In vitro study of alphavirus assembly

Eunmee M Hong, Purdue University

Abstract

Alphavirus virions possess a T=4 icosahedral nucleocapsid core (NC), enveloped in a host-derived membrane whose glycoprotein components are also present in a matching icosahedral lattice. The NC consists of the positive-strand RNA genome of ∼12 kb surrounded by 240 copies of a single species of a 30-kDa capsid protein (CP). The assembly of NC has been investigated using an in vitro assembly system. Core-like particles (CLPs) can be assembled in vitro by using single-stranded nucleic acid and recombinant CP that is expressed and purified from E. coli . The three dimensional structures of in vitro-assembled CLPs of Ross River virus (RRV) and western equine encephalitis virus (WEEV) were solved at 30 Å resolution. They contain the same T=4 icosahedral symmetry, similar to the NC found in mature virions. This indicates that alphaviruses preassemble their icosahedral NC in the cytoplasm and that this in vitro assembly system is a reliable representation for alphavirus NC assembly. Additional studies on assembly determinants were done using this in vitro assembly system. The C-terminal two thirds of the CP, residues 81 to 264 in Sindbis virus (SINV) has been previously shown to have all the RNA-CP and CP-CP contacts required for core assembly. The Helix I region, which is located in the N-terminal dispensible region of the CP, has been proposed to stabilize the core by forming a coiled-coil in the CP dimer through the interaction of residues 81 to 264. We examined the ability of heterologous alphavirus CPs to dimerize and form phenotypically mixed CLPs using the in vitro assembly system. The CPs of SINV and RRV do not form phenotypically mixed CLPs but SINV and WEEV CPs do form mixed CLPs. In contrast, an N-terminal truncated SINV CP (residues 81-264) forms phenotypically mixed CLPs when it is mixed with full-length heterologous CPs, suggesting that the region which controls the mixing is present in the N-terminal 80 residues of the SINV CP. We mapped the determinant that is responsible for phenotypic mixing onto Helix I by using domain swapping. Thus, discrimination of the CP partner in core assembly appears dependent on Helix I sequence compatibility. This study suggests that Helix I provides one of the important interactions during NC formation, and may play a regulatory role during the early steps in the assembly process.

Degree

Ph.D.

Advisors

Kuhn, Purdue University.

Subject Area

Molecular biology

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