Identification of a Novel Regulatory Sequence of Actin Nucleation Promoting Factor Encoded by Autographa californica Multiple Nucleopolyhedrovirus*

Background: The regulatory mechanism of baculoviral actin nucleation promoting factor (NPF) remains elusive. Results: Baculoviral NPF harbors a multifunctional regulatory sequence (MRS) at its N terminus. Conclusion: Baculoviral NPF is modulated by the host proteasome and viral nucleocapsid protein C42 through its N-terminal MRS. Significance: Identification of a novel regulatory mechanism of viral NPF. Actin polymerization induced by nucleation promoting factors (NPFs) is one of the most fundamental biological processes in eukaryotic cells. NPFs contain a conserved output domain (VCA domain) near the C terminus, which interacts with and activates the cellular actin-related protein 2/3 complex (Arp2/3) to induce actin polymerization and a diverse regulatory domain near the N terminus. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) nucleocapsid protein P78/83 is a virus-encoded NPF that contains a C-terminal VCA domain and induces actin polymerization in virus-infected cells. However, there is no similarity between the N terminus of P78/83 and that of other identified NPFs, suggesting that P78/83 may possess a unique regulatory mechanism. In this study, we identified a multifunctional regulatory sequence (MRS) located near the N terminus of P78/83 and determined that one of its functions is to serve as a degron to mediate P78/83 degradation in a proteasome-dependent manner. In AcMNPV-infected cells, the MRS also binds to another nucleocapsid protein, BV/ODV-C42, which stabilizes P78/83 and modulates the P78/83-Arp2/3 interaction to orchestrate actin polymerization. In addition, the MRS is also essential for the incorporation of P78/83 into the nucleocapsid, ensuring virion mobility powered by P78/83-induced actin polymerization. The triple functions of the MRS enable P78/83 to serve as an essential viral protein in the AcMNPV replication cycle, and the possible roles of the MRS in orchestrating the virus-induced actin polymerization and viral genome decapsidation are discussed.


Actin polymerization induced by nucleation promoting factors (NPFs) is one of the most fundamental biological processes in eukaryotic cells. NPFs contain a conserved output domain (VCA domain) near the C terminus, which interacts with and activates the cellular actin-related protein 2/3 complex (Arp2/3) to induce actin polymerization and a diverse regulatory domain near the N terminus. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) nucleocapsid protein P78/83 is a virus-encoded NPF that contains a C-terminal VCA domain and induces actin polymerization in virus-infected cells. However,
there is no similarity between the N terminus of P78/83 and that of other identified NPFs, suggesting that P78/83 may possess a unique regulatory mechanism. In this study, we identified a multifunctional regulatory sequence (MRS) located near the N terminus of P78/83 and determined that one of its functions is to serve as a degron to mediate P78/83 degradation in a proteasome-dependent manner. In AcMNPV-infected cells, the MRS also binds to another nucleocapsid protein, BV/ODV-C42, which stabilizes P78/83 and modulates the P78/83-Arp2/3 interaction to orchestrate actin polymerization. In addition, the MRS is also essential for the incorporation of P78/83 into the nucleocapsid, ensuring virion mobility powered by P78/83-induced actin polymerization. The triple functions of the MRS enable P78/83 to serve as an essential viral protein in the AcMNPV replication cycle, and the possible roles of the MRS in orchestrating the virus-induced actin polymerization and viral genome decapsidation are discussed.
Actin is one of the most abundant and evolutionarily conserved molecules in eukaryotic cells. Morphologically, globular actin (G-actin) can nucleate and polymerize to filamentous actin (F-actin) to form a functional actin cytoskeleton. Actin polymerization is mediated by actin nucleators, and the actinrelated protein 2/3 complex (Arp2/3), which consists of seven subunits, is one of the most important nucleators that nucleates G-actin to Y-branched F-actin (reviewed in Ref. 1). Arp2/3 activity in nucleating G-actin heavily depends on nucleation promoting factors (NPFs) 4 (reviewed in Ref. 2). The conserved output region of NPFs is the C-terminal VCA domain, which includes a verprolin homology motif (V), an amphipathic connector region (C), and an acidic peptide (A) that interact with Arp2/3 through the P40 subunit (reviewed in Ref. 2; see also Ref. 3). In addition to the conserved VCA domain, NPFs harbor diverse N-terminal regulatory sequences that confer a variety of regulation mechanisms and functions. Therefore, an understanding of NPF regulation, especially the identification and characterization of their N-terminal regulatory sequences, is central to determining the mechanism and function of actin polymerization. Currently, at least eight NPFs (Class I) have been identified in mammalian cells based on different N-terminal sequences (reviewed in Ref. 4). One of the most studied NPFs is Wiskott-Aldrich syndrome protein (WASP) (5). The N terminus of WASP contains a GTPase-binding domain that binds to the VCA domain via intramolecular interaction and keeps WASP in an inactive state (6). Upon stimulation, the small GTPase Cdc42 can competitively bind to the GTPasebinding domain and subsequently abolish the intramolecular interaction to release the VCA domain, thus activating WASP to direct Arp2/3 to initiate actin polymerization (6).
Pathogens also encode NPFs to promote host actin polymerization to assist in their replication. Pathogen-derived NPFs such as ActA encoded by Listeria monocytogenes (7), RickA encoded by Rickettsia sp. (8), and BimA encoded by Burkholderia thailandensis contain conserved VCA domains and different N-terminal regulatory sequences that enable the NPFs to precisely control actin polymerization in pathogen multiplication (9). Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most studied baculovirus. After AcMNPV entry into the host cell, the virus induces host actin polymerization to aid in its replication: during the early infection phase, the viral nucleocapsid induces cytoplasmic actin polymerization to propel nucleocapsid migration toward the nucleus for replication (10,11); after the nucleocapsid reaches the nuclear membrane, the cytoplasmic F-actin tail depolymerizes and detaches from the nucleocapsid, allowing the nucleocapsid alone to enter the nucleus (11); during the late infection phase, the virus promotes nuclear actin polymerization to assist in nucleocapsid assembly (12,13). P78/83, a viral nucleocapsid protein encoded by AcMNPV ac9, is the NPF responsible for virus-induced actin polymerization (12). Sequence comparison with other well characterized NPFs has revealed a classic VCA domain located near the C terminus of P78/83 (14). In contrast, its N terminus exhibits no similarity with any identified NPFs, suggesting that P78/83 may possess a unique regulation mechanism.
Our previous work revealed that another viral protein, BV/ODV-C42 (C42), is functionally related to P78/83. In addition to its role in mediating the nuclear relocation of P78/83 in virus-infected cells (15), P78/83 fails to initiate actin polymerization in the absence of C42 (16), suggesting that C42 plays a key role in regulating the NPF activity of P78/83. In the present study, we identified a multifunctional regulatory sequence (MRS) located at the N terminus of P78/83. This MRS serves as a degron to mediate P78/83 degradation by the host proteasome. In virus-infected cells, the MRS is masked by C42, and P78/83 is thus exempt from proteasomal degradation, ensuring its NPF activity in mediating actin polymerization. In addition to modulating P78/83 stability, the MRS is also required for P78/83 incorporation into the viral nucleocapsid, which ensures the nucleocapsid integrity and viral mobility powered by P78/83-induced actin polymerization.

EXPERIMENTAL PROCEDURES
Cell Culture, Transfection, and Virus Manipulation-Sf9 cells were maintained at 27°C in Grace's medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen). The proteasome inhibitor PS-341 (LC Laboratories) was added to the medium 4 h prior to cell lysis for Western blot or immunoprecipitation (IP) assays. Viral supernatants were filtered through a 0.45-m syringe filter (Millipore), followed by incubation with fresh Sf9 cells to initiate secondary infection for virus titration or ultracentrifugation at 120,000 ϫ g for 150 min at 4°C to collect purified virions. Virus titration and infectivity assays were performed as described previously (15).
Preparation of an ac9 Knock-out Bacmid-To remove ac9 from the bacmid (bMON14272; Invitrogen), the -red recombination system was employed, as described previously (15). The recombinant bacmid (vAc ac9ko ) was verified by PCR and sequencing (data not shown).

Construction of Plasmids and Recombinant
Bacmids-A standard molecular cloning protocol was used to prepare the indicated plasmid constructs. Genes were cloned into pIZ-V 5 / flag/myc (Invitrogen) for transient expression, and a Bac-to-Bac protocol was employed to prepare recombinant bacmids. In brief, gene expression cassettes were cloned into pFastBacdual (Invitrogen), and the resulting shuttle vectors were transformed to DH10B Escherichia coli cells harboring bMON14272, vAc c42ko (15), or vAc ac9ko to generate transposed bacmids.
Western Blot and IP Assay-Cells were washed with PBS and lysed in radioimmune precipitation assay buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 50 mM Tris, pH 8.0) with cOmplete protease inhibitor mixture (Roche). Total proteins were quantified by Quickstart Bradford (Bio-Rad), and lysates containing 100 g of total proteins were mixed with 2ϫ Laemmli buffer before performing SDS-PAGE. The proteins were transferred to nitrocellulose membranes, which were blocked in 0.5% nonfat dry milk and incubated with the indicated antibodies (anti-V 5 was purchased from Invitrogen; anti-flag (M2) was purchased from Sigma; anti-tubulin and anti-ubiquitin were purchased from Cell Signaling; anti-EGFP and anti-actin were purchased from Santa Cruz; anti-myc and anti-P78/83 were purchased from Abmart) overnight at 4°C. After incubation with HRP-conjugated secondary antibodies (Jackson Labs), the membranes were developed using enhanced chemiluminescence (Pierce). For the IP assay, 2000 g of total protein was incubated with 2 g of the indicated antibodies and 50 l of protein G-agarose (Millipore) overnight at 4°C. The immunoprecipitated agarose was extensively washed with radioimmune precipitation assay buffer, mixed with 2ϫ Laemmli buffer, and subjected to the Western blot assay.
F-actin Staining-Cells grown on coverslips were washed with PBS, fixed in 3.7% formaldehyde for 10 min at room temperature, and permeabilized with 0.1% Triton X-100 for 5 min. The cells were then incubated with a 1:40 dilution of rhodamine-phalloidin (Invitrogen) at room temperature inside a covered container for 20 min. After being extensively washed with PBS, the samples were subjected to fluorescence microscopy observation using a BX53 microscope.

Baculoviral NPFs Exhibit Different Protein Abundance when
Transiently Expressed in Insect Cells-Viral genes encoding P78/83 and its baculoviral homologs are essential genes that exist in all sequenced Lepidoptera nuclear polyhedrosis virus (NPV) genomes (17). To compare the expression pattern of different baculoviral NPFs, we transiently expressed three P78/83 baculoviral homologs using the same vector in Sf9 cells, AcMNPV ORF9 (P78/83), Bombyx mori NPV (BmNPV) ORF2 (Bm2), and Helicoverpa armigera NPV (HearNPV) ORF2 (Ha2), which represent NPFs encoded by group I NPV (AcMNPV and BmNPV) and group II NPV (HearNPV). All three baculoviral NPFs were tagged with the FLAG epitope, and Western blotting using an anti-flag antibody demonstrated a strong band at ϳ45 kDa and a weak band at ϳ60 kDa only for Ha2, as we reported previously (18). P78/83 and Bm2 were not detected (Fig. 1A). This phenotype indicated that baculoviral NPFs show dramatically different protein abundance when expressed in virus-free cells.
To reveal how aa 40 -143 modulate P78/83 protein abundance, aa 40 -143 were fused to enhanced GFP (EGFP) at either the N terminus (40 -143-GFP) or C terminus (GFP-40 -143) (Fig. 1E). The subsequent expression cassettes were transiently expressed in Sf9 cells. Fluorescence microscopy indicated that both fusion proteins generated an extremely low level of EGFP fluorescence, in contrast to cells expressing native EGFP (GFP) or GFP-1-40 (aa 1-40 of P78/83 fused with EGFP) (Fig. 1E). A Western blot assay also provided consistent evidence that both fusion proteins were barely detectable in Sf9 cells (Fig. 1E). A degron is generally defined as a minimal element within a protein that is sufficient for recognition and degradation by proteolytic machinery (19). One of the key properties of degrons is that they are transferable, which means that genetically engineered fusions of such sequences confer instability on otherwise long-lived proteins (reviewed in Refs. 20 and 21). The phenotype of the fusion of aa 40 -143 to EGFP resulted in fusion protein degradation, demonstrating that aa 40 -143 serve as a degron that directly mediates protein degradation in insect cells as well as in mammalian cells (data not shown).
P78/83 Degradation Is Proteasome-dependent-The proteasomal degradation pathway is the most frequently used pathway for protein degradation. We next determined whether a chemical blocker of the proteasome could influence P78/83 protein abundance. Western blot assays demonstrated that the proteasome blocker PS-341, but not the lysosome inhibitor chloroquine (data not shown), could effectively increase P78/83 protein abundance in a concentration-dependent manner ( Fig.  2A). Similar to P78/83, Bm2 protein abundance also appeared to be increased by PS-341, in contrast to Ha2, which exhibited no significant change in protein abundance in response to PS-341 (Fig. 2B).
Because aa 40 -143 serve as a degron to mediate protein degradation, to assess whether PS-341 can inhibit the degron-mediated protein degradation, an EGFP fusion protein (GFP-40 -143) and P78/83 ⌬40 -143 were transiently expressed in Sf9 cells in the presence or absence of PS-341. A Western blot assay demonstrated that PS-341 significantly increased the protein abundance of GFP-40 -143 but did not exert a significant influence on P78/83 ⌬40 -143 (Fig. 2C). This finding suggested that aa 40 -143 modulate protein abundance by promoting protein degradation via a proteasome-dependent pathway, which can be inhibited by PS-341.
Because most substrates of proteasomal degradation are ubiquitinated proteins, to determine whether aa 40 -143 contain important ubiquitination residues responsible for P78/83 degradation, all potential lysine ubiquitination sites within the region were mutated. The resulting mutants were expressed in Sf9 cells and compared with P78/83 and P78/83 ⌬40 -143 . Western blotting demonstrated that none of these ubiquitination site mutants exhibited significantly increased stability in comparison with P78/83 (Fig. 2D). Accordingly, the levels of ubiquitination of P78/83 in the presence or absence of aa 40 -143 were the same, implying that aa 40 -143 contains no important ubiquitination sites (Fig. 2, E and F).
C42 Modulates P78/83 Protein Abundance-We next investigated the P78/83 expression pattern in AcMNPV-infected cells. Unlike the previous result that P78/83 is highly unstable in virus-free cells, P78/83 appeared to be a stable protein in AcMNPV-infected cells (Fig. 3A), suggesting that certain unidentified viral products can protect P78/83 from degradation during AcMNPV infection.
C42 protects P78/83 from degradation by binding to aa 40 -143 of P78/83-Previous data demonstrated that C42 harbors the nuclear localization sequence (NLS) KRKK at its C terminus, the removal of which results in the loss of the capacity of C42 to relocate P78/83 to the nucleus by co-transportation  (15,23). We therefore speculated that C42 could possibly modulate P78/83 protein abundance by changing its subcellular distribution. When P78/83 was co-expressed with C42 (C42-V 5 ) or a C42 NLS mutant (C42 ⌬nls -V 5 ) in Sf9 cells, both generated abundant P78/83, as indicated by equally strong bands on Western blots (Fig. 3E). This finding is in sharp contrast to the co-expression of P78/83 and EGFP (Fig. 3E), indicating that the mechanism by which C42 modulates P78/83 protein abundance is independent of the change in its subcellular localization.
A co-IP assay demonstrated that P78/83 failed to interact with P40 in the absence of C42; in contrast, PS-341 partially A series of C42 truncations tagged with the V 5 epitope were generated as described by the diagram. The resulting C42 truncations were co-expressed with P78/83 in Sf9 cells. At 48 hpt, the cells were harvested and subjected to a co-IP assay using an anti-V 5 antibody. The immunoprecipitated proteins and whole cell lysates were probed with the indicated antibodies. C, C42-P78/83 interaction is essential for P78/83 stability. EGFP, C42-V 5 , and C42 ⌬1-10 -V 5 were co-expressed with P78/83 in Sf9 cells, respectively. At 48 hpt, cells were harvested, and the proteins were subjected to a Western blot assay using an anti-P78/83 antibody. D, the expression pattern of P78/83 in bacmid-transfected cells. Two micrograms of bMON14272 (wild-type AcMNPV bacmid), vAc c42ko (a c42 knock-out bacmid), vAc c42ko-c42 (a c42 knock-out bacmid with a rescued full-length c42), or vAc c42ko-c42⌬1-10 (a c42 knock-out bacmid with a rescued c42 without aa 1-10 at N terminus) were transfected to Sf9 cells, respectively. At 24 hpt, the cells were harvested, and the proteins were subjected to a Western blot assay using an anti-P78/83 antibody. The viral major capsid protein VP39 served as the exogenous reference to indicate bacmid transfection efficiency. E, the influence of P78/83-C42 subcellular localization on P78/83 stability. C42-V 5 and the C42 NLS mutant (C42 ⌬nls -V 5 ) were co-expressed with P78/83 in Sf9 cells. At 48 hpt, the cells were harvested, and the proteins were subjected to a Western blot assay using the indicated antibodies. F and G, characterization of the P78/83-C42 interaction. A series of flag-tagged P78/83 truncations (F) and deletion mutants (G) were co-expressed with C42-V 5 in Sf9 cells. At 48 hpt, the cells were harvested and subjected to a co-IP assay using an anti-V 5 antibody. The immunoprecipitated proteins and whole cell lysates (WCL) were probed with the indicated antibodies.
restored the P78/83-P40 interaction, and C42 maximized the P78/83-P40 interaction (Fig. 4A). These results could easily be attributed to the fact that C42 is essential for P78/83 to be at a measurable level to interact with P40 (Fig. 4A).
P78/83-C42 Interaction Is Pivotal for Nuclear Actin Polymerization and AcMNPV Replication-Previous reports have shown that both P78/83 and C42 are essential for nuclear actin polymerization and AcMNPV replication (12,16). In this study, C42 was demonstrated to stabilize P78/83 and subsequently enhance the P78/83-Arp2/3 interaction level. We next explored how C42 influences nuclear actin polymerization and AcMNPV replication by disrupting the C42-P78/83 interaction.
We continued to determine the infectivity of these bacmids. After transfection of these bacmids into Sf9 cells, viral supernatants were collected at 120 hpt. Viral infectivity assays showed that the supernatants from vAc c42ko-ac9nls -and vAc c42ko-c42⌬1-10-ac9nlstransfected cells failed to infect fresh Sf9 cells at 120 h postinfection (hpi) (Fig. 4D), indicating that the P78/83-C42 interaction is also essential for AcMNPV replication. The replication deficiency of vAc c42ko-ac9nls and vAc c42ko-c42⌬1-10-ac9nls is due, at least in part, to the instability of P78/83 and the resulting failure of nuclear actin polymerization, which is a key step for viral nucleocapsid assembly in the AcMNPV replication cycle (12,13).
AA 40 -143 Are Required for P78/83 Incorporation into the Viral Nucleocapsid-Because P78/83 is a viral structural protein distributed at the basal region of the nucleocapsid (25), one possible function of P78/83 degradation is that it may generate a leak in the enclosed nucleocapsid and that the viral genome may escape through that leak (decapsidation), resulting in viral DNA replication. An easy way to verify this hypothesis is to check whether P78/83 ⌬40 -143 (the nondegradable version of P78/83), as a nucleocapsid protein, can block viral genome decapsidation.

DISCUSSION
Most NPFs contain a conserved VCA domain near their C terminus that plays a similar role in binding and activation of the Arp2/3 to initiate actin polymerization. However, the diverse N-terminal regulatory domains confer different regulation mechanisms to NPFs and subsequently enable the precise control of actin polymerization to exert different functions in eukaryotic cells. Therefore, the identification of NPF N-terminal regulatory sequences and investigation of their regulation mechanisms are key to understanding the control of actin polymerization. In this study, we identified an MRS of the AcMNPV-encoded NPF that is novel not only because it presents a completely different regulatory mechanism from the well defined intramolecular conformational switch model employed by WASP and other NPFs but also because of its multiple roles in assisting in virus replication.
Actin polymerization plays a crucial role in regulating a variety of biological functions in eukaryotic cells. The modulation of NPF stability is one of the major ways of orchestrating actin polymerization. Reicher et al. (26) reported that WASP could be ubiquitinated and degraded upon T-cell antigen receptor FIGURE 5. The MRS is essential for P78/83 incorporation into the viral nucleocapsid. A and C, diagram of bacmids. Two expression cassettes expressing EGFP and either NLS-tagged wild-type P78/83 (ac9nls) or P78/83 with aa 40 -143 deleted (ac9⌬40 -143nls), both controlled by the ac9 promoter (P ac9 ), were transposed into vAc ac9ko (A) or bMON14272 (C), respectively. B, virus infectivity assay. vAc ac9ko-ac9nls-gfp and vAc ac9ko-ac9⌬40 -143nls-gfp were transfected into Sf9 cells. At 120 hpt, viral supernatants were collected and used to infect fresh Sf9 cells. Cells expressing EGFP were captured at 24 hpt and 24 hpi using an Olympus IX-51 fluorescence microscope. D, the impact of the MRS on P78/83 nucleocapsid incorporation. Viral stocks of vAc ac9nls-gfp and vAc ac9⌬40 -143nls-gfp were used to infect fresh Sf9 cells at a multiplicity of infection of 5. At 24 hpi, viral supernatants were collected and subjected to ultracentrifugation at 120,000 ϫ g for 150 min at 4°C to harvest the purified virions before Western blotting; the infected cells were lysed, and 100 g of cell lysate was subjected to Western blotting with the indicated antibodies. WCL, whole cell lysates.
activation, which subsequently enhances T cell activation. King et al. (27) also showed that ␤1-integrin can facilitate plateletderived growth factor receptor trafficking and fibroblast chemotaxis by modulating actin polymerization through stabilizing Neural-WASP. As a virus-encoded NPF, P78/83 has been fully proven to be responsible for AcMNPV-induced actin polymerization (11,12). However, the molecular mechanism responsible for the regulation of virus-induced actin polymerization remains obscure. In this study, the identified MRS of P78/83 indicates that the modulation of P78/83 stability by the host proteasome and viral protein C42 is a major mechanism for orchestrating AcMNPV-induced actin polymerization.
It has been reported that P78/83 is located at the end of the nucleocapsid, which contains the basal structure (25). The requirement of the MRS for P78/83 integration into the nucleocapsid suggests that the MRS is buried in the nucleocapsid, preventing the cellular proteolytic machinery from interacting with and degrading P78/83. Conversely, the VCA domain of P78/83 is proposed to be exposed on the nucleocapsid surface to induce cytoplasmic actin polymerization, providing the viral mobility that propels the nucleocapsid to migrate to the nucleus after AcMNPV entry into the host cytoplasm (10, 11). When the nucleocapsid transports across the nuclear mem-brane, the F-actin tail detaches from the nucleocapsid, allowing the nucleocapsid to enter the nucleus through the tight nuclear pore (11). It is possible that the MRS may be exposed to the cellular proteolytic machinery at this phase because of unidentified reasons, leading to P78/83 degradation, which subsequently ends P78/83-induced actin polymerization and detaches the nucleocapsid from the F-actin tail. After the nucleocapsid enters the nucleus, the viral genome may be released from the nucleocapsid leak generated by P78/83 degradation, resulting in viral genome replication and gene transcription. After the viral mRNAs migrate out of the nucleus and P78/83 is translated in the cytoplasm, simultaneously expressed C42 binds to the MRS of P78/83 (22,23), preventing the cellular proteolytic machinery from degrading P78/83 and relocating P78/83 to the nucleus (15). In the nucleus, P78/83-C42 interaction through the MRS ensures P78/83 protein abundance and induces nuclear actin polymerization to assist in nucleocapsid assembly (13). The proposed working model for the MRS of P78/83 is summarized in Fig. 6.
In summary, we identified a novel regulatory sequence of baculovirus-encoded NPF. The interplay between the host proteolytic machinery and viral protein C42 through the MRS presents a novel regulatory model for NPF and helps to shed light on the decapsidation mechanism of the viral nucleocapsid.