Atsuko Negishi

Development and Characterization of Novel Empty Adenovirus Capsids and Their Impact on Cellular Gene Expression

ABSTRACT Adenovirus (Ad) has been extensively studied as a eukaryotic viral vector. As these vectors have evolved from first-generation vectors to vectors that contain either very few or no viral genes (“gutless” Ad), significant reductions in the host innate immune response upon infection have been observed. Regardless of these vector improvements an unknown amount of toxicity has been associated with the virion structural proteins. Here we demonstrate the ability to generate high particle numbers (1011 to 1012) of Ad empty virions based on a modification of Cre/lox gutless Ad vectors. Using a battery of analyses (electron microscopy, atomic force microscopy, confocal images, and competition assays) we characterized this reagent and determined that it (i) makes intact virion particles, (ii) competes for receptor binding with wild-type Ad, and (iii) enters the cell proficiently, demonstrating an ability to carry out essential steps of viral entry. To further study the biological impact of these Ad empty virions on infected cells, we carried out DNA microarray analysis. Compared to that for recombinant Ad, the number of mRNAs modulated upon infection was significantly reduced but the expression signatures were similar. This reagent provides a valuable tool for studies of Ad in that researchers can examine the effect of infection in the presence of the virion capsid alone.

Quantitative manipulation of DNA and viruses with the nanomanipulator scanning force microscope

The nanoManipulator, a modified scanning force microscope with a direct manipulation interface that allows natural and controlled manipulation of nanometer-sized samples, was used to investigate DNA and adenoviruses. During a manipulation, the scanning tip is coupled to a hand-held stylus via a force-feedback loop. Accordingly, the tip follows the movement of the users hand; moreover, the user is able to feel the topography of the sample. For imaging fragile biological samples, a magnetically driven intermittent contact (MDIC) mode in fluid was implemented and added to the microscope. In this mode, a cantilever with a magnetic particle glued onto its back is driven by an external, oscillating magnetic field. In contrast to regular intermittent contact (tapping) mode imaging in liquids, where the whole liquid cell is excited (resulting in many resonance peaks in the spectrum), in this mode the cantilever is driven directly and the spectrum usually displays a single, easily identifiable resonance peak. Using this set-up, adenoviruses were manipulated in liquids and 1100 bp DNA molecules were manipulated in ambient conditions while recording lateral force data. In these experiments, the rupture force of DNA was measured directly for the first time. In an effort to measure the interaction force between a virus and a substrate, adenoviruses were pushed over a silicon substrate. A force of ∽25 nN was measured to move the virus on this surface. Copyright