Tight correlation between the effects of TNPO3 depletion and CPSF6-358 on transduction by a panel of 27 HIV-1 CA mutants
To determine if HIV-1 determinants for sensitivity to CPSF6-358 track with those for TNPO3-dependence, the infectivity of each of the 27 previously characterized HIV-1 CA mutants [8] was assessed on cells expressing CPSF6-358 (Figure 3C). HeLa TZM-bl cells were challenged with a bicistronic, lentiviral vector encoding CPSF6-358 and puromycin acetyl transferase (PAT), or with the parental, control vector encoding only PAT. Pools of the two transduced cell populations were selected with puromycin (Figure 3B) and challenged with VSV G-pseudotyped, 3-part, lentiviral vectors, in which the GFP-encoding reporter genome and the viral structural proteins are provided by separate plasmids.
Transduction efficiency by vector bearing wild-type CA was reduced 26-fold by CPSF6-358 (Figure 3C). 11 CA mutants were inhibited to similar extent as the wild type (represented by white bars in Figure 3C). Vectors bearing the CA mutants G89V/A92E or P90A/A92E displayed greater sensitivity to CPSF6-358 than the wild type (gray bars, up to 60-fold lower in the presence of CPSF6-358, as compared to control cells). Fourteen CA mutants were insensitive to the inhibitory effect of CPSF6-358 (black bars).
As compared with HIV-1 CA determinants for sensitivity to TNPO3 depletion [8], sensitivity to inhibition by CPSF6-358 tracked closely (R2 = 0.883, p < 0.0001; Figure 3D). In contrast, no significant correlation for the effect of CPSF6-358 was found when the 27 CA mutants were tested on the CA-dependent HIV-1 restriction factors human TRIM5Cyp (hTRIMCyp) or rhesus monkey TRIM5α (rhTRIM5α) (Additional file 1: Figure S2) [32, 33]. Such tight correlation in the behavior of the CA mutants is consistent with a common mechanism of action for HIV-1 inhibition by TNPO3 KD and CPSF6-358.
Cytoplasmic localization of CPSF6-358 is required to inhibit HIV-1
The three pools of cells with CPSF6-358 targeted to different cellular compartments were then challenged with HIV-1 vectors bearing WT CA, A105T CA, or P90A/A92E CA (Figure 4C). Virus bearing the A105T CA mutant was resistant to the inhibitory effects of CPSF6-358, whether the HIV-1 inhibitor was localized to the nucleus or to the cytoplasm. Amino acid A105 is in fact localized to the pocket where CPSF6 binds to HIV-1 CA and makes direct contact with the SR-protein [23]. Alteration of the A105 side chain in the A105T mutant would be expected to disrupt the interaction between CA and CPSF6 and the consequent viral restriction would be abolished. Infection with WT or P90A/A92E CA mutant viruses was inhibited when CPSF6-358 was detected in the cytosol. When CPSF6-358 was targeted to the nucleus by fusion to the SV40 NLS, HIV-1 transduction was no longer inhibited, indicating that CPSF6-358 needs to be in the cytoplasm to inhibit HIV-1 infection.
HIV-1 is inhibited when full-length CPSF6 is targeted to the cytoplasm
TNPO3 is required for CPSF6 localization to the nucleus and HIV-1 permissiveness
As a further test of the model, stable CPSF6 knockdown and control knockdown cells were transfected with siRNA targeting TNPO3 or the firefly luciferase gene as a control (Figure 6A), and infectivity of HIV-1 vectors carrying WT CA or CA mutants A105T or P90A/A92E was tested. When CPSF6 was depleted from the cells, TNPO3 KD did not inhibit HIV-1 infectivity (Figure 6D), indicating that CPSF6 was required for the antiviral effect of TNPO3 KD.
The four stable cell lines were then tested for the ability to restrict HIV-1 bearing WT, A105T or P90A/A92E CA (Figure 7C). Conditions where CPSF6 localized to the cytoplasm, resulted in a block on the replication of HIV-1 bearing WT or P90A/A92E mutant CA. These results demonstrate that the decrease in HIV-1 infectivity associated with TNPO3 KD is a consequence of the re-localization of CPSF6 to the cytosol.
Cytoplasmic CPSF6 stabilizes the HIV-1 CA core
After fusion of HIV-1 with the target cell, the virion core is released into the cytoplasm from which it can be precipitated by ultracentrifugation. CPSF6 binds HIV-1 CA [23] and CPSF6-358 inhibits HIV-1 in a CA-specific manner, with the block occurring at a step before the virus reaches the nucleus (Additional file 1: Figure S1). CPSF6-358 might inhibit HIV-1 infectivity by altering the kinetics of CA core uncoating, consequently delaying the nuclear import of the PIC.
Finally, the effect of TNPO3 KD on the stability of the CA cores was assessed (Figure 8B). WT cores were stabilized when TNPO3 was knocked down, while the CA mutant A105T was not altered. As a positive control, destabilization of the CA core mediated by rhTRIM5α was assessed [35]. Both WT and A105T CA cores were destabilized when rhTRIM5α was expressed (Figure 8C). These results indicate that retention of CPSF6 in the cytoplasm, either via deletion of its NLS or KD of TNPO3, inhibits HIV-1 replication by causing hyperstabilization of the CA core, and presumably delaying transit of the PIC to the nucleus.
TNPO3 KD inhibits HIV-1 in a step before nuclear import
In previous works, when the effect of TNPO3 on HIV-1 replication was assessed, some research groups showed that TNPO3 promotes HIV-1 replication at a step before nuclear import, while an equal number claimed that it acts after nuclear entry [5, 6, 8, 9, 12–17]. The assay for HIV-1 nuclear import that was used by all of these investigators was PCR-based detection of 2-LTR circles [26]. These circular viral cDNAs are generated by cellular enzymes that promote the covalent joining of the LTR termini [36]. In the work here, the PCR products amplified using standard primers flanking the 2-LTR circle junction were examined in detail. As previously described, 2-LTR circles with consensus sequence, deletion of the termini, point mutations and PBS or PPT insertions were observed [27, 28]. We also identified abundant products (between 45 and 58% of the total) amplified from circles produced during opposite-strand joining autointegration events. When we sequenced the unintegrated viral products present in the cell after HIV-1 infection, we saw between 600 and 5000 times more autointegrants compared to 2-LTR circles. Differences in the assessment of 2-LTRs could easily be explained by interference from the abundant autointegration products.
When 2-LTR PCR products were cloned and sequenced, the bona fide 2-LTR circles with consensus LTR junctions were significantly reduced when TNPO3 was depleted. We then designed new primers that are able to discriminate consensus 2-LTR circles from autointegration events. Using this PCR assay and the deep-sequencing analysis of unintegrated viral products, we again demonstrated a significant reduction of 2-LTR circles as a result of TNPO3 depletion. Lack of specificity of the standard 2‐LTR circle PCR therefore explains why different research groups have not obtained similar data. Indeed, all studies that reported no difference in the level of 2‐LTR circles by TNPO3 depletion used primers flanking the circle junction to detect 2‐LTR circles [8, 12, 13, 15, 16]. Many studies that have quantified the 2-LTR circles using primers that flank the circle junction should be revisited with the new PCR assay.
TNPO3 prevents CPSF6 from inhibiting HIV-1 replication
A model that describes the mechanism of action of TNPO3 in HIV-1 replication has remained elusive for several years. Based on their observation that TNPO3 interacts with HIV-1 IN, Christ et al. hypothesized that TNPO3 has a direct role in nuclear import of the viral preintegration complex [6]. Then several groups demonstrated that HIV-1 capsid is the primary determinant of TNPO3-dependence [8, 19]. However, more recent reports suggest that TNPO3 might affect HIV-1 nuclear import only indirectly [9, 14]. Zhou et al. proposed that TNPO3 promotes HIV-1 replication after the PIC has been imported into the nucleus, by displacing and exporting to the cytosol the viral CA still associated with the nuclear PIC [13]. In our previous work, analyzing a panel of CA mutants, we hypothesized that TNPO3 might alter the stability of the CA core [8].
Interestingly, Lee et al. recently characterized a truncated form of CPSF6, CPSF6-358, capable of restricting HIV-1 infectivity in a CA-dependent manner similarly to TNPO3 [7]. CPSF6-358 lacks the C-terminal RS-like domain, a NLS region commonly used by cargo proteins for a TNPO3-dependent nuclear import. Lee et al. showed that full-length CPSF6 is strictly localized to the nucleus of NIH3T3 cells, while the truncated protein is also present in the cytosol, suggesting that the RS-domain is required for the nuclear localization of the protein [7]. Here we showed that CPSF6 is indeed capable of inhibiting HIV-1 replication, but only when it accumulates in the cytosol, as occurs when TNPO3 is disrupted. Our data suggest that when CPSF6 accumulates in the cytosol, it binds to CA via the pocket where N74 and A105 are located [7]. This stabilizes the HIV-1 CA core, causing a delay in uncoating and in nuclear import of the viral cDNA.
Since results obtained in our study are based on the artificial alteration of CPSF6 localization, the question now remains: is there a physiological condition where CPSF6 accumulates in the cytoplasm to control HIV-1 replication? A recent study reported that, in response to Toll-like receptor-3 (TLR-3) stimulation, macrophages counteract HIV-1 by upregulating microRNA-155; this has the result that several factors important to the early steps of HIV-1 replication - including TNPO3 - are down-regulated [37]. Further studies are required to find an answer to this question. However, we can hypothesize two scenarios: CPSF6 could become cytoplasmic and restrict HIV-1 infection in specific cell types or after particular stimuli; CPSF6 is a factor required for proper CA core uncoating in certain conditions.
CA core stability is tightly regulated by host cellular factors
Since the discovery of the interaction between HIV-1 CA and the host peptidyl-prolyl isomerase cyclophilin A (CypA) [38], several host factors that bind HIV-1 CA and alter the early steps of virus replication have been identified [7, 9, 33, 39]. The nuclear pore protein Nup358 seems to be directly involved in promoting the nuclear import of the HIV-1 PIC, while CypA and the tripartite motif 5 (TRIM5) proteins have been demonstrated to regulate the stability of the CA cores. The TRIM5 proteins specifically recognize the CA core lattice and accelerate uncoating, causing a premature release of the reverse transcription complex (RTC) and consequently inhibits viral cDNA synthesis [35, 40]. CypA has opposite effects on HIV-1 replication in different cell lines: in Jurkat T cells CypA stabilizes CA cores promoting reverse transcription, in HeLa cells CypA destabilizes A92E CA mutant cores blocking viral replication in a step between reverse transcription and integration [41–43]. The impact of these cellular factors on HIV-1 replication, together with the inhibition of reverse transcription by CA mutants that alter core stability [44], demonstrates how crucial is the optimal stability of the CA cores for productive infection. Recent work also demonstrated a direct correlation between HIV-1 reverse transcription and core uncoating [3]. In this study, we characterized a new host factor that alters CA core stability, CPSF6. The SR-protein specifically binds a pocket located in the N-terminal domain of HIV-1 CA previously described as the target of a compound capable of destabilizing CA cores, and stabilizes the cores [23, 45].
Since the identification of TNPO3 as a factor required for HIV-1 infection many studies have attempted to understand the specific function of this karyopherin in viral replication. Experiments in this manuscript demonstrate that TNPO3 is not directly required for nuclear import of the viral PIC. Instead, TNPO3 promotes HIV-1 infection indirectly, by inhibiting an inhibitor of HIV-1, CSPF6. In the presence of TNPO3, CPSF6 localizes to the nucleus. When TNPO3 is depleted, CPSF6 accumulates in the cytoplasm where it stabilizes HIV-1 CA cores and prevents their disassembly.
Cell lines, tissue culture, and drugs
293T and TZM-bl cells were grown in Dulbecco's modified Eagle medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (PAA), 20 mM L-glutamine, 1000 U/ml penicillin, and 1000 mg/ml Streptomycin (GIBCO). Raltegravir (RAL, Santa Cruz) was used at a final concentration of 10 μM.
pWPTs-GFP is an HIV-1-based transfer vector with EGFP expression under the control of the EF1α promoter [46]. p8.9NdSB is a minimal HIV-1 packaging plasmid for gag and pol expression [47]. pMD2-G encodes the vesicular stomatitis virus G protein (VSV-G). pNL4-3.GFP.E− bears HIV-1 proviral sequence with an env-inactivating mutation and EGFP in place of nef[48]. pAPM and pAHM are HIV-1 based knockdown vector in which a single transcript driven by the spleen focus-forming virus (SFFV) LTR contains a miR30 framework modified to target a gene of interest and the puromycin N-acetyltransferase gene or hygromycin B phosphotransferase, respectively [49]. pAIB is an HIV-1-based transfer vector expressing the protein of interest from the SFFV LTR followed by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) cassette and the drug resistance cassette, blasticidin-S-deaminase [50]. CPSF6 and CPSF6-358 were subcloned from pLPC-CPSF6-FL-HA and pLPC-CPSF6-358-HA (kindly provided by Vineet KewalRamani) into pAIB by cutting pAIB and pLPC with NotI/XbaI and BglII/NotI respectively [7]. pCR®II-TOPO® (Invitrogen) is a plasmid containing single 3′-thymidine overhangs for TA cloning and the topoisomerase I enzyme covalently bound to the vector.
Knockdown vector cloning and siRNA transfection
AHM-CPSF6 vectors targeting three different sequences of CPSF6 were designed as previously described [8]. Three 97-mer oligonucleotides were synthesized and PAGE purified: ts10, 5′-TGCTGTTGACAGTGAGCGCGGAAAGA GAATTGCATTATATTAGTGAAGCCACAGATGTAAT ATAATGCAATTCTCTTTCCATGCCTACTGCCTCGG A-3′; ts13, 5′- TGCTGTTGACAGTGAGCGAGCAATC TCAAGCAGTGCTATTTAGTGAAGCCACAGATGTAA ATAGCACTGCTTGAGATTGCCTGCCTACTGCCTCG GA-3′; ts37, 5′-TGCTGTTGACAGTGAGCGATTGGA TCTGAAGCATCTTCAATAGTGAAGCCACAGATGTA TTGAAGATGCTTCAGATCCAACTGCCTACTGCCTC GGA-3′. TZM-bl cells were transduced with the AHM-CPSF6 vectors (ts10, ts13 and ts37). Efficiency of the CPSF6 KDs was analyzed by western blot and AHM-CPSF6 ts10 was selected since it was the most potent of the three constructs. The APM-TNPO3 KD vector and the TNPO3 siRNA transfection procedure were previously described [8].
Cloning and mutagenesis
To express a form of CPSF6 that was resistant to CPSF6 KD ts 10, silent mutations were introduced into the CPSF6 cDNA (ntCPSF6) by overlapping PCR using the oligonucleotides ntCPSF6 fwd, 5′- CAAATGTTGTCTA TACATATACTGGAAAACGTATAGCGCTTTATATTG GAAATCTAACATGGTGGAC -3′; ntCPSF6 rev, 5′- G TCCACCATGTTAGATTTCCAATATAAAGCGCTATA CGTTTTCCAGTATATGTATAGACAACATTTG -3′.
NLS and NES were cloned at the C-terminus of CPSF6, ntCPSF6 and CPSF6-358. PCR used pAIB-CPSF6-HA, pAIB-ntCPSF6-HA and pAIB-CPSF6-358-HA as templates and the primers SSFV (fwd), 5′-C TCACTCGGCGCGCCAGTC -3′ coupled with SV40 NLS (rev), 5′- TGTGTGGCGGCCGCCTATACCTTTC TCTTCTTTTTTGGGGCGTAGTCGGGCACGTC -3′; or PKI NES (rev), 5′- TGTGTGGCGGCCGCCTAATT TATATCGAGTCCAGCTAGCTTCAATGCCAGGGCG TAGTCGGGCACGTC -3′. The PCR products were subcloned into pAIB-CPSF6-HA, pAIB-ntCPSF6-HA and pAIB-CPSF6-358-HA vectors digesting with NotI and XbaI.
Tranduction and transfection of mammalian cells
To express the different forms of CPSF6, TZM-bl cells were transduced with pAIB encoding CPSF6-358, CPSF6-358-NLS, CPSF6-358-NES, CPSF6, CPSF6-NLS or CPSF6-NES. To express rhTRIM5 or hTRIMCyp, cells were transduced with pAIB encoding the respective proteins. Cells were selected with blasticidin (10 μg/mL) starting two days after transduction.
To generate stable KDs, TZM-bl cells were transduced with pAHM microRNA-based shRNA vectors targeting either control or CPSF6 mRNA. Cells were selected with 200 μg/mL of hygromycin B two days after transduction. To generate the rescue cells, CPSF6 KD cells were transduced with the pAIB vector encoding for ntCPSF6 or ntCPSF6-NLS. 2 days after transduction, the cells were selected with 10 μg/mL blasticidin.
For transfection of siRNA, Lipofectamine RNAiMAX was complexed with 100 nM of Gene Solution siRNA (Qiagen) targeting TNPO3 following manufacturer's instruction.
Production of viruses and vectors
Viruses and minimal vectors were produced by transfection of 293T cells using Polyethylenimine (PEI) (Sigma, Inc) as previously described [8].
Western blot analysis
We used rabbit anti-TNPO3 antibody (ab71388, abcam), rabbit anti-CPSF6 antibody (Novus biological), human anti-p24 (NIBSC) and mouse anti-actin antibody (Sigma). The secondary antibodies were HRP-linked donkey anti-human IgG (Jackson), HRP-linked donkey anti-rabbit IgG and HRP-linked sheep anti-mouse IgG (GE Healthcare Life Sciences).
2-LTR circles, late RT and autointegration PCR
Low molecular weight DNA was extracted from 4 × 106 cells using the QIAprep Spin Miniprep Kit (Qiagen), following the manufacturer's instructions.
Quantitative PCR for NL4.3 GFP E- 2-LTR circles was detected with Sybr green (Invitrogen) or TaqMan probes. 2-LTR circle detection with primers external to the circle junction (Figure 1B) was made with primers: NL4.3_2 fwd, 5′-GAGATCCCTCAGACCCTTTTAG-3′; MH536, 5′- TCCACAGATCAAGGATATCTTGT C-3′. The primers used for detection of 2-LTR circles with perfect junction (Figure 1F) are: Junct2 fwd, 5′-C AGTGTGGAAAATCTCTAGCAGTAC-3′; or Junct4 fwd, 5′- CAGTGTGGAAAATCTCTAGCAGTACTG-3′; both coupled with J2 rev, 5′-GCCGTGCGCGCTT CAGCAAGC-3′. 2-LTR circles qPCRs with TaqMan probe detection method were made using the primers [26]: MH535, 5′-AACTAGGGAACCCACTGCTTAA G-3′; and MH536. The TaqMan probes external or overlapping with the junction were, respectively: MH603, 5′-(FAM)-ACACTACTTGAAGCACTCAAGG CAAGCTTT-(TAMRA)-3′ [26]; JunctPro, 5′- (FAM)-C TCTAGCAGTACTGGAAGGGCTA-(TAMRA)-3′.
Sybr green 2-LTR circle PCR reaction contained 1x Sybr green mix (10 mM Tris pH 8.3, 10 mM KCl, 2.5mM NH4SO4, 5 mM MgCl2, 0.1 mg/ml BSA, 0.2 mM dNTPs, 1× Sybr green), 300 nM each primer, 6 μl of template low-molecular weight DNA, and 0.2 μl of Hot Start Taq Polymerase (Promega) in a volume of 20 μl. After initial incubation at 95°C for 2 min to activate the Hot Start Taq Polymerase, 40 cycles of amplification and acquisition were carried out at 95°C for 6 s, followed by 10 s at 55°C, 30 s at 72°C and 6 s at 80°C. TaqMan 2-LTR circle PCR reaction mix contained 1× TaqMan Universal Master Mix (Applied Biosystems), 50 nM primer forward and reverse, 100 nM TaqMan probe and 6 μl of template low-molecular weight DNA in a volume of 20 μl. After an initial incubation at 95°C for 10 min, 40 cycles of amplification were carried out at 95°C for 15 s followed by 1 min and 30 s at 60°C. qPCR reactions were made using the CFX96™ thermal cycler (Biorad). Where indicated, cells were treated 1 hour before infection with 10 μM Raltegravir.
Late RT levels were assessed by qPCR as previously described [8].
Autointegration events were measured by two-step nested PCR as described [31]. The first amplification step used 200 ng of genomic DNA, 1X PCR buffer, 1.5 mM MgCl2, 0.2 mM of dNTPs, 0.5 μl Hot Start Taq polymerase, 0.2 μM of primers PBS- (5′ - TTTCCGGTCCCTGTT CGGGCGCCA- 3′), NY199/primer B- (5′ – CTACCTTG TTATGTCCTGCTTG- 3′) and NY200/primer A + (5′- CTCTACAGCACTTGGCACTAGC- 3′) in a final volume of 25 μl. After initial incubation at 95°C for 5 min, 24 cycles of amplification were carried out at 95°C for 30 s followed by 30 s at 60°C and 3 min at 72°C; a final step of elongation was made at 72°C for 7 min. The PCR product was then diluted 1:100 and used in the second step of amplification carried out with the same conditions and primers (NL4.3_2 fwd and MH536) as the qPCR for the detection of 2-LTR circles using primers external to the circle junction.
Cloning and sequencing of PCR products
2-LTR circle PCR products were cloned into pCR®II-TOPO® by TOPO TA cloning (Invitrogen), following the manufacturer's instructions. The plasmids were transformed into One Shot Mach1-T1 competent cells and plated in petri dishes. Single colonies were picked and used to stab a 96 well agar plate. Sequencing was carried out by Beckman Genomics (UK).
High-throughput sequencing of viral DNA byproducts
Genomic libraries were prepared using the TruSeq® DNA Sample Prep kit V2 (Illumina) following manufacturer's instructions. Briefly, 1 μg of genomic DNA was sheared with the Covaris 2 system (Covaris). The DNA fragments were then end-repaired, extended with an 'A' base on the 3′ end, ligated with indexed paired-end adaptors and PCR amplified. PCR amplification was carried out as follows: initial denaturation at 98°C for 30 sec, followed by 8 cycles consisting of 98°C for 10 sec, 60°C for 30 sec and 72°C for 30 sec, then a final elongation at 72°C for 5 min. Four different genomic libraries were pooled and sequenced in one lane of an Illumina HiSeq2000 sequencer using a 2 x 95bp paired end indexing protocol. Demultiplexed fastq files were obtained for each sample using the Illumina CASAVAv1.8.1 software and processed by a custom pipeline running on the Vital-IT (http://www.vital-it.ch). Center for high-performance computing of the SIB Swiss Institute of Bioinformatics. Specifically, the mapping of fastq reads has been performed with BWA [51] with duplicate removal by samtools [52] against the HIV genome in order to quantify the amount of virus for each sample. The same fastq reads have then been mapped against the human genome to eliminate DNA cell contamination (>90%). Remaining reads have been finally processed with Blat [53] and custom made Python scripts against HIV genome to capture and count the occurrence of junction structures as circles and autointegrants.
Average coverage of the HIV genome is ~5000 and the number of detected junctions (90% autointegrants) ranges from 30000 to 100000 according to the viral quantity (Table S1).
The complete high throughput sequencing dataset has been submitted to the Sequence Read Archive (SRA) /ci(http://www.ncbi.nlm.nih.gov/sra) under accession number SRA056122.
Immunofluorescence assay
TZM-bl cells expressing specific KD vectors or a version of the HA-tagged CPSF6 protein were fixed for 10 min with 3% paraformaldhyde, quenched for 10 min with 0.1 M glycine, permeabilized for 10 min with 0.2% Triton-X 100, and blocked for 20 min with 1% BSA in PBS. Cells were then incubated for 1 hour either with anti-HA (Covance) or anti-CPSF6 antibodies (Novus Biologicals) diluted in PBS with 1% BSA. After 5 washes, the cells were incubated with anti-mouse (HA) or anti-rabbit (CPSF6) Alexa Fluor 488-conjugated secondary antibodies for 1 hour, washed again, incubated for 1 min in Hoechst and mounted with Mowiol. The slides were viewed with a Zeiss LSM 510 confocal laser-scanning microscope.
Cellular fractionation
Control and TNPO3 KD TZM-bl cells were resuspended in 1 ml of ice-cold hypotonic buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, protease inhibitors) and lysed in a 7-ml Dounce homogenizer by 15 stokes with pestle B. The lysate was centrifuged at 1,000 rpm for 5 min at 4°C and the supernatant was used as cytoplasmic fraction. The nuclear pellet was resuspended in 1 ml of sucrose buffer (0.25 mM sucrose, 10 mM MgCl2, protease inhibitors), layered over a 1 ml sucrose cushion (0.88 mM sucrose, 0.5 mM MgCl2, protease inhibitors) and centrifuged at 3,500 rpm for 10 min at 4°C. The nuclear pellet was finally resuspended in 1× SDS-PAGE loading buffer.
Fate of capsid assay
Fate of capsid assay was performed as previously described [35]. TZM-bl cells expressing an empty vector, CPSF6-358, rhTRIM5α or the TNPO3 KD were seeded onto T75 flasks. 24 hours later, the confluent cells were incubated with 10 ml of Env- HIV-1, pseudotyped with VSV G, and bearing either WT or A105T mutant CA, for 30 min at 4°C and then shifted to 37°C. After 4 hours, the virus was removed, the cells were washed and returned to 37°C for 12 hours. Cells were detached with pronase (7 mg/ml in DMEM) for 5 min at 4°C, washed 3 times with ice cold PBS and finally resuspended in 2.5 ml of hypotonic lysis buffer (10 mM Tris–HCl, pH 8.0, 10 mM KCl, 1 mM EDTA). After 5 min incubation on ice, the cells were lysed in a 7-ml Dounce homogenizer by 15 stokes with pestle B. The lysate was cleared by centrifugation for 3 min at 3,000 rpm at 4°C to remove the nuclear fraction. 100 μl of the cleared lysate was collected to determine the viral input in the assay, while 2 ml was layered on top of a 7-ml 50% sucrose gradient and centrifuged for 2 hours at 30,000 rpm at 4°C using a Beckman SW41 rotor. After centrifugation, 100 μl of the top-most part were collected (supernatant) and the pellet resuspended in 1× SDS-PAGE loading buffer. All samples were analyzed by WB using antibody against p24.
Ethics statement
All experimental protocols were approved by the ethical committees of the University of Geneva and the University of Massachusetts.
We thank Massimo Pizzato for technical assistance and suggestions and Vineet Kewalramani for reagents. This work was supported by NIAID/NIH/USA grant RO1AI59159, NIDA/NIH/USA Grant DP1DA034990, and Swiss National Science Foundation grant 3100A0-128655 to J.L. The funders had no role in study design; in the collection, analysis, and interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication.
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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July 2015 Concert
July 2015 Concert
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July 18, 2015 | Concert | 53' 35"
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