Analysis of Interactions Between Viral Replicase Proteins and Plant Intracellular Membranes
-
Last Update: 2021-01-19
-
Source: Internet
-
Author: User
Search more information of high quality chemicals, good prices and reliable suppliers, visit
www.echemi.com
Replication of the genome of positive-strand RNA plant viruses takes place in membrane-bound complexes that contain viral replicase proteins, viral RNA, and host proteins. Many viral replicase proteins play a crucial role in the assembly of replication complexes at intracellular membranes. They are integral membrane proteins that interact directly with the membranes and bring other proteins and the viral RNA to the complex via protein—protein or protein—RNA interactions. In this chapter, we describe subcellular fractionation methods that determine whether viral proteins are integral membrane proteins
in planta
. Differential centrifugation techniques are used to produce membrane-enriched fractions, which can then be analyzed for the presence of viral replicase proteins by immunoblotting. Confirmation of the membrane-association is obtained by membrane flotation assays and treatment of membrane-enriched fractions with high salt or high pH followed by detection of the viral proteins. Because many plant viruses replicate in association with the endoplasmic reticulum (ER), we also discuss two techniques to specifically analyze the interaction of viral proteins with these membranes. These techniques are continuous sucrose-gradient fractionation in the presence or absence of 3 mM Mg
2+
and glycosylation assays.
This article is an English version of an article which is originally in the Chinese language on echemi.com and is provided for information purposes only.
This website makes no representation or warranty of any kind, either expressed or implied, as to the accuracy, completeness ownership or reliability of
the article or any translations thereof. If you have any concerns or complaints relating to the article, please send an email, providing a detailed
description of the concern or complaint, to
service@echemi.com. A staff member will contact you within 5 working days. Once verified, infringing content
will be removed immediately.