ReportTwo-hybrid analysis of Ty 3 capsid subdomain interactions

Background: The yeast retrotransposon Ty3 forms stable virus-like particles. Gag3, the major structural protein, is composed of capsid, spacer and nucleocapsid domains. The capsid domain of Gag3 was previously modeled as a structure similar to retrovirus capsid. Findings: Two-hybrid analysis was used to understand the interactions that contribute to particle assembly. Gag3 interacted with itself as predicted based on its role as the major structural protein. The N-terminal subdomain (NTD) of the capsid was able to interact with itself and with the C-terminal subdomain (CTD) of the capsid, but interacted less well with intact Gag3. Mutations previously shown to block particle assembly disrupted Gag3 interactions more than subdomain interactions. Conclusions: The findings that the NTD interacts with itself and with the CTD are consistent with previous modeling and a role similar to that of the capsid in retrovirus particle structure. These results are consistent with a model in which the Gag3-Gag3 interactions that initiate assembly differ from the subdomain interactions that potentially underlie particle stability. Findings The Ty3 retrotransposon in budding yeast forms viruslike particles (VLPs) comprised of precursor Gag3 and Gag3-Pol3 polyproteins [1,2]. Previous alanine-scanning mutagenesis indicated that the N-terminal domain (NTD) of the structural polyprotein Gag3 plays an important role in VLP formation [3]. During maturation, Gag3 is processed into 24 kDa capsid (CA), 27 kDa CA-spacer (SP), 3 kDa SP, and 7 kDa nucleocapsid (NC) protein by the Ty3 protease. Unlike most retrovirus cores, these cytoplasmic particles remain stable after proteolytic maturation. Two-hybrid analysis [4] was used to better understand the contributions of Gag3 subdomains to formation and stability of the Ty3 VLP. Fusions of Gag3 and derivatives to the C-terminus of the Gal4-BD tagged with c-Myc were expressed from the high-copy, TRP1-marked pGBK vector (Clontech, Palo Alto, CA, USA). Fusions of Gag3 and derivatives to the C-terminus of the Gal4-AD tagged with HA were expressed from the LEU2-marked highcopy plasmid pGAD T7 (pGAD). These fusions were constructed by amplifying the appropriate regions from Ty3 Gag3 subclones in pGEM (Invitrogen, Carlsbad, CA, USA) using polymerase chain reaction (PCR) primers containing NdeI and BamHI sites at the 5' and 3' outside ends, respectively and ligating fragments to the pGBK and pGAD vectors linearized with NdeI and BamHI. Constructs (Table 1) were confirmed by DNA sequence analysis (GeneWiz, South Plainfield, NJ, USA). Fusion proteins in these vectors are expressed under the constitutive ADH1 promoter. Two-hybrid plasmids and negative control vector plasmids were transformed into yeast strain yAH109 (MATa trp1-901 leu2-3, 112 ura3-52 his3200 gal4Δ gal80Δ LYS2::GAL1UAS/TATAHIS3 GAL2UAS/ TATA-ADE2, URA3::MEL1UAS/TATA-lacZ), which has ADE2, HIS3, LacZ, and MEL1 reporters regulated by the GAL4 responsive upstream activating sequences (UASs) (Clontech). Preliminary tests showed that expression of ADE2, which results in cream-colored colonies and growth in medium lacking adenine, provided the most reliable detection of two-hybrid interaction (data not shown). The yAH109 transformants containing pairwise combinations of pGAD and pGBK plasmids were selected on medium lacking tryptophan and leucine. Four * Correspondence: sbsandme@uci.edu 1 Department of Biological Chemistry, University of California, Irvine, CA, USA † Contributed equally Full list of author information is available at the end of the article BioMed Central © 2010 Zhang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Zhang et al. Mobile DNA 2010, 1:14 http://www.mobilednajournal. com/content/1/1/14 Page 2 of 7 isolates from each transformation were restreaked and then replica plated onto complete synthetic medium lacking adenine, tryptophan and leucine and grown at 30°C for evaluation of color development and growth. We first tested Gag3, CA, p27, and NC fusions in both vectors in all combinations. A mutant deleted for SP residues 208 to 232 (ΔSP) was also assayed (K Christiansen, MZ, VB and SBS, unpublished results). AD-CA transformants were not recovered as stable colony isolates in repeated attempts. Other constructs were shown to be positive for expression of the appropriate fusion by immunoblot analysis using rabbit polyclonal antibodies against Gal4 AD and Gal4 BD (Upstate Biotechnology, Lake Placid, NY, USA) or CA [5] (data not shown) [6]. With the exception of p27, which produced a slight amount of background growth in cells with both vectors, these domains were negative for reporter activation when expressed from either vector in the presence of the other vector (Figure 1, Additional files 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). Homotypical interactions (except for CA) and heterotypical interactions were assayed (Figure 1). Gag3, ΔSP, and p27 interacted in both vector contexts and in all comTable 1: Two-hybrid plasmids Vector/plasmid NdeI/BamHI insert Gag3 residues Mutation(s) pGBK T7 (with Gal4-BD): pMZ2667 Gag3 1-290 WT pMZ2668 Gag3 ΔSP Δ207-233 WT pMZ2669 p27 1-233 WT