A new function for oncoproteins of DNA tumor viruses

oncoproteinsOncogenes of DNA tumor viruses encode proteins that cause cells to divide incessantly, eventually leading to formation of a tumor. These oncoproteins have now been found to antagonize the innate immune response of the cell (link to paper).

Most cells encountered by viruses are not dividing, and hence do not efficiently support viral DNA synthesis. The genomes of adenoviruses, polyomaviruses, and papillomaviruses encode proteins that cause cells to divide. This effect allows for efficient viral replication, because a dividing cell is producing the machinery for DNA synthesis. Under certain conditions, infections by these viruses do not kill cells, yet they continue to divide due to the presence of viral oncoproteins. Such incessant division gives the cells new properties – they are called transformed cells – and they may eventually become a tumor.

These so-called viral oncoproteins include large T antigen (of SV40, a polyomavirus); E6 and E7 (papillomavirus), and E1A (adenovirus). These viral proteins kick cells into mitosis by inactivating cell proteins (such as Rb, pictured) that are normally involved in regulating cell growth. The cells divide, and in the process produce proteins involved in DNA replication, which are then used for viral replication. These oncoproteins accidentally cause tumors: the replication of none of these viruses is dependent on transformation or tumor formation.

Cells transformed with T, E6/E7, or E1A proteins are commonly used in laboratories because they are immortal. An example is the famous HeLa cell line, transformed by human papillomavirus type 18 (which originally infected Henrietta Lacks and caused the cervical tumor that killed her). Another commonly used transformed cell line is 293 (human embryonic kidney cells transformed by adenovirus E1A). It’s been known for some time that when DNA is introduced into normal (that is, not transformed) cells, they respond with an innate response: interferons are produced. In contrast, when DNA is introduced into the cytoplasm of a transformed cell, there is no interferon response.

To understand why HeLa and HEK 293 cell lines did not respond to cytoplasmic DNA, the authors silenced the viral oncogenes by disrupting them with CRISPR/Cas9. The altered cells produced interferon in response to cytoplasmic DNA. Furthermore, they produced new transformed lines by introducing genes encoding E6, E7, E1A, or T into normal mouse embryonic fibroblasts. These new transformed cells failed to respond to cytoplasmic DNA.

Cytoplasmic DNA is detected in cells by an enzyme called cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase) together with an adaptor protein known as STING (stimulator of interferon genes). When cytoplasmic DNA is detected by this system, the antiviral interferons are produced. The viral oncoproteins were found to directly bind STING, but not cGAS. A five amino acid sequence within E1A and E7 proteins was identified that is responsible for overcoming the interferon response to cytoplasmic DNA. When this sequence was altered, interaction of the oncoprotein with cGAS was reduced, and antagonism of interferon production in response to cytoplasmic DNA was blocked.

These findings provide a new function for the oncoproteins from three DNA tumor viruses: antagonism of the interferon response to cytoplasmic DNA. Normally DNA is present in the cell nucleus, and when it is detected in the cytoplasm, this is a signal that a virus infection is underway. The cytoplasmic DNA is sensed by the cGAS-STING system, leading to interferon production and elimination of infection. A herpesvirus protein has been identified that binds to STING and blocks interferon responses to cytoplasmic DNA. Clearly antagonism of the cGAS-STING DNA sensing system is of benefit to DNA viruses.

An interesting question is what selection pressure drove the evolution of viral oncogenes. One hypothesis, described above, is that they are needed to induce a cellular environment that supports viral DNA synthesis. The other idea, favored by the authors of this new work, is that oncogenes arose as antagonists of innate immune signaling. But I can’t imagine these DNA viruses without oncogenes, because they would not be able to replicate very efficiently. Could both functions have been simultaneously selected for? Why not – the same five amino acid sequence that binds cGAS also binds cellular proteins (such as Rb), disrupting their function and leading to uncontrolled cell growth!

TWiV 392: Zika virus!

Four virologists discuss our current understanding of Zika virus biology, pathogenesis, transmission, and prevention, in this special live episode recorded at the American Society for Microbiology in Washington, DC.

You can find TWiV #392 at microbe.tv/twiv, or listen/watch below.

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TWiV 382: Everyone’s a little bit viral

TWiVOn episode #382 of the science show This Week in Virology, Nels Elde and Ed Chuong join the TWiV team to talk about their observation that regulation of the human interferon response depends on regulatory sequences that were co-opted millions of years ago from endogenous retroviruses.

You can find TWiV #382 at microbe.tv/twiv, or listen below.

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TWiV 353: STING and the antiviral police

On episode #353 of the science show This Week in Virology, the TWiVniacs discuss twenty-eight years of poliovirus shedding by an immunodeficient patient, and packaging of the innate cytoplasmic signaling molecule cyclic GMP-AMP in virus particles.

You can find TWiV #353 at www.microbe.tv/twiv.

TWiV 336: Brought to you by the letters H, N, P, and Eye

On episode #336 of the science show This Week in Virology, the TWiVsters explore mutations in the interferon pathway associated with severe influenza in a child, outbreaks of avian influenza in North American poultry farms, Ebolavirus infection of the eye weeks after recovery, and Ebolavirus stability on surfaces and in fluids.

You can find TWiV #336 at www.microbe.tv/twiv.

How influenza virus infection might lead to gastrointestinal symptoms

influenza virusHuman influenza viruses replicate almost exclusively in the respiratory tract, yet infected individuals may also have gastrointestinal symptoms such as vomiting and diarrhea. In mice, intestinal injury occurs in the absence of viral replication, and is a consequence of viral depletion of the gut microbiota.

Intranasal inoculation of mice with the PR8 strain of influenza virus leads to injury of both the lung and the intestinal tract, the latter accompanied by mild diarrhea. While influenza virus clearly replicates in the lung of infected mice, no replication was observed in the intestinal tract. Therefore injury of the gut takes place in the absence of viral replication.

Replication of influenza virus in the lung of mice was associated with alteration in the populations of bacteria in the intestine. The numbers of segmented filamentous bacteria (SFB) and Lactobacillus/Lactococcus decreased, while numbers of Enterobacteriaceae increased, including E. coli. Depletion of gut bacteria by antibiotic treatment had no effect on virus-induced lung injury, but protected the intestine from damage. Transferring Enterobacteriaceae from virus-infected mice to uninfected animals lead to intestinal injury, as did inoculating mice intragastrically with E. coli.

To understand why influenza virus infection in the lung can alter the gut microbiota, the authors examined immune cells in the gut. They found that Mice lacking the cytokine IL-17A, which is produced by Th17 helper T cells, did not develop intestinal injury after influenza virus infection. However these animals did develop lung injury.

Th17 cells are a type of helper T cells (others include Th1 and Th2 helper T cells) that are important for microbial defenses at epithelial barriers. They achieve this function in part by producing cytokines, including IL-17A. Th17 cells appear to play a role in intestinal injury caused by influenza virus infection of the lung. The number of Th17 cells in the intestine of mice increased after influenza virus infection, but not in the liver or kidney. In addition, giving mice antibody to IL-17A reduced intestinal injury.

There is a relationship between the intestinal microbiome and Th17 cells. In mice treated with antibiotics, there was no increase in the number of Th17 cells in the intestine following influenza virus infection. When gut bacteria from influenza virus-infected mice were transferred into uninfected animals, IL-17A levels increased. This effect was not observed if recipient animals were treated with antibiotics.

A key question is how influenza virus infection in the lung affects the gut microbiota. The chemokine CCL25, produced by intestinal epithelial cells, attracts lymphocytes from the lung to the gut. Production of CCL25 in the intestine increased in influenza virus infected mice, and treating mice with an antibody to this cytokine reduced intestinal injury and blocked the changes in the gut microbiome.

The helper T lymphocytes that are recruited to the intestine by the CCL25 chemokine produce the chemokine receptor called CCR9. These CCR9 positive Th cells increased in number in the lung and intestine of influenza virus infected mice. When helper T cells from virus infected mice were transferred into uninfected animals, they homed to the lung; after virus infection, they were also found in the intestine.

How do CCR9 positive Th cells from the lung influence the gut microbiota? The culprit appears to be interferon gamma, produced by the lung derived Th cells. In mice lacking interferon gamma, virus infection leads to reduced intestinal injury and normal levels of IL-17A. The lung derived CCR9 positive Th cells are responsible for increased numbers of Th17 cells in the gut through the cytokine IL-15.

These results show that influenza virus infection of the lung leads to production of CCR9 positive Th cells, which migrate to the gut. These cells produce interferon gamma, which alters the gut microbiome. Numbers of Th17 cells in the gut increase, leading to intestinal injury. The altered gut microbiome also stimulates IL-15 production which in turn increases Th17 cell numbers.

It has been proposed that all mucosal surfaces are linked by a common, interconnected mucosal immune system. The results presented in this study are consistent with communication between the lung and gut mucosa. Other examples of a common mucosal immune system include the prevention of asthma in mice by the bacterium Helicobacter pylori in the stomach, and vaginal protection against herpes simplex virus type 2 infection conferred by intransal immunization.

Do these results explain the gastrointestinal symptoms that may accompany influenza in humans? The answer is not clear, because influenza PR8 infection of mice is a highly artificial model of infection. It should be possible to sample human intestinal contents and determine if alterations observed in mice in the gut microbiome, Th17 cells, and interferon gamma production are also observed during influenza infection of the lung.

TWiV 269: Herpesvirus stops a nuclear attack

On episode #269 of the science show This Week in Virology, the complete TWiV team reviews evidence for sensing of herpesviral DNA in the nucleus by the cell protein IFI16.

You can find TWiV #269 at www.microbe.tv/twiv.

TWiV 255: Longhorns go viral

On episode #255 of the science show This Week in Virology, Vincent and Rich visit the University of Texas at Austin and meet up with Bob and Chris to talk about their work on influenza virus and microRNAs.

You can find TWiV #255 at www.microbe.tv/twiv.

Thirty years in my laboratory at Columbia University

Racaniello labThirty years ago this month I arrived in the Department of Microbiology at Columbia University’s College of Physicians and Surgeons (P&S) to start my own laboratory. Thirty is not only a multiple of ten (which we tend to celebrate), but also a long time to be at one place. It’s clearly time to reminisce!

After studying influenza viruses with Peter Palese in New York City, in 1979 I headed to David Baltimore‘s laboratory at MIT. It was not long after Baltimore had received the Nobel Prize in Physiology or Medicine for discovering retroviral reverse transcriptase. In his laboratory I first encountered poliovirus, which would hold my interest for many years to come. The moratorium on recombinant DNA research had just been lifted, and it was now possible to clone complete viral DNA genomes. My first project was to make a DNA copy of poliovirus RNA, clone it into a bacterial plasmid, and determine its sequence. The result gave us the first glimpse of the viral genome. I then found that a DNA copy of poliovirus RNA is infectious in mammalian cells, a story that I have documented elsewhere.

The next step in my career was to have my own laboratory. With these two papers in hand I was able to obtain several respectable job offers, including one in the Microbiology Department at P&S. The department chair was Harold S. Ginsberg, an adenovirologist. My decision to accept his offer was influenced by the strong virology component of the department, which included Saul Silverstein and Hamish Young. I moved back to New York City in September 1982 with a DNA copy of the poliovirus genome in hand. In the last few weeks in the Baltimore laboratory, I had cloned a DNA copy of another poliovirus strain – type 2 Lansing – which had the interesting ability to infect mice. I spent the first few years in my new laboratory studying this virus and how it caused paralysis in mice. We found that the type 2 Lansing viral capsid was important for the ability to infect mice. Later, we narrowed this down to 8 amino acids. The type 1 Mahoney strain of poliovirus – which I had studied in the Baltimore laboratory – cannot infect mice. However, if we substituted 8 amino acids of the Mahoney capsid with the corresponding sequence from the Lansing genome, the recombinant virus could infect mice.

Our sequence analysis of poliovirus RNA had revealed an unusually long 5′-noncoding region. We began to carry out experiments to understand how such a viral RNA could be translated, and found that this long sequence enabled translation in the absence of a cap protein. This observation lead to the discovery by others that the poliovirus 5′-noncoding region contains an internal ribosome entry site (IRES). In the ensuing years our interest in translation continued. Examples include our finding that cell proteins bind to the poliovirus 5′-noncoding region, now known to participate in regulation of translation and genome replication, and understanding the inhibition of cell translation by poliovirus. Years later we developed a functional assay for the IRES in yeast, allowing identification of cell proteins needed for internal ribosome binding.

There is one area of research that has received the most attention in my laboratory, and on which we have published most extensively: the interaction of viruses with cell receptors. Towards the end of my stay in the Baltimore laboratory I became interested in how poliovirus attaches to and enters cells.  I came to Columbia with a strong interest in identifying the cell receptor for poliovirus, which we subsequently achieved. This finding lead to a series of studies on virus attachment to cells and virus entry. We produced transgenic mice susceptible to poliovirus, and used them to study aspects of poliovirus replication and pathogenesis, including how the virus attaches to its cellular receptor, regulation of viral tissue tropism, and the basis for attenuation of the Sabin vaccine strains.

The finding that poliovirus tropism is regulated by the interferon response lead to a change in the direction of our research. Beginning in the early 2000s we began studying how poliovirus interacted with the innate immune response. We found that poliovirus is relatively resistant to the antiviral effects of interferon, a property conferred by the viral 2Apro proteinase. How poliovirus is sensed by the innate immune system has also become a focus of our work. With the looming prospect of poliovirus eradication, and subsequent prohibition of work on the virus, we have also turned our attention to rhinoviruses, agents of respiratory illness. One focus has been to establish a mouse model for rhinovirus infection.

This story would not be complete without mentioning my foray into science communication. I have written a virology textbook and a blog about viruses, and began three science podcasts, including This Week in Virology, This Week in Parasitism, and This Week in Microbiology. My use of social media to teach microbiology to the world has been documented in a Social Media and Microbiology Education and in my Peter Wildy Prize Address.

None of this work would have been possible without the participation of 24 Ph.D. students (Nicola La Monica, Cathy Mendelsohn, Eric Moss, Robert O’Neill, Mary Morrison, Ruibao Ren, Elizabeth Colston, Michael Bouchard, Suhua Zhang, Alan Dove, Sa Liao, Yanzhang Dong, Yi Lin, Brian McDermott, Melissa Stewart Kim, Steven Kauder, Julie Harris, Amy Rosenfeld, Juliet Morrison, Angela Rasmussen, Jennifer Drahos, and Esther Francisco), 7 postdoctoral fellows (Gerardo Kaplan, Marion Freistadt, Michael Shepley, Ornella Flore, Juan Salas-Benito, and Scott Hughes), and many technicians and undergraduate students. My laboratory currently consists of postdoctoral scientist Rea Dabelic, and graduate students Ashlee Bennett and Michael Schreiber (pictured above).

Counting my time here, together with my Ph.D. and postdoctoral years, I’ve been working on viruses for 37 years. I do not know how much longer I will be doing the same, but it’s safe to say that it won’t be for another 37 years. But whenever I stop directing virology research, I will continue to write, podcast, and teach – you can expect nothing less from Earth’s virology professor.

TWiV 180: Throwing IFIT at flu and holding a miR to HCV

On episode #180 of the science show This Week in Virology, Vincent, Alan, and Rich review association of an interferon-induced protein with severe influenza, and stabilization of HCV RNA by a microRNA.

You can find TWiV #180 at www.microbe.tv/twiv.