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Dynamic Nuclear Actin Assembly by Arp2/3 Complex and a Baculovirus WASP-Like Protein

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Science  20 Oct 2006:
Vol. 314, Issue 5798, pp. 464-467
DOI: 10.1126/science.1133348

Abstract

Diverse bacterial and viral pathogens induce actin polymerization in the cytoplasm of host cells to facilitate infection. Here, we describe a pathogenic mechanism for promoting dynamic actin assembly in the nucleus to enable viral replication. The baculovirus Autographa californica multiple nucleopolyhedrovirus induced nuclear actin polymerization by translocating the host actin-nucleating Arp2/3 complex into the nucleus, where it was activated by p78/83, a viral Wiskott-Aldrich syndrome protein (WASP)–like protein. Nuclear actin assembly by p78/83 and Arp2/3 complex was essential for viral progeny production. Recompartmentalizing dynamic host actin may represent a conserved mode of pathogenesis and reflect viral manipulation of normal functions of nuclear actin.

Baculoviruses are enveloped, double-stranded (ds) DNA viruses that primarily infect the larvae of lepidopteran insects. They are widely used as protein expression vectors and are being developed for mammalian gene delivery and as environmentally benign pesticides (1). The best-studied baculovirus is Autographa californica multiple nucleopolyhedrovirus (AcMNPV). During infection, AcMNPV induces a series of actin rearrangements in host cells, the most striking of which is the accumulation and polymerization of actin within the nucleus (2, 3). Monomeric actin (G-actin) is driven to accumulate in the nucleus by early viral gene products (4), and nuclear actin is polymerized into filaments (F-actin) by the products of late viral genes (2, 4). Both steps are essential for progeny production (57). Actin is also present in the nucleus in uninfected cells, even in polymeric form (8, 9), and plays a role in diverse nuclear processes (10). However, little is known about the active forms and the regulation of nuclear actin. We investigated the process of nuclear actin polymerization induced by AcMNPV.

To establish the timing of nuclear recruitment and polymerization of actin, we infected Trichoplusia ni TN-368 cells expressing either enhanced green fluorescent protein (EGFP)–actin or mCherry-actin with AcMNPV and followed actin localization by time-lapse microscopy. Diffuse fluorescent actin began to accumulate in nuclei at 10 to 20 hours post-infection (hpi), and an apparent equilibrium between nuclear and cytoplasmic actin was reached within 2 hours (Fig. 1A and movies S1 to S3). The initial diffuse signal corresponded to G-actin, because it did not react with the F-actin probe phalloidin in fixed cells (4) and was not disrupted by the actin depolymerizing agent latrunculin A (latA) in live cells. Nuclear G-actin began to polymerize 2.0 ± 0.4 hours (mean ± SD, n = 7) after nuclear entry, as indicated by the conversion of diffuse actin into distinct structures and the reversion of these structures back to diffuse signal within minutes after latA treatment (Fig. 1A and movie S1). Polymerization proceeded rapidly until most of the cytoplasmic actin accumulated within the nucleus (Fig. 1A and movies S1 to S3). The uniformity of timing between nuclear recruitment and polymerization of actin indicates that actin rearrangements are precisely controlled.

Fig. 1.

AcMNPV induces dynamic nuclear actin polymerization that is required for viral replication. (A) EGFP-actin fluorescence in live, infected TN-368 cells imaged at the indicated times (in hours:minutes) post-infection [multiplicity of infection (MOI) of 20]. Scale bar indicates 10 μm. (B) EGFP-actin fluorescence in the nucleus of a TN-368 cell at 12 hpi (MOI = 20) at the indicated times before and after photobleaching. Arrow indicates bleached region. Scale bar, 10 μm. (C) Fluorescence recovery versus time in cells photobleached at 12 (triangles), 18 (circles), or 24 hpi (squares) or at 24 hpi in the presence of jasplakinolide (solid line; 1 μM, added at 20 hpi). (D) Replication of AcMNPV in Sf9 cells in the absence (squares) or the presence of 1 μM jasplakinolide added at 0 (triangles) or 15 hpi (circles). pfu, plaque-forming units.

We next assessed the dynamics of nuclear F-actin by monitoring fluorescence recovery after photobleaching (FRAP) in infected TN-368 cells expressing EGFP-actin. FRAP was performed by photobleaching a small region in the nucleus during the peak of viral replication (12 to 24 hpi). In most cells (63%), fluorescence recovered quickly, with a mean half-time of 22 ± 6 s (n = 10) (Fig. 1, B and C, and movie S4), similar to the rate in highly dynamic structures in migrating cells (11). When nuclear actin filaments were stabilized with the depolymerization inhibitor jasplakinolide (jasp), fluorescence did not recover, demonstrating that recovery was due to filament turnover (Fig. 1C). Conversely, when filaments were depolymerized by using latA, fluorescence loss in photobleaching (FLIP) was observed in the nucleus, presumably due to rapid diffusion of EGFP-actin monomers (movie S5). In some cells (37%), nuclear F-actin failed to recover fluorescence after photobleaching, suggesting that it could vary from a highly dynamic to a stable state. Nevertheless, dynamic actin was essential for viral replication, because treatment of infected cells with jasp prevented production of budded virus (Fig. 1D). Given the rapid turnover of nuclear F-actin in infected cells, we reasoned that factors regulating cytoplasmic actin assembly might be involved in NPV-induced nuclear actin dynamics.

One such regulator is the Arp2/3 complex, which is activated to nucleate branched actin filaments by proteins called nucleation-promoting factors (NPFs) (12). Diverse NPV species encode a capsid-associated protein, called p78/83 in AcMNPV, that contains domains conserved in Wiskott-Aldrich syndrome protein (WASP) family NPFs (13). These include proline-rich regions, WASP–homology 2 (WH2 or W) domains that bind G-actin, and a connector and acidic region (CA) that binds Arp2/3 complex (Figs. 2A and 3A and fig. S1). Because the WCA fragment of NPFs is sufficient to activate the Arp2/3 complex (12), we purified a truncation of p78/83 containing the WCA region (p78-WCA) and assessed whether it could activate Arp2/3 in vitro. Purified p78-WCA stimulated actin polymerization with Arp2/3 complex in a concentration-dependent manner but had no effect in the absence of Arp2/3 (Fig. 2B). Moreover, p78-WCA induced the organization of filaments into y branches, a hallmark of Arp2/3 activation (fig. S2). Capsid-associated p78/83 also activated Arp2/3 complex to accelerate actin polymerization but had no effect in the absence of Arp2/3 (Fig. 2C). Thus, p78/83 is a viral NPF for the Arp2/3 complex.

Fig. 2.

p78/83 activates Arp2/3 complex in vitro and localizes with Arp2/3 complex and F-actin in the nucleus in infected cells. (A) Domain organization of AcMNPV p78/83 depicting proline rich (P), WH2 (W), and connector and acidic (C and A) regions. (B and C) Pyrene-actin polymerization assays with 2 μM actin (7% pyrene labeled), 20 nM Arp2/3 complex, and indicated concentrations of glutathione S-transferase (GST)–p78–WCA or AcMNPV nucleocapsid (NC). (D) Arp2/3 complex and p78/83 in infected or uninfected TN-368 cells fixed at 22 hpi. Arp2/3 complex was stained by transfection with a plasmid expressing yellow fluorescent protein (YFP)–tagged T. ni ARPC3/p21 (p21-YFP) and by immunofluorescence using GFP antibodies. p78/83 was visualized by immunofluorescence using a p78/83 antibody (fig. S3). Scale bar, 10 μm. (E) Arp2/3 complex, p78/83, and actin in infected TN-368 cells either transfected with p21-YFP (top) or not transfected (bottom). Cells were fixed at 22 hpi and stained with 4′,6′-diamidino-2-phenylindole (DAPI), rhodamine-(top), or Alexa 488 (Invitrogen, Carlsbad, CA)–phalloidin (bottom) and GFP or p78/83 antibodies.

Fig. 3.

Mutations in p78/83 cause defects in NPF activity and viral replication. (A) Alignment of the CA region of AcMNPV p78/83 with human WASP, N-WASP, and WAVE/Scar1 (WASP family verprolin-homologous protein/suppressor of cyclic adenosine monophosphate). Conserved residues are outlined in gray, and acidic A region residues are bold. Residues in p78/83 that were mutated to alanine are colored. (B) Pyrene-actin polymerization assays performed as in Fig. 2B with 500 nM of the indicated GST-p78-WCA proteins. (C) Growth of wild-type (WT) and viable p78/83 mutant viruses in Sf9 cells.

The biochemical activity of p78/83 and Arp2/3 complex suggested that they could nucleate actin polymerization during infection. To learn where these factors acted, we determined their localization in infected TN-368 cells. Both Arp2/3 complex and p78/83 were localized in the nucleus by 22 hpi (Fig. 2D). Most cells exhibiting nuclear p78/83 and Arp2/3 also showed strong nuclear F-actin staining (Fig. 2E). Neither Arp2/3 complex (Fig. 2D) nor exogenously expressed p78/83 (fig. S3) localized to the nucleus in uninfected cells, indicating that both required other viral products for nuclear accumulation. The localization of host Arp2/3 complex and p78/83 in infected cells suggests that they function in the nucleus to initiate actin polymerization.

Nuclear F-actin is essential for NPV replication (5, 7). The p78/83 gene is also essential (14), consistent with the hypothesis that p78/83 NPF activity is required for nuclear actin polymerization and progeny production. To test this, we mutated conserved residues in the p78/83 CA region (Fig. 3A) that, when mutated in host NPFs, caused defects in Arp2/3 activation (15, 16). Purified mutant p78-WCA proteins exhibited actin polymerizing activities in vitro ranging from wild-type [for Asp364→Ala364 (D364A)] to moderately defective [Ile358→Ala358 (I358A) and Asp384Glu385→Ala384Ala385 (DE384-5AA)] and to severely defective [Trp387→ Ala387 (W387A) and Arg368Arg369Arg370→ Ala368Ala369Ala370 (RRR368-7AAA)] (Fig. 3B), similar to the corresponding host WASP mutants (15, 16).

To correlate p78/83 activity in vitro with its role in viral replication, we introduced each of the above mutations into the viral genome. We constructed an AcMNPV E2 baculovirus shuttle vector (bacmid), called WOBpos, that could be propagated in Escherichia coli even when it contained lethal mutations (fig. S4). WOBpos-derived AcMNPV was equivalent to wild type with respect to replication in cells and infectivity in the insect host (fig. S4). We engineered p78/83 point mutations and a deletion mutation (Dp78/83) by homologous recombination with WOBpos in E. coli (fig. S4). Equal amounts of wild-type and mutant bacmids were transfected into Spodoptera frugiperda Sf9 cells, and culture supernatants were tested for infectious virus. Of the mutants, only D364A (wild-type activity) and I358A (moderately defective) yielded viable progeny (table S1). D364A replicated with kinetics indistinguishable from those of the wild type, whereas I358A showed delayed viral production and reduced titer (Fig. 3C and table S1). The remaining mutants failed to produce progeny virus (table S1), in correlation with their severely reduced NPF activities. Viral viability could be rescued by cotransfection with a plasmid expressing wild-type p78/83 (table S1). Furthermore, dsRNA-mediated silencing of the ARPC3/p21 subunit of Arp2/3 complex (to 50% of normal amounts) caused a substantial reduction in viral titer (fig. S5). Thus, the ability of p78/83 to activate Arp2/3 complex is essential for AcMNPV replication.

To determine whether p78/83 activation of Arp2/3 complex is required for nuclear actin polymerization, we observed the distributions of F-actin, p78/83, and Arp2/3 complex in TN-368 cells transfected with wild-type or p78/83 mutant bacmids. Mutant p78/83 proteins were produced at quantities similar to those of wild type and localized to nuclei (Fig. 4A and fig. S6). Arp2/3 complex also localized to nuclei in cells transfected with the Dp78/83 mutant (fig. S7), indicating that p78/83 is not necessary for its nuclear translocation. We never observed accumulation of nuclear F-actin in cells transfected with Dp78/83, W387A, DE384-5AA, or RRR368-70AAA mutants, which had low NPF activity and were inviable (Fig. 4, A and B; fig. S6; and table S1). Nuclear F-actin staining was observed, however, in cells transfected with wild-type bacmid and with the viable D364A and I358A mutants. Thus, p78/83 activation of Arp2/3 complex is necessary for nuclear actin polymerization during NPV infection. p78/83 and Arp2/3 complex were not sufficient, however, because artificially targeting each to the nucleus in uninfected cells by appending a nuclear localization signal did not cause nuclear actin polymerization (fig. S7), probably because other viral factors are required for nuclear G-actin accumulation (4).

Fig. 4.

p78/83 activation of Arp2/3 complex is required for NPV-induced nuclear actin polymerization. (A) F-actin and p78/83 in TN-368 cells transfected with the indicated bacmids. Cells were fixed 24 hpt and stained with DAPI, Alexa 488–phalloidin, and p78/83 antibodies. Scale bar, 10 μm. (B) Quantification of the imaging in (A). (Top) Scatter plot of the ratio of average nuclear-to-cytoplasmic F-actin intensity in individual transfected cells (n from 65 to 250) for each p78/83 variant. (Bottom) Percentage of transfected cells with nuclear-to-cytoplasmic F-actin ratio >1.5 for each p78/83 variant. (C) Transmission electron microscopy of viral particles formed in the nuclei of TN-368 cells transfected with WT (left) or DE284-5AA (right) bacmids. Nucleocapsids (nc), envelope (e), free membranes (m). Scale bar, 200 nm.

To investigate the mechanism by which p78/83 and Arp2/3 participate in viral replication, we examined virions in cells at 48 hours posttransfection (hpt) with wild-type or DE384-5AA mutant bacmids by using electron microscopy. The wild-type bacmid produced characteristic preoccluded (Fig. 4C) and occluded virions containing multiple nucleocapsids neatly aligned and surrounded by tight membranous envelopes. Although the DE384-5AA mutant bacmid produced many apparently normal nucleocapsids, there was a higher frequency of nucleocapsids of aberrant length (Fig. 4C). We also observed striking defects in virion organization. Most mutant nucleocapsids lacked envelopes or were misaligned within envelopes, and there were abundant membranes without associated nucleocapsids. These observations point to a key role for nuclear actin polymerization in coordinating nucleocapsid morphogenesis and membranecapsid interactions during virion assembly.

AcMNPV has evolved exquisite control over the actin cytoskeleton of its host cell, manipulating both activity and localization of the Arp2/3 complex to promote dynamic nuclear actin polymerization that is essential for proper virion processing and infectivity. Given the conservation of p78/83 among lepidopteran NPVs (13, 17), it is likely that these viruses use the same mechanism for nuclear actin polymerization. It seems quite possible that other unrelated pathogens have also evolved similar strategies for exploiting nuclear actin (18). Because pathogens rarely invent cell biological processes, preferring to adapt existing pathways to their own needs, we suggest that AcMNPV may have co-opted existing nuclear functions and regulatory mechanisms of actin to facilitate its replication.

Supporting Online Material

www.sciencemag.org/cgi/content/full/314/5798/464/DC1

Materials and Methods

Figs. S1 to S7

Table S1

Movies S1 to S5

References and Notes

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