AP1 enhances polyomavirus DNA replication by promoting T-antigen-mediated unwinding of DNA

Simian virus 40 (SV40)-infected CV1 cells transiently exposed to hypoxia show a burst of viral replication immediately after reoxygenation. DNA precursor incorporation and analysis of growing daughter strands by alkaline sedimentation demonstrated that SV40 DNA synthesis began with a lag of about 3 to 5 min after reoxygenation followed by a largely synchronous viral replication round. Viral RNA-DNA primers complementary to the SV40 origin region were not detectable before 3 min upon reoxygenation. A distinct form of circular closed, supercoiled SV40 DNA was detectable as soon as 3 min after reoxygenation but not under hypoxia. Sensitivity to the DNA nucleaseBal 31 and migration behavior in chloroquine-containing agarose gels suggested that this DNA species was highly underwound compared to other SV40 topoisomers and was probably related to the highly underwound form U DNA first described by Dean et al. (F. B. Dean, P. Bullock, Y. Murakami, C. R. Wobbe, L. Weissbach, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 84:16–20, 1987), in vitro. 3′-OH ends of presumed RNA-DNA primers could be detected in form U by 3′ end labeling with T7 polymerase. Addition of aphidicolin to the cells before reoxygenation led to a pronounced accumulation of form U DNA containing RNA-DNA primers. In vivo pulse-chase kinetic studies performed with aphidicolin-treated SV40-infected cells showed that form U is an initial intermediate of SV40 DNA replication which matures into higher-molecular-weight replication intermediates and into SV40 form I DNA after removal of the inhibitor. These results suggest that in vivo initiation of SV40 replication is arrested by hypoxia before origin unwinding and primer synthesis.

Section: Discussionmentioning

confidence: 99%

Hypoxia Blocks In Vivo Initiation of Simian Virus 40 Replication at a Stage Preceding Origin Unwinding

Riedinger

Betteraey

Probst

1999

“…Although transcription and DNA replication are distinct biological processes, they frequently share cis-and trans-acting components. Replication origins from fungal and animal replicons contain transcription factor binding sites (9,10,39), and there is considerable evidence that proteins bound at these sites are involved in stimulation of DNA replication as well as transcriptional regulation (2,3,9,21,22,29). In contrast, AL1 is the only example of a protein with dual roles in transcription and replication in plants.…”

Section: Discussionmentioning

confidence: 99%

cis elements that contribute to geminivirus transcriptional regulation and the efficiency of DNA replication

Eagle

Hanley‐Bowdoin

1997

The A genomic component of the geminivirus tomato golden mosaic virus (TGMV) contains a 5 intergenic sequence that includes the overlapping AL61 promoter and positive-strand origin of DNA replication. The TGMV AL1 protein negatively regulates its own transcription and mediates origin recognition by binding to a repeated motif shared by the AL61 promoter and the viral origin. We examined a series of truncated or mutated 5 intergenic regions in transient expression and replication assays to identify other DNA sequences that contribute to TGMV promoter and origin function. These experiments revealed that negative regulation of the AL61 promoter is complex, involving multiple cis-acting sequences and the AL1 and AL4 proteins, which acted through different DNA elements. We also found that mutation of the TATA box motif in the AL61 promoter reduced overall transcriptional activity and AL1-mediated repression, confirming the importance of this sequence in promoter function. Mutation of a G-box consensus sequence was highly detrimental to AL61 transcription and abolished AL1 sensitivity, suggesting that AL1 interferes with transcriptional activation. Cotransfection experiments showed that the TATA box and G-box motif mutations also impaired viral DNA replication in the presence of a wild-type origin but had no effect in its absence, demonstrating that these transcriptional motifs also function as replication efficiency elements.

“…Other transcription factors have been implicated in the regulation of viral DNA replication (18). Furthermore, it was shown recently that AP-1 enhances viral DNA replication by promoting unwinding of DNA in vitro in polyomavirus infection (17). It is not known whether AP-1 has a similar role during the replication of RNA viruses.…”

Section: Fig 5 Time Course Of Junb Induction In Ev1 (A)-and Ev7 (B)mentioning

confidence: 99%

Echovirus 1 replication, not only virus binding to its receptor, VLA-2, is required for the induction of cellular immediate-early genes

Huttunen

Heino

Hyypiä

1997

Induction of immediate-early genes c-jun, junB, and c-fos was demonstrated during echovirus 1 infection in a human osteogenic sarcoma (HOS) cell line. Tenfold induction was seen at 10 h postinfection, corresponding approximately to the end of the first replication cycle of the virus. Echovirus 1 uses VLA-2 integrin as its cellular receptor, and ligand binding by integrin is known to trigger signal transduction pathways ultimately activating immediate-early genes. In the present study, however, VLA-2 binding alone was not sufficient to induce their expression; viral replication was needed. This conclusion was based on the observations that no stimulation of the immediate-early genes occurred in the MG-63 cell line where the virus attached only to VLA-2 but was not able to replicate and that induction of these genes was observed when the HOS cells were infected with echovirus type 7, known to use a different cellular receptor. Induction was not seen in the presence of the antiviral compound WIN 54954, which evidently inhibits the uncoating but not receptor binding of echovirus 1, suggesting that viral replication triggers the activation of the immediate-early genes. The induction of these genes may have a role in viral replication and in the pathogenesis of infection.