Adenovirus Protein VII Functions throughout Early Phase and Interacts with Cellular Proteins SET and pp32

Abstract

Adenovirus protein VII is the major component of the viral nucleoprotein core. It is a highly basic nonspecific DNA-binding protein that condenses viral DNA inside the capsid. We have investigated the fate and function of protein VII during infection. "Input" protein VII persisted in the nucleus throughout early phase and the beginning of DNA replication. Chromatin immunoprecipitation revealed that input protein VII remained associated with viral DNA during this period. Two cellular proteins, SET and pp32, also associated with viral DNA during early phase. They are components of two multiprotein complexes, the SET and INHAT complexes, implicated in chromatin-related activities. Protein VII associated with SET and pp32 in vitro and distinct domains of protein VII were responsible for binding to the two proteins. Interestingly, protein VII was found in novel nuclear dot structures as visualized by immunofluorescence. The dots likely represent individual infectious genomes in association with protein VII. They appeared within 30 min after infection and localized in the nucleus with a peak of intensity between 4 and 10 h postinfection. After this, their intensity decreased and they disappeared between 16 and 24 h postinfection. Interestingly, disappearance of the dots required ongoing RNA synthesis but not DNA synthesis. Taken together these data indicate that protein VII has an ongoing role during early phase and the beginning of DNA replication.The adenovirus nucleoprotein core consists of doublestranded genomic DNA, the highly basic viral proteins VII, V, and (mu), as well as protein IVa2 and the 55-kDa terminal protein (1,5,19,25,31,33,34,42). Protein VII is the major protein component of the core with an estimated 1,070 copies present per virion (9) and is primarily responsible for establishing viral chromatin structure. It can potently condense DNA in vitro and in vivo and also repress transcription (3,6,21,35). This is consistent with the highly condensed configuration of viral chromatin found within the virion. When delivered to the nucleus, the chromatin is silent prior to stimulation by the viral transcriptional activator E1A (13).We and others have reported that protein VII from infectious viral particles enters the nucleus along with viral DNA and remains associated with it, suggesting that the protein VII-DNA complex is the substrate for transcriptional activation by E1A during early phase (8,15,18,21). Moreover we found that protein VII can associate with E1A protein in vitro (21).In this report we have studied the fate and function of protein VII during the early phase of infection. We demonstrate that virus-derived "input" protein VII persists in the nucleus throughout early phase and the beginning of viral DNA replication, suggesting that it has an ongoing role in gene regulation and perhaps DNA replication. During this period protein VII is found in discrete nuclear dot structures and viral DNA continues to associate with protein VII.We have also investigated the association of protein VII with cel...

Regulation of Adeno-Associated Virus DNA Replication by the Cellular TAF-I/Set Complex

“…In this context, the likely activity carried out by TAF-I is to remodel this adenovirus core, thereby making the template DNA accessible to the transcription and replication apparatus. Consistent with this function, both ANP32A and the TAF-I/Set histone chaperone are present on adenovirus DNA during the first phases of viral infection (46). Taken together, these findings clearly suggest a role for the TAF-I complex in the replication of viral DNA genomes by the host cell machinery.…”

Assembly of Helper-Dependent Adenovirus DNA into Chromatin Promotes Efficient Gene Expression

“…The observation that both VII and histones can be found bound to the same DNA molecule (31) suggests that the processes of core VII eviction and histone placement may be coincident. The cellular chaperones implicated in removing VII from the incoming viral DNA, TAFI-III also known as B23/nucleophosmin (45), pp32 (77), SET (37), and NAP-1 (30), have also been implicated in the removal of protamines from incoming sperm DNA and in the assembly of sperm DNA into chromatin (38,52,60). HIRA, which we showed is necessary for placement of H3.3 on vector DNA (Fig.…”

“…During wild-type Ad infection, the major DNA-binding protein, protein VII, enters the nucleus with the Ad DNA (9). However, conflicting data suggest that VII either stably associates with Ad DNA throughout the early phase of infection (9,77) or is evicted within a few hours (70), and eviction of VII may require active transcription (10). Few studies have addressed whether Ad DNA in the nucleus directly interacts with histones or assembles into chromatin; indeed, conflicting data suggest that Ad DNA is (5,13,14,66) or is not (76) assembled into chromatin.…”

The Adenovirus L4 33-Kilodalton Protein Binds to Intragenic Sequences of the Major Late Promoter Required for Late Phase-Specific Stimulation of Transcription

“…This pattern required viral DNA synthesis but no, or very little, synthesis of late proteins (33). One interpretation of these observations is that synthesis of progeny viral DNA molecules, which are not associated with viral core proteins, in contrast to the templates for transcription of viral early genes (9,24,63), is sufficient for transcription to the end of the ML transcription unit and that utilization of the L4 polyadenylation site is initially favored. Additional experiments will clearly be necessary to elucidate the mechanisms by which the L4 33-kDa mRNA and protein are made prior to the appearance of the full panoply of ML mRNAs in infected cells.…”