Sindbis virions fuse efficiently with receptor-free liposomes, showing that receptor interaction is not a mechanistic requirement for fusion.

Fusion is strictly low-pH dependent, optimal at pH 5.0 with a threshold at pH 6.0 and undetectable at neutral pH, supporting entry by fusion from within acidic endosomes.

Fusion requires both cholesterol and sphingolipid in the target membrane, with cholesterol driving low-pH binding and sphingolipid catalysing the fusion step.

Low pH dissociates the E2-E1 heterodimer and generates a trypsin-resistant E1 homotrimer that kinetically precedes fusion, identifying the E1 trimer as the fusion-active conformation.

Wild-type 6K expressed from a Sindbis replicon strongly increases membrane permeability to hygromycin B in BHK cells, while interfacial-domain mutants permeabilize less, linking the conserved pretransmembrane interfacial motif to viroporin activity.

The Sindbis capsid (core) protein fold from residue 114 to 264 is homologous to chymotrypsin-like serine proteinases, with His 141, Asp 163 and Ser 215 positioned as a canonical catalytic triad.

The C-terminal tryptophan remains in the P1 substrate site after autocatalytic cis cleavage, leaving the proteinase inactive - a single-turnover, self-limiting activity.

Model building of the core protein dimer indicates the nucleocapsid has T=4 quasisymmetry.

Sindbis 6K expressed in Xenopus oocytes localized to both the plasma membrane and the endoplasmic reticulum membrane.

No intrinsic channel properties of 6K were detected in oocytes; the currents observed were endogenous oocyte conductances upregulated downstream of reticular calcium-store depletion.

Host SMURF1 and the Sindbis capsid protein co-immunoprecipitate from infected mouse embryonic fibroblasts and HeLa cells.

SMURF1 is required for targeting the Sindbis capsid protein to autophagosomes and for its proteasome-independent degradation; capsid ubiquitination was not detected and SMURF1's role in selective autophagy does not require its ligase activity.

A tetracysteine tag at capsid position Q94 permits biarsenical dye labelling without detectable effects on virus growth, particle morphology or protein distribution, validating the imaging system.

Tagged capsid protein is diffusely cytoplasmic with three distinct classes of foci: motile E2-positive internal puncta, non-motile G3BP/nsP3-positive structures, and plasma-membrane puncta.

Capsid puncta at the plasma membrane colocalize with E2, recruit newly synthesized capsid, require the E2-capsid interaction (abolished by E2 Y400K) and are not replication complexes, consistent with sites of assembly and egress.

The 6K protein is heavily acylated with long-chain fatty acids in hydroxylamine-sensitive ester bonds and is detectable in purified virion preparations.

6K is present in virions at low stoichiometry, 0.08-0.12 relative to E1 plus E2, although in infected cells 4K plus 6K are roughly equimolar with the glycoproteins.

E1 carries internal signal sequences, can be inserted into the ER membrane independently of the pE2 signal sequence, and is efficiently transported to the plasma membrane in the absence of pE2/E2.

BioID2 proximity labelling from the Sindbis capsid protein identified an interaction with host IRAK1.

The capsid-IRAK1 interaction is conserved across arthritogenic (SINV, Ross River, chikungunya) and encephalitic (EEEV, VEEV) alphaviruses.

The alphaviral capsid protein specifically inhibits IRAK1-dependent signalling, providing a route to evade innate immune detection before viral gene expression.

The E2-E1 glycoproteins of Semliki Forest virus, eastern equine encephalitis virus and Sindbis virus interact with the ligand-binding domains of VLDLR and ApoER2.

Ectopic expression of VLDLR or ApoER2 confers cellular attachment and internalization of alphavirus virus-like particles.

6K has previously been shown to form cation-selective ion channels when inserted into planar lipid bilayers, via a transmembrane helix shared with TF.

The Vpu channel blocker 5-N,N-hexamethylene amiloride significantly reduces SINV release, indicating that 6K's ion channel activity promotes virus budding.

Live imaging of miniSOG-tagged 6K with mCherry-E2 places 6K in the ER and on Golgi and post-Golgi vesicles together with E2 in the secretory pathway.

In the absence of 6K, type II cytopathic vacuoles - which originate from the Golgi apparatus - are not detected, with a concomitant reduction in glycoprotein transport to the plasma membrane.

The TF protein is produced by a (-1) programmed ribosomal frameshift at a heptanucleotide slip site in the 6K coding region - a property of the mRNA and the ribosome rather than an activity of the polyprotein.

Cryo-electron microscopy of vitrified Sindbis particles at 35 A resolution shows columnar spike trimers on a T=4 lattice standing on a polyhedral lipid bilayer that surrounds the nucleocapsid, with 240 copies of each spike protein per virion.

The envelope lattice is complementary to the nucleocapsid, with spike trimers near the fivefold axes interacting tightly with capsid triplets and those on threefold axes more loosely.

Insertion mutants NB1 and NB12 map near the PE2 cleavage site and NB2 between E2 residues 69 and 74; PE2 cleavage was completely or partially blocked in the former two.

NB1 and NB2 are defective for binding to chicken embryo fibroblast and BHK-21 cells despite normal virion assembly and release, and mild trypsin digestion of NB1 virions cleaves PE2 and partially restores binding.

PE2 cleavage and domains in the N-terminal portion of E2 are important determinants of alphavirus binding and penetration.

Lactoperoxidase-mediated surface iodination of wild-type-infected cells labels E1 and E2 in the same relative amounts as in iodinated virions, whereas PE2, though present in membranes, is never iodinatable at the surface.

Cells infected with the maturation-defective mutant ts-20 display significant viral protein, mainly E1, at the plasma membrane by both ferritin-antibody labelling and surface iodination, while ts-23-infected cells display none.