Biological Effects Of The Glycan Loop Deletions Of Zika Virus Envelope Protein

Zika virus(ZIKV)belongs to genus flavivirus,family flaviviridae,which include dengue virus,yellow fever virus,Japanese encephalitis virus and West Nile virus.ZIKV is the pathogen of ZIKV disease that mainly prevalent in Africa,the Americas,Asia and the Pacific.ZIKV infection only causes mild and typical self-limitating diseases.The genome of ZIKV is a single-stranded RNA molecule of positive polarity comprising about 11,000 nucleotides,including 3 structural proteins(C,prM/M and E)and 7 non-structural proteins(NS1,NS2 A,NS2B,NS3,NS4 A,NS4B and NS5).Envelope protein is responsible for receptor binding,membrane fusion and other important functions,which is also the main antigens to induce protective antibodies.The E protein consists of three ectodomains(Domain?(D?),D?,D?)connected by short flexible loops,D?acts as a bridge berwween D?and D?.The tip of the D?contains the fusion loop,which interacts with the host membrane during membrane fusion.In most flaviviruses,D?conrtains the putative receptor-binding site and has important roles in fusion.Of note,ZIKV envelope protein has a single glycosylation site at N154 in the D?,which protrudes from the surface,because the ZIKV E-D?has a longer ‘150 loop'(residues 145-160,the glycan loop is the residues 145-165)compared with other flaviviruses.The glycan loop region varies among not only ZIKV strains,but also other flaviviruses,which suggests that the difference in glycan loop influence virus transmission and pathogenesis.ZIKV acquired adaptive mutations during its evolution,which significantly enhanced its infectivity and transmissibility,probably resulting in the world pandemic and thousands of neonatal microcephaly.By phylogenetic analysis we found that there were different amino acid motif deletions in the envelope glycanloop of ZIKV African strains.Meanwhile,If the glycan loop deletions maybe also an adaptive mutation of ZIKV,so its functions on biological effects and specific mechanisms of glycan loop deletions are also scientific issues that needed to be clarified urgently.In this study,we firstly identified the distributions of ZIKV strains that contains the amino acid motif deletions in the glycan loop among all the ZIKV isolates worldwide by phylogenetic analysis and make sure their roles in evolution.Then we generated recombinant ZIKV containing different deletions in the glycan loop based an Asian ZIKV infectious cDNA clone by standard reverse genetics.The glycan loop deletion-mutants were constructed and recombinanted,then through a series of in vivo and in vitro experiments to identify their biological functions.Finally,the possible mechanism was clarified by analyzing the structure of the glycan loop deletion-mutants.Main results are described below in two parts.1.Construction and identification of full-length cDNA clone of ZIKV glycan loop deletion-mutants.Firstly,in order to confirm the strains that contained the glycan loop deletions among African strains and Asian strains and the corresponding deletion motifs,the genome sequences of ZIKV envelope were aligned and phylogenetic tree was established by phylogenetic analysis.The results showed that there were 4to 7 amino acid deletions in the envelope glycan loop in different evolutionary branches of ZIKV,especially in newly isolated Asian epidemic strains.We can speculate that the glycan loop deletions may play a crucial role in the evolution of ZIKV.Then to obtain the recombinant ZIKV strains contains the glycan loop deletions and evaluated their basic biological characterization,we introduced the specific amino acid deletions in the envelope glycan loop into ZIKV strainFSS13025,generated the recombinant ZIKV and recombinanted the mutant viruses.Then we found that the glycan loop deletions did not affect viral protein expression by indirect immunofluorescence.The growth curve analysis showed that the glycan loop deletions did not change the replication ability of ZIKV.By plague forming assay we found similar plague morphologies between the wild type and mutant viruses.Finally,we found that the ZIKV envelope protein contained the glycan loop deletions resulting in non-glycosylation modification by glycosylation test.Our results indicated that the glycan loop deletion affected the basic biological characterization of ZIKV to some extent.2.Infection and pathogenicity evaluation of recombinant ZIKV with glycan loop deletions.To clarify the infection and pathogenoicity ability of the recombinant ZIKV contained the glycan loop deletions,we explored the neuroinvasiveness and neurovirulence of the mutant virus in different mouse models,and mosquito model was used to detect the infectivity of the mutant virus.The results showed that the glycan loop deletion enhanced the neurovirulence of ZIKV to the suckling mice,resulting in more severe microcephaly.However,the pathogenicity of the glycan loop mutants was significantly reduced in A129 mice model and BALB/c mice model.In addition,the glycan loop mutants had lower infectivity to Aedes aegypti than WT by oral infection,and still could pass through the midgut barrier of mosquitoes.Finally,we further identified and analyzed the structural characteristics of the recombinant ZIKV,and found that the glycan loop deletions induced a ‘JEV-like' conformation alteration,including unusual ‘holes' and remarkable change to the top topology of the glycan loop,thus might enhancing the binding to specific receptors in neural cells.In conclusion,we found ZIKV envelope glycan loop deletions not only existedin African lineage,also appeared in the contemporaneous Asian lineage,which may play crucial roles in ZIKV adaptive evolution.Furthermore,the glycan loop deletions resulted reduction of the neuroinvasiveness in adult mice and infection ability in mosquitoes.While the glycan loop deletions enhanced neurovirulence in suckling mice and leading more severe microcephaly,which may be related to its ‘JEV-like' alteration in ZIKV envelope protein structure.These results layed an important foundation for our deepen understanding of the pathogenic mechanism of ZIKV.At the same time,the glycan loop deletions in envelope protein represent as per se biomarker for monitoring the neurovirulence of ZIKV,highlighting the necessity and importance of extensive and long time surveillance for the reemergence of these glycan loop deletions.