Live Blogger: Brenna Saladin
Editors: Varsha Shankar and Ryan Schildcrout
This piece was written live during the 8th annual RNA Symposium, “Unmasking the Power of RNA: From Structure to Medicine” hosted by the University of Michigan’s Center for RNA Biomedicine. Follow MiSciWriters’ coverage of this event on Twitter with the hashtag #umichrna.
Viruses are a routine occurrence in everyday life. The common cold, the flu, and most recently the COVID-19 pandemic has kept viruses at the forefront of the public mind. One of the key aspects of the viral life cycle and infectious mechanism is the fact that they rely on the host in order to replicate and spread infection. Viruses do not contain their own machinery for replication, but rather they hijack host cell machinery to do their bidding. Our speaker today studies one of the beginning steps of this process: reverse transcription initiation. This is the process by which the retroviruses, such as human immunodeficiency virus (HIV), create the DNA template from viral RNA that encodes the necessary components for infection and proliferation in host cells. The D’ Souza lab studies these processes so that we have a better understanding of the mechanisms of viral infection, and therefore the potential to create drugs to combat them.
Dr. D’Souza starts her talk by describing what her lab studies, including internal ribosome entry sites, ribosomal recoding, mRNA transcription in viruses and eukaryotes, and retroviral reverse transcription initiation, which is what she is going to focus on in the talk today. Specifically, Dr. D’Souza will cover how her lab utilized different structural discovery techniques such as Cryo-EM and NMR to determine the different structures of proteins central to HIV reverse transcription initiation.
Dr. D’Souza begins by including background about the current knowledge on how reverse transcriptase initiation occurs for retroviruses like human immunodeficiency virus, or HIV. On the 5 prime end of retroviral RNA (which is the beginning of the long RNA molecule that is reverse transcribed into DNA) there is a specific structural component termed the U5-PBS structure that anneals to a host cellular tRNAlys to create a U5-PBS-tRNAlys complex. This complex is recognized by the HIV reverse transcriptase (RT) to trigger transcription initiation. Another viral protein, termed the nucleocapsid (NC), is a chaperone that is also necessary to bind this complex to assist in creating the optimal structure for transcription initiation. Dr. D’Souza ends her introduction by emphasizing that retroviruses have a dimeric genome (two genomes) which means that two of these initiation complexes are forming at a time.
Dr. D’Souza describes how her lab struggled at first to determine the secondary structure of the U5-PBS complex. The primary structure of the U5-PBS is the RNA sequence of bases, while the secondary structure describes how these bases interact with each other and what shapes are formed. The D’Souza lab first utilized cross-linking the different protein components to the RNA for better elucidation of Cryo-EM structures. They also needed to ensure they could observe two helices (two helix shapes in the RNA) that make up the U5-PBS structure to fully determine and analyze the secondary structure using NMR methods. After determining this structure, which includes 5 modules (M1-M5) of GNRA tetraloops (G stands for guanine, N stands for any nucleotide, R stands for either a guanine or adenine, and A stands for adenine), they titrated (or added) in tRNAlys and saw that the bottom helix 1 of the secondary structure came apart and the tRNAlys bound 18 nucleotides to the U5-PBS sequence near the 3 prime end, forming the 18bp primer for the HIV RT.
In small-angle X-ray scattering (SAXS) (which is another structural technique) profiles they saw that these 5 modules from the U5-PBS primer dock onto different regions of the tRNA along with other regions of the secondary structure of U5-PBS to form a pre-initiation complex which is in a repressed state. This led them to wonder how the reverse transcriptase was able to recognize the 18bp primer that was hidden in this pre-initiation complex. Using NMR, they discovered that the HIV RT actually interacts with helix 2 of the U5-PBS. This was an important discovery, as previous knowledge suggested that there was only one RT involved in complex binding at the primer region. What this discovery indicates is that two RTs are binding to the primer before initiation: one binds at the top in helix 2 first and then a second binds to the 18 bp primer.
This led them to ask about the importance of this interaction of the second RT with helix 2 of the U5-PBS. Using mutational analysis, they were able to see that this interaction is critical for activity; mutations in the U-rich loop of the U5-PBS that interacts with the RT, prevents RT binding, and leads to no initiation of reverse transcription occurring. Since this second binding location for the RT at helix 2 has not been previously thought to occur, it has not been a target for drugs to inhibit HIV. Because of its importance for transcription initiation to occur, this provides other avenues of research for HIV treatment and prevention.
To further determine the structure of this second RT binding to helix 2, they used a combination of Cryo-EM and NMR. Previous structures of the HIV RT complexing with U5-PBS indicate two structures, a bound state (PDB: 6B19) or an apo (unbound) state (PDB: 1LO). They discovered that the state of binding of RT to helix 2 is similar to an apo-closed state rather than an enzymatic open state. The NMR analysis also demonstrated that the binding interactions occur in the thumb domain of the complex structure. Specifically, the interactions occur in the U-rich loop at the top and near the AAAA sequence towards the bottom of helix 2. The D’Souza lab is currently working on mutational analysis to determine how changes to these binding regions could affect reverse transcription initiation.
Bound State HIV RT PDB: 6B19 APO State HIV RT PDB: 1DLO
Beyond the RT, the nucleocapsid protein of HIV (NC) is also important for transcription initiation. The D’Souza lab’s data indicate that the two NC complexes that bind the U5-PBS affect the formation of M4 and M3 on helix 1. However, the two NCs bind to regions away from these modules. One NC binds to the D stem of the U5-PBS-tRNAlys complex, while the second binds near the core triples. It is currently not known how binding to these regions affects the formation of M4 and M3 on helix 1, but they know that the two NC complexes are important for structural stability of the U5-PBS-tRNAlys complex. In Cryo-EM grids, analyzing the complexes without the NC’s the complexes are difficult to resolve, but when you add two NC’s the structure is more stabilized and easier to determine.
To round out the end of her talk, Dr. D’Souza reminds us how the dimeric genome plays an important role in these processes. She proposes that the two U-5 PBS regions from the two sets of the genome can form dimers that create a larger structure for RT initiation regulation. This could lead to a potential for larger initiation complexes than previously thought. D’Souza ends her talk discussing the final model for initiation, where the NC’s bind first, followed by RT binding to helix 2 and then a second RT binding the primer region. She ends with the question of how a potentially larger complex could form with the dimeric genome, in which there are two U5-PBS regions interacting with all of these factors.
Overall, these data provide great insight into the structure of the HIV initiation complex and further our understanding of these processes. By knowing the intricacies of such necessary components needed for structure formation and binding interactions, there is potential for better creation of HIV pharmaceuticals to target initiation and stop the viral life cycle.
Victoria D’Souza’s background lies in Biochemistry and Structural Biology, where she earned her Bachelors of Science and Masters of Science at the University of Mumbai. She went on to University of Maryland Baltimore County where she earned her Ph.D. in Biochemistry, followed by a postdoc in Structural Biology at Howard Hughes Medical Institute. Currently, she is a Professor of Molecular and Cellular Biology at Harvard University, where her lab specializes in studying viral biology and the mechanisms of RNA-mediated processes that viruses utilize to drive replication. D’Souza has many accolades, but most notably is a Howard Hughes Medical Institute Faculty Scholar.
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