Prenylation and HDV Assembly

Within the 19 amino acids unique to L-HDAg lies a four-amino acid sequence motif conserved across HDV isolates. This motif, consisting of a cysteine situated exactly three amino acids from the C terminus, is termed a 'CXXX box' (where C = cysteine,andX=anyamino acid) (Maltese 1990). CXXXboxes are found in a variety of proteins and are significant because they represent the substrates recognized by a family of enzymes called prenyltransferases.

Prenyltransferases catalyze the covalent attachment of a prenyl lipid to the CXXX box cysteine, a process termed prenylation (Zhang and Casey 1996). Farnesyltransferase (FTase) adds a 15-carbon prenyl lipid (farnesyl) and geranylgeranyltransferase-I (GGTase I) adds a 20-carbon prenyl lipid (geranylgeranyl). These prenyl lipids are both derived from mevalonic acid. Of note, a second class of GGTases, GGTase type II, has a more complex substrate recognition and catalyzes the transfer of geranylgeranyl to cysteine residues contained in C-terminal motifs such as CC or CXC (Pereira-Leal and Seabra 2001).

Examples of farnesylated proteins include lamin B, Ras, and the Batten disease CLN3 protein, whereas the y-subunit of G proteins and the Rab proteins are examples of geranylgeranylated proteins (Farnsworth et al. 1989; Hancock et al. 1989; Novick and Brennwald 1993; Pullarkat and Morris 1997; Yamane et al. 1990). One effect of prenylation is to promote membrane association of the modified protein (Casey 1995). Prenylation can also play a major role in protein-protein interactions (Hoffman et al. 2000; Pfeffer and Aivazian 2004). It was hypothesized that the conserved CXXX box in large delta antigen was a substrate for prenylation and that such a lipid modification could help mediate interaction with the membrane-associated HBsAg required for HDV morphogenesis.

Labeling studies with [3H]-mevalonate - the metabolic precursor of prenyl lipids - have demonstrated both in in vitro translation reactions (Glenn et al. 1992) and in intact cells (Glenn et al. 1992; Hwang et al. 1992) that large delta antigen is indeed subject to prenylation.

The specific type of prenyl lipid added is farnesyl (Otto and Casey 1996). That such modification is critical to the HDV assembly process was indicated by site-directed mutagenesis studies. Indeed, genetic disruption of the delta antigen CXXX box-such as by substitution of the CXXX box cysteine with serine-abolishes prenylation of large delta antigen. The same mutation also abrogates large delta antigen's ability to interact with, and form secreted particles with, HBsAg (Glenn et al. 1992; Hwang and Lai 1993). This was the first demonstration that a viral protein could be modified by prenylation, and highlighted a novel mechanism of virion assembly.

Although genetically disrupting the delta antigen CXXX box would not be practical in natural infections, achieving a similar end result - namely prevention of prenylation - pharmacologically might be translated into a practical clinical strategy. As detailed below, this approach has been progressively eval uated, first with virus-like particles (VLPs), then complete infectious virions, and most recently in an in vivo mouse model of HDV

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