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 411: Chicken runs

The TWiVeroos examine a reverse spillover of Newcastle disease virus vaccines into wild birds, and identification of a protein cell receptor for murine noroviruses.

You can find TWiV #411 at microbe.tv/twiv, or listen below.

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Wolbachia in an insect cell. Image credit: PLoS/Scott O’Neill.

Viruses are tidily categorized into three groups according to the hosts they infect – bacteriophages, eukaryotic viruses, and archaeal viruses. Viruses do not infect hosts in another domain of life, and therefore lateral gene transfer is limited (giant DNA viruses might be exceptions). Now there is evidence for lateral gene transfer between eukaryotes and bacteriophages.

Proof of this unusual movement of DNA comes from studies of the obligate intracellular bacteria Wolbachia, which infects 40% of arthropods (pictured). Wolbachia are in turn infected with a bacteriophage called WO; nearly all sequenced Wolbachia genomes contain integrated WO DNA. Analysis of complete WO genome sequences revealed the presence of mutiple eukaryotic genes (link to paper) that comprise about half of the phage genome!

Ten different protein domains were identified in the eukaryotic genes of WO phage with four functions: toxins, host-microbe interactions, host cell suicide, and protein secretion through membranes.

One eukaryotic gene in phage WO is a black widow spider toxin called latrotoxin-CTD. Sequence analysis suggests that the spider toxin gene was transferred to phage WO within a Wolbachia genome (these bacteria are known to infect widow spiders).

It is not surprising that a virus of a bacterium that infects a eukaryotic cell might acquire eukaryotic genes, but the exact mechanism of gene transfer is unknown. Eukaryotic DNA might enter the WO genome while the particles are in the insect cell cytoplasm, or during packaging of viral DNA in the presence of animal DNA. Another possibility is transfer of eukaryotic DNA to the Wolbachia genome, and then to phage WO.

The fact that eukaryotic-like DNA sequences make up half of the phage WO genome suggests that they serve important functions for the virus. The functions ascribed to these eukaryotic genes suggest roles in cell lysis, modification of host proteins, and toxicity.

There are other examples of phage-infected obligate intracellular bacteria of Chlamydia, aphids, and tsetse flies. A study of these viral genomes should reveal whether lateral gene transfer between metazoans and bacteriophages is a common mechansim for augmenting functions of the viral genome.

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

TWiV 410: Hurricane Zika

Sharon and Scott join the TWiV team to talk about their work on dengue antibody-dependent enhancement of Zika virus infection, and identifying the virus in mosquitoes from Miami.

You can find TWiV #410 at microbe.tv/twiv, or listen below.

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autophagyYoshinori Ohsumi has been awarded the 2016 Nobel Prize for Physiology or Medicine for his work on autophagy, a process of eukaryotic cells for degrading and recycling cellular components. Because of his research, we now understand the importance of autophagy in health and human disease. It is another example of the serendipity of science and yes, it is relevant to virology!

The word autophagy was coined by Christian de Duve in 1963 to describe a process that he and others had previously described: when stressed, cells would sequester portions of the cytoplasm in double-membraned vesicles called autophagosomes. These would then fuse with lysosomes (which de Duve had discovered) and the contents were degraded (illustrated; image credit).

In subsequent years it was suggested that autophagy might have roles in human disease, but little progress was made on understanding how the process worked: how it was triggered, what proteins were involved, and its function in health and pathogenesis.

As a young Assistant Professor at Tokyo University in the early 1990s, Ohsumi found that autophagy occurred in the yeast Saccharomyces cerevisiae. He decided to produce yeast strains lacking the proteases that would be involved in digesting the contents of the autophagic vesicles, with the idea that these vesicles would accumulate under stress (under normal conditions the autophagosome exists for a short period of time, making its study difficult).

When Ohsumi stressed the yeasts lacking the vacuolar (equivalent of mammalian lysosome) proteases, autophagosomes accumulated in the cytoplasm which were readily visible by light microscopy. He used this clear phenotype to isolate yeast mutants that could no longer accumulate such vesicles, and identified 15 genes that are essential for induction of autophagy in eukaryotic cells.

Later Ohsumi elucidated the functions of these genes in autophagy induction, and found homologues in mammalian cells. His work stimulated great interest in autophagy, and it became a highly studied pathway. We now understand that autophagy is not only important for embryogenesis and cell differentiation, but plays roles in neurodegenerative diseases, cancer, and in defenses against bacterial and viral infections.

As might be expected, virus infection is a stress that triggers autophagy, which may impact the outcome of infections: it can have both antiviral functions, and it can also stimulate virus replication. As might be expected for a cellular process that degrades cytosolic contents, autophagy can lead to clearance of viruses. Consequently a number of viral genomes encode proteins that inhibit autophagy, including herpes simplex virus. Degradation of viral proteins by autophagy can also provide peptides for presentation to the cellular adaptive immune system, further enhancing clearance.

Autophagy may also benefit virus replication. For example, non-lytic cell to cell spread of poliovirus depends on release of virus particles from autophagosomes, and autophagy of lipids provides metabolic energy for dengue virus replication.

When autophagy was first described in the 1950s, no one knew its significance. Nevertheless, a number of scientists, including Ohsumi, continued to study autophagy because they were curious. The unexpected result was the elucidation of a pathway that has substantial roles in a variety of human diseases.

The lesson is clear: let scientists pursue their curiosity. It’s fine to target specific research problems, like curing cancer or diabetes, but don’t ignore fundamental research on problems that don’t seem to be directly relevant to human diseases. I’m concerned (as are many others) that the US science establishment is moving away from fundamental research (whose benefits may not be apparent for a long time) to translational research.

How do we explain the trend from fundamental to translational research? I don’t have all the answers, but I think part of the problem is that the US Congress likes to spend taxpayer money on targeted problems, like Alzheimer’s disease. But tell them that you want to study a process in cells that looks interesting, but you are not sure what it means, and you can see their reluctance to support this type of work.

TWiV 409: A Nef is enough

Jeremy joins the TWiVeroids to tell the amazing story of how the function of the HIV-1 protein called Nef was discovered and found to promote infection by excluding the host protein SERINC from virus particles.

You can find TWiV #409 at microbe.tv/twiv, or listen below.

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poliovirusI cannot let September pass without noting that 34 years ago this month, I arrived at Columbia University to start my laboratory to do research on poliovirus (pictured). That virus is no longer the sole object of our attention – we are wrapping up some work on poliovirus and our attention has shifted elsewhere. But this is a good month to think about the status of the poliovirus eradication effort.

So far this year 26 cases of poliomyelitis have been recorded – 23 caused by wild type virus, and three caused by vaccine-derived virus. At the same time in 2015 there were 44 reported cases of polio – small progress, but, in the words of Bill Gates, the last one percent is the hardest.

One of the disappointments this year is Nigeria. It was on the verge of being polio-free for one year – the last case of type 1 poliovirus in Nigeria had been recorded in July of 2014. In August the government reported that 2 children developed polio in the Borno State. The genome sequence of the virus revealed that it had been circulating undetected in this region since 2011. Due to threats from militant extremists, it has not been possible for vaccination teams to properly cover this area, and surveillance for polioviruses has also been inefficient. The virus can circulate freely in a poorly immunized population, and as only 1% of infections lead to paralysis, cases of polio might have been missed.

The conclusion from this incident is that the declaration that poliovirus is no longer present in any region is only as good as the surveillance for the virus, which can never be perfect as all sources of infection cannot be covered.

Of the 26 cases of polio recorded so far in 2016, most have been in Afghanistan and Pakistan (9 and 14, respectively). It is quite clear that conflict has prevented vaccination teams from immunizing the population: in Pakistan, militants have attacked polio teams during vaccination campaigns.

Recently 5 of 27 sewage samples taken from different parts of the province of Balochistan in Pakistan have tested positive for poliovirus. Nucleotide sequence analysis revealed that the viruses originated in Afghanistan. The fact that such viruses are present in sewage means that there are still individuals without intestinal immunity to poliovirus in these regions. In response to this finding, a massive polio immunization campaign was planned for the end of September in Pakistan. This effort would involve 6000 teams to reach 2.4 million children. Apparently police will be deployed to protect immunization teams (source: ProMedMail).

The success of the polio eradication program so far has made it clear that if vaccines can be deployed, circulation of the virus can be curtailed. If immunization could proceed unfettered, I suspect the virus would be gone in five years. But can anyone predict whether it will be possible to curtail the violence in Pakistan, Afghanistan, and Nigeria that has limited polio vaccination efforts?

TWiV 408: Boston Quammens

Four years after filming ‘Threading the NEIDL’, Vincent and Alan return to the National Emerging Infectious Diseases Laboratory BSL4 facility at Boston University where they speak with science writer David Quammen.

You can find TWiV #408 at microbe.tv/twiv, or watch/listen here.

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by David Tuller, DrPH

David Tuller is academic coordinator of the concurrent masters degree program in public health and journalism at the University of California, Berkeley.

‘The PACE trial is a fraud.’ Ever since Virology Blog posted my 14,000-essord investigation of the PACE trial last October, I’ve wanted to write that sentence. (I should point out that Dr. Racaniello has already called the PACE trial a “sham,” and I’ve already referred to it as “doggie-poo.” I’m not sure that “fraud” is any worse. Whatever word you use, the trial stinks.)

Let me be clear: I don’t mean “fraud” in the legal sense—I’m not a lawyer–but in the sense that it’s a deceptive and morally bankrupt piece of research. The investigators made dramatic changes from the methodology they outlined in their protocol, which allowed them to report purported “results” that were much, much better than those they would have been able to claim under their originally planned methods. Then they reported only the better-looking “results,” with no sensitivity analyses to analyze the impact of the changes—the standard statistical approach in such circumstances.

This is simply not allowed in science. It means the reported benefits for cognitive behavior therapy and graded exercise therapy were largely illusory–an artifact of the huge shifts in outcome assessments the authors introduced mid-trial. (That’s putting aside all the other flaws, like juicing up responses with a mid-trial newsletter promoting the interventions under investigation, failing to obtain legitimate informed consent from the participants, etc.)

That PACE suffered from serious methodological deficiencies should have been obvious to anyone who read the studies. That includes the reviewers for The Lancet, which published the PACE results for “improvement” in 2011 after what editor Richard Horton has called “endless rounds of peer-review,” and the journal Psychological Medicine, which published results for “recovery” in 2013. Certainly the deficiencies should have been obvious to anyone who read the trenchant letters and commentaries that patients routinely published in response to the egregious errors committed by the PACE team. Even so, the entire U.K. medical, academic and public health establishments refused to acknowledge what was right before their eyes, finding it easier instead to brand patients as unstable, anti-science, and possibly dangerous.

Thanks to the efforts of the incredible Alem Matthees, a patient in Perth, Australia, the U.K.’s First-Tier Tribunal last month ordered the liberation of the PACE trial data he’d requested under a freedom-of-information request. (The brief he wrote for the April hearing, outlining the case against PACE in great detail, was a masterpiece.) Instead of appealing, Queen Mary University of London, the home institution of lead PACE investigator Peter White, made the right decision. On Friday, September 9, the university announced its intention to comply with the tribunal ruling, and sent the data file to Mr. Matthees. The university has a short window of time before it has to release the data publicly.

I’m guessing that QMUL forced the PACE team’s hand by refusing to allow an appeal of the tribunal decision. I doubt that Dr. White and his colleagues would ever have given up their data willingly, especially now that I’ve seen the actual results. Perhaps administrators had finally tired of the PACE shenanigans, recognized that the study was not worth defending, and understood that continuing to fight would further harm QMUL’s reputation. It must be clear to the university now that its own reputational interests diverge sharply from those of Dr. White and the PACE team. I predict that the split will become more apparent as the trial’s reputation and credibility crumble; I don’t expect QMUL spokespeople to be out there vigorously defending the unacceptable conduct of the PACE investigators.

Last weekend, several smart, savvy patients helped Mr. Matthees analyze the newly available data, in collaboration with two well-known academic statisticians, Bruce Levin from Columbia and Philip Stark from Berkeley.  Yesterday, Virology Blog published the group’s findings of the single-digit, non-statistically significant “recovery” rates the trial would have been able to report had the investigators adhered to the methods they outlined in the protocol. That’s a remarkable drop from the original Psychological Medicine paper, which claimed that 22 percent of those in the favored intervention groups achieved “recovery,” compared to seven percent for the non-therapy group.

Now it’s clear: The PACE authors themselves are the anti-science faction. They tortured their data and ended up producing sexier results. Then they claimed they couldn’t share their data because of alleged worries about patient confidentiality and sociopathic anti-PACE vigilantes. The court dismissed these arguments as baseless, in scathing terms. (It should be noted that their ethical concerns for patients did not extend to complying with a critical promise they made in their protocol—to tell prospective participants about “any possible conflicts of interest” in obtaining informed consent. Given this omission, they have no legitimate informed consent for any of their 641 participants and therefore should not be allowed to publish any of their data at all.)

The day before QMUL released the imprisoned data to Mr. Matthees, the PACE authors themselves posted a pre-emptive re-analysis of results for the two primary outcomes of physical function and fatigue, according to the protocol methods. In the Lancet paper, they had revised and weakened their own definition of what constituted “improvement.” With this revised definition, they could report in The Lancetthat approximately 60 % in the cognitive behavior and graded exercise therapy arms “improved” to a clinically significant degree on both fatigue and physical function.

The re-analysis the PACE authors posted last week sought to put the best possible face on the very poor data they were required to release. Yet patients examining the new numbers quickly noted that, under the more stringent definition of “improvement” outlined in the protocol, only about 20 percent in the two groups could be called “overall improvers.”. Solely by introducing a more relaxed definition of “improvement,” the PACE team—enabled by The Lancet’s negligence and an apparently inadequate “endless” review process–was able to triple the trial’s reported success rate..

So now it’s time to ask what happens to the papers already published. The editors have made their feelings clear. I have written multiple e-mails to Lancet editor Richard Horton since I first contacted him about my PACE investigation, almost a year before it ran. He never responded until September 9, the day QMUL liberated the PACE data. Given that the PACE authors’ own analysis showed that the new data showed significantly less impressive results than those published in The Lancet, I sent Dr. Horton a short e-mail asking when we could expect some sort of addendum or correction to the 2011 paper. He responded curtly: “Mr. Tuller–We have no such plans.”

The editors of Psychological Medicine are Kenneth Kendler of Virginia Commonwealth University and Robin Murray of Kings College London. After I wrote to the journal last December, pointing out the problems, I received the following from Dr. Murray, whose home base is KCL’s Department of Psychosis Studies: “Obviously the best way of addressing the truth or otherwise of the findings is to attempt to replicate them. I would therefore like to encourage you to initiate an attempted replication of the study. This would be the best way for you to contribute to the debate…Should you do this, then Psychological Medicine will be most interested in the findings either positive or negative.”

This was not an appropriate response. I told Dr. Murray it was “disgraceful,” given that the paper was so obviously flawed. This week, I wrote again to Dr. Murray and Dr. Kendler, asking if they now planned to deal with the paper’s problems, given the re-analysis by Matthees et al. In response, Dr. Murray suggested that I submit a re-analysis, based on the released data, and Psychological Medicine would be happy to consider it. “We would, of course, send it out to referees for scientific scrutiny in the same manner as we did for the original paper,” he wrote.

I explained that it was his and the journal’s responsibility to address the problems, whether or not anyone submitted a re-analysis. I also noted that I could not improve on the Matthees re-analysis, which completed rebutted the results reported in Psychological Medicine’s paper. I urged Dr. Murray to contact either Dr. Racaniello or Mr. Matthees to discuss republishing it, if he truly wished to contribute to the debate. Finally, I noted that the peer-reviewers for the original paper had okayed a study in which participants could be disabled and recovered simultaneously, so I wasn’t sure if the journal’s assessment process could be trusted.

(By the way, Kings College London, where Dr. Murray is based, is also the home institution of PACE investigator Trudie Chalder as well as Simon Wessely, a close colleague of the PACE authors and president of the Royal College of Psychiatrists*. That could explain Dr. Murray’s inability or reluctance to acknowledge that the “recovery” paper his journal peer-reviewed and published is meaningless.)

Earlier today, the PACE authors posted a blog on The BMJ site, their latest effort to salvage their damaged reputations. They make no mention of their massive research errors and focus only on their supposed fears that releasing even anonymous data will frighten away future research participants. They have provided no evidence to back up this unfounded claim, and the tribunal flatly rejected it. They also state that only researchers who present  “pre-specified” analysis plans should be able to obtain trial data. This is laughable, since Dr. White and his colleagues abandoned their own pre-specified analyses in favor of analyses they decided they preferred much later on, long after the trial started.

They have continued to refer to their reported analyses, deceptively, as “pre-specified,” even though these methods were revised mid-trial. The following point has been stated many times before, but bears repeating: In an open label trial like PACE, researchers are likely to know very well what the outcome trends are before they review any actual data. So the PACE team’s claim that the changes they made were “pre-specified” because they were made before reviewing outcome data is specious. I have tried to ask them about this issue multiple times, and have never received an answer.

Dr. White, his colleagues, and their defenders don’t yet seem to grasp that the intellectual construct they invented and came to believe in—the PACE paradigm or the PACE enterprise or the PACE cult, have your pick—is in a state of collapse. They are used to saying whatever they want about patients—Internet Abuse! Knife-wielding! Death threats!!–and having it be believed. In responding to legitimate concerns and questions, they have covered up their abuse of the scientific process by providing non-answers, evasions and misrepresentations—the academic publishing equivalent of “the dog ate my homework.” Amazingly, journal editors, health officials, reporters and others have accepted these non-responsive responses as reasonable and sufficient. I do not.

Now their work is finally being scrutinized the way it should have been by peer reviewers before this damaging research was ever published in the first place. The fallout is not going to be pretty. If nothing else, they have provided a great gift to academia with their $8 million** disaster—for years to come, graduate students in the U.S., the U.K. and elsewhere will be dissecting PACE as a classic case study of bad research and mass delusion.

*Correction: The original version of the post mistakenly called the organization the Royal Society of Psychiatrists.

**Correction: The original version of the post stated that PACE cost $8 million, not $6.4 million. In fact, PACE cost five million pounds, so the cost in dollars depends on the exchange rate used. The $8 million figure is based on the exchange rate from last October, when Virology Blog published my PACE investigation. But the pound has fallen since the Brexit vote in June, so the cost in dollars at the current exchange rate is lower.