Japanese encephalitis virus NS1' protein depends on pseudoknot secondary structure and is cleaved by caspase during virus infection and cell apoptosis.

Japanese encephalitis virus (JEV) is a flavivirus with a complex life cycle involving mosquito vectors that mainly target birds and pigs, and causes severe encephalitis in children in Asia. Neurotropic flaviviruses of the JEV serogroup have a particular characteristic of expressing a unique nonstructural NS1' protein, which is a prolongation of NS1 at the C terminus by 52 amino acids derived from a pseudoknot-driven-1 translation frameshift. Protein NS1' is associated with virus neuro-invasiveness. In this study, the need of the pseudoknot structure for NS1' synthesis was confirmed. By using a specific antibody against the prolonged peptide, NS1' was found to be absent from the JEV SA14-14-2 vaccine strain, resulting from a single nucleotide silent mutation in the pseudoknot. A partial cleavage of NS1' at a specific site of its C-terminal appendix recognized by caspases and inhibited by caspase inhibitors suggests a unique feature of intracellular NS1'.

Sun J ，Yu Y ，Deubel V 《-》

The A66G back mutation in NS2A of JEV SA14-14-2 strain contributes to production of NS1' protein and the secreted NS1' can be used for diagnostic biomarker for virulent virus infection.

Japanese encephalitis virus (JEV) is the most common cause of the prevalent encephalitis in Asia-Pacific region and poses a serious risk to public health. Here, we developed a reliable reverse genetics system based on the JEV SA14-14-2 strain to further explore the mechanism for the synthesis of NS1' protein and to investigate the function of NS1' protein during virus infection. NS1' is an additional form of NS1 protein with 52 amino acid carboxy-terminal extension and is expressed by the members of the Japanese encephalitis (JE) serogroup due to the translation frameshift. A66G substitution in NS2A gene of JEV SA14-14-2 strain contributed to recover the GC-rich pseudoknot and resulted in the formation of the NS1'. The NS1' protein has no significant effect on the virus replication properties in BHK-21 cells. Animal experiments demonstrated that the NS1' protein had a rather minor effect on neurovirulence of JEV SA14-14-2 strain. But the NS1'-expressing virus (rA66G) could induce a higher humoral immune response than the NS1'-non-expressing virus (rSA14-14-2). NS1' protein can be detected in the serum of JEV rA66G infected animal and in the cultural media of that infected mammalian cells. Interesting, only the dimer of NS1' can be detected in the cultural media of the infected BHK-21 cells and the amount of the secreted NS1' was in agreement with that of the secreted virion. In comparison with the live-attenuated JE vaccine strain which is incapable of formation of NS1', most of the virulent JEV strains produce the NS1' protein. And the secreted NS1' may serve as an early surrogate biomarker for viremia to distinguish the field infection from the vaccine inoculation. In total, in the present study, we identified the nt 66 in the viral NS2A gene as one of the critical site for the -1 programmed ribosomal frameshift to produce the NS1' protein and demonstrated the secreted NS1' could be used for diagnostic biomarker during JEV infection.

Wang J ，Li X ，Gu J ，Fan Y ，Zhao P ，Cao R ，Chen P ... -  《-》

Japanese encephalitis virus NS1 and NS1' proteins induce vimentin rearrangement via the CDK1-PLK1 axis to promote viral replication.

Xie S ，Yang X ，Yang X ，Cao Z ，Wei N ，Lin X ，Shi M ，Cao R ... -  《-》

Japanese encephalitis virus NS1 and NS1' protein disrupts the blood-brain barrier through macrophage migration inhibitory factor-mediated autophagy.

Zhang L ，Nan X ，Zhou D ，Wang X ，Zhu S ，Li Q ，Jia F ，Zhu B ，Si Y ，Cao S ，Ye J ... -  《-》

A single nucleotide mutation in NS2A of Japanese encephalitis-live vaccine virus (SA14-14-2) ablates NS1' formation and contributes to attenuation.

Ye Q ，Li XF ，Zhao H ，Li SH ，Deng YQ ，Cao RY ，Song KY ，Wang HJ ，Hua RH ，Yu YX ，Zhou X ，Qin ED ，Qin CF ... -  《-》