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.

Keywords: alphavirus; virus assembly; virus budding.

FIG 1

Revertant virus growth. Plaque-purified virus stocks from the revertant selection were used to infect BHK cells at a multiplicity of 1 PFU/cell (see also Table 1). The cells were then incubated at 37°C, the media harvested at the indicated times, and virus was quantitated by plaque assay. (A) Growth curves shown are the average of two independent experiments. (B) Comparison of WT and revertant virus growth at 24 hpi (data from panel A). The dotted line indicates a threshold set at 10⁹ PFU/ml, 1 log lower than the growth of the WT virus. Data shown are the average of two experiments, with the range indicated by the bars.

FIG 2

Growth of mutants derived from infectious clones. Viral RNAs were generated from infectious clone constructs containing the indicated revertant mutations (see also Table 1). BHK cells were electroporated with WT or mutant RNA and cultured at 37°C, and the media were harvested at the indicated times. Infectious virus production was quantitated by plaque assay. (A) Growth curves shown are the average of two experiments. (B) Comparison of virus growth at 24 hpi, with the range indicated by the bars (data from panel A).

FIG 3

Assembly of HH rescue mutants. BHK cells were infected with WT, H348/352A, or the indicated H348/352A rescue mutants at a multiplicity of 10 PFU/cell for 5 h and then pulse-labeled for 30 min with [³⁵S]methionine-cysteine and chased for the indicated times. Cell lysates and media were harvested at each time point, and samples were immunoprecipitated with an E1/E2 polyclonal antibody and analyzed by SDS-PAGE and fluorography. Media samples were immunoprecipitated in the absence of detergent to allow retrieval of intact virus particles. The data shown are representative of two experiments.

FIG 4

TEM of SFV-infected cells. BHK cells were infected with WT SFV (A), the H348/352A mutant (B), the HH E1-T317I mutant (C), the HH E2-T402K mutant (D), or the HH C-D180N mutant (E). Infection was continued for 8 h, and the cells were fixed and processed for TEM. A representative image of each sample is shown. Arrows indicate nucleocapsids/capsid proteins at the plasma membrane, arrowheads indicate budded virus particles, and asterisks indicate replication spherules. Scale bars, 200 nm.

FIG 5

Thermostability of HH rescue mutants. Virus stocks of WT, H348/352A, or the indicated H348/352A rescue mutants were diluted to titers of 1 × 10⁸ PFU/ml and incubated at 50°C for the indicated times. Infectious virus was quantitated by a plaque assay. The results shown are means and standard deviations from three to four independent experiments.

FIG 6

Location of SFV E2-T402K mutation. Shown are the E1 (yellow), E2 (green), and capsid (teal) proteins extracted from the atomic model built for the alphavirus surface glycoprotein shell and nucleocapsid shell from CHIKV, as based on the CHIKV cryo-EM reconstructions of CHIKV VLPs to 5-Å resolution (PDB access 3J2W). (A) The E2 residues H348 and H352 in the juxtamembrane D-loop are indicated by black sticks, the E2 and E1 transmembrane helices are indicated by arrows, and the E2 endodomain rescue mutation is in the boxed region. (B) Expanded view of the CHIKV E2-capsid interaction from the boxed region in panel A (at a slightly tilted angle for clarity). The capsid is shown in a surface view; E2-T402 (which is K402 in the revertant) is shown as a blue stick, and capsid D254 is shown in red. Note that for clarity the SFV residue numbering is used here for CHIKV E2 and capsid. All images were prepared using PyMOL software (37).

FIG 7

Interactions of E1-T317 in the glycoprotein lattice. Shown are segments corresponding to E1 (yellow) and E2 (green) extracted from the atomic model built for the alphavirus surface glycoprotein shell from CHIKV, based on the cryo-EM reconstructions of CHIKV VLPs to 5-Å resolution (PDB access 3J2W). (A) CHIKV E1 interactions at the 5-fold axis of symmetry. The highlighted residues are from E1 belonging to five different trimeric E2/E1 spikes coming together at one vertex. E1-I317 is shown as a black stick image, and S323 and S295 on the adjacent E1′ are shown as sticks with the side chain oxygen atoms in red. Note that the E1 residue numbering is the same for SFV and CHIKV, but these residues are T317, T295, and N323 in WT SFV. (B) Expanded view (at a slightly tilted angle for clarity) of the CHIKV E1 domain III interactions involving these three residues. CHIKV E1-I317 is in position to interact with the adjacent E1′ via S323 and S295. All images were prepared using PyMOL software (37).