Determinants in alphavirus assembly

Rushika Michelle Perera, Purdue University

Abstract

Alphaviruses are members of the Togaviridae family and constitute a group of widely distributed positive strand RNA viruses. The virus particles have an internal nucleocapsid core consisting of 240 copies of a single capsid protein, a host derived lipid bilayer and a glycoprotein shell consisting of two transmembrane glycoproteins, E1 and E2. Analysis of nucleocapsid core assembly using a well established in vitro core assembly system has suggested a dimeric assembly model for the core that places 120 intercapsomeric dimers in an arrangement similar to the T = 4 native cores. This dimerization is nucleic acid dependant and involves extensive protein-protein and protein-RNA contacts that occur between residues 81–264 of the capsid protein. Through the analysis of mutant capsid protein, it has also been shown that assembly defective capsid proteins that retain the ability to bind nucleic acid, are capable of dimerization, yet cannot proceed along the assembly pathway unless the dimer intermediates are stabilized by chemical cross linking reagents. It has recently been shown that this cross-link mimics the function of a coiled-coil motif that has been identified in the N-terminal one third of the capsid protein. This motif termed helix I, is a conserved stretch of 18 amino-acids, and is embedded within a low complexity sequence enriched with basic and proline residues. In Sindbis virus, helix I spans residues 38 to 55 and has three conserved leucine residues that form two and a half heptad repeat motifs. Complete or partial deletion of helix 1, or single site substitutions of the conserved leucine residues caused a significant decrease in virus replication. These viral mutants were sensitive to elevated temperatures compared to the wild-type virus and failed to accumulate cores in the cytoplasm although protein translation and processing remained unaffected. The mutant capsid proteins showed a trans-negative dominant phenotype in in vitro assembly reactions involving both mutant and wild-type capsid proteins. Synthetic peptides corresponding to helix I, also inhibited wild-type core assembly in vitro. Replacing helix I with an unrelated sequence corresponding to the dimeric, coiled coil sequence from the GCN4 transcription factor, produced a virus that was wild-type in phenotype. However, a similar substitution of helix I with the trimeric coiled coil sequence of GCN4 produced a virus that was ∼100-fold reduced in virus replication compared to the wild-type virus. It is proposed that helix I functions as a dimeric coiled coil sequence, and is involved in coiled coil interactions that stabilize subviral intermediates formed through the interaction of the C-terminal domain of the capsid protein and the genomic RNA during the assembly of the alphavirus nucleocapsid core. Studies pursuing direct evidence for the interaction of this core with the glycoprotein E2 are also described.

Degree

Ph.D.

Advisors

Kuhn, Purdue University.

Subject Area

Molecular biology|Biophysics|Microbiology

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