Virus World

Drug hunters are finding that ancient virus-like artifacts in the human genome could offer new avenues to treat neurodegeneration, cancer, autoimmunity and even aging with antibodies, vaccines and antiretroviral agents. A new wave of therapies is taking aim at virus-like elements spread throughout the human genome. These genomic parasites, which have accumulated over the course of human evolution, are embedded in the vast expanse of DNA sequences that dwell in the spaces between our genes — what scientists call the ‘dark genome’. Although they were largely ignored in the past, growing scientific evidence indicates that these normally dormant elements — including retroviruses, transposons and other repetitive sequences — can reactivate, triggering inflammation, cancer and other disease-related cellular damage.

Human endogenous retroviruses (HERVs) are ancestral viral relics that constitute nearly 8% of the human genome. Although normally silenced, the most recently integrated provirus HERV-K (HML-2) can be reactivated in certain cancers. Here, we report pathological expression of HML-2 in malignant gliomas in both cerebrospinal fluid and tumor tissue that was associated with a cancer stem cell phenotype and poor outcomes. Using single-cell RNA-Seq, we identified glioblastoma cellular populations with elevated HML-2 transcripts in neural progenitor–like cells (NPC-like) that drive cellular plasticity. Using CRISPR interference, we demonstrate that HML-2 critically maintained glioblastoma stemness and tumorigenesis in both glioblastoma neurospheres and intracranial orthotopic murine models.

Additionally, we demonstrate that HML-2 critically regulated embryonic stem cell programs in NPC-derived astroglia and altered their 3D cellular morphology by activating the nuclear transcription factor OCT4, which binds to an HML-2–specific long-terminal repeat (LTR5Hs). Moreover, we discovered that some glioblastoma cells formed immature retroviral virions, and inhibiting HML-2 expression with antiretroviral drugs reduced reverse transcriptase activity in the extracellular compartment, tumor viability, and pluripotency. Our results suggest that HML-2 fundamentally contributes to the glioblastoma stem cell niche. Because persistence of glioblastoma stem cells is considered responsible for treatment resistance and recurrence, HML-2 may serve as a unique therapeutic target.

Published in JCI (July 3, 2023):

https://doi.org/10.1172/JCI167929 

Researchers assess the impact of SARS-CoV-2 infection on endogenous retroviruses of the LTR69 subfamily. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which is otherwise known as the coronavirus disease 2019 (COVID-19), continues to pose significant worldwide health and economic effects. There remains an urgent need to better comprehend the intricate relationship between SARS-CoV-2, infected host cells, and the pathophysiology of the disease. Recent research suggests that transposable elements (TEs) play a crucial role in the host's response to COVID-19 and the development of illness. In a recent study posted to the bioRxiv* preprint server, researchers assess the impact of SARS-CoV-2 infection on endogenous retroviruses of the LTR69 subfamily.

About the study

In the present study, researchers examine the effect of SARS-CoV-2 on the expression profiles of TEs in virus-exposed or -infected cells. To this end, the team investigated publicly available poly(A)-enriched messenger ribonucleic acid (mRNA)-seq data from cell lines and COVID-19 patients to determine the impact of COVID-19 on TE activity. Initially, data collected from SARS-CoV-2-infected and uninfected Calu-3 cells were employed to detect TEs with differential expression. To examine the enhancer activity of long terminal repeat (LTR)-103 and LTR69 in the absence and presence of SARS-CoV-2, the team analyzed publicly available ChIP-seq data associated with Histone H3 Lysine 27 acetylation (H3K27ac) in A549-angiotensin-converting enzyme 2 (ACE2) cells. To determine whether any of the SARS-CoV-2-activated LTR69 repeats elicit regulatory influences, their potential enhancer activities were examined. Five representative candidates were inserted into enhancer reporter vectors. To explore the mechanisms that may be involved in LTR69-Dup69 activation following SARS-CoV-2 infection, the viral nucleotide sequence was examined for binding sites associated with transcription factors known to be active in infected cells.

Results

Solo-LTRs found in two human endogenous retroviruses (HERV) subfamilies, LTR103_Mam and LTR69, were considerably up-regulated by SARS-CoV-2 infection. Similarly, A549-ACE2 lung cells infected with SARS-CoV-2 and bronchoalveolar lavage fluid (BALF) obtained from non-deceased and deceased SARS-CoV-2 patients exhibited higher LTR69 expression. In contrast, there was no remarkable enhancement in LTR69 expression in SARS-CoV-2-infected Calu-3 cells or in peripheral blood mononuclear cells (PBMCs) of surviving COVID-19 patients. The transcription start site (TSS) profile plot over LTR69 loci revealed enrichment of H3K27Ac marks in infected cells as compared to uninfected cells. No considerable enrichment of enhancer marks was observed on the LTR103_Mam loci. Subsequent investigations were primarily focused on individual LTR69 loci, for which the MACS2 peak calling method detected a minimum of one significant H3K27Ac peak. There were 12 distinct peaks associated with H3K27Ac on 15 LTR69 loci following SARS-CoV-2 infection.  LTR12C_GBP2 boosted the expression of Gaussia luciferase relative to the vector control that lacked an LTR repeat. Dup69 had a comparable boosting impact, while the remaining LTR69 elements exhibited no remarkable modulatory impact or even lowered reporter gene expression. Dup69 resides in an intron of protein tyrosine phosphatase receptor type N2 (PTPRN2), which is approximately 500 nucleotides upstream of a long non-coding RNA gene called ENSG00000289418, according to an examination of the respective gene locus. PTPRN2 encodes a tyrosine phosphatase receptor that is a significant autoantigen in type 1 diabetes. In A549-ACE2 and Calu-3 cells, lncRNA expression rose by a factor of 25.2 and 3.6, respectively.

In addition, PTPRN2 expression increased by an average of 4.1 times in A549-ACE2 cells following SARS-CoV-2 infection, whereas PTPRN2 mRNA was undetectable in Calu-3 cells. Interestingly, a previous study discovered the up-regulation of PTPRN2 in whole blood samples of COVID-19 patients. Collectively, these results indicate that SARS-CoV-2 infection activates an LTR69 repeat that responded to interferon regulatory transcription factor (IRF)-3 and p65/RelA functions as an enhancer element and may influence the expression of nearby genes. Several probable binding sites for nuclear factor kappa B (NF-κB) subunits, signal transducer and activator of transcription 1 (STAT1), and IRF3 were identified. Furthermore, p65/RelA and an active mutant of IRF3, but not STAT1, may augment the LTR69-mediated increase in reporter gene expression. Consistent with the activation of IRF3 and NF-κB upon innate sensing, synthetic double-stranded RNA analog polyI:C dramatically boosted LTR69_Dup69 activity.

Conclusions

The study findings showed the differential expression and activation of distinct mobile genetic elements in response to SARS-CoV-2 infection. Specifically, the team identified and validated the infection-induced upregulation of the LTR69 subfamily of endogenous retroviruses. LTR69-Dup69 was also found to possess enhancer activity and exhibit sensitivity to the transcription factors IRF3 and p65/RelA. LTR69 is considered a TE that is activated in SARS-CoV-2-infected cells and can regulate host gene expression, thus contributing to the outcome of COVID-19. However, additional research is required to confirm this finding.

Research cited available in bioRxiv  (March 21, 2023):

 https://doi.org/10.1101/2023.03.21.533610 

Using a next generation sequencing analysis to examine human endogenous retrovirus (HERV) integration sites, researchers from Kumamoto University, the National Institute of Genetics (Japan), and the University of Michigan (U.S.) have discovered that these ancient retroviruses can undergo retrotransposition (DNA sequence insertion with RNA mediation) into iPS cells. The team believes that their discovery places a spotlight on a possible risk that HERVs pose when using iPS cells in regenerative medicine. The study of ancient retroviruses embedded in our genome requires knowledge about our coexistence with viral threats throughout history. We know that HERVs occupy approximately 8% of the human genome and obtain mutations and deletions over long periods. HERVs are also expressed in early embryos and play several physiological roles in human development. For example, HERV-W and HERV-FRD Env proteins are important for placental formation, and HERV-K is thought to protect host cells from exogenous retrovirus infection. However, uncontrollable HERV-K expression is also thought to be associated with various diseases, including various cancers and neurological diseases, but the details of this association is not well known in humans.

Since no one has yet discovered replication competent HERVs in our genome, it is thought that they are from an extinct (fossil) virus. In their current work, the research team from Japan and the US discovered that HERV-K is expressed in SOX2-expressing cells, such as those in early embryos, cancer stem cells and iPS cells. They also found that some HERV-K are newly integrated into the host genome in the absence of Env, the viral envelope glycoprotein. This integration was dependent on reverse transcriptase, integrase and protease, thus the researchers hypothesized that the HERV-K embedded in our genome is actually not from a fossil virus, but moves on the genome through the synthesis of proviral DNA reverse transcription. Interestingly, when the researchers compared the HERV-K integration sites between iPS and fibroblast cells from the same donor, they found new HERV-K integration sites in iPS cells.

However, the new integration sites were rarely preserved and disappeared during long-term culturing. HERV-K is likely to be randomly integrated into genome, thus the possibility remains that HERV-K retrotransposed-cells predominantly survive depending on their integration site. The movement of HERV-K on the genome might cause cancer and neurological diseases by altering the gene expression profile. The researchers believe that the risk of HERV-K transposition is low in iPS cells but suggest that monitoring HERV-K integration sites should be seriously considered to improve the safety of regenerative medicine using iPS cells. This research was published online on 14 April 2022 in the Journal of Virology.

Research cited published in J. Virology (April 14, 2022):

https://doi.org/10.1128/jvi.00356-22 

Researchers discovered a potential link between "endogenous retroviruses" present in the human genome and the development of neurodegenerative diseases.

Summary: Researchers discovered a potential link between “endogenous retroviruses” present in the human genome and the development of neurodegenerative diseases. Their study found that these ancient viral remnants might influence the spread of protein aggregates commonly associated with certain dementias. While these retroviruses don’t trigger neurodegeneration, they may exacerbate the disease process. This discovery offers new potential therapeutic avenues, such as suppressing gene expression or neutralizing viral proteins.

Key Facts:

1. “Endogenous retroviruses”, ancient viral remnants in human DNA, might contribute to neurodegenerative disease progression.
2. HERV-W and HERV-K, two such retroviruses, were found to aid in the transport of tau aggregates, protein clumps associated with diseases like Alzheimer’s.
3. Potential therapeutic approaches include suppressing the retroviruses or neutralizing their proteins, possibly with antibodies.

Source: DZNE

Genetic remnants of viruses that are naturally present in the human genome could affect the development of neurodegenerative diseases. Researchers at DZNE come to this conclusion on the basis of studies on cell cultures. They report on this in the journal Nature Communications. In their view, such “endogenous retroviruses” could contribute to the spread of aberrant protein aggregates – hallmarks of certain dementias – in the brain. Thus, these viral relicts would be potential targets for therapies. It has been suspected for some time that viral infections contribute to the genesis and development of neurodegenerative diseases. Laboratory studies by DZNE scientists now suggest a mechanism that, although related to viruses, does not require infection by external pathogens. According to this study, the culprits would be “endogenous retroviruses” that are naturally present in the human genome. “During evolution, genes from numerous viruses have accumulated in our DNA. Most of these gene sequences are mutated and normally muted,” explained Ina Vorberg, research group leader at DZNE and a professor at the University of Bonn. “However, there is evidence that endogenous retroviruses are activated under certain conditions and contribute to cancer and neurodegenerative diseases. Indeed, proteins or other gene products derived from such retroviruses are found in the blood or tissue of patients.”

Experiments with Tau Aggregates 

Vorberg followed this trail together with colleagues from Bonn and Munich. Using cell cultures, the researchers simulated the situation in which human cells produce certain proteins from the envelope of endogenous retroviruses. Specifically, this involved HERV-W and HERV K – both viruses are present in the human genome but are usually dormant. However, studies indicate that HERV-W is activated in multiple sclerosis and HERV-K in the neurological disease “amyotrophic lateral sclerosis” (ALS) and in frontotemporal dementia (FTD). Now, Vorberg’s team found that the viral proteins facilitate the transport of so-called tau aggregates from cell to cell. “Tau aggregates” are tiny protein clumps that occur in the brains of people affected by certain neurodegenerative diseases – these include Alzheimer’s disease and FTD. “Certainly, conditions in the brain are much more complex than our cellular model system can replicate them. Nevertheless, our experiments show that endogenous retroviruses can influence the spread of tau aggregates between cells,” Vorberg said. “Endogenous retroviruses would thus not be triggers of neurodegeneration, but could fuel the disease process once it is already underway.”

Viral Transport Mediators

The current research and earlier studies by Vorberg’s team suggest that viral proteins serve as transport mediators for tau aggregates because they insert into the cell membrane and into the membrane of so-called extracellular vesicles: These are small fat bubbles that are naturally secreted by cells. “For the transport of tau aggregates from cell to cell, we see two pathways in particular. Transfer between cells that are in direct contact, and transport within vesicles that act as cargo capsules, so to speak, and pass from one cell to another to eventually merge with it,” Vorberg explained. “In both scenarios, membranes have to fuse. Proteins from the envelope of viruses can promote this process. That’s because many viruses are adapted to fuse with host cells. “This happens by means of special proteins that viruses carry on their surfaces. If precisely these proteins are incorporated into the cell membrane and the membrane of extracellular vesicles, it is understandable that the tau aggregates then spread more easily.”

Starting Points for Therapy

In the course of the natural aging process, the regulation of genes can change – originally “dormant” endogenous retroviruses could be “awakened” as a result. Indeed, the symptoms of most neurodegenerative diseases do not manifest until older age. This raises two conceivable approaches to therapy. “On the one hand, one could try to specifically suppress gene expression, that is, to inactivate the endogenous retroviruses again. That would get to the root of the problem,” Vorberg said. “But you could also start elsewhere and try to neutralize the viral proteins – for example, with antibodies.”

Searching for Antibodies

In the opinion of the researchers, it is likely that dementia patients with tau aggregates carry increased amounts of such antibodies. If it were possible to isolate these and reproduce them using biotechnological methods, it might be possible to develop a passive vaccine. Thus, in collaboration with DZNE colleagues in Berlin and Bonn, Vorberg’s team aims to specifically search for such antibodies in patients. In addition, the scientists are considering antiviral drugs. In cell culture, they have already found that such agents can actually stop the spread of protein aggregates. “This is another approach we intend to pursue,” said Vorberg.

Research cited published in Nature (Aug. 18, 2023):

https://doi.org/10.1038/s41467-023-40632-z 

A discovery gives experts new ideas for developing vaccines to treat or even prevent cancer.

Liu and colleagues uncover the ways in which derepression of human endogenous retrovirus triggers cellular senescence and tissue aging; the findings provide fresh insights into therapeutic strategies for alleviating aging.

Summary

DNA "echoes" of viruses that infected our ancestors millions of years ago could help the immune system to identify and kill cancer cells, according to new research from Crick scientists. The new study, published in Genome Research, looked at endogenous retroviruses, fragments of DNA in the human genome that were left behind by viruses that infected our ancestors. 

Over millions of years, our ancestors were infected with countless viruses and their DNA now makes up more of our genome than human genes. Approximately 8 percent of the human genome is made up of retroviral DNA, while known genes only make up 1-2 percent.

"This viral DNA typically lies dormant, as it is either non-functional or our bodies have evolved to suppress it," explains Crick Group Leader Dr. George Kassiotis, who led the study. "However, when a cell becomes cancerous, some of these suppression mechanisms can fail and this ancient viral DNA can be reactivated. In this study, we looked for viral DNA that is reactivated by cancer and produces products that the immune system can see. The hope is that if we can train the immune system to spot these, we can selectively target cancer cells."

Genes are pieces of DNA that contain instructions to produce proteins, which perform important functions in the cell or the body. These instructions are transcribed into RNA "messenger" molecules before the proteins are produced. However, this transcription process can be influenced by DNA outside the gene, including endogenous retroviruses. To study the effects of endogenous retroviruses on transcription, the team looked at patient samples from 31 different cancer types using a technology called "RNASeq' that can read short, random fragments of RNA. However, as each "read" only delivers a small part of the sequence in an unknown order, it takes up to 50 million "reads" per sample to build a complete picture of transcriptional activity.

"Piecing together a full transcriptional profile is a monumental task," says George. "It's been likened to trying to read a magazine that's been shredded into millions of pieces, when you don't even know what the magazine was supposed to be about or what language it's in. All you have is random fragments, so to piece them together you need to see where they overlap."  The team used RNA sequencing data from 768 patient samples, with almost 40 billion reads to piece together. Even using sophisticated algorithms, a desktop computer would need to run constantly for 24 years to stitch this data together. To speed things up significantly, the researchers turned to the Crick's specialist Scientific Computing team. Running the analysis on the in-house High Performance Computing cluster, they got results far quicker.

From the full transcriptional data, the team developed a catalog of over 130,000 different RNA transcripts produced by endogenous retroviruses, more than half of which had not been previously discovered. Of these, there were roughly 6,000 transcripts that were specifically found in cancer samples and not healthy tissue. Many of these were specific to the type of cancer, with most individual cancers expressing high levels of a few hundred transcripts. 

"We focused on melanoma-specific transcripts and applied an algorithm to predict which could code for material that is visible to the immune system," explains George. "We found 14 candidate transcripts from 8 different regions of the genome that could produce unique cancer antigens. Together with the Proteomics team at the Crick and Nicola Ternette's lab in Oxford, we inspected mass spectrometry data to see which of these antigens were present in real patient samples. This narrowed it down to nine unique peptides that could be visible to the immune system. We hope this approach could form the basis of future cancer therapies, if we can vaccinate the immune system to recognize and attackcancer cells presenting these peptides."

George is one of the scientific co-founders of Ervaxx, a spin-out company from the Crick that aims to take this science into the clinic to help patients. Ervaxx is expanding on these foundational insights to create a pipeline of off-the-shelf, cancer-specific vaccines and other immunotherapies, including a lead product candidate focused on treating melanoma patients.

Published on Genome Research on September 19, 2019: 

https://doi.org/10.1101/gr.248922.119