Abstract
Actin, nucleation-promoting factors (NPFs), and the actin-related protein 2/3 complex (Arp2/3) are key elements of the cellular actin polymerization machinery. With nuclear actin polymerization implicated in ever-expanding biological processes and the discovery of the nuclear import mechanisms of actin and NPFs, determining Arp2/3 nucleo-cytoplasmic shuttling mechanism is important for understanding the function of nuclear actin. A unique feature of alphabaculovirus infection of insect cells is the robust nuclear accumulation of Arp2/3, which induces actin polymerization in the nucleus to assist in virus replication. We found that Ac34, a viral late gene product encoded by the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is involved in Arp2/3 nuclear accumulation during virus infection. Further assays revealed that the subcellular distribution of Arp2/3 under steady-state conditions is controlled by chromosomal maintenance 1 (CRM1)-dependent nuclear export. Upon AcMNPV infection, Ac34 inhibits CRM1 pathway and leads to Arp2/3 retention in the nucleus.
Highlights
Actin polymerization is an evolutionarily conserved biological process in eukaryotic cells
P40 was selected to represent actin-related protein 2/3 complex (Arp2/3) because P40 appeared to be the most abundant protein detected by either Western blot or fluorescence microscopy (Arp2 and P20 were less abundant than P40; Arp2 could only be detected by Western blot; other subunits were barely detected by Western blot or fluorescence microscopy when transiently expressed in Sf9 cells)
We prepared plasmid-based expression constructs encoding P40 tagged with a V5 epitope (P40-V5) at its C-terminus or P40 fused to enhanced green fluorescent protein at its N-terminus (EGFP-P40) to monitor the Arp2/3 dynamics during Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection
Summary
Actin polymerization is an evolutionarily conserved biological process in eukaryotic cells. The key elements of cellular actin polymerization machinery include, but are not limited to, actin, nucleation promoting factors (NPFs), and the actin-related protein 2/3 complex (Arp2/3). Activated by NPFs, Arp2/3 initiates globular actin (G-actin) polymerization into filamentous actin (F-actin) (Reviewed in [4]). Under steady-state conditions, Arp2/3 and other actin polymerization elements are predominantly localized in the cytoplasm. Increasing evidence has shown that actin polymerization elements are present in the nucleus and play important roles ranging from chromatin remodeling to transcription regulation (Reviewed in [5, 6]). The nuclear import mechanisms of actin and N-WASP, one of the best characterized NPFs, were previously determined [7,8,9,10], whereas nucleo-cytoplasmic shuttling mechanism of Arp2/3 remains enigmatic
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