EV71 2A Protease inhibits P-body formation to promote viral RNA synthesis

Several viruses were proved to inhibit the formation of RNA processing bodies (P-bodies); however, knowledge regarding whether enterovirus blocks P-body formation remains unclear, and the detailed molecular mechanisms and functions of picornavirus regulation of P-bodies are limited. Here we show the crucial role of 2A protease in inhibiting P-bodies to promote viral replication during enterovirus 71 infection. Moreover, we found that the activity of 2A protease is essential to inhibit P-body formation, which was proved by the result that infection of EV71-2A C110S , the 2A protease activity-inactivated recombinant virus, failed to block the formation of P-bodies. Furthermore, we showed DDX6, a scaffolding protein of P-bodies, interacted with viral RNA to facilitate viral replication rather than viral translation, by using a Renilla luciferase mRNA reporter system and capturing the nascent RNA assay. Altogether, our data firstly demonstrate that the 2A protease of enterovirus inhibits P-body formation to facilitate viral RNA synthesis by recruiting the P-body components to viral RNA. IMPORTANCE Processing bodies (P-bodies) are constitutively present in eukaryotic cells and play an important role in the mRNA cycle, including regulating gene expression and mRNA degradation. P-bodies are the structure that viruses to manipulate to facilitate their survival. Here, we show that the 2A protease alone was efficient to block P-body formation during enterovirus 71 infection and its activity was essential. When the assembly of P-bodies was blocked by 2A, DDX6 and 4E-T which were required for P-body formation bound to viral RNA to facilitate viral RNA synthesis. We propose a model revealing that EV71 manipulates P-body formation to generate an environment that is conducive to viral replication by facilitating viral RNA synthesis: 2A protease blocked P-body assembly to make it possible for virus to take advantage of P-body components.

Polyamine Analog Diethylnorspermidine Restricts Coxsackievirus B3 and Is Overcome by 2A Protease Mutation In Vitro

Enteroviruses, including Coxsackievirus B3 (CVB3), are pervasive pathogens that cause significant disease, including cardiomyopathies. Unfortunately, no treatments or vaccines are available for infected individuals. We identified the host polyamine pathway as a potential drug target, as inhibiting polyamine biosynthesis significantly reduces enterovirus replication in vitro and in vivo. Here, we show that CVB3 is sensitive to polyamine depletion through the polyamine analog diethylnorspermidine (DENSpm), which enhances polyamine catabolism through induction of polyamine acetylation. We demonstrate that CVB3 acquires resistance to DENSpm via mutation of the 2A protease, which enhances proteolytic activity in the presence of DENSpm. Resistance to DENSpm occurred via mutation of a non-catalytic site mutation and results in decreased fitness. These data demonstrate that potential for targeting polyamine catabolism as an antiviral target as well as highlight a potential mechanism of resistance.

Immunohistochemical Detection of Enteroviruses in Pancreatic Tissues of Patients with Type 1 Diabetes Using a Polyclonal Antibody Against 2A Protease of Coxsackievirus

The need for antiserum for immunohistochemical (IHC) detection of enterovirus (EV) in formaldehyde fixed and paraffin-embedded (FFPE) specimens is increasing. The standard monoclonal antibody against EV-envelope protein (VP1), clone 5D8/1, was proven to cross-react with other proteins. Another candidate marker of EV proteins is 2A protease (2Apro), which is coded by the EV gene and translated by host cells during EV replication. We raised polyclonal antiserum by immunizing rabbits with an 18-mer peptide of Coxsackievirus B1 (CVB1)-2A protease (2Apro) and examined the specificity and sensitivity for EV on FFPE tissue samples. ELISA study showed a high titer of antibody for CVB1-2Apro. IHC demonstrated that antiserum against 2Apro reacted with CVB1-infected Vero-cells. Confocal microscopy demonstrated that 2Apro labelled by the antibody located in the same cell with VP1 stained with 5D8/1. IHC demonstrated dense positive reactions pancreatic islets of EV-associated fulminant type 1 diabetes (FT1DM), and located in the same cell stained positive with 5D8/1. Specificity of IHC staining FT1DM pancreas was confirmed by absorption with an excessive concentration of immunized peptide. In conclusion, our study provides a new polyclonal antiserum against CVB1 2Apro which may be useful for detection of EV-infected human tissues stored as archive of FFPE tissue samples.

Characterization of Recombinant Poliovirus 2A Protease; A Potential Anti-Viral Drug Target

Poliovirus 2A protease (PV2Apro) plays a vital role in viral replication and down-regulation of host cell protein synthesis. In order to understand more concerning PV2Apro, the protein was over-expressed in bacteria following amplification using sense and antisense primers and cloning in pET15b. Several expression hosts were tested and BL21 (DE3) pLysS cells gave the best expression of PV2Apro with minimal unwanted protein expression following IPTG induction. The 2Apro protein was purified to homogeneity using column chromatography, its solubility determined and its molecular weight and composition determined by MALDI-TOF mass spectrometry. The protease was found in the insoluble fraction and the purified protein had a slightly lower molecular weight than predicted. Moreover, three dimensional structure was modelled using template 1z8r with 58% identity and validated using ramachandran plot. Results revealed that most of the residues lie in favoured and allowed regions. These findings could help in a better understanding of PV2Apro structure and inhibition thus, highlighting potential targets for antiviral drug development.

Structural view of the 2A protease from human rhinovirus C15

The majority of outbreaks of the common cold are caused by rhinoviruses. The 2A protease (2Apro) of human rhinoviruses (HRVs) is known to play important roles in the propagation of the virus and the modulation of host signal pathways to facilitate viral replication. The 2Aprofrom human rhinovirus C15 (HRV-C15) has been expressed inEscherichia coliand purified by affinity chromatography, ion-exchange chromatography and gel-filtration chromatography. The crystals diffracted to 2.6 Å resolution. The structure was solved by molecular replacement using the structure of 2Aprofrom coxsackievirus A16 (CVA16) as the search model. The structure contains a conserved His–Asp–Cys catalytic triad and a Zn2+-binding site. Comparison with other 2Aprostructures from enteroviruses reveals that the substrate-binding cleft of 2Aprofrom HRV-C15 exhibits a more open conformation, which presumably favours substrate binding.