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The cytoplasmic domains of many membrane proteins contain sorting signals that mediate their endocytosis from the plasma membrane. VZV gB contains three consensus internalization motifs within its cytoplasmic domain: YMTL (aa 818-821), YSRV (aa 857-860), and LL (aa 841-842). To determine whether VZV gB is internalized from the plasma membrane, and whether these motifs are required for its endocytosis, we compared the internalization of native gB to that of gB containing mutations in each of the predicted internalization motifs. VZV gB present on the surface of transfected cells associated with clathrin and was efficiently internalized to the Golgi apparatus within 60 min at 37 degrees C. VZV gB containing the mutation Y857 failed to be internalized, while gB-Y818A was internalized but did not accumulate in the Golgi. These data indicate that the internalization of VZV gB, and its subsequent localization to the Golgi, is mediated by two tyrosine-based sequence motifs in its cytoplasmic domain., (Copyright 2001 Academic Press.)

Normal herpesvirus assembly and egress depend on the correct intracellular localization of viral glycoproteins. While several post-Golgi transport motifs have been characterized within the cytoplasmic domains of various viral glycoproteins, few specific endoplasmic reticulum (ER)-to-Golgi transport signals have been described. We report the identification of two regions within the 125-amino-acid cytoplasmic domain of Varicella-Zoster virus gB that are required for its ER-to-Golgi transport. Native gB or gB containing deletions and specific point mutations in its cytoplasmic domain was expressed in mammalian cells. ER-to-Golgi transport of gB was assessed by indirect immunofluorescence and by the acquisition of Golgi-dependent posttranslational modifications. These studies revealed that the ER-to-Golgi transport of gB requires a nine-amino-acid region (YMTLVSAAE) within its cytoplasmic domain. Mutations of individual amino acids within this region markedly impaired the transport of gB from the ER to the Golgi, indicating that this domain functions by a sequence-dependent mechanism. Deletion of the C-terminal 17 amino acids of the gB cytoplasmic domain was also shown to impair the transport of gB from the ER to the Golgi. However, internal mutations within this region did not disrupt the transport of gB, indicating that its function during gB transport is not sequence dependent. Native gB is also transported to the nuclear membrane of transfected cells. gB lacking as many as 67 amino acids from the C terminus of its cytoplasmic domain continued to be transported to the nuclear membrane at apparently normal levels, indicating that the cytoplasmic domain of gB is not required for nuclear membrane localization.

The safety and immunogenicity of a novel hepatitis B virus (HBV) vaccine containing recombinant PreS2 and S antigens combined with MF59 adjuvant (HBV/MF59) was evaluated in healthy adults (N=230) who were randomized to receive 2 or 3 immunizations of either the study vaccine or a licensed control vaccine (Recombivax HB). After a single immunization, 105 of 118 (89%) recipients of HBV/MF59 achieved protective serum levels of anti-HBs antibody (> 10 mIU/ml), compared with 13 of 110 (12%) recipients of licensed vaccine (P < 0.001). The geometric mean titer (GMT) after 2 doses of HBV/MF59 given 2 months apart (13,422 mIU/ml) was more than 5-fold higher than that following 3 doses of licensed vaccine given over 6 months (2,346 mIU/ml; P < 0.001). The GMT following 3 injections of HBV/MF59 (249,917 mIU/ml) was 100-fold higher than licensed vaccine (P < 0.001). Anti-PreS2 antibodies were elicited in over 90% of the subset of HBV/MF59 recipients tested. Both vaccines were well tolerated; transient, mild-to-moderate local inflammation was the major postinjection reaction.

The varicella-zoster virus (VZV) genes ORF47 and ORF66 are predicted to encode serine/threonine protein kinases, which are homologs of herpes simplex virus 1 (HSV-1) UL13, and US3. When mutants were constructed by inserting stop codons into ORF47 and ORF66, the recombinants ROka47S and ROka66S, as well as intact ROka replicated in tissue culture. In contrast, inoculation of human thymus/liver or skin implants in SCID-hu mice showed that ORF47 protein was required for viral growth in human T cells and skin. Eliminating ORF66 expression inhibited VZV infectivity for T cells partially but did not impair replication in skin compared with ROka. Infectivity for T cells and skin was restored when ROka47S virus was complemented by insertion of ORF47 into a distant, noncoding site. The ORF47 gene product is the first VZV protein identified as necessary for T cell tropism. It also is essential for skin infectivity in vivo, as is glycoprotein C. Expression of ORF66 did not compensate for the absence of the ORF47 protein. The requirement for ORF47 expression in T cells and skin indicates that this gene product, which is dispensable in vitro, has a critical role within differentiated cells that are essential targets for VZV pathogenesis in vivo.