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About viruses and viral disease
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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Oncogenes 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 293Â 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!
A major new feature of the fourth edition of Principles of Virology is the inclusion of 26 video interviews with leading scientists who have made significant contributions to the field of virology. For the chapter on Transformation and Oncogenesis, Vincent spoke with Nobel Laureate J. Michael Biship, of the University of California, San Francisco, about his career and his work on oncogenes.
Today it is well known that viruses may contain DNA (poxvirus, mimivirus) or RNA (influenza virus, Zika virus), but for many years it was thought that genomes were only made of DNA. The surprise at finding only RNA in a virus is plainly evident in a 1953 letter from Harriett Ephrussi-Taylor to James D. Watson (pictured, Cold Spring Harbor Archives Repository*).
While DNA was discovered in the late 1800s, its role as genetic material was not proven until the famous experiments of McLeod, Avery, and McCarty, published in 1944. They showed that DNA from a strain of Pnemococcus bacteria that formed smooth colonies, when added to a rough colony former, produced smooth colonies.
By this time many viruses had been identified, and it was assumed that their genetic information was DNA. The ‘kitchen blender’ experiments of Hershey and Chase in 1952 proved that the genetic information of bacteriophage T2 is DNA. Watson and Crick proposed the double-helical structure of DNA in 1953, and a few years later published the Central Dogma, which suggested that information flowed in biological systems from DNA to RNA to protein.
Amidst all these experimental findings, which gave rise to the field of molecular biology,  comes the note in 1953 from Ephrussi-Taylor to Watson. Under the heading TOP SECRET she writes:
Burnet swears, from work in his lab, that flu virus has principally, if not exclusively RNA. Suspects the same for polioviruses. ??
During her career, Dr. Ephrussi-Taylor carried out work on bacterial transformation by DNA and was knowledgeable about its history as genetic material. Frank Macfarlane Burnet was an Australian immunologist who worked on influenza virus early in his career.
By the 1950s many viruses had been isolated which we now know have genomes of DNA (bacteriophage, poxvirus) or RNA (yellow fever virus, poliovirus, influenza virus). But it was the first virus discovered – tobacco mosaic virus, in the 1890s – that lead the way to establishing RNA as genetic material. Wendell Stanley produced crystals of TMV in 1935 and found that they contained 5% RNA. But Stanley and others thought TMV was a protein, and that the RNA was either a contaminant, or played a structural role.
A structural role for RNA was reinforced as late as 1955 when Heinz Fraenkel-Conrat separately purified TMV protein and RNA. When he mixed the two components together, they formed infectious, 300 nm rods. When the RNA was omitted, noninfectious aggregates formed. This finding reinforced the belief that RNA helped form virus particles.
This view changed when Fraenke-Conrat gave his wife, Beatrice Singer, the task of purifying TMV RNA until it had lost all infectivity. To everyone’s surprise she found that TMV RNA itself was infectious, proving in 1957 that it was the viral genetic material. However, RNA also has a structural role in TMV virus particles, as it organizes the capsid protein (yellow in illustration at left) into regularly repeated subunits.
Demonstration of infectivity of RNA from animal viruses soon followed, for mengovirus, a picornavirus, in 1957 and for poliovirus in 1958 (the latter done at my own institution, the College of Physicians and Surgeons of Columbia University!).
By the early 1950s the idea that RNA could be viral genetic material was clearly in the minds of virologists, hence Ephrussi-Taylor’s amusing letter on influenza virus and poliovirus.
*Thanks to @infectiousdose for finding this amazing letter.
Vincent, Rich, and Kathy and their guests Clodagh and Ron recorded episode #291 of the science show This Week in Virology at the 33rd annual meeting of the American Society for Virology at Colorado State University in Ft. Collins, Colorado.
You can find TWiV #291 at www.microbe.tv/twiv.
On episode #259 of the science show This Week in Virology, Vincent and Rich join Jackie at the University of Texas, Austin to talk about her work on mouse mammary tumor virus.
You can find TWiV #259 at www.microbe.tv/twiv.