ABSTRACT
Hepatitis C virus (HCV) infection remains a major health problem worldwide. HCV entry into host cells and membrane fusion are achieved by two envelope glycoproteins, E1 and E2. We report here the 3.5-Å resolution crystal structure of the N-terminal domain of the HCV E1 ectodomain, which reveals a complex network of covalently linked intertwined homodimers that do not harbour the expected truncated class II fusion protein fold.
Subject(s)
Hepacivirus/chemistry , Viral Envelope Proteins/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Gene Expression , HEK293 Cells , Humans , Molecular Sequence Data , Protein Folding , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Viral Envelope Proteins/geneticsABSTRACT
Single-wavelength anomalous dispersion of S atoms (S-SAD) is an elegant phasing method to determine crystal structures that does not require heavy-atom incorporation or selenomethionine derivatization. Nevertheless, this technique has been limited by the paucity of the signal at the usual X-ray wavelengths, requiring very accurate measurement of the anomalous differences. Here, the data collection and structure solution of the N-terminal domain of the ectodomain of HCV E1 from crystals that diffracted very weakly is reported. By combining the data from 32 crystals, it was possible to solve the sulfur substructure and calculate initial maps at 7â Å resolution, and after density modication and phase extension using a higher resolution native data set to 3.5â Å resolution model building was achievable.
Subject(s)
Hepacivirus/chemistry , Viral Envelope Proteins/chemistry , Amino Acid Sequence , Cloning, Molecular , Molecular Sequence Data , Protein Conformation , Viral Envelope Proteins/geneticsABSTRACT
Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress.
Subject(s)
Diarrhea Virus 1, Bovine Viral/chemistry , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/isolation & purification , Base Sequence , Cloning, Molecular , Crystallization/methods , Crystallography, X-Ray/methods , Molecular Sequence Data , Protein Conformation , Viral Envelope Proteins/geneticsABSTRACT
Enveloped viruses have developed various adroit mechanisms to invade their host cells. This process requires one or more viral envelope glycoprotein to achieve cell attachment and membrane fusion. Members of the Flaviviridae such as flaviviruses possess only one envelope glycoprotein, E, whereas pestiviruses and hepacivirus encode two glycoproteins, E1 and E2. Although E2 is involved in cell attachment, it has been unclear which protein is responsible for membrane fusion. We report the crystal structures of the homodimeric glycoprotein E2 from the pestivirus bovine viral diarrhea virus 1 (BVDV1) at both neutral and low pH. Unexpectedly, BVDV1 E2 does not have a class II fusion protein fold, and at low pH the N-terminal domain is disordered, similarly to the intermediate postfusion state of E2 from sindbis virus, an alphavirus. Our results suggest that the pestivirus and possibly the hepacivirus fusion machinery are unlike any previously observed.
Subject(s)
Diarrhea Virus 1, Bovine Viral/physiology , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolism , Virus Internalization , Amino Acid Sequence , Animals , Cattle , Conserved Sequence , Epitopes/chemistry , Epitopes/immunology , HEK293 Cells , Humans , Models, Molecular , Molecular Sequence Data , Protein Multimerization , Protein Stability , Protein Structure, Secondary , Protein Structure, Tertiary , Structure-Activity RelationshipABSTRACT
Matrix proteins associated with the viral membrane are important in the formation of the viral particle and in virus maturation. The 1.0 A crystal structure of the ecotropic Gammaretrovirus Moloney murine leukemia virus (M-MuLV) matrix protein reveals the conserved topology of other retroviral matrix proteins, despite undetectable sequence similarity. The N terminus (normally myristylated) is exposed and adjacent to a basic surface patch, features likely to contribute to membrane binding. The four proteins in the asymmetric unit make varied contacts. The M-MuLV matrix structure is intermediate, between those of the lentiviruses and other retroviruses. The protein fold appears to be maintained, in part, by the conservation of side chain packing, which may provide a useful tool for searching for weak distant similarities in proteins.
Subject(s)
Moloney murine leukemia virus/chemistry , Viral Matrix Proteins/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Sequence Homology, Amino AcidABSTRACT
The Gag polyprotein is key to the budding of retroviruses from host cells and is cleaved upon virion maturation, the N-terminal membrane-binding domain forming the matrix protein (MA). The 2.8-A resolution crystal structure of MA of equine infectious anemia virus (EIAV), a lentivirus, reveals that, despite showing no sequence similarity, more than half of the molecule can be superimposed on the MAs of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV). However, unlike the structures formed by HIV-1 and SIV MAs, the oligomerization state observed is not trimeric. We discuss the potential of this molecule for membrane binding in the light of conformational differences between EIAV MA and HIV or SIV MA.
