Robert N. Kirchdoerfer

Photo of Robert N. Kirchdoerfer
Assistant Professor (also Institute for Molecular Virology)
B.S., University of Wisconsin-Madison, Genetics and Biochemistry
Ph.D., The Scripps Research Institute
Postdoctoral, The Scripps Research Institute (Saphire laboratory)
Postdoctoral, The Scripps Research Institute (Ward laboratory)
Phone: (608) 262-6191
Email: rnkirchdoerf@wisc.edu

Structural and biochemical exploration of RNA virus entry and replication

image of SARS-CoV nsp12 polymerase complex

SARS-CoV nsp12 polymerase complex: SARS-CoV non-structural protein (nsp) 12 contains the RNA polymerase active site of the multi-subunit viral RNA synthesis complex. However, nsp12 (yellow) shows greater polymerization activity when bound to the viral nsp7 and nsp8 co-factors (blue and green). Single-particle cryoEM can reveal these complexes at high-resolution. The study of these protein complexes less than 200 kDa in size remains a challenge for cryoEM, but improved sample preparation and data processing strategies are providing access to these complexes for the first time.

SARS-CoV spike protein binds to human ACE2: A domain within the trimeric SARS-CoV spike protein samples downwards and upwards conformations. Only in the upwards conformation can the spike protein recognize its host receptor ACE2 (orange). One of the strengths of single-particle cryo-electron microscopy is the capability to structurally observe multiple conformations and bound receptors within a single sample.

Coronaviruses are RNA viruses that infect a large number of avian and mammalian host species including humans. Most notable amongst the coronavirus family are highly pathogenic SARS-CoV and MERS-CoV which crossed into humans from animal reservoirs. Additionally, many coronaviruses cause significant pathogenesis in animal livestock

Coronaviruses are faced with many challenges to enter and replicate in host cells. Coronaviruses are encased in a lipid envelope and must fuse their envelope membranes with the host cell membrane. To accomplish this, they use specialized protein machines called viral fusion proteins. For some viruses like coronaviruses, these fusion proteins also recognize receptors on the host cell and are the target of the host antibodies. By studying these viral fusion proteins, we can better understand how viruses choose their target host cells and work their way inside. We use high-resolution cryo-electron microscopy and biophysical experiments to examine viral fusion proteins and their interactions with host receptors and antibodies. This will illuminate viral evolution and allow us to develop effective vaccines against these viruses.

Once inside host cells, viruses must copy their RNA genomes and produce instructions to create new viral proteins. Coronaviruses use a large multi-subunit complex of viral proteins containing a plethora of enzyme activities for these processes. Coronaviruses are unusual amongst RNA viruses in that they couple their RNA polymerases with an exonuclease to correct errors that occur during the course of viral replication. In addition to increasing the fidelity of viral replication, this exonuclease also makes coronaviruses naturally resistant to many nucleoside analog antivirals. We use a combination of structural biology, biochemistry and cell-based assays to study the mechanisms of viral RNA synthesis. This work will inform the design and characterization of novel antiviral drugs for treating emerging viral infections.

Image for movie of SARS-CoV spike protein binds to human ACE2

 

 

SARS-CoV spike protein binds to human ACE2: A domain within the trimeric SARS-CoV spike protein samples downwards and upwards conformations. Only in the upwards conformation can the spike protein recognize its host receptor ACE2 (orange). One of the strengths of single-particle cryo-electron microscopy is the capability to structurally observe multiple conformations and bound receptors within a single sample.

Click for movie