A new mechanism for nuclear import by actin-based propulsion used by a baculovirus nucleocapsid

Abstract

The transport of macromolecules into the nucleus is mediated by soluble cellular receptors of the importin β superfamily and requires the Ran-GTPase cycle. Several studies have provided evidence that there are exceptions to this canonical nuclear import pathway. Here, we report a new unconventional nuclear import mechanism exploited by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). We found that AcMNPV nucleocapsids entered the nucleus of digitonin-permeabilized cells in the absence of exogenous cytosol or under conditions that blocked the Ran-GTPase cycle. AcMNPV contains a protein that activates the Arp2/3 complex and induces actin polymerization at one end of the rod-shaped nucleocapsid. We show that inhibitors of Arp2/3 blocked nuclear import of nucleocapsids in semi-permeabilized cells. Nuclear import of nucleocapsids was also reconstituted in purified nuclei supplemented with G-actin and Arp2/3 under actin polymerization conditions. Thus, we propose that actin polymerization drives not only migration of baculovirus through the cytoplasm but also pushes the nucleocapsid through the nuclear pore complex to enter the cell nucleus. Our findings point to a very distinct role of actin-based motility during the baculovirus infection cycle.

Pressurized DNA state inside herpes capsids—A novel antiviral target

“…This finding is consistent with a recent study demonstrating that untegumented C-capsids and PLOS PATHOGENS viral capsids exposing inner tegument proteins on their surface had a similar degree of binding to NPCs [41,72]. The reconstituted capsid-nuclei system describes the main infection step of viral DNA ejection into a host cell [41][42][43][44]. Although the term 'infection' usually refers to both viral genome transport into the cell and subsequent replication of the virus, the primary infection in vivo by HSV-1 and HSV-2 is mostly latent; that is, the herpes genome is translocated into the host nucleus without subsequent genome replication [73].…”

“…This subsequently prevents infection. In order to demonstrate this mechanism of action (MOA) and avoid the issue of limited compound cell penetration, we use a reconstituted isolated nucleus system that accurately reproduces capsid-nuclei binding and nuclear transport of the herpes genome into living cells, which leads to infection [41][42][43][44] (the term "infection" denotes the introduction of viral nucleic acid into a host cell by a virus [45]). Drug delivery, including cell membrane permeability, is a challenge in the development of most antiviral agents (e.g.…”

Viral mechanisms for docking and delivering at nuclear pore complexes

“…Exactly how AcMNPV targets NPCs for transport is not understood but translocation of intact nucleocapsids (30 x 250-300 nm) appears to occur independently of the Ran-GTPase cycle, and requires actin-based propulsion mediated by Arp2/3, as well as nucleocapsidassociated Ac132 [169,170]. Nucleocapsids cross NPCs lengthwise with the distal end containing the actin-assembly inducing VP78/83.…”

Nuclear pore complex tethers to the cytoskeleton

“…An antibody against a myosin heavy chainlike polypeptide was shown to label NPCs in Drosophila culture cells (Berrios et al, 1991), which could indicate a possible mechanism for linking the NPC to Factin. However, there is currently little structural, biochemical or functional evidence for such a linkage, other than for nucleoplasmic actin inside amphibian oocyte nuclei (Hofmann et al, 2001;Kiseleva et al, 2004), and for interesting, but unusual, roles in viral transport (Au et al, 2016). Likewise there is some evidence from electron microscopy that cytoplasmic intermediate filaments link to the NPC (Djabali, 1999), but no biochemical or functional data proving such a link.…”