Significant progress has been made in understanding early events in adenovirus (Ad) entry as well as inhibition of these processes by immune effectors. In contrast, late events in Ad entry including microtubule (MT)-dependent cytoplasmic transport to the nucleus are poorly understood. As a number of viral and nonviral pathogens exploit MT-dependent transport as a key component of their lifecycles, this common entry pathway is a likely restriction point for inhibition by immune mechanisms. The goal of this proposal is to study the mechanisms by which effectors of the adaptive (a neutralizing antibody) and innate (defensin peptides) immune systems inhibit late events in Ad entry. Preliminary studies have shown that a neutralizing antibody (NAb) increases the affinity of Ad for MTs and that this increased affinity may contribute to inhibition of infection. Using confocal microscopy of fixed cells and of live cells in real time, the impact of NAb binding on the MT-dependent cytoplasmic translocation of fluorescently labeled Ad will be determined. Studies have shown that the Ad capsid undergoes partial disassembly during entry; therefore, a biochemical approach will be used to study the effects of partial uncoating on the affinity of Ad for MTs and on the ability of the NAb to alter this affinity. In addition, we will determine whether NAb binding alters the specificity of the Ad capsid for cytoplasmic MT-dependent motors. Finally, the human antibody repertoire will be analyzed for specificities equivalent to this NAb as a first approach to determine whether NAbs with similar mechanisms of action are elicited by natural Ad infections. In addition to this adaptive immune effector, our preliminary studies have shown that defensin peptides, innate immune effectors, also inhibit late events in Ad entry. To understand the mechanism of this inhibition, we will study the ability of defensins to bind directly to Ad, determine whether defensins alter the affinity of Ad for MTs, and, using confocal microscopy, we will study the impact of defensins on the cellular localization of Ad during entry. We propose to identify ways in which the human immune system blocks adenovirus infection by preventing the virus, once it has entered the cell, from reaching the nucleus. Many viruses infect cells through a pathway that is similar to that used by adenovirus; therefore, these studies are likely applicable to other viral systems. In addition, an understanding of the ways in which the immune system blocks infection may guide the design of antiviral drugs. ? ? ?
| Smith, Jason G; Silvestry, Mariena; Lindert, Steffen et al. (2010) Insight into the mechanisms of adenovirus capsid disassembly from studies of defensin neutralization. PLoS Pathog 6:e1000959 |
| Silvestry, Mariena; Lindert, Steffen; Smith, Jason G et al. (2009) Cryo-electron microscopy structure of adenovirus type 2 temperature-sensitive mutant 1 reveals insight into the cell entry defect. J Virol 83:7375-83 |
| Smith, Jason G; Cassany, Aurelia; Gerace, Larry et al. (2008) Neutralizing antibody blocks adenovirus infection by arresting microtubule-dependent cytoplasmic transport. J Virol 82:6492-500 |
| Smith, Jason G; Nemerow, Glen R (2008) Mechanism of adenovirus neutralization by Human alpha-defensins. Cell Host Microbe 3:11-9 |
