Negative strand non-segmented (NNS) RNA viruses include potent human and animal pathogens, e.g. Ebola, measles and vesicular stomatitis virus (VSV). To transcribe and replicate their genome, these viruses package multiple copies of an RNA dependent RNA polymerase (RdRP), within each virion. While the virion morphology varies among NNS RNA viruses, the basic mechanism of transcription and replication is shared. Specifically, the genome template in NNS RNA viruses consists of a single molecule of negative sense RNA, typically encoding more than 5 genes and fully encapsidated with the nucleoprotein N (N-RNA). RdRP polymerases transcribe the genome sequentially by initiating transcription at or near the 3' end and moving toward the 5' end. While the N-RNA bound with RdRPs is the deadly engine within many major human pathogens, its transcription mechanism is distinct from cellular DNA based RNA polymerases and remains poorly understood. VSV is the only member of the NNS RNA family with a robust in vitro transcription and a strong genome replication producing ~10,000 progeny virions from a single infected cell within 10 hrs. Our lab has measured the location of RdRPs within the bullet shaped VSV virions and found them asymmetrically localized at the 5' end of the genome template. We also measured the dissociation constant of VSV RdRP polymerases from their genome template and found them to be tightly bound with a 20 pM dissociation constant. How the RdRPs find the 3' end of the template and sustain transcription is therefore nontrivial. We investigated possible mechanisms using Monte Carlo simulations, which concluded that the mechanism of RdRP transcription is distinct from cellular DNA dependent RNA polymerases. Monte Carlo simulations predicted that sliding of non-transcribing RdRPs is critical for initiation and sustainability of transcription by RdRPs. Our lab is now focused on visualizing the sliding of RdRPs and investigating the mechanism of RdRP redistribution along genome templates.
Human immunodeficiency virus (HIV) hijacks endosomal sorting complexes required for transport (ESCRT) to facilitate release from the host plasma membrane. ESCRT assembly is initiated by recruitment of ALIX and TSG101, which bind directly to the viral Gag protein and then recruit the downstream ESCRT-III and VPS4 factors to complete the budding process. The temporal and spatial details of ALIX and TSG101 recruitment are not yet well understood. Our lab is focused on establishing the sequence of events that underlie ALIX recruitment to HIV virus like particles (VLP). We also create tools and methodologies that can be used to define how other ESCRT components are recruited during HIV budding. This information is critical for understanding the mechanism of HIV budding and will also inform our understanding of membrane protein trafficking and cell division because ESCRTs facilitate MVB vesicle formation and the abscission stage of cytokinesis.
ALIX binds weakly, but directly to the YPXL motif within p6Gag, NCGag and also to the CHMP4 subset of ESCRT-III proteins. Although ALIX has been reported to accumulate at budding sites in parallel with the viral Gag protein, we have recently used quantitative live imaging to show that ALIX is actually recruited transiently following completion of Gag assembly. We find that as the virus buds most of the recruited ALIX is recycled back into the cytosol, and only a fraction remains within the virus. Further, we find that the remaining ALIX molecules within released virus like particles (VLPs) are located asymmetrically within the VLP. Our lab is now focused on understanding how ALIX is recruited at the end of Gag assembly and released during last stages of budding.
Our lab focuses on understanding the mechanism of assembly and replication of enveloped RNA viruses. It is possible that the earliest form of life was RNA based. Indeed the first self replicating molecule made by man was the Spigelman's monster which consisted of ~200 nt long RNA that replicates using a replicase. A significant section of human pathogenic viruses have an RNA genome, these include HIV, Influenza, Rabies, measles and Ebola. All these viruses have to deliver their RNA genome into a host cell and replicate it to produce infection progeny viruses. It is therefore not surprising that we have evolved a variety of tools to neutralize these processes ranging from simple RNA digestion enzymes that cleave any naked non cellular RNA to more elaborate innate immune responses and specific antibodies that help identify these virions to the immune system for destruction. The battle between the RNA viruses and eukaryotic cells is likely as old as Biology and the interface between these pathogens and their cellular antiviral responses is among the fastest evolving fronts on the planet. Developing a detailed mechanistic understanding of viral assembly and replication therefore provides both a window into this exciting fast evolving world and also a road map for developing future antiviral responses.
Research Assistant Professor
National Institute of Health
National Science Foundation