Alphaviruses are members of a group of small enveloped RNA viruses that includes important human pathogens such as Chikungunya virus and the equine encephalitis viruses. The virus membrane is covered by a lattice composed of 80 spikes, each a trimer of heterodimers of the E2 and E1 transmembrane proteins. During virus endocytic entry, the E1 glycoprotein mediates the low-pH-dependent fusion of the virus membrane with the endosome membrane, thus initiating virus infection. While much is known about E1 structural rearrangements during membrane fusion, it is unclear how the E1/E2 dimer dissociates, a step required for the fusion reaction. A recent Alphavirus cryo-electron microscopy reconstruction revealed a previously unidentified D subdomain in the E2 ectodomain, close to the virus membrane. A loop within this region, here referred to as the D-loop, contains two highly conserved histidines, H348 and H352, which were hypothesized to play a role in dimer dissociation. We generated Semliki Forest virus mutants containing the single and double alanine substitutions H348A, H352A, and H348/352A. The three D-loop mutations caused a reduction in virus growth ranging from 1.6 to 2 log but did not significantly affect structural protein biosynthesis or transport, dimer stability, virus fusion, or specific infectivity. Instead, growth reduction was due to inhibition of a late stage of virus assembly at the plasma membrane. The virus particles that are produced show reduced thermostability compared to the wild type. We propose the E2 D-loop as a key region in establishing the E1-E2 contacts that drive glycoprotein lattice formation and promote Alphavirus budding from the plasma membrane.

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.