Purification and Proteomic Analysis of Alphavirus Particles from Sindbis Virus Grown in Mammalian and Insect Cells.

Raquel Hernandez,Davis Ferreira,Trevor Glaros,Gabrielle Rizzo

doi:10.21769/bioprotoc.3239

Abstract

Current mass spectrometry (MS) methods and new instrumentation now allow for more accurate identification of proteins in low abundance than previous protein fractionation and identification methods. It was of interest if this method could serve to define the virus proteome of a membrane-containing virus. To evaluate the efficacy of mass spec to determine the proteome of medically important viruses, Sindbis virus (SINV), the prototypical alphavirus was chosen for evaluation. This model system was chosen specifically because the alphaviruses contain members which are human pathogens, this virus is well defined biochemically and structurally, and grows to high titers in both vertebrate and non-vertebrate host cells. The SINV proteome was investigated using this method to determine if host proteins are specifically packaged into infectious virions. It was also of interest if the SINV proteome, when grown in multiple host cells representing vertebrate and mosquito hosts, incorporated specific host proteins from all hosts. Observation of recurrent or distinctive proteins in the virus proteome aided in the determination of proteins incorporated into the virion as opposed to those bound to the particle exterior. Mass spectrometry analysis identified the total protein content of purified virions within limits of detection. The most significant finding was that in addition to the host proteins, SINV non-structural protein 2 (nsP2) was detected within virions grown in all host cells examined. This analysis identified host factors not previously associated with alphavirus entry, replication, or egress, identifying at least one host factor integrally involved in alphavirus replication. Key to the success of this analysis is the method of virus purification which must deliver measurably infectious virus free of high levels of contaminants. For SINV and other members of the alphavirus family, this is accomplished by isopycnic centrifugation through potassium tartrate, followed by a high salt wash.

Highlights

[Background] The terms proteome and proteomics were coined by Marc Wilkins to describe the systematic evaluation of proteins in a model system using a detailed study of structure, function and regulation of its biology including aberrations which lead to disease (Wilkins et al, 1996 and 2009)
Virus proteomes have been under investigation long before the field of proteomics evolved in an attempt to understand the mechanisms of virus-host interactions in vitro and to evaluate virus
Sindbis virus was chosen for this investigation because it is a member of the Alphavirus genus, Family Togaviridae, which contains a significant number of human pathogens of medical importance, it is structurally stable, grows to high titers, is well described in the literature and can be grown in vertebrate and non-vertebrate host cells

Summary

Introduction

[Background] The terms proteome and proteomics were coined by Marc Wilkins to describe the systematic evaluation of proteins in a model system using a detailed study of structure, function and regulation of its biology including aberrations which lead to disease (Wilkins et al, 1996 and 2009). It is important to make the serial dilutions of virus immediately prior to infection of the plaque assay flasks/plates to limit the amount of virus binding to the tubes.

Results

Conclusion