Abstract
Although HCMV encodes many genes required for the replication of its DNA genome, no HCMV-encoded orthologue of the origin binding protein, which has been identified in other herpesviruses, has been identified. This has led to speculation that HCMV may use other viral proteins or possibly cellular factors for the initiation of DNA synthesis. It is also unclear whether cellular replication factors are required for efficient replication of viral DNA during or after viral replication origin recognition. Consequently, we have asked whether cellular pre-replication (pre-RC) factors that are either initially associated with cellular origin of replication (e.g. ORC2), those which recruit other replication factors (e.g. Cdt1 or Cdc6) or those which are subsequently recruited (e.g. MCMs) play any role in the HCMV DNA replication. We show that whilst RNAi-mediated knock-down of these factors in the cell affects cellular DNA replication, as predicted, it results in concomitant increases in viral DNA replication. These data show that cellular factors which initiate cellular DNA synthesis are not required for the initiation of replication of viral DNA and suggest that inhibition of cellular DNA synthesis, in itself, fosters conditions which are conducive to viral DNA replication.
Highlights
Replication from the single origin of lytic replication of HCMV requires the proteins from 11 loci of the HCMV genome, 6 core replication proteins and 5 transacting or cell survival factors in oriLyt amplification assays [1]
As a proof of principle, we tested the effect of siRNA knock down of HCMV UL44, an essential viral DNA polymerase accessory protein [5], on viral DNA replication
The complex assembles from its components in a stepwise manner onto cellular DNA replication origin. This culminates in phosphorylation of the minichromosome maintenance proteins (MCMs) which leads to origin melting and recruitment of the DNA polymerase [41]
Summary
Replication from the single origin of lytic replication (oriLyt) of HCMV requires the proteins from 11 loci of the HCMV genome, 6 core replication proteins and 5 transacting or cell survival factors in oriLyt amplification assays [1]. The core viral replication proteins are a single stranded binding protein (UL57); the helicaseprimase complex (UL70, UL102 and UL105) [2]; the viral DNA polymerase (UL54) and the polymerase accessory factor (UL44) [3][4]. UL44, essential for virus DNA replication [5,6], localises to viral replication compartments in infected cells [7] and been shown to interact with a potential replication initiator UL84 [8] HCMV does not encode a functional origin binding protein (OBP) and, whilst it has been suggested that this role is carried out by UL84 and IE86 [9], whether this process involves cellular DNA replication proteins is not known. Once in S phase, cells arrest and do not replicate their DNA. Both advance into S phase and cell cycle arrest have been attributed to IE86 [14][15][16][17]
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