Transcription factors NFI and NFIII/oct-1 function independently, employing different mechanisms to enhance adenovirus DNA replication

Abstract

Initiation of adenovirus DNA replication is strongly enhanced by two transcription factors, nuclear factor I (NFI) and nuclear factor III (NFIII/oct-1). These proteins bind to two closely spaced recognition sequences in the origin. We produced NFI and NFIII/oct-1, as well as their biologically active, replication-competent DNA-binding domains (NFI-BD and the POU domain), in a vaccinia virus expression system and purified these polypeptides to apparent homogeneity. By DNase I footprinting and gel retardation, we show that the two proteins, as well as their purified DNA-binding domains, bind independently and without cooperative effects to their recognition sequences. By using a reconstituted system consisting of the purified viral proteins (precursor terminal protein-DNA polymerase complex (pTP-pol) and DNA-binding protein, we show that NFIII/oct-1 or the POU domain stimulates DNA replication in the absence of NFI or NFI-BD and vice versa. When added together, the enhancing effect of the two transcription factors was independent and nonsynergistic. Interestingly, stimulation by NFI or NFI-BD was strongly dependent on the concentration of the pTP-pol complex. At low pTP-pol concentrations, NFI or NFI-BD stimulated up to 50-fold, while at high concentrations, the stimulation was less than twofold, indicating that the need for NFI can be overcome by high pTP-pol concentrations. In contrast, stimulation by NFIII/oct-1 or the POU domain was much less dependent on the pTP-pol concentration. These data support a model in which NFI enhances initiation through an interaction with pTP-pol. Glutaraldehyde cross-linking experiments indicate contacts between pTP-pol and NFI but not NFIII/oct-1. The site of interaction is located in the NFI-BD domain.

A Zoonotic Adenoviral Human Pathogen Emerged through Genomic Recombination among Human and Nonhuman Simian Hosts

“…For HAdVs, these include the core origin and the NF-I and NF-III motifs. The latter two elements reflect multiple observations that HAdVs utilize two host transcription factors, nuclear factors I and III, as part of the viral DNA replication complex for optimal replication (35)(36)(37)(38). Except for variations in the genotypes of species D, all HAdVs have identical or highly similar NF-I and NF-III motifs.…”

Genomic analysis of HAdV-B14 isolate from the outbreak of febrile respiratory infection in China

“…Another 10 nt conserved motif (ATAATATACC) within the 5′ ITR of HAdV is directly involved in the interaction of the terminal protein precursor (pTP) and DNA polymerase during viral DNA replication [11], and this was seen in HAdV-B14 CHN. Several cellular transcription factors, including nuclear factor 1 (NF1), nuclear factor III (NFIII), specificity protein 1 (Sp1), and activating transcription factors (ATFs) are known to enhance virus replication, and are important for efficient growth [12,13]. The binding motifs for these factors are localized within ITRs of AdVs; to identify the binding motifs for these host transcription factors, we generated multiple ITR sequence alignments using various HAdV-B species (Table 1).…”

Domain organization of the adenovirus preterminal protein

“…This is followed by elongation of the nascent DNA strand by adenovirus DNA polymerase (Adpol), with fork movement being accompanied by DNA strand displacement (7,9,14,35). In addition to pTP and Adpol, virus-coded single-stranded DNA binding protein (DBP) and two cellular factors, nuclear factor I (NFI or CTF1) and nuclear factor III (NFIII or Oct1) are required for virus DNA replication (24,29,43). Our understanding of the mechanisms involved in DNA replication has been considerably enhanced through the overexpression of these proteins and the development of in vitro assay systems using purified components (reviewed in reference 7).…”

“…pTP and Adpol form a stable heterodimer which binds to bp 9 to 18 of the genome (25,32,39,41). The heterodimer is guided into position at the core origin of replication through specific associations with NFI (1)(2)(3)24) and NFIII (6), which bind to auxiliary sequences in the inverted terminal repeats at bp 19 to 39 and 40 to 51, respectively. Binding of NFI to its recognition sequence is enhanced by DBP through an alteration in the structure of DNA (4,40).…”