Molecular mechanisms of viral gene expression.
Nonsegmented negative-strand (NNS) RNA viruses include some of the most significant human pathogens extant (rabies, ebola, respiratory syncytial, measles, mumps, Nipah viruses). Vesicular stomatitis virus (VSV) has served as an important prototype to understand gene expression in NNS RNA viruses. As a post-doctoral fellow, I developed an approach to recover infectious virus from cDNA (Whelan et al, PNAS 1995), and since the establishment of my group in 2002 we have played a key role in understanding the structure and function of the VSV replication machinery. The goal of such studies is to ultimately inform the development of inhibitors against this group of important pathogens, and to advance the use of VSV as a vaccine vector, an oncolytic agent, and a neuronal tracer.
Major contributions from my group in understanding gene expression in NNS RNA viruses include defining the unusual enzymes within the 250 kDa large polymerase protein that are responsible for mRNA cap addition (Li et al, PNAS 2006; Li et al JVI 2008). Using negative-stain electron microscopy we obtained the first structural insights into the L protein (Rahmeh et al, PNAS 2010) and defined how the cofactor P necessary to permit access to the N protein coated genomic RNA template alters the conformation of L (Rahmeh et al PNAS 2012). These studies led us to pursue higher resolution structural studies that culminated in the first atomic structural model of an NNS RNA virus L protein (Liang et al, Cell 2015), and determined the complete structure of Rabies virus L (Horwitz et al, PNAS 2020).
We extended our structural and functional studies into the segmented negative strand RNA viruses that also use a single large protein to catalyze all the steps in viral gene expression. Working with Machupo virus – an arenavirus related to Lassa fever virus and itself an agent that causes a hemorrhagic fever - we developed the first in vitro assay to biochemically characterize the activities of its large polymerase protein as well as define its overall architecture (Kranzusch et al, PNAS 2010) and subsequently resolve the mechanism by which Z a small viral protein cofactor, suppresses the activity of L (Kranzusch and Whelan, PNAS 2011). Building on those advances we also demonstrated that the polymerase of these segmented negative-strand RNA viruses is licensed by small viral encoded RNAs for recognition and use of specific promoters for gene expression (Pyle and Whelan, PNAS 2019).
Cell entry pathways.
By manipulating the genome of vesicular stomatitis virus we generate recombinant autonomously propogating viruses in which the single VSV attachment and fusion glycoprotein (G) has been replaced by the entry machinery of representatives from every family of enveloped viruses. We have employed those viruses in genome wide loss of function screens to probe the cellular factors for viral entry. We identified Neimann-Pick C1 as the receptor for Ebola virus (Carette et al Nature 2011), LAMP1 as the receptor for Lassa virus (Jae et al, Science 2013), and defined Neuropilin 2 and CD63 in the entry of Lujo virus (Raaben et al, Cell Host and Microbe 2017). We extended this work to identify the entry pathway of an extinct virus (Robinson et al, PloS Pathogens 2018). Our core objective here is to hone a platform that allows the rapid identification of host factors coopted during the entry and assembly of any enveloped virus so that it can be deployed as new emerging infections arise, and as a vaccine candidates.
As vaccine candidates we have advanced VSV-Zika, VSV-Oropouche, VSV-SARS, VSV-MERS and VSV-SARS-CoV-2. In addition to serving as vaccine candidates, these recombinant viruses allow for rapid mapping of escape mutations to envelope protein directed inhibitors including monoclonal antibodies and small molecules.