Accurate single-day titration of adenovirus vectors based on equivalence of protein VII nuclear dots and infectious particles

Abstract

Protein VII is an abundant component of adenovirus particles and is tightly associated with the viral DNA. It enters the nucleus along with the infecting viral genome and remains bound throughout early phase. Protein VII can be visualized by immunofluorescent staining as discrete dots in the infected cell nucleus. Comparison between protein VII staining and expression of the 72kDa DNA-binding protein revealed a one-to-one correspondence between protein VII dots and infectious viral genomes. A similar relationship was observed for a helper-dependent adenovirus vector expressing green fluorescent protein. This relationship allowed accurate titration of adenovirus preparations, including wild-type and helper-dependent vectors, using a 1-day immunofluorescence method. The method can be applied to any adenovirus vector and gives results equivalent to the standard plaque assay.

Figure 1

Protein VII and DBP staining of low MOI infection. Cells were infected with wild-type dl309 for 4 hours and stained for protein VII, or were infected for 24 hours in the presence of hydroxyurea and stained for DBP expression. Prior to mounting, the cells were counterstained with DAPI (light gray).

Figure 2

Protein VII and DBP staining of a high MOI infection. Cells were infected with wild-type dl309 for 4 hours and stained for protein VII, or were infected for 24 hours in the presence of hydroxyurea and stained for DBP expression. Cells were counterstained with DAPI (light gray). Arrows indicate the position of uninfected cells.

Figure 3

Titers of ARM using protein VII staining and plaque assay. Data are from Table 3. Use of 293 cells or HeLa cells is indicated.

Figure 4

Structure of gAd.HSU/GFP. CMV, human cytomegalovirus promoter; GFP, green fluorescent protein; CRE, cyclic AMP response element; NFκB, binding site for NFκB; AdITR, adenovirus inverted terminal repeat; stuffer DNA, human intronic DNA sequences.

Figure 5

Protein VII staining and GFP expression of cells infected with gAd.HSU/GFP. Cells were infected for 4 hours and stained for protein VII (Protein VII and Protien VII + DAPI), or were infected for 24 hours in the presence of hydroxyurea and analyzed for GFP expression (GFP and GFP + phase). Cells were counterstained with DAPI (light gray). Phase, phase contrast microscopy.

Figure 6

Quantification of protein VII and gAd.HSU/GFP infectivity. Data are from Table 4.