The Autographa californica Multiple Nucleopolyhedrovirus ac54 Gene Is Crucial for Localization of the Major Capsid Protein VP39 at the Site of Nucleocapsid Assembly

Abstract

Baculovirus DNAs are synthesized and inserted into preformed capsids to form nucleocapsids at a site in the infected cell nucleus, termed the virogenic stroma. Nucleocapsid assembly ofAutographa californicamultiple nucleopolyhedrovirus (AcMNPV) requires the major capsid protein VP39 and nine minor capsid proteins, including VP1054. However, how VP1054 participates in nucleocapsid assembly remains elusive. In this study, the VP1054-encoding gene (ac54) was deleted to generate theac54-knockout AcMNPV (vAc54KO). In vAc54KO-transfected cells, nucleocapsid assembly was disrupted, leading to the formation of abnormally elongated capsid structures. Interestingly, unlike cells transfected with AcMNPV mutants lacking other minor capsid proteins, in which capsid structures were distributed within the virogenic stroma,ac54ablation resulted in a distinctive location of capsid structures and VP39 at the periphery of the nucleus. The altered distribution pattern of capsid structures was also observed in cells transfected with AcMNPV lacking BV/ODV-C42 or in cytochalasind-treated AcMNPV-infected cells. BV/ODV-C42, along with PP78/83, has been shown to promote nuclear filamentous actin (F-actin) formation, which is another requisite for nucleocapsid assembly. Immunofluorescence using phalloidin indicated that the formation and distribution of nuclear F-actin were not affected byac54deletion. However, immunoelectron microscopy revealed that BV/ODV-C42, PP78/83, and 38K failed to integrate into capsid structures in the absence of VP1054, and immunoprecipitation further demonstrated that in transient expression assays, VP1054 interacted with BV/ODV-C42 and VP80 but not VP39. Our findings suggest that VP1054 plays an important role in the transport of capsid proteins to the nucleocapsid assembly site prior to the process of nucleocapsid assembly.

Importance: Baculoviruses are large DNA viruses whose replication occurs within the host nucleus. The localization of capsids into the capsid assembly site requires virus-induced nuclear F-actin; the inhibition of nuclear F-actin formation results in the retention of capsid structures at the periphery of the nucleus. In this paper, we note that the minor capsid protein VP1054 is essential for the localization of capsid structures, the major capsid protein VP39, and the minor capsid protein 38K into the capsid assembly site. Moreover, VP1054 is crucial for correct targeting of the nuclear F-actin factors BV/ODV-C42 and PP78/83 for capsid maturation. However, the formation and distribution of nuclear F-actin are not affected by the lack of VP1054. We further reveal that VP1054 interacts with BV/ODV-C42 and a capsid transport-related protein, VP80. Taken together, our findings suggest that VP1054 plays a unique role in the pathway(s) for transport of capsid proteins.

FIG 1

Characterization of the ac54-knockout bacmid. (A) Strategy for the construction of the recombinant viruses vAc54KO, vAc54:2HA, and vAcWT. (B) Sf9 cells were transfected with vAc54KO, vAc54:2HA, and vAcWT bacmid DNA and observed at 72 h p.t. (C) Virus growth curves. The supernatants of transfected cells were harvested at different time points, and viral titers were determined by TCID₅₀ endpoint dilution assays. Each data point represents the average titer of three independent biological repeats; error bars indicate standard deviations. (D) Real-time PCR analysis of viral DNA synthesis in Sf9 cells. Total DNA was extracted from Sf9 cells transfected with vAc54KO, vIE1KO, or v38KKO bacmid DNA at indicated time points, further digested with the restriction enzyme DpnI to eliminate input bacmid DNA, and quantified by real-time PCR. Data are shown as mean values ± standard deviations.

FIG 2

Electron micrographs of Sf9 cells transfected with vAc54KO or infected with wt AcMNPV. (A) Nucleus of a vAc54KO-transfected Sf9 cell at 24 h p.t. No capsid structures (with or without DNA content) exist in the VS. Black arrowheads indicate normal-appearing nucleocapsids in the RZ. (B) Partial view of the VS magnified from the cell shown in panel A, showing a large quantity of abnormal electron-dense bodies (indicated with white arrows) at the edge of the electron-dense mats. (C) Magnified view from panel A. Abnormally long, electron-lucent tubules can be found in the RZ. (D and E) Partial view of an Sf9 cell infected with wt AcMNPV at 24 h p.i. Nucleocapsids with DNA content are found in the VS (D), and ODVs are found in the RZ (E). Black arrowheads indicate normal nucleocapsids. (F to H) Immunoelectron microscopy analysis of vAc54KO-transfected Sf9 cells at 72 h p.t. (cells were labeled with BrdU at 12 h p.t.). Antiserum against BrdU (F), P6.9 (G), or VP39 (H) was used as the primary antibody. VS, virogenic stroma; RZ, ring zone; Nu, nucleus; nm, nuclear membrane. Scale bars, 2 μm (A) and 500 nm (B to H).

FIG 3

Subcellular localization of the major capsid protein VP39. Sf9 cells were transfected with vAc54KO (A), vAc54:2HA (B), or v38KKO (C) bacmid DNA. Transfected cells were fixed at 36 h p.t. and incubated with antisera against VP39 (rabbit) and IE-1 (mouse) as the primary antibody mixture. The primary antibodies were visualized with Alexa Fluor 555-conjugated goat anti-rabbit (VP39; red) and Alexa Fluor 647-conjugated donkey anti-mouse (IE1; green) antibodies. Hoechst 33342 was used to visualize DNA-rich regions of the nucleus (blue).

FIG 4

Subcellular localization of total actin and F-actin in Sf9 cells transfected with different recombinant viruses. (A) Total actin localization. Sf9 cells were transfected with vAc54KO, vAc54:2HA, or v38KKO bacmid DNA. At 36 h p.t., the cells were fixed, incubated with an anti-β-actin antibody and visualized with Alexa Fluor 647-conjugated donkey anti-mouse antibody (red). Hoechst 33342 was used to visualize DNA-rich regions of the nucleus (blue). (B) F-actin localization. Sf9 cells were infected with AcMNPV or transfected with bAc54KO, bMON14272 (indicated as WT in the figure), b38KKO, or bPP78/83KO bacmid DNA. At 36 h p.i. or 48 h p.t., cells were fixed and stained with both phalloidin-FITC (red) and Hoechst 33342 (blue).

FIG 5

Subcellular localization of the minor capsid proteins PP78/83, BV/ODV-C42 (C42), and 38K, as indicated. Sf9 cells were transfected with vAc54KO, vAc54:2HA, or v38KKO bacmid DNA and fixed at 36 h p.t. (A) Transfected cells were incubated with antisera against PP78/83 (rabbit) and IE-1 (mouse) as the primary antibody mixture. The primary antibodies were visualized with Alexa Fluor 555-conjugated goat anti-rabbit (PP78/83; red) and Alexa Fluor 647-conjugated donkey anti-mouse (IE1; green) antibodies. (B) Transfected cells were incubated with antiserum against BV/ODV-C42 and visualized with Alexa Fluor 555-conjugated goat anti-rabbit (red). (C) vAc54KO- or vAc54:2HA-transfected cells were incubated with antiserum against 38K and visualized with Alexa Fluor 555-conjugated goat anti-rabbit (red). Hoechst 33342 was used to visualize DNA-rich regions of the nucleus (blue).

FIG 6

Immunoelectron microscopy analysis of Sf9 cells transfected with vAc54KO or vAcWT bacmid DNA at 72 h p.t. using antisera against different nucleocapsid structural proteins. For the experiments shown in panels A to C, Sf9 cells were transfected with vAc54KO bacmid DNA. (A) Antiserum against PP78/83 was used as the primary antibody. The entire nucleus was equally stained with gold particles. (B) Antiserum against BV/ODV-C42 was used as the primary antibody. A minimum BV/ODV-C42 signal is visible in the VS; in the RZ, gold particles specifically colocalize with microvesicles but not with capsid structures. (C) Antiserum against 38K was used as the primary antibody. 38K mainly colocalizes with microvesicles; only a background level of gold particles was observed in the VS or on capsid structures. For the experiments shown in panels D to F, Sf9 cells were transfected with vAcWT. Signals for PP78/83 (D), BV/ODV-C42 (E), and 38K (F) specifically localize to the nucleocapsids. Arrowheads, gold particles; Nu, nucleus; nm, nuclear membrane; mv, microvesicles; C, capsid structures. Scale bars, 500 nm (A to C) and 200 nm (D to F).

FIG 7

VP1054 is able to interact with BV/ODV-C42 and VP80. (A and B) Sf9 cells were cotransfected with pIB-nGFP-VP1054 and pIB-BV/ODV-C42:FLAG or pIB-VP80:FLAG, as indicated. Cells cotransfected with pIB-nGFP and the corresponding FLAG-tagged expression plasmid were used as negative controls. (C) Sf9 cells were cotransfected with pIB-nGFP-VP1054 and pIB-VP39:FLAG. Cells cotransfected with pIB-nGFP-VP1054 and pIB-BV/ODV-C42 were used as a positive control. (D to F) Sf9 cells were cotransfected with pIB-VP1054:HA and pIB-BV/ODV-C42:FLAG, pIB-VP80:FLAG, or pIB-VP39:FLAG, as indicated. Cells cotransfected with pIB/V5-His and the corresponding FLAG-tagged expression plasmid were used as negative controls. For all cotransfections, cells were collected at 36 h p.t., lysed, immunoprecipitated with anti-FLAG or anti-HA antibody, and subjected to Western blot analysis.