The Functional Oligomeric State of Tegument Protein GP41 Is Essential for Baculovirus Budded Virion and Occlusion-Derived Virion Assembly

Abstract

gp41, one of the baculovirus core genes, encodes the only recognized tegument (O-glycosylated) protein of the occlusion-derived virion (ODV) phenotype so far. A previous study using a temperature-sensitive Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) mutant showed that GP41 plays a crucial role in budded virion (BV) formation. However, the precise function of GP41 in the baculovirus replication cycle remains unclear. In this study, AcMNPV GP41 was found to accumulate around the ring zone (RZ) region within the infected nucleus and finally assembled into both BVs and ODVs. Deletion of gp41 from the AcMNPV genome showed that BVs were no longer formed and ODVs were no longer assembled, suggesting the essential role of this gene in baculovirus virion morphogenesis. In infected cells, besides the 42-kDa monomers, dimers and trimers were detected under nonreducing conditions, whereas only trimeric GP41 forms were selectively incorporated into BVs or ODVs. Mutations of all five cysteines in GP41 individually had minor effects on GP41 oligomer formation, albeit certain mutations impaired infectious BV production, suggesting flexibility in the intermolecular disulfide bonding. Single mutations of key leucines within two predicted leucine zipper-like motifs did not interfere with GP41 oligomerization or BV and ODV formation, but double leucine mutations completely blocked oligomerization of GP41 and progeny BV production. In the latter case, the usual subcellular localization, especially RZ accumulation, of GP41 was abolished. The above findings clearly point out a close correlation between GP41 oligomerization and function and therefore highlight the oligomeric state as the functional form of GP41 in the baculovirus replication cycle.IMPORTANCE The tegument, which is sandwiched between the nucleocapsid and the virion envelope, is an important substructure of many enveloped viruses. It is composed of one or more proteins that have important functions during virus entry, replication, assembly, and egress. Unlike another large DNA virus (herpesvirus) that encodes an extensive set of tegument components, baculoviruses very likely exploit the major tegument protein, GP41, to execute functions in baculovirus virion morphogenesis and assembly. However, the function of this O-glycosylated baculovirus tegument protein remains largely unknown. In this study, we identified trimers as the functional structure of GP41 in baculovirus virion morphogenesis and showed that both disulfide bridging and protein-protein interactions via the two leucine zipper-like domains are involved in the formation of different oligomeric states. This study advances our understanding of the unique viral tegument protein GP41 participating in the life cycle of baculoviruses.

Keywords: GP41; baculovirus; function; leucine zipper; oligomerization; tegument protein; virion assembly.

FIG 1

Transcription, expression, and localization of AcMNPV GP41 in infected cells. (A) Time course analysis of AcMNPV gp41 transcription. Cells were infected with the control virus AcBac-egfp-ph and analyzed at the indicated time points. Total RNA was extracted, and gp41, ie1, and vp39 were detected by RT-PCR. The negative control (NC) was an RT-PCR mixture without reverse transcriptase. (B) Western blot analysis of the GP41 expression profile. Cells were infected with control virus and analyzed at the indicated time points. The expression profile of GP41 was detected by use of a specific anti-GP41 pAb. The major viral capsid protein VP39 and the host protein GAPDH were used as controls. (C) Subcellular localization of GP41 in infected cells by immunofluorescence microscopy. Sf9 cells were infected with control virus and fixed at the indicated time points. The boxed views in the tetramethyl rhodamine isocyanate (TRITC) and 4′,6-diamidino-2-phenylindole (DAPI) images were merged and enlarged and are shown in the last row. FITC, fluorescein isothiocyanate. (D) Subcellular localization of GP41 in transfected or infected cells. Sf9 cells were transfected with plasmid pIZ/V5-gp41. At 24 h p.t., cells were infected with the control virus AcBac-egfp-ph or mock infected. The localization of GP41 was detected by immunofluorescence microscopy at 24 h p.t. or 36 h p.i. Enhanced green fluorescent protein (EGFP) (green) was used as an indicator of successful virus infection, and an anti-GP41 pAb was used to detect the localization of GP41 in the infected cells (red). The nuclei were stained with Hoechst 33258 (blue). Bars, 5 μm for the enlarged images and 10 μm for the others.

FIG 2

Construction and characterization of gp41 knockout and gp41 repaired AcMNPV bacmids. (A) Schematic representations of the recombinant bacmids AcΔgp41, AcΔgp41-ph, and AcΔgp41-gp41R-ph. (B) Transfection-infection assay. Sf9 cells were transfected with bacmid DNA of AcΔgp41-ph or AcΔgp41-gp41R-ph, and at 96 h p.t., the supernatants were used to infect uninfected Sf9 cells. Fluorescence was observed at the indicated time points. (C) EM analysis of nucleocapsid assembly and BV formation. Sf9 cells were transfected or infected with recombinant bacmid AcΔgp41-ph (a and b) or AcΔgp41-gp41R-ph (c and d). At 48 h p.t. or 48 h p.i., cells were fixed and observed by TEM. Panels b and d are enlarged pictures of the boxed regions in panels a and c. White arrows, nucleocapsids; red arrow, nucleocapsids enclosed within a cytoplasmic vesicle; black arrows, mature BVs. Nu, nucleus; Cyto, cytoplasm. Bars, 500 nm.

FIG 3

EM analysis of ODV morphogenesis and GP41 localization. (A) EM analysis of virion morphogenesis in recombinant virus-transfected cells. Sf9 cells were transfected with AcΔgp41-ph (a to f) or AcΔgp41-gp41R-ph (g to l) and fixed at 48, 72, and 96 h p.t. Ultrathin sections of the cells were observed by TEM. For each panel on the left, the right picture show an enlarged view of the boxed region. White arrows and red arrows indicate nucleocapsids and MVs, respectively. Nu, nucleus; Cyto, cytoplasm; NCs, nucleocapsids. Bars, 500 nm. (B) IEM analysis of GP41 localization in infected cells and virions. Sf9 cells were infected with control virus at an MOI of 5 TCID₅₀ units/cell and harvested at 24, 48, and 72 h p.i. The cells were probed with anti-GP41 pAb as the primary antibody and goat anti-rabbit IgG coated with gold particles (10 nm) as the secondary antibody. Cell sections were also detected with preimmune rabbit serum as a control group. The samples were observed by TEM. Arrowheads indicate the location of GP41. Bars, 200 nm.

FIG 4

Oligomerization of GP41 in infected cells and virions. (A) Detection of GP41 on BVs and ODVs. BVs harvested from control virus AcBac-egfp-ph-infected cells and ODVs from AcBac-egfp-ph-infected larvae were purified and analyzed by Western blotting using anti-GP41 pAb. The major viral capsid protein VP39 and the BV- and ODV-specific envelope proteins GP64 and PIF5, respectively, were used as controls. (B) Analysis of GP41 oligomerization in infected cells. Sf9 cells were infected with control virus (WT) or the gp41 repaired virus (Rep) at an MOI of 5 TCID₅₀ units/cell. Cells were collected at 36 h p.i., treated with nonreducing buffer or reducing buffer, and subjected to Western blot analysis using anti-GP41 pAb. (C) Subcellular fractionation of virus-infected cells. Sf9 cells were infected with AcBac-egfp-ph at an MOI of 5 TCID₅₀ units/cell. Cytoplasmic (Cy) and nuclear (Nu) components were separated at 36 h p.i., and GP41 was detected under nonreducing conditions. Lamin B and GAPDH were detected under reducing conditions to indicate the fractionation efficiency. Wc, whole-cell lysates. (D) Analysis of GP41 oligomerization in virions. BVs and ODVs were purified and subjected to Western blotting under nonreducing conditions, using a method similar to that described above.

FIG 5

Construction of cysteine and leucine zipper mutant recombinant viruses. (A) Prediction of GP41 oligomerization motifs. The leucine zipper-like coiled-coil domain was predicted by PCOILS and is presented by use of helical wheel projection software. The key leucine residues are presented as green diamonds. (B) Construction of cysteine and leucine zipper-like motif mutant recombinant viruses. The cysteines and key leucines were mutated individually or in combination by site-directed mutagenesis. The ph gene and each of the mutant gp41 genes were coinserted into the ph locus of the AcΔgp41 bacmid to construct AcΔgp41-gp41^x-ph.

FIG 6

Characterization of cysteine mutant recombinant viruses. (A) Transfection-infection assay. Sf9 cells were transfected with the five cysteine mutants, and fluorescence was observed at 96 h p.t. and 96 h p.i. (B) One-step growth curve analysis of cysteine mutants. Sf9 cells were infected separately with the five single cysteine mutants, WT virus, or repaired virus at an MOI of 5 TCID₅₀ units/cell. Virus titers of supernatants collected at different time points were determined by endpoint dilution assay. The experiments were replicated twice. Error bars show standard deviations. (C) Sf9 cells were infected with the five cysteine mutants and the gp41 repaired recombinant virus and subjected to Western blot analysis under nonreducing (NR) or reducing (R) conditions.

FIG 7

Characterization of leucine zipper-like motif mutant recombinant viruses. (A) Transfection-infection assay. Sf9 cells were transfected with leucine zipper-like motif mutant recombinant bacmids, and at 96 h p.t. the transfection supernatants were used to infect healthy Sf9 cells. Fluorescence was observed at 96 h p.t. and 96 h p.i. (B) One-step growth curve assay of mutated viruses. Sf9 cells were infected with single leucine mutant viruses or the gp41 repaired virus (AcΔgp41-gp41R-ph) at an MOI of 5 TCID₅₀ units/cell. The supernatants of infected cells were harvested at the indicated time points, and virus titers were determined by endpoint dilution assay. The experiments were replicated twice. Error bars show standard deviations. (C) EM analysis of GP41 leucine zipper-like motif mutants. Cells were infected with single leucine mutant viruses or transfected with double leucine mutant bacmid DNA. At 96 h p.t. and 96 h p.i., cells were harvested for EM analysis. Black arrows indicate OBs, red arrows indicate MVs, and white arrows indicate nucleocapsids. Bars, 500 nm.

FIG 8

Oligomerization and subcellular localization of leucine zipper-like motif mutants. (A) Oligomerization of leucine zipper-like motif mutant GP41. Sf9 cells were infected with single-site mutants or transfected with double-site mutants and collected at 36 h p.i. or 48 h p.t. After being treated under nonreducing (NR) or reducing (R) conditions, cell samples were analyzed by Western blotting using anti-GP41 pAb. GAPDH was detected as a control. (B) Subcellular localization of leucine zipper-like motif mutants. Cells were infected or transfected with leucine zipper-like motif mutants and fixed at 36 h p.i. or 48 h p.t. The locations of GP41 mutants were detected by immunofluorescence microscopy with anti-GP41 pAb. The enlarged fields show the boxed regions of the TRITC and DAPI channels. Bars, 5 μm for the enlarged images and 10 μm for the others.

FIG 9

Proposed model for GP41 transportation, oligomerization, and function in the AcMNPV life cycle. (A) Working model for oligomerization of WT GP41 and different mutants. (a) Oligomeric states of WT GP41. GP41 recruits other GP41 monomers to form relaxed trimers via leucine zipper-like motifs. Afterwards, disulfide bonds form and bind the trimers tightly and stably. (b) Oligomeric states of cysteine or leucine zipper mutants. In single cysteine mutants, one disulfide bond is broken, and other disulfide bonds together with leucine zipper-like motifs loosely maintain the trimeric structure. In single leucine mutants, although the hydrophobic interaction is partially interrupted, disulfide bonds still maintain a relatively stable trimeric structure. However, a double leucine mutation totally interrupts the interactions of GP41 monomers. (B) Transportation and function of GP41 in infected cells. Three forms (monomers, dimers, and trimers) of GP41 are synthesized in the cytoplasm and transported into the nucleus (step 1; black arrows). The nuclear import and RZ accumulation of GP41 likely happen with the help of certain still-unknown viral/host proteins. Within the infected nucleus, GP41 trimers may interact with MVs and nucleocapsids to accomplish ODV envelopment and embedding (step 2; pink arrows), promote efficient nuclear egress of nucleocapsids, or facilitate cytoplasmic transport of nucleocapsids to form BVs (step 3; blue arrows). VS, virogenic stroma; ER, endoplasmic reticulum; Golgi, Golgi apparatus.