ABOVE: LURKING VIRUSES: Particles of the human endogenous retrovirus HERV-K (colored in red) budding off a human teratocarcinoma cell (colored in green) in vitro.

© DR KLAUS BOLLER, SCIENCE PHOTO LIBRARY

In February 2005, an HIV patient in an unusually difficult situation walked into a neuroimmunology clinic at Johns Hopkins University, where he was seen by the specialist Avindra Nath. The patient had not only immune deficiency resulting from HIV infection, but amyotrophic lateral sclerosis (ALS) as well, and the neurodegenerative disease was causing his condition to deteriorate rapidly. For several months, the patient had noticed his hands and feet becoming increasingly sore and weak, making tasks such as eating with utensils or opening a window all but impossible. When Nath saw him, the 29-year-old had difficulty climbing stairs and couldn’t get up from a seated position on the floor without assistance.

The patient was initially reluctant to take a combination of antiretroviral drugs to treat his HIV due to his rapidly progressing ALS symptoms, Nath recalls. “He said he would likely die from ALS before he died from HIV infection, so why take the [antiretrovirals]?” But the doctor had read some clinical reports about other HIV-ALS patients’ ALS symptoms improving after a course of antiretrovirals, and Nath suggested he take them. The patient agreed and, like the other patients Nath had read about previously, rapidly began to feel better. When Nath saw him a year later, the patient no longer complained of weakness or muscle twitching, and his gait was normal. “This patient’s symptoms [had] totally reverted,” Nath tells The Scientist. “It’s one thing seeing it in the literature, but another thing to witness it yourself.”

The experience piqued Nath’s interest in the potential role of viruses in ALS. Digging further into published research on the disease, he also found a handful of blood analyses indicating the activity of reverse transcriptase, an enzyme that converts the RNA genome of a retrovirus into DNA. But when scientists had looked for infectious retroviruses in those blood samples, they hadn’t found any. Nath reasoned that if the culprit wasn’t an exogenous virus, it could be one that’s already present in the human genome.

Hervs through the ages Over the course of evolution, several groups of ancient viruses colonized our ancestors’ genomes, leaving thousands of fragments of viral code in modern-day human DNA. The bulk of HERVs integrated during primate evolution. Subsequent mutations in these sequences have rendered older insertions nonfunctional, but some of the younger and more intact sequences from HERVs have been linked to disease. PDF See full infographic: WEB the scientist staff Around 8 percent of our genetic code stems from HERVs, the bulk of which integrated during primate evolution. HERV-K viruses colonized the genomes of ancient primates as early as 55 million years ago (mya). Many of the youngest and most preserved elements, such as those in the HERV-K (HML-2) group, can produce viral proteins and have been linked to ALS.

The HERV-W group, which invaded the genome starting around 25 mya, was first detected in multiple sclerosis patients and named MS-associated retrovirus (MSRV) for its connection to the myelin-degenerating condition.

HERV-Fc, the youngest member of the HERV-F viruses, integrated into the genome more than 20 mya and has also been linked with multiple sclerosis.

HERV-L elements have been detected in all placental mammals, and are thought to have integrated between 100 million and 150 mya. They represent the oldest HERVs in the human genome, are not known to produce any proteins, and so far have not been linked to disease.

Like many other animal species, humans carry viral remnants in their genomes, left behind from the integration of retrovirus sequences into the germline DNA of our ancestors over the course of millions of years. Today, these human endogenous retroviruses (HERVs) exist as 450,000 fragments of biological code, representing 39 major viral groups, broken up and scattered throughout the genome. Nath asked his colleague Jeffrey Rothstein, now the director of Johns Hopkins’s Robert Packard Center for ALS Research, for some samples of postmortem brain tissue of ALS patients, and began to search them for RNA transcripts of HERV sequences.

The work yielded one match: HERV-K, the youngest group of viral insertions in humans. The transcripts were specifically associated with ALS, Nath and his colleagues found; they were not present in the brains of healthy individuals who died in accidents or in the brains of Parkinson’s patients.1 Since then, Nath, who now heads the Section of Infections of the Nervous System at the National Institutes of Health in Bethesda, Maryland, and others have been steadily accumulating evidence that these viral sequences are expressed in a subset—about 30 percent—of ALS patients.

The vast majority of the HERV protein-coding sequences scattered across the genome have over time accumulated many mutations that render them inactive. That HERVs can cause damage to their hosts in modern times has long been dismissed as a fringe idea. Over the past three decades, however, research has implicated several of the younger, more intact HERV insertions in a range of diseases, including ALS, multiple sclerosis (MS), cancer, and schizophrenia.

“Though they’ve mostly been ignored by the medical research community for quite a long time, they can actually have a large effect on the human body in more than one disease setting,” says Molly Hammell, a geneticist who studies HERVs at Cold Spring Harbor Laboratory in New York. Since this idea has come to light, some researchers have launched clinical studies to evaluate HERVs’ roles in diseases.

Skeptics are still wary of the evidence that HERVs are involved in pathogenesis at all. Although there is a correlation, few studies address a possible causative role. “We know that they’re expressed,” says virologist George Kassiotis of the Francis Crick Institute in London, “but whether that expression really causes or really contributes todisease, that remains to be proven.”

Rise of the Phoenix proteins

In 1967, University College London virologist Robin Weiss noticed a viral envelope protein emerging not just from chicken cells that he had infected with a Rous sarcoma virus, which is known to cause cancer in poultry, but also in control cells that had not been exposed to the virus. Together with geneticist Jim Payne, the former director of the now-defunct Houghton Poultry Research Station in the UK, he conducted classical Mendelian cross-breeding experiments that pointed to the protein’s heritability, identifying the virus as avian leukosis sarcoma virus, an endogenous retrovirus in chicken genomes.2 “These things are inherited, just like Mendel’s peas,” says Weiss, noting that Peter Vogt of the Scripps Research Institute came to this conclusion around the same time.

Initially, journals were reluctant to publish the result, says Weiss, who recently retired. At the time, reverse transcriptase—which made the concept of a retrovirus integrating into the genome plausible—wasn’t even known to exist yet. After the enzyme’s discovery in 1970, the idea that viral protein-coding genes could live in host DNA gained traction as additional endogenous retroviruses were found in mice and other animals. Many of these viruses proved to be active, giving rise to infectious viral particles capable of inserting new pieces of DNA into the animals’ genomes—and causing diseases, including cancer and autoimmune disorders.

Though they’ve mostly been ignored by the medical research community for quite a long time, they can actually have a large effect on the human body in more than one disease setting. —Molly Hammell, Cold Spring Harbor Laboratory

Since discovery of endogenous retroviruses in humans in the 1980s, there has been a contentious debate as to whether HERVs behave like endogenous retroviruses in animals. While scientists have established that most HERVs are dormant, some researchers believe that the youngest insertions of a HERV-K virus called the HML-2 virus, which invaded our lineage as recently as 670,000 years ago, can produce retroviral particles that infect other cells. In 2006, a group of French researchers succeeded in reconstructing the ancestral HML-2 virus in the lab based on a consensus sequence from all its snippets in the human genome. The resulting “Phoenix” element, as they called it, was capable of infecting other mammalian cells in culture.3 And in 2015, a team of Stanford University biologists captured electron micrographs of HML-2 viral particles—containing proteins and nucleic acids—budding off cultured human blastocysts,4 a phenomenon that researchers now think occurs naturally during development.

To date, however, there is no in vivo evidence that such particles can infect other cells or result in new HERV-K insertions in the human genome. In fact, Ralf Tönjes of Germany’s Paul Ehrlich Institute discovered in 1999 that two nearly intact insertions of HERV-K viruses have inactivating mutations, “leading to a nonfunctional retrovirus,” he explains.5

Even if HERVs can’t assemble into infectious particles, they can still have a significant influence on human biology. (See infographic.) For instance, many HERV sequences can be transcribed and even translated in human cells, with hundreds of HERV genes likely having the capacity to yield viral proteins, says Cornell University geneticist Cedric Feschotte. Some of these proteins may have taken on beneficial roles in humans. For instance, HERV-encoded syncytin 1 is produced in early development, and is thought by some researchers to function in the formation of the placenta. Moreover, humans appear to have co-opted many viral promoters to help drive the expression of our own genes.

While Kassiotis doesn’t believe that HERV particles trigger disease, in rare cases, the mere presence of HERVs in the genome can cause problems, he says. For example, when normally silenced viral promoters lie upstream of an oncogene, and happen to be activated during cancer, they can sometimes contribute to accelerating the disease, explains Kassiotis.

But other researchers are steadily accumulating evidence that our viral hitchhikers may be involved in much more than that—perhaps playing a direct role in the pathology of other ailments, in particular, ALS and MS.

HERVs and neurological disease

In 1985, Hervé Perron, then a doctoral student at the University Hospital of Grenoble, France, came across a Nature paper that postulated a retroviral cause of MS. He decided to look for evidence of retroviruses in tissue samples from MS patients as part of his thesis. Sure enough, he found fragments of viral proteins in cultures grown from cells inside donors’ cerebrospinal fluid.6 In further experiments, he and others found the same viral particles in macrophages isolated from the blood of MS patients, but not in cells taken from control samples. Together with University College London’s Jeremy Garson, who had developed a technique to detect the RNA of unknown retroviruses, and other colleagues, Perron characterized the viral particle for the first time in 1997 and showed that the viral genetic code closely matched an endogenous family of retroviruses—the first hint that the element was endogenous.7 Perron called it the MS-associated retrovirus (MSRV), and it was later found to belong to a larger group named HERV-W.

Parsing the HERV-disease link Current research suggests that viral hitchhikers in human DNA may play roles in cancer, inflammation, and neurodegenerative disorders. The mechanisms that underpin these connections between human endogenous retroviruses (HERVs) and disease are just beginning to emerge. Transcription of viral RNA can signal the presence of foreign DNA in cells, triggering defensive immune reactions. Scientists have also proposed that synthesis of the HERV envelope protein—which once enclosed the viral capsid of its retroviral ancestors—exerts pathogenic effects. In other contexts, such as certain cancers, researchers think that the disease state activates HERVs, rather than the other way around. PDF See full infographic: WEB © nicolle rager fuller (1) Activation of viral promoters: Ancient retroviral infections have left viral promoters throughout the human genome. Although our bodies have coopted many of them to drive the expression of our own genes, a lot of those promoters are kept silenced through epigenetic repression. Reactivation of these elements can result in abnormal expression of nearby oncogenes or tumor-suppressor genes. (2) Expression of viral genes: Under some circumstances, such as cancer, many regions of the genome that are normally silenced can awaken. This can activate the transcription of HERVs, causing viral RNA to accumulate in the cytoplasm. According to the “viral mimicry” theory, these molecules alert cellular RNA-sensing pathways to the viral material, triggering an immune response. (3) Translation of viral proteins: Some viral RNAs are translated into proteins, which can be secreted and travel to other cells. It’s unclear what effects these proteins have, but some researchers hypothesize they activate surface receptors and ultimately initiate immune reactions.

Just as Weiss’s results had experienced 16 years earlier, these findings were met with a chilly reception. “People had the dogma that in humans, there are very few of such elements in the genome, and they are all junk DNA that is completely inactivated by mutations,” Perron recalls.

But a series of studies has uncovered more associations between MS and HERV-W sequences as well as those of another viral group known as HERV-F. In the brains of deceased MS patients, for instance, researchers discovered fragments of viral proteins within the lesions that form in the central nervous system due to a loss of axonal myelin sheaths. And virologist Antonina Dolei of the University of Sassari in Italy and other researchers have uncovered a strong correlation between the blood levels of HERV-W RNA and the severity of MS symptoms: levels increase around the onset of the disease and as symptoms get worse, but decrease during remissions and under effective therapies. “The virus is present in the right place at the right times,” she says.

By now, researchers “have a very well-argued case for an association,” says MS researcher Tove Christensen of Aarhus University in Denmark, “but we haven’t yet come to the point of showing that it’s actually causative.” Weiss is particularly skeptical of the idea of a cause-effect relationship. “I would say that maybe it’s the autoimmune activation that’s activating the virus, and not the virus that’s causing the autoimmune disease,” he says, though he adds that “the virus in turn may exacerbate the disease.”

The idea that HERVs might play a role in MS was supported by the detection of a HERV-K virus in pathological tissue in ALS, as shown by Nath and others. But a lot more work is needed to establish causation, Nath cautions. Understanding the mechanisms at play “is key not only for establishing its role in ALS,” he says, “but also for developing targeted therapy.”

Mechanistic insights into HERVs

In figuring out the possible mechanisms underlying HERV-disease connections, Nath says he thinks one of the most pressing questions is whether they’re capable of forming infectious retroviral particles. An infectious retrovirus would help explain the progressive nature of ALS symptoms, from motor extremities to the central nervous system. “If it’s not HERV-K being transmitted [from cell to cell], then there’s got to be some other factor that’s being transmitted to activate it,” he says.

So far, there’s no evidence for this type of viral behavior in ALS or in MS. Rather, researchers have focused on HERV envelope proteins, because the homologous proteins in HIV are known to have neurotoxic effects. In 2015, Nath’s team created a transgenic mouse line in which the animals produced the envelope protein of HERV-K. The mice subsequently developed ALS-like symptoms, including spasticity, weakness, and muscle atrophy.8

How this occurred is a mystery. Nath hypothesizes that the HERV-K envelope protein can cause dysfunction in the nucleolus, the membraneless structure within the nucleus that hosts ribosome biogenesis. “And [this] messes up the entire protein synthesis machinery in the cell itself . . . that eventually leads to toxicity,” Nath speculates. (See infographic.)

MS researchers similarly suspect that the HERV-W envelope protein, which activates the immune-linked toll-like receptor 4 on microglia and macrophage cells, could drive the disease’s pathology. In fact, Perron is so convinced of its role that in 2006 he and others launched the Geneva-based biotech company GeNeuro to develop novel therapies based on antibodies that specifically target HERV envelope proteins. In a mouse model, Perron’s team found in 2013 that injecting the animals with the HERV-W envelope protein contributed to the development of motor defects and inflammation of the nervous system.9 While not providing evidence of causation, the experiments showed that the protein “is capable of initiating an autoimmune process that results in inflammation and tissue destruction in the central nervous system,” writes Robert Glanzman, the company’s chief medical officer, in an email.

In subsequent experiments in murine and human cell cultures conducted by German researchers, the protein appeared to stifle the maturation process of oligodendroglial precursor cells, which would normally help repair degraded myelin sheaths. And in other research from the same group that has yet to be published, the envelope protein also inhibited microglia cells from scavenging myelin debris—another process critical to myelin repair—and stimulated them to secrete proinflammatory cytokines, Glanzman adds.

HERV proteins in neurodegenerative disease The discovery of viral proteins in the eroded brains of MS and ALS patients has prompted researchers to investigate the role of HERVs in these diseases. Although this research is becoming more widespread, the mechanisms are still unclear and remain hypothetical. Multiple sclerosis The HERV-W envelope protein binds toll-like receptor 4 on microglia, triggering the cells to secrete proinflammatory cytokines (1). At the same time, the protein also inhibits these cells from scavenging myelin debris (2), a mechanism important for rebuilding myelin sheaths that are damaged in MS, and prevents oligodendrocyte precursor cells (OPCs)—which normally help remyelinate damaged axons—from maturing (3). Combined, these two pathways create an inflammatory environment that contributes to the development of lesions in the brain, while also impairing the ability of local cells to repair the damage. Researchers haven’t yet discovered what triggers the production of HERV-W in the first place. PDF See full infographic: WEB © nicolle rager fuller ALS Experiments in mice have shown that activation of the most recently integrated HERV in the human genome, known as HERV-K, in specific regions of the nervous system causes motor neuron deterioration. This could explain the neurodegeneration seen in ALS, although it is still unclear exactly how HERV-K is involved. Researchers speculate that the envelope protein of HERV-K causes disruption of the machinery in the nucleolus responsible for producing ribosomes, and this in turn results in cell death (1) .This process is thought to spread from cell to cell—in accordance with the progressive deterioration seen in ALS—through factors that stimulate the production of the viral envelope protein, through the secretion of the protein (2), or possibly through the spread of HERV-K itself, though there is no evidence that the endogenous virus can behave in this way. PDF See full infographic: WEB © nicolle rager fuller

The envelope protein is not the only way that HERVs can mess with the immune system. Even in the protein’s absence, excess HERV RNA and other HERV-derived nucleic acids can trigger the body’s immune response, alerting cellular sensors that detect cytoplasmic DNA, explains Feschotte, who receives funding from GeNeuro. And “when these sensors get overwhelmed, it triggers autoimmune reactions,” he says. “That’s very well characterized.”

A key question that remains unanswered is why these disease processes would occur in only some people. The vast majority of HERV sequences are present in everyone. But people can carry variable numbers of particular HERV fragments, and a few HERV snippets are found only in some human genomes—both factors that can in theory contribute to individual susceptibility to HERV-driven pathologies.

In addition, work by Christensen’s team at Aarhus University suggests that the host’s individual genetic makeup may come into play. In 2011, the researchers demonstrated a preponderance of certain single-nucleotide polymorphisms around a particular locus of HERV-Fc1, a member of the HERV-F group, on the X chromosome in MS patients, compared with healthy individuals.10 And other research suggests that environmental factors—such as Epstein-Barr virus, a common infection thought to play a role in MS—can activate the expression of HERV-W. Hammell and colleagues have found that the aggregation of the TDP-43 protein, an RNA- and DNA-binding protein known to accumulate in the vast majority of ALS patients, induces the expression of an endogenous retrovirus in Drosophila.11

Rush to the clinic

Although the mechanisms remain murky, researchers are already eyeing HERVs as possible therapeutic targets for several diseases. Some researchers are now conducting trials with ALS and MS patients testing the antiretrovirals that have proven effective in preventing HIV’s reproduction and entry into the genome.

The ALS patient Nath treated in 2005 inspired him to launch a small pilot study in which five individuals with both HIV and ALS each received a different combination of up to 16 antiretrovirals. Similar to his earlier observations, Nath noted that three patients experienced a reversal of motor symptoms associated with ALS, and the other two experienced a notable slowing of their ALS progression.12 However, Nath says, “it is quite possible that the antiretroviral had an effect on the ALS only because of downregulation of HIV.” He is now planning another study to investigate whether a combination retroviral therapy can affect the levels of HERV-K in the blood and cerebrospinal fluid of 20 ALS patients without HIV.

Other diseases associated with HERVs

There is copious research on the involvement of HERVs in ALS, MS, and cancer. But researchers have also identified tenuous links between the endogenous viruses and a handful of other conditions.

Disorder Evidence

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), a neurological disorder characterized by progressive

weakness and sensory disturbances in the legs and arms HERV-W envelope protein is found in the serum of some CIDP patients, and viral RNA is present in peripheral blood mono-nuclear cells. 1

The protein is also found in peripheral nerve lesions. In Schwann cells and immune cells of blood serum, the protein induces the secretion of proinflammatory factors. Schizophrenia and bipolar disorder HERV-W RNA is present in the cerebrospinal fluid and brains of some deceased schizophrenia patients. 3

One particular HERV-K insertion serves as a genetic enhancer for the schizophrenia-linked gene PRODH. 4

Transcription of the HERV-W envelope (env) gene is elevated in bipolar disorder and schizophrenia compared with healthy controls.5 Type 1 diabetes HERV-W envelope protein is detected in the blood serum of some type 1 diabetes patients, and its corresponding RNA is found in peripheral blood mononuclear cells. 6

Transgenic mice expressing the HERV-W env gene display hyperglycemia and decreased levels of insulin. Addiction One specific variant of HERV-K, which is in close proximity to a gene involved in dopaminergic activity in the brain, is more common in drug addicts than in controls.7

Julian Gold, a virologist at Queen Mary University of London, is interested in using antiretrovirals to treat MS, but a 2014 pilot study of 20 MS patients proved disappointing: the antiretroviral drug raltegravir didn’t help reduce the number of new lesions, the most visible sign of myelin loss, that appeared in MRI scans of the patients’ brains.13 Gold thinks this is because he used a single drug, and not a combination therapy that is typically effective in HIV. In March, he concluded a separate trial in 40 ALS patients, this time using a combination therapy known as Triumeq. “The trial clearly shows that it’s safe and well tolerated,” says Gold, who is also director of Albion Centre, a public health-care facility in Sydney, Australia. The efficacy results are not yet published, but Gold says the initial data make him optimistic about this approach in ameliorating symptoms in some MS patients.

But Glanzman is skeptical, as he posits another reason for Gold’s disappointing MS trial—the use of an antiretroviral drug to target what Glanzman thinks is an inactive virus. If the HERVs that have been linked to these diseases are incapable of replicating, “trying to approach them with an antiviral treatment is not going to be effective,” says Glanzman. Instead, he and his GeNeuro colleagues are developing an envelope-targeting antibody called GnbAC1 that binds to a surface subunit of the envelope protein to block its interactions with toll-like receptor 4 carried by microglia and macrophage cells and has been recently shown to promote myelination in rat nerve cell cultures. Last March, the company concluded a Phase 2b trial with 270 MS patients in Europe that showed preliminary evidence of the drug’s effectiveness in treating the disease. In results that were presented at a conference on MS research in Berlin last fall, patients who got the antibody at the highest dose experienced 31 percent less shrinkage of cortical tissue, a hallmark of MS, compared with patients treated with placebo for six months. Treated patients also experienced a 63 percent reduction in the formation of “T1 black holes,” the brain lesions associated with the most severe damage to the central nervous system.

It’s early days, Glanzman says, but so far the results look promising. “Now we have the clinical data to support that if you block the protein, you have beneficial effects.” The company is now following some of these patients to see if the changes observed in their brains translate to a clinical benefit; results are expected later this year.

The company recently made an agreement to work together with Nath on treatments for ALS with a similar antibody approach, and is starting to work on other diseases that have been linked to HERVs. (See table.) For instance, unpublished research from GeNeuro suggests that HERV-derived proteins are toxic to the pancreases of mice, Glanzman says. After a study finding that 70 of 100 patients with type 1 diabetes had evidence of a HERV-W envelope protein in the pancreas,14 they initiated a one-year trial to see if an antibody against the HERV-W envelope protein is safe in type 1 diabetes patients. The company also plans to test HERV-targeting therapies to treat a condition known as chronic inflammatory demyelinating polyneuropathy as well as inflammatory psychosis, which encompasses some forms of schizophrenia and bipolar disorder.

Other researchers have begun cancer trials that aim to improve immunotherapy treatments by epigenetically boosting the expression of HERVs to coax the immune system into killing the tumor cells. For example, George Washington University’s Katherine Chiappinelli recently showed that administering compounds that remove the methylation around HERV genes triggered the immune system to attack tumor cells in a mouse model of ovarian cancer. “The cancer cell thinks it has a virus, because it sees the viral RNA,” she explains, triggering an immune response that helped the mice survive much longer than controls.

Despite the growing link between HERVs and disease, and the positive results from early-stage trials, the idea that the viruses of the human genome can become destructive is still not widely accepted, Nath notes. “The way most of these things work is some people think it’s great science, some people are going to be skeptics, so it takes some time before other labs can reproduce your results,” he says. “Publishing one or two papers, it gets people excited about the field, but it takes much, much longer for people to accept the concept.”

Katarina Zimmer is a freelance science writer living in New York City.

References

Correction (January 7): The original version of the infographic in this story mistakenly depicted baboons instead of bonobos. In addition, a previous version of this story erroneously mischaracterized the diabetes trial conducted by GeNeuro as a four-year trial designed to test whether the HERV-W antibody will improve symptoms. Rather, it is a one-year trial intended to evaluate safety in patients with diabetes type 1. The Scientist regrets the errors.