PMID:27852864

Citation	Ramsey, J, Renzi, EC, Arnold, RJ, Trinidad, JC and Mukhopadhyay, S (2017) Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions. J. Virol. 91
Abstract	Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a -1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding.
Links	PubMed PMC5244351 Online version:10.1128/JVI.02000-16
Keywords	Amino Acid Sequence; Animals; Cell Line; Cell Membrane/metabolism; Cricetinae; Gene Expression; Membrane Proteins/chemistry; Membrane Proteins/genetics; Membrane Proteins/metabolism; Mutation; Protein Interaction Domains and Motifs; Protein Processing, Post-Translational; Protein Transport; Sindbis Virus/physiology; Sindbis Virus/ultrastructure; Viral Proteins/chemistry; Viral Proteins/genetics; Viral Proteins/metabolism; Virion/physiology; Virion/ultrastructure; Virus Release; Virus Replication
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
SINDV:POLS		GO:0020002: host cell plasma membrane	ECO:0000315:		C		Structural protein Sindbis virus, Figure 5 centrifugal separation of cellular membrane components from virus-infected cells, analyzed by western blot against virus proteins (specifically the TF protein)	complete
SINDV:POLSF		GO:0019031: viral envelope	ECO:0000315:		C		Figure 6C demonstrates that the E1, E2, and TF proteins (all products of the frameshifted polyprotein) are found in the virion envelope of Sindbis virus.	complete

References

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