Molecular determinants of Arc oligomerization and formation of virus-like capsids

Abstract

Expression of activity-regulated cytoskeleton-associated protein (Arc) is critical for long-term synaptic plasticity, memory formation, and cognitive flexibility. The ability of Arc to self-associate and form virus-like capsid structures implies functionally distinct oligomeric states. However, the molecular mechanism of Arc oligomerization is unknown. Here, we identified a 28-amino-acid region necessary and sufficient for Arc oligomerization. This oligomerization region is located within the second coil of a predicted anti-parallel coiled-coil in the N-terminal domain (NTD). Using alanine scanning mutagenesis, we found a 7-amino-acid motif critical for oligomerization and Arc-mediated transferrin endocytosis in HEK cells. Intermolecular fluorescence lifetime imaging in hippocampal neurons confirmed selfassociation mediated by the motif. To quantify oligomeric size, we performed a single-molecule photobleaching analysis of purified Arc wild-type and mutant. This analysis revealed a critical role for the NTD motif in the formation of higher-order Arc oligomers (30-170 molecules). Moreover, assembly of higher-order wild-type Arc oligomers was significantly enhanced by addition of GFP RNA. Purified wild-type Arc formed virus-like capsids, as visualized by negative-stain EM, and was estimated by light scattering analysis to contain 40-55 Arc units. In contrast, mutant Arc formed a homogenous dimer population as demonstrated by single-molecule TIRF imaging, size-exclusion chromatography with multi-angle light scattering analysis, small-angle X-ray scattering analysis, and single-particle 3D EM reconstruction. Thus, the dimer appears to be the basic building block for assembly. Herein, we show that the NTD motif is essential for higher-order Arc oligomerization, assembly of virus-like capsid particles, and facilitation of oligomerization by exogenous RNA. SIGNIFICANCEArc protein is rapidly expressed in neurons in response to synaptic activity and plays critical roles in synaptic plasticity, postnatal cortical developmental, and memory. Arc has diverse molecular functions, which may be related to distinct oligomeric states of the protein. Arc has homology to retroviral Gag protein and self-assembles into retrovirus-like capsid structures that are capable of intercellular transfer of RNA. Here, we identified a motif in the N-terminal coiled-coil domain of mammalian Arc that mediates higher-order oligomerization and formation of virus-like capsids. The basic building block is the Arc dimer and exogenous RNA facilitates further assembly. The identified molecular determinants of Arc oligomerization will help to elucidate the functional modalities of Arc in the mammalian brain. Recent advances highlight a structural and functional relationship between Arc and retroviralGag polyprotein. Arc was identified in a computational search for domesticated retrotransposons harboring Gag-like protein domains (Campillos et al., 2006). Biochemical studies showed that mammalian Arc has a positively charged N-terminal domain (NTD) and a ne...

Structures of virus-like capsids formed by the Drosophila neuronal Arc proteins

“…S10), the hydrophobic face of which is likely to mediate oligomerization or binding to cellular proteins or membranes. Tetrapod Arc has an extended ~200 amino acid N-terminal domain that may bind RNA (Eriksen et al, 2019). In dArc, the N terminus occludes openings in the CA shell at the five-fold and two-fold axes.…”

Structures of virus-like capsids formed by the Drosophila neuronal Arc proteins

“…This suggests that some copies of the N-terminal region constitute the spike, while others protrude inwards. Tetrapod Arc has an extended ~200 amino acid Nterminal domain that may bind RNA and/or negatively charged membranes 16 . We speculate that this domain is similarly located both outside and inside the capsid, allowing membrane binding and RNA packaging to be facilitated by the same domain.…”

Herpes Simplex Virus type 1 neuronal infection triggers disassembly of key structural components of dendritic spines

“…Notably there was no increase in Arc expression at 8 hpi in total homogenate fraction, and no significant differences between soluble and synaptoneurosomal fraction regarding HSV-1 infection ( Figure 5A). It is known that Arc protein is capable of oligomerization [34,35]. We also see high molecular weight complex (supplementary Figure 1) therefore, the low osmolarity of the synaptoneurosomal buffer must be unable to completely solubilize oligomeric Arc or Arc interaction complexes.…”

“…This might be a stimulating idea to test, whether Arc is acetylated at 8 hpi, and therefore accumulated. There is also the possibility of Arc oligomerization under high up-regulation during HSV-1 infection ( Supplementary Figure 1) due to the known low solubility of Arc oligomers [35,42,55]. Remarkably, in 2013, Naghavi and colleagues demonstrated that Us3, a viral Ser/ Thr kinase inactivates GSK3 at ~9 hpi, through phosphorylation of Ser9 [56].…”