Mutations That Increase DNA Binding by the Processivity Factor of Herpes Simplex Virus Affect Virus Production and DNA Replication Fidelity

Abstract

The interactions of the herpes simplex virus processivity factor UL42 with the catalytic subunit of the viral polymerase (Pol) and DNA are critical for viral DNA replication. Previous studies, including one showing that substitution of glutamine residue 282 with arginine (Q282R) results in an increase of DNA binding in vitro, have indicated that the positively charged back surface of UL42 interacts with DNA. To investigate the biological consequences of increased DNA binding by UL42 mutations, we constructed two additional UL42 mutants, including one with a double substitution of alanine for aspartic acid residues (D270A/D271A) and a triple mutant with the D270A/D271A and Q282R substitutions. These UL42 mutants exhibited increased and prolonged DNA binding without an effect on binding to a peptide corresponding to the C terminus of Pol. Plasmids expressing any of the three UL42 mutants with an increased positive charge on the back surface of UL42 were qualitatively competent for complementation of growth and DNA replication of a UL42 null mutant on Vero cells. We then engineered viruses expressing these mutant proteins. The UL42 mutants were more resistant to detergent extraction than wild-type UL42, suggesting that they are more tightly associated with DNA in infected cells. All three UL42 mutants formed smaller plaques on Vero cells and replicated to reduced yields compared with results for a control virus expressing wild-type UL42. Moreover, mutants with double and triple mutations, which contain D270A/D271A mutations, exhibited increased mutation frequencies, and mutants containing the Q282R mutation exhibited elevated ratios of virion DNA copies per PFU. These results suggest that herpes simplex virus has evolved so that UL42 neither binds DNA too tightly nor too weakly to optimize virus production and replication fidelity.Processivity factors of DNA polymerases promote longchain DNA synthesis by preventing dissociation of the DNA polymerase from the primer/template. Processivity factors also can influence DNA replication fidelity, as indicated by numerous in vivo and in vitro studies (1-3, 5, 6, 11, 12, 18, 28, 36). A major class of processivity factors known as "sliding clamps" includes proliferating cell nuclear antigen (PCNA) of eukaryotic cells (23) and gp45 of T4 bacteriophage (27). Sliding clamps are homodimers or homotrimers that encircle DNA and interact with the catalytic subunits (Pols) of their cognate DNA polymerases to promote processive DNA synthesis.A second class of processivity factors includes those encoded by herpesviruses and is exemplified by herpes simplex virus (HSV) UL42. UL42 forms a heterodimer with the HSV Pol. Both subunits are essential for production of infectious virus and for viral DNA replication (20,26). UL42 can stimulate long-chain DNA synthesis by Pol, and template challenge experiments established that this stimulation is due to increased processivity (15). In addition to its interaction with Pol, which is mediated by the C terminus of Pol, UL42 also binds DNA directly...

DNA-Binding Properties of African Swine Fever Virus pA104R, a Histone-Like Protein Involved in Viral Replication and Transcription

“…(C) The specific recognition of the serum used was tested against whole infected-cell extracts by Western blotting. pA104R-DNA interactions were not detected under high-ionic-strength conditions (Ն2 M NaCl), indicating that pA104R-binding activity involves ion pair formation, as reported for other viral DNA-binding proteins (26)(27)(28).…”

DNA Replication Fidelity of Herpes Simplex Virus

“…These studies suggest that UL42 interacts with DNA via interaction between the basic charged residues located on the surface of the molecule and the negatively charged DNA. Recombinant viruses expressing UL42 with increased DNA binding exhibits impaired phenotypes, including the formation of smaller plaques, decrease replication of progeny and synthesis of DNA with higher ratio of DNA copies per plaque forming unit (PFU) (Jiang et al, 2009). As expected the mutants are more mutagenic than the control virus expressing wild type UL42 (Jiang et al, 2009).…”

“…Recombinant viruses expressing UL42 with increased DNA binding exhibits impaired phenotypes, including the formation of smaller plaques, decrease replication of progeny and synthesis of DNA with higher ratio of DNA copies per plaque forming unit (PFU) (Jiang et al, 2009). As expected the mutants are more mutagenic than the control virus expressing wild type UL42 (Jiang et al, 2009). Similarly mutants with decreased DNA binding also have defective phenotypes and are highly mutagenic (Jiang et al, 2007a), and that mutants with no detectable DNA binding are deleterious in viral growth and DNA synthesis (Jiang et al, 2007b).…”

Effect of a caspase inhibitor, zVADfmk, on the inhibition of breast cancer cells by herpes simplex virus type 1

“…PCR was performed with the iQ SYBR Green Supermix (BioRad, Hercules, CA). Oligonucleotide primers were DBP-3261 (5 0 -GGTGCAGAAAGGTGTTGTTG-3 0 ) and DBP-3315, 31 32 Each PCR run contained a series of pUL29-OriL DNA dilutions (1 Â 10 3 -1 Â 10 8 copies per 5 ml) and a negative control DNA.…”