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Monoclonal antibody against EV71 3Dpol inhibits the polymerase activity of RdRp and virus replication.

ABSTRACT: BACKGROUND:Enterovirus A 71 (EV71) is a neurotropic virus that may lead to acute flaccid paralysis, encephalitis, cardiopulmonary failure or even death. No vaccine and defensive drug controlling EV71 is currently available, novel and efficient antiviral drug or vaccine is therefore urgently needed. 3Dpol (RNA-dependent RNA polymerase (RdRp)) has been an important target for anti-EV71 drug development. METHODS:A panel of monoclonal IgG antibodies (mAbs) against EV71 3Dpol were generated by traditional cell fusion methods. And the antibody affinity and specificity to EV71 3Dpol were evaluated by Enzyme-linked Immunosorbent Assay (ELISA), Indirect Fluorescent Assay (IFA) and Western blotting. Antiviral activities of these antibodies were also determined in vitro and in vivo. RESULTS:Two mAbs towards EV71 3Dpol were able to effectively suppress EV71 replication in Vero-1008 cell when intracellarly delivered. And they also dampened the RNA polymerase activity of 3Dpol in vitro. More importantly, these mAbs provided partial protection in EV71-challenged neonatal murine challenge model. CONCLUSIONS:These results showed that two of mAbs against EV71 3Dpol inhibited EV71 replication and could be utilized as promising therapeutic drug candidate.

SUBMITTER: Li Y

PROVIDER: S-EPMC6343263 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Monoclonal antibody against EV71 3D<sup>pol</sup> inhibits the polymerase activity of RdRp and virus replication.

Li Yaoming Y Yu Jie J Qi Xiuwen X Yan Huimin H

BMC immunology 20190122 1

<h4>Background</h4>Enterovirus A 71 (EV71) is a neurotropic virus that may lead to acute flaccid paralysis, encephalitis, cardiopulmonary failure or even death. No vaccine and defensive drug controlling EV71 is currently available, novel and efficient antiviral drug or vaccine is therefore urgently needed. 3D<sup>pol</sup> (RNA-dependent RNA polymerase (RdRp)) has been an important target for anti-EV71 drug development.<h4>Methods</h4>A panel of monoclonal IgG antibodies (mAbs) against EV71 3D<s ...[more]

PMID: 30669993

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Project description:Chikungunya fever, a mosquito-borne disease manifested by fever, rash, myalgia, and arthralgia, is caused by chikungunya virus (CHIKV), which belongs to the genus Alphavirus of the family Togaviridae Anti-CHIKV IgG from convalescent patients is known to directly neutralize CHIKV, and the state of immunity lasts throughout life. Here, we examined the epitope of a neutralizing mouse monoclonal antibody against CHIKV, CHE19, which inhibits viral fusion and release. In silico docking analysis showed that the epitope of CHE19 was localized in the viral E2 envelope and consisted of two separate segments, an N-linker and a ?-ribbon connector, and that its bound Fab fragment on E2 overlapped the position that the E3 glycoprotein originally occupied. We showed that CHIKV-E2 is lost during the viral internalization and that CHE19 inhibits the elimination of CHIKV-E2. These findings suggested that CHE19 stabilizes the E2-E1 heterodimer instead of E3 and inhibits the protrusion of the E1 fusion loop and subsequent membrane fusion. In addition, the antigen-bound Fab fragment configuration showed that CHE19 connects to the CHIKV spikes existing on the two individual virions, leading us to conclude that the CHE19-CHIKV complex was responsible for the large virus aggregations. In our subsequent filtration experiments, large viral aggregations by CHE19 were trapped by a 0.45-?m filter. This virion-connecting characteristic of CHE19 could explain the inhibition of viral release from infected cells by the tethering effect of the virion itself. These findings provide clues toward the development of effective prophylactic and therapeutic monoclonal antibodies against the Alphavirus infection.IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Neither a specific antiviral drug nor a commercial vaccine for CHIKV infection are available. Here, we show a detailed model of the docking between the envelope glycoprotein of CHIKV and our unique anti-CHIKV-neutralizing monoclonal antibody (CHE19), which inhibits CHIKV membrane fusion and virion release from CHIKV-infected cells. Homology modeling of the neutralizing antibody CHE19 and protein-protein docking analysis of the CHIKV envelope glycoprotein and CHE19 suggested that CHE19 inhibits the viral membrane fusion by stabilizing the E2-E1 heterodimer and inhibits virion release by facilitating the formation of virus aggregation due to the connecting virions, and these predictions were confirmed by experiments. Sequence information of CHE19 and the CHIKV envelope glycoprotein and their docking model will contribute to future development of an effective prophylactic and therapeutic agent.

Project description:Many RNA viruses encode a proof-reading deficient, low-fidelity RNA-dependent polymerase (RdRp), which generates genetically diverse populations that can adapt to changing environments and thwart antiviral therapies. 3Dpol, the RdRp of the foot-and-mouth disease virus (FMDV), is responsible for replication of viral genomes. The 3Dpol N terminus encodes a nuclear localization signal (NLS) sequence,MRKTKLAPT, important for import of the protein to host nucleus. Previous studies showed that substitutions at residues 18 and 20 of the NLS are defective in proper incorporation of nucleotides and RNA binding. Here, we use a systematic alanine scanning mutagenesis approach to understand the role of individual residues of the NLS in nuclear localization and nucleotide incorporation activities of 3Dpol We identify two residues of 3Dpol NLS, T19 and L21, that are important for the maintenance of enzyme fidelity. The 3Dpol NLS alanine substitutions of T19 and L21 results in aberrant incorporation of nucleoside analogs, conferring a low fidelity phenotype of the enzyme. A molecular dynamics simulation of RNA- and mutagen (RTP)-bound 3Dpol revealed that the T19 residue participates in a hydrogen bond network, including D165 in motif F and R416 at the C terminus of the FMDV 3Dpol and RNA template-primer. Based on these findings and previous studies, we conclude that at least the first six residues of theMRKTKLAPT sequence motif play a vital role in the maintenance of faithful RNA synthesis activity (fidelity) of FMDV 3Dpol, suggesting that the role of the NLS motif in similar viral polymerases needs to be revisited.IMPORTANCE In this study, we employed genetic and molecular dynamics approaches to analyze the role of individual amino acids of the FMDV 3Dpol nuclear localization signal (NLS). The NLS residues were mutated to alanine using a type A full-genome cDNA clone, and the virus progeny was analyzed for defects in growth and in competition with the parental virus. We identified two mutants in 3Dpol, T19A and L21A, that exhibited high rate of mutation, were sensitive to nucleotide analogs, and displayed reduced replicative fitness compared to the parental virus. Using molecular dynamics simulation, we demonstrated that residues T19 and L21 played a role in the structural configuration of the interaction network at the 3Dpol palm subdomain. Cumulatively, our data suggest that the T19 and L21 3Dpol amino acids are important for maintaining the fidelity of the FMDV polymerase and ensuring faithful replication of the FMDV genome.

Dataset Information

Monoclonal antibody against EV71 3Dpol inhibits the polymerase activity of RdRp and virus replication.

Publications

Monoclonal antibody against EV71 3D<sup>pol</sup> inhibits the polymerase activity of RdRp and virus replication.

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