The SHBsAg Protein and the Assembly of HDV Particles

It is worthy of note that the members of the Hepadnaviridae family closest to HBV, namely the Woodchuck hepatitis virus (WHV) and the Woolly monkey hepatitis B virus (WMHBV), can assist in HDV propagation because their small envelope proteins (S-WHsAg and S-WMHBsAg, respectively) are competent for HDV RNP envelopment (Barrera et al. 2004; Ponzetto et al. 1984; Ryu et al. 1992). Experimental transmission of HDV has been achieved in woodchucks, and this animal model has been useful to study the interactions between HDV and the helper Hepadnavirus. In contrast, the envelope protein of a more distantly related Hepadnavirus, namely the Duck hepatitis B virus (DHBV), is unable to package the HDV RNP (O'Malley and Lazinski 2005). Therefore, determinants that are specific for HDV maturation on the S-HBsAg protein should be present on S-WHsAg and absent on the small DHBV envelope protein (S-DHBsAg). Compared to S-HBsAg or S-WHsAg, the S-DHBsAg polypeptide appears to lack the region corresponding to the antigenic loop between TMD2 and the carboxyl terminal hydrophobic domain (Fig. 3). When part of this domain (from residues 107 to 147) was experimentally deleted on S-HBsAg, it led to a drastic reduction in the capacity of the mutant for HDV maturation (O'Malley and Lazinski 2002). Interestingly, this deletion mutant was competent for the envelopment of the singly expressed L-HDAg protein, suggesting that the hindrance observed in RNP envelopment may rather reflect a lesser flexibility of the envelope, which could no longer accommodate an RNP, than a lack of binding. On one hand, S-DHBsAg cannot package the RNP and cannot interact with L-HDAg, and on the other hand, a S-HBsAg mutant that mimics S-DHBsAg in lacking the antigenic loop, is also deficient for packaging the RNP, while competent for L-HDAg interaction. Hence, it would be interesting to swap subdomains between the two proteins to precisely identify determinants of L-HDAg interaction. The deficiency in HDV maturation observed with the antigenic loop-deleted S-HBsAg, could also be explained, at least in part, by its lack of N-linked carbohydrates, since the removal of the glycosylation site (Asn146) was shown to prevent glycosylation, and partially inhibit HDV assembly (Sureau et al. 2003; Wang et al. 1996).

Considering the topology of the S-HBsAg protein at the ER membrane, it was expected that regions most likely to contain an RNP binding site will be exposed to the cytosolic face of the membrane (Fig. 3). The S-HBsAg loop, from residues 24 to 80, thus appeared as a good candidate because its disposal to the cytosol had been experimentally established. A genetic analysis of residues 24 to 59 revealed that a S-HBsAg mutant carrying a deletion of residues 24-28 was not affected for SVP secretion or L-HDAg packaging, but was partially deficient for HDV virion assembly (Jenna and Sureau 1998). It thus might reflect a hindrance in the envelope flexibility to accommodate an RNP. The same study also revealed that the 28-59 domain does not contain motifs essential for HDV maturation. Interestingly, a determinant of HBV virion assembly was shown to reside in a region that overlaps with the 28-59 sequence (Loffler-Mary et al. 2000). When mutational analysis was conducted on the carboxyl terminus of S-HBsAg, it was initially found that the tryptophan residue at position 196 was a determinant of HDV assembly (Jenna and Sureau 1999). When reexamined in a recent study, it was observed that the carboxyl domain of S-HBsAg contained a conserved tryptophan-rich domain of which Trp196, Trp199 and Trp201 were demonstrated to be critical for HDV assembly and for interaction with L-HDAg (Komla-Soukha and Sureau 2006). The entire carboxyl terminus of S-HBsAg (residues 164-226) is highly hydrophobic; it includes eight tryptophan residues, and it is predicted to con tain two TMDs located at positions 173-193 and 202-222 (Persson and Argos 1994). They bracket a short sequence (194-201), including Trp196, Trp199 and Trp201, which presents a low degree of flexibility (Fig. 3). Hydrophobic-ity and secondary structure predictions are compatible with the orientation of the tryptophan residues at the cytosolic side of the ER membrane in a position potentially adequate for interaction with the RNP. However, a topological model of S-HBsAg obtained by epitope mapping of monoclonal antibodies raised against HBV particles, proposed that the 187-207 region would not be buried inside the S-HBsAg particles, but would, instead, lie on the surface (Chen et al. 1996; Paulij et al. 1999). One could speculate that after synthesis at the ER membrane, the loop is initially disposed to the cytosolic face, and is translocated to the outside of the viral membrane after budding. The two topologies may also coexist at the virion surface. The fact that motifs identified as essential to HDV assembly, such as Trp196, Trp199 and Trp201, are dispensable for subviral particle secretion and yet strictly conserved among HBV, WHV and WMHBV isolates, suggests that the selection pressure that has led to their conservation concerns functions other than those involved in subviral particle assembly, for instance the processes of HBV maturation or entry. But they can also be conserved on S-HBsAg because the corresponding DNA coding sequence also encodes critical domains of the HBV polymerase. Interestingly, the Trp196 codon is included in the DNA sequence that codes for the YMDD motif of the polymerase catalytic domain. This motif is crucial to the activity of the enzyme and only in lamivudine-resistant virus is YMDD converted to YVDD, YSDD or YIDD (Torresi 2002; Torresi et al. 2002). The latter mutation results in a W196S mutation in S-HBsAg. As a consequence, patients infected with a YIDD mutant, are expected to be resistant to HDV superinfection because the W196S mutation in the S-HBsAg prevents RNP packaging (Komla-Soukha and Sureau 2006). For a better understanding of the HDV maturation process, the determinants of incorporation of L-HDAg proteins into SVPs need to be sorted from those involved in RNP envelopment. In the former case, assembly should proceed through colocalization of L-HDAg and S-HBsAg followed by a specific interaction between these two partners, whereas in the latter case, assembly is likely to depend also on the constraints exerted on the envelope to accommodate a 19 nm RNP. The capacity of S-HBsAg to modulate its intrinsic membrane bending force is suggested by the fact that in nature HBV manages to assemble three types of particles, namely the 22 nm spheres, the filaments that are 22 nm in diameter and up to several hundred nanometers in length, and the 42 nm Dane particles. Therefore, flexibility of the viral envelope is another characteristic of HBV that is beneficial to HDV (Fig. 3). In the light of a recent study that measured the concentration of HDAg proteins at up to six million copies per infected cell, it seems that the encounter between L-HDAg and S-HBsAg, or any cellular factor involved in this process, should be facilitated (Gudima et al. 2002). However, the possibility of obtaining S-HBsAg mutants that are deficient for both HDV maturation and L-HDAg packaging, while being permissive for SVP secretion, strongly suggests that the RNPs cannot be passively incorporated in the budding vesicles. In favor of a direct interaction between S-HBsAg and HDV RNPs are the following observations: (a) S-HBsAg and L-HDAg have been reported to bind to each other in a far Western binding assay (Hwang and Lai 1993); and (b) synthetic peptides specific for HBV envelope proteins have been shown to bind both L- and S-HDAg proteins in an in vitro assay (de Bruin et al. 1994; Hourioux et al. 1998). At present, we are left with a tryptophan-rich domain as a binding motif candidate on S-HBsAg, and a carboxyl terminal proline-rich domain as a possible ligand on the partner L-HDAg. Since examples of protein interaction mediated by a proline-rich sequence binding to a tryptophan-rich motif have been reported (Kay et al. 2000; Simon et al. 1998), it would be interesting to investigate this hypothesis in the process of HDV maturation.

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