Study of the structure-function relationship of viral proteins by using biochemical, biophysical and cell biology techniques. During the last years our research has focused on the study of the envelope proteins of the Hepatits B and C virus. These proteins play an essential role in the process of viral particles recognitionand, subsequently, by the possible prevention of the viral infection,

Methodologies and Techniques:
Protein purification. Primary structure analysis. Molecular and enzymatic characterization. Spectroscopical analysis (UV-absorbance; circular dichroism; fluorescence emission) of proteins. Cloning and expression in yeast and bacteria. Site-directed mutagenesis. Protein engineering.

Hepatitis B virus

(Image obtained from Scripps Research Institute)

The Hepatitis B surface antigen (HBsAg) is the base of the current vaccine against HBV infection. It is a macromolecular complex structure that forms lipoprotein particles. The main component is the small protein (S). Although these exist two minor proteins with the same C-terminal sequence that the S protein and amino terminal additions (preS domains), called protein M (middle, preS2+S) and L (large, preS1+ preS2+S).

(Image obtained from “Glebe, D.World J. Gastroenterol. (2007), 13: 22-38”)

The most recent results have been obtained with the preS region that has been related to the viral infectivity. This hydrophilic region has been expressed in Escherichia coli, both as the complete sequence (preS) and different deletion mutants, and as the separated domains (preS1 and preS2). The purification of these proteins in high quantities has allowed us to characterize their interaction with model systems constituted by phospholipids vesicles. Although both preS1 and preS2 domains interact with acidic phospholipids, the preS1 region seems to be responsible for the observed effects. Studies with preS1 deletion mutants indicate that the amino terminal end of this region is dispensable for the aggregation and destabilization of lipid vesicles.

            Different studies allow us to conclude that the preS domains of hepadnaviruses have similar properties and that they might be involved in the first stages of the infection process. On the other hand, the role of the myristoylation of the amino terminal glycine on the function of preS domains has been studied by coexpression of the preS domains and the yeast N-myristoyltransferase enzyme. The myristoylated protein reconstituted into PC vesicles is able to interact with model membrane systems and also to enter in duck hepatocytes in a specific way. Then, this domain seems to have all the necessary information to interact with the receptor and enter into the cell via endocytosis. Currently, we are using this model system to study the fusion and viral content release mechanism into the hepatocyte.

Hepatitis C virus

The genome of Hepatitis C virus (HCV) encodes two envelope proteins, E1 and E2, that could be responsible for the binding and entry of the virus into hepatic cells. Then, these proteins are the main candidates to develop vaccines against the HCV infection. We are studying different aspects currently unknown, such as the structure and the structure-funcion relathionship of E1 and E2, toward the understanding of the molecular biology of Hepatitis C.

(Imagen obtained from Louis E. Henderson (Frederick Cancer Research Center)

1.EXPRESSION AND PURIFICATION OF RECOMBINANT FORMS OF E1 AND E2 

Attempts to produce correctly folded HCV envelope proteins in Escherichia coli or in the metylotrophic yeast Pichia pastoris have been unsatisfactory. However, several forms of E1 and E2 ectodomains have been successfully obtained in the baculovirus-insect cells expression system.

 Putative glycosylation sites of E1 and E2. The glycosylation sites were identified by searching the consense sequence of N-glycosylation in the available sequences of E1 and E2 in the data base EMBL. The glycosylation position is indicated by numbers. At the bottom the percentage of conservation is indicated. The transmembrane domains are indicated in black. (Thesis of J. Daniel Tello).

By using this system we have obtained the E2 ectodomain, E2(661), that includes the amino acids 384-661, without the transmembrane region. Different studies indicate that recombinant E2(661) is correctly folded and able to recognize antibodies from HCV-infected patients. E2(661) is also able to interact with acidic phospholipid vesicles both at neutral and acidic pH, inducing the destabilization of the model membrane systems. 

            Furthermore, we have cloned and expressed chimeric fusion proteins with the E1 and E2 complete ectodomains (E1E2 and E2E1). The structural and functional characterization of these chimeric proteins indicate that ectodomains of E1 and E2 are folded in the recombinant proteins in a similar way to that they have on the virus surface. Also, these chimeric forms possess a fusogenic activity and they are able to interact with cellular receptors that are involved in the entry mechanism of the virus. All these results indicate that these proteins are a good model to study the first stages of the infective cycle of HCV and to develop a possible vaccine against the viral infection.

2.  MECHANISM OF THE HEPATITIS C VIRUS ENTRY IN THE CELL

There are some studies that indicate that the non covalent heterodimers E1E2 may have a role in the virus entry into the cell; specifically, E2 seems to be the protein involved in the interaction with cellular receptors. Besides their role in cellular receptor binding, the viral envelope proteins E1 and E2 must induce fusion of the viral and host cell membranes. It has been proposed that HCV-E2 is a Class II fusion protein. All the recombinant proteins obtained with the baculovirus system are able to interact with the plasma membrane of hepatic cells (Huh-7, HepG2, Hep3B). The interaction is mediated by the binding to the membrane receptors CD81 and SR-BI. After this interaction, the recombinant proteins are captured by different endocytosis mechanisms involving the actin cytoskeletal and clathrin coated vesicles. Part of the endocyted proteins is present in early endosomes, although they are also present in lysosomes, where they are probably degraded. Currently, we continue with the study and characterization of the endocytosis mechanism of HCV by using different biochemical and cell biology methodologies.