Rad51 and Rad52 are involved in homologous recombination of replicating herpes simplex virus DNA

Abstract

Replication of herpes simplex virus 1 is coupled to recombination, but the molecular mechanisms underlying this process are poorly characterized. The role of Rad51 and Rad52 recombinases in viral recombination was examined in human fibroblast cells 1BR.3.N (wild type) and in GM16097 with replication defects caused by mutations in DNA ligase I. Intermolecular recombination between viruses, tsS and tsK, harboring genetic markers gave rise to ∼17% recombinants in both cell lines. Knock-down of Rad51 and Rad52 by siRNA reduced production of recombinants to 11% and 5%, respectively, in wild type cells and to 3% and 5%, respectively, in GM16097 cells. The results indicate a specific role for Rad51 and Rad52 in recombination of replicating herpes simplex virus 1 DNA. Mixed infections using clinical isolates with restriction enzyme polymorphisms in the US4 and US7 genes revealed recombination frequencies of 0.7%/kbp in wild type cells and 4%/kbp in GM16097 cells. Finally, tandem repeats in the US7 gene remained stable upon serial passage, indicating a high fidelity of recombination in infected cells.

Figure 1. The HSV-1 genome and markers for recombination experiments.

A, The bipartite HSV-1 genome. TR_L and TR_S denote terminal repeats and IR_L and IR_S are internal repeats . The a sequence repeats (black bars) is located at the genome ends and in between the internal repeats. The tsS mutation is mapped to the UL9 gene and the tsK mutation is mapped to the ICP4 gene. A cluster of glycoprotein genes gG (US4) and gI (US7) in the U_S segment is used to examine recombination during serial propagation of virus. B, Enlargement of the glycoprotein gene cluster. The location of the 21 nucleotide tandem repeats is highlighted. The primer pair in the US4 gene generates a 269 bp PCR product. PflMI cleaves at nucleotide 55 in the PCR product for clade C, and DdeI cleaves at nucleotide 172 for clades B and C. The primer pair in US7 generates a 410 bp PCR product. SacI cleaves at nucleotide 222 for clade C and PleI at nucleotide 355 for clades B and C. The distance between the markers in US4 and US7 is approximately 3 kbp.

Figure 2. HSV-1 tsK and tsS recombination in 1BR.3.N and GM16097.

A, Different mixtures of temperature-sensitive virus were added at the m.o.i. indicated to 1BR.3.N cells either mock-transfected or transfected with siRNA against Rad51 and Rad52. Percentage of recombinant virus was determined as the titer obtained by plaque assay on GMK-AH1 cells at 39°C (non-permissive temperature) divided by the titer obtained at 32°C (permissive temperature). Recombinant virus detectable in our assay contain the U_L-segment (wt UL9 gene) from tsK virus and U_S-segment (wt ICP4 gene) from tsS virus. Values represent average from three independent experiments and error bars indicate standard deviation. B, Recombination in GM16097 cells examined as described above. C, Western blots demonstrating knock-down of Rad51 and Rad52 proteins in 1BR.3.N and GM16097 cells.