N-terminal sequences from Autographa californica nuclear polyhedrosis virus envelope proteins ODV-E66 and ODV-E25 are sufficient to direct reporter proteins to the nuclear envelope, intranuclear microvesicles and the envelope of occlusion derived virus

Abstract

Baculovirus occlusion-derived virus (ODV) derives its envelope from an intranuclear membrane source. N-terminal amino acid sequences of the Autographa californica nuclear polyhedrosis virus (AcMNPV) envelope proteins, ODV-E66 and ODV-E25 (23 and 24 amino acids, respectively) are highly hydrophobic. Recombinant viruses that express the two N-terminal amino acid sequences fused to green fluorescent protein (23GFP or 24GFP) provided visual markers to follow protein transport and localization within the nucleus during infection. Autoflourescence was first detected along the cytoplasmic periphery of the nucleus and subsequently localized as foci to discrete locations within the nucleus. Immunoelectron microscopy confirmed that these foci predominantly contained intranuclear microvesicles and the reporter fusion proteins were also detected in cytoplasmic membranes near the nucleus, and the outer and inner nuclear membrane. Therefore, these defined hydrophobic domains are sufficient to direct native and fusion proteins to induced membrane microvesicles within a baculovirus-infected cell nucleus and the viral envelope. In addition, these data suggest that movement of these proteins into the nuclear envelope may initiate through cytoplasmic membranes, such as endoplasmic reticulum, and that transport into the nucleus may be mediated through the outer and inner nuclear membrane.

Figure 1

Genetic constructs, in vitro translation, and membrane copurification. (A) Genetic constructs. Numbers indicate positions of amino acids based upon ODV-E66. (B) In vitro translation and membrane copurification. P, microsomal membrane pellet fraction; S, supernatant fraction.

Figure 4

Comparison of N-terminal domains of ODV-E66, ODV-E25, and fusion constructs. Blocked and shaded amino acids indicate hydrophobic domain; underlined (dashed) amino acids were directly N-terminal sequenced; underlined (solid) amino acids indicate N-, S-rich region, boxed amino acids indicate amino acids added by cloning strategy; asterisk indicates charged amino acids. OpMNPV, Orgyia pseudotsugata nuclear polyhedrosis virus; BmMNPV, Bombyx mori nuclear polyhedrosis virus.

Figure 2

Fluorescence and ImmunoGold (Janssen) localization of pVL1393–23GFP infected cells. (A and B) 23GFP autofluorescence in pVL1393-23GFP-infected Sf9 cells. (a and b) GFP autofluorescence and phase contract double exposure. (A and a) Forty-eight hours postinfection. (B and b) Seventy-two hours postinfection. Exposure time for cells at different time points postinfection was constant and set for exposure of the 72-hr postinfection. (C) ImmunoGold labeling (48 hr postinfection) using antiserum to GFP (1:1500; CLONTECH) and secondary gold conjugate. Fixation and ImmunoGold labeling of infected were done as described in Hong et al. (5). (), Intranuclear microvesicles, (↔), cytoplasmic membranes condensed near the nuclear envelope. Protein was detected as in C. C, cytoplasm; n, nucleus.

Figure 3

ImmunoGold localization of ODV-E25, 23β-gal, 23GFP, 24GFP, and ODV-E66. All data are presented from Sf9 infected cells at 48 hr postinfection and secondary antibody linked to gold. (A) 23β-gal recombinant virus (anti-β-gal); (B) AcMNPV (E2 strain; anti-ODV-E25 1:1000). (C) 23GFP recombinant virus (anti-GFP). (D) 24GFP recombinant virus (anti-GFP). (E) pVL-E66 recombinant virus (anti-ODV-E66, ref. 5). n, nucleus; C, cytoplasm; m, microvesicles; ➯, labeling of ODV envelope; ➤, labeling of the nuclear envelope; →, labeling of cytoplasmic membranes; ↔, condensation of cytoplasmic membranes to the periphery of the nucleus. (Bar = 1 μm.)

Figure 5

Models of protein transport into membranes within the nucleus.