Interference can be an advantage

Individual protein molecules are typically too small to see, but amazingly, the new technique of mass photometry can pinpoint the location of individual proteins, and accurately measure their mass. When illuminated by a laser, protein molecules scatter a tiny amount of light, too tiny for most light microscopes to detect. However, if the molecule is close to a glass surface, the light scattered by the molecule and the light reflected by the glass interfere with each other, producing a characteristic dot that can be seen in the microscope. Large proteins make darker dots and smaller proteins make lighter dots.

Mass photometry video of Gag molecules diffusing in a membrane. Each dot is a single Gag molecule, dimer, or trimer.

HIV-1 Gag protein in action

B-HIVE researchers are using mass photometry to explore the early steps of assembly of HIV-1 virions. They coated the glass slide with a lipid membrane, and then added HIV-1 Gag protein and genomic RNA to the solution above the membrane. They then watched to see what bound to the membrane. The prevailing opinion in past research hypothesized that many Gag proteins assemble on the genomic RNA, then bind to the cell membrane. However, recent work from B-HIVE researchers showed that Gag is chaperoned molecule-by-molecule by ribosomes and transfer RNA, and probably doesn’t form big assemblies in the cell cytoplasm. The new mass photometry work is perfectly consistent with this new view. B-HIVE researchers watched as individual Gag polyproteins bound to the membrane, diffusing randomly in the local area once bound. When they measured the masses of these diffusing molecules, they found mostly individual Gag proteins, dimers, and trimers.

Artistic conception of the assembly of Gag protein into a lattice on an infected cell membrane, viewed from the inside of the cell. Gag molecules are colored yellow and red to highlight trimeric associations in the lattice. Each molecule includes only the matrix and capsid domains from PDB entries 7ovq and 4usn.

What mass photometry tells us about HIV-1

These results add additional support to a new conception of how HIV-1 assembles on an infected cell’s membrane. Gag molecules associate one-by-one with the membrane, building a growing lattice by adding new molecules at the edges. Careful analysis of the mass photometry experiments implicated the trimer of matrix protein as the interaction that kick-starts the process of assembly and chaperones growth at the edge of larger Gag lattices. This adds additional nuance to the previous view that the hexamer of capsid drives assembly, and highlights matrix protein as an attractive target for drugs to block viral assembly in infected cells.

To read more about this work, see:

Anne X-Z Zhou, John A Hammond, Kai Shen, David P Millar, James R Williamson (2023) Early HIV-1 gag assembly on lipid membrane with vRNA. BioRxiv 2023.01.27.525415

Yisong Deng, John A Hammond, Raymond Pauszek, Stosh Ozog, Ilean Chai, Jessica Rabuck-Gibbons, Rajan Lamichhane, Scott C Henderson, David P Millar, Bruce E Torbett, James R Williamson (2021) Discrimination between functional and non-functional cellular gag complexes involved in HIV-1 assembly. J Mol Biol 433, 166842.

How did you get interested in science?

Growing up in a family where my father is a chemist, I was exposed to science at a very early age. However, it was watching a fictional story about the discovery of a miraculous drug that can reverse aging by ten years that truly captivated me. Looking back, it seems somewhat cliché, but that is truly how it all began. That initial interest led me to enroll in advanced physics and chemistry courses during middle and high school, and eventually pursue a major in chemical biology in college.

During my time as an undergraduate research assistant, there was a pivotal day when I obtained a positive result for my drug target identification project. I can still vividly recall the overwhelming excitement I felt at that very moment. It was then that I realized, for a fleeting instant, I held this small piece of human knowledge exclusively. That taste of exploring the frontiers of science solidified my certainty in pursuing a career as an independent researcher.

Having recently completed my PhD training, I find myself increasingly grateful for the decision I made. I am drawn to the very essence of being one researcher among countless others striving to contribute to our collective understanding and unravel the unknown. And it is just satisfying to know that there are so many “unknown” lies waiting.

Tell us about the lab where you did this work.

I did this work at the Williamson Lab, located at the Scripps Research Institute in the United States. The Scripps Research Institute is an esteemed independent institution, renowned for its excellence in scientific research with 5 Nobel Laureates. In addition to that, it also offers an incomparable ocean view.

I had the opportunity to join the Williamson Lab after transferring from another lab during my third year of graduate school. I was fortunate to have Dr. Williamson as my mentor for this challenging and captivating project, while also benefiting from the co-advisement of Dr. Millar. This arrangement was a perfect fit for me, as I was hoping to expand my knowledge and skills in RNA and single molecule techniques, both of which were available through the labs I worked in. The labs I was a part of upheld strong work ethics and played a pivotal role in nurturing my scientific growth.

The colleagues in my lab became like a second family to me. We shared a deep passion for science, celebrated our achievements together, and provided support during difficult times. Without the insightful guidance from Dr. Williamson and Dr. Millar, as well as the love and support from my lab mates, my graduate school experience would not have been as smooth and rewarding.

What were the biggest challenges with this study?

The biggest challenge in this study is to find a suitable membrane system to characterize this very early stage of Gag assembly in the presence of viral RNA. Extensive research has been conducted on Gag assembly and the structural features of the immature virion, providing considerable knowledge on the assembled lattice. However, the initial assembly strategy employed by HIV-1 remains inconclusive due to the complexity of the process, which involves intricate interactions among the lipid membrane, Gag protein, and viral RNA. Moreover, capturing the very early stages of assembly is challenging due to the rapid and highly coordinated nature of this process within infected cells.

To address this challenge, we embarked on a comprehensive investigation, comparing various in vitro methods and model membrane systems, aiming to identify a system that aligned with our research objectives. Our goal was to select a system compatible with single molecule analysis, enabling us to observe the behavior of individual assembly sites, while also ensuring that the assembly process did not propagate too far. After an exhaustive search and nearly two years of trials, we ultimately decided to utilize the recently acquired mass photometer instrument at Scripps to conduct our experiments.

In our experimental setup, we employed lipid bilayers to mimic the cellular membrane. It is a good mimic for the cytoplasm and its lacking the inherent curving ability could potentially restrict further assembly to some extent. The excitement peaked when we obtained proof-of-concept data during a late-night experiment during the equipment demonstration period, confirming that this setup precisely met our requirements. I think our final results demonstrate that the two-year search period for this method and setup was well worth every moment we invested.

What are you working on now?

I am now continuing my scientific training as a postdoctoral researcher at the Bartel Lab in the Whitehead Institute. My current project involves studying mRNA conformation and how it regulates translation efficiency.

Anne X-Z Zhou