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(Received for publication, August 21, 1997)
From the Department of Biochemistry & Molecular Biology, University
of Kansas Medical Center, Kansas City, Kansas 66160
Polyprotein processing of dengue virus type 2, a
positive strand RNA virus, is carried out by the host signal peptidase
and a novel two-component viral proteinase of the serine proteinase family, NS2B/NS3(Pro), in the endoplasmic reticulum. Using an in
vitro processing system, we examined the cis and
trans cleavages of the 2B/3 and 4B/5 sites by
NS2B/NS3(Pro), respectively. Lysates of BHK-21 cells coexpressing NS2B
and NS3(Pro) mediated trans cleavage of the 4B/5 site
in vitro, and the protease activity was associated with the
membrane fraction. To study the role of membranes in the protease
activity of NS2B/NS3(Pro), labeled precursors, NS2B-NS3(Pro), and the
mutant ndNS2B-NS3(Pro) in which the functional hydrophilic domain of
NS2B was deleted, were analyzed using a coupled in vitro
transcription/translation system (TnT). The results showed that
cotranslational addition of microsomal membranes to the TnT reaction
markedly enhanced the cis cleavage of the 2B/3 site in a
dose-dependent manner. NS2B synthesized in the presence of
membranes also facilitated trans cleavage of the 2B/3 site in the mutant precursor. The cleavage products, NS2B and NS3(Pro), were
membrane-associated. Furthermore, this membrane requirement was
dictated by the hydrophobic regions of NS2B. Deletion of hydrophobic regions of NS2B, leaving only the conserved hydrophilic domain of 40 amino acids, resulted in highly efficient processing of the 2B-3 site
in vitro in the absence of microsomal membranes.
Volume 272, Number 49,
Issue of December 5, 1997
pp. 30715-30723
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Cotranslational Membrane Insertion of the Serine Proteinase
Precursor NS2B-NS3(Pro) of Dengue Virus Type 2 Is Required for
Efficient in Vitro Processing and Is Mediated through the
Hydrophobic Regions of NS2B
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