Cytoplasmic domain signal sequences that mediate transport of varicella-zoster virus gB from the endoplasmic reticulum to the Golgi

Abstract

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.

FIG. 1

VZV gB cytoplasmic domain mutations. The 125-aa residues (from aa 744 to 868) that make up the cytoplasmic domain of VZV gB are shown. The lines bisecting the cytoplasmic domain are located immediately after the terminal amino acids of the truncation mutants used in this study. These mutants are named according to the number of deleted C-terminal amino acids. Substitution mutations are indicated by lines and brackets, and the amino acids replacing the native residue(s) are noted. The regions in red (aa 818 to 826), blue (aa 833 to 851), and green (aa 852 to 860) denote separate internal deletion mutations.

FIG. 2

Microscopic localization of VZV gB. Native gB or gB containing cytoplasmic domain C-terminal truncations was expressed in mammalian cells by transfection. Cells were also infected with native VZV as positive control. MeWo cells are shown; similar results were obtained with HEp-2 cells. Transfected or infected cells were incubated with both anti-VZV gB MAbs and WGA (a lectin that concentrates in the Golgi). The intracellular localization of gB and WGA was analyzed by laser-scanning confocal microscopy. Arrows mark the location of the Golgi in selected cells.

FIG. 3

Microscopic localization of VZV gB. Native gB or gB containing cytoplasmic domain C-terminal truncations was expressed in mammalian cells by transfection. Transfected cells were coincubated with anti-VZV gB MAbs and anticalnexin antibodies (a marker of the ER). The intracellular localization of gB and calnexin was analyzed by laser-scanning confocal microscopy.

FIG. 4

Proteolytic cleavage of VZV gB cytoplasmic domain truncation mutants. Native gB (wt [wild type]) or gB containing cytoplasmic domain C-terminal truncation mutations was expressed in mammalian cells, immunoprecipitated, and resolved by SDS-PAGE under reducing conditions. Truncation mutants are designated by the number of amino acids removed from the C terminus. Mock-transfected cells (−) were included as a negative control. Native gB migrates as a 110-kDa species and as a doublet of about 60 kDa (resulting from its proteolytic cleavage in the Golgi). Molecular masses are given in kilodaltons.

FIG. 5

ER-to-Golgi transport of VZV gB containing deletions in its cytoplasmic domain. Native gB (wt [wild type]) or gB containing cytoplasmic domain deletion mutations was expressed in mammalian cells, pulse-labeled, and then immunoprecipitated immediately (0-h chase) or after a 4-h chase. Treatment of immunoprecipitated gB with endo H is denoted by +. Mock-transfected cells (M) were processed identically as a negative control. The immunoprecipitated proteins were resolved by nonreducing SDS-PAGE on 8% gels.

FIG. 6

Quantitation of ER-to-Golgi transport of VZV gB cytoplasmic domain deletion mutants. The C-terminal 54 aa of the VZV gB cytoplasmic domain (aa 815 to 868) are listed. Native (wild-type [wt]) gB contains this entire region, whereas the segments of this region retained by the various deletion mutants are represented by the black bars. The proportion of each mutant transported to the Golgi (as a percentage of native gB transport) was quantitated and is depicted by the stippled bars.

FIG. 7

Quantitation of ER-to-Golgi transport of VZV gB mutants containing substitutions in the C-terminal 17 aa of its cytoplasmic domain. The C-terminal 17 aa of the VZV gB cytoplasmic domain (aa 852 to 868) are listed. Boldface letters represent substitution mutations within this region. The proportion of each mutant transported to the Golgi (as a percentage wild-type [wt] gB transport) was quantitated and is shown by the stippled bars.

FIG. 8

Quantitation of ER-to-Golgi transport of VZV gB mutants containing deletions or substitutions in the region between aa 816 and 837 of the cytoplasmic domain. Amino acid residues 816 to 837 of the VZV gB cytoplasmic domain are listed (Native gB). Amino acids 818 to 826 are underlined. Boldface letters represent substitution mutations within this region. The proportion of each mutant transported to the Golgi (as a percentage wild-type [wt] gB transport) was quantitated and is shown by the stippled bars.