TY  - JOUR
TI  - Purification of HCV-remodeled and Control ER Membranes
AU  - Paul, David  
AU  - Bartenschlager, Ralf  
VL  - 4
IS  - 10
PY  - 2014
DA  - 2014/05/20
SP  - e1134
C1  - Bio-protocol 2014;4:e1134
DO  - 10.21769/BioProtoc.1134
UR  - https://doi.org/10.21769/BioProtoc.1134
AB  - As for all positive strand RNA viruses, hepatitis C virus (HCV) RNA replication is tightly associated with rearranged host cell membranes, termed viral replication factories. However, up to now little is known about both viral and cellular constituents of viral replication factories. Here, we describe a protocol to specifically isolate HCV-remodeled host cell membranes and endoplasmic reticulum (ER) membranes of naïve cells, by using a functional NS4B HA-tagged subgenomic replicon and a C-terminally HA-tagged calnexin-overexpressing cell line, respectively. Post-nuclear whole cell membrane fractions are first enriched by density gradient centrifugation, followed by HA-specific affinity tag purification. Upon elution under native conditions, purified samples can be subject to a variety of biochemical and functional assays.
KW  - Hepatitis c virus
KW  - Double membrane vesicles
KW  - Affinity purification
KW  - Plus-strand RNA virus
KW  - Replication factory
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False