Multimodal Imaging of Host-Viral Lifecycle (Major Collaborator-Driven Research Project)

UC Davis: RH Cheng, F Chuang, T Huser, G McNerney, P Soonsawad; 

Mount Sinai School of Medicine: B Chen, W Hübner

PUBLISHED IN SCIENCE March 27, 2009

JOURNAL ARTICLE: Quantitative 3D Video Microscopy of HIV Transfer Across T Cell Virological Synapses

NSF CBST Press release

CBST Photos and Videos

Huser Group's Announcement

The investigation of complex biological processes ideally requires imaging that provides fast data acquisition (on the order of diffusion time constants for 5-50 nm sized objects), the ability to locate and track molecular structures with minimal interference by large exogenous probes, high contrast, high spatial

resolution, and unlimited time constraints on the imaging process. Since no single microscopic technique can provide all of these attributes at once, we are exploring the use of multi-modal imaging. In this project we are investigating the combination of several advanced optical analysis techniques that include dynamic spinning-disk confocal fluorescence microscopy, deconvolution microscopy, static high-resolution structured illumination fluorescence microscopy, and coherent anti-Stokes Raman scattering (CARS) label-free microscopy – in parallel with static electron microscopy to develop a much better understanding of viral infection, production, and transmission pathways using as models of human immunodeficiency virus (HIV) and echovirus (EV1).

In collaboration with researchers at the Mount Sinai School of Medicine in New York, CBST has produced the first real-time video images of direct cell-to-cell transmission of human immunodeficiency virus (HIV). Prof. Benjamin Chen from the Division of Infectious Disease at Mt. Sinai was recently successful in fusing green fluorescent protein (GFP) to Gag - a key structural protein of HIV. In contrast to previous attempts to engineer similar proteins, the resultant virus proved to be competent and able to infect other CD4+ lymphocytes. Using conventional fluorescence microscopy, Dr. Chen was able to demonstrate HIV transmission through direct cell contact -- challenging the current belief that free virus is the principal mode of infection. Using the 3D spinning disk confocal microscope at CBST, we are able to observe this process in much greater detail (more than 10 times the useful information in terms of spatial and temporal resolution), and reconstruct video images to focus on the structure and behavior of the virological synapse, which forms at the interface of cells in contact. By uncovering the different modes of Gag movements in the donor cell, we may be able to identify new targets or strategies for antiviral therapy.

With echovirus 1 (EV1) (a cousin of poliovirus) we are imaging the internalization of viral particles by cells into vesicles through macropinocytosis, which later mature into multivesicular bodies (MVBs). The process of EV1’s RNA separation from its capsid and leakage into the cytoplasm from the MVB is poorly understood, and our techniques should help produce various insights into this process. This approach allows us to image the infection process during the entire viral lifecycle and validate specific steps in this process by comparison to high-resolution cryo-electron microscopy. We are able to image the viral infection process in real-time, from the uptake of a virus, penetration of the nucleus, multiplication, and ultimate budding off of the new virus particles from the host. In addition to providing crucial information about the dynamics of the viral lifecycle, it will allow us to image the efficacy and action of novel antiviral drugs. It should also be noted that because CARS imaging is based on near-infrared lasers, it allows for deep tissue penetration, providing a route for future in vivo imaging.