Among the three related L, M, and S envelope proteins of the hepatitis B virus (HBV), the L and S polypeptides are required for virion production. Whereas the pivotal function of the pre-S region of L in nucleocapsid envelopment has been established, the contribution of its S domain and the S protein is less clear. In this study, we evaluated the role of the cytosolic S loop, common to L and S, in HBV assembly by performing mutagenesis experiments. To distinguish between the effect of the mutations on either envelope or virion formation, we investigated the ability of the mutants to assemble into secretable subviral empty envelopes and to replace the wild-type proteins in virion maturation, respectively. Virion production was found to be blocked by each of the secretion-competent deletion and substitution mutants SDelta35-39, SDelta40-46, SDelta50-56, and Svarsigma56-59, while an insertion within the loop is tolerated. Surprisingly, single mutations of the arginines terminating the loop had an opposite effect: while a conservative exchange of Arg-73 still allowed virion formation, the same mutation of Arg-79 did not. The critical sequences and/or structural requirements of the cytosolic S loop involved in nucleocapsid envelopment primarily act in the S background. These findings can be related to a model for a synergistical function of both L and S proteins in HBV morphogenesis.

The large L envelope protein of the hepatitis B virus has the peculiar capacity to adopt two transmembrane topologies. The N-terminal preS domain of L initially remains in the cytosol while the S domain is cotranslationally inserted into the endoplasmic reticulum membrane. The preS region of about half of the L molecules' is posttranslationally translocated to the lumenal space. We now demonstrate that the repression of cotranslational translocation of preS is conferred by a preS1-specific sequence. By analysis of L deletion mutants, the cytosolic anchorage determinant was mapped to amino acid sequence 70 to 94 of L. The intrinsic potential of this determinant to suppress cotranslational translocation was confirmed by transfer to the HBV middle envelope protein. In searching for cellular factors potentially involved in this novel process, we identified the cytosolic heat shock protein Hsc70 as a specific binding partner of L. The interaction site(s) for the chaperone was mapped to amino acids 63 to 107 of L using coimmunoprecipitation and in vitro binding analyses. Deletion of the cytosolic anchorage determinant almost completely abolished ATP-dependent Hsc70 binding. Therefore, interaction between Hsc70 and L is likely to be responsible for the suppression of cotranslational translocation of the preS domain.

Little is known about host cell factors necessary for hepatitis B virus (HBV) assembly which involves envelopment of cytosolic nucleocapsids by the S, M and L transmembrane viral envelope proteins and subsequent budding into intraluminal cisternae. Central to virogenesis is the L protein that mediates hepatocyte receptor binding and envelopment of capsids. To serve these topologically conflicting roles, L protein exhibits an unusual dual membrane topology, disposing its N-terminal preS domain inside and outside of the virion lipid envelope. The mixed topology is achieved by posttranslational preS translocation of about half of the L protein molecules across a post-endoplasmic reticulum membrane. Here we identify and characterize a preS-specific sequence that confers the suppression of cotranslational translocation even of a model reporter. This cytosolic anchorage sequence specifically binds the cognate heat shock protein Hsc70, thus indicating chaperone participitation in HBV morphogenesis. Conversely, the M envelope protein needs the assistance of the chaperone calnexin for proper folding and trafficking. Calnexin selectively binds to the N-glycan, specific for M, rather than to the N-glycan, common to all three envelope proteins. As inhibition of the calnexin-M interaction blocks the secretion of viral envelopes, we propose an essential role for calnexin, as well as for Hsc70, in chaperoning HBV assembly.

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