ABSTRACT
We have developed a system for the preparation of La Crosse virus (LAC) and Hantaan virus (HTN) pseudotypes using a murine leukemia virus vector. After concentration, the pseudotypes were present in quantities sufficient to analyze cell tropism and neutralization. Cells resistant to LAC could not be infected with the MLV (LAC) pseudotypes, and the pseudotypes were sensitive to neutralizing monoclonal antibodies prepared against LAC glycoproteins, as well as to inhibition by a soluble form of the virus cell-attachment protein, G1. Perhaps because of lower expression of the HTN glycoproteins at the cell surface, MLV (HTN) pseudotypes were present at lower titers. However, they were also sensitive to appropriate neutralizing antibodies. This pseudotype system will be useful for analysis of the entry process of the Bunyaviridae, and for neutralization studies with some Bunyaviruses whose high virulence normally requires specialized containment facilities.
Subject(s)
Genetic Vectors , Hantaan virus/physiology , La Crosse virus/physiology , Leukemia Virus, Murine/genetics , Animals , Antibodies, Monoclonal , Cell Line , Cell Membrane/virology , Cricetinae , Hantaan virus/genetics , Humans , La Crosse virus/genetics , Mice , Neutralization TestsABSTRACT
Proteolytic processing is required for the activation of numerous viral glycoproteins. Here we show that the envelope glycoprotein from the Zaire strain of Ebola virus (Ebo-GP) is proteolytically processed into two subunits, GP1 and GP2, that are likely covalently associated through a disulfide linkage. Murine leukemia virions pseudotyped with Ebo-GP contain almost exclusively processed glycoprotein, indicating that this is the mature form of Ebo-GP. Mutational analysis identified a dibasic motif, reminiscent of furin-like protease processing sites, as the Ebo-GP cleavage site. However, analysis of Ebo-GP processing in LoVo cells that lack the proprotein convertase furin demonstrated that furin is not required for processing of Ebo-GP. In sharp contrast to other viral systems, we found that an uncleaved mutant of Ebo-GP was able to mediate infection of various cell lines as efficiently as the wild-type, proteolytically cleaved glycoprotein, indicating that cleavage is not required for the activation of Ebo-GP despite the conservation of a dibasic cleavage site in all filoviral envelope glycoproteins.
Subject(s)
Ebolavirus/metabolism , Protein Processing, Post-Translational , Viral Envelope Proteins/metabolism , 3T3 Cells , Animals , Binding Sites , Cell Line , Cell Line, Transformed , Cricetinae , Ebolavirus/genetics , Endopeptidases/metabolism , Epitopes , Furin , Humans , Mice , Mutagenesis, Site-Directed , Oxidation-Reduction , Protein Denaturation , Rabbits , Subtilisins , Viral Envelope Proteins/geneticsABSTRACT
Tva is the cellular receptor for subgroup A avian leukosis and sarcoma virus (ALSV-A). The viral interaction domain of Tva is determined by a 40-residue, cysteine-rich module closely related to the ligand binding domain of the human low-density lipoprotein receptor (LDLR). In this report, we examined the role of the LDLR-like module of Tva in envelope binding and viral infection by mutational analysis. We found that the entire LDLR module in Tva is essential for efficient binding to the viral envelope protein. However, the 17 N-terminal residues of this module can be deleted without affecting receptor function, suggesting that the major determinants for viral entry are located at the C terminus of the module. The effect on viral infection of many amino acid substitutions and deletions in the LDLR module is context dependent, suggesting that the residues important for viral entry are dispersed throughout the LDLR module. In addition, we found that all 27 mutations at residues D46, E47, and W48 greatly reduced envelope binding. These results are discussed in relation to a recently elucidated structure for an LDLR module.
Subject(s)
Avian Leukosis Virus/physiology , Avian Sarcoma Viruses/physiology , Receptors, Virus/metabolism , Viral Envelope Proteins/metabolism , Virus Replication/physiology , Amino Acid Sequence , Amino Acid Substitution , Animals , Avian Proteins , Binding Sites , Cells, Cultured , DNA Mutational Analysis , Humans , Molecular Sequence Data , Receptors, LDL/genetics , Receptors, LDL/metabolism , Receptors, Virus/genetics , Sequence DeletionABSTRACT
Studies analyzing Ebola virus replication have been severely hampered by the extreme pathogenicity of this virus. To permit analysis of the host range and function of the Ebola virus glycoprotein (Ebo-GP), we have developed a system for pseudotyping these glycoproteins into murine leukemia virus (MLV). This pseudotyped virus, MLV(Ebola), can be readily concentrated to titers which exceed 5 x 10(6) infectious units/ml and is effectively neutralized by antibodies specific for Ebo-GP. Analysis of MLV(Ebola) infection revealed that the host range conferred by Ebo-GP is very broad, extending to cells of a variety of species. Notably, all lymphoid cell lines tested were completely resistant to infection; we speculate that this is due to the absence of a cellular receptor for Ebo-GP on B and T cells. The generation of high-titer MLV(Ebola) pseudotypes will be useful for the analysis of immune responses to Ebola virus infection, development of neutralizing antibodies, analysis of glycoprotein function, and isolation of the cellular receptor(s) for the Ebola virus.
