Continuous DNA replication is required for late gene transcription and maintenance of replication compartments in gammaherpesviruses

Late gene transcription in herpesviruses is dependent on viral DNA replication in cis but the mechanistic basis for this linkage remains unknown. DNA replication results in demethylated DNA, topological changes, removal of proteins and recruitment of proteins to promoters. One or more of these effects of DNA replication may facilitate late gene transcription. Using 5-azacytidine to promote demethylation of DNA, we demonstrate that late gene transcription cannot be rescued by DNA demethylation. Late gene transcription precedes significant increases in DNA copy number, indicating that increased template numbers also do not contribute to the linkage between replication and late gene transcription. By using serial, timed blockade of DNA replication and measurement of late gene mRNA accumulation, we demonstrate that late gene transcription requires ongoing DNA replication. Consistent with these findings, blocking DNA replication led to dissolution of DNA replication complexes which also contain RNA polymerase II and BGLF4, an EBV protein required for transcription of several late genes. These data indicate that ongoing DNA replication maintains integrity of a replication-transcription complex which is required for recruitment and retention of factors necessary for late gene transcription.
Author summary Herpesviruses exhibit both latent and lytic replication cycles. Gammaherpesviruses such as Kaposi’s sarcoma-associated herpesvirus and Epstein Barr virus undergo lytic replication when they reactivate from latency. During this process, when infectious virions are produced, an orderly cascade of gene expression occurs. Late lytic genes, which primarily encode structural components of the virion, are only transcribed after replication of the DNA genome has occurred. Unlike early lytic genes, late gene transcription is tightly linked to viral DNA replication; if viral DNA replication is blocked, late gene mRNA accumulation is severely inhibited. The mechanism by which late gene transcription is linked to DNA replication has remained elusive. In this paper we show that a process of continuous DNA replication is required. If one blocks DNA replication, further transcription also ceases, indicating that concurrent DNA replication is required to maintain late transcription. We also show that when DNA replication is blocked, the nuclear complexes in which herpesviruses are replicating dissociate. These replication complexes also serve as factories of viral transcription. When the complexes disperse, proteins required for transcription dissociate from the DNA replication machinery. These data indicate that ongoing DNA replication is necessary to maintain the physical and functional integrity of these structures. Our study provides new insight into this linkage that ensures coordination between viral replication and late gene expression.