Department of Medical Microbiology and Hygiene, University of Mainz, Augustusplatz, D-55101 Mainz, Germany
Correspondence
Reinhild Prange
prange{at}mail.uni-mainz.de
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ABSTRACT |
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MAIN TEXT |
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At present, little is known about the mechanism by which the dual topology of the HBV L protein is established. During biogenesis, the L protein, together with the related middle (M) and small (S) envelope proteins, are expressed from a single open reading frame by differential translation initiation. As a result, the sequence of S is repeated at the C termini of M and L, which contain the additional preS2 domain or preS2 and preS1 domains, respectively (reviewed by Heermann & Gerlich, 1991). All three proteins are co-translationally integrated into the endoplasmic reticulum (ER) membrane, most likely directed by a signal-anchor and stop-transfer sequence encoded within the first and second transmembrane (TM) segments (TM1 and TM2) of their S domains (Eble et al., 1987
). These signals also direct co-translational translocation of the upstream preS2 region of M into the ER lumen (Eble et al., 1990
). In contrast, the preS2 plus preS1 (preS) domain of L initially remains cytosolic. During maturation, about half of the L molecules post-translationally translocate their preS region into the ER, thereby generating a dual topology that is maintained in the virion envelope (Bruss et al., 1994
) (Fig. 1
). By orientating the preS domain at both the cytosolic (inside the virus) and luminal (outside the virus) location, L serves its topological opposing functions in the virus life cycle, capsid envelopment and receptor binding, respectively.
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A series of mutant and chimeric proteins was constructed and their topological features were analysed by transient expression in COS-7 cells. As a reliable assay, trypsin protection experiments of microsomes were used to monitor the partial preS post-translocation of L across membranes (Lambert & Prange, 2001). Upon expression of an HA-tagged version of the L gene, driven by plasmid pNI2.LHA, L appeared in its characteristic doublet of a 39 kDa non-glycosylated (p39) and a 42 kDa single-glycosylated (gp42) form as a consequence of partial N-glycosylation at Asn-309 in its S domain. While the preS domain of newly synthesized L chains is almost fully sensitive to cleavage with trypsin, over time it becomes increasingly protected due to its partial post-translocation into the ER, yielding up to 5060 % resistant chains at steady state, as summarized in Fig. 1
. Trypsin treatment in the absence of detergent (NP-40) yielded two fractions of L: trypsin-resistant full-length molecules with preS post-translationally translocated into the ER and trypsin-sensitive chains with preS orientated to the cytosol where they are cleaved to a 25 kDa non-glycosylated (T) and a 28 kDa single-glycosylated (gT) fragment, corresponding to the C-terminal S parts. Upon disruption of microsomes with NP-40, trypsin completely converted L to these fragments. The post-translational mode of preS translocation has been further evidenced by the absence of N-linked glycosylation of preS carrying two modification-competent glycosylation sites (Asn-4 and Asn-123) (Löffler-Mary et al., 1997
).
Previously, we showed that among the four membrane-spanning segments of L, only TM2 is needed for preS post-translocation (Lambert & Prange, 2001). While these results largely excluded an HBV-specific envelope structure serving as a preS conduit, there still remained the possibility that lateral interactions of TM2 domains by helical packing might form an autonomous translocation channel, as, for example, exemplified by the M2 transmembrane proton channel protein of influenza A (Sansom et al., 1998
). In favour of this assumption, database searching identified a leucine-rich heptad motif within TM2 (Fig. 2
A), a candidate self-interaction motif (Gurezka et al., 1999
; Scholze et al., 2002
), which might form a pore structure. To assess whether the leucine zipper-type motif comprised within Leu-247, Leu-254 and Leu-261 might contribute to preS reorientation, double and triple substitutions by alanine were made by PCR-directed mutagenesis. Upon synthesis in COS-7 cells, each of the L247/254A, L247/261A, L254/261A and L247/254/261A mutants were obtained in a doublet that is characteristic for the non- and single-glycosylated chains of wild-type (wt) L and hence for co-translational insertion into the ER membrane (Fig. 2A
). The analysis of their topological properties, however, revealed that all double mutants displayed the wt translocational phenotype, occurring in a fraction of
4050 % of polypeptides that were protected from trypsin by the microsomal membrane unless NP-40 was present. The triple mutant supported preS post-translocation even to a greater extent. Accordingly, the leucine zipper-like motif is not required for preS post-translocation but appears to affect its degree.
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To gain additional insight into the action of the topogenic signals of L, we performed domain-exchange experiments by shuffling mammalian and avian HBV envelope subdomains. First, a chimera was generated by substituting the S region of L with the corresponding domain of DHBV. For construction of preS(h) : : S(d), the DHBV S region was amplified from plasmid pDHBV (Wildner et al., 1991) prior to cloning into pNI2.LHA. In accordance with the calculated molecular mass, preS(h) : : S(d) was expressed as a membrane-bound 37 kDa polypeptide (Fig. 3
A). Surprisingly, two higher molecular mass forms were found in addition. These bands disappeared on deglycosylation with peptide:N-glycosidase F (PNGase F), indicating that they represented N-glycosylated versions. Because the DHBV S domain is known to lack N-glycans, the modification of preS(h) : : S(d) must have occurred at Asn-4 and Asn-123 within preS. Given that both glycosylation sites of preS(h) : : S(d) were on the luminal side of the ER membrane, this observation implicated an unexpected co-translational mode of preS translocation. Consistent with this, the majority of preS(h) : : S(d) glycosylated chains (
95 %) were protected from trypsin in intact microsomes. Non-glycosylated p37, however, was fully accessible to trypsin. This might be due to inefficient co-translational preS import in the ER and/or retrograde preS dislocation out of the ER.
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As evident from Fig. 3, faster-migrating polypeptides appeared in the case of preS(h) : : S(d), which probably represented preS2(h) : : S(d) and S(d) forms derived from internal translational initiation. Such forms were missing in preS(d) : : S(h) because the S promoter of DHBV located within preS is inactive in non-hepatic cell lines (Welsheimer & Newbold, 1996
). This difference, however, is unlikely to account for the divergent behaviour of the two chimeras because co-translational polypeptide translocation into the ER is known to proceed independently from interprotein subunit interaction.
Given that the mixing of the two hepadnaviral L subdomains in either order blocked the correct (re)folding of L, we conclude that preS- and S-specific topogenic determinants of these hepadnavirus genera cannot act synergistically and in concert. To account for the co-translational preS phenotype of preS(h) : : S(d), the activity of the HBV-specific CAD element in preventing co-translational preS translocation through interaction with Hsc70 appeared to be abrogated by the DHBV S domain. This might imply that the DHBV S domain has a high preference to orient its N terminus towards the ER lumen, thereby enforcing co-translational N-tail translocation even in the presence of CAD. Such a strong orientation effector function might explain why the DHBV L protein unlike the corresponding protein of HBV engages a stretch of four lysines located within its preS domain in addition to Hsc70-binding elements to block co-translational preS translocation (Swameye & Schaller, 1997). Reversibly, these binary DHBV preS-specific determinants might dominantly interfere with the topogenic function of the HBV S-specific signals in such a way that the TM2-directed post-translational N-tail reorientation is inhibited in the preS(d) : : S(h) chimera. Taken together, our finding that the specialized topogenic determinants of the DHBV and HBV L proteins were functionally non-interchangeable and failed to cross-operate provides further evidence for distinct folding pathways used by the two hepadnaviral proteins to acquire their dual topology.
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ACKNOWLEDGEMENTS |
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Received 20 October 2003;
accepted 30 January 2004.
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