The Interaction Between the Hdv Rnp and the HBV Envelope Proteins

In general, the assembly of enveloped viruses occurs in a specific subcellular compartment where all the structural components colocalize (Garoff et al. 1998). Newly synthesized structural proteins are prevented from initiating budding reactions until all virion components are present at the site of assembly. This rule, however, does not apply to HBV because for this virus, budding is driven by the envelope proteins, irrespective of the presence of the nucleocapsid. This is obviously an important consequence for HDV, which can utilize the budding machinery to its profit.

Overall, the production of progeny HDV virions by an infected cell involves two processes, which are independent of each other: the formation of the RNP and that of the viral envelope. They are directed by distinct viral species, and they are spatially separated, occurring in the nucleus and at the ER membrane, respectively. Therefore, a critical step of the HDV life cycle is the encounter of the RNP with the HBV budding system. The RNP comprises both the S-HDAg and L-HDAg proteins associated with the viral RNA, and it is thought to shuttle between the cell nucleus and the cytoplasm (Lee et al. 2001; Tavanez et al. 2002). Export from the nucleus has been shown to rely on a specific nuclear export signal (NES) located in the 19 amino acid carboxyl terminus of L-HDAg (Lee et al. 2001), which also contains a carboxyl terminal CXXQ motif (where C = cysteine, Q = glutamine and X = any amino acid) for farnesylation (Glenn et al. 1992). The farnesyl group is covalently bound to the cysteine residue at position 211, and probably serves to anchor the RNP in the ER membrane where the envelope proteins assemble (Otto and Casey 1996). As an indirect proof of this phenomenon, treatment of HDV-producing cells with a farnesyl transferase inhibitor prevents assembly of RNPs into enveloped particles (Bordier et al. 2002; see also the chapter by J.S. Glenn, this volume). Interestingly, when expressed with the HBV envelope proteins in the absence of HDV RNA and S-HDAg, L-HDAg protein can be packaged and secreted in the SVPs (Chang et al. 1991; Chen et al. 1992; Ryu et al. 1992). This strongly suggests that L-HDAg mediates the incorporation of the RNP in the HBV envelope. Since deletions in the L-HDAg polypeptide can be performed between amino acids 2 and 195 without preventing packaging with S-HBsAg (Chang et al. 1994; Chen et al. 1992), the 19 amino acid carboxyl terminus is likely to constitute the packaging signal. Support for such a role comes also from the observation that its appending to the carboxyl terminus of a foreign protein, namely cHRas, leads to the co-secretion of the latter with SVPs (Lee et al. 1995). Ras is similar in size to L-HDAg and naturally farnesylated at its carboxyl terminus but cannot be packaged as such by S-HBsAg. In addition, a farnesylated S-HDAg could not be assembled with S-HBsAg indicating that the farnesyl group, per se, is not sufficient (Lee et al. 1994). The carboxyl terminus of L-HDAg is likely to mediate an interaction with S-HBsAg but, surprisingly, its amino acid sequence is not well conserved among the different HDV genotypes, except for the farnesylation signal CXXQ, for a tryptophan residue at position 196 and for the presence of at least five proline residues (Radjef et al. 2004). A mutational analysis of this domain has shown that substitution of Ala for Trp196 had no effect on packaging, whereas mutation of the proline residues at positions 201,204,205 and 208 (positions in L-HDAg genotype I) were detrimental (O'Malley and Lazinski 2005). Although Pro-201 and Pro-205 reside in the NES, it was demonstrated that the lack of packaging with S-HBsAg was not due to a deficient NES in the corresponding mutants but to a probable defect in S-HBsAg interaction. Whether L-HDAg binds directly to S-HBsAg or not, whether binding occurs during budding or beforehand, and whether the free form of L-HDAg binds to the envelope proteins in addition to the RNP-associated form, remain unknown (Sheu et al. 1996).

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