Vincent R. Racaniello, PhD

Much of our work of the past 17 years has been on poliovirus, the causative agent of poliomyelitis. Poliovirus infection begins when the virus binds to a cell surface receptor and subsequently releases the viral nucleic acid into the cell. Poliovirus is a particularly good model for studying these interactions. The three dimensional structure of poliovirus has been determined by X-ray crystallography, the cell receptor for poliovirus has been identified, and genetic manipulation of the virus is possible with infectious cDNA copies of the viral genome. One goal of our research is to use these experimental tools to identify regions of the capsid and Pvr that initially interact and lead to the structural transitions associated with uncoating of the viral RNA. These questions are addressed by the construction and analysis of mutants of the viral capsid and the cellular receptor. To learn more about how poliovirus causes disease, we have established a new mouse model for poliomyelitis. Mice are normally resistant to poliovirus infection, but transgenic mice expressing Pvr develop poliomyelitis after inoculation with virulent strains of the virus. This mouse model is being used to address questions on the pathogenesis of poliomyelitis, such as how poliovirus infection is restricted to specific cell types such as neurons, how the virus spreads in the infected animal, and the basis for the attenuation phenotype of the live poliovirus vaccines. We are using a similar approach to establish mouse models for diseases caused by other picornaviruses, including echoviruses, rhinoviruses, and enteroviruses. To establish a mouse model for rhinovirus infection, we first examined the ability of the virus to replicate in cultured mouse cells. Rhinovirus type 16 replicates in mouse L cells that express the viral receptor, human intercellular adhesion molecule 1 (ICAM-1). Infection leads to no discernible cytopathic effect, and low virus titers are produced. A variant virus, 16/L, was isolated after passage of rhinovirus in ICAM-1 L cells. Virus 16/L produces higher virus titers and viral RNA, and efficiently kills ICAM-1 L cells. Adaptation to mouse cells maps to two changes in viral protein 2C. How changes in this protein improve replication in mouse cells is currently under investigation. The poliovirus receptor is a member of the immunoglobulin superfamily of proteins and consists of an NH2-terminal signal sequence, three extracellular Ig-like domains, a transmembrane domain, and a cytoplasmic tail. Sequence homologs of pvr have been identified in humans (nectin-1 and nectin-2), monkeys (agm1 and agm2), mice (nectin-1 and nectin-2), and rats (pE4). Only pvr, agm1 and agm2 encode proteins that can function as cell receptors for poliovirus. Pvr, Nectin-1, and Nectin-2 are entry cofactors for alphaherpesviruses. To understand the cell function of members of the Pvr family, we inactivated the nectin-2 gene in the mouse. Nectin-2 is a component of cell-cell adherens junctions, and interacts with afadin, an actin-binding protein. Disruption of both alleles of the murine nectin-2 gene resulted in morphologically aberrant spermatozoa with defects in nuclear and cytoskeletal morphology and mitochondrial localization. As a result, homozygous null males are sterile. The structural defects observed in spermatozoa of mice lacking Nectin-2 suggest a role for this protein in organization and reorganization of the cytoskeleton during spermiogenesis. The interaction of Pvr family members with the cytoskeleton might play a role in axonal transport of poliovirus in the infected animal.