Flossie Wong-Staal, Ph.D. (1946- 2020). Have you heard that name? Unfortunately, you likely haven’t – even though Wong-Staal was hugely instrumental in figuring out the molecular biology of HIV – because she was often overshadowed by more, uh, “large personality” types. While her colleagues argued contentiously in the media about virus discovery & tests first-nests, Wong-Staal carried out a staggering array of firsts of her own. Among other accomplishments, she was the first person to successfully clone it (isolate its genetic instructions and stick them in other cells to study in the lab). Clone in hand, she was able to obtain the first full sequences of the HIV genome, and she discovered 2 of its regulatory genes, tat & rev. She also was the first to discover that the HIV virus is highly genetically variable – it mutates frequently so that, in addition to differences between viruses in different people, people can have slightly different versions of the virus within their own bodies! Wong-Staal warned that this could make vaccine development challenging and necessitate the need for combination therapies. Her premonitions would prove prescient as, decades later, we still don’t have an HIV vaccine, and combo therapies are the treatment of choice.
Last week I attended a virtual conference on SARS-CoV-2, the virus that causes the coronavirus diseases COVID-19. And it struck me that a LOT – I mean really a lot – of the scientists started their talks by saying something like, “before this, I was *not* a coronavirus researcher – instead I was/am an HIV researcher.” At other points, scientists pointed to ways in which HIV research largely paved the way for a lot of the research that is being done on SARS-CoV-2. And a lot of that HIV research was carried out by a scientist at the NIH (National Institutes of Health) and later at the University of California, San Diego (UCSD) named Flossie Wong-Staal. I was incredibly saddened to hear that she passed away earlier this month, on July 8 2020, from (non-COVID) pneumonia at the age of 73.
I just learned of her death a few days ago – it didn’t get much news attention, just like Flossie herself never got that much news attention outside of the HIV/AIDS research-sphere. Part of me wanted to rush out a tribute, but I decided to delay and really write a more thorough post on Flossie as a person and as a scientist. I hope you will read – and I hope you will remember. And, in addition to remembering Flossie herself, I want you to remember that a lot of the most important science takes place out of the spotlight. And, each contribution you make as a scientist, no matter how big or small it might seem at the time, can have tremendous impact in the future in ways you could never imagine.
First, let’s just clear up some terminology and basic “what we know now about HIV/AIDS” info. Similarly to the whole SARS-CoV-2 vs. COVID-19 dealiobob, HIV/AIDS has different names for the virus and the disease it causes. The virus is HIV, which stands for Human Immunodefficiency Virus – and the disease it causes is Acquired ImmunoDeficiency Syndrome (AIDS). This immune deficiency comes about because HIV infects a kind of immune cell called T cells. It can deplete the patient’s T cells, leaving them vulnerable to opportunistic infections which can be deadly. Also, another confusing thing is that when the virus was discovered by different groups, they named it different things – so Gallo & Wong-Staal’s group called it HTLV-III, Barre-Sinoussi et al called it LAV, and Levy et al. called it ARV. Flossie would show they were all HIV and in 1986 an international committee made the name HIV for all strains of the “AIDS virus.” That helped clear up confusion, but if you look to the early papers, which I’m going to show you a couple of, the names are confusing! So be warned…
Similarly to SARS-CoV-2, HIV is an RNA virus – it carries its genetic blueprint (genome) in the form of RNA, not DNA like we do. However, unlike SARS-CoV-2, which just gets into our cells and makes RNA copies of itself without even getting near our DNA, HIV is able to make a DNA copy of itself and stick it into our own genome. This type of virus is called a retrovirus, because it “goes back” from RNA to DNA, which is something our own cells never do with our own RNA. This special “RNA to DNA copying” part is called Reverse Transcription (because going from DNA to RNA is called transcription). The “sticking that DNA into our DNA” part is called integration, and it’s part of why HIV is so damn hard to get rid of. By sticking itself into our own DNA, the virus can hang out in a latent form for years and years, just biding its time until the time is right. When that time’s right, it can get the cell to start making lots of copies of it & ship those copies out & go infect more cells.
As you might imagine, all that requires a lot of coordination, and thus the HIV genome is fairly complex & sophisticated. If scientists wanted any hope of conquering, or at least subduing, it, they’d need a better sense of what it contains & how it works. And they’d need a way to study potential treatments in a lab – in cells in a dish as well as in animal models. This meant they’d need a molecular clone of it (the viral genome in a manipulatable form). Enter Flossie Wong-Staal.
Wong-Staal herself entered the US in 1964, at the age of 18, in order to attend college. She was born Yee Ching Wong in China in 1947 and her family fled to Hong Kong in 1952. (She got the name Flossie as a child because an American nun/teacher in Hong Kong said she needed an English name and she didn’t want a “boring” one so her dad took the name from that of a recent typhoon). The first woman in her family to work outside the home or pursue an advanced education, she left Hong Kong as a teenager to attend UCLA, where she earned a BS in bacteriology followed by a PhD in molecular biology.
She then moved to the NIH’s National Cancer Institute (NCI) in 1973 to work with Dr. Robert Gallo studying retroviruses. It was here were her first breakthroughs would be carried out. Those breakthroughs started even before the HIV work. Among other early accomplishments, she the first person to clone and sequence a pathogenic human retrovirus. She was initially focused on studying oncogenic retroviruses – retroviruses that can cause cancer, such as HTLV-1, which, as she provided the molecular evidence for, can cause a form of leukemia (an idea that was largely considered heretical at the time). That work was groundbreaking and view-changing, but it was her HIV work that would come to largely shape her career and legacy.
Working with Gallo, she was part of a team that co-discovered HIV (a second team, at the Pasteur Institute in Paris, including Françoise Barré-Sinoussi (another amazing female scientist) also made this discovery – first – and it became this big deal…). That breakthrough was only one (important) step in scientists’ contribution to the fight against AIDS. They still needed to figure out how this virus was able to cause the devastating disease, and in order to do that they needed to see what it contained.
Wong-Staal was able to clone and genetically map the HIV virus. In terms of significance, this meant that they’d have a sequence in hand to help in the development of blood tests for the virus and to figure out the function of the HIV virus’ different parts. But what did this mean in terms of actual, practical, at the bench work? How did she do it?
Let’s think about what she’d need to do. First, she’d need to collect T cells from an HIV patient and grow those T cells in a dish in the lab so she could get a lot of viral genetic information. Then she’d need to isolate that information from all the cells’ own DNA and stick it in a circular piece of DNA called a plasmid vector to serve as a “vehicle” to put it in other cells.
For a long time it was considered impossible to grow T cells in the lab, but Gallo’s lab had previously found that you could get them to grow if you added “T-cell growth factor” (interleukin 2 (IL-2)). They also had an immortalized T-cell line in the lab (H9) that they could infect with virus to make lots of it. The HIV virus reverse transcribes its RNA before integrating. And it actually makes multiple DNA copies that can kinda hang out. So she was looking for this unintegrated DNA. Which involved a lot of careful fishing.
To do the isolation, she took the infected cells, extracted their DNA, and separated the DNA by length using a sucrose gradient (basically spin them really fast in a tube filled with sugar and the bigger pieces will sink further). She then took the separated fractions and did something called a Southern blot to find which fractions contained the viral DNA. This technique works by using labeled probes to find complementary sequences. So you take your DNA, run it through an agarose gel to separate them even further by size and kinda concentrate them, and then you transfer that DNA out of the gel and onto a membrane that you can add that probe to and see if it binds. http://bit.ly/blotcompass
Problem is, you need to know what you’re looking for – the way such probes work is that they take advantage of specific, one-to-one base pairing – the DNA letter A binds to T and C to G. This is why each strand of double-stranded DNA can serve as a template for making the other, complementary strand. So if you have probes that are complementary to the DNA you’re looking for, the probes will only bind to corresponding sequences. So she’d need some viral sequences to make probes to look for viral sequences! So she made probes by using oligo(dT) primers to make cDNA from virally-infected cells – sorry this is technical and I don’t have time to go into it but wanted to put this out there for folks who might be wondering. If you’re really curious, I explain cDNA here http://bit.ly/2DP5ngD
For the rest of you, just know that she had a way to look for which DNA pieces had the virus. Once she figured out which fraction the viral genome was in, she could take it and stick it into a phage plasmid. A phage is a virus that infects bacteria and some phages have nice circular extracellular chromosomes called plasmids that you can insert DNA into. To do the inserting, she digested the DNA pieces she’d isolated and the plasmid (λ) with a restriction enzyme. Restriction enzymes (aka site-specific DNA endonucleases) recognize short (6-8 letter long) DNA sequences and cut them. And then you can ligate (stitch together) complementary cut pieces. http://bit.ly/restrictionenzymecloning
She used specific restriction enzyme called Sst I which is known to cut in the Long Terminal Repeats (LTRs) which flank the beginning and end of the virus (the virus has these same sequences on either side to help it get in and out of our DNA and it makes a way for Flossie to make sure she gets the virus’ ends!). She then put those plasmids into phages (a technique called a phage library), screened them to see which phages had the viral DNA in them, then isolated the plasmids those “hits” contained.
Now she needed to check they really had the *full* HIV genome. So she did some more tests like seeing how big they were and seeing if they bound samples from virus-infected cells but not those of non-infected cells. She isolated one phage, λBH-10, which contained the full HIV genome (but I think it was missing a bit of one of the leader sequences according to a later paper of hers), as well as 2 other phages, λBH-8 & λBH-5 that seemed to represent 2 halves of an HIV genome (but a second clone of it).
They reported this first HIV clone in November 1984. https://bit.ly/39t1jkl
Now, she had to show that this genome really was the whole HIV, and that it was capable of causing AIDS. A few months later, in July 1985, her group did just that (she credits one of her postdocs, Amanda Fisher, with this breakthrough). They stuck the HIV genome into a different plasmid which she stuck into bacteria and used a protofusion transfection technique to get that plasmid into heathy T cells. Those healthy T-cells became unhealthy and they produced active viral particles they could see under the electron microscope. As further proof of infectivity, they could also detect reverse transcriptase activity in the liquid the infected cells were grown in, indicating the presence of replicating virus. And further, further, staining the cells with antibodies specific to viral surface proteins detected them in the infected but not the uninfected cels.
So basically, they show, for the first time, that they have a “biologically active” clone that produces identical effects to the whole virus, showing that the viral genome (once in cells) is all that’s needed.
This was incredibly important because the retroviral research field had been long beset with challenges due to contaminating viruses. Scientists would think they’d discovered something, only to find out it was a contaminant introduced from somewhere in the lab. A lot of the viral searching was done by looking for reverse transcriptase activity – they’d add RNA templates and radio labeled DNA primers to a sample from a patient and see if DNA copies of those RNA templates would get made. If they did, that would mean that there was reverse transcriptase present, which our cells don’t have but retroviruses do. So that would indicate that a retrovirus was present in the patient – at least in theory… It turned out to frequently be an artifact of a contaminant, leading to human retroviruses being mockingly referred to as “rumor viruses” in the early days.
Because of these problems, the scientific community was very skeptical about retroviruses and there was a high barrier to establishing legitimacy. A barrier that Flossie would surpass and keep going.
In 1999, Flossie gave a really great interview to Victoria Harden, Ph.D., Director, Office of NIH History, and Caroline Hannaway, Ph.D., Historical Contractor, NIH. You can read the transcript here: https://bit.ly/330bRGi
In it she refers to those years as “the highlight of [her] career, that period of discovery, intense discovery” – and she makes sure to make clear that she wasn’t working alone, giving shout outs to her “fantastic team” which included “Beatrice [Beatrice Hahn, M.D.], George [George Shaw, M.D.], Sasha [Surresh Arya, Ph.D.], Mandy [Amanda Fisher, Ph.D.], Lee [Lee Ratner, M.D.] and Mark [Mark Feinberg, Ph.D.].” Note that when you’re looking at papers, like the ones I show in the figs, the “last author” is usually the “principal investigator” who’s typically the person who runs the lab and is like the boss and helps design the experiments, mentor their lab members, etc. and then the first person on the paper is the postdoc or grad student, etc. who actually did the work (so, for example, when my work hopefully eventually gets published, my name will be first and then my PI’s name will be last). In this spirit of emphasizing many people being involved, I should also note that, soon after Flossie, other groups published HIV clones and later, soon after Flossie, other groups published HIV genome sequences.
With clone in hand, Flossie and her lab members (and researchers around the world) were able to make all sorts of discoveries. There’s no possible way I could explain them all. But here are a few highlights, starting with her discovery of a couple of “trans-activating” genes in HIV.
HIV, like other viruses, was known to have “cis-regulatory elements” which are stretches of the DNA or RNA that act as signals for various processes like transcription (making messenger RNA (mRNA) copies of the protein instructions held in DNA genes) or translation (making protein based on those mRNA instructions). Trans-regulatory elements are things like proteins that can recognize & bind to those cis elements and affect those processes.
Most retroviruses known at the time used only *cellular* trans-elements. So, for example, the viruses have cis-regulatory sequences that get bound by cellular transcription factors to enhance or discourage transcription. Flossie showed that, in addition to using the cells’ regulatory factors, HIV made its own from genes her group found. They discovered 2 of them, called tat (trans-activation of transcription) & rev (regulator of expression of virion proteins) (which was initially called Art and then Trs…)
Flossie, in work she credits to one of her lab members, Suresh Arya, discovered the gene tat (trans-activation of transcription) which encodes for a protein that binds to a folded-up section of the viral RNA called TAR (Trans-Activation Response) and enhances transcription – although it looks like another team, Joseph Sodroski et. al, actually found the protein made from it slightly before, but called it Art? and didn’t know the gene it came from – I’m not sure, sorry. But Flossie’s group definitely helped figure out it’s importance! (for the nerds among us: It acts as part of a complex that includes a kinase that phosphorylates the polymerase to un-stall it. This has been worked out in large part by Andrew Rice and his lab).
Tat acts at a transcriptional level, but Flossie, in work largely carried out by one of her postdocs, Mark Feinberg, also found a trans-regulatory element that acts at a post-transcriptional level, rev.
As I mentioned above, proteins are made from messenger RNA (mRNA) copies of DNA genes. But usually those RNA copies aren’t “letter for letter” – instead, they’re “edited” by cutting out parts of it in a process called splicing. Rev is a protein that binds to a cis element in HIV, another folded up bit of DNA, called RRE [Rev Response Element] to allow incompletely processed viral RNA to be exported out of the nucleus so that proteins can be made from them. At first glance, this might sound like it would be a bad thing but, for HIV (at the right time) it’s a good thing. I can’t get into it all here, but basically one of the tricks HIV uses is that it packs a lot of genetic info into not a lot of letters. So it can make different proteins by processing the RNA copies made from its genes differently. For instance, if it doesn’t splice out some of the parts that normally get spliced out (parts called introns), it can combine the instructions for multiple and/or different proteins on its mRNAs.
Early in the HIV cycle, the virus only wants to make its regulatory proteins, so it lets the cell splice them normally. But later in the HIV cycle, when the virus is rearing up to make copies to ship out, it needs to start making all of those structural proteins that make up the “packaging” that the virus travels in. So Rev revs up! Those structural proteins and other “late proteins” have their instructions in incompletely-spliced genes so Rev helps them get out into the cytoplasm (general cellular interior) where they can get made into proteins.
One of the structural proteins that gets the most attention is the envelope protein gp120. It’s kinda like the coronavirus Spike protein in that it sticks out from the viral membrane & is responsible for docking onto cellular receptors. In the case of HIV, the envelope protein binds to the CD4 receptor, which is located on T cells. Similarly to how Spike is the target of vaccines & monoclonal antibody treatments, with the hope that antibodies can bind to Spike to prevent it from docking on our cells, scientists hoped that vaccines would induce patients to make antibodies that could block the HIV virus from docking.
But Flossie poured some water on those hopes… Flossie showed that there was a lot of genetic variation in the virus. In addition to differences between HIV clones taken from different patients, clones from the same patient showed “micro variations” – basically there were minor genetic differences that could build up over time. She made these findings by using methods like restriction enzyme mapping. This involves using restriction enzymes that recognize and cut different DNA sequences to cut HIV clones and analyze the size of the resulting pieces. Even single letter differences, if they happen to occur in the recognition sequence of one of the restriction enzymes she chose, could lead to a different distribution of fragments, so she could detect the presence of differences.
And she found a lot of differences – especially in the envelope gene – and more changes showed up over time. So, a patient might have one variant early on and make antibodies against that variant. But then that variant would mutate so that the patient’s own antibodies didn’t work against it anymore. This sort of virus evolution in response to external pressures (such as the antibodies) is referred to as “antigenic drift” (antigen is a term for a thing an antibody binds to).
Similar to what was happening at the surface in response to antibody pressure, variation also could arise in parts of the virus when they’re exposed to other pressures – like pharmaceutical drugs, which can often lead to rapid resistance developing.
For example, scientists would treat a patient with a single reverse transcriptase inhibitor. And it would work for a while. But then a random mutation in one of the viral copies would lead it to make a version of reverse transcriptase that wasn’t affected by that inhibitor. So that virus would have a growth advantage and proliferate.
This is why HIV is now treated with “combination therapy” – such as a “cocktail” of several reverse transcriptase and/or protease inhibitors (the protease inhibitors prevent the virus from processing its proteins into their mature form). A virus might randomly develop resistance to one of them, but it’s a lot less likely to develop resistance to all 4 because that’d take at least 4 random mutations in the “right spots” occurring when the virus was incapacitated. Flossie was instrumental in showing that combination therapy can be effective.
Note: thankfully, SARS-CoV-2 doesn’t appear to mutate nearly as much – they’re completely different viruses and HIV has a lot more “ways” to mutate than SARS-CoV-2. For example, at one stage of its lifecycle, 2 copies of the HIV genome pair up, which can allow for swappage, etc. Additionally, SARS-CoV-2’s RNA dependent RNA polymerase (RdRP) (its RNA to RNA copier) has a proofreading capability so fewer mutations get through than with HIV, whose reverse transcriptase is highly error-prone so the virus often mutates before integration. I just wanted to stick this note in here so people know this HIV case isn’t the “norm” for all viruses, but scientists are considering the likely benefits of combination therapies for COVID-19. Now, back to the HIV story. Because Flossie wasn’t done yet!
Over the course of 20 years, Wong-Staal would co-author more than 100 scientific papers with Gallo. https://bit.ly/3fZdPdL
But Gallo would get embroiled in a whole public and legal dispute over patent stuff regarding HIV testing and who really discovered HIV. And Wong-Staal didn’t want to get caught up in it. As she says in the interview: “I have even thought, “Bob, let other people do something first. We don’t have to make all the discoveries and do everything first.” But he does have this attitude that the lab’s goal is to win, to achieve, and I think that turns off a lot of people.”
She also had a desire to get out from under Gallo’s shadow: “I was at a stage of my career where I felt that, much as I admire Bob as a leader and as a scientist, his visibility was really overshadowing me. I think we had complementary expertise, and people recognized that I was doing molecular biology and he was not. But still, I think the association sometimes worked against me.”
So, in 1990, she moved to the University of California, San Diego (UCSD), where she continued to study HIV as the Florence Riford Chair in AIDS Research. In 1994, she started, and was chosen to lead, UCSD’s Center for AIDS Research, the same year she was elected to the National Academies’’ Institute of Medicine. At UCSD she became more involved in the clinical aspects of HIV, searching for treatments and, she wasn’t afraid to say it, cures. A lot of scientists thought it’d be impossible to actually cure AIDs, since it integrates into our genome. There’s a thinking that the best we might be able to accomplish would be a sort of “permanent remission” where the virus is there but in the latent phase. Flossie hoped that she could actually irradiate the virus with gene therapy treatments. If you want to hear more about this, I encourage you to watch her talk at CSHL’s HIV meeting in 2016: https://bit.ly/3g1KDTp
While at UCSD, she co-founded a biotech company called Immusol, focused on AIDS treatments. She retired in 2002 as Professor Emerita but continued her biotech career taking on a role as Chief Scientific Officer of Immusol. She and her husband and co-worker Jeffrey McKelvy changed the name to iTherX Pharmaceuticals when they switched their focus to researching treatments for another deadly virus, hepatitis C (the company is no longer active, but I don’t know the story).
Flossie was named by The Scientist as the most-cited female scientist of the 1980s and, among other honors, she was inducted into the National Women’s Hall of Fame in 2019. https://bit.ly/3fZdPdL
She is survived by her husband, 2 daughters, several siblings, and 4 grandchildren and, according to a memorium post by the NIH, the family is asking people to donate to Doctors Without Borders in Flossie’s name. I know money times are tough for a lot of people right now, but if anyone wants to donate, here’s a link: https://bit.ly/2WVP8HY
That video I pointed you to earlier was just one in a great series of talks by a lot of prominent HIV researchers as part of a meeting called “HIV/AIDS Research: Its History and Future” which was held at CSHL in 2016. You can view all the videos here: https://bit.ly/3g1KDTp
I had the absolute privilege of being able to attend in person and it was one of the greatest experiences of my lifetime. It was only a couple months after I arrived at CSHL for grad school and I remember walking by someone and thinking “wait a sec – was that Harold Varmus?!” it was – and I wish I’d been just as excited to have seen Flossie Wong-Staal. But, honestly, I don’t think I’d ever heard of her. I certainly wouldn’t have been able to have a double-take moment. I wish I’d known more about her. I wish I’d gone up to her and asked to have a picture taken or at least to tell her thank you and that she’s an inspiration.
For more information:
In Memoriam: Flossie Wong-Staal, Ph.D., NIH: https://bit.ly/3fZdPdL
Dr. Flossie Wong-Staal Oral History 1997, NIH: https://bit.ly/330bRGi
Flossie Wong-Staal, Who Unlocked Mystery of H.I.V., Dies at 73, Faye Flam, NYT: https://nyti.ms/3fUO6Dn