Alphaviruses are enveloped viruses with highly organized structures. The nucleocapsid (NC) core contains a
capsid protein lattice enclosing the plus-sense
RNA genome, and it is surrounded by a
lipid bilayer containing a lattice of the E1 and E2 envelope
glycoproteins.
Capsid protein is synthesized in the cytoplasm and particle budding occurs at the plasma membrane (PM), but the traffic and assembly of viral components and the exit of virions from host cells are not well understood. To visualize the dynamics of
capsid protein during
infection, we developed a Sindbis virus infectious clone tagged with a tetracysteine motif. Tagged
capsid protein could be fluorescently labeled with biarsenical
dyes in living cells without effects on virus growth, morphology, or
protein distribution. Live cell imaging and colocalization experiments defined distinct groups of capsid foci in infected cells. We observed highly motile internal puncta that colocalized with E2
protein, which may represent the transport machinery that
capsid protein uses to reach the PM. Capsid was also found in larger nonmotile internal structures that colocalized with cellular G3BP and viral nsP3. Thus, capsid may play an unforeseen role in these previously observed G3BP-positive foci, such as regulation of cellular stress granules. Capsid puncta were also observed at the PM. These puncta colocalized with E2 and recruited newly synthesized
capsid protein; thus, they may be sites of virus assembly and egress. Together, our studies provide the first dynamic views of the alphavirus
capsid protein in living cells and a system to define detailed mechanisms during
alphavirus infection.