The coiled-coil domain of the adenovirus type 5 protein IX is dispensable for capsid incorporation and thermostability

Abstract

The 14.4-kDa hexon-associated protein IX (pIX) acts as a cement in the capsids of primate adenoviruses and confers a thermostable phenotype. Here we show that deletion of amino acids 100 to 114 of adenovirus type 5 pIX, which eliminates the conserved coiled-coil domain, impairs its capacity to self-associate. However, pIXDelta100-114 is efficiently incorporated into the viral capsid, and the resulting virions are thermostable. Deletion of the central alanine-rich domain, as in pIXDelta60-72, does not impair self-association, incorporation into the capsid, or the thermostable phenotype. These data demonstrate, first, that the self-association of pIX is dispensable for its incorporation into the capsid and generation of the thermostability phenotype and, second, that the increased thermostability results from pIX monomers binding to different hexon capsomers rather than capsid stabilization by pIX multimers.

FIG. 1.

Conserved amino acid regions in pIX. (A) Amino acid sequence alignment of all available primate Ad pIX proteins (CLUSTAL W) (19). Fully conserved residues are shaded black. Residues that occur in more than 50% of the sequences are shaded gray. Lines under the aligned sequences indicate the deletions mentioned in the text. (B) DNA sequence of the human Ad type 5 pIX gene. Deletions of the three conserved domains were introduced by site-directed mutation PCR. The lines depict the DNA sequence deletions. (C) Schematic representation of the pIX variants used for coprecipitation assays in this study. Molecular weights are in thousands.

FIG. 2.

Assay to measure pIX incorporation into the capsid. (A) Cultured 911 cells were transfected with the various pIX expression plasmids. Twenty hours posttransfection, the cells were infected with hAd5dl313 viruses. Two days later, protein extracts were prepared and analyzed by Western blotting. The blots were probed with anti-pIX or antiactin sera. The negative control (neg.control) is a lysate from untransfected 911 cells. (B) From parallel cultures of transfected and pIX-expressing cells, the progeny hAd5dl313 viruses were harvested, purified, and assayed for the presence of the pIX variants in the capsids by Western analysis. The negative control is hAd5dl313 virus lysate propagated on normal 911 cells; the positive control is wt hAd5. (C) pIX self-association assay. To test the self-association of pIX molecules, an expression plasmid containing the pIX.MYC gene and a plasmid containing a gene encoding one of the Flag-tagged pIX deletion mutants were cotransfected into 911 cells. The cell lysates of these cells were used for coprecipitation with Flag-beads. As a negative control we used 911 cells that were transfected with the pIX.MYC expression plasmid only. A protein lysate from 9ll cells transfected with the pIX.Flag plasmid was used as a positive control. (D) Western blot analysis of the pIX proteins associating with pIX-Flag. After coprecipitation the samples were analyzed by Western blotting with anti-pIX serum for detection of the pIX variants. (E) Western blot analysis of 911 cells transfected with plasmids coding for pIX.Flag, wt pIX, pIXΔ100-114, and pIXΔ100-114-Flag. The blots were probed with anti-pIX or antiactin sera. (F) Western blot analysis of the pIX proteins associating with the Flag-tagged pIX and pIXΔ100-114. The blots were probed with anti-pIX serum.

FIG. 3.

Virus stability assay. For the thermostability assay deletions were made in the conserved alanine stretch (Δ60-72) and in the conserved coiled-coil domain (Δ100-114) of the viral backbone. Viruses were propagated on 911 cells and purified by CsCl gradient centrifugation. (A) The presence of the deletions was tested by PCR with primers that flanked the pIX gene in the virus backbone. PCR on hAd5.LUC, which contains wt pIX, should reveal a band of 569 bp, PCR on hAd5.ΔpIX.LUC viruses should reveal a band of 171 bp, PCR on hAd5.pIXΔ60-72.LUC should reveal a band of 528 bp, and PCR on hAd5.pIXΔ100-114.LUC should reveal a band of 525 bp. As internal control, a PCR on the fiber gene was performed with the same samples. Water was used in both PCRs as a negative control. (B) The incorporation levels of the mutant pIX were assayed by Western blotting. As positive and negative controls, wt Ad5 and Ad5 ΔpIX, respectively, were included. (C) Thermostability of wt and mutant viruses. Aliquots of the vectors were incubated in a water bath at 45°C for 4, 6, 8, or 10 min. Residual infectious virus titers were estimated by determining the capacity of the virus to induce luciferase activity in U2OS cell 24 h after infection. The results are presented as percentages of residual luciferase activity. ○, ▵, +, and *, wt hAd5.LUC, hAd5.ΔpIX.LUC, hAd5.pIXΔ60-72.LUC, and hAd5.pIXΔ100-114.LUC, respectively. Each bar represents the cumulative mean ± standard deviation of triplicate analyses.

FIG. 4.

Models for pIX-hexon interaction. (A) Three hexon capsomers are kept together via a pIX trimer. In this model a pIX molecule binds to a single hexon capsomer and two other pIX molecules. (B) Three hexon capsomers are kept together via hexon capsomer-pIX-hexon capsomer interactions; there is no multimerization of pIX molecules required. (C) Three hexon capsomers bind more strongly to each other as result of a conformational change induced by pIX.