ABSTRACT
TER94 is a multifunctional AAA+ ATPase crucial for diverse cellular processes, especially protein quality control and chromatin dynamics in eukaryotic organisms. Many viruses, including coronavirus, herpesvirus, and retrovirus, coopt host cellular TER94 for optimal viral invasion and replication. Previous proteomics analysis identified the association of TER94 with the budded virions (BVs) of baculovirus, an enveloped insect large DNA virus. Here, the role of TER94 in the prototypic baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) life cycle was investigated. In virus-infected cells, TER94 accumulated in virogenic stroma (VS) at the early stage of infection and subsequently partially rearranged in the ring zone region. In the virions, TER94 was associated with the nucleocapsids of both BV and occlusion-derived virus (ODV). Inhibition of TER94 ATPase activity significantly reduced viral DNA replication and BV production. Electron/immunoelectron microscopy revealed that inhibition of TER94 resulted in the trapping of nucleocapsids within cytoplasmic vacuoles at the nuclear periphery for BV formation and blockage of ODV envelopment at a premature stage within infected nuclei, which appeared highly consistent with its pivotal function in membrane biogenesis. Further analyses showed that TER94 was recruited to the VS or subnuclear structures through interaction with viral early proteins LEF3 and helicase, whereas inhibition of TER94 activity blocked the proper localization of replication-related viral proteins and morphogenesis of VS, providing an explanation for its role in viral DNA replication. Taken together, these data indicated the crucial functions of TER94 at multiple steps of the baculovirus life cycle, including genome replication, BV formation, and ODV morphogenesis.
IMPORTANCE TER94 constitutes an important AAA+ ATPase that associates with diverse cellular processes, including protein quality control, membrane fusion of the Golgi apparatus and endoplasmic reticulum network, nuclear envelope reformation, and DNA replication. To date, little is known regarding the role(s) of TER94 in the baculovirus life cycle. In this study, TER94 was found to play a crucial role in multiple steps of baculovirus infection, including viral DNA replication and BV and ODV formation. Further evidence showed that the membrane fission/fusion function of TER94 is likely to be exploited by baculovirus for virion morphogenesis. Moreover, TER94 could interact with the viral early proteins LEF3 and helicase to transport and further recruit viral replication-related proteins to establish viral replication factories. This study highlights the critical roles of TER94 as an energy-supplying chaperon in the baculovirus life cycle and enriches our knowledge regarding the biological function of this important host factor.
INTRODUCTION
In recent years, accumulating studies have revealed that transient endoplasmic reticulum 94 (TER94, also termed CDC48 in yeast and plants or VCP/P97 in metazoans) plays crucial roles in diverse cellular processes. As a member of the ATPases associated with diverse cellular activities (AAA+) ATPase family, TER94 utilizes the energy of ATP hydrolysis to remodel substrate proteins (1, 2) and functions in endoplasmic reticulum-associated protein degradation, mitochondrion-associated degradation, autophagy, DNA damage response, membrane fusion, NF-κB activation, and DNA replication (3–5). TER94 is an approximately 97-kDa protein in Xenopus laevis oocytes that contains two ATPase domains and forms hexamers (∼612 kDa) to implement its versatile cellular functions by cooperating with its cofactors (6, 7). These multifarious regulatory cofactors bind to TER94 at distinct binding sites or domains and recruit TER94 to specific cellular pathways. As a multifunctional hub connecting diverse cellular pathways and regulating protein homeostasis of cells, TER94 has been shown to be associated with numerous human neurodegenerative diseases and has aroused considerable interest as a target for controlling cancer cells (8–10).
Numerous studies have revealed that viruses can also hijack and exploit TER94 at different levels to establish productive infection in host cells. For example, TER94 is rearranged and colocalizes with viral proteins in viral replication organelles of enterovirus 71-infected cells (11). Conversely, knockdown of TER94 expression blocks the escape of the coronavirus infectious bronchitis virus from endosomes during the entry process (12). TER94 is also required for West Nile virus and poliovirus replication and possibly functions in the cellular secretion pathway for the latter (13, 14). Influenza virus, Rift Valley fever virus, and mouse mammary tumor virus depend on TER94 for the proper expression or transportation of viral proteins to virus assembly sites (15–18). In addition, a recent study on human cytomegalovirus showed that TER94 is required for viral protein expression and the onset of virus replication in human cells, further highlighting the potential antiviral activity of a TER94-specific inhibitor (16). However, the detailed mechanism of how TER94 functions in virus infection processes remains obscure.
Baculoviruses are insect-specific large DNA viruses that have been widely used as biological pesticides and protein expression vectors (19). Most baculoviruses have a unique biphasic life cycle characterized by the production of two distinct types of progeny virion phenotypes, budded virions (BVs) and occlusion-derived virions (ODVs). ODVs infect exclusively epithelium cells of the insect midgut to initiate primary infection, whereas BVs are subsequently produced and infect other cell types within larval bodies to spread systemic infection (20, 21). Following the entry of BV/ODV into cells, the incoming nucleocapsids will be transported into the nucleus, where the viral genome is released and DNA replication is initiated (22, 23). In the infected nuclei, a virus-induced subnuclear structure termed the virogenic stroma (VS) serves as a viral factory for successive viral DNA replication, gene transcription, and nucleocapsid assembly (19). A small fraction of progeny nucleocapsids will egress from the nuclear envelope and bud from the plasma membrane to form BVs, whereas others retained within the nucleus are enveloped to form mature ODVs at the ring zone region (19).
During these processes, many host factors are utilized by the baculovirus. For example, at the early stage of infection, host RNA polymerase II and transcription machinery are involved in baculovirus early gene transcription (19, 24, 25). Host cytoskeleton components, including actin and tubulin, are responsible for transporting baculovirus virions during virus entry and egress (26–31). The components of the endosomal sorting complex required for transport (ESCRT) and its regulator VPS4, an ATPase, were also found to be required for baculovirus entry and egress (32, 33). Other ATPases, such as N-ethylmaleimide-sensitive factor (NSF) and ATAD3A, also participate in baculovirus infection (34, 35). Moreover, recent transcriptomic and proteomic studies have revealed further possible interactions between baculoviruses and hosts, in addition to the cellular proteins associated with the virions. One such virion-associated host protein is TER94, which potentially plays a role in the baculovirus life cycle (36). Furthermore, a recent study showed that TER94 played a role in baculoviral replication; however, the precise mechanism remains unclear (37).
In this study, we investigated the detailed functions of TER94 in the infection process of the prototype baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). We confirmed TER94 as a nucleocapsid component of both BV and ODV and found the crucial role of TER94 for genomic DNA replication, nucleocapsid trafficking in the cytoplasm for further BV budding, and ODV envelopment within AcMNPV-infected nuclei. Moreover, we demonstrated that TER94 is recruited by the viral early proteins late expression factor 3 (LEF3) and helicase (P143), which are crucial for DNA replication, to the VS region of infected nuclei and is essential for proper formation of VS structure. These data support the multiple crucial functions of TER94 during AcMNPV infection.
RESULTS
TER94 is associated with nucleocapsids of both BV and ODV.A previous report showed that TER94 is a BV component of Helicoverpa armigera nucleopolyhedrovirus (36). To detect the presence of TER94 in baculovirus virions, BVs from AcMNPV-infected Spodoptera frugiperda (Sf9) cells and ODVs from AcMNPV-infected larvae were harvested. The purified BVs and ODVs were separated into envelope (E) and nucleocapsid (NC) fractions, respectively, and analyzed using Western blotting. As shown in Fig. 1, a specific band of approximately 100 kDa was detected in BV and BV-NC lanes using a TER94 polyclonal antibody (PAb). The specificity of TER94 PAb has been verified prior to use (data not shown). However, in ODV and ODV-NC samples, apart from the 100-kDa protein, an additional band of approximately 90 kDa was also observed. The BV-specific envelope protein GP64 and ODV-specific envelope protein per os infectivity factor 5 (PIF5) were detected as controls for BV- and ODV-E fractions, respectively, and the major capsid protein VP39 was used as a control for NC fractions of both BV and ODV (Fig. 1). The calculated molecular mass of Sf9-TER94 (UniProtKB A0A2H1V392) is approximately 90 kDa. As TER94 may contain posttranslational modifications such as SUMOylation (SUMO stands for small ubiquitin-like modifier), ubiquitylation, and phosphorylation (38, 39), we surmised that the approximately 100-kDa protein detected by anti-TER94 in BV and ODV samples may represent a posttranslationally modified form of TER94. These results demonstrated that TER94 is a component of both AcMNPV BV and ODV and is exclusively associated with viral nucleocapsids.
Localization of TER94 on BVs and ODVs. BVs or ODVs harvested from wild-type virus AcMNPV-WT-infected cells or larvae were purified by ultracentrifugation and subjected to envelope- and nucleocapsid fractionation. TER94, the major capsid protein VP39, BV envelope-specific protein GP64, and ODV envelope-specific protein PIF5 were detected by Western blotting. E, envelope fractions; NC, nucleocapsid fractions.
TER94 is rearranged in Sf9 cells and incorporated into BV/ODV upon AcMNPV infection.Next, the subcellular localization of TER94 in healthy and virus-infected cells was detected using immunofluorescence microscopy. As shown in Fig. 2, in healthy cells TER94 was evenly distributed in the nucleus and cytoplasm, with a larger amount of TER94 apparent in the cytoplasm than in the nucleus. In comparison, upon AcMNPV infection, TER94 exhibited dynamic distribution, being accumulated in the central region of the nucleus at 6 and 12 h postinfection (p.i.) and then translocated to the ring zone, the locus for ODV assembly, at 24 and 48 h p.i.
The subcellular localization of TER94 in Sf9 cells. Sf9 cells were infected by AcBac-egfp-ph at an MOI of 5 TCID50 units/cell and fixed at 6, 12, 24, and 48 h p.i. Uninfected cells were also harvested and analyzed. The localization of TER94 was detected using anti-TER94 as the primary antibody and Alexa 555-labeled goat anti-rabbit IgG as the secondary antibody. Nuclei were stained by Hoechst 33258. EGFP indicates the virus infection. Bars, 5 μm for the enlarged images and 10 μm for the others.
To further establish the precise localization of TER94 in cells, immunoelectron microscopy (IEM) was performed. In uninfected cells, many gold particles were observed in the darkly stained regions within the nucleus, which are considered to reflect host chromosomes (40, 41) (Fig. 3a, red arrows). Gold particles were also evenly distributed in the cytoplasm and around the nuclear envelope (data not shown). In the infected cells, in addition to the similar distribution of TER94 in the cytoplasm and on the nuclear envelope as observed in uninfected cells (data not shown), gold particles were accumulated on the chromatin-like structures in the VS at 12 h p.i. (Fig. 3b, red arrows), which serves as the factory for viral DNA replication and nucleocapsid assembly (19). During BV formation, nucleocapsids were found to egress from the nuclear envelope and bud through the plasma membrane, with gold particles evident on these nucleocapsids, which were destined to become BVs (Fig. 3c and d, red arrows). For nucleocapsids which were retained in the nucleus to form ODVs, TER94 was found in both the immature and finely assembled ODVs, being associated with mainly the nucleocapsids (Fig. 3e and f, red arrows). The IEM results were consistent with the Western blotting results in Fig. 1, confirming that TER94 is associated with nucleocapsids of both BV and ODV. In addition, the distribution of TER94 on the nuclear envelope (data not shown), especially at the nucleocapsid egress sites (Fig. 3c, white arrows), implied a role of TER94 at the step of nucleocapsid egress from the nucleus to the cytoplasm for BV formation.
IEM analysis of the localization of TER94. Sf9 cells were infected by AcBac-egfp-ph at an MOI of 5 TCID50 units/cell. The uninfected cells and infected cells at 12, 24, and 48 h p.i. were fixed and subjected to IEM. The localization of TER94 was probed using anti-TER94 as a primary antibody and 12-nm colloidal gold anti-rabbit IgG as a secondary antibody. White and red arrows indicate gold particles on the envelope and nucleocapsids, respectively. Nu, nucleus; NE, nuclear envelope; Cyto, cytoplasm. Bars, 200 nm.
TER94 is required for AcMNPV DNA replication and infectious BV production.To investigate whether TER94 is involved in the AcMNPV infection process, a TER94-specific inhibitor, N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ), was used to block the ATPase activity of TER94 (42). The cell viability assay showed that DBeQ exhibited no cytotoxicity toward Sf9 cells at a concentration of ≤9.0 μM (Fig. 4A). Further detection of cell apoptosis showed that DBeQ treatment did not induce significantly more (or even less) apoptosis than in the dimethyl sulfoxide (DMSO) group at all time points, while a commonly used apoptosis inducer, camptothecin (CPT) (43–45), stimulated extensive apoptosis (Fig. 4B). After excluding the cytotoxicity of DBeQ, cells were infected with AcBac-egfp-ph at a multiplicity of infection (MOI) of 5 50% tissue culture infective dose (TCID50) units/cell. The BV production upon DBeQ or DMSO treatment was measured at different time points. As shown in Fig. 4C, compared with DMSO treatment (shown as 0 μM), DBeQ concentrations of 4.5 and 9.0 μM resulted in approximately 3- and 60-fold reductions of BV genomic copy numbers in the cell culture supernatant at 24 h p.i., respectively (P < 0.001). At 48 h p.i., BV genomic copy numbers were reduced by approximately 2- and 20-fold under 4.5 and 9.0 μM DBeQ treatment (P < 0.001), respectively. At 72 h p.i., although the BV genomic copy numbers under 4.5 μM DBeQ treatment showed no significant discrepancy with that of the DMSO group, 9.0 μM DBeQ treatment reduced BV genomic copy numbers over 20-fold (P < 0.001). The BV titers were further determined by endpoint dilution assay. The one-step growth curves also showed a dose-dependent inhibitory effect of DBeQ (4.5 and 9.0 μM) on infectious BV production from 24 to 72 h p.i. (Fig. 4D) (P < 0.001).
The functions of TER94 in BV production and viral DNA replication. (A) Cell viability assay under DBeQ treatment. Sf9 cells were incubated with different concentrations of DBeQ for 24 h, and the cell viability was determined by Cell Counting Kit-8. (B) Detection of cell apoptosis in Sf9 cells under DBeQ treatment. Sf9 cells were infected by AcMNPV-WT at an MOI of 5 TCID50 units/cell and then cultured with 0, 4.5, or 9.0 μM DBeQ or 10.0 μM CPT. Apoptosis was detected by measuring caspase-3 activity at 24, 48, and 72 h p.i. (C and D) BV production under DBeQ treatment. Sf9 cells were infected by AcBac-egfp-ph at an MOI of 5 TCID50 units/cell and cultured with 0, 4.5, or 9.0 μM DBeQ. Supernatants were collected at the indicated time points and measured using qPCR for analysis of BV production (C) or by endpoint dilution assay for one-step growth curve analysis (D). (E) Cell viability in RNAi assay. Sf9 cells were transfected with dster94 or dsegfp and infected by AcBac-egfp-ph at an MOI of 5 TCID50 units/cell. Cell viability was detected at 24, 48, and 72 h p.i. (F) Detection of cell apoptosis in RNAi assay. The dster94- or dsegfp-transfected Sf9 cells were infected by AcMNPV-WT at an MOI of 5 TCID50 units/cell. Apoptosis was measured at 24, 48, and 72 h p.i. (G) BV production following RNAi. Sf9 cells were transfected with dster94 or dsegfp for 24 h and then infected by AcBac-egfp-ph at an MOI of 5 TCID50 units/cell. RNAi efficiency was detected by Western blotting, and BV production at 24, 48, and 72 h p.i. was quantified by qPCR. (H) Viral DNA replication under DBeQ treatment. Sf9 cells were transfected with bAcgp64− and cultured in medium containing 9.0 μM DBeQ or DMSO as a control. Cells were collected to quantify viral genomic copy numbers by qPCR at 24 and 48 h p.t. All results represent means ± standard deviations (n = 3 independent experiments). Statistical significance was analyzed using the Student t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. The insignificant differences were not labeled.
Furthermore, the requirement of TER94 for BV production was confirmed by RNA interference (RNAi) assay. The expression of ter94 was knocked down using synthesized double-stranded RNA (dsRNA). An egfp-specific dsRNA (dsegfp) was synthesized and used as a control. At 24 h after dsRNA transfection, cells were infected with AcBac-ph-egfp at an MOI of 5 TCID50 units/cell. Cell viability showed that transfection of dster94 neither decreased cell viability (Fig. 4E) nor induced obvious apoptosis (Fig. 4F) compared to the control group (transfection of dsegfp) at all infection time points. However, BV genomic copy numbers in dster94-transfected cells decreased by about 2-fold at 24 h p.i. and 17-fold at 48 h p.i. (P < 0.001). At 72 h p.i., the BV genomic copy numbers in dster94-transfected cells increased only minimally, whereas the BV genomic copy numbers in dsegfp-transfected cells were almost 26-fold (P < 0.05) higher than those in dster94-transfected cells (Fig. 4G, upper panel). The RNAi efficiency was detected at the corresponding time points using Western blotting, and the results showed that TER94 was efficiently depleted (Fig. 4G, lower panel).
To explore whether TER94 is involved in viral DNA replication, a gp64-deleted recombinant bacmid, bAcgp64−, which blocks BV budding but does not affect viral DNA replication, was used for viral DNA replication assays (46). Following transfection with bAcgp64− bacmid DNA, cells were treated with DMSO (shown as 0 μM) or DBeQ (9.0 μM). At 24 and 48 h posttransfection (p.t.), total cellular DNA was isolated and viral genomic copy numbers were measured using quantitative PCR (qPCR). As shown in Fig. 4H, the number of viral genomic DNA copies in the DBeQ treatment group was reduced about 5-fold (P < 0.01) and 10-fold (P < 0.05) at 24 and 48 h p.t. compared to that following DMSO treatment, respectively, confirming that TER94 is involved in viral DNA replication.
TER94 is required for BV and ODV morphogenesis.To investigate whether TER94 plays a role in AcMNPV virion morphogenesis, the infected cells were treated with DBeQ or DMSO and analyzed by electron microscopy (EM) at the indicated time points. At a relatively early stage of virus infection, newly assembled nucleocapsids egress from the nucleus and bud at the plasma membrane to form BVs (47). In DMSO-treated cells, naked nucleocapsids were observed trafficking in the cytoplasm or budding at the plasma membrane; only a small fraction of nucleocapsids was trapped in cytoplasmic vacuoles at 36 h p.i. (Fig. 5A-a and -a′, red arrows). In contrast, a large number of nucleocapsids were trapped within cytoplasmic vacuoles adjoining or near the nuclear envelope upon DBeQ treatment, with little nucleocapsid budding observed at the plasma membrane (Fig. 5A-c and -c′, red arrows). By 72 h p.i., almost no nucleocapsids could be observed in the cytoplasm or budding at the plasma membrane in the DMSO group (Fig. 5A-b), indicating the successful formation of BV. However, in the DBeQ-treated cells, nucleocapsids were still trapped around the nuclear envelope, indicating that further BV formation was blocked (Fig. 5A-d, red arrows). To better evaluate the effect of DBeQ on nucleocapsid trafficking, nucleocapsids trapped in vacuoles were quantified from 27 randomly chosen EM sections for each group. As shown in Fig. 5B, many more nucleocapsids were trapped in cytoplasmic vacuoles under DBeQ treatment at both 36 and 72 h p.i. than in the DMSO control groups (P < 0.001).
Functional analysis of TER94 in BV formation and ODV morphogenesis. (A to C) Sf9 cells were infected with AcBac-egfp-ph at an MOI of 5 TCID50 units/cell and cultured with 9.0 μM DBeQ or with DMSO. At 36 and 72 h p.i., cells were fixed and subjected to EM. (A) For BV formation, nucleocapsids were observed in the cytoplasm (a, a′, and b) under DMSO or 9.0 μM DBeQ (c, c′, and d) treatment. (B) Nucleocapsids trapped in cytoplasmic vacuoles were quantified in 27 cell sections for DBeQ and DMSO treatment at 36 and 72 h p.i. Statistical significance was analyzed using the Student t test: ***, P < 0.001. (C) For ODV morphogenesis, nucleocapsids were observed in the nucleus under DMSO (a and b) or 9.0 μM DBeQ (c and d) treatment. Red arrows indicate nucleocapsids. Nu, nucleus; Cyto, cytoplasm. Bars, 500 nm.
Next, ODV morphogenesis in the infected nucleus was also examined. As shown in Fig. 5C, in the DMSO-treated group, a large number of finely assembled ODVs were observed in the infected nucleus at 36 h p.i. (Fig. 5C-a, red arrows), with the ODVs having been embedded into occlusion bodies (OBs) at 72 h p.i. (Fig. 5C-b, red arrows). Under DBeQ treatment, numerous newly formed nucleocapsids were attached to microvesicles and appeared to begin envelopment at 36 h p.i. However, these ODVs appeared to be immature, within which a large number of nucleocapsids were arranged in a disordered manner (Fig. 5C-c, red arrows). Until 72 h p.i., the ODVs remained immature and could not be processed into smaller, mature ODVs (Fig. 5C-d, red arrows). Concurrently, ODV occlusion into OBs could be hardly observed until the very late stage of infection following DBeQ treatment (Fig. 5C-c and -d, as well as data not shown). The subcellular localization of TER94 in infected cells under DBeQ treatment was also detected by IEM, with the results showing similar distribution as that in untreated cells (data not shown). Taken together, these results implied that the ATPase activity of TER94 is essential for its proper functions in BV and ODV morphogenesis.
TER94 colocalizes with replication-related viral proteins within virogenic stroma upon infection.To identify the potential proteins interacting with TER94 or its cofactors, coimmunoprecipitation (co-IP) assay and mass spectrometric analysis were performed. Notably, many of the identified interacting proteins were replication-related viral proteins, with highest abundance of LEF3 and helicase (data not shown). As TER94 was localized to VS at the early stage of infection and involved in viral DNA replication (Fig. 3 and 4H), we decided to investigate the interaction between TER94 and viral replication-related proteins. Six viral proteins including DNA polymerase, helicase, LEF1, LEF2, LEF3, and immediate early gene 1 (IE1) are required for DNA replication (48). The six viral proteins fused with enhanced green fluorescent protein (EGFP) were expressed by transfection of recombinant plasmids into Sf9 cells separately, and their colocalization with TER94 was detected in AcMNPV-infected cells. As shown in Fig. 6A (EGFP channel), upon AcMNPV infection, all six proteins were accumulated, at least partially, in the subnuclear structures representing VS, which was consistent with previous reports (49, 50). The distribution patterns of the six proteins appeared to be in accordance with their DNA replication-associated functions (51–54). Moreover, in cells expressing LEF3EGFP, TER94 was found to aggregate in VS and exhibited a distribution pattern highly similar to that of LEF3EGFP. The remaining five viral proteins partially colocalized with TER94 in the infected nucleus but did not induce substantial VS aggregation of TER94.
Colocalization of TER94 with viral proteins in infected cells. (A) Immunofluorescence microscopy analysis of the subcellular localization of TER94 and viral proteins in infected cells. Sf9 cells were transfected with plasmids expressing LEF3EGFP, helicaseEGFP, DNAPolEGFP, IE1EGFP, LEF1EGFP, LEF2EGFP, or EGFP for 24 h and infected by AcMNPV-WT at an MOI of 5 TCID50 units/cell. Cells were fixed at 14 h p.i. and subjected to immunofluorescence microscopy analysis. TER94 was detected using anti-TER94; cell nuclei were stained by Hoechst 33258. Bars, 5 μm for the enlarged images and 10 μm for the others. (B) IEM analysis of the colocalization of TER94 and LEF3. Sf9 cells transfected with pIZ/V5-lef3HA for 24 h were infected by AcBac-egfp-ph and fixed at 14 h p.i. for IEM analysis. Cells in subpanels a and b were probed with anti-HA or anti-TER94 as a primary antibody and 12-nm colloidal gold anti-mouse/rabbit IgG as a secondary antibody. Cells in subpanel c were probed using both anti-HA and anti-TER94 as primary antibodies and 12-nm and 18-nm colloidal gold anti-mouse/rabbit IgG as a secondary antibody, respectively. The 12-nm colloidal gold-labeled LEF3HA and 18-nm colloidal gold-labeled TER94 in subpanel c are marked as red and blue dots in subpanel d, respectively. Bars, 200 nm. (C) The interactions of TER94 with LEF3 and helicase by co-IP experiment. Sf9 cells were infected by Ac-lef3flag or Ac-helicaseflag for 36 h. The infected cells were further treated with DMSO or DBeQ for 12 h and then cross-linked by DSP. The cross-linked samples were subjected to co-IP with anti-FLAG MAb and analyzed by Western blot analysis with anti-TER94 PAb. M, marker; WCL, whole-cell lysates; IP, immunoprecipitates. Arrows indicate the TER94 detected by anti-TER94 PAb.
Furthermore, the detailed localization of TER94 and LEF3 in infected nuclei was detected by IEM. Toward this end, LEF3 fused with a C-terminal hemagglutinin (HA) tag (LEF3HA) was transiently expressed in Sf9 cells and detected using the tag antibody, whereas the endogenous TER94 was detected using its specific antibody. Labeling of LEF3HA or TER94 individually showed that both were localized in the electron-dense regions within VS and on nucleocapsids at 14 h p.i. (Fig. 6B-a and -b). Labeling TER94 with 18-nm gold particles (blue dots in Fig. 6B-d) and LEF3HA with 12-nm gold particles (red dots in Fig. 6B-d) simultaneously showed the colocalization of TER94 and LEF3HA in VS, especially on the chromatin-like structures (Fig. 6B-c and -d).
According to a previous report, TER94 interacts with its cargos in a highly dynamic manner (55). To detect the associations of TER94 with LEF3 and helicase, recombinant AcMNPV containing FLAG-tagged lef3 or helicase was used to infect Sf9 cells. Prior to the coimmunoprecipitation (co-IP) assay, DBeQ and a cross-linking agent, dithiobis(succinimidylpropionate) (DSP), were used to “freeze” the TER94 complexes (55). As shown in Fig. 6C, in Ac-lef3flag-infected cells, TER94 could be immunoprecipitated by LEF3 only in the presence of DBeQ treatment, while TER94 could be immunoprecipitated by helicase in the absence of DBeQ treatment (DMSO group) in Ac-helicaseflag-infected cells, suggesting the presence of interactions of TER94 with LEF3 and helicase.
TER94 is recruited to subnuclear structures by interacting with LEF3 and helicase.A previous report showed that cotransfection of four elements including LEF3, helicase, IE1, and homologous region (hr) induced subnuclear structures in Bombyx mori cells (56). However, we found that coexpressing only two of these, LEF3 and helicase, was sufficient to form similar subnuclear structures that excluded host chromatin from the central nucleus in Sf9 cells (Fig. 7A). Notably, TER94 was recruited to the subnuclear structures and colocalized with LEF3EGFP and helicasemCherry. As a control, transient expression of LEF3EGFP or helicasemCherry alone yielded nuclear localization of LEF3EGFP and cytoplasmic distribution of helicasemCherry, which were consistent with the previous report (57). Separate expression of these two proteins did not affect the even distribution of TER94 in the nucleus (Fig. 7A).
Interaction of TER94 with viral proteins. (A) Coexpression of LEF3 and helicase induces subnuclear structures in Sf9 cells. Sf9 cells were transfected with the indicated plasmids and fixed at 36 h p.t. Localization of TER94 was detected using anti-TER94 antibody and Alexa 647-labeled goat anti-rabbit IgG. Nuclei were stained by Hoechst 33258. (B) Interaction of TER94 with viral proteins as detected by BiFC assay. Sf9 cells were transfected with plasmids expressing Vn-TER94 and Vc-X (X represents viral proteins) or Vc-TER94 and Vn-X (X represents viral proteins). Cells were monitored under fluorescence microscopy at 36 h p.t. Bars, 10 μm.
Furthermore, the interactions of TER94 with viral replication-related proteins were detected by bimolecular fluorescence complementation assay (BiFC), in which the N-terminal (Vn) and C-terminal (Vc) regions of fluorescent protein Venus were fused with the two target proteins, respectively, as these would produce fluorescence if the two target proteins interacted with each other (58). As shown in Fig. 7B, LEF3 and helicase could interact with TER94, and fluorescent dot structures formed in the nucleus when cells expressed the LEF3 and TER94 BiFC pair, whereas fluorescent tubular structures formed in the cytoplasm when Vn-helicase and Vc-TER94 were expressed. Apart from LEF3 and helicase, IE1 and LEF1 also showed reciprocal interactions with TER94. However, the interactions of LEF2 and DNA polymerase with TER94 occurred in only one direction. IE2-TER94 did not produce fluorescence in both directions. Together, these results suggested that TER94 could interact with LEF3, helicase, IE1, and LEF1 and was recruited to VS or subnuclear structures by LEF3-helicase.
TER94 is involved in baculovirus-induced virogenic stroma formation and expansion.To explore whether TER94 is involved in the correct formation of baculovirus-induced VS structure, the localization of several well-known indicator proteins for VS, including IE1, DNA-binding protein (DBP), LEF3, and helicase, was detected following AcMNPV infection. As previous studies showed that IE1 localizes to specific foci and is associated with the viral DNA synthesis region in the infected cells, IE1 was considered an indicator of VS. During viral DNA replication, IE1 foci gradually expand and colocalize with LEF3 and DBP and finally develop into VS (49, 59). In the present study, the function of TER94 in VS morphogenesis was studied through use of DBeQ. As shown in Fig. 8, in DMSO-treated cells, IE1EGFP and DBPEGFP formed large foci in the nucleus at 14 h p.i., indicating the formation of VS and synthesis of viral DNA. LEF3EGFP and helicaseEGFP were also localized to the nucleus and accumulated in VS. However, under DBeQ treatment, only a few small foci were observed in the nuclei of IE1EGFP- and DBPEGFP-expressing cells at 14 h p.i. The localization pattern of LEF3EGFP in DBeQ-treated cells was similar to that in uninfected cells, being dispersed across the whole nucleus (Fig. 7A). However, the nuclear import of helicaseEGFP upon infection was severely impaired under DBeQ treatment. These results indicated that inhibition of TER94 blocked the proper transportation and localization of DNA replication-associated proteins and impaired VS formation and expansion.
Functional analysis of TER94 in VS morphogenesis. Sf9 cells transfected with plasmids expressing viral proteins fused with EGFP were infected by AcMNPV-WT at an MOI of 5 TCID50 units/cell and then cultured in the presence of DBeQ (9.0 μM) or DMSO. Cells were fixed at 14 h p.i., and immunofluorescence microscopy analysis was performed using anti-TER94 antibody. Nuclei were stained by Hoechst 33258. Bars, 3.3 μm for the enlarged images and 10 μm for the others.
DISCUSSION
Virus-host interaction occurs at multiple steps of infection, during which some host factors are recruited and utilized by viruses and finally packaged into progeny virions. TER94 and its homologs constitute highly abundant and conserved AAA+ ATPases that have been found in both the cytoplasm and nucleoplasm of vertebrate cells, including mammalian cells and Xenopus laevis kidney epithelial cells (60, 61). In the present study, insect cellular TER94 was detected as being exclusively associated with the nucleocapsid fractions of both AcMNPV BV and ODV (Fig. 1). Further functional studies indicated that TER94 plays crucial roles at multiple steps of baculovirus infection, including viral DNA replication, BV formation, and ODV morphogenesis.
A critical role of TER94 is to mediate membrane biogenesis, especially during nuclear envelope reassembly (4, 62). During BV formation, the nuclear envelope evaginates and blebs to facilitate the egress of nucleocapsids from the nucleus. Then, the nude nucleocapsids escape from the nuclear envelope-derived vacuoles (egress vacuoles) and bud at the plasma membrane to form BVs. In our study, inhibition of TER94 resulted in the retention of nucleocapsids in the egress vacuoles and dramatically reduced the subsequent BV production (Fig. 4 and 5). Notably, ESCRT-III, the key regulator for membrane budding and scission, and soluble NSF, another member of the AAA+ ATPase family that functions in mediating the membrane fusion pathway, were found to be required for AcMNPV nucleocapsid egress from the nuclear membrane (33, 34). Impairment of the function of NSF or ESCRT-III components resulted in the aggregation of nucleocapsids in the perinuclear spaces between inner and outer nuclear membranes (33, 34). Moreover, TER94 is associated with ESCRT-III and NSF in nuclear envelope reformation in vertebrate cells and is proposed to be recruited to the highly curved membrane to assist in membrane fission (63–65).Therefore, we deduced that TER94, ESCRT-III, and NSF might be recruited to the nucleocapsid egress sites and act in concert during BV formation, although this process requires further investigation.
The envelopment of ODV also requires the processing of the nuclear envelope to form the precursor of the ODV envelope (47, 66). In addition, the last step of ODV morphogenesis requires splicing of the large and immature ODVs to small and mature ones (66). Inhibition of TER94 did not affect the association of nucleocapsids with microvesicles but impaired the last step of ODV maturation, as the TER94 inhibitor DBeQ blocked the membrane invagination of immature ODVs (Fig. 5C). In a study of nuclear envelope assembly, TER94 was shown to function at two distinct but sequential steps: formation of the closed nuclear envelope and the subsequent nuclear envelope expansion (62). As the egress vacuole and ODV envelope are derived from the nuclear envelope, we propose that the role of TER94 in baculovirus BV and ODV morphogenesis appears to be highly correlated with its function in nuclear envelope reassembly.
Another important function of TER94 lies in participating in both cellular and viral genome replication (5, 67–69). In the present study, TER94 was found to be recruited to the chromatin-like structure in VS (Fig. 3) and was crucial for AcMNPV genome replication (Fig. 4H). Although the precise mechanisms of baculoviral VS formation and viral DNA replication remain obscure, it is considered that the generation of VS is regulated by sequential steps: IE1 binds to DNA replication origins and then LEF3 and helicase cluster with IE1 to form the basic fine structure of VS (49, 56). The subsequent participation of DBP and viral DNA synthesis trigger the expansion of VS (70, 71). In a study of Bombyx mori nucleopolyhedrovirus, four viral elements including IE1, LEF3, helicase, and hr could induce the subnuclear structure (56). However, we found that coexpression of AcMNPV LEF3 and helicase alone could induce a similar subnuclear structure that excludes host chromatin to the nuclear margin and relocates TER94 to this structure (Fig. 7A). The interaction of LEF3 with TER94 in the BiFC assay formed smaller fluorescent structures in chromatin-free regions (Fig. 7B), which also implied a role of TER94 in the subnuclear structure and VS formation. Subsequent inhibition of TER94 by DBeQ revealed that although small foci of IE1 and DBP could be observed, the further expansion of these foci was blocked, indicating the deficiency of VS formation. In addition, LEF3 accumulation in the subnuclear domain and nuclear transport of helicase were severely impaired (Fig. 8). Thus, we proposed that TER94 was recruited by LEF3 and helicase to the DNA replication foci to assist with the construction of VS. Moreover, recent studies revealed essential functions of TER94 in disassembly of the eukaryotic replication complex at the end of DNA replication. Therefore, the finding that TER94 interacted with the AcMNPV helicase (Fig. 6C and Fig. 7B) implied that baculovirus may utilize TER94 and other host factors to disassemble the viral replisome and terminate replication, which is similar to eukaryotic replication mechanisms (5, 67, 72, 73).
Based on our findings, we proposed a model that depicts the three steps in which TER94 is involved during baculovirus infection (Fig. 9A). (i) During viral DNA replication, TER94 interacts with LEF3 and helicase and is recruited to VS by these proteins to participate in VS morphogenesis and viral genome replication. (ii) During BV formation, TER94 in the nuclear envelope facilitates the release of nucleocapsids from the egress vacuoles to form BVs. (iii) TER94 is also essential for processing the immature ODV envelope for the final maturation of ODV. For all three steps, the energy-consuming property of TER94 is essential, as the ATP-competitive inhibitor DBeQ could block these processes. A more detailed model to describe the role of TER94 in VS morphogenesis is presented in Fig. 9B. At the immediate early stage of infection, IE1 binds to the viral genome and forms initial foci in the nucleus, followed by the participation of other replication-associated proteins, including LEF3, helicase, and DBP. In addition, TER94 is recruited by the LEF3-helicase complex to the foci and in turn helps other viral proteins to aggregate and form subnuclear compartments. The viral DNA synthesis in the subnuclear compartments promotes their development into VS. It is possible that TER94 may be packaged into the capsids together with some replication-related viral proteins, such as LEF3 and helicase. Moreover, TER94 was identified to interact with certain viral nucleocapsid or tegument proteins (data not shown); therefore, it might specifically attach to nucleocapsids via interacting with these viral proteins to function during BV formation and ODV morphogenesis. However, we could not exclude the possibility that as an abundant host protein, TER94 may be accidentally incorporated into virions.
Proposed functional models of TER94 in AcMNPV infection. (A) Functions of TER94 in the AcMNPV life cycle. (1) At the early stage of infection, TER94 interacts with viral proteins LEF3 and helicase and is taken into VS to participate in viral DNA replication. (2) In the BV production process, nucleocapsids egress from the nucleus by blebbing from the nuclear envelope and then escape from the vacuoles to transport to the budding sites. TER94 may help to disrupt the vacuolar membrane, as nucleocapsids were restricted in the egress vacuoles when cells were treated with DBeQ. (3) In ODV morphogenesis, nucleocapsids interact with nuclear envelope-derived microvesicles and are enveloped by the membrane to form ODVs. TER94 may splice the ODV envelope and promote the maturation of ODVs. (B) Roles of TER94 in VS morphogenesis. Upon release of the viral genome into the nucleus, IE1 binds to the viral DNA and forms foci. Other replication-associated proteins, including LEF3 and helicase, which recruit TER94, join the foci and form subnuclear compartments. The subsequent DNA replication promotes the development of VS. VS, virogenic stroma; ER, endoplasmic reticulum.
In mammalian cells, TER94 is known to interact with different cofactors to exert its distinct functions (38). For example, p47 recruits TER94 to membrane fusion pathways (4, 74). Additional well-studied cofactors, ubiquitin fusion degradation 1 (UFD1) and nuclear pore localization 4 (NPL4), are required for replisome assembly, and both p47 and UFD1-NPL4 are utilized in nuclear envelope reformation (62, 73). However, whether these cofactors are involved in and differentially required for TER94-associated baculoviral DNA replication, nucleocapsid egress, and ODV envelopment remains to be determined. In summary, the study sheds light on the critical roles of the host AAA+ ATPase TER94 in baculovirus progeny virion morphogenesis and the viral replication process.
MATERIALS AND METHODS
Cells, viruses, and drugs.Sf9 cells were cultured with Grace’s insect medium (pH 6.0; Gibco-BRL) containing 10% fetal bovine serum (Gibco-BRL) at 27°C. Wild-type virus AcMNPV-WT, recombinant AcMNPV AcBac-egfp-ph, and a gp64-null bacmid, bAcgp64−, used in this study have been described in previous reports (46, 75, 76). Recombinant baculoviruses Ac-lef3flag and Ac-helicaseflag were constructed using the Bac-to-Bac baculovirus expression system (Invitrogen). Briefly, lef3 and helicase genes fused with a FLAG tag at their 5′ end were PCR amplified and inserted into pFastBacDual transfer vector to generate donor plasmids pFBD-lef3flag and pFBD-helicaseflag. Then, the lef3flag and helicaseflag fragments were transposed into AcMNPV bacmid to generate recombinant bacmids, respectively. Infectious baculoviruses Ac-lef3flag and Ac-helicaseflag were produced by transfection of recombinant bacmids into Sf9 cells. DMSO and the TER94-specific inhibitor DBeQ were purchased from Sigma-Aldrich. CPT was purchased from Beyotime.
Cloning of ter94.Total RNA of Sf9 cells was extracted and cDNA was synthesized according to a previous report (76). The open reading frame (ORF) of ter94 was amplified by PCR using ter94-specific primers ter94-F and ter94-R (Table 1). The PCR products were digested by restriction enzymes and inserted into the pIZ/V5-His vector (Invitrogen), generating the plasmid pIZ/V5-ter94. The resulting plasmid was sequenced to eliminate unintended mutations.
Primers used in this study
Preparation of polyclonal antibody.To prepare a TER94-specific antibody, the ORF of ter94 was amplified by PCR using primers ter94-F/R (Table 1) with plasmid pIZ/V5-ter94 as the template. The amplified fragment was inserted into the pET28a+ vector to produce the prokaryotic expression plasmid pET28a-ter94. Following DNA sequencing, pET28a-ter94 plasmids were electroporated into Escherichia coli strain BL21 to express TER94 proteins. The purified TER94 proteins were used to immunize rabbits to elicit the polyclonal antibody anti-TER94 as previously described (77).
Western blot analysis.To analyze the localization of TER94 in virions, BVs or ODVs harvested from AcMNPV-WT-infected cell culture or larvae were purified as previously described (76). The purified BVs and ODVs were lysed on ice using 1% NP-40 lysis buffer for 30 min and centrifuged at 20,000 × g for 30 min. The supernatant containing the viral envelope and pellet containing nucleocapsids were collected separately. Both fractions were analyzed by Western blotting using anti-TER94 (1:5,000), anti-VP39 (1:5,000), anti-GP64 (1:5,000), and anti-PIF5 (1:4,000) antibodies as primary antibody (76) and horseradish peroxidase-labeled goat anti-rabbit immunoglobulin (1:5,000; Pierce) antibody as secondary antibody. Protein signals were detected by chemiluminescence reaction using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific).
Immunofluorescence microscopy.Sf9 cells (1 × 106) were seeded into a 35-mm glass-bottom dish and infected by AcBac-egfp-ph at an MOI of 5 TCID50 units/cell or mock infected following cell adhesion. Immunofluorescence microscopy analysis was performed as previously described (78). Briefly, cells were fixed at indicated time points as shown in Fig. 2 and then detected with anti-TER94 PAb (1:500) as a primary antibody and Alexa 555-labeled goat anti-rabbit (1:500; Abcam) as a secondary antibody. Nuclei were stained with Hoechst 33258 (Beyotime). Cell samples were monitored using fluorescence microscopy.
EM and IEM.For EM analysis, Sf9 cells were cultured in 35-mm dishes and infected by AcBac-egfp-ph at an MOI of 5 TCID50 units/cell for 1 h. After removing the medium containing virus, cells were washed three times with fresh medium and then cultured in culture medium containing DMSO or DBeQ (9.0 μM). Cells were harvested at 36 and 72 h p.i. and processed for EM analysis as previously reported (76).
To follow the subcellular localization of TER94 as shown in Fig. 3, Sf9 cells (2 × 106) were seeded in 60-mm dishes and infected with AcBac-egfp-ph at an MOI of 5 TCID50 units/cell or mock infected. Infected cells were fixed at 12, 24, and 48 h p.i. and subjected to IEM analysis according to a previous report (76). To detect the colocalization of TER94 and LEF3HA as shown in Fig. 6B, Sf9 cells (2 × 106) were transfected with pIZ/V5-lef3HA, which expresses LEF3 with a hemagglutinin (HA) tag fused at its C terminus, and infected with AcBac-egfp-ph at an MOI of 5 TCID50 units/cell. Cells were harvested at 14 h p.i. and subjected to IEM analysis. Ultrathin sections were immunostained with anti-TER94 (1:50) or anti-HA (1:100; Sigma) antibodies as a primary antibody and 12- or 18-nm colloidal gold-labeled goat anti-rabbit/mouse IgG (1:50; Jackson) as a secondary antibody. Sections were observed under transmission EM (FEI Tecnai G2 20 Twin) at 200-kV acceleration voltage.
qPCR analysis.To quantify viral DNA accumulation, Sf9 cells (1 × 106) were seeded in a 6-well plate and transfected with 5 μg bAcgp64− using 10 μl Cellfectin II reagent (Invitrogen) for each dish. After incubation for 6 h, the supernatant was removed and cells were washed three times with phosphate-buffered saline and cultured in medium containing DMSO or DBeQ (9.0 μM). The total genomic DNA of each dish was extracted using the AxyPrep multisource genomic DNA miniprep kit (Axygen) and dissolved in 50 μl double-distilled water (ddH2O) at 12, 24, and 48 h p.t. A 2-μl aliquot of each DNA sample was digested using 10 U of DpnI restriction enzyme (New England BioLabs) overnight to eliminate residual bacmid DNA. The products were used for qPCR to analyze the viral genomic copy numbers.
To quantify BV production, Sf9 cells were infected with AcBac-egfp-ph at an MOI of 5 TCID50 units/cell for 1 h, after which cells were washed three times and then cultured in Grace’s culture medium containing DSMO or DBeQ (4.5 or 9.0 μM). Supernatants were collected at 0, 24, 48, and 72 h p.i., and BV was titrated by endpoint dilution assay. Viral genomic DNA was quantified by qPCR using primers vp80-F/R as described previously (76).
RNAi assay.The RNAi assay was performed using ter94-specific dsRNA (dster94) and egfp-specific dsRNA (dsegfp). To produce gene-specific dsRNAs, the 1,036-bp ter94 fragment and 720-bp egfp fragment were amplified by PCR using primers dster94-F/R or dsegfp-F/R (Table 1) and pIZ/V5-ter94 or AcBac-egfp-ph as the templates, respectively. The PCR products were purified and used as the templates in the dsRNA synthesis system. The dsRNAs were synthesized and purified using the MEGAscript RNAi kit (Life Technologies) according to the manufacturer’s instructions.
Sf9 cells (5 × 105 cells/well) were seeded in a 24-well tissue culture plate and transfected with 7.5 μg/well dster94 or dsegfp using Cellfectin II. Transfected cells were infected by AcBac-egfp-ph at an MOI of 5 TCID50 units/cell at 24 h p.t. Supernatants were harvested at 24, 48, and 72 h p.i. to analyze BV production by qPCR. Cells were collected to detect the RNAi efficiency by Western blot analysis at the corresponding time points.
Cell viability assay.To detect the cell viability under DBeQ treatment, Sf9 cells were seeded in a 96-well plate and cultured with 0 to 10 μM DBeQ. After incubation for 24 h, cell viability was determined with Cell Counting Kit-8 (Beyotime, China) according to the manufacturer’s instructions. To measure the cell viability in the RNAi assay, Sf9 cells transfected with dsegfp and dster94 were seeded in a 96-well plate. At 24 h p.t., cells were infected by AcBac-egfp-ph at an MOI of 5 TCID50 units/cell. The cell activities were measured by Cell Counting Kit-8 at 24, 48, and 72 h p.i., separately.
Detection of apoptosis.Sf9 cells were seeded in a 96-well plate and infected by AcMNPV-WT at an MOI of 5 TCID50 units/cell in the presence of DBeQ, CPT, or DMSO. Caspase-3 activity was measured with the GreenNuc caspase-3 assay kit for live cells (Beyotime, China) according to the manufacturer’s instructions at the indicated time points. Briefly, 10 μl caspase-3 substrate Asp-Glu-Val-Asp (DEVD)-DNA dye was added into each well and incubated for 30 min. Fluorescence was detected after incubation. To detect cell apoptosis in RNAi assay, dsegfp- and dster94-transfected cells were seeded in a 96-well plate and infected by AcMNPV-WT. The cell apoptosis at indicated time points was measured with a similar method.
Co-IP assay.Sf9 cells (2 × 107 cells/well) were seeded in 10-cm dishes and infected by Ac-lef3flag or Ac-helicaseflag at an MOI of 5 TCID50 units/cell. At 36 h p.i., DMSO or DBeQ (9.0 μM) was added into the cell culture. After incubation for 12 h, cells were cross-linked with 1 mM DSP (Thermo Scientific) at room temperature for 30 min and then quenched by 25 mM Tris (pH 7.4) for 10 min according to a previous report (55). The cross-linked cells were then lysed with cell lysis buffer (Beyotime, China) with protease inhibitor cocktail (Roche) for 5 min on ice. Cell lysates were centrifuged at 20,000 × g for 15 min to remove debris. For immunoprecipitation, supernatant was collected and mixed with anti-FLAG monoclonal antibody (MAb) (Life Technologies) and Protein G Plus/protein A-agarose (Millipore) overnight at 4°C. After pelleting, agarose beads were washed five times with cell lysis buffer and treated with SDS-PAGE loading buffer (50 mM Tris-HCl, 2% SDS, 0.1% bromophenol blue, 10% glycerol, 5% β-mercaptoethanol). The IP samples were heated at 95°C for 10 min and then subjected to Western blot analysis as described above.
BiFC assay.DNA fragments of the N-terminal (Vn, residues 1 to 173) and C-terminal (Vc, residues 174 to 239) regions of fluorescent protein Venus were amplified by PCR using primers Vn-F/R1 and Vc-F/R1, respectively, according to a previous report (58). The DNA fragments were inserted into pIZ/V5-his separately to construct plasmids pIZ/V5-Vn and pIZ/V5-Vc. The ORFs of helicase, lef3, ie1, lef1, lef2, the DNA polymerase gene, and ie2 of AcMNPV were amplified by PCR using primers helicase-F1/R1, lef3-F1/R1, ie1-F1/R1, lef1-F1/R1, lef2-F1/R1, dnapol-F1/R1, and ie2-F1/R1, respectively, and the DNA fragments were in-frame inserted into pIZ/V5-Vn and pIZ/V5-Vc to produce pIZ/V5-Vn/Vc-X. The ter94 gene was amplified and inserted into pIZ/V5-Vn and pIZ/V5-Vc to produce pIZ/V5-Vn/Vc-ter94. Sf9 cells seeded on glass coverslips in a 24-well plate were transfected with BiFC plasmid pairs (0.5 μg of each plasmid per well) and observed under fluorescence microscopy at 36 h p.t.
Analysis of protein subcellular localization.Viral gene helicase with mCherry fused at its N terminus was inserted into pIZ/V5-his, generating pIZ/V5-helicasemCherry. The viral gene ie1, lef1, lef2, lef3, helicase, DNA polymerase gene, or dbp was fused with egfp at its C terminus to generate pIZ/V5-ie1egfp, pIZ/V5-lef1egfp, pIZ/V5-lef2egfp, pIZ/V5-lef3egfp, pIZ/V5-helicaseegfp, pIZ/V5-dnapolegfp, or pIZ/V5-dbpegfp, respectively. The ORFs of egfp and mCherry were cloned into pIZ/V5-his to construct the control plasmids pIZ/V5-egfptaa and pIZ/V5-mCherrytaa, respectively. Sf9 cells seeded on coverslips were transfected with the indicated plasmids in Fig. 6A and infected with AcMNPV-WT at an MOI of 5 TCID50 units/cell. Subcellular localization of viral proteins and TER94 was detected at 14 h p.i. To follow the subcellular localization of proteins in the absence of virus infection, the indicated plasmids in Fig. 7A were transfected and imaged by fluorescence microscopy at 36 h p.t. To detect the subcellular localization of proteins under DBeQ treatment as shown in Fig. 8, transfected cells were infected by AcMNPV-WT at an MOI of 5 TCID50 units/cell at 24 h p.t., cultured with DBeQ (9.0 μM) or DMSO for 14 h, and then imaged using fluorescence microscopy.
ACKNOWLEDGMENTS
This work was supported by grants from the National Natural Science Foundation of China (grant no. 31621061 and 31572334).
We thank Ding Gao, Anna Du, Pei Zhang, Bichao Xu, and Juan Min from the Core Facility and Technical Support facility of the Wuhan Institute of Virology for technical assistance.
FOOTNOTES
- Received 29 September 2019.
- Accepted 19 December 2019.
- Accepted manuscript posted online 2 January 2020.
- Copyright © 2020 American Society for Microbiology.
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