Kinjal Majumder

Finding how a Virus Navigates the Nucleus of a Cell

December 22, 2020 By Becca Wolf in Research Tags: parvovirus

Kinjal Majumder and David Pintel | photo by Roger Meissen, Bond LSC

By Becca Wolf | Bond LSC

Four years of hard work certainly paid off for Kinjal Majumder.

Majumder, a former postdoctoral fellow in the Pintel lab at Bond Life Sciences Center, spent the past four years looking at how the parvovirus, Minute Virus of Mice (MVM), is getting to the sites in the nucleus that it needs to replicate.

Parvovirus is used as a model to study virus behavior because it is a simple, single stranded DNA virus. MVM infects mouse cells and transformed human cells.

The work builds off a paper the lab published back in 2018 where Majumder and the Pintel lab looked at where a virus goes inside the nucleus of a cell. The results of this study were published in PLOS Pathogens in October and make progress towards understanding how viruses can localize to distinct sites to set up replication centers.

“This was the next logical step in the laundry list of questions to try and figure out how it’s getting there,” Majumder said. “This is important because we’re interested in the basic mechanisms of how a virus does what it does. So, using the parvoviruses as a model system, we can slowly chip away at these questions.”

Based on their previous work, Majumder had a good idea of where the virus went once in a cell, but he had to figure out how it was happening. He found that the non-structural protein (NS1) of the virus carries the viral DNA to sites on the cell’s genome which are prone to incurring DNA breaks, after which it can start to replicate. The genome is the set of genetic material of an organism, so it is quite important for the cell.

The NS1 protein is critical for creating new copies of the virus and binds to damaged cellular DNA breaks in a cell, though it is not yet clear why.

“When the virus goes in the cell, it seeks regions where there is broken DNA like a heat seeking missile. The reason we think the virus is doing that is because broken DNA means that the cell has repair and replication proteins at those regions. The virus can then hijack and use the proteins to replicate itself,” Majumder said.

NS1 plays a key role in this process by binding and transporting the viral DNA to these cellular break sites to initiate replication.

Majumder used many resources that MU has to offer to complete this project. To create the break in DNA in the cell nucleus, he used a laser on the confocal microscopes at the Advanced Light Microscopy Core in Bond LSC. He also collaborated with the MU Genomics Technology Core Facility, Informatics Research Core Facility and the Lewis Cluster in the Engineering department.

“It was really nice to have all of my resources within one building,” Majumder said. “I would go in the morning and stripe the cells, and then two minutes later I would be processing them upstairs for the next stage of the experiments, so it really expedited our research. And of course, Drs. Jurkevich and Baker, who run the Advanced Light Microscopy Core were always a great help in the laser micro-irradiation assays. It really made the project go smoother than anticipated. These experiments were first started by Dr. Matthew Fuller in the Pintel lab in 2016, who is also a coauthor on the paper.”

Since this project, Majumder has moved to the University of Wisconsin-Madison where he is starting his own lab in the Institute for Molecular Virology. The distance has not stopped him from continuing this work.

“Dave [Pintel] has been an incredibly supportive mentor. We Zoom twice a week and we talk about experiments, things that are going on in the Pintel lab, things that are starting to happen in my lab, and stuff that we can work on together going forward,” Majumder said.

Pintel has enjoyed continuing this professional relationship.

“He’s a terrific young scientist, he got a number of grants and awards while he was here, some, I think, that are really hard to get. He got a terrific job is in a terrific place,” Pintel said. “I’m very happy that I could support him and that he has something strong for him to take forward as he starts his career.”

Despite Majumder now working over 400 miles away, this project is not yet done.

“We haven’t fully completed the second step yet,” Majumder said. “We figured out that NS1 is dragging the viral DNA to the cellular site of DNA damage, but that’s only half of the picture. We’re looking at the virus’ side of the puzzle, we don’t know what’s on the host side. We want to find out what’s on the cellular side or what cellular protein is creating this bridge. That would be the next step.”

Majumder and Pintel, together with graduate student Maria Boftsi, are already beginning to study this process on a human parvovirus called Adeno-Associated Virus (AAV).

“We’re trying to do a similar thing in a different parvovirus. This is a parvovirus that people use a lot for gene therapy applications so there is a little more medical interest in it,” Pintel said. “This is a human virus and we already know that some things are a little bit different. But we’re getting close to getting an idea of what’s happening.”

Going forward, Majumder hopes to look at more viral systems in addition to these two in order to get a basic idea on how different viruses can navigate the nuclear environment.

“We can expand into more pathogenic systems like cancer causing viruses such as Hepatitis B virus or human papillomavirus,” Majumder said. “Those viruses are more complicated, so they have a more sophisticated machinery to modify the nucleus for their purposes. They employ many different mechanisms to take over the nucleus, so trying to dissect things out in them is more challenging. Those would be the future, long-term directions of these studies.”

Find out more about this study in “The NS1 protein of the parvovirus MVM Aids in the localization of the viral genome to cellular sites of DNA damage” published in PLOS Pathogens.

#IAmScience Kinjal Majumder

September 18, 2020 By Lauren Hines in #IAmScience Tags: NIH, parvovirus

By Lauren Hines | Bond LSC

It was another day in the lab. Kinjal Majumder, a postdoctoral fellow in the David Pintel lab at Bond Life Sciences Center, was working on his research and stopped to check his email. At that moment, he found out he just won a $700,000 grant from the National Institutes of Health.

He felt relieved.

“I wish I could say something more high minded about it, but honestly, at that point, you’re like, ‘Oh, thank God. I got the award,’” Majumder said. “Then I thought, ‘Alright. I’m gonna go back to my experiments.’”

Majumder won the NIH Pathway to Independence award from the National Institute of Allergy and Infectious Diseases that helps postdoctoral research fellows transition from working under a research mentor to starting their own lab. The application process takes time, so Majumder had to wait almost a year and a half from the time he submitted his application.

“It’s very humbling and honestly I am still processing it,” Majumder said. “The way these awards work is that you apply, you hope for the best, you walk away and try not to think about the possible outcomes.”

According to 2019 data from NIAID, applicants have a 19.1% success rate in receiving the award.

“No, I’m not surprised at all,” said Maria Boftsi, graduate student in the Pintel lab. “I think he’s worth it. He works very hard. He loves research. He loves being in the lab and doing experiments.”

Even when Majumder first started at Bond LSC, he showed promise.

“He’s a very imaginative guy, and he’s got a lot of energy,” said David Pintel, endowed professor in medical research Molecular Microbiology and Immunology. “He’s very forward thinking and he’s very creative. Those are the kind of scientists that can get to another level because they do experiments that most people wouldn’t think of.”

When he first joined Pintel’s lab, they were trying to figure out where a virus goes once it enters a cell.

The lab studies a small, single stranded DNA virus called, Minute Virus of Mice, which is a parvovirus. According to Majumder, it’s a good prototype to study how small DNA viruses in general navigate the host cell’s environment to establish infection.

“So, when we started asking where a virus localizes in the nucleus of a host cell, I realized that I possessed the expertise in the techniques we need to answer those basic questions in virology [techniques I had learned during my Ph.D. at Washington University],” Majumder said. “We jumped into it headfirst and experiments started working.”

Researchers originally thought viruses set up replication centers in the nucleus of cells by randomly interacting with the factors they need to replicate. However, Majumder and a previous graduate student in the lab, Matt Fuller, started to test whether viruses went to specific places in the nucleus, particularly on the cellular genome where factors already exist to facilitate virus replication.

“For a lot of reasons, it seemed a reasonable idea although people hadn’t thought about that before, certainly in our lab,” Pintel said. “So, they started doing the experiments, and they found that’s actually what happens.”

Majumder has been working at Bond LSC for the past five years doing many collaborations.

“He has a lot of knowledge and lab experience so whenever I have troubles and I need to troubleshoot experiments, I will talk to him,” Boftsi said. “He will share thoughts, and he’ll give me advice. His advice is always helpful.”

Majumder has been studying virology ever since he first started as a post doctorate in the Pintel lab. His interest began in graduate school thanks to his colleagues in the immunology graduate program at Washington University in St. Louis.

“I had several friends in graduate school who were virologists and studied how the immune system responds to different types of viral infection. Every time they presented at departmental seminar, they insisted that viruses were super cool,” Majumder said. “Eventually I decided that, ‘Hey, maybe I’ll check this out and see what the fuss is about.’”

Turns out it was worth the hype.

Now, Majumder is going to use the grant money to further his research on studying how viruses establish infection in host cells, usurp host proteins to replicate and spread to neighboring cells.

“In this proposal, I want to study the nuts and bolts of this process and find out what are the proteins that the virus is hijacking from the cell,” Majumder said. “How is it using those proteins to replicate in the cell, and then how is it further damaging the cell’s DNA so that it creates new viral replication centers?”

Other than it being “really cool science,” as Majumder puts it, this research can eventually be applicable to human diseases. Some parvoviruses can even be modified to target cancer cells.

Majumder is planning on continuing this research in his own lab at the University of Wisconsin-Madison at the Institute for Molecular Virology and McArdle Laboratory for Cancer Research starting in October 2020.

“I think his future students will be very lucky working with him because he likes sharing his knowledge, and he really loves what he’s doing,” Boftsi said. “I think this is something that he will pass to his future students.”

Majumder received help from Bond LSC investigators David Pintel, Christian Lorson, Ron Mittler, Trupti Joshi, the MMI department faculty, the MU Postdoc Association and the MMI postdoctoral fellows in the assembly of his K99/R00 application. Majumder also relied on the Genomics Technology Core and the Advanced Light Microscopy Core at the Bond LSC for experimental support. Majumder was funded for his postdoctoral research by a Ruth L. Kirschstein Postdoctoral Individual National Service Award by the NIH.

Studying Drug Resistance, One Connection at a Time

June 1, 2020 By Becca Wolf in Research Tags: drugs, virology

Kinjal Majumder | photo by Mariah Cox, Bond LSC

By Becca Wolf | Bond LSC

Where you are creates opportunities.

The structure and placement of labs encourages researchers to collaborate and talk to each other and often, connections and friendships are formed.

For Kinjal Majumder, a virologist and postdoctoral fellow in the David Pintel lab at Bond Life Sciences Center, that has meant bouncing many ideas off friends and colleagues from neighboring labs. Little did he know that one connection would lead him to interesting findings in a field outside of virology.

Those results may help make progress toward understanding the machinery behind drug resistance and toxicity in the body.

“The big picture is that it will help future studies on how drugs are metabolized by our body,” Majumder said.

The connection started with Andrew Huber, the first author of the paper, who completed his Ph.D. while in the lab of Dr. Stefan Sarafianos at Bond LSC. He graduated two years ago, moved to the lab of Dr. Taosheng Chen at St. Jude Children’s Research Hospital (SJCRH) in Memphis, Tenn. and began his postdoctoral work.

Dr. Chen’s lab focuses on mechanisms of cellular drug metabolism, one of which functions through the pregnane X receptor (PXR) protein.

PXR is found in the liver and is activated by drugs and other substances in the blood. Once activated, it binds to DNA and activates genes responsible for detoxifying the system. However, this system often leads to metabolism and reduced efficacy of administered drugs such as chemotherapies and antibiotics.

“When Andrew was in the Sarafianos lab, we’d talk science often and we collaborated on how different viral proteins bind to host and virus DNA, and how this can aid viral life cycles,” Majumder said, “When he moved on to his postdoctoral work, he saw that he can start applying similar techniques to study how the liver metabolizes toxins. So that’s when we set up this collaboration to pursue these things together.”

Majumder studies how viral DNA and proteins interact in virus-infected cells. One technique he uses is called chromatin immunoprecipitation assay (ChIP) which determines where proteins of interest can bind to DNA molecules. Majumder freezes protein and DNA that are interacting together in the process and he pulls that complex down with an antibody. “It’s like going fishing,” he said.

In order to apply ChIP and other techniques to study PXR signaling, Majumder drove down to SJCRH in Memphis to work with Huber on these assays.

Once in Memphis, Majumder and his collaborators worked and conducted experiments around the clock, but they also found time to explore the city and have fun.

“We go into lab and try to get done as many experiments as we can,” Majumder said, “And whenever we have a long break we go out on the town. It’s usually a really fun way to go and experience the life and scientific culture of a new place.”

It is a lot to pack into one week, but Majumder and his collaborators make the most of it, finding a way to effectively balance their studies on PXR with soaking up life in Memphis.

Majumder compares PXR to a light dimmer switch. When activated, it “brightens” by increasing drug metabolism, leading to decreased drug efficacy. PXR inhibitors work to “dim” this effect. At St. Jude, Dr. Chen’s group found that of the 434 amino acids that make up PXR, only one mutation is needed to “turn a dimmer into a brightener.”

“We sort of got lucky, we thought that you’d have to make a lot of changes in order to make PXR do something different,” Majumder said, “Without making too many changes — Dr. Chen’s group just switched one thing — and that was sufficient enough to turn it into a brightener. So that was a happy and unexpected outcome.”

Once these results were reviewed, the Cellular and Molecular Life Sciences journal accepted their results and published them this March in an article titled “Mutation of a single amino acid of pregnane X receptor switches an antagonist to agonist by altering AF-2 helix positioning.”

Looking ahead, Majumder and his collaborators hope to make more progress in understanding how drugs are metabolized in the body.

All of this would not have been possible if it wasn’t for the connections Majumder made at Bond LSC a few years ago.

“It was so cool to have this opportunity to collaborate with someone from the LSC that I have collaborated with before and apply our experimental techniques to study a new system,” Majumder said, “It feels very encouraging that now the work can be expanded upon and applied to other systems and settings.”

In a viral game, the fight isn’t fair

October 30, 2017 By Samantha Kummerer in Research Tags: viruses

Lab explores how parvo wins in tug of war with cells

Kinjal Majumder and David Pintel examine the protein levels in mouse cells during MVM infection. Each black band represents the amount of viral protein in infected cells over time. | Photo by Samantha Kummerer, Bond LSC

By Samantha Kummerer | Bond LSC

At the start of any tug of war game, the battle is even. But it doesn’t stay that way for long. After a back and forth, the inevitable happens — the stronger team gives the rope one last tug and send the losers toppling over, claiming their dominance.

This is a game cells and viruses know well. In their version of tug of war, the virus eventually overtakes the cell and not only topples it but causes a consequence far worse than a few scraped knees.

This is how post-doctoral fellow Kinjal Majumder thinks of the interaction between parvoviruses and the dividing cells it conquers.

Majumder and others in the lab of David Pintel at Bond Life Sciences Center recently gained insight into how the virus achieves victory over the cell. These findings could improve human therapies and even play a role in treating cancer.

Meet the Champion

Parvoviruses are some of the smallest, simplest viruses. With only two genes, it has fewer DNA base pairs than most other viruses, up to ten times smaller in some cases. The virus’ size and simplicity, however, do not make it any easier to understand.

Pintel’s lab works at the “nitty gritty” level of the virus to study its basic molecular mechanism.

The group understands a little about how the virus operates but is working on the why.

Like a sly culprit, the virus uses its tiny nature to sneak inside the cell. The cell recognizes the presence of the virus as a foreign piece of DNA and responds to try to remove it. Once the cell responds, the virus begins replicating inside until it overtakes the cell. But, the tug of war game ends up more one-sided, so perhaps viewing the virus as a ruthless conqueror would be more accurate.

Latest Developments

Majumder’s experiments specifically focus on DNA damage response, an intrinsic function of cells. The DNA damage response constantly works to protect us from cancer by continuously repairing broken DNA to prevent harmful mutations in cells . This response uses a network of cellular pathways to monitor and provide checkpoints in the cell cycle to prevent damage from being passed on to the next generation of cells. But parvoviruses also tricks cells to begin a DNA damage response, which they use to eventually take over the host cell.

In July, the lab published the latest finding from a series of papers exploring a type of these parvoviruses called minute virus of mice (MVM). The discovery began when the team found that the virus stops cells from dividing within infected cells.

To do this, MVM uses the DNA damage response to stop cells from dividing, but still allows virus replication to continue. The group developed a system to examine how the virus took over the cell. Further experiments revealed the virus transcriptionally regulates cell cycle genes.

The team used CRISPR to target a cellular gene that the virus must inactivate for it to replicate. Expression of this gene is required for the cell to divide. They discovered the virus actually blocks the transcription of this gene so it cannot make its protein. Blocking this function also prevents the cell from dividing. Without cell division, the virus is free to rapidly replicate inside the cell.

Post-doctoral fellow, Kinjal Majumder, points to the results of a recent experiment. When MVM is mutated at particular sites, the protein levels produced by the virus decrease, but the virus continues to replicate efficiently. | Photo by Samantha Kummerer. Bond LSC

Majumder said parvo’s manipulation of infected cell cycles is different from other viruses because it can only replicate in cells that are actively synthesizing DNA. It eventually halts the process of cell division in infected cells by dysregulating transcription factors that regulate cell cycle gene expression. That’s what makes this discovery unique.

Endless Possibilities

This discovery only scratches the surface.

Majumder said the lab constantly thinks of new experiments to explore parvovirus biology. The simplicity of its DNA expedites the process of culturing and growing the virus, leaving more time for what Majumder calls the “fun experiments.”

“We try to be thorough and confident of our findings, so we attack experiments from many different angles,” Majumder said.

Imagine viewing an object from multiple angles under varying light conditions. The change in perspective reveals something different about it with each new look. This approach expands their understanding of parvoviruses.

Majumder explained the lab makes use of everything from high-resolution imaging and CRISPR technology to proteomics and deep sequencing to study the tug-of-war between MVM and the DNA damage response.

“The thing about being a postdoc is you kind of have to be a jack of all trades,” Majumder said about his ability to conduct the range of experiments.

Wider Implications

While parvoviruses are not a deadly threat to humans, understanding it has major implications for humans.

Parvoviruses are used to develop gene therapy tools to treat disorders like muscular dystrophy and spinal muscular atrophy. The Pintel lab collaborates with labs such as the Lorson and Sarafianos lab in Bond LSC, to explore its therapeutic potential.

Majumder explained the lab is also interested in understanding how the virus could improve cancer treatment. Parvo’s tendency to replicate in dividing cells links it to how cells divide uncontrollably in cancer. Majumder explained those scientists are trying to use that function of the virus to target cancer cells.

This makes work in the Pintel lab that much more important. But, before the virus can fully improve humans’ conditions, researchers better grasp its capabilities. And that means more of the daily experiments within the Pintel lab.

“What we learn from studying a simple virus can be expanded to more complicated viruses because there are some viruses that can make a dozen different proteins, so that’s a more complicated system,” Majumder said. “Before you can get to the more complicated systems, you need to be able to understand the one that makes just a few proteins.”

For now, scientists will play their own tug of war as they go back and forth in their findings and experiments to uncover the mystery surrounding this small, unique virus.