Oleg Iourin

Structure of a Pestivirus Envelope Glycoprotein E2 Clarifies Its Role in Cell Entry

Summary 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.

Atomic Resolution Structure of Moloney Murine Leukemia Virus Matrix Protein and Its Relationship to Other Retroviral Matrix Proteins

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.

Unexpected structure for the N-terminal domain of hepatitis C virus envelope glycoprotein E1

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.

Structure of Equine Infectious Anemia Virus Matrix Protein

ABSTRACT 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-Å 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.

The identification of abnormal glycoforms of serum transferrin in carbohydrate deficient glycoprotein syndrome type i by capillary zone electrophoresis

One of the biochemical characteristics of carbohydrate deficient glycoprotein syndromes is the presence of abnormal glycoforms in serum transferrin. Both glycoform heterogeneity and variable site occupancy may, in principle, lead to the generation of a range of glycoforms which contain different numbers of sialic acid residues, and therefore variable amounts of negative charge. Capillary zone electrophoresis was used to resolve the glycoforms of normal human serum transferrin and also of a set of glycoforms which were prepared by digesting the sugars on the intact glycoprotein with sialidase. The sugars on the intact glycoprotein were also modified by a series of exoglycosidase enzymes to produce a series of neutral glycoforms which were also analysed by capillary zone electrophoresis. The oligosaccharide population of human serum transferrin was analysed by a series of mixed exoglycosidase digests on the released glycan pool and quantified using a novel HPLC strategy. Transferrin was isolated from carbohydrate deficient glycoprotein syndromes type I serum and both the intact glycoforms and released sugars were resolved and quantified. The data presented here confirm the presence of a hexa-, penta- and tetra-sialoforms of human serum transferrin in both normal and carbohydrate deficient glycoprotein syndrome type I serum samples. Consistent with previous reports carbohydrate deficient glycoprotein syndrome type I transferrin also contained a di-sialoform, representing a glycoform in which one of the two N-glycosylation sites is unoccupied, and a non-glycosylated form where both remain unoccupied. This study demonstrates that capillary zone electrophoresis can be used to resolve quantitatively both sialylated and neutral complex type glycoforms, suggesting a rapid diagnostic test for the carbohydrate deficient glycoprotein syndromes group of diseases.

Cloning and characterization of hIF2, a human homologue of bacterial translation initiation factor 2, and its interaction with HIV-1 matrix

The cDNA for a human homologue (hIF2) of bacterial (bIF2) and yeast (yIF2) translation initiation factor two (IF2) has been identified during a screen for proteins which interact with HIV-1 matrix. The hIF2 cDNA encodes a 1220-amino-acid protein with a predicted relative molecular mass of 139 kDa, though endogeneous hIF2 migrates anomalously on SDS/PAGE at 180 kDa. hIF2 has an extended N-terminus compared with its homologues, although its central GTP-binding domain and C-terminus are highly conserved, with 58% sequence identity with yIF2. We have confirmed that hIF2 is required for general translation in human cells by generation of a point mutation in the P-loop of the GTP-binding domain. This mutant protein behaves in a transdominant manner in transient transfections and leads to a significant decrease in the translation of a reporter gene. hIF2 interacts directly with HIV-1 matrix and Gag in vitro, and the protein complex can be immunoprecipitated from human cells. This interaction appears to block hIF2 function, since purified matrix protein inhibits translation in a reticulocyte lysate. hIF2 does not correspond to any of the previously characterized translation initiation factors identified in mammals, but its essential role in translation appears to have been conserved from bacteria to humans.

Pushing the limits of sulfur SAD phasing: de novo structure solution of the N-terminal domain of the ectodomain of HCV E1

The sulfur SAD phasing method was successfully used to determine the structure of the N-terminal domain of HCV E1 from low-resolution diffracting crystals by combining data from 32 crystals.

Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus

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.