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.

Poliovirus capsid protein VP3 can penetrate vascular endothelial cells.

The poliovirus (PV) enters the central nervous system (CNS) via the bloodstream, suggesting the existence of a mechanism to cross the blood-brain barrier. Here, we report that PV capsid proteins (VP1 and VP3) can penetrate cells, with VP3 being more invasive. Two independent parts of VP3 are responsible for this function. Both peptides can penetrate human umbilical cord vascular endothelial cells, and one peptide of VP3 could also penetrate peripheral blood mononuclear cells. In an in vitro blood-brain barrier model using rat-derived astrocytes, pericytes, and endothelial cells, both peptides were observed to traverse from the blood side to the brain side at 6 h after administration. These results provide insights into the molecular mechanisms underlying PV invasion into the CNS.

An efficient trans complementation system for in vivo replication of defective poliovirus mutants.

The picornavirus genome encodes a large, single polyprotein that is processed by viral proteases to form an active replication complex. The replication complex is formed with the viral genome, host proteins, and viral proteins that are produced/translated directly from each of the viral genomes (viral proteins provided in cis). Efficient complementation in vivo of replication complex formation by viral proteins provided in trans, thus exogenous or ectopically expressed viral proteins, remains to be demonstrated. Here, we report an efficient trans complementation system for the replication of defective poliovirus (PV) mutants by a viral polyprotein precursor in HEK293 cells. Viral 3AB in the polyprotein, but not 2BC, was processed exclusively in cis. Replication of a defective PV replicon mutant, with a disrupted cleavage site for viral 3Cpro protease between 3Cpro and 3Dpol (3C/D[A/G] mutant) could be rescued by a viral polyprotein provided in trans. Only a defect of 3Dpol activity of the replicon could be rescued in trans; inactivating mutations in 2CATPase/hel, 3B, and 3Cpro of the replicon completely abrogated the trans-rescued replication. An intact N-terminus of the 3Cpro domain of the 3CDpro provided in trans was essential for the trans-active function. By using this trans complementation system, a high-titer defective PV pseudovirus (PVpv) (>107 infectious units per mL) could be produced with the defective mutants, whose replication was completely dependent on trans complementation. This work reveals potential roles of exogenous viral proteins in PV replication and offers insights into protein/protein interaction during picornavirus infection. IMPORTANCE: Viral polyprotein processing is an elaborately controlled step by viral proteases encoded in the polyprotein; fully processed proteins and processing intermediates need to be correctly produced for replication, which can be detrimentally affected even by a small modification of the polyprotein. Purified/isolated viral proteins can retain their enzymatic activities required for viral replication, such as protease, helicase, polymerase, etc. However, when these proteins of picornavirus are exogenously provided (provided in trans) to the viral replication complex with a defective viral genome, replication is generally not rescued/complemented, suggesting the importance of viral proteins endogenously provided (provided in cis) to the replication complex. In this study, I discovered that only the viral polymerase activity of poliovirus (PV) (the typical member of picornavirus family) could be efficiently rescued by exogenously expressed viral proteins. The current study reveals potential roles for exogenous viral proteins in viral replication and offers insights into interactions during picornavirus infection.

Hacia la erradicación de la poliomielitis: logros y retos en México

Desde la implementación de las estrategias globales de erradicación, la incidencia de parálisis poliomielítica ha disminuido dramáticamente. Cuatro estrategias han contribuido notablemente: a) Altas coberturas de inmunización con vacuna oral de polio (VOP), b) Inmunización suplementaria durante los Días Nacionales de Vacunación, c) Vigilancia epidemiológica efectiva de casos de parálisis flácida aguda (PFA), y d) Bloqueos vacunales en zonas de alto riesgo. Sólo quedan tres países polioendémicos, no obstante, cualquier país corre el riesgo potencial de importación del virus de algunas de estas áreas, de la liberación accidental del virus resguardado en laboratorios de diagnóstico clínico o investigación, o de la presencia de virus circulantes derivados de vacuna en el medio ambiente. Este documento pretende exponer los antecedentes históricos que hicieron posible la eliminación de la enfermedad en México, así como los retos para lograr un mundo libre de poliomielitis.

Since the strategies to eradicate polio were implemented, the incidence of paralytic polio has dropped dramatically. Four main strategies have greatly contributed: a) High immunization coverage rate with oral polio vaccine (OPV), b) Supplementary immunization activities during the National Immunizations Days c) An effective epidemiological surveillance system for acute flaccid paralysis (AFP) and d) Intensified immunization activities in high risk areas. Three countries remain polio endemic, nevertheless, any country has a potential risk of the virus importation from one of these endemic areas; an accidental release of poliovirus from a research or clinical laboratory, or from having a circulating vaccine-derived poliovirus in the environment. The present document aims to provide an historical background that made possible the disease elimination in Mexico. Moreover, we discuss the challenges that every country needs to face in order to achieve a polio-free world.