The goal of this project is to determine the mechanism of assembly of the envelope of vesicular stomatitis virus (VSV), the prototype rhabdovirus, which has been widely used to study virus assembly. Like many enveloped viruses, VSV has a matrix (M) protein, which serves as a bridge between the nucleocapsid and the virus envelope. The mechanism of how nucleocapsids and M proteins are assembled at the cytoplasmic surface of host membranes to form virus budding sites is a central question in virus assembly. According to virology textbooks, assembly at the plasma membrane is mediated through a transmembrane network of protein- protein interactions of M protein with nucleocapsids and the envelope glycoprotein (G protein). However, most of the available evidence in the literature argues against this textbook model of virus assembly. The research in this project has developed novel methods of electron microscopy analysis and new biophysical and biochemical tools, which have provided evidence for a new model for assembly of VSV envelopes, as follows: A key step is movement of viral nucleocapsids to membrane-proximal regions of the cytoplasm, where they associate with membrane microdomains that are enriched not in M protein, but rather in G protein. 2) These membrane microdomains serve as sites where association of M protein with nucleocapsids occurs. 3) Importantly, one or more of these steps is regulated by host factors that affect virus assembly. This model will be tested by the following aims:
Aim 1 is to test the hypothesis that a key step in the initiation of virus assembly is association of viral nucleocapsids with membrane microdomains that are enriched in G protein. These experiments will use new methods of confocal and electron microscopy analysis to study previously described virus mutants, in which assembly of nucleocapsids into virions is defective. The model will also be tested by complementary biochemical approaches and a modification of the """"""""microdomain proteomics"""""""" approach to identify host proteins associated with sites of virus assembly.
In Aim 2 the mechanism of assembly of M protein into nucleocapsid-M protein (NCM) complexes will be tested. These experiments will use novel stopped-flow light scattering approaches capable of measuring the rapid rates of these protein-protein interactions.
This Aim will also analyze the ability of mutant M proteins to be recruited into NCM complexes in vivo. These experiments will involve complementation of temperature-sensitive M protein mutants, which have been shown previously to be defective in assembly of M protein into virions. These experiments not only provide an in vivo assay for assembly of NCM complexes, but also have the potential to provide important new tools for studying the initiating steps in virus assembly.
Vesicular stomatitis virus (VSV) is an animal virus that has been studied for many years as a prototype for many important human viral pathogens. One of the important areas where study of VSV has yielded novel insights is the question of how viruses assemble their outer membrane (or envelope). Recent technological advances in viral genetics, structural biology, and image analysis of microscopy data have revealed that our textbook understanding of VSV envelope assembly is likely to be incorrect. The goal of this project is to test new ideas about how VSV assembles its envelope. This will provide fundamental new information on the molecular basis of how viruses like VSV cause disease, and should contribute to development of novel viruses for use as vaccines and for the treatment of human cancer.
|Yacovone, Shalane K; Ornelles, David A; Lyles, Douglas S (2016) The border-to-border distribution method for analysis of cytoplasmic particles and organelles. Cell Tissue Res 363:351-60|
|Yacovone, Shalane K; Smelser, Amanda M; Macosko, Jed C et al. (2016) Migration of Nucleocapsids in Vesicular Stomatitis Virus-Infected Cells Is Dependent on both Microtubules and Actin Filaments. J Virol 90:6159-70|
|Lyles, Douglas S (2013) Assembly and budding of negative-strand RNA viruses. Adv Virus Res 85:57-90|
|Johnson, John B; Lyles, Douglas S; Alexander-Miller, Martha A et al. (2012) Virion-associated complement regulator CD55 is more potent than CD46 in mediating resistance of mumps virus and vesicular stomatitis virus to neutralization. J Virol 86:9929-40|
|Dancho, Brooke; McKenzie, Margie O; Connor, John H et al. (2009) Vesicular stomatitis virus matrix protein mutations that affect association with host membranes and viral nucleocapsids. J Biol Chem 284:4500-9|
|Agnihothram, Sudhakar S; Dancho, Brooke; Grant, Kenneth W et al. (2009) Assembly of arenavirus envelope glycoprotein GPC in detergent-soluble membrane microdomains. J Virol 83:9890-900|
|Swinteck, B Dancho; Lyles, Douglas S (2008) Plasma membrane microdomains containing vesicular stomatitis virus M protein are separate from microdomains containing G protein and nucleocapsids. J Virol 82:5536-47|
|Connor, John H; McKenzie, Margie O; Parks, Griffith D et al. (2007) Antiviral activity and RNA polymerase degradation following Hsp90 inhibition in a range of negative strand viruses. Virology 362:109-19|
|Connor, John H; McKenzie, Margie O; Lyles, Douglas S (2006) Role of residues 121 to 124 of vesicular stomatitis virus matrix protein in virus assembly and virus-host interaction. J Virol 80:3701-11|