Dimerization of the hepatitis C virus nonstructural protein 4B depends on the integrity of an aminoterminal basic leucine zipper.
The hepatitis C virus (HCV) nonstructural (NS) protein 4B is known for protein-protein interactions with virus and host cell factors. Only little is known about the corresponding protein binding sites and underlying molecular mechanisms. Recently, we have predicted a putative basic leucine zipper (bZIP) motif within the aminoterminal part of NS4B. The aim of this study was to investigate the importance of this NS4B bZIP motif for specific protein-protein interactions. We applied in silico approaches for 3D-structure modeling of NS4B-homodimerization via the bZIP motif and identified crucial amino acid positions by multiple sequence analysis. The selected sites were used for site-directed mutagenesis within the NS4B bZIP motif and subsequent co-immunoprecipitation of wild-type and mutant NS4B molecules. Respective interaction energies were calculated for wild-type and mutant structural models. NS4B-homodimerization with a gradual alleviation of dimer interaction from wild-type towards the mutant-dimers was observed. The putative bZIP motif was confirmed by a co-immunoprecipitation assay and western blot analysis. NS4B-NS4B interaction depends on the integrity of the bZIP hydrophobic core and can be abolished due to changes of crucial residues within NS4B. In conclusion, our data indicate NS4B-homodimerization and that this interaction is facilitated by the aminoterminal part containing a bZIP motif.
Welker
Martin-Walter
MW
Klinikum der Johann Wolfgang Goethe-Universität, Medizinische Klinik 1, Theodor-Stern-Kai 7, Frankfurt am Main 60590, Germany.
Welsch
Christoph
C
Meyer
Aline
A
Antes
Iris
I
Albrecht
Mario
M
Forestier
Nicole
N
Kronenberger
Bernd
B
Lengauer
Thomas
T
Piiper
Albrecht
A
Zeuzem
Stefan
S
Sarrazin
Christoph
C
D000595:Amino Acid Sequence; D047468:Immunoprecipitation; D016350:Leucine Zippers / Q000502:physiology; D008969:Molecular Sequence Data; D011485:Protein Binding; D055503:Protein Multimerization / Q000235:genetics / Q000502:physiology; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism*
C080107:NS4B protein, flavivirus; D017361:Viral Nonstructural Proteins