Deborah S. Parris

Characterization of a herpes simplex virus type 2 deoxyuridine triphosphate nucleotidohydrolase and mapping of a gene conferring type specificity for the enzyme

The herpes simplex virus type 2 (HSV-2)-induced deoxyuridine triphosphate nucleotidohydrolase (dUTPase) was purified approximately 600 +/- 43-fold using a combination of affinity, hydrophobic, absorption, and ion-exchange chromatography techniques. The only substrate for the dUTPase was dUTP with a Km of 3.6 +/- 1.1 microM. There was no apparent divalent cation requirement, but the HSV-2-induced dUTPase was inhibited by EDTA (0.1 mM) and this inhibition was reversed by either Co2+ (0.5 mM) or Mg2+ (0.5 mM). The HSV-2-induced dUTPase was distinguished from the HSV-1-induced and cellular dUTPases based upon differences in sensitivity to substrate inhibition, thermostability, and electrophoretic migration in nondenaturing polyacrylamide gels. Analysis of HSV-1 temperature-sensitive (ts) mutants demonstrated that ts A15 and ts K13 did not induce significant amounts of dUTPase activity at the permissive or nonpermissive temperatures. Mutants with defects in HSV-induced DNA polymerase or in the major DNA binding protein induced dUTPase at both temperatures. In contrast ts mutants defective in the alpha polypeptide VP175 (ICP4) did not induce normal levels of dUTPase at the nonpermissive temperature. The location of a gene encoding for the type specificity of the HSV induced dUTPase was mapped to the left 20% of the genome in Us in the region 0.060 to 0.100 or from 0.148 to 0.204.

Bortezomib-induced unfolded protein response increases oncolytic HSV-1 replication resulting in synergistic antitumor effects

Background: Bortezomib is an FDA-approved proteasome inhibitor, and oncolytic herpes simplex virus-1 (oHSV) is a promising therapeutic approach for cancer. We tested the impact of combining bortezomib with oHSV for antitumor efficacy. Experimental Design: The synergistic interaction between oHSV and bortezomib was calculated using Chou–Talalay analysis. Viral replication was evaluated using plaque assay and immune fluorescence. Western blot assays were used to evaluate induction of estrogen receptor (ER) stress and unfolded protein response (UPR). Inhibitors targeting Hsp90 were utilized to investigate the mechanism of cell killing. Antitumor efficacy in vivo was evaluated using subcutaneous and intracranial tumor xenografts of glioma and head and neck cancer. Survival was analyzed by Kaplan–Meier curves and two-sided log-rank test. Results: Combination treatment with bortezomib and oHSV (34.5ENVE), displayed strong synergistic interaction in ovarian cancer, head and neck cancer, glioma, and malignant peripheral nerve sheath tumor (MPNST) cells. Bortezomib treatment induced ER stress, evident by strong induction of Grp78, CHOP, PERK, and IRE1α (Western blot analysis) and the UPR (induction of hsp40, 70, and 90). Bortezomib treatment of cells at both sublethal and lethal doses increased viral replication (P < 0.001), but inhibition of Hsp90 ablated this response, reducing viral replication and synergistic cell killing. The combination of bortezomib and 34.5ENVE significantly enhanced antitumor efficacy in multiple different tumor models in vivo. Conclusions: The dramatic synergy of bortezomib and 34.5ENVE is mediated by bortezomib-induced UPR and warrants future clinical testing in patients. Clin Cancer Res; 20(14); 3787–98. ©2014 AACR.

Evidence against a Simple Tethering Model for Enhancement of Herpes Simplex Virus DNA Polymerase Processivity by Accessory Protein UL42

ABSTRACT The DNA polymerase holoenzyme of herpes simplex virus type 1 (HSV-1) is a stable heterodimer consisting of a catalytic subunit (Pol) and a processivity factor (UL42). HSV-1 UL42 differs from most DNA polymerase processivity factors in possessing an inherent ability to bind to double-stranded DNA. It has been proposed that UL42 increases the processivity of Pol by directly tethering it to the primer and template (P/T). To test this hypothesis, we took advantage of the different sensitivities of Pol and Pol/UL42 activities to ionic strength. Although the activity of Pol is inhibited by salt concentrations in excess of 50 mM KCl, the activity of the holoenzyme is relatively refractory to changes in ionic strength from 50 to 125 mM KCl. We used nitrocellulose filter-binding assays and real-time biosensor technology to measure binding affinities and dissociation rate constants of the individual subunits and holoenzyme for a short model P/T as a function of the ionic strength of the buffer. We found that as observed for activity, the binding affinity and dissociation rate constant of the Pol/UL42 holoenzyme for P/T were not altered substantially in high- versus low-ionic-strength buffer. In 50 mM KCl, the apparent affinity with which UL42 bound the P/T did not differ by more than twofold compared to that observed for Pol or Pol/UL42 in the same low-ionic-strength buffer. However, increasing the ionic strength dramatically decreased the affinity of UL42 for P/T, such that it was reduced more than 3 orders of magnitude from that of Pol/UL42 in 125 mM KCl. Real-time binding kinetics revealed that much of the reduced affinity could be attributable to an extremely rapid dissociation of UL42 from the P/T in high-ionic-strength buffer. The resistance of the activity, binding affinity, and stability of the holoenzyme for the model P/T to increases in ionic strength, despite the low apparent affinity and poor stability with which UL42 binds the model P/T in high concentrations of salt, suggests that UL42 does not simply tether the Pol to DNA. Instead, it is likely that conformational alterations induced by interaction of UL42 with Pol allow for high-affinity and high-stability binding of the holoenzyme to the P/T even under high-ionic-strength conditions.

3′ to 5′ Exonuclease Activity of Herpes Simplex Virus Type 1 DNA Polymerase Modulates Its Strand Displacement Activity

ABSTRACT Using a minicircle DNA primer-template, the wild-type catalytic subunit of herpes simplex virus type 1 (HSV-1) DNA polymerase (pol) was shown to lack significant strand displacement activity with or without its processivity factor, UL42. However, an exonuclease-deficient (exo−) pol (D368A) was capable of slow strand displacement. Although UL42 increased the rate (2/s) and processivity of strand displacement by exo− pol, the rate was slower than that for gap-filling synthesis. High inherent excision rates on matched primer-templates and rapid idling-turnover (successive rounds of excision and polymerization) of exo-proficient polymerases correlated with poor strand displacement activity. The results suggest that the exo activity of HSV-1 pol modulates its ability to engage in strand displacement, a function that may be important to the viability and genome stability of the virus.

Characterization of the RNA Silencing Suppression Activity of the Ebola Virus VP35 Protein in Plants and Mammalian Cells

ABSTRACT Ebola virus (EBOV) causes a lethal hemorrhagic fever for which there is no approved effective treatment or prevention strategy. EBOV VP35 is a virulence factor that blocks innate antiviral host responses, including the induction of and response to alpha/beta interferon. VP35 is also an RNA silencing suppressor (RSS). By inhibiting microRNA-directed silencing, mammalian virus RSSs have the capacity to alter the cellular environment to benefit replication. A reporter gene containing specific microRNA target sequences was used to demonstrate that prior expression of wild-type VP35 was able to block establishment of microRNA silencing in mammalian cells. In addition, wild-type VP35 C-terminal domain (CTD) protein fusions were shown to bind small interfering RNA (siRNA). Analysis of mutant proteins demonstrated that reporter activity in RSS assays did not correlate with their ability to antagonize double-stranded RNA (dsRNA)-activated protein kinase R (PKR) or bind siRNA. The results suggest that enhanced reporter activity in the presence of VP35 is a composite of nonspecific translational enhancement and silencing suppression. Moreover, most of the specific RSS activity in mammalian cells is RNA binding independent, consistent with VP35s proposed role in sequestering one or more silencing complex proteins. To examine RSS activity in a system without interferon, VP35 was tested in well-characterized plant silencing suppression assays. VP35 was shown to possess potent plant RSS activity, and the activities of mutant proteins correlated strongly, but not exclusively, with RNA binding ability. The results suggest the importance of VP35-protein interactions in blocking silencing in a system (mammalian) that cannot amplify dsRNA.

Functional Interaction between the Herpes Simplex Virus Type 1 Polymerase Processivity Factor and Origin-Binding Proteins: Enhancement of UL9 Helicase Activity

ABSTRACT The origin (ori)-binding protein of herpes simplex virus type 1 (HSV-1), encoded by the UL9 open reading frame, has been shown to physically interact with a number of cellular and viral proteins, including three HSV-1 proteins (ICP8, UL42, and UL8) essential for ori-dependent DNA replication. In this report, it is demonstrated for the first time that the DNA polymerase processivity factor, UL42 protein, provides accessory function to the UL9 protein by enhancing the 3′-to-5′ helicase activity of UL9 on partially duplex nonspecific DNA substrates. UL42 fails to enhance the unwinding activity of a noncognate helicase, suggesting that enhancement of unwinding requires the physical interaction between UL42 and UL9. UL42 increases the steady-state rate for unwinding a 23/38-mer by UL9, but only at limiting UL9 concentrations, consistent with a role in increasing the affinity of UL9 for DNA. Optimum enhancement of unwinding was observed at UL42/UL9 molecular ratios of 4:1, although enhancement was reduced when high UL42/DNA ratios were present. Under the assay conditions employed, UL42 did not alter the rate constant for dissociation of UL9 from the DNA substrate. UL42 also did not significantly reduce the lag period which was observed following the addition of UL9 to DNA, regardless of whether UL42 was added to DNA prior to or at the same time as UL9. Moreover, addition of UL42 to ongoing unwinding reactions increased the steady-state rate for unwinding, but only after a 10- to 15-min lag period. Thus, the increased affinity of UL9 for DNA most likely is the result of an increase in the rate constant for binding of UL9 to DNA, and it explains why helicase enhancement is observed only at subsaturating concentrations of UL9 with respect to DNA. In contrast, ICP8 enhances unwinding at both saturating and subsaturating UL9 concentrations and reduces or eliminates the lag period. The different means by which ICP8 and UL42 enhance the ability of UL9 to unwind DNA suggest that these two members of the presumed functional replisome may act synergistically on UL9 to effect initiation of HSV-1 DNA replication in vivo.

Herpes Simplex Virus Type 1 Suppresses RNA-Induced Gene Silencing in Mammalian Cells

ABSTRACT RNA-induced silencing is a potent innate antiviral defense strategy in plants, and suppression of silencing is a hallmark of pathogenic plant viruses. However, the impact of silencing as a mammalian antiviral defense mechanism and the ability of mammalian viruses to suppress silencing in natural host cells have remained controversial. The ability of herpes simplex virus type 1 (HSV-1) to suppress silencing was examined in a transient expression system that employed an imperfect hairpin to target degradation of transcripts encoding enhanced green fluorescent protein (EGFP). HSV-1 infection suppressed EGFP-specific silencing as demonstrated by increased EGFP mRNA levels and an increase in the EGFP mRNA half-life. The increase in EGFP mRNA stability occurred despite the well-characterized host macromolecular shutoff functions of HSV-1 that globally destabilize mRNAs. Moreover, mutant viruses defective in these functions increased the stability of EGFP mRNA even more than did the wild-type virus in silenced cells compared to results in control cells. The importance of RNA silencing to HSV-1 replication was confirmed by a significantly enhanced virus burst size in cells in which silencing was knocked down with small inhibitory RNAs directed to Argonaute 2, an integral component of the silencing complex. Given that HSV-1 encodes several microRNAs, it is possible that a dynamic equilibrium exists between silencing and silencing suppression that is capable of modulating viral gene expression to promote replication, to evade host defenses, and/or to promote latency.

Changes in subcellular localization reveal interactions between human cytomegalovirus terminase subunits.

BackgroundDuring herpesvirus replication, terminase packages viral DNA into capsids. The subunits of herpes simplex virus terminase, UL15, UL28, and UL33, assemble in the cytoplasm prior to nuclear import of the complex.MethodsTo detect similar interactions between human cytomegalovirus terminase subunits, the orthologous proteins UL89, UL56, and UL51 were expressed in HEK-293 T cells (via transfection) or insect cells (via baculovirus infection) and subcellular localizations were detected by cellular fractionation and confocal microscopy.ResultsIn both cell types, UL56 and UL89 expressed alone were exclusively cytoplasmic, whereas UL51 was ~50% nuclear. Both UL89 and UL56 became ~50% nuclear when expressed together, as did UL56 when expressed with UL51. Nuclear localization of each protein was greatest when all three proteins were co-expressed.ConclusionsThese results support inclusion of UL51 as an HCMV terminase subunit and suggest that nuclear import of human cytomegalovirus terminase may involve nuclear import signals that form cooperatively upon subunit associations.

Processing of Lagging-Strand Intermediates In Vitro by Herpes Simplex Virus Type 1 DNA Polymerase

ABSTRACT The processing of lagging-strand intermediates has not been demonstrated in vitro for herpes simplex virus type 1 (HSV-1). Human flap endonuclease-1 (Fen-1) was examined for its ability to produce ligatable products with model lagging-strand intermediates in the presence of the wild-type or exonuclease-deficient (exo−) HSV-1 DNA polymerase (pol). Primer/templates were composed of a minicircle single-stranded DNA template annealed to primers that contained 5′ DNA flaps or 5′ annealed DNA or RNA sequences. Gapped DNA primer/templates were extended but not significantly strand displaced by the wild-type HSV-1 pol, although significant strand displacement was observed with exo− HSV-1 pol. Nevertheless, the incubation of primer/templates containing 5′ flaps with either wild-type or exo− HSV-1 pol and Fen-1 led to the efficient production of nicks that could be sealed with DNA ligase I. Both polymerases stimulated the nick translation activity of Fen-1 on DNA- or RNA-containing primer/templates, indicating that the activities were coordinated. Further evidence for Fen-1 involvement in HSV-1 DNA synthesis is suggested by the ability of a transiently expressed green fluorescent protein fusion with Fen-1 to accumulate in viral DNA replication compartments in infected cells and by the ability of endogenous Fen-1 to coimmunoprecipitate with an essential viral DNA replication protein in HSV-1-infected cells.

The herpes simplex virus type 1 DNA polymerase processivity factor, UL42, does not alter the catalytic activity of the UL9 origin-binding protein but facilitates its loading onto DNA

The herpes simplex virus type 1 UL42 DNA polymerase processivity factor interacts physically with UL9 and enhances its ability to unwind short, partially duplex DNA. In this report, ATP hydrolysis during translocation of UL9 on single-stranded (ss) or partially duplex DNA was examined in the presence and absence of UL42 to determine the effect of UL42 on the catalytic function of UL9. Our studies reveal that a homodimer of UL9 is sufficient for DNA translocation coupled to ATP hydrolysis, and the steady-state ATPase catalytic rate was greater on partially duplex DNA than on ss DNA in the presence or absence of UL42. Although UL42 protein increased the steady-state rate for ATP hydrolysis by UL9 during translocation on either partially duplex or ss DNA, UL42 had no significant effect on the intrinsic ATPase activity of UL9. UL42 also had no effect on the catalytic rate of ATP hydrolysis when UL9 was not limiting but enhanced the steady-state ATPase rate at only subsaturating UL9 concentrations. At subsaturating UL9 to DNA ratios, stoichiometric concentrations of UL42 were shown to increase the amount of UL9 bound to ss DNA at equilibrium. These data support a model whereby UL42 increases the ability of UL9 to load onto DNA, thus increasing its ability to assemble into a functional complex capable of unwinding duplex DNA.