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!

Virus Watch: Cancer Killing Viruses

Guest host Lynda Coughlan reviews how oncolytic viruses, which specifically kill tumor cells, are designed and how they work.

TWiV 395: The cancer thief

Vincent, Rich and Kathy speak with Stephen Russell about his career and his work on oncolytic virotherapy – using viruses to treat cancers. Recorded before an audience at ASV 2016 at Virginia Tech in Blacksburg, Virginia.

You can find TWiV #395 at microbe.tv/twiv, or listen/view below.

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TWiV 359: A Blossom by any other name

On episode #359 of the science show This Week in Virology, Vincent speaks with Blossom about her laboratory’s research on Kaposi’s sarcoma-associated herpesvirus, including how it transforms cells, the switch between lytic and latent replication, and its interaction with the innate immune system of the host.

You can find TWiV #359 at www.microbe.tv/twiv.

TWiV 337: Steamer

On episode #337 of the science show This Week in Virology, Vincent meets up with Michael and Steve to discuss their finding of a transmissible tumor in soft-shell clams associated with a retrovirus-like element in the clam genome.

You can find TWiV #337 at www.microbe.tv/twiv.

Therapeutic teamwork: Coupling oncolytic viruses with immunotherapy to destroy tumor cells

NDV infected glioma cellsThis article was written for extra credit by a student in my recently concluded virology course.

by Nayan Lamba

A recent study by scientists at the Ludwig Center for Cancer Immunotherapy offers a new, multifaceted therapy for destroying tumors. A team of researchers led by Dmitriy Zamarin combined checkpoint blockade, a technique aimed at enhancing antitumor immune responses, with oncolytic viral therapy, a technique that uses viruses to kill tumor cells. By employing the two immunotherapies together, the researchers had more success in destroying tumor cells than they have had while investigating each therapy independently.

While checkpoint blockade has been effective as a therapy against some tumors, its major drawback seems to be an inability to destroy strongly immunosuppressing tumors that evade immune system detection. While many oncolytic viruses have also had success as antitumor agents, their impact thus far has been limited to locally restricted tumors. Scientists major concern about oncolytic viruses has thus been whether they must be injected at all possible tumor sites in order to combat metastatic tumors. In an innovative approach aimed at overcoming the weaknesses of the two independent therapies, Zamarin and his team were able to destroy previously resistant tumor cells.

The researchers initially injected mice with melanoma tumor cells, followed by injection of Newcastle disease virus (NDV) directly into these tumor sites. Newcastle disease virus is an avian virus that is non-pathogenic in humans and capable of inducing a robust immune response. Initial injection of NDV resulted in a pronounced inflammatory response in the mice, with increased activation of both innate and adaptive players of immunity. What is perhaps most striking about the data is that despite direct injection of NDV at a local tumor site, increased inflammation was also observed in sites distal and contralateral to tumor injection. The researchers noted tumor growth delay at sites both local and distant to sites of injection, indicating a potential for use of NDV in targeting metastatic tumors. While this observation was certainly promising, the researchers noted that complete, long-term destruction of distant tumor cells was seen in only 10% of animals. The researchers attributed this low level to the increased immunosuppression performed by the distant tumors. This suggestion was based on their observation that the distant tumor sites exhibited increased activity of CTLA-4 cells, which down-regulate the immune system.

While a more traditional, unilateral approach employing oncolytic viruses would have stopped here, the researchers instead proceeded to couple NDV injection with antibodies to CTLA-4 cells. They hypothesized that because NDV caused an increased level of inflammation at distant tumor sites, the anti-CTLA-4 antibodies might have more of an effect if they had been administered without NDV. Indeed, the researchers found that NDV coupled with anti-CTLA-4 resulted in long-term tumor suppression at sites both local and distant to NDV injection. By increasing the inflammatory response via NDV injection, they made the immune cells more receptive to the anti-CTLA-4 antibodies. Through a combination of oncolytic virus therapy and checkpoint blockade, the researchers overcame the limitations faced when each one is employed independently.

What is perhaps most promising about this therapy is that it has also proved effective against tumors that have previously shown resistance to oncolytic viral therapies. For example, TRAMP C2 prostrate adenocarcinoma cells previously showed resistance to lysis by NDV in vitro, unlike the melanoma cells discussed above. Yet, despite this resistance, when both therapies were employed on the adenocarcinoma cells in vivo, researchers noted distant tumor regression and long-term survival, just as they did with the melanoma cells. When they examined these adenocarcinoma cells in vitro, they noted an increased inflammatory response. They noted an up-regulation of MHC I molecules in all cells, even cells that were not infected with NDV. MHC I cells are important players of the immune system, responsible for presenting fragments of virus at the surface of infected cells so that the body can recognize when a cell is infected and subsequently destroy that cell. By demonstrating that a tumor cell does not need to be permissive to a virus in order to be a target for therapy, Zamarin’s approach greatly expands the potential applicability of NDVs and other oncolytic viruses. It seems that what is most important to tumor suppression is the virus-generated inflammatory response and the increased tumor immogenicity that this approach facilitates.

The clinical potential of this double-therapy in humans is particularly exciting to me, especially in light of the fact that both oncolytic therapies and checkpoint blockade have independently been successful in combating tumors in humans. This observation certainly suggests that the two therapies may prove even more effective when combined in humans. Indeed, such a clinical trial is already underway in humans; a current study is studying the effects of ipilimumab, a CTLA-4 target, administered with a herpes simplex oncolytic virus, in the treatment of melanoma. It will be interesting to see how the double-therapy plays out in a human population, and how these results affect the future use of oncolytic viruses.

TWiV 218: Monkeys turning valves and pushing buttons

On episode #218 of the science show This Week in Virology, Vincent, Alan, and Welkin discuss how endogenous retroviruses in mice are held in check by the immune response.

You can find TWiV #218 at www.microbe.tv/twiv.

TWiV 214: This is your brain on polyomavirus

On episode #214 of the science show This Week in Virology, Vincent, Alan, and Kathy discuss how coagulation factor X binding to adenovirus activates the innate immune system, and a novel polyomavirus associated with brain tumors in raccoons.

You can find TWiV #214 at www.microbe.tv/twiv.

TWiV 142: Viral oinkotherapy

seneca valley virusHosts: Vincent Racaniello, Rich Condit, and Alan Dove

Vincent, Rich, and Alan discuss a method for identifying viruses of individual environmental bacteria, and the using a picornavirus for oncotherapy.

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Weekly Science Picks

Alan – Germlines podcast
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Listener Pick of the Week

AngusScience Weekly with Alok Jha

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TWiV 124: Viruses that make you better

Rabbits Myxomatosis Trial WardangIsland 1938Hosts: Vincent Racaniello, Dickson DespommierAlan DoveRich Condit, and Grant McFadden

On episode #124 of the podcast This Week in Virology, Vincent, Dickson, Alan, Rich, and Grant discuss a tanapoxvirus protein that inhibits tumor necrosis factor, purging tumors with myxoma virus, and destruction of the last known stocks of smallpox virus.

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Weekly Science Picks

Grant – A Short History of Nearly Everything by Bill Bryson
Dickson – An Inquiry into the Causes and Effects of the Variolae Vaccinae by Edward Jenner
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Selinah (Topsy Foundation) thanks Meghan!
Alan – Draft report (pdf) on WHO’s H1N1 response and NY Times summary
Vincent – 10 stunning science visualizations

Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv, or call them in to 908-312-0760. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.