Recombinant Measles Viruses Efficiently Entering Cells through Targeted Receptors

Abstract

We sought proof of principle that one of the safest human vaccines, measles virus Edmonston B (MV-Edm), can be genetically modified to allow entry via cell surface molecules other than its receptor CD46. Hybrid proteins consisting of the epidermal growth factor (EGF) or the insulin-like growth factor 1 (IGF1) linked to the extracellular (carboxyl) terminus of the MV-Edm attachment protein hemagglutinin (H) were produced. The standard H protein gene was replaced by one coding for H/EGF or H/IGF1 in cDNA copies of the MV genome. Recombinant viruses were rescued and replicated to titers approaching those of the parental strain. MV displaying EGF or IGF1 efficiently entered CD46-negative rodent cells expressing the human EGF or the IGF1 receptor, respectively, and the EGF virus caused extensive syncytium formation and cell death. Taking advantage of a factor Xa protease recognition site engineered in the hybrid H proteins, the displayed domain was cleaved off from virus particles, and specific entry in rodent cells was abrogated. These studies prove that MV can be engineered to selectively eliminate cells expressing a targeted receptor and provide insights into the mechanism of MV entry.Measles virus (MV) is a nonsegmented negative-strand RNA virus of the family Paramyxoviridae, genus Morbillivirus. It remains one of the leading causes of infant death in developing countries, but live attenuated vaccines derived from the MV strain Edmonston B (MV-Edm) have almost completely eliminated measles-related fatalities in countries that have adopted compulsory vaccination (13). We aim to transform MV-Edm, an extremely cost-effective public health tool, into a replicating vector for cytoreductive therapy. Restriction of cell entry through the use of receptor-specific virus attachment is the approach that we are developing to achieve selective MV replication in target cells. To this end, we describe here the production of dual tropic MV-Edm derivatives that specifically enter rodent cells through a targeted receptor.The MV envelope is composed of two glycoproteins: the hemagglutinin (H) and the fusion (F) protein. The H protein binds directly to the cellular receptor (17), and the F protein, containing a putative hydrophobic fusion peptide, executes fusion between the viral and the cell membrane at a neutral pH (50). A cellular receptor for MV-Edm is the type I glycoprotein CD46 (18, 34), a ubiquitous regulator of complement activation expressed in humans and Old World monkeys. CD46 is a major determinant of virus tropism, and its expression allows MV binding, entry, and replication in normally nonsusceptible rodent cells (6,18,33). The CD46 extracellular part consists of four complement control protein domains (CCP) and other small repeats (30). The viral H protein binds initially the two most distal domains, CCP 1 and 2 (6, 32), followed by secondary contacts with CCP 4 (12, 16). Several CCP 1 and 2 amino acids important for virus binding have been identified (5,22), and the crystal structure of these domains (8) reveals an...

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