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

Abstract

, 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 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. 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.

The Major Hurdle for Effective Baculovirus Transduction into Mammalian Cells Is Passing Early Endosomes

“…BHK-21, HCM3, HEK 293, HEK 293T, HeLa, HepG2, RD, SH-SY5Y, and Vero cells were obtained from the American Type Culture Collection (ATCC). Hep2, PK15, and Spodoptera frugiperda 9 (Sf9) cell lines were kept by our laboratory (39). Sf9 cells were cultured at 27°C in Grace's insect medium (pH 6.0; Gibco-BRL) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL).…”

“…Detection of virus binding. Cells were precooled on ice for 10 min and incubated with Ac-BPegfp at an MOI of 0.4, 5, or 20 TCID 50 units/cell for 1 h. The virus-containing medium was removed, the cells were washed with cold PBS 3 times to remove the unbound virus, and the cell-bound viral genome was quantified by qPCR with primers against the viral gene vp80 as previously reported (39).…”

“…(Sf9 cells). Immunofluorescence microscopy was performed using anti-VP39 antibody and Alexa Fluor 488 -goat anti-rabbit antibody (Abcam) as previously described (39). Cells were counterstained with Hoechst 33258 to stain the nuclei and imaged with a fluorescence microscope.…”

Dissecting the Cell Entry Pathway of Baculovirus by Single-Particle Tracking and Quantitative Electron Microscopic Analysis

“…The primers (vp39-mCherry For-1, Rev-1, For-1, and Rev-1) used for overlap extension PCR (OE-PCR) amplification are shown in Table 1. The recombinant virus vAc VP39-mCherry was generated by transforming Escherichia coli DH10Bac (Thermo Fisher Scientific) and transfecting the indicated bacmids into Sf9 cells according to the manufacturer's instructions (Bac-to-Bac baculovirus expression system; Thermo Fisher Scientific) (79). Purified virus vAc VP39-mCherry was analyzed by Western blotting with an anti-VP39 antibody (a gift from Zhihong Hu, Wuhan Institute of Virology, Chinese Academy of Sciences, China) or anti-mCherry antibody (TaKaRa) as the primary detection antibody.…”

“…Sf9 cells were incubated with the wild-type AcMNPV at an MOI of 1,000 for 30 min at 4°C, allowing the virus to attach to the cell surface. The cells were treated with drugs as described above, after which they were fixed in 2.5% glutaraldehyde in PBS at 4°C for 1 h. Cells were processed as described before (79,88). Ultrathin sections of these cells were examined by FEI Tecnai G 2 20 TWIN transmission electron microscopy.…”