Re‐oxygenation of hypoxic simian virus 40 (SV40)‐infected CV1 cells causes distinct changes of SV40 minichromosome‐associated replication proteins

Abstract

Hypoxia interrupts the initiation of simian virus 40 (SV40) replication in vivo at a stage situated before unwinding of the origin region. After re-oxygenation, unwinding followed by a synchronous round of viral replication takes place. To further characterize the hypoxia-induced inhibition of unwinding, we analysed the binding of several replication proteins to the viral minichromosome before and after re-oxygenation. T antigen, the 34-kDa subunit of replication protein A (RPA), topoisomerase I, the 48-kDa subunit of primase, the 125-kDa subunit of polymerase d, and the 37-kDa subunit of replication factor C (RFC) were present at the viral chromatin already under hypoxia. The 70-kDa subunit of RPA, the 180-kDa subunit of polymerase a, and proliferating cell nuclear antigen (PCNA) were barely detectable at the SV40 chromatin under hypoxia and significantly increased after re-oxygenation. Immunoprecipitation of minichromosomes with T antigen-specific antibody and subsequent digestion with micrococcus nuclease revealed that most of the minichromosome-bound T antigen was associated with the viral origin in hypoxic and in re-oxygenated cells. T antigen-catalysed unwinding of the SV40 origin occurred, however, only after re-oxygenation as indicated by (a) increased sensitivity of re-oxygenated minichromosomes against digestion with single-stranded DNA-specific nuclease P1; (b) stabilization of RPA-34 binding at the SV40 minichromosome; and (c) additional phosphorylations of RPA-34 after re-oxygenation, probably catalysed by DNA-dependent protein kinase. The results presented suggest that the subunits of the proteins necessary for unwinding, primer synthesis and primer elongation first assemble at the SV40 origin in form of stable, active complexes directly before they start to work.Keywords: hypoxia; DNA unwinding; SV40; large T antigen; replicon initiation.DNA replication in mammalian cells is subject to a regulation, which depends on the O 2 tension in the cellular environment. This regulation results in inhibition of cellular replicon initiation when the concentration of O 2 falls below 0.1%. Re-oxygenation after several hours of hypoxia causes a burst of new initiations within a few minutes. So far, this regulatory phenomenon has been demonstrated for Ehrlichascites, HeLa and CCRF cells [1][2][3] and it may be a general mechanism, which adapts the cellular DNA replication to the supply of O 2 and other nutrients. This seems to be of particular significance during embryonic growth, wound healing or tumour cell propagation.The mechanism leading from re-oxygenation to replicon initiation is largely obscure. The remarkably fast resumption of initiations after re-oxygenation suggests that the O 2 -dependent replication control acts very directly on the replication apparatus.O 2 -Dependent regulation of replicon initiation was also demonstrated for viral replication in simian virus 40 (SV40)-infected CV1 cells [4,5]. As the replication of SV40 is relatively well investigated, this virus seems to be well suited to exami...

Parvovirus Infection-Induced DNA Damage Response

“…The nonstructural protein of parvoviruses, such as the NS1 protein of H-1PV and MVM and the large Rep proteins of AAV2, are able to induce a DDR without the presence of viral genomes [10,19,34,68,123], suggesting that they may create damaged cellular DNA though their endonuclease activity. The UV-inactivated AAV2 genome triggered ATR-Chk1 signaling [67,86]. However, during infection of MVM, MVC, B19V and coinfection of AAV2 and adenovirus, the DDR signaling is majorly activated from the DNA replication events [33–35,68], likely due to the specific nicking of viral DNA or generation of viral DNA intermediates that mimic damaged DNA.…”

“…The DDR triggered either by expression of the largest nonstructural viral proteins of parvoviruses [10,17,19,123] or by infection with autonomous parvoviruses [33–35] is able to arrest infected cells at S phase or G2/M phase. UV-inactivated AAV2-induced DDR not only arrests cells at G2/M [67], but also triggers apoptosis in tumor cells [86]. Viral infection-induced apoptosis was also observed following DDR activation during MVC infection, which activates the ATM-p53 signaling pathway [35].…”

“…Infection of UV-inactivated AAV2 triggers G2/M arrest of host cells and activates p84N5, a proapoptotic protein that further activates caspase-6 and induces p53-independent apoptosis in several cancer cell lines [67,86]. Notably, AAV2 can replicate autonomously (to a limited extent) in UV-treated host cells [87], suggesting that DDR signaling may trigger replication of AAV2 DNA.…”

Kinetic behavior of the reaction between hydroxyl radical and the SV40 minichromosome

“…To provide a better model system for chromatin, we have most recently used as a DNA substrate the minichromosomal form of the mammalian virus SV40. The SV40 minichromosome has found frequent use as a model for DNA in cellular chromatin (van Holde 1988, Riedinger et al 2002. It contains a closed-circular double-stranded DNA molecule 5243 bp in length associated with an approximately equal mass of histone proteins (Blasquez et al 1988).…”

Analyzing changes of chromatin‐bound replication proteins occurring in response to and after release from a hypoxic block of replicon initiation in T24 cells

“…before and after triggering initiation, we found that a large number of polypeptides taking part in viral replication were bound to the SV40 minichromosome already under hypoxia. However, the multiprotein complexes necessary for unwinding, primer synthesis and elongation lacked essential components and remained incomplete as long as hypoxia lasted [8]. Reoxygenation triggered fast completion to a functional complex, indicating a specific influence of O 2 ‐dependent cellular changes on critical steps of the assembly of a functional viral replication machinery.…”