Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone.

RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3'-to-5' unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our understanding of enteroviruses and the two types of RNA remodeling activities.

Crystal structures of enterovirus 71 (EV71) recombinant virus particles provide insights into vaccine design.

Hand-foot-and-mouth disease (HFMD) remains a major health concern in the Asia-Pacific regions, and its major causative agents include human enterovirus 71 (EV71) and coxsackievirus A16. A desirable vaccine against HFMD would be multivalent and able to elicit protective responses against multiple HFMD causative agents. Previously, we have demonstrated that a thermostable recombinant EV71 vaccine candidate can be produced by the insertion of a foreign peptide into the BC loop of VP1 without affecting viral replication. Here we present crystal structures of two different naturally occurring empty particles, one from a clinical C4 strain EV71 and the other from its recombinant virus containing an insertion in the VP1 BC loop. Crystal structure analysis demonstrated that the inserted foreign peptide is well exposed on the particle surface without significant structural changes in the capsid. Importantly, such insertions do not seem to affect the virus uncoating process as illustrated by the conformational similarity between an uncoating intermediate of another recombinant virus and that of EV71. Especially, at least 18 residues from the N terminus of VP1 are transiently externalized. Altogether, our study provides insights into vaccine development against HFMD.

Percutaneous transhepatic variceal embolization combined with endoscopic ligation for the prevention of variceal rebleeding.

OBJECTIVE: To compare the efficiency of percutaneous transhepatic variceal embolization (PTVE) plus endoscopic variceal ligation (EVL) with EVL alone in the treatment of esophageal variceal bleeding. METHODS: Cirrhotic patients with recent esophageal variceal bleeding from January 2007 to December 2011 were collected and assigned to PTVE + EVL (N = 41) or EVL (N = 47) groups. We performed chart reviews and prospective follow-up to determine variceal rebleeding, recurrence of varices and survival. RESULTS: During the follow-up period, recurrence of esophageal varices (EV) occurred in 8 patients (19.5%) in the PTVE + EVL group and 23 (48.9%) in the EVL group (P = 0.004). The time to recurrence of EV was 9.2 ± 2.7 months and 4.9 ± 2.1 months, respectively. Three patients (7.3%) in the PTVE + EVL group and 12 (25.5%) in the EVL group experienced rebleeding from all sources (P = 0.023). One patient (2.4%) in the PTVE + EVL group and 7 (14.9%) in the EVL group experienced rebleeding from EV (P = 0.024). Multivariate Cox analysis indicated that the treatment method was the only predictor of rebleeding. There was no significant difference in the survival rate between the two groups. CONCLUSION: With adequate and permanent obliteration of EV and their feeding veins, the combination of PTVE with cyanoacrylate and EVL is more effective than EVL alone in the prevention and treatment of EV recurrence and rebleeding.

MDCT angiography to evaluate the therapeutic effect of PTVE for esophageal varices.

AIM: To evaluate the role of multi-detector row computed tomography (MDCT) angiography for assessing the therapeutic effects of percutaneous transhepatic variceal embolization (PTVE) for esophageal varices (EVs). METHODS: The subjects of this prospective study were 156 patients who underwent PTVE with cyanoacrylate for EVs. Patients were divided into three groups according to the filling range of cyanoacrylate in EVs and their feeding vessels: (1) group A, complete obliteration, with at least 3 cm of the lower EVs and peri-/EVs, as well as the adventitial plexus of the gastric cardia and fundus filled with cyanoacrylate; (2) group B, partial obliteration of varices surrounding the gastric cardia and fundus, with their feeding vessels being obliterated with cyanoacrylate, but without reaching lower EVs; and (3) group C, trunk obliteration, with the main branch of the left gastric vein being filled with cyanoacrylate, but without reaching varices surrounding the gastric cardia or fundus. We performed chart reviews and a prospective follow-up using MDCT images, angiography, and gastrointestinal endoscopy. RESULTS: The median follow-up period was 34 mo. The rate of eradication of varices for all patients was 56.4% (88/156) and the rate of relapse was 31.3% (41/131). The rates of variceal eradication at 1, 3, and 5 years after PTVE were 90.2%, 84.1% and 81.7%, respectively, for the complete group; 61.2%, 49% and 42.9%, respectively, for the partial group; with no varices disappearing in the trunk group. The relapse-free rates at 1, 3 and 5 years after PTVE were 91.5%, 86.6% and 81.7%, respectively, for the complete group; 71.1%, 55.6% and 51.1%, respectively, for the partial group; and all EVs recurred in the trunk group. Kaplan-Meier analysis showed P values of 0.000 and 0.000, and odds ratios of 3.824 and 3.603 for the rates of variceal eradication and relapse free rates, respectively. Cyanoacrylate in EVs disappeared with time, but those in the EVs and other feeding vessels remained permanently in the vessels without a decrease with time, which is important for the continued obliteration of the feeding vessels and prevention of EV relapse. CONCLUSION: MDCT provides excellent visualization of cyanoacrylate obliteration in EV and their feeding veins after PTVE. It confirms that PTVE is effective for treating EVs.