Trafficking of ODV-E66 is mediated via a sorting motif and other viral proteins: facilitated trafficking to the inner nuclear membrane

Abstract

The N-terminal 33 aa of the envelope protein ODV-E66 are sufficient to traffic fusion proteins to intranuclear membranes and the ODV envelope during infection with Autographa californica nucleopolyhedrovirus. This sequence has two distinct features: (i) an extremely hydrophobic sequence of 18 aa and (ii) positively charged amino acids close to the C-terminal end of the hydrophobic sequence. In the absence of infection, this sequence is sufficient to promote protein accumulation at the inner nuclear membrane. Covalent cross-linking results show that the lysines of the motif are proximal to FP25K and/or BV/ODV-E26 during transit from the endoplasmic reticulum to the nuclear envelope. We propose that the 33 aa comprise a signature for sorting proteins to the inner nuclear membrane (sorting motif) and that, unlike other resident proteins of the inner nuclear membrane, ODV-E66 and sortingmotif fusions do not randomly diffuse from their site of insertion at the endoplasmic reticulum to the nuclear envelope and viral-induced intranuclear membranes. Rather, during infection, trafficking is mediated by protein-protein interactions.

Fig. 1.

Orientation of 23-GFP and E66. (A) Construct 1 is the native sequence E66. Construct 2 shows the added N-GlcNAc acceptor sequence (underlined). Constructs 3 and 4 show an equivalent sequence modification of 23-GFP. Lanes 1-4 show in vitro translation of E66_G, and lanes 5-8 show an equivalent experiment using 23-GFP_G. -, translation in the presence of microsomal membranes; +, paired reaction with the addition of enzyme; ^*, unmodified protein; arrow, glycosylated protein. (B) Schematic orientation of protein in the ER, ONM, pore membrane, and INM.

Fig. 3.

Localization of SM mutants. Clones from each group were transiently expressed in Sf9 cells, and a representative Z section is shown. ER was identified by using antibody to calreticulin and DNA labeled with 4′,6-diamidino-2-phenylindole, whereas the SM fusion was detected by GFP autofluorescence. If two clones are represented within the same group, the results were visually indistinguishable.

Fig. 4.

The SM sequence is proximal to FP25K and E26. (A) Sequence of the SM cassette. Placement of the unique lysines (^*), position of the T7 epitope tag, and C-terminal (His)₆ are shown. (B) Sf9 cells were infected with the _polhSM-cassette virus, cells were collected at 33 h after infection, and nuclei were purified and exposed to BS³. 0, No cross-linker; X, addition of cross-linking reagent. The SM cassette and cross-linked adduct were detected by using antibody to T7 (lanes 1-8). Lanes 1 and 2 show total nuclear extract, whereas lanes 3-8 show product precipitated with antibodies: FP25K (lanes 3 and 4), E26 (lanes 5 and 6), and normal rabbit serum (lanes 7 and 8). Disrupted nuclear extracts were treated with Talon beads to purify the SM cassette and cross-linked complex, and proteins were separated by using SDS/PAGE and Western blotted. The identity of the proteins was determined by using antibodies to FP25K (lane 9) or T7 epitope (lane 10). (C) The SM cassette was translated in the presence of infected Sf9 cell microsomes (33 h after infection) and [³⁵S]Met, and then treated with BS³. The proteins were precipitated by using antibodies to T7, FP25K, E26, ODV-E25, p39, and polyhedrin (lanes 11-16, respectively) and separated by SDS/PAGE, and protein was detected by using incorporated [³⁵S]Met.

Fig. 2.

Localization of SM fusion in Sf9 cells during transient expression. The SM-fusion construct 33-GFP was transiently expressed in Sf9 cells, and a representative Z image through the center of the nucleus is shown. Colocalization of 33-GFP with lamin (A), calreticulin (B), and calnexin (C) is shown. (D) 23-GFP was transiently expressed in Sf9 cells, fixed, and prepared for electron microscopy. The cells were probed with antibody to GFP and gold-conjugated secondary antibody; ER, ONM, and INM localization is noted. (E and F) Z sections showing localization of 33-GFP transiently expressed in Sf9 cells and colocalization with lamin (row 1) and calnexin (row 2) under conditions of semi-(E) or full (F) permeabilization.

Fig. 5.

Comparison of SM with resident INM proteins. (A complete description of this figure and associated references are available in supporting information.) The TM and flanking sequence of the hydrophobic sequence most likely to influence INM localization are shown. The ΔG for membrane insertion was calculated by using the White-Wimley (octanol interface) scale. The orientation is shown with placement of the positively charged amino acids on the cytoplasmic/nucleoplasmic face noted.