Unraveling the Mystery of HHV-6: An interview with Dr. Bhupesh K. Prusty

The virus's interactions with HERVs, its role in ME/CFS, why blood banks should screen for HHV-6, and the future of HHV-6 research

Bhupesh K. Prusty, Ph.D., is Senior Researcher/ Principal Investigator and Senior Lab Manager on the Faculty of Medicine’s Institute for Virology and Immunobiology at Julius-Maximilians-Universität Würzburg (in Bavaria, Germany). For more than a decade, he has studied Human Herpes Virus 6 (HHV-6), publishing more than 20 research reports on the virus. Currently, the focus of his research involves studying the molecular biology of HHV-6/HHV-7 encoded small non-coding RNAs and their potential role in human diseases. In December 2022, we discussed how he became interested in HHV-6, its interactions with human endogenous retroviruses (HERVs), his work on ME/CFS, why blood banks should screen for HHV-6 and his thoughts about future research.

Neenyah Ostrom: How did you first become interested in studying HHV-6? What first drew your attention to this virus?

Bhupesh K. Prusty: My fascination towards this virus began during my post-doctoral days in the laboratory of Prof. Harald zur Hausen at German Cancer Research Center (DKFZ), in Heidelberg, Germany. During those days, we were interested in understanding infectious triggers behind childhood leukemia. Harald had the hypothesis that children who grew up in a very protected environment away from common childhood infections frequently develop leukemia. Hence, based on epidemiological data, Harald hypothesized that common infections during childhood help to prevent more complex diseases like leukemia. My task was to analyze some of the very common viral infectious triggers, including herpesviruses and human endogenous retroviruses (HERVs) in tumor cells.

One day I was analyzing some of the tumor cell lines infected with HHV-6 under an electron microscope and I observed some very interesting structures. I saw that fully matured HHV-6 viral particles, inside and outside cells, were coated with flower-like small vesicular structures. (1) Later on, by using gold-labelled secondary antibodies, we found out that these small vesicles contain HERV envelope proteins that coat the HHV-6 viral particles.

This was a life-changing observation for me. I became fascinated with HHV-6. Subsequently, I learned how these viruses integrate into human telomeres [a region of repetitive DNA sequences at the end of a chromosome that protect them from becoming shortened during each cell division; telomeres maintain the lifespan of chromosomes] and how they are inherited from parents to offspring in a Mendelian fashion. All these factors made me curious to learn more about these viruses.

My interest in this virus has been greatly influenced by some of the best science philosophers with whom I have worked. Harald was one of them. Later I met Bob [Robert K. Naviaux, M.D., Ph.D., Professor of Genetics at UCSD School of Medicine], who often asks me questions about HHV-6, the answers of which extend beyond our imagination. One day Bob asked me why we get infected with HHV-6 very early during our childhood and mostly from shared foods from our mothers. Why does the virus stay in our body for the rest of the life? There must be some benefit to us from these latent viruses. Questions like this keep me thinking about HHV-6.

And how did you first become involved in studying ME/CFS?  

My research has always been focused on translational aspects of science [translational science connects different areas of research to each other]. During my initial days as a semi-independent group leader at University of Würzburg [Germany], I had the opportunity to work with some very curious young minds. One day we were discussing HHV-6, and the difficulties of growing the virus in cell culture and finding cells where the virus frequently reactivates from latency. During these discussions, we decided that we must do something to make our life easier in the lab while studying virus reactivation. And we came up with an elegant solution. We thought of taking HHV-6 BAC (bacterial artificial chromosome) with a fluorophore tagged in it  [a fluorophore is a chemical that glows when stimulated] and prepare virus particles that will carry these fluorophore expression cassettes. Later, we targeted these viruses into some cells where the virus became latent after infection. As the virus genome integrated into the sub-telomeric region of the human chromosome, the fluorophore was no longer expressed during viral latency. Once the virus was reactivated, the fluorophore was expressed and the cells with the reactivated virus glowed under a fluorescent microscope.

So now we could see and differentiate the cells with a reactivated HHV-6. (2)  Because of our curiosity about mitochondria, we created some HeLa cells carrying latent HHV-6A. These HeLa cells had genetically altered mitochondria, expressing another fluorophore in it. We observed that mitochondria often fragments [breaks up] as soon as the HHV-6A reactivates.

Mitochondrial fragmentation is a key phenomenon that is involved in the development and progression of many human neurodegenerative disorders. So, we thought of taking our research to a disease model. We came across ME/CFS, as the literature was already flooded with articles showing mitochondrial dysfunction and HHV-6 infection in patients. Therefore, we picked ME/CFS as our disease model to continue our research work.

Until this time, I was carrying out hobby HHV-6 research with limited support from HHV-6 Foundation, USA. But now I had to face the reality of science, which is that funding is a huge barrier to overcome. There was no funding for HHV-6 research and moreover, ME/CFS was taboo for funding agencies. During this time, I spoke to Dr. Zaher Nahale from Solve ME/CFS Initiative and he encouraged me to apply for a pilot grant. I applied for the Ramsay Research Award and succeeded in getting $30,000. With this funding, we started our journey to connect the dots between HHV-6 and ME/CFS.

Why do you think ME/CFS is shunned by funding agencies?

Everyone knows that it is a hard task to solve a disease like ME/CFS. It will be a challenging and very high-risk task to solve this disease. No funding agency wants to take the risk of supporting a failure. Moreover, the majority of funding agencies provide money based on a proven track record of research and basic, pre-established data. They want to see data first. There is hardly any funding agency that wants to support innovative and challenging subjects like ME/CFS. If I already have done the experiments and have results, why would I ask for funding again? Another important truth about this disease is lack of mortality (that also means it does not make the headline of a newspaper or TV show). If ME/CFS killed hundreds of people overnight like SARS, MERS or Ebola, then there would be millions of dollars of funding for it.

What do you think is the most impactful/important finding you’ve published so far?

One of the key aspects of studying herpesvirus reactivation is to characterize the pathogenic role of the reactivated virus in disease progression—how does the reactivated virus cause disease? Virologists always argue that the virus needs to be fully active and infectious to cause a disease. That means one should observe and detect widespread viral DNA, RNA, and proteins in affected patients, and the virus reactivation should cause an immunological response in the patient’s body.

But I do not agree with this argument. Instead, I argue that once the virus reactivates, there can be two consequences. Either the virus will succeed and take over the host cell, leading to viremia. Or the host will succeed and keep the reactivated virus under check. There can be a third intermediate stage, where the virus immediately after reactivation starts producing minimal genetic material before it can be fully controlled by the host. And this minimal viral activity is enough to damage a cell at least for a few days to weeks.

This minimal damage process may not be harmful for every cell type. But those cells whose numbers are limited (like neurons) or those cells whose function is critical for the host (like neurons, cardiac muscles, etc.) can be greatly affected by minimal virus reactivation. In a groundbreaking research report published in 2022 in Nature, my team characterized the entire process of mitochondrial dysfunction during early stages of virus reactivation. (3) We not only characterized the effect of one HHV-6 miRNA [miRNAs are “micro RNAs” that do not code for proteins, unlike other RNAs] on the host cell, we also showed that this unique feature of viral miRNA-mediated regulation of human miRNAs are well beyond host-virus interaction. This knowledge alters the fundamentals of the infection process and opens new horizons for understanding the role of small RNAs in human diseases.

Do you think that HHV-6 Variant A and Variant B are different enough that they might be considered separate viruses? Are A and B as different from each other as each is from HHV-7?

HHV-6A and HHV-6B are already considered as two distinct viruses. In a landmark paper published in 2014, HHV-6 biologists have agreed to consider these two viruses as distinct viruses. (4)

Do you know any viruses other than HHV-6 that are able to disrupt so many different organ systems? There are reports in the literature about HHV-6 being involved in: aging (via telomere shortening); blood cell disorders (GvH disease, Hodgkin’s disease, non-Hodgkin’s lymphoma, leukemia, lymph node atrophy and inflammation, thrombocytopenia); cardiac system dysfunction (cardiomyopathy); CNS diseases (Alzheimer’s disease, autism, convulsions, encephalopathy, PMI, viral meningitis);  immune system and autoimmune illnesses (ME/CFS, AIDS, lupus, MS [childhood & adult]); liver dysfunction (hepatitis); lung disease (pneumonitis); reproductive system disorders (cervical cancer, intrauterine infection, miscarriage); and intriguingly, mental illnesses (bipolar disorder, catatonia, autism spectrum disorders [it’s hard to know in which organ system to place autism], major depressive disorder).

The answer to all this confusion lies in the fact that HHV-6 can infect every nucleated cell type and undergo latency. It can’t produce virus particles in all these cell types, but it can reactivate and cause cellular damage and inflammation in every cell type. Depending upon where the virus reactivates, and how it reactivates (secondary infection, other cellular factors, immunological alteration), it can have a different consequence and hence can play a role in different types of diseases. It is not necessary for HHV-6 to be the sole cause of any disease. It is potentially a major contributing factor for the disease. The majority of viruses can, in fact, infect more than one cell type. All the herpesviruses infect more than one cell type. The recent Nature paper on SARS-CoV-2 shows virus signature throughout the body. (5) So, it is quite possible that many viruses, including HERVs, can play roles in more than one disease.

Do you believe blood banks should screen for HHV-6? The Red Cross has discouraged ME/CFS patients from donating blood for nearly 30 years.

HHV-6 screening should be carried out just like HIV screening. Its prevalence is much higher than HIV. We have published several clinical case reports showing the lethal consequences of reactivated HHV-6 after organ transplantation. The latent virus can reactivate and complicate the transplant recipient’s life. Hence it is crucial to check for HHV-6 routinely. In a recent bioRxiv paper, Lareau et al. have shown the potentials of reactivation of HHV-6 in donor blood during CAR-T cell therapy. [Chimeric antigen receptor (CAR) T-cell therapy involves engineering T-cells in the lab so that they attack a particular cancer target; it's most often used to treat blood cell cancers like lymphoma.] (6) I am sure this issue occurs more commonly, but many of us simply have not recognized the consequences of this virus reactivation.

Are you aware of antiviral treatments that are effective against HHV-6?  Different research papers describe differing responses to various medications, leading one to think there’s no consensus in how to treat active HHV-6 infection.

Most of the current antivirals are designed to target HHV-6 replication machinery. That means they are designed to inhibit virus DNA replication. As I explained before, virus DNA replication may not be the key factor in several disease conditions. Hence, the current antivirals may not be sufficient for every disease in which HHV-6 is involved. Our strategy is to develop other, alternate methodologies. For example, using antisense RNAi against HHV-6 miR-aU14. We are also developing other types of antivirals that are independent of virus replication machinery.

What do you think about the Aswad publication that places HHV-6’s integration into the human immune system around 50,000 years ago? (7)

This is a very interesting work from our collaborator’s lab in Berlin. But this work is focused on inherited HHV-6. It is not always necessary that only inherited HHV-6 plays a role in disease. We all agree now that HHV-6 infection and integration is a frequent event and happens most of the time when the virus enters a cell.

Do you think there are interactions between HHV-6 that is latent and HHV-6 that is integrated into the genome? How do you see the superantigen HERV-K interacting with either/both?  

Yes, inherited HHV-6 can interact with non-inherited HHV-6. But in my scientific opinion, HHV-6 is mostly integrated during latency. The link to HERVs is always fascinating for me. As I mentioned above, we have some evidence for a close interaction and interplay between HHV-6 and HERVs. More research is needed to unravel this mystery.

You are probably aware of a 2009 paper by Albert Tai, Dharam Ablashi, et al. that shows cells with active HHV-6A infections directly transactivate HERV-K18, resulting in production of a superantigen. (8) These researchers suggested that this HHV-6A/HERV-K18 could be very important in MS—has this study’s implications held up over a decade-plus?

I think this concept has a lot of potential. Co-functioning of more than one virus (HHV-6:HERV, HHV-6:HIV, HHV-6:EBV) can have a robust impact on disease development. Recently, we published a review article which aims to highlight this type of co-viral infection scenarios and explains how small non-canonical peptides potentially originating from viruses can influence host immune response. (9) The fact that one virus infection can positively influence another virus’s growth makes it possible that a combinatorial effect of two viruses can have a greater role in diseases like MS. In 2011-2013, I published several papers on co-infection models of HHV-6. I showed that HHV-6 infection and reactivation can be influenced even by an obligate intracellular bacterium like Chlamydia. Our current work explains this in more detail at the organelle level. We are now able to explain how mitochondria play a key role in these types of co-infections. When one pathogen fragments mitochondria, another pathogen being suppressed by healthy mitochondria can get an opportunity to come up and affect the host cell.

Other researchers who have studied HHV-6 and the immune system (Gallo, Lusso, Knox, Ablashi, and others) have suggested that, in AIDS patients, HHV-6 may actually do more damage to the immune system than HIV does. Do you think that’s possible? Have you considered studying the interaction between HHV-6 and HIV or other viruses?

I fully agree with this. I intended to do more work in this direction. Unfortunately, there is no funding to do this work now-a-days. I even tried to propose this work to be done at UCSD [University of California San Diego] in 2018 but without much success.

Can you give us a hint about what is next on your list of HHV-6 studies you’d like to perform?

The list is big and never ending. At this moment, we are employing modern high throughput technologies to understand key deciding factors in HHV-6 reactivation. We are carrying out molecular studies to link virus reactivation to mitochondrial alterations, immune dysfunction. We are also carrying out studies to understand potential role of immunoglobulins in ME/CFS and Long Covid development. In this regard, we are also studying co-infection of HHV-6 and SARS-CoV-2. Some of our studies are also focused to identify cell types that are more susceptible to virus reactivation and disease development. Our plan is also to develop organoid as well as animal models to understand HHV-6 and other herpesvirus (like EBV, HSV-1) reactivations. We already have a mouse model in collaboration with other groups linking HHV-6 infection to ME/CFS-like conditions.

[Follow Dr. Prusty on Twitter: @BhupeshPrusty]

BIBLIOGRAPHY

1.     Prusty, Bhupesh et al.; “Transcription of HERV-E and HERV-E-related sequences in malignant and non-malignant human haematopoietic cells”; Journal of Virology 382:1; 5 December 2008; Pages 37-45 doi: 10.1016/j.virol.2008.09.006; Figure 1A.
2.     Prusty, Bhupesh et al.; “HHV-6 Encoded Small Non-coding RNAs Define an Intermediate and Early Stage in Viral Reactivation.” npj Genomic Med 3, 25 (2018). https:// doi: 10.1038/s41525-018-0064-5
3.     Hennig, T., Prusty, A.B., Kaufer, B.B., et al. Selective inhibition of miRNA processing by a herpesvirus-encoded miRNA. Nature 605, 539–544 (2022). https:// doi.org/10.1038/s41586-022-04667-4
4.     Ablashi D, Agut H, Alvarez-Lafuente R, Clark DA, Dewhurst S, DiLuca D, Flamand L, Frenkel N, Gallo R, Gompels UA, Höllsberg P, Jacobson S, Luppi M, Lusso P, Malnati M, Medveczky P, Mori Y, Pellett PE, Pritchett JC, Yamanishi K, Yoshikawa T. Classification of HHV-6A and HHV-6B as Distinct Viruses. Arch Virol. 2014 May;159(5):863-70. doi: 10.1007/s00705-013-1902-5. Epub 2013 Nov 6. PMID: 24193951; PMCID: PMC4750402.
5.     Stein, Sydney R. et al.; “SARS-CoV-2 infection and persistence in the human body and brain at autopsy”; December 14, 2022; Nature 612, pages 758–763.
6.     Lareau, Caleb A et al.; “Latent Human Herpesvirus 6 is Reactivated in Chimeric Antigen Receptor T-cells”; August 25, 2022; oRxiv doi: 10.1101/2022.08.12.503683
7.     Aswad, Amr et al.; 2020. "Evolutionary History of Endogenous Human Herpesvirus 6 Reflects Human Migration Out of Africa"; Mol. Biol. Evol. 38(1):96-107. Advance Access publication July 28, 2020. doi:10.1093/molbev/msaa190
8.     Tai AK, Luka J, Ablashi D, and Huber, B; “HHV-6A infection induces expression of HERV-K18-encoded superantigen,” Journal of Clinical Virology, September 2009, PMID: 19505843 DOI: 10.1016/j.jcv.2009.05.019
9.     Lodha M, Erhard F, Dölken L, and Prusty BK; “The Hidden Enemy Within: Non-canonical Peptides in Virus-Induced Autoimmunity”; Front. Microbiol., 10 February 2022; https://doi.org/10.3389/fmicb.2022.840911

ABOUT THE AUTHOR

Neenyah Ostrom was the first reporter in the United States to report weekly for a decade on ME/CFS. Her reporting on the Chronic Fatigue Syn­drome epidemic from 1988-1997 is getting increased atten­tion thanks to Robert F. Kennedy Jr.  He discusses her work exten­sively in his 2022 best seller, The Real Anthony Fauci. Ostrom’s groundbreaking reporting on Chronic Fatigue Syn­drome and AIDS appeared in the New York Native from 1988 to 1997.

Ostrom is the author of four books about the Chronic Fatigue Syndrome epidemic: What Really Killed Gilda Radner? Frontline Reports On The Chronic Fatigue Syndrome Epidemic (1991; TNM Inc., New York, NY), 50 Things Everyone Should Know About The Chronic Fatigue Syndrome Epidemic And Its Link To AIDS (1992; TNM Inc. and St. Martin’s Press, New York, NY; published in Japanese by Shindan-to-Chiryo, 1993; and in French by Les Edi­tions Logiques, 1994), and America’s Biggest Cover-Up: 50 More Things Everyone Should Know About The Chronic Fatigue Syn­drome Epidemic And Its Link To AIDS (1993; TNM Inc., New York, NY); and America’s Biggest Cover-Up: 50 More Things Everyone Should Know About The Chronic Fatigue Syn­drome Epidemic And Its Link To AIDS, Updated 2nd Edition (2022, available as a Kindle ebook and paperback on Amazon.com). Her most recent book, Ampligen: The Battle for a Promising ME/CFS Drug (2022) is available as a Kindle ebook and paperback on Amazon.com

 In 1995, Ostrom and New York Native were rec­ognized as having reported one of the top 25 most-censored stories in the U.S. press by 1995’s Censored: The News That Didn’t Make The News And Why (The 1995 Project Censored Yearbook by Sonoma State University Professor Carl Jensen, introduction by Michael Crichton; pub­lished by Four Walls Eight Win­dows, New York, NY, 1995).

Ostrom is ghostwriter/editor of seven popular science books. Additionally, she was an editor of Total Breast Health: The Power Food Solution For Health And Wellness by Robin Keuneke, which was chosen as a Publishers Weekly “Best Book of 1998” in the category of Breast Health (Kensing­ton Publishing Corp., April 1998).