Search

Alphavirus Purification Using Low-speed Spin Centrifugation   

Download PDF How to cite Favorites Q&A Share your feedback Cited by

In this protocol

Original research article

A brief version of this protocol appeared in:
Journal of Virology
Feb 2017

Abstract

Chemical and sedimentation procedures are used to purify virus particles. While these approaches are successful for wild-type viruses, they are often not feasible for purifying mutant viruses with assembly defects. We combined two published methods (Atasheva et al., 2013; Moller-Tank et al., 2013), to generate a protocol that uses low-speed centrifugation to purify both wildtype and mutant enveloped virus particles at high yield with minimal handling steps. This protocol has successfully been used to purify alphavirus particles for imaging and structural studies (Wang et al., 2015; Ramsey et al., 2017).

Keywords: Enveloped virus purification, Centrifugation, Assembly mutants

Background

Virus purification is traditionally based on chemical precipitation (e.g., PEG) or density gradient centrifugation. Centrifugation protocols involve pelleting particles at high speeds (> 100,000 x g) or through sedimentation matrices such as cesium, Nycodenz, Iodixanol, sucrose, glycerol, or tartrate. After sedimentation in the gradient, the purified virus sample usually requires additional steps to remove the gradient matrix, concentrate the purified particles, and buffer exchange into a stabilizing buffer for downstream applications. These approaches include dialysis, centrifugation through a centrifugal filter, or PEG precipitation. While these approaches will produce purified particles, there are several drawbacks: (1) overall yields can be low, (2) time required for the purification can extend to a week, (3) morphologically heterogeneous particles are not purified with equal efficiency, (4) particles can be damaged in the process, and (5) assembly mutants often do not survive the purification process making certain downstream analyses challenging.

The protocol described here uses a gentle approach to purify enveloped virus particles. We used minimal centrifugal force to reduce damage to particles which is observed as increased morphological heterogeneity in negative-stain transmission electron microscopy (TEM) or total loss of fragile particles by TEM or infectivity assays. In addition, we wanted to reduce the manipulation of purified virions post-purification. By merging two protocols from the literature (Atasheva et al., 2013; Moller-Tank et al., 2013), we are able to purify different Alphaviruses (Sindbis [Ramsey et al., 2017], Ross River [Wang et al., 2015], Chikungunya [Mukhopadhyay and Wang, unpublished data] and assembly mutants of each) via low-speed centrifugation (LSC). No additional steps for gradient matrix removal, sample concentration, or buffer exchange are necessary. We are also able to use this protocol to purify viruses from different cell lines (mammalian and arthropod). These purified particles have been used for cryo-EM, mass spectrometry, and protease cleavage studies.

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Rayaprolu, V., Ramsey, J., Wang, J. C. and Mukhopadhyay, S. (2018). Alphavirus Purification Using Low-speed Spin Centrifugation. Bio-protocol 8(6): e2772. DOI: 10.21769/BioProtoc.2772.
Q&A

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.

You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.