Exploring Hantavirus Replication and Transcription Mechanisms by cryoEM/cryoET

Secondary Supervisor(s): Professor Jonathan Grimes (University of Oxford), Professor Corinne Smith

University of Registration: University of Warwick

BBSRC Research Themes: Understanding the Rules of Life (Microbiology, Structural Biology)

Project Outline

Hantaviruses are a family of segmented, negative-sense RNA viruses that can undergo zoonotic transmission from rodents to humans, causing severe diseases such as Hantavirus Pulmonary Syndrome (HPS) and Hemorrhagic Fever with Renal Syndrome (HFRS). Currently, there are no FDA-approved treatments or vaccines against hantaviruses, and the molecular mechanisms involved in their viral life cycle are poorly understood. In this project, we will investigate how hantaviruses hijack the host cell machinery to continue their viral life cycle. Our study will examine these processes in both the vector species (rodent cell lines) and the post-zoonosis species (human cell lines).

Central to the viral life cycle is the viral polymerase, a multifunctional protein responsible for both transcription (viral mRNA production) and replication (copying the viral genome). To initiate transcription, the viral polymerase utilizes a process called "cap-snatching," where it binds to and cleaves host mRNA in the cytoplasm, using it as a primer for transcription. How, when, or where the virus gains access to the host mRNA inside an infected cell is not known. Building on previous work in the Keown lab investigating the polymerase in vitro, we will now develop a pipeline to identify the polymerase in the complex cellular environment. We have generated a panel of nanobodies—small antibodies specific to the polymerase—which will allow us to track the polymerase inside the cell.

We will first develop a detailed understanding of the infection time course using fluorescence microscopy to monitor viral infection. Next, we will vitrify infected cells at specific time points during infection, followed by focused-ion beam milling to create then lamellae of the infected cells. Using fluorescence microscopy, we will identify regions with high concentrations of polymerase and subsequently image these regions with cryo-electron tomography. For the first time we will be able to directly image the sites of infection and identify the interaction partners inside a cell. In collaboration with partners in Oxford and Germany, these results will contribute to a broader study aimed at understanding the lifecycle of these viruses.

The Keown, Smith, and Grimes labs provide a supportive and collaborative environment for developing scientists. We will provide academic and pastoral support for the members of our labs such that our students are able to achieve their goals.

References

1. Keown, J. R., Carrique, L., Nilsson-Payant, B., Fodor, E. & Grimes, J. M. Structural Characterization of the Full-length Hantaan Virus Polymerase. bioRxiv 2023.06.09.544421 (2023) doi:10.1101/2023.06.09.544421.

Understanding the molecular basis of Nairovirus genome replication and transcription

Secondary Supervisor(s): Dr Nicole Robb

University of Registration: University of Warwick

BBSRC Research Themes: Understanding the Rules of Life (Microbiology, Structural Biology)

Project Outline

Nairoviruses are potent human and animal pathogens that have been identified by global agencies, including the World Health Organization, as potential causes of future pandemics. Animal-infecting nairoviruses, such as the Nairobi Sheep Disease virus, cause significant loss of animal life and economic hardship. The serious threat posed by these viruses necessitates a detailed understanding of their lifecycle to identify potential targets for inhibition.

In the Keown lab, we investigate how nairoviruses replicate and transcribe their viral genome. The genetic material of nairoviruses consists of three segments of single-stranded RNA. Each RNA segment is coated by many copies of viral nucleoprotein with each end of the genome bound by a single enzyme called the polymerase. The polymerase is the enzyme which performs both replication (copying the genome) and transcription (producing viral mRNA). Polymerase activity is regulated by interactions with RNA, viral proteins, and host proteins.

Using purified proteins and synthetic RNA, we aim to determine the structures of the polymerase to better understand its molecular movements during transcription and replication. We will make extensive use of single-particle cryo-electron microscopy (cryoEM). To complement and inform these structural studies, we will characterize the function of the polymerase using in vitro fluorescence functional assays and minireplicon activity assays. In addition to these techniques, you will gain experience in molecular biology, protein expression, and single-molecule fluorescence assays.

The Keown and Robb labs provide a supportive and collaborative environment for developing scientists. We will provide academic and pastoral support for the members of our labs such that our students are able to achieve their goals.