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Protein interactions involved in alphavirus assembly

thesis
posted on 2024-09-02, 20:19 authored by Ulrica Skoging Nyberg

Viruses have to protect their genomes during transport from the infected to an uninfected host cell. This is achieved by forming a protein shell around the nucleic acid. Virus assembly is a complex process that involves interactions with the viral genome, the viral structural proteins and for enveloped viruses a lipid membrane. The assembly mechanisms differ between virus families and are in most cases poorly understood. This thesis describes protein interactions involved in alphavirus assembly.

Alphaviruses are among the best-characterised enveloped viruses. They have positive single-stranded RNA genomes encapsidated by 240 copies of the capsid (C) protein, forming a nucleocapsid (NC). This is surrounded by a lipid bilayer derived from the host cell plasma membrane during budding. 80 spike complexes composed of three heterodimers of the envelope proteins, El and E2, traverse the membrane and interact with the nucleocapsid.

The capsid protein has an autoprotease activity and cleaves itself from the growing polypeptide chain. Its enzymatic activity is special in that it only works in one reaction. The last Trp residue remains bound to the substrate pocket after cleavage and functions as a built-in inhibitor. Analysis of a set of mutants showed that the Trp residue was not only important as an inhibitor but also for correct folding and stability of the capsid protein.

The assembly of alphaviral particles depends on the interaction between transmembrane spike proteins and the nucleocapsid. A structural model of the interaction was made and tested by a set of mutants. The interaction was found to involve a conserved Tyr-X-Leu motif in the E2 cytoplasmic tail. The motif bound into a hydrophobic cavity on the capsid protein. The E2 Tyr residue interacted with a Tyr and a Trp residue in the first half of the pocket while the Leu interaction in the other half was less specific according to the model.

Modelling of the cavity from different alphaviruses suggest that the cavity has evolved to fit an internal capsid motif rather than the spike. The SFV motif Met-X-Ile was indeed found to be important for nucleocapsid assembly. Mutants of this motif did not form intracellular nucleocapsids. However, virus was released in wild type amounts indicating that NCs could form at the site of budding. A possible switch mechanism for nucleocapsid assembly involving the spike-binding cavity are discussed.

The results in this thesis have furthered our knowledge about the mechanisms involved in alphavirus assembly. It is clear that the interactions between the spike and capsid proteins are specific and possess a central role in the assembly process.

List of scientific papers

I. Skoging U, Liljestrom P (1998). "Role of the C-terminal tryptophan residue for the structure-function of the alphavirus capsid protein" J Mol Biol 279(4): 865-72
https://pubmed.ncbi.nlm.nih.gov/9642067

II. Skoging U, Vihinen M, Nilsson L, Liljestrom P (1996). "Aromatic interactions define the binding of the alphavirus spike to its nucleocapsid" Structure 4(5): 519-29
https://pubmed.ncbi.nlm.nih.gov/8736551

III. Skoging-Nyberg U, Liljestrom P (2000). "A conserved leucine in the cytoplasmic domain of the Semliki Forest virus spike protein is important for budding" Arch Virol 145(6): 1225-30
https://pubmed.ncbi.nlm.nih.gov/10948994

IV. Skoging-Nyberg U, Liljestrom P (2000). "An M-X-I motif of Semliki Forest virus capsid protein affects nucleocapsid assembly" (Submitted)

History

Defence date

2000-12-08

Department

  • Department of Microbiology, Tumor and Cell Biology

Publication year

2000

Thesis type

  • Doctoral thesis

ISBN-10

91-628-4329-X

Number of supporting papers

4

Language

  • eng

Original publication date

2000-11-17

Author name in thesis

Skoging Nyberg, Ulrica

Original department name

Microbiology and Tumor Biology Center (MTC)

Place of publication

Stockholm

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