Functional Interaction of the Adenovirus IVa2 Protein with Adenovirus Type 5 Packaging Sequences

Abstract

Adenovirus type 5 (Ad5) DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent on the cis-acting packaging domain located between nucleotides 230 and 380. Seven AT-rich repeats that direct packaging have been identified within this domain. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans-acting factor(s) that plays a role in packaging. Both cellular and viral proteins that interact with adenovirus packaging elements in vitro have been identified. In this study, we characterized a group of recombinant viruses that carry site-specific point mutations within a minimal packaging domain. The mutants were analyzed for growth properties in vivo and for the ability to bind cellular and viral proteins in vitro. Our results are consistent with a requirement of the viral IVa2 protein for DNA packaging via a direct interaction with packaging sequences. Our results also indicate that higher-order IVa2-containing complexes that form on adjacent packaging repeats in vitro are the complexes required for the packaging activity of these sites in vivo. Chromatin immunoprecipitation was used to study proteins that bind directly to the packaging sequences. These results demonstrate site-specific interaction of the viral IVa2 and L1 52/55K proteins with the Ad5 packaging domain in vivo. These results confirm and extend those previously reported and provide a framework on which to model the adenovirus assembly process.Adenovirus (Ad) assembly has been studied by using pulsechase kinetic analyses, through the characterization of temperature-sensitive virus mutants blocked at different stages of assembly at the restrictive temperature, by studies of virus mutants defective in the expression of proteins involved in the packaging process, and by studies of mutants defective in the packaging sequences (2-4, 6-8, 12, 25, 31, 32). The results suggest that adenovirus assembly follows an ordered series of assembly and processing events analogous to those found in the assembly of bacteriophages. Whether the viral genome is involved in nucleating the initiating steps of capsid morphogenesis or whether viral DNA is inserted into an empty, preformed capsid has not been resolved. It is clear, however, that the viral genome contains cis-acting sequences that mediate the packaging process (reviewed in reference 20). Adenovirus DNA packaging occurs in a polar manner from the left end of the genome (1, 28). The genomes of adenovirus type 3 (Ad3), Ad5, and Ad16, representatives of adenovirus subgroups B and C, harbor conserved packaging signals (6,10,19,22). These conserved cis-acting packaging domains suggest similar mechanisms of selective and polar DNA packaging for all adenovirus subgroups.Ad5 DNA encapsidation is dependent on cis-acting sequences located between nucleotides (nt) 230 and 380 (Fig. 1A) (6,7,13,25). Seven repeated sequences (termed A-repeats due to their AT-ric...

Dependence of the Encapsidation Function of the Adenovirus L1 52/55-Kilodalton Protein on Its Ability To Bind the Packaging Sequence

“…Furthermore, both proteins bind to the Ad5 packaging sequence located at the left end of the genome (19,20,30). We examined the domains of the L1 52/55-kDa protein required for the interaction with the IVa2 protein and the DNA and the importance of those interactions for the encapsidation function of the L1 52/55-kDa protein.…”

“…More recently, the IVa2 protein was found to bind directly to the A repeats in the packaging domain in vitro and in vivo (19,20,30). The IVa2 protein is present in assembly intermediates and mature virions (7,11,28).…”

“…A more pronounced phenotype occurs in infections with the pm8001 mutant virus, which is unable to produce the L1 52/55-kDa protein. This virus has normal early and late viral gene expression and DNA replication but produces empty capsids with no associated viral DNA (7).Recently, we and others demonstrated that the L1 52/55-kDa protein binds to the packaging sequence in vivo by using chromatin immunoprecipitation (ChIP) assays (19,20). This interaction might be mediated by another protein, since purified L1 52/55-kDa protein did not bind to the packaging sequence in vitro even in the presence of the IVa2 protein, which is known to bind to the packaging sequence (20).…”

“…The packaging sequence contains at least seven functional units, called A repeats (3,4,13,22). At least two viral proteins, L1 52/55-kDa and IVa2, are involved in packaging sequence interactions (19,20,30,32).The IVa2 protein is a viral product that was first reported to be a transcriptional activator of the adenovirus major late promoter (16,17,26). More recently, the IVa2 protein was found to bind directly to the A repeats in the packaging domain in vitro and in vivo (19,20,30).…”

“…The packaging sequence contains at least seven functional units, called A repeats (3,4,13,22). At least two viral proteins, L1 52/55-kDa and IVa2, are involved in packaging sequence interactions (19,20,30,32).…”