Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Pivot Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Target Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Query: EC:3.4.21.69 (
APC
)
16,337
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
Mature
protein C
of tick-borne encephalitis virus (TBEV) is cleaved from the polyprotein precursor by the viral NS2B/3 protease (NS2B/3(pro)). We showed previously that replacement of the NS2B/3(pro) cleavage site at the C terminus of
protein C
by the foot-and-mouth disease virus (FMDV) 2A StopGo sequence leads to the production of infectious virions. Here, we show that infectious virions can also be produced from a TBEV mutant bearing an inactivated 2A sequence through the expression of the FMDV
3C protease
(3C(pro)) either in cis or in trans (from a TBEV replicon). Cleavage at the C terminus of
protein C
depended on the catalytic activity of 3C(pro) as well as on the presence of an optimized 3C(pro) cleavage site. Passage of the TBEV mutants bearing a 3C(pro) cleavage site either in the absence of 3C(pro) or in the presence of a catalytically inactive 3C(pro) led to the appearance of revertants in which
protein C
cleavage by NS2B/3(pro) had been regained. In three different revertants, a cleavage site for NS2B/3(pro), namely RR*C, was now present, leading to an elongated
protein C
. Furthermore, two revertants acquired additional mutations in the C terminus of
protein C
, eliminating two basic residues. Although these latter mutants showed wild-type levels of early RNA synthesis, their foci were smaller and an accumulation of
protein C
in the cytoplasm was observed. These findings suggest a role of the positive charge of the C terminus of
protein C
for budding of the nucleocapsid and further support the notion that TBEV
protein C
is a multifunctional protein.
...
PMID:Generation and genetic stability of tick-borne encephalitis virus mutants dependent on processing by the foot-and-mouth disease virus 3C protease. 2213 10
The replication of tick-borne encephalitis virus (TBEV), like that of all flaviviruses, is absolutely dependent on proteolytic processing. Production of the mature proteins C and prM from their common precursor requires the activity of the viral NS2B/3 protease (NS2B/3(pro)) at the C-terminus of
protein C
and the host signal peptidase I (SPaseI) at the N-terminus of protein prM. Recently, we have shown in cell culture that the cleavage of
protein C
and the subsequent production of TBEV particles can be made dependent on the activity of the foot-and-mouth disease virus
3C protease
, but not on the activity of the HIV-1 protease (HIV1(pro)) (Schrauf et al., 2012). To investigate this failure, we developed an in vitro cleavage assay to assess the two cleavage reactions performed on the C-prM precursor. Accordingly, a recombinant modular NS2B/3(pro), consisting of the protease domain of NS3 linked to the core-domain of cofactor NS2B, was expressed in E. coli and purified to homogeneity. This enzyme could cleave a C-prM protein synthesised in rabbit reticulocyte lysates. However, cleavage was only specific when protein synthesis was performed in the presence of canine pancreatic microsomal membranes and required the prevention of signal peptidase I (SPaseI) activity by lengthening the h-region of the signal peptide. The presence of membranes allowed the concentration of NS2B/3(pro) used to be reduced by 10-20 fold. Substitution of the NS2B/3(pro) cleavage motif in C-prM by a HIV-1(pro) motif inhibited NS2B/3(pro) processing in the presence of microsomal membranes but allowed cleavage by HIV-1(pro) at the C-prM junction. This system shows that processing at the C-terminus of
protein C
by the TBEV NS2B/3(pro) is highly membrane dependent and will allow the examination of how the membrane topology of
protein C
affects both SPaseI and NS2B/3(pro) processing.
...
PMID:NS2B/3 proteolysis at the C-prM junction of the tick-borne encephalitis virus polyprotein is highly membrane dependent. 2272 84
