Research Program
Nanoscale Cell Biology

Project #
NCB9

Participating Faculty:	L. Pollack, G. Whittaker, B. Crane and S. Daniel
NBTC Students/Postdocs:	Jessica Lamb, Shoshannah Roth, Ken Gee
Other Students/Postdocs:	Ikenna Madu, Xiangjie Sun, Suzette Pabit, Brian Zoltowski, Sandrine Belouzard, Sudhir Prabhu, Li Li

Objectives
Viruses are the ultimate nano-machines, programmed to invade cells to deliver genomic materials.  This invasion can be triggered by exposure to low pH, as found in the endosome of host cells. The goal of this project is to unravel critical steps in this attack. To achieve this goal, we will employ two distinct microfluidic methods to alter the pH around whole virus particles and the isolated proteins responsible for fusion of viral and host membranes. This year’s efforts will focus on characterizing the conformational dynamics of influenza hemagglutinin (HA), subtype H2: an important and poorly characterized fusion protein. We have chosen the H2 subtype in particular as it is a known human pathogen, but is not currently circulating in the population and as such is a high priority virus for the impending flu pandemic.

First, the conformational dynamics of H2 will be measured by time- resolved small angle x-ray scattering (SAXS). Preliminary scattering data indicate that the relevant time scales for pH activation of HA are accessible to our microfluidic tools. Coupled studies of other macromolecular systems demonstrate the feasibility of reconstructing transient states from time resolved SAXS data, potentially with enough detail to unravel the fusion mechanism.

Second, using methods developed within this program for effecting a rapid pH jump, we aim to study the fusion of virus-like-particles containing H2 with cell membrane mimics contained within microfluidic channels.  Fluorescent reporters will allow us to monitor the time-scale for fusion of single particles.  Control of composition of model membranes will reveal the relative effect of different lipid components on membrane fusion.

Methods
To study the conformational dynamics of influenza H2, we plan to use a four-port, rapid microfluidic mixer in conjunction with small angle x-ray scattering. Conformational changes will be triggered by a rapid (sub-millisecond) change in pH. Since the H2 protein undergoes a reversible conformational change, this mixer provides the flexibility of either increasing or decreasing the pH, to study this reversible conformational change. Time scales ranging from 0.5 through 500 ms are accessible to this technology.

We also plan to use total internal reflection microscopy (TIRFM) to examine fluorescent markers indicating two sequential steps in viral infection (a): membrane fusion and (b): transfer of vesicle contents into the ‘host cell’ mimic, consisting of bilayers suspended within microfluidic channels.  To obtain the time scale for H2-enabled fusion of virus like particles, VLPs, they will be suitably labeled with dyes in the membrane and within the vesicle. Fusion will be triggered by a rapid decrease in the pH of the surrounding solution. Distinct fluorescence markers present within the samples can be used to monitor the events of interest.

Summary
Using the methods outlined above (rapid pH jump, time resolved SAXS, and fluorescence microscopy within microchannels), we plan to characterize the time scale and structural dynamics that accompany viral fusion. Our focus is on the influenza virus H2 protein, which may pose a significant future threat to human health. Characterization of these important proteins is currently limited.

Accomplishments

• Time resolved small angle x-ray scattering detects conformational change of light sensitive protein
• Architecture of fusion loop of VSV G protein identified
• Baculovirus vectors tested for robust expression system