ABSTRACT
The adenovirus IVa2 protein has been implicated as a transcriptional activator of the viral major late promoter (MLP) and a key component in the packaging of the viral genome. IVa2 functions in packaging through its ability to form a complex with the viral L1 52/55kDa protein, which is required for encapsidation. IVa2, alone and in conjunction with another viral protein, the L4 22K protein, binds to the packaging sequence on the viral genome and to specific elements in the promoter. To define the DNA binding domain on IVa2 and determine its contribution to the viral life cycle, we created a mutant protein that lacks a putative helix-turn-helix motif at the extreme C-terminus. Characterization of this mutant protein showed that while MLP activity is relatively unaffected, it is unable to bind to and package DNA.
Subject(s)
Adenoviruses, Human/physiology , DNA, Viral/genetics , DNA-Binding Proteins/chemistry , Genome, Viral , Viral Nonstructural Proteins/chemistry , Viral Proteins/chemistry , Amino Acid Sequence , Cell Line , DNA, Viral/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Electrophoretic Mobility Shift Assay , Helix-Turn-Helix Motifs , Humans , Molecular Sequence Data , Promoter Regions, Genetic , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Virion/physiology , Virus AssemblyABSTRACT
Assembly of infectious adenovirus particles requires seven functionally redundant elements at the left end of the genome, termed A repeats, that direct packaging of the DNA. Previous studies revealed that the viral IVa2 protein alone interacts with specific sequences in the A repeats but that additional IVa2-containing complexes observed during infection require the viral L4 22-kDa protein. In this report, we purified a recombinant form of the 22-kDa protein to characterize its DNA binding properties. In electrophoretic mobility shift assay analyses, the 22-kDa protein alone did not interact with the A repeats but it did form complexes on them in the presence of the IVa2 protein. These complexes were identical to those seen in extracts from infected cells and had the same DNA sequence dependence. Furthermore, we provide data that the 22-kDa protein enhances binding of the IVa2 protein to the A repeats and that multiple binding sites in the packaging sequence augment this activity. These data support a cooperative role of the IVa2 and 22-kDa proteins in packaging and assembly.
Subject(s)
Adenoviridae/physiology , DNA, Viral/metabolism , Viral Proteins/metabolism , Virus Assembly , Animals , Base Sequence , DNA, Viral/chemistry , Electrophoretic Mobility Shift Assay , Mice , Mice, Inbred BALB C , Viral Proteins/chemistry , Viral Proteins/isolation & purificationABSTRACT
During adenovirus virion assembly, the packaging sequence mediates the encapsidation of the viral genome. This sequence is composed of seven functional units, termed A repeats. Recent evidence suggests that the adenovirus IVa2 protein binds the packaging sequence and is involved in packaging of the genome. Study of the IVa2-packaging sequence interaction has been hindered by difficulty in purifying the protein produced in virus-infected cells or by recombinant techniques. We report the first purification of a recombinant untagged version of the adenovirus IVa2 protein and characterize its binding to the packaging sequence in vitro. Our data indicate that there is more than one IVa2 binding site within the packaging sequence and that IVa2 binding to DNA requires the A-repeat consensus, 5'-TTTG-(N(8))-CG-3'. Furthermore, we present evidence that IVa2 forms a multimeric complex on the packaging sequence. These data support a model in which adenovirus DNA packaging occurs via the formation of a IVa2 multiprotein complex on the packaging sequence.
