Biology subjects

Wahaab, A.

Publications and source records attributed to Wahaab, A..

3 recordsLinked to original sources

bioRxiv2024

DDX3 regulates the cap-independent translation of the Japanese encephalitis virus via its interactions with PABP1 and the untranslated regions of the viral genome

The translation of global cellular proteins is almost completely repressed in cells with flavivirus infection, while viral translation remains efficient. The mechanisms of flaviviruses evade host translational shutoff are largely unknown. Here, we identified viral elements and host factors associated with JEV evasion of host shutoff. JEV 5'UTR lacked IRES or IRES-like activity, while noncapped 5'UTR initiated translation in the presence of 3'UTR. Furthermore, the elements DB2 and sHP-SL within 3'UTR were involved in the regulation of cap-independent translation, which is conserved in the genus Orthoflavivirus. By RNA affinity purification and mass spectrometry analysis, cellular DDX3 and PABP1 were identified as key factors in regulating cap-independent translation of JEV via their interactions with DB2 and sHP-SL RNA structures. Mechanistically, we revealed that DDX3 could bind to both 5'UTR and 3'UTR of the JEV genome to establish a closed-loop architecture, recruit eIF4G/eIF4A to form the DDX3/PABP1/eIF4G/eIF4A tetrameric complex via its interaction with PABP1, thereby recruiting 43S PIC to the 5'-end of the JEV genome to start translation. Our findings demonstrated a noncanonical translation strategy employed by JEV and further revealed the regulatory roles of DDX3 and PABP1 in this mechanism. These results expand our knowledge of the translation initiation regulation in flaviviruses under the state of host translational shutoff, which provides a conserved antiviral target against orthoflavivirus.

microbiology↗

bioRxiv2023

NS2B-D55E and NS2B-E65D Variations are Responsible for Differences in NS2B-NS3 Protease Activities Between Japanese Encephalitis Virus Genotype I and III in Fluorogenic Peptide Model

Japanese Encephalitis Virus (JEV) NS2B-NS3 is a protein complex composed of NS3 proteases and a NS2B cofactor. The N-terminal protease domain (180 residues) of NS3 (NS3(pro)) interacts directly with a central 40-amino acid hydrophilic domain of NS2B (NS2B(H)) to form an active serine protease. In this study, the recombinant NS2B(H)-NS3(pro) proteases were prepared in E. coli and used to compare the enzymatic activity between genotype I (GI) and III (GIII) NS2B-NS3 proteases. The GI NS2B(H)-NS3(pro) was able to cleave the sites at internal C, NS2A/NS2B, NS2B/NS3 and NS3/NS4A junctions that were identical to the sites proteolytically processed by GIII NS2B(H)-NS3(pro). Analysis of the enzymatic activity of recombinant NS2B(H)-NS3(pro) proteases using a model of fluorogenic peptide substrate revealed that the proteolytical processing activity of GIII NS2B(H)-NS3(pro) was significantly higher than that of GI NS2B(H)-NS3(pro). There were eight amino acid variations between GI and GIII NS2B(H)-NS3(pro), which may be responsible for the difference in enzymatic activities between GI and GIII proteases. Therefore, recombinant mutants were generated by exchanging NS2B(H) and NS3(pro) domains between GI and GIII NS2B(H)-NS3(pro) and subjected to protease activity analysis. Substitution of NS2B(H) significantly altered the protease activities, as compared to the parental NS2B(H)-NS3(pro), suggesting that NS2B(H) played an essential role in regulation of NS3(pro) protease activity. To further identify the amino acids responsible for the difference in protease activities, multiple substitution mutants including the individual and combined mutations at the variant residue 55 and 65 of NS2B(H) were generated and subjected to protease activity analysis. Replacement of NS2B-55 and NS2B-65 of GI to GIII significantly increased the enzymatic activity of GI NS2B(H)-NS3(pro) protease, whereas mutation of NS2B-55 and NS2B-65 of GIII to GI remarkably reduced the enzymatic activity of GIII NS2B(H)-NS3(pro) protease. Overall, these data demonstrated that NS2B-55 and NS2B-65 variations in hydrophilic domain of NS2B co-contributed to the difference in NS2B(H)-NS3(pro) protease activities between GI and GIII. These observations gain an insight into the role of NS2B in regulation of NS3 protease activities, which is useful for understanding the replication of JEV GI and GIII viruses.

microbiology↗

bioRxiv2020

Mosquito defensins enhance Japanese encephalitis virus infection by facilitating virus adsorption and entry within mosquito

Japanese encephalitis virus (JEV) is a viral zoonosis which can cause viral encephalitis, death and disability. Culex is the main vector of JEV, but little is known about JEV transmission by this kind of mosquito. Here, we found that mosquito defensin facilitated the adsorption of JEV on target cells via both direct and indirect pathways. Mosquito defensin bound the ED III domain of viral E protein and directly mediated efficient virus adsorption on the target cell surface, Lipoprotein receptor-related protein 2 expressed on the cell surface is the receptor affecting defensin dependent adsorption. Mosquito defensin also indirectly down-regulated the expression of an antiviral protein, HSC70B. As a result, mosquitos defensin enhances JEV infection in salivary gland while increasing the possibility of viral transmission by mosquito. These findings demonstrate that the novel effects of mosquito defensin in JEV infection and the mechanisms through which the virus exploits mosquito defensin for infection and transmission.

microbiology↗