On episode #288 of the science show This Week in Virology, the Twivsters discuss how reverse transcriptase encoded in the human genome might produce DNA copies of RNA viruses in infected cells.

You can find TWiV #288 at www.twiv.tv.

Vincent Racaniello

Photo by Chris Suspect

Ten years ago this month I wrote the first post at virology blog, entitled Are viruses living? Thanks to EE Giorgi for pointing out the ten year anniversary, and also for publishing an interview with me at her blog, Chimeras.

Here is how this blog got started: in June 2004 the second edition of our virology textbook, Principles of Virology, had just been published. While the textbook had so far done well, its audience was limited, and I wanted to find ways to better spread information about viruses. At the time I had a hosting account that I used to publish a website for our cub scout pack, and while visiting the administration page, I noticed an option to install blogging software. The idea then came to me to start blogging about viruses, so I looked for a good domain name. All of the virology names were taken except for virology.ws, so I bought that, and set up the blog. An artist made the logo, using an image of poliovirus bound to its cellular receptor; this structure was the product of a collaboration between my lab and those of Jim Hogle and Alasdair Steven. Then I wrote my first post. Discussing whether or not viruses are living seemed like a good introductory topic, and I used some ideas that had been published in our textbook.

To my surprise, after a few months the post began to attract comments, and to this day it remains one of the most commented posts on virology blog. My views on whether or not viruses are living have certainly evolved; a more accurate summary of my thoughts on this subject would be The virus and the virion.

I like to think that blogging has been a pathway to all of my other efforts to communicate information about viruses. Blogging brought me into the world of social media, leading me to start accounts on Twitter, Facebook, and Google Plus. Four years after virology blog, I started my first podcast, This Week in Virology, which is approaching one million downloads each year (we now have four science shows, including This Week in Parasitism, This Week in Microbiology, and Urban Agriculture). I began teaching an undergraduate virology course at Columbia University in 2010, and I have used video recordings of my lectures to teach virology at iTunes University and Coursera. I have had wonderful opportunities to interview virologists at colleges and scientific meetings; some of these can be found at my YouTube channel. I believe that I have shown that scientists can effectively communicate their field to the general public, and I hope I have inspired some of my colleagues to emulate my efforts.

For the first 20 years of my career I taught virology to roughly 200 students every year, for a total reach of four thousand people. My blogging, podcasting, and online teaching now reach millions in over 170 countries. It all started with a blog.

I have been lucky to reach so many people, in different ways, with information about viruses. But I still love blogging, and I will be writing about viruses here as long as I my brain and body permit. My sincere thanks to everyone who has visited virology blog and has been part of this engaged and excited community.

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vesicular stomatitis virusMany years ago a claim was made that cells infected with respiratory syncytial virus contained infectious DNA copies of the viral genome. When this paper was published, retroviral reverse transcriptase had been discovered, which explained how DNA copies of retroviral RNA genomes were made in infected cells. Although the respiratory syncytial viral genome is RNA, it does not encode a reverse transcriptase, and how a DNA copy of this genome could be made in infected cells was unknown. The observation was initially met with great fanfare, and was suggested to account for why some RNA virus infections persist, and even to explain autoimmune diseases. However the findings were never duplicated, and the authors fell into scientific obscurity. Now it appears that they might not have been entirely wrong.

It is now quite clear that DNA copies of viral RNA are made in infected cells. A DNA copy of lymphocytic choriomeningitis virus (LCMV) RNA has been detected in mouse cells, and this DNA can be integrated into cellular DNA. LCMV DNA was only detected in cells that produce a retrovirus, implicating reverse transcriptase in this process. DNAs of various RNA viruses, including bornaviruses and filoviruses, have been found integrated into the genome of many animals. An explanation for the genesis of these DNA copies is provided by studies of cells infected with vesicular stomatitis virus.

Vesicular stomatitis virus (pictured) is an enveloped virus with a genome of (-) strand RNA. In infected cells the (-) strand RNA is copied by the viral RNA polymerase to form 5 mRNAs which encode the viral proteins. Infection of various human cell lines resulted in the production of DNA complementary to VSV RNA. The DNAs are single stranded and appear to be produced from the viral mRNAs, not the viral genome.

How might VSV DNA be produced in virus infected cells? About 20% of the human genome consists of a mobile genetic element called LINE-1 (Long Interspersed Nuclear Element). These elements, also called retrotransposons, encode a reverse transcriptase. This enzyme converts LINE-1 mRNA into DNA, which can then integrate elsewhere in the cell genome – hence the name mobile genetic element. LINE-1 encoded reverse transcriptase can also produce DNA copies of cellular mRNAs and other mobile elements that do not encode their own reverse transcriptase. Thanks to LINEs, our genomes are littered with mobile genetic elements.

To determine if LINE-1 could make VSV in infected cells, the authors took advantage of their observation that not all human cells produce VSV DNA after infection. Introduction of LINE-1 DNA into one of these cell lines enabled it to produce DNA copies of VSV mRNAs. This result shows that LINE-1 can make VSV DNA in infected cells. Whether LINE-1 actually accomplishes this process in unmodified, VSV infected cells remains to be proven.

The authors also show that viral DNA is produced in cells infected with two other RNA-containing viruses, echovirus and respiratory syncytial virus. The latter of course is the virus used to make the claim nearly 40 years ago that viral DNA is present in infected cells. Whether that viral DNA is infectious, however, is not known. No complete copies of the VSV RNA genome have been observed in infected cells, only copies of viral mRNAs.

It seems likely that in cells infected with RNA viruses, reverse transcriptase encoded by LINE-1 could produce DNA copies of viral RNAs. After entering the nucleus these viral DNAs could become integrated into cellular DNA. If a germline cell were infected, the integrated viral DNA would be passed on to subsequent generations, explaining the presence of DNA copies of various RNA viruses in animal genomes.

A key question is whether the production of DNA copies of viral RNAs is an accident, or benefits the virus or host. There is yet no answer to this question. The authors suggest that DNA copies of viral RNA might contribute to innate immune sensing of viral infection. VSV replication is not altered by the presence or absence of viral DNA, but there could be a role for viral DNA during infection of a host animal.

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Back in 1974, before it was possible to determine the sequence of a viral genome, before we knew much about the origin of viruses and their ability to move genes from organism to organism, Lewis Thomas wrote the following incredibly prescient words in The Lives of a Cell:

The viruses, instead of being single-minded agents of disease and death, now begin to look more like mobile genes. We live in a dancing matrix of viruses; they dart, rather like bees, from organism to organism, from plant to insect to mammal to me and back again, and into the sea, tugging along pieces of this genome, strings of genes from that, transplanting grafts of DNA, passing around heredity as though at a great party. They may be a mechanism for keeping new, mutant kinds of DNA in the widest circulation among us. If this is true, the odd virus disease, on which we must focus so much of our attention in medicine, may be looked on as an accident, something dropped.

When Thomas wrote these words we knew that bacteriophages could move pieces of DNA from bacterium to bacterium, but we had no idea of the global scale of this movement. We did not know that most viruses could carry genes from cell to cell, nor did we appreciate that viruses could be beneficial. I am amazed by the accuracy of his words written at a time when we knew so little.

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On episode #287 of the science show This Week in Virology, Matt Frieman updates the TWiV team on MERS-coronavirus, and joins in a discussion of whether we should further regulate research on potentially pandemic pathogens.

You can find TWiV #287 at www.twiv.tv.

SofosbuvirThe Federal Drug Administration of the US approves new drugs solely on the basis of safety and effectiveness, with no value assessment. Pharmaceutical companies may set their drug prices based mainly on what the market will bear. Nevertheless, the announcement that Gilead Sciences would price their just-approved, anti-hepatitis C virus (HCV) drug sofosbuvir (Solvaldi) at $84,000 for 12 weeks of treatment was met with considerable complaints.

Solvaldi is a member of a class of antiviral drugs called nucleoside analogs. They act as chain terminators and inhibit viral RNA synthesis. When the viral RNA polymerase is copying the viral RNA, to enable the production of more virus particles, it normally uses the pool of ATP, UTP, GTP, and CTP to produce more RNA. When Solvaldi is incorporated into the growing RNA chain by the viral enzyme, no additional triphosphates can be added, because the drug contains a fluorine atom at the 2′-position of the ribose. Its presence inhibits addition of the next nucleoside by the polymerase to the 3′-OH. Viral RNA synthesis therefore stops, and production of virus particles is inhibited. For more information on chain terminators, see my virology lecture on antivirals.

Gilead believes that the price of the drug is fully justified: a spokesperson said “We’re just looking at what we think was a fair price for the value that we’re bringing into the health care system and to the patients.”

It could cost up to $300,000 to treat patients with chronic HCV infection using less effective and more difficult to tolerate regimens. The potential benefit of a cure for patients with liver disease is clear, as the virus is the main reason that nearly 17,000 Americans are waiting for a liver transplant. The need for a well-tolerated, effective regimen is equally critical for people infected with HIV and HCV, because having both infections accelerates liver damage.

Despite these arguments, the high price will be a significant barrier for many, especially those in limited and fixed-budget programs such as Medicare and Medicaid. A panel of experts in San Francisco estimated that switching HCV infected Californians to Sovaldi would raise annual drug expenditures in the state by at least $18 billion.

Gilead has agreed to help U.S. patients pay for Sovaldi if they cannot afford it, or help patients obtain drug coverage. The company also plans to charge substantially less for a course of treatment in India ($2000 for the 12 week course), Pakistan, Egypt ($990 for the 12 week course), and China, where most people infected with HCV live. These deals have prompted some to ask if the US is being forced to subsidize the cost of the drug worldwide. I personally do not object to helping other countries solve their HCV problem.

What is a fair price for a drug that can eliminate HCV infection? Gilead paid more than $11 billion in 2011 to acquire the company that developed Sovaldi, and it is reasonable for them to recoup that investment. Andrew Hill of the Department of Pharmacology and Therapeutics at Liverpool University estimates the manufacturing cost of a 12 week course of treatment with this drug to be $150 to $250 per person. The answer to our fair price question must lie somewhere between these extremes.

There are parallels between Sovaldi (and other new anti-HCV drugs in the pipeline) and the initially expensive antivirals that were introduced ~20 years ago to treat HIV. Anti-retrovirals revolutionized the treatment of a chronic, lethal infection that is major global health problem, and the anti-HCV drugs could have the same effect. But there are also important differences: based on the number of infected individuals, HCV is a much larger public health threat than HIV. Furthermore, the new HCV antivirals can eliminate the virus completely, whereas anti-HIV drugs only suppress virus replication, so they must be taken (and paid for) for life.

At some point in the future competition among pharmaceutical companies and manufacturers of generic drugs should make it possible to treat everyone infected with HCV with affordable, curative antivirals. If the cost and efficiency of diagnosis and drug delivery keeps pace, it might be possible to eradicate HCV. That accomplishment might well be priceless.

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On episode #286 of the science show This Week in Virology, Vincent and Alan meet up with Julie and Paul at the General Meeting of the American Society for Microbiology in Boston, to talk about their work on the pathogenesis of poliovirus and measles virus.

You can find TWiV #286 at www.twiv.tv.

NP evolutionEvolution proceeds by selection of mutants that arise by error-prone duplication of nucleic acid genomes. It is believed that mutations that are selected in a gene are dependent on those that have preceded them, an effect known as epistasis. Analysis of a sequence of changes in the influenza virus nucleoprotein provides clear evidence that stability explains the epistasis observed during evolution of a protein.

Evolutionary biologist John Maynard Smith used an analogy with a word game to explain how epistasis constrains the evolution of a protein. In this game, single letter changes are made to a four letter word to convert it to another valid word:

WORD->WORE->GORE->GONE->GENE

Although all the intermediates are valid words, the sequence of changes is important. For example, the G in GENE, if introduced into WORD would produce GORD which is not a word. D must be changed to E before W is changed to G. In a similar way mutations in a gene are likely to depend on the changes that have previously taken place.

Whether similar constraints affect protein evolution has been studied with the nucleoprotein (NP) of influenza virus. Between 1968 and 2007, 39 mutations appeared in the NP RNA of influenza virus H3N2. Because sequences of this viral RNA are available each year, it was possible to deduce the order in which these changes appeared in the viral genome (illustrated; figure credit). Plasmids encoding 39 different NP proteins were then constructed which represent viral NP sequences present from 1968 through 2007. All of the NP proteins were found to support similar levels of viral RNA synthesis.

The 39 mutations were then introduced singly into the NP RNA, and RNA synthesis was measured. Three of the altered proteins had large decreases in activity. Their presence also substantially reduced the growth of infectious viruses. However when these NP changes were combined with the amino acid changes that preceded it during evolution, replication was normal. The three NP changes that reduce viral RNA synthesis and replication also decrease the thermal stability of the protein.

These findings show that, from 1968-2007, three amino acid changes were fixed in the influenza virus NP protein whose deleterious effects on protein stability were compensated by previously accumulated changes in the protein. The three amino acids are located in a part of the protein that harbors sequences recognized by T cells. These changes likely allow the virus to escape the host immune response.

Protein stability clearly mediates the epistasis observed in the influenza virus NP protein. It will be important to determine which other protein properties determine the sequence of mutations that are fixed in a viral genome. Influenza viruses are ideal for this work because sequences of all of the viral RNAs are determined for multiple isolates on an annual basis. Studies of what regulates epistasis for other RNA and DNA viruses are also needed to provide an understanding of the constraints of viral evolution.

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Ian Lipkin, Columbia University, New York, and Lyle Petersen, Centers for Disease Control and Prevention, Fort Collins, Colorado, discuss recently emerged pathogens, and how to prepare should their range expand. When asked if MERS-coronavirus would cause the next pandemic, Ian Lipkin responded ‘I don’t have a crystal ball’.

Recorded at the Annual Meeting of the American Society for Microbiology, Boston, MA on 19 May 2014.

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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.

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