The TWiV team reveal the origin of the poxvirus membrane, and how a retrovirus drove the development of the placenta of a lizard.
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Show notes at microbe.tv/twiv
The protein syncytin, which is essential for formation of the placenta, originally came to the genome of our ancestors, and those of other mammals, via a retrovirus infection. Placental structures have also developed in non-mammalian vertebrates. The Mabuya lizard (pictured: image credit), which emerged 25 million years ago, has a placenta very much like those in mammals, and its development was likely driven by capture of a retroviral gene.
There aren’t enough human organs to meet the needs for transplantation, so we have turned to pigs. Unfortunately pig cells contain porcine endogenous retroviruses, PERVS, which could infect the transplant recipient, leading to tumor formation. But why worry? Just use CRISPR to purge the PERVs.
The genomes of many species on Earth are littered with endogenous retroviruses. These are DNA copies of retroviral genomes from previous infections that are integrated into germ line DNA and passed from parent to offspring. About 8% of the human genome consists of ERVs. The pig genome is no different – it contains PERVs (an acronym made to play with). The genome of an immortalized pig cell line called PK15 contains 62 PERVs. Human cells become infected with porcine retroviruses when they are co-cultured with PK15 cells.
The presence of PERVS is an obvious problem for using pig organs for transplantation into humans – a process called xenotransplantation. The retroviruses produced by pig cells might infect human cells, leading to problems such as immunosuppression and tumor formation. No PERV has ever been shown to be transmitted to a human, but the possibility remains, especially with the transplantation of increasing numbers of pig organs into humans.
The development of CRISPR/Cas9 gene editing technology made it possible to remove PERVs from pigs, potentially easing the fears of xenotransplantation. This technology was first used to remove all 62 copies of PERVS from the PK15 cell line. But having PERV-free pig cells doesn’t help humans in need of pig organs – for that you need pigs.
To make pigs without PERVs, CRISPR/Cas9 was used to remove the PERVs from primary (that is, not immortal) pig cells in culture. Next, the nuclei of these PERV-less cells was used to replace the nucleus of a pig egg cell. After implantation into a female, these cells gave rise to piglets lacking PERVs.
In theory such PERV-less piglets can be used to supply organs for human transplantation, eliminating the worrying about infecting humans with pig retroviruses. But first we have to make sure that the PERV-free pigs, and their organs, are healthy. The more we study ERVs, the more we learn that they supply important functions for the host. For example, the protein syncytin, needed to form the placenta, is a retroviral gene, and the regulatory sequences of interferon genes come from retroviruses. There are likely to be many more examples of essential functions provided by ERVs. It would not be a good idea to have transplanted pig organs fail because they lack an essential PERV!
Paul Bieniasz joins the TWiV team to talk about the co-option, millions of years ago, of an endogenous retrovirus envelope protein by hominid ancestors for host defense against viral infection.
You can find TWiV #439 at microbe.tv/twiv, or listen. below.
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Did you know that the evolution of ancient retroviruses, millions of years ago, can be traced by studying their genomes in the chromosomes of contemporary animals? Ted Diehl and Welkin Johnson join the TWiV team to tell us how they did it with mammals. All without a single wet experiment! They also join in the discussion about virus dispersal by hand dryers.
You can find TWiV #386 at microbe.tv/twiv, or listen below.
On episode #320 of the science show This Week in Virology, Vincent speaks with John Coffin about his career studying retroviruses, including working with Howard Temin, endogenous retroviruses, XMRV, chronic fatigue syndrome and prostate cancer, HIV/AIDS, and his interest in growing cranberries.
You can find TWiV #320 at www.microbe.tv/twiv.
On episode #279 of the science show This Week in Virology, Vincent, Alan, and Kathy reveal how a retrovirus in the human genome keeps embryonic stem cells in a pluripotent state, from where they can differentiate into all cells of the body.
You can find TWiV #279 at www.microbe.tv/twiv.
About eight percent of human DNA is viral – remnants of ancestral infections with retroviruses. These endogenous retroviral sequences do not produce infectious viruses, and most are considered to be junk DNA. But some of them provide important functions. The protein called syncytin, which is essential for formation of the placenta, originally came to the genome of our ancestors, and those of other mammals, via a retrovirus infection. Another amazing role of endogenous retroviruses is that they regulate the stem cells that are the precursors of all the cells in our body.
The genetic material of retroviruses is RNA, but during infection it is converted to DNA which then integrates into the chromosome of the cell. If the infected cell happens to be a germ cell, then the viral DNA, now called called an endogenous retrovirus, becomes a permanent part of the animal and its offspring. One of our endogenous retroviruses, called HERV-H, infected human ancestors about 25 million years ago. HERV-H has been found to be important for the properties of human embryonic stem cells.
Embryonic stem cells (ES cells), which are derived from the inner cell mass of a blastocyst (which forms 4-5 days after implantation), are pluripotent – they can differentiate into every cell type in the human body. Being pluripotent means expressing a very different set of genes compared with somatic cells – the cells of skin, muscle, organs, to name a few. The genes that are expressed in ES cells are controlled by a small number of key proteins that regulate mRNA synthesis. If these proteins – just four – are produced in a differentiated cell, it will turn into an ES cell – an induced, pluripotent embryonic stem cell, or iPSC. This observation garnered Shinya Yamanaka the Nobel Prize in 2012.
The first clue that HERV-H might be important for the pluripotency of ES cells was the finding that this DNA is preferentially expressed in human ES cells (the figure [credit] shows the expression of HERV-H in ES and two other cell types). When the levels of HERV-H RNAs are reduced (by RNA interference) in ES cells, the morphology of the cells changes – they become fibroblast-like, a sign of differentiation. In contrast, when fibroblasts are reprogrammed to become iPSCs, the levels of HERV-H RNAs rise. These findings suggest that HERV-H is essential for keeping ES cells pluripotent, and for making somatic cells pluripotent.
The HERV-H DNA in our genome is flanked by viral sequences called long terminal repeats, or LTRs. These provide initiation sites for the synthesis of viral mRNAs. In human ES cells the HERV-H LTRs appear to be enhancing the transcription of nearby human genes that are important for maintaing pluripotency. In an interesting twist, the HERV-H viral RNA is important for this activity: it appears to bind proteins involved in the regulation of mRNAs important for pluripotency. This observation explains why reducing HERV-H viral RNA leads to loss of pluripotency.
The HERV-H RNA made in human ES cells is not translated into protein because it contains many mutations that have accumulated over the past 25 million years. Therefore HERV-H is a long, non-coding RNA (lncRNA), a relatively recently discovered class of regulatory RNAs. There are about 35,000 lncRNAs in human cells that are involved in controlling a variety of processes such as splicing, translation and epigenetic modifications. Now we know that endogenous retroviruses can also produce lncRNAs.
Without endogenous retroviruses, humans might not be recognizable as the Homo sapiens that today walk the Earth. They might also be egg-layers – but the eggs would be white. Viruses don’t just make us sick.
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.
Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease or Lou Gehrig’s disease, is a fatal disorder of unknown etiology. The disease involves degeneration of motor neurons, leading to paralysis, respiratory failure, and death within five years. A viral etiology for ALS has been suggested but never proven. Retroviruses have been considered because they cause motor neuron disease in mice, and HIV-1 and HTLV-1 cause ALS-like symptoms in humans. Sera from some ALS patients have been shown to contain elevated levels of reverse transcriptase, an enzyme found in retrovirus particles. RNAs encoding this enzyme have now been found in the brains of ALS patients, and their origin appears to be the human endogenous retrovirus HERV-K.
Reverse transcriptase is a retroviral enzyme that makes a DNA copy of the viral RNA genome. It is present in the virion, a property that can be used to assay for reverse transcriptase activity. In one study of ALS patients, serum or cerebrospinal fluid is first centrifuged at low speeds to remove cells and debris. The cell-free extracts are then subjected to high speed centrifugation to pellet* viral particles. These are disrupted and an RNA template, a primer, and the precursors of DNA are added, and the mixture is incubated. A PCR reaction is then done; if any DNA has been made by reverse transcription, it will be amplified. In one study, 50% of ALS patients’ sera contained reverse transcriptase activity, at levels comparable to those found in AIDS patients. RT activity was detected in 7% of control sera.
What is the source of RT activity in sera from ALS patients? It does not appear to be from any known human exogenous retrovirus, including HIV-1, HIV-2, HTLV-1, HTLV-2, or HTLV-3. A recent study explored the possibility that the enzyme activity was due to the presence of the gammaretrovirus XMRV. However, the authors did not detect XMRV in DNA or RNA extracted from peripheral blood mononuclear cells of 20 ALS patients.
Human endogenous retroviruses are integrated into the human genome, but do not produce infectious virions and therefore are only transmitted through the germline. Although HERVs are highly mutated, some produce mRNA, viral proteins, and particles. At least 8% of the human genome consists of HERVs. One HERV, called HERV-K, may have integrated into the germline after the divergence of humans and chimpanzees.
The expression of HERV-K in ALS patients was examined in two ways: by PCR amplification of RNA extracted from brain tissues, using primers that amplify the pol gene (encoding RT), and by immunohistochemistry, using antibodies directed against the HERV-K reverse transcriptase protein. All samples used in the study were from patients who died of ALS (n=28), chronic systemic disease (n=10), accidental death (n=10) or Parkinson’s disease (n=12). HERV-K pol mRNA was detected in tissues from ALS patients and those who died of chronic systemic disease, but not from the accidental death or Parkinson’s patients. Levels of pol mRNA were highest in brains of ALS patients. Staining of brain sections with antibody to HERV-K pol revealed that the RT protein is produced only in neurons.
These observations raise a number of immediate questions. Is the RT activity detected in the serum of ALS patients from HERV-K? Why don’t all ALS patients have RT activity in their serum, and why is there such a wide range of HERV-K pol mRNA in the brain? Are the higher levels of HERV-K pol and RT observed in ALS patients a cause or a consequence of the disease? Answers to these questions will require more extensive epidemiological studies, and experiments to determine how HERV-K expression might lead to disease.
*pellet is jargon: the material at the bottom of a tube produced by centrifugation is a pellet, but scientists often use it as a verb, to indicate the separation of insoluble material by centrifugation.
[I used a photo of Lou Gehrig in a Columbia University uniform not only because I work at Columbia, but for several years I lived across the street from Gehrig’s birthplace, on 94th street and First Avenue in New York City. On a building across the street from my apartment was a plaque with the inscription ‘Lou Gehrig was born here’. I’d see it every day on my way to the lab. Years later, when I returned to look for the plaque, it had been stolen.]
McCormick AL, Brown RH Jr, Cudkowicz ME, Al-Chalabi A, & Garson JA (2008). Quantification of reverse transcriptase in ALS and elimination of a novel retroviral candidate. Neurology, 70 (4), 278-83 PMID: 18209202
Douville R, Liu J, Rothstein J, & Nath A (2011). Identification of active loci of a human endogenous retrovirus in neurons of patients with amyotrophic lateral sclerosis. Annals of neurology, 69 (1), 141-51 PMID: 21280084