Importance of the C2, N7, and C8 positions to the mutagenic potential of 8-Oxo-2'-deoxyguanosine with two A family polymerases.

8-Oxo-2'-deoxyguanosine (OdG) is a prominent DNA lesion produced from the reaction of 2'-deoxyguanosine (dG) with reactive oxygen species. While dG directs the insertion of only dCTP during replication, OdG can direct the insertion of either dCTP or dATP, allowing for the production of dG → dT transversions. When replicated by Klenow fragment-exo (KF-exo), OdG preferentially directs the incorporation of dCTP over dATP, thus decreasing its mutagenic potential. However, when replicated by a highly related polymerase, the large fragment of polymerase I from Bacillus stearothermophilus (BF), dATP incorporation is preferred, and a higher mutagenic potential results. To gain insight into the reasons for this opposite preference and the effects of the C2, N7, and C8 positions on OdG mutagenicity, single-nucleotide insertions of dCTP and/or dATP opposite dG, OdG, and seven of their analogues were examined by steady state kinetics with both KF-exo and BF. Results from these studies suggest that the two enzymes behave similarly and are both sensitive not only to steric and electronic changes within the imidazole ring during both dCTP and dATP incorporation but also to the presence of the C2-exocyclic amine during dATP incorporation. The difference in incorporation preference opposite OdG appears to be due to a somewhat increased sensitivity to structural perturbations during dCTP incorporation with BF. Single-nucleotide extensions past the resulting base pairs were also studied and were not only similar between the two enzymes but also consistent with published ternary crystallographic studies with BF. These results are analyzed in the context of previous biochemical and structural studies, as well as stability studies with the resulting base pairs.

Herpes simplex virus tegument ICP0 is capsid associated, and its E3 ubiquitin ligase domain is important for incorporation into virions.

Herpes simplex virus (HSV) immediate-early (IE) protein ICP0 is a multifunctional regulator of HSV infection. ICP0 that is present in the tegument layer has not been well characterized. Protein compositions of wild-type and ICP0 null virions were similar, suggesting that the absence of ICP0 does not grossly impair virion assembly. ICP0 has a RING finger domain with E3 ubiquitin ligase activity that is necessary for IE functions. Virions with mutations in this domain contained greatly reduced levels of tegument ICP0, suggesting that the domain influences the incorporation of ICP0. Virion ICP0 was resistant to removal by detergent and salt and was associated with capsids, features common to inner tegument proteins.

Reversible conformational change in herpes simplex virus glycoprotein B with fusion-from-without activity is triggered by mildly acidic pH.

BACKGROUND: The pre-fusion form of the herpes simplex virus (HSV) fusion protein gB undergoes pH-triggered conformational change in vitro and during viral entry (Dollery et al., J. Virol. 84:3759-3766, 2010). The antigenic structure of gB from the fusion-from-without (FFWO) strain of HSV-1, ANG path, resembles wild type gB that has undergone pH-triggered changes. Together, changes in the antigenic and oligomeric conformation of gB correlate with fusion activity. We tested whether the pre-fusion form of FFWO gB undergoes altered conformational change in response to low pH. RESULTS: A pH of 5.5 - 6.0 altered the conformation of Domains I and V of FFWO gB, which together comprise the functional region containing the hydrophobic fusion loops. The ANG path gB oligomer was altered at a similar pH. All changes were reversible. In wild type HSV lacking the UL45 protein, which has been implicated in gB-mediated fusion, gB still underwent pH-triggered changes. ANG path entry was inactivated by pretreatment of virions with low pH. CONCLUSION: The pre-fusion conformation of gB with enhanced fusion activity undergoes alteration in antigenic structure and oligomeric conformation in response to acidic pH. We propose that endosomal pH triggers conformational change in mutant gB with FFWO activity in a manner similar to wild type. Differences apart from this trigger may account for the increased fusion activity of FFWO gB.