DNA and Protein Interactions of the Small Subunit of Herpes Simplex Virus Type 1 DNA Polymerase

Franz, Cornelia; Kühn, Frank; Knopf, Charles W.

doi:10.1006/viro.1998.9491

Cited by 3 publications

(2 citation statements)

References 34 publications

(65 reference statements)

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“…In contrast to the results reported herein, Franz et al (11) reported that UL42 binding to a 159-bp DNA fragment was resistant to 150 mM ammonium sulfate, although they did not quantify the amount of binding observed. It is not clear why their results are different from those we have shown.…”

Section: Discussioncontrasting

confidence: 99%

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

Chaudhuri

Parris

2002

J Virol

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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.The DNA polymerase of herpes simplex virus type 1 (HSV-1) is a heterodimer composed of a catalytic subunit (Pol), encoded by the UL30 gene, and a double-stranded (ds) DNA binding protein, encoded by the UL42 gene (6,13,15,31). The UL42 protein stimulates the activity of Pol on nicked and/or gapped DNA templates in high-salt buffer and increases the processivity of Pol on singly primed single-stranded (ss) DNA templates (12,15,17). The results of biochemical and genetic analyses have revealed that both Pol and UL42 protein are required for extensive leading and lagging strand DNA synthesis in cell-free (in vitro) assays (10,25,35,36) and for viral DNA replication in infected cells (in vivo) (20,22,27,33). The carboxyl-terminal (C-terminal) 227-amino-acid domain of Pol is suffici...

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Section: Discussioncontrasting

confidence: 99%

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

Chaudhuri

Parris

2002

J Virol

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“…This is documented by a linear incorporation rate of the Pol holoenzyme during the polymerization reaction (Fig. 4), and an increased processivity as determined by analyzing the DNA extension products [47]. In conclusion, the functional assessment of recombinant Pol showed that the VV vector system is capable to synthesize the herpesviral enzyme with all its functions known at the time when these studies were performed.…”

Section: Discussionmentioning

confidence: 97%

Expression of herpes simplex virus type 1 DNA polymerase by recombinant vaccinia virus

2009

Self Cite

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We have studied expression of the catalytic subunit of a phosphonoacetic acid-resistant (PAA(r)) DNA polymerase (Pol) of herpes simplex virus type 1 (HSV-1) strain ANG by recombinant vaccinia virus (VV) engineered with the dominant Ecogpt selection system. In agreement with the vector construction recombinant Pol expression was regulated like a VV late function. De novo-synthesis of the 136-kDa Pol polypeptide was detectable as early as 6 h postinfection, peaked between 10 and 12 h, and correlated with specific polymerase activity. Compared with HSV-1 lytic infection, the recombinant Pol protein exhibited a reduced stability with a half-life of 7 h. Whereas the Pol-associated exonuclease activities, determined from lysates of recombinant VV- and HSV-1-infected cells, were almost identical, the polymerizing activity of recombinant Pol ceased after 10 min of incubation, in correlation with the fact that Pol depends on its cofactor for optimal chain elongation. Kinetics of cellular localization, tracked by a monospecific Pol antibody, revealed that the catalytic subunit initially assembled to a few dot-like nuclear sites, reminiscent of HSV-1 DNA replication compartments. Later during infection, the localization of recombinant Pol matched with that found in lytically HSV-1-infected cells. This study demonstrates that nuclear transport and localization of the Pol subunit is independent of herpesviral functions, and neither requires the presence of herpesviral DNA sequences. Recombinant VV provides a promising alternative to explore protein interactions of the herpesviral replication machinery in their authentic cellular environment.

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A high resolution melting (HRM) technology-based assay for cost-efficient clinical detection and genotyping of herpes simplex virus (HSV)-1 and HSV-2

Lieveld

Carregosa

Benoy³

et al. 2017

Journal of Virological Methods

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Genital herpes can be caused by two very similar viruses, herpes simplex virus (HSV)-1 or HSV-2. These two HSV types cannot be distinguished clinically, but genotyping is recommended in the first-episodes of genital herpes to guide counselling and management. Quantitative polymerase chain reaction (qPCR) is the preferred diagnostic method for HSV typing. However, commercial qPCR methods use expensive fluorescent labeled probes for detection. Furthermore, most low-cost methods are not able to differentiate between HSV-1 and -2. The aim of this study was to develop a high resolution melting (HRM) technology-based assay for sensitive HSV-1 and HSV-2 detection and genotyping. Using a panel of 46 clinical specimens, the performance of the HRM assay was compared to two commercial HSV tests: the HRM assay detected HSV in all 23 positive samples, with no false positive results (100% concordance with HSV I/II Real-TM assay). Additionally, the HRM assay correctly genotyped both HSV types in a subset of these clinical samples, as determined by the Realstar HSV PCR Kit. The HSV HRM assay provides a cost-effective alternative method to conventional more expensive assays and can be used in routine clinical specimens, in cases where it is particularly necessary to detect and distinguish HSV-1 from -2.

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DNA and Protein Interactions of the Small Subunit of Herpes Simplex Virus Type 1 DNA Polymerase

Cited by 3 publications

References 34 publications

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

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

Expression of herpes simplex virus type 1 DNA polymerase by recombinant vaccinia virus

A high resolution melting (HRM) technology-based assay for cost-efficient clinical detection and genotyping of herpes simplex virus (HSV)-1 and HSV-2

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