Structure of infective Getah virus at 2.8 Å resolution determined by cryo-electron microscopy

Getah virus (GETV), a member of the genus alphavirus, is a mosquito-borne pathogen that can cause pyrexia and reproductive losses in animals. Although antibodies to GETV have been found in over 10% of healthy people, there are no reports of clinical symptoms associated with GETV. The biological and pathological properties of GETV are largely unknown and antiviral or vaccine treatments against GETV are still unavailable due to a lack of knowledge of the structure of the GETV virion. Here, we present the structure of infective GETV at a resolution of 2.8 Å with the atomic models of the capsid protein and the envelope glycoproteins E1 and E2. We have identified numerous glycosylation and S-acylation sites in E1 and E2. The surface-exposed glycans indicate a possible impact on viral immune evasion and host cell invasion. The S-acylation sites might be involved in stabilizing the transmembrane assembly of E1 and E2. In addition, a cholesterol and a phospholipid molecule are observed in a transmembrane hydrophobic pocket, together with two more cholesterols surrounding the pocket. The cholesterol and phospholipid stabilize the hydrophobic pocket in the viral envelope membrane. The structural information will assist structure-based antiviral and vaccine screening, design, and optimization.

. Cryo-EM data collection and processing, block-based reconstruction, model building, and refinement statistics. Supplementary Video S1. GETV caused mobility impairments in pelvic limbs in newborn mice.

Supplementary Video S2. Molecular dynamics simulation of interactions in the Wild
Type E1-E2 system. Twenty pregnant mice were randomly divided into four groups, and mice in the first three groups were inoculated oronasally with 100 µl (10 6 TCID50/mL) GETV V1 strain at embryonic day 6 (E6, early-gestation), E10 (middle-gestation), and E14 (late-gestation), respectively. Mice in the fourth group were inoculated with an equal volume of DMEM at E6 as a control. Abortions (a, embryonic were expelled from the uterus before parturition), mummies (b, embryos were lost before farrowing), and stillbirths (c, embryos were lost around the time of birth, and might be prepartum or intrapartum) arising from GETVinfection were dominant in the E6, E10, and E14 groups. Some weak (d) and normal (e) newborns were found in E10 and E14 groups. Forty 7-day-old newborn and forty 2-month-old adult mice were randomly divided into two groups, inoculated oronasally with 10 µl (10 6 TCID50/mL) GETV strain GETV-V1 or control DEME. Mice were held under clinical observation after inoculation. a and b, No GETV antigen was detected in the hippocampal dentate gyrus of the brain or spinal cord from 7-day-old mice 3 DPI. c and d, No GETV antigen was found in 2-month-old mice 3 DPI.

Supplementary
Monoclonal antibody against capsid protein of GETV was used for immuno-histochemical assays. The protein contents of purified GETV samples were further analyzed with protein mass spectrometry. Representative peptide spectra from Capsid (a), E3 (b), E2 (c) and E1 (d).

Supplementary
e, Four GETV-encoded structural proteins previously described were identified by mass spectrometry. f, Capsid, E1, and E2 that were highly abundant in SDS-PAGE analyses were displayed a higher score of sequence coverage, 81.3%, 78.5%, and 63.3%, respectively. Only one small peptide from the structural protein E3 was identified, and sequence coverage is 10.9%. Peptide from the structural protein 6K was not detected.
g, The 6K gene produced two distinct protein products, 6K and transframe (TF). This occurred via a (−1) ribosomal frameshift site that is highly conserved across the alphaviruses. The 6K and TF proteins each contain an identical N-terminal transmembrane domain (first 49 amino acids) and unique C-terminal ends 3 . h, Peptide from protein TF was not detected.
Supplementary Figure S6. Mass spectrometry of GETV non-structural proteins. a, Representative peptide spectrum of non-structural-protein 1 (nsP1). b and c, Only three peptides were identified in nsP1, and sequence coverage for nsP1 is 7.9%.
Peptides from nsP2, nsP3 or nsP4 were not detected.

Micrographs were captured in a Thermo Fisher Scientific Titan Krios Cryo-Transmission
Electron Microscope equipped with a Gatan K3 direct electron detector camera. Beaminduced shifting that blurred the captured images were corrected using MotionCor2 4 .

Supplementary
The secondary structure elements are displayed above the sequences. Fully conserved residues and similar residues are shaded and shown in red. Structural polyprotein sequences and the markers for key residues are the same as in Supplementary Figure   S14.

structural elements of alphaviruses capsid protein.
Sequence alignment of capsid proteins from representative members of alphaviruses.
The secondary structure elements are displayed above the sequences.

E1 protein.
The protein sequence coverage bar graphically represents the protein sequence. The colored areas represent portions of the protein sequence that match peptides. The protein sequence coverage bar is colored to indicate the confidence of the peptide sequence identification: green, high-confidence peptides; yellow, medium-confidence peptides; pink, low-confidence peptides. Single-letter abbreviation "P" above the sequence indicates the identified palmitoyl sites by mass spectrometry, and the blue arrowhead points to the position of the C433 palmitoylation site identified in the cryo-EM density map.

E2 protein.
Single-letters abbreviation "P" and "S" above the sequence indicate the identified   Figure S13.