Knockdown of DNA Ligase IV/XRCC4 by RNA Interference Inhibits Herpes Simplex Virus Type I DNA Replication

Abstract

Herpes simplex virus has a linear double-stranded DNA genome with directly repeated terminal sequences needed for cleavage and packaging of replicated DNA. In infected cells, linear genomes rapidly become endless. It is currently a matter of discussion whether the endless genomes are circles supporting rolling circle replication or arise by recombination of linear genomes forming concatemers. Here, we have examined the role of mammalian DNA ligases in the herpes simplex virus, type I (HSV-1) life cycle by employing RNA interference (RNAi) in human 1BR.3.N fibroblasts. We find that RNAi-mediated knockdown of DNA ligase IV and its co-factor XRCC4 causes a hundred-fold reduction of virus yield, a small plaque phenotype, and reduced DNA synthesis. The effect is specific because RNAi against DNA ligase I or DNA ligase III fail to reduce HSV-1 replication. Furthermore, RNAi against DNA ligase IV and XRCC4 does not affect replication of adenovirus. In addition, high multiplicity infections of HSV-1 in human DNA ligase IVdeficient cells reveal a pronounced delay of production of infectious virus. Finally, we demonstrate that formation of endless genomes is inhibited by RNAi-mediated depletion of DNA ligase IV and XRCC4. Our results suggests that DNA ligase IV/XRCC4 serves an important role in the replication cycle of herpes viruses and is likely to be required for the formation of the endless genomes early during productive infection. Herpes simplex virus type I (HSV-1)2 has a 152-kb linear double-stranded genome with direct repeats, a sequences, presented at the genomic termini (1). Once the genomic DNA is delivered through the nuclear pores, it rapidly becomes converted to an endless state (2-4). The endless genomes may be circular molecules, products of rolling circle replication, and/or concatemers formed by recombination or intermolecular ligation. It has been a commonly held opinion that circular genomes are replicated from the viral origins of replication, oriS and oriL, by theta type replication and that later a switch into a rolling circle mode of replication will take place (1). As a result, the products of viral DNA synthesis appear as linear concatemers, which are the immediate precursors for the cleavage packaging process. This view has recently received strong support by the observation that a genetically modified HSV-1 genome undergoes fusion of the termini rapidly upon infection and in the presence of inhibitors of viral DNA synthesis (4). Alternatively, it has been proposed that circularization does not take place during the productive phase of wild-type HSV-1 infection (5, 6). Instead, the linear genomes are substrates for replication, and concatemers are formed later by recombination. Circular molecules, on the other hand, are proposed to be unique to latently infected neurons.To provide some insights into the issues raised above, it would be beneficial to identify the enzymes required for replication and recombination of viral genomes. HSV-1 encodes seven gene products directly involved in DNA synthe...

Contributions of Nucleotide Excision Repair, DNA Polymerase η, and Homologous Recombination to Replication of UV-irradiated Herpes Simplex Virus Type 1

“…MRC5 monolayers at 30% confluence on 24-well plates were transfected with 150 pmol of Rad51, Rad52 (a pool containing 37.5 pmol of each flexitube siRNA), or Rad54 siRNAs by using Oligofectamine as described before (2). At 72 h after transfection, the cells were infected with HSV-1 or UV/HSV-1 at an m.o.i.…”

“…BHK-21 cells were cultured in Glasgow minimum essential medium (Invitrogen) supplemented with 10% fetal bovine serum. HSV-1 (Glasgow strain 17 synϩ), UV-irradiated HSV-1 (UV/HSV-1), and calicheamicin-treated HSV-1 (calicheamicin/HSV-1) titers were determined by plaque assay on the indicated cell line, as previously described (2).…”

“…The replisome is loaded on the origins of replication oriS and oriL by a sequence-specific superfamily II DNA helicase termed OBP or UL9 protein (6 -9), and it is capable of processive leading strand DNA synthesis coupled to discontinuous synthesis of lagging strand intermediates (5). Processive polymerization is dependent on the UL42 protein, which is a monomer folded as the proliferating cell nuclear antigen (PCNA) 2 protomer but with no sequence similarity to the cellular processivity factor (10). The UL30 subunit is a proofreading family B DNA polymerase (11).…”

“…Herpes simplex virus has a linear double-stranded genome, which, upon entry into the eukaryotic nucleus, circularizes and becomes replicated by a replisome consisting of six virus-encoded proteins (1)(2)(3)(4). The herpes simplex virus replisome is composed of a DNA polymerase made up from the UL30 and UL42 gene products, a helicase-primase complex encoded by the UL5, UL8, and UL52 genes and a single-stranded DNAbinding protein ICP8, which is a product of the UL29 gene (4,5).…”

Argininosuccinate synthetase 1 depletion produces a metabolic state conducive to herpes simplex virus 1 infection

“…An siRNA targeting GFP was used as a negative control, and siRNAs targeting the viral proteins VP16 and pUL30 (16) were used as positive controls. An siRNA targeting the cellular protein XRCC4, known to be important during HSV-1 infection (17), also served as a positive control. Sample images from the ICP4-stained secondary infection plates show that each of the three positive control siRNAs effectively decreased virus yield (Fig.…”