In a study published in Nature on Oct. 13, researchers led by Dr. ZHANG Xinzheng at the Institute of Biophysics of the Chinese Academy of Sciences (CAS), in cooperation with Dr. XIANG Ye at Tsinghua University, have reported two cryo-EM structure of Venezuelan equine encephalitis virus (VEEV) and VEEV complexed with its receptor low-density lipoprotein receptor class A domain-containing？3 (LDLRAD3) protein, revealing the assembly mechanism of alphaviruses and VEEV host-cell receptor binding mechanism.

VEEV is an RNA enveloped alphavirus that infects humans and equines, it can be transmitted by mosquitoes, jumping from animals to humans causing progressive central neurological disease and complications, sometimes lethal. Due to no vaccine or effective therapeutic drug for this virus. Recently, the receptor for VEEV was identified as the LDLRAD3 protein, however, the high-resolution 3D structure of VEEV virus and VEEV bound to its receptor haven't been revealed, nor the specific molecular mechanism of the binding.

The researchers solved the structures of VEEV and VEEV complexed with LDLRAD3 at pseudo atomic resolutions using their previously developed "block-based" reconstruction method. The structures revealed that the extracellular region domain？1 of LDLRAD3 protein, inserted between the cleft formed by two adjacent spike-in proteins E1/E2 of VEEV by hydrophobic and polar interactions, binds at a molar ratio of 1:1.

Interestingly, the same cleft that binds LDLRAD3 is also used by chikungunya virus, Ross River virus, Mayaro virus, and O'nyong nyong virus for binding their host cell receptor Mxra8, both bind broadly similarly in the B domain of E2, but Mxra8 has a three amino acid bulge that prevents the formation of a salt bridge interaction with R64 of E2. Although the binding area is the same, critical binding sites of the two receptors are different, suggesting that VEEV enters the host via different binding receptor with other alphaviruses.

As the first pseudo atomic resolution structure of VEEV, the structure also revealed the detailed interaction between the cytoplastic tail of glycoprotein E2 and the nucleocapsid protein. This interaction was believed to be essential for the nucleocapsid core to locate the glycoprotein shell of E1/E2 during alphavirus assembly and was never been resolved.

Furthermore, mutagenesis study was performed on LDLRAD3 to verify the key residues involved in the interaction, in which the R41A mutation could enhance the binding ability of the receptor to the virus by approximately 10-fold, suggesting that LDLRAD3 could be used as a potential scaffold to study the inhibitor that blocks the cell host entry of VEEV.

This work was supported by grants from the Ministry of Science and Technology of China and the Spring Wind Foundation of Tsinghua University, the National Natural Science Foundation of China, the Strategic Key Research Program of CAS, and the Frontier Science Key Research and Development Program of CAS.

Overall structure of the VEEV VLP-LDLRAD3-D1 complex and their detailed interactions. (Image by Institute of Biophysics)