Hepatitis B virus assembly is sensitive to changes in the cytosolic S loop of the envelope proteins.

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.