In vitro reconstitution reveals membrane clustering and RNA recruitment by the enteroviral AAA+ ATPase 2C.

Enteroviruses are a vast genus of positive-sense RNA viruses that cause diseases ranging from common cold to poliomyelitis and viral myocarditis. They encode a membrane-bound AAA+ ATPase, 2C, that has been suggested to serve several roles in virus replication, e.g. as an RNA helicase and capsid assembly factor. Here, we report the reconstitution of full-length, poliovirus 2C's association with membranes. We show that the N-terminal membrane-binding domain of 2C contains a conserved glycine, which is suggested by structure predictions to divide the domain into two amphipathic helix regions, which we name AH1 and AH2. AH2 is the main mediator of 2C oligomerization, and is necessary and sufficient for its membrane binding. AH1 is the main mediator of a novel function of 2C: clustering of membranes. Cryo-electron tomography reveal that several 2C copies mediate this function by localizing to vesicle-vesicle interfaces. 2C-mediated clustering is partially outcompeted by RNA, suggesting a way by which 2C can switch from an early role in coalescing replication organelles and lipid droplets, to a later role where 2C assists RNA replication and particle assembly. 2C is sufficient to recruit RNA to membranes, with a preference for double-stranded RNA (the replicating form of the viral genome). Finally, the in vitro reconstitution revealed that full-length, membrane-bound 2C has ATPase activity and ATP-independent, single-strand ribonuclease activity, but no detectable helicase activity. Together, this study suggests novel roles for 2C in membrane clustering, RNA membrane recruitment and cleavage, and calls into question a role of 2C as an RNA helicase. The reconstitution of functional, 2C-decorated vesicles provides a platform for further biochemical studies into this protein and its roles in enterovirus replication.

Structural and functional analysis of natural capsid variants suggests sialic acid-independent entry of BK polyomavirus.

BK polyomavirus (BKPyV) is an opportunistic pathogen that uses the b-series gangliosides GD1b and GT1b as entry receptors. Here, we characterize the impact of naturally occurring VP1 mutations on ganglioside binding, VP1 protein structure, and virus tropism. Infectious entry of single mutants E73Q and E73A and the triple mutant A72V-E73Q-E82Q (VQQ) remains sialic acid dependent, and all three variants acquire binding to a-series gangliosides, including GD1a. However, the E73A and VQQ variants lose the ability to infect ganglioside-complemented cells, and this correlates with a clear shift of the BC2 loop in the crystal structures of E73A and VQQ. On the other hand, the K69N mutation in the K69N-E82Q variant leads to a steric clash that precludes sialic acid binding. Nevertheless, this mutant retains significant infectivity in 293TT cells, which is not dependent on heparan sulfate proteoglycans, implying that an unknown sialic acid-independent entry receptor for BKPyV exists.

Structural Insight into Non-Enveloped Virus Binding to Glycosaminoglycan Receptors: A Review.

Viruses are infectious agents that hijack the host cell machinery in order to replicate and generate progeny. Viral infection is initiated by attachment to host cell receptors, and typical viral receptors are cell-surface-borne molecules such as proteins or glycan structures. Sialylated glycans (glycans bearing sialic acids) and glycosaminoglycans (GAGs) represent major classes of carbohydrate receptors and have been implicated in facilitating viral entry for many viruses. As interactions between viruses and sialic acids have been extensively reviewed in the past, this review provides an overview of the current state of structural knowledge about interactions between non-enveloped human viruses and GAGs. We focus here on adeno-associated viruses, human papilloma viruses (HPVs), and polyomaviruses, as at least some structural information about the interactions of these viruses with GAGs is available. We also discuss the multivalent potential for GAG binding, highlighting the importance of charged interactions and positively charged amino acids at the binding sites, and point out challenges that remain in the field.