5-23-2024
We are proud to report that HARC completed the cryo-EM structure of the HIV Rev/RRE nuclear export complex. The complex contains six Rev subunits, the 350nt RRE RNA, nuclear export factor Crm1, RanGTP, and a FG-containing fragment of nuclear pore protein Nup214 (Amber Smith, Yifan Cheng & Alan Frankel, unpublished data). The HIV research community has conducted research on Rev and RRE for over 30 years. But what is all the fuss about? And why is this complex important for viral/host biology and to the HIV research community?
The discovery of HIV-1 Rev and the Rev response element (RRE) was a hot topic during early HIV research. The RRE gene was first identified in 1986 at nucleotides 7709–8063 of the HXB2 HIV-1 reference strain (1). During this time, then postdoctoral fellow, Michael Emerman worked on Rev in the lab of Luc Montagnier at the Viral Oncology unit of the Pasteur Institute. He and his colleagues used in situ hybridization and immunoprecipitation experiments to demonstrate that Rev was necessary to express the HIV envelope (Env) protein in the cytoplasm of cells (2). With limited technology available at the time, Dr. Emerman showed that Rev transported viral RNA from the nucleus to the cytoplasm where Env was translated. He discovered a novel mechanism of HIV viral RNA nuclear export.
I conducted an interview with Dr. Emerman about his early work on Rev, with hopes of learning more about the significance of completing the Rev/RRE cryo-EM structure. Dr. Emerman is a Professor at The Fred Hutch Cancer Center in Seattle WA, and an accomplished scientist.
Michael Emerman (ME): The discovery of Rev was important because people were interested in expressing the HIV-1 envelope (Env) protein. HIV-1 Env was going to be the target for vaccine development, but it was difficult to express Env in cells. Some studies suggested that you needed this little open reading frame, called Rev, to express Env.
The function of Rev was not known at the time. Ideas were going around in the HIV research community that Rev played a role in translation, splicing or RNA stability.
Eventually there was a meeting at Cold Spring Harbor, at the Banbury Center, where every idea of how Rev worked got shut down. And so, it ended up being this big mystery… and that was the point!
I had an idea that Rev was a transport protein because we were able to express Env with the cytoplasmic vaccinia virus. Vaccinia expressed Env like gangbusters - you could see the band on a Coomassie gel without doing any further precipitation or anything. But if you tried to express the HIV equivalent with a plasmid in cells, it wouldn't make any Env protein. You needed Rev to get Env protein, but you didn't need Rev to get Env RNA.
We got lots of RNA, but the RNA was in the wrong place. It was in the nucleus, instead of being in the cytoplasm. And to get into the cytoplasm you needed this nucleotide element, called the Rev response element (RRE). The RRE had to be on the RNA transcript, but not in the protein. We showed that the element was necessary to get the RNA from the nucleus to the cytoplasm.
ME: We used a lot of the standard molecular biology techniques of the time. While we did not have the high-throughput techniques used now, the experimental approach was to simplify the problem as much as possible so that it could be tackled with what you had. The interesting thing about this paper was that there was an important problem: How do I express the HIV-1 Env protein? The idea of this project wasn't really to determine how Rev worked, but rather we needed to learn about the function of Rev to get to the next step - to understand the structure of Env. None of the ideas about the function of Rev were completely consistent with the data that was available. So that's why it was a place where you could come up with a hypothesis and you could step in and say, I think I know what's going on. My hypothesis was that Rev acted as a transport and that's what this paper showed. There isn't anything super complicated in this paper. I was simply putting the pieces of a puzzle together, and then I had to interpret all the data. What was important to me, was that there was an important problem, and I could make an impact by solving that problem.
ME: Luc Montagnier was in control of a big unit at Pasteur. He was not a Molecular Biologist but considered himself to be a Cell Biologist. He basically allowed us to do whatever we wanted in the lab. The first time Luc heard about the project was when I gave him the paper to read. I asked where he wanted his name on the paper, but he didn't think that he should be an author, since he had nothing to do with the paper.
The last author, Keith Peden, was a visiting scientist at Pasteur. He was British and he had to temporarily leave the US to renew his visa at the time. He was with us for like a year - he was brilliant!
But I talked to a lot of people at Pasteur. It was a rich scientific environment. I was surrounded by people with lots of expertise in different areas. Also, the group was very interactive; we all shared the same office, got coffee, and went for lunch together. We talked about our projects, and there was a lot of discussion about ideas in the lab. When you read the acknowledgements, there’s a long list of people who I thank for reading the manuscript for me.
ME: My paper on Rev was all conceptual. Everything was an idea.
And now, to be able to see structures of this idea and actual physical molecules interacting in this way… is amazing! We can do simple experiments that you know are consistent with a particular hypothesis. But when you see the things that you predicted interact with each other, and how they're interacting with each other in new ways that we could never have predicted from simple experiments… that is exciting!
ME: We published a Nature paper on Env soon after we published our work on Rev. The Nature paper showed which amino acid residues of Env were critical for binding to CD4 (3).
We had to use information from the Rev paper to complete the Env paper. We worked on both papers at the same time.
ME: We characterized many HIV proteins in the late 80’s and early 90’s. At the time everything was unknown. There was no treatment for HIV. No vaccine. We did not know of anything that could be done to treat people living with HIV. The goal was always that the basic research we were doing would help clarify targets that could be used for therapeutic or preventive interventions.
ME: Yes, I do! But it’s a different type of excitement now that I am a mentor. When I was a postdoc and then as a PI for the first decade, I did the experiments with my own hands, and I was the first person to see the outcome of an experiment. But now that I am more senior, I'm helping the people in my lab make their own discoveries. It's through the people that I'm training where my excitement comes from.
ME: I really liked looking back and re-reading this paper. It showed me how different publishing was then vs. now.
I will not be able to publish the Rev paper today. The images were not qualitative. There was no quantification. Each cell looked different in the figures. The experiments were not done in primary cells, but they were done in an artificial cell line. The Discussion section was mainly speculation, which almost all of it ended up being right. But I am sure reviewers will not let me get away with any of that today. It was fun to read!
References:
1) Sodroski J, Goh WC, Rosen C, Dayton A, Terwilliger E, Haseltine W.
A second post-transcriptional trans-activator gene required for HTLV-III replication.
Nature. 1986 May;321(6068):412-7. doi: 10.1038/321412a0. PMID: 3012355
2) Emerman M, Vazeux R, Peden K.
The rev gene product of the human immunodeficiency virus affects envelope-specific RNA localization.
Cell. 1989 Jun 30;57(7):1155-65. doi: 10.1016/0092-8674(89)90053-6. PMID: 2736624
3) Cordonnier A, Montagnier L, Emerman M.
Single amino-acid changes in HIV envelope affect viral tropism and receptor binding.
Nature. 1989 Aug 17;340(6234):571-4. doi: 10.1038/340571a0. PMID: 2475780
By Zelda Love, Ph.D.
