Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Summary

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

Abstract

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Introduction

Infection of the peripheral nervous system (PNS) by alphaherpes viruses such as herpes simplex virus (HSV) -1, -2, and pseudorabies virus (PRV) involves several intricate and highly regulated steps throughout the viral life cycle. Transport within neurons of the PNS is critical during the events of both primary viral infection and subsequent inter-host spread. The molecular mechanisms that modulate two components of the viral life cycle; directed transport of viral assemblies away from cell bodies within axons (anterograde transport) and subsequent transmission of virions to susceptible cells (anterograde spread) are important to understanding herpesvirus pathogenesis....

Protocol

Section 1 - Environmental Control Conditions for Live-cell Fluorescence Microscopy

1. 30 min prior to any imaging experiment connect and begin warming the microscope stage top incubator.
   1. Turn on microscope, transmitted and epifluorescent light sources, and automated hardware controls. Open the microscope control software, NIS Elements, to connect to the microscope and begin cooling the EM-CCD camera.
   2. Attach Lens warmer to appropriate objective (either 60X or 100X oil immersion). Note: Select objective lens based on experiment type. The 100X differential interference contrast (DIC) is best for tracking of anterograde tra....

Results

Anterograde Transport

Application of this protocol to infections of dissociated SCG cultures with PRV 348, a recombinant PRV strain expressing GFP-Us9 and gM-mCherry membrane fusion proteins, has facilitated the visualization of the anterograde transport of virions (Figure 3 and Supplemental Movie 4). Incorporation of these fusion proteins into viral particles results in their detection on transporting puncta, and the aforementioned imaging conditions minimize th.......

Discussion

The goal of live cell imaging is observing biological processes as they occur without significant alteration by the act of observation. This goal is achieved by optimizing three variables: environmental control, speed of imaging, and fluorescence illumination. These inter-dependent variables must be balanced to achieve viable imaging conditions. The protocols presented utilize specific conditions to produce the representative results. We will briefly discuss environmental controls and observational damage before detailin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35 mm glass bottom culture dish	MatTek Corporation; Ashland, MA	P35G-1.5-20-C
35 mm μ-Dish	Ibidi USA LLC, Verona, WI	81156
Microscope body	Nikon Instruments Inc.	Nikon Eclipse Ti
Motorized microscope X-Y stage	Prior Scientific; Rockland, MA	H117 ProScan Flat Top
Motorized filter wheels	Prior Scientific	HF110 10 position filter wheel
Fluorescent illumination source	Lumen Dynamics; Mississauga, Ontario Canada	X-Cite 120Q
Multi-band Fluorescence filter sets	Chroma Technology Corp.; Bellows Falls, VT	89000 Sedat Quad - ET 89006 ECFP/EYFP/mCherry - ET
EM-CCD camera	Andor Technology USA; South Windsor, CT	iXon3 897
Chamlide stage top environmental incubator	Live Cell Instruments; Seoul, South Korea	TC-L-10
Objective lens heater	Bioptecs; Butler, PA	150803 Controller 150819-12-08 Heater
Analysis software	Nikon Instruments Inc.; Melville, NY	NIS Elements