TWiV 428: Lyse globally, protect locally

The TWiVsters explain how superspreader bacteriophages release intact DNA from infected cells, and the role of astrocytes in protecting the cerebellum from virus infection.

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TWiV 426: I’m Axl, and I’ll be your cervid today

The sages of TWiV explain how chronic wasting disease of cervids could be caused by spontaneous misfolding of prion protein, and the role of the membrane protein Axl in Zika virus entry into cells.

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TWiV 412: WO, open the borders and rig the infection

The TWiVome reveal the first eukaryotic genes found in a bacteriophage of Wolbachia, and how DNA tumor virus oncogenes antagonize sensing of cytoplasmic DNA by the cell.

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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.

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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.

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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.

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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.

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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.

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