Adenovirus Protein VII Condenses DNA, Represses Transcription, and Associates with Transcriptional Activator E1A

Abstract

Adenovirus protein VII is the major protein component of the viral nucleoprotein core. It is highly basic, and an estimated 1070 copies associate with each viral genome, forming a tightly condensed DNA-protein complex. We have investigated DNA condensation, transcriptional repression, and specific protein binding by protein VII. Xenopus oocytes were microinjected with mRNA encoding HA-tagged protein VII and prepared for visualization of lampbrush chromosomes. Immunostaining revealed that protein VII associated in a uniform manner across entire chromosomes. Furthermore, the chromosomes were significantly condensed and transcriptionally silenced, as judged by the dramatic disappearance of transcription loops characteristic of lampbrush chromosomes. During infection, the protein VII-DNA complex may be the initial substrate for transcriptional activation by cellular factors and the viral E1A protein. To investigate this possibility, mRNAs encoding E1A and protein VII were comicroinjected into Xenopus oocytes. Interestingly, whereas E1A did not associate with chromosomes in the absence of protein VII, expression of both proteins together resulted in significant association of E1A with lampbrush chromosomes. Binding studies with proteins produced in bacteria or human cells or by in vitro translation showed that E1A and protein VII can interact in vitro. Structure-function analysis revealed that an N-terminal region of E1A is responsible for binding to protein VII. These studies define the in vivo functions of protein VII in DNA binding, condensation, and transcriptional repression and indicate a role in E1A-mediated transcriptional activation of viral genes.The adenovirus nucleoprotein core consists of doublestranded genomic DNA, three highly basic viral proteins VII, V, and (mu), as well as protein IVa2 and the 55-kDa terminal protein (1,8,33,42,(52)(53)(54)61). Protein VII is the major protein component of the core, with an estimated 1,070 copies present per virion (20). Along with , it is bound noncovalently to the DNA in a sequence-independent manner (2, 6, 36, 55). Protein V contacts the DNA as well and also acts as a bridge between protein VII and the outer capsid (19,55). Protein IVa2 makes sequence-specific contacts with the viral DNA packaging sequence and is thought to play a role in DNA packaging (64). Salt-extracted preparations of the core contain only DNA and protein VII, suggesting that this protein is the most tightly DNA bound of all core proteins (60). Similarly, Sarkosyl preparations of the core contain predominantly DNA and protein VII (8).Structural features of the DNA-protein complex within the adenovirus capsid and during infection remain largely unknown. DNA within the capsid is in a highly compact configuration, and electron microscopy studies of purified viral core reveal structures reminiscent of beads on a string, or higherorder chromatin compaction, depending on the method of preparation (8,18,45,49,50,60,63). Nuclease digestion of core preparations results in discrete populations of prote...

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

“…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.…”

“…We and others have reported that protein VII from infectious viral particles enters the cell along with viral DNA (8,15,18,21). We wanted to examine the appearance of this input protein VII and also the new production of pre-protein VII and mature protein VII during infection.…”

“…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).…”

“…pGSTE1A289 and pGSTVII were described previously (21). pGST CAS YXXP, a gift from Amy Bouton, contains the YXXP domain of CAS in pGEX-2TK (7).…”

“…HeLa cells were grown on 22-by 22-mm glass coverslips (Fisher) at 37°C in DMEM supplemented with 10% NCS for 24 h prior to infection. Cells were infected for 1 h as previously described (21). Cells were then washed two times with 37°C PBS to remove unadsorbed virus, followed by incubation at 37°C in DMEM-10% NCS for the indicated times.…”