The Alphavirus E2 Membrane-Proximal Domain Impacts Capsid Interaction and Glycoprotein Lattice Formation

Abstract

Alphaviruses are small enveloped RNA viruses that bud from the host cell plasma membrane. Alphavirus particles have a highly organized structure, with a nucleocapsid core containing the RNA genome surrounded by the capsid protein, and a viral envelope containing 80 spikes, each a trimer of heterodimers of the E1 and E2 glycoproteins. The capsid protein and envelope proteins are both arranged in organized lattices that are linked via the interaction of the E2 cytoplasmic tail/endodomain with the capsid protein. We previously characterized the role of two highly conserved histidine residues, H348 and H352, located in an external, juxtamembrane region of the E2 protein termed the D-loop. Alanine substitutions of H348 and H352 inhibit virus growth by impairing late steps in the assembly/budding of virus particles at the plasma membrane. To investigate this budding defect, we selected for revertants of the E2-H348/352A double mutant. We identified eleven second-site revertants with improved virus growth and mutations in the capsid, E2 and E1 proteins. Multiple isolates contained the mutation E2-T402K in the E2 endodomain or E1-T317I in the E1 ectodomain. Both of these mutations were shown to partially restore H348/352A growth and virus assembly/budding, while neither rescued the decreased thermostability of H348/352A. Within the alphavirus particle, these mutations are positioned to affect the E2-capsid interaction or the E1-mediated intertrimer interactions at the 5-fold axis of symmetry. Together, our results support a model in which the E2 D-loop promotes the formation of the glycoprotein lattice and its interactions with the internal capsid protein lattice. IMPORTANCE Alphaviruses include important human pathogens such as Chikungunya and the encephalitic alphaviruses. There are currently no licensed alphavirus vaccines or effective antiviral therapies, and more molecular information on virus particle structure and function is needed. Here, we highlight the important role of the E2 juxtamembrane D-loop in mediating virus budding and particle production. Our results demonstrated that this E2 region affects both the formation of the external glycoprotein lattice and its interactions with the internal capsid protein shell.

Emerging Chikungunya Virus Variants at the E1-E1 Interglycoprotein Spike Interface Impact Virus Attachment and Inflammation

“…The copyright holder for this preprint this version posted September 14, 2021. ; https://doi.org/10.1101/2021.09. 13.460192 doi: bioRxiv preprint particle stability, and immunogenicity [24,26,27]. Considering this multi-functionality throughout the viral replication cycle, it is likely that there are additional roles for E1 and further details of its known functions yet to be elucidated.…”

A molecular understanding of alphavirus entry

“…The alphavirus E2 protein facilitates receptor engagement [ 5 ], whereas E1 principally mediates membrane fusion after viral entry [ 5 , 6 ]. The carboxyl terminus of E2 also interacts with the capsid core, which stabilizes the virion [ 7 , 8 ]. The 6K protein is thought to promote glycoprotein maturation, spike assembly, and act as a viroporin [ 9 ].…”

Near-atomic resolution Cryo-EM structure of Mayaro virus identifies key structural determinants of alphavirus particle formation

“…As previously described, the outer GP shell is linked to NC in the mature virus particle by direct interaction between the Y-X-L motif of the E2 cytosolic tail and hydrophobic CP binding pocket. The correct orientation of E2 endodomain presented at the cell surface is critical for NC interaction, and it has been suggested interactions just above the viral membrane between residues in E2 subD and E1 are important for correct positioning of the CP binding site (Byrd and Kielian 2019). Within E2 subD we observe E1-E2…”

“…Deletion of E1 residues D151,H152 significantly reduced MAYV growth, presumably through disruption of an interface between two quasi-3-fold spike trimers. Recently, a revertant mutation to the assembly-impaired SFV E2 H348A/H352A double mutant was mapped to an inter-trimer contact point in EI domain III at the five-fold axis, providing additional evidence this region is an important determinant of alphavirus particle formation (Byrd and Kielian, 2019). Our cryo-EM structure can be used as a guide to perform mutagenesis experiments to determine relative contributions of the interfacial residues around the five-fold axis to the stability of the virus particle.…”