Viral polyproteins

Many viruses encode their proteins in the form of a polyprotein. Cellular ribosomes begin at one promoter in the viral genome, and then build one long protein chain with all of the viral proteins connected together one after the other. Later in the viral replication cycle, when the time is right, the polyprotein is clipped into its functional proteins, often by a protease that is itself included in the polyprotein. This is an economical way to encode proteins in the small genome (only one promoter is needed), and the polyprotein itself can have different functions than the individual proteins.

HIV-1 builds two types of polyproteins. Gag polyprotein includes the structural components of the virus. When it is newly made and still a polyprotein, it orchestrates the budding of new virions from the cell surface. Later, as the virus matures, it is clipped into functional pieces that build the infectious structures of the virion. HIV-1 also builds a longer polyprotein that includes Gag plus an extra Pol section that includes the three viral enzymes: reverse transcriptase and integrase, which play essential roles in viral replication, and protease, which is the enzyme that clips the polyproteins into functional proteins. Protease has posed a persistent chicken-and-egg paradox in the study of HIV-1. Protease is active as a dimer, but each Gag-Pol polyprotein only contains one protease chain. How does the protease clip itself out of the polyprotein to form the active dimer?

Tricks of the trade

This process has remained a mystery because Gag-Pol is difficult to study – it is difficult to purify, and the protein is flexible, posing challenges for structure determination. Recent work from B-HIVE researchers has addressed both these challenges. First, a new approach to produce and purify the Pol portion of the polyprotein provided enough material to study. This portion includes protease connected to reverse transcriptase connected to integrase. Second, a focused classification method was developed and used to probe the cryo-EM data to generate structures of pieces that are held together through flexible linkers.

The cryo-EM structure determination reveals that the reverse transcriptase portion of the polyprotein forms a dimer that is very similar to the structure of mature reverse transcriptase. In initial maps, protease and integrase were not visible, but focused classification was able to locate a full protease connected in a subset of the molecules visible in the micrographs. PDB entry 7sjx includes the structure of reverse transcriptase and protease, as seen in this map. In an even smaller subset of the data, a portion of integrase is also visible. Protease, and especially integrase, are presumably so flexible in the remaining molecules that they don’t give a strong signal in the final maps.

What the structure tells us about HIV-1

The dimeric structure of both the reverse transcriptase portion of Pol and the protease portion came as a surprise. Biochemical testing reveals that both are active while part of Pol, although protease is a bit more sluggish than in the mature form. The structure reveals that these functional dimers form very early during the formation of new HIV-1 virions, indicating that it may not need to extricate itself from Pol before it starts work in cleaving other sites in Gag and Pol.

(Top) Cryo-EM structure of HIV-1 Pol polyprotein, which resolves positions for dimeric reverse transcriptase linked to dimeric protease with atomic-level precision. Explore the structure at the Protein Data Bank in PDB ID 7sjx. (Bottom) Artistic depiction of a cross-section through an immature HIV-1 virion. Gag and Gag-Pol proteins (tan) extend inward from the membrane, bound to the genomic RNA (yellow). The location of the Pol structure is highlighted in bright magenta in one Gag-Pol dimer, and also shown with an asterisk.

Jerry Joe Harrison

How did you get interested in science?

I grew up in a rural area in Ghana without any hospital and so people relied on herbal medicine for the treatment of various diseases. I always wondered how germs caused diseases and what is in plants that allowed them to cure them. I was also very curious about the universe and asked questions about it. Unfortunately, I couldn’t get any meaningful answers to many of the lingering questions in my mind at the time. Fortunate enough to go to school, science classes were very interesting and provided the opportunity to understand some of these questions. I really enjoyed these classes and always wanted to know more about how and why things work. Out of this desire to understand scientific questions, I decided to major in science during my senior year in high school, against strong advice from my parents and some of my teachers. But even at that young age, I stood my ground and took chemistry, mathematics, biology and physics courses. Going through these courses provided very intriguing answers to many of my questions, but the more I learned about science, the more I wanted to know more. Finally, at the University of Ghana, my medicinal chemistry classes were pivotal in answering some of the questions I had from the time of my childhood, for example what’s in plants that allows them to cure diseases, and they provided an important foundation on the principles of drug action, drug discovery, and drug development. In a nutshell, my interest in science has been borne out of natural curiosity, and that continues to drive my passion for science.

Tell us about the lab where you did this work.

I came to the United States as a Fulbright Scholar to pursue a Ph.D. in Medicinal Chemistry at Rutgers university from the university of Ghana where I was a faculty in the department of chemistry. I met Eddy Arnold at Rutgers in the medicinal chemistry department where he is a faculty. Eddy has been a world leader in HIV structural biology and therapeutics development for HIV, having been one of the pioneers in the field. Eddy’s work begin with his groundbreaking results determining the structure of HIV-1 reverse transcriptase with nucleic acid substrates in the early 90s, and he went on to help develop two of the drugs currently being used to treat HIV, and he contributed immensely to the current understanding of the biology of HIV. I was fascinated by the structures of enzymes from my medicinal chemistry classes in the university and had learned about the structure of HIV-1 reverse transcriptase and drugs used to target the virus.  When I learned about Eddy’s research, I was immediately drawn to his lab. He was gracious enough to allow me to undertake my Ph.D. thesis research in his lab where we solved the structure of HIV-1 Pol. Eddy’s lab continues to contribute to the field of structural biology of viruses that include HIV by asking fundamental biological questions about these viruses, and he continues to apply structural biology tools to the discovery of inhibitors against viruses including HIV.

What were the biggest challenges in determining this structure?

I believe it is not through a lack of effort that this structure was elusive for more than 40 years since HIV was discovered as the etiologic agent for AIDS. The fact is that no one had been able to figure out a way to express and purify this protein in reasonable quantity and purity for structural studies, and so naturally establishing a method to purify this protein was a huge challenge. When I took over the project, I wasn’t sure how things were going to turn out because many had tried but not succeeded in being able to address the challenges. I was excited when I was able to figure out how to express and purify this protein in bacteria by using a media that has come to be known as the JJH media with low pH and high Mg2+ content. Solving the structure was a huge challenge as well. This protein is extremely flexible, even though the individual domains that make up the polyprotein are largely folded in the structure. It took several tries and painstaking effort to finally be able to solve the structure and to refine an atomic model that informed us about the biology.

What are you working on now?

I am currently at the University of Ghana, Department of Chemistry as a senior lecturer, where I teach courses in general chemistry, organic chemistry, and chemical kinetics. I am also continuing with the polyprotein work with Eddy Arnold. There are still lingering questions that need answers urgently and which we believe will aid the development of the next generation of HIV therapeutics.

Dario Passos

How did you get interested in science?

As a child, my curiosity and desire to create new things led me to tinker with any electronic device I could get my hands on. I vividly remember my dad leaving the house with his toolbox as I disassembled everything from TVs and radios to watches and toys. The inner workings of these devices fascinated me, and I spent countless hours exploring and experimenting with them. My love for science extended beyond electronics to the natural world as well. I spent countless afternoons collecting and studying insects, marveling at the intricacies of their anatomy and behavior.

Through my natural inclination towards experimentation and exploration, I discovered my passion for science. In college, I realized that I could use my skills and curiosity to contribute to scientific progress and drive positive change in our society. It’s exciting to think about the possibilities that lie ahead as I continue to explore the frontiers of scientific knowledge and push the boundaries of what’s possible.

Tell us about the lab where you did this work?

The Salk Institute is one of the most prestigious research institutions in the world, and I was fortunate enough to work there for 7 years under the guidance of Dr. Lyumkis. My time at Salk was an unforgettable experience that allowed me to work on cutting-edge projects alongside some of the most talented and innovative scientists in the field.

Dr. Lyumkis’ commitment to excellence was an inspiration for all of us to push ourselves to the limit. He provided us with the freedom and resources to explore new ideas and develop groundbreaking research. Being part of such a dynamic and exciting environment was a truly transformative experience, and it shaped the way I approach scientific inquiry to this day.

Collaborating with some of the best minds in the field was an opportunity I will always be grateful for. I learned so much from my colleagues and was constantly inspired by their passion and dedication to the pursuit of scientific discovery. Through our collective efforts, we were able to make significant contributions to the field and advance the boundaries of what was previously thought possible.

My time at Salk was an incredible journey that allowed me to grow both personally and professionally. It was an honor to be a part of such an extraordinary institution, and I am proud of the work we accomplished during my time there.

What were the biggest challenges in determining this structure?

The road to unraveling the structure of the Pol polyprotein from HIV was far from easy. It took years of dedicated work from Jerry Joe and Eddy Arnold to find optimal conditions to preserve the delicate structure of the polyprotein and make it suitable for structural characterization. Even then, we had to employ every technique at our disposal to extract the most information possible from the cryo-EM data. After numerous experiments and optimizations, we were able to answer a critical question that has intrigued scientists for decades. How does the protease, which is part of the Pol polyprotein and is dependent on dimerization, become active in the first place? We were thrilled to discover that Pol polyprotein has uniquely evolved to accommodate an architecture that promotes the dimerization of proteases along a dimerized scaffold. It was an incredible achievement, made possible through the hard work and productive collaboration of our dedicated teams.

What are you working on now?

I am currently part of the structural biology team in a pharmaceutical company in San Diego, where I continue using and developing cryo-EM methods to solve structures that can be used to understand and/or treat diseases.