Viral disease update

2011

J Virol

The origin-specific replication of the herpes simplex virus 1 genome requires seven proteins: the helicaseprimase (UL5-UL8-UL52), the DNA polymerase (UL30-UL42), the single-strand DNA binding protein (ICP8), and the origin-binding protein (UL9). We reconstituted these proteins, excluding UL9, on synthetic minicircular DNA templates and monitored leading and lagging strand DNA synthesis using the strand-specific incorporation of dTMP and dAMP. Critical features of the assays that led to efficient leading and lagging stand synthesis included high helicase-primase concentrations and a lagging strand template whose sequence resembled that of the viral DNA. Depending on the nature of the minicircle template, the replication complex synthesized leading and lagging strand products at molar ratios varying between 1:1 and 3:1. Lagging strand products (ϳ0.2 to 0.6 kb) were significantly shorter than leading strand products (ϳ2 to 10 kb), and conditions that stimulated primer synthesis led to shorter lagging strand products. ICP8 was not essential; however, its presence stimulated DNA synthesis and increased the length of both leading and lagging strand products. Curiously, human DNA polymerase ␣ (p70-p180 or p49-p58-p70-p180), which improves the utilization of RNA primers synthesized by herpesvirus primase on linear DNA templates, had no effect on the replication of the minicircles. The lack of stimulation by polymerase ␣ suggests the existence of a macromolecular assembly that enhances the utilization of RNA primers and may functionally couple leading and lagging strand synthesis. Evidence for functional coupling is further provided by our observations that (i) leading and lagging strand synthesis produce equal amounts of DNA, (ii) leading strand synthesis proceeds faster under conditions that disable primer synthesis on the lagging strand, and (iii) conditions that accelerate helicase-catalyzed DNA unwinding stimulate decoupled leading strand synthesis but not coordinated leading and lagging strand synthesis.

mentioning

confidence: 99%

Coordinated Leading and Lagging Strand DNA Synthesis by Using the Herpes Simplex Virus 1 Replication Complex and Minicircle DNA Templates

Stengel

2011

J Virol

“…(7). Herpes simplex virus 1 (HSV 1 ) encodes seven proteins essential for viral DNA replication: a) the heterodimeric DNA polymerase-processivity factor complex (UL30-UL42), b) the heterotrimeric helicase-primase (UL5-UL8-UL52), c), an origin-binding protein (UL9), and a single-stranded DNA binding protein (UL29/ICP8) (8–10).…”

mentioning

confidence: 99%

“…Herpes viruses are complex DNA viruses that are responsible for a variety of indications, including oral and genital herpes sores, chickenpox, viral encephalitis, etc . Herpes simplex virus 1 (HSV) encodes seven proteins essential for viral DNA replication: (a) the heterodimeric DNA polymerase–processivity factor complex (UL30/UL42), (b) the heterotrimeric helicase-primase (UL5-UL8-UL52), (c) an origin binding protein (UL9), and (d) a single-stranded DNA binding protein (UL29/ICP8). − In addition to polymerase activity, UL30 also possesses 3′–5′ exonuclease activity that proofreads the just-incorporated nucleotide. , …”

mentioning

confidence: 99%

Effects of Acyclovir, Foscarnet, and Ribonucleotides on Herpes Simplex Virus-1 DNA Polymerase: Mechanistic Insights and a Novel Mechanism for Preventing Stable Incorporation of Ribonucleotides into DNA

Vashishtha

2016

Biochemistry

We examined the impact of two clinically approved anti-herpes drugs, acyclovir and Forscarnet (phosphonoformate) on the exonuclease activity of the herpes simplex virus-1 DNA polymerase, UL30. Acyclovir triphosphate and Foscarnet, along with the closely related phosphonoacetic acid, did not affect exonuclease activity on single-stranded DNA. Furthermore, blocking the polymerase active site due to either binding of Foscarnet or phosphonoacetic acid to the E-DNA complex or polymerization of acyclovir onto the DNA also had minimal effect on exonuclease activity. The inability of the exonuclease to excise acyclovir from the primer 3′-terminus results from the altered sugar structure directly impeding phosphodiester bond hydrolysis as opposed to inhibiting binding, unwinding of the DNA by the exonuclease or transfer of the DNA from the polymerase to the exonuclease. Removing the 3′-hydroxyl or the 2′-carbon from the nucleotide at the 3′-terminus of the primer strongly inhibited exonuclease activity, although addition of a 2′-hydroxyl did not affect exonuclease activity. The biological consequences of these results are two-fold. First, the ability of acyclovir and Foscarnet to block dNTP polymerization without impacting exonuclease activity raises the possibility that their effects on herpes replication may involve both direct inhibition of dNTP polymerization along with exonuclease mediated destruction of herpes DNA. Second, the ability of the exonuclease to rapidly remove a ribonucleotide at the primer 3´-terminus in combination with the polymerase not efficiently adding dNTPs onto this primer provides a novel mechanism by which the herpes replication machinery can prevent incorporation of ribonucleotides into newly synthesized DNA.

“…Herpes viruses comprise a large family of complex DNA viruses, eight of which can infect humans. 2 Although generally benign, they can cause a variety of diseases including oral lesions (HSV1), genital lesions (HSV2), chicken pox and shingles ( Varicella zoster ), etc. 2 HSV encodes seven proteins essential for DNA replication: the heterotrimeric helicase–primase (UL5–UL8–UL52), the heterodimeric DNA polymerase–processivity factor complex (UL30–UL42), single-stranded DNA binding protein (UL29/ICP8), and an origin binding protein (UL9).…”

mentioning

confidence: 99%

“…Herpes viruses comprise a large family of complex DNA viruses, eight of which can infect humans . Although generally benign, they can cause a variety of diseases including oral lesions (HSV1), genital lesions (HSV2), chicken pox and shingles (Varicella zoster), etc .…”

mentioning

confidence: 99%

Polymerase and Exonuclease Activities in Herpes Simplex Virus Type 1 DNA Polymerase Are Not Highly Coordinated

Vashishtha

2015

Biochemistry

The herpes polymerase–processivity factor complex consists of the catalytic UL30 subunit containing both polymerase and proofreading exonuclease activities and the UL42 subunit that acts as a processivity factor. Curiously, the highly active exonuclease has minimal impact on the accumulation of mismatches generated by the polymerase activity. We utilized a series of oligonucleotides of defined sequence to define the interactions between the polymerase and exonuclease active sites. Exonuclease activity requires unwinding of two nucleotides of the duplex primer–template. Surprisingly, even though the exonuclease rate is much higher than the rate of DNA dissociation, the exonuclease degrades both single- and double-stranded DNA in a nonprocessive manner. Efficient proofreading of incorrect nucleotides incorporated by the polymerase would seem to require efficient translocation of DNA between the exonuclease and polymerase active sites. However, we found that translocation of DNA from the exonuclease to polymerase active site is remarkably inefficient. Consistent with inefficient translocation, the DNA binding sites for the exonuclease and polymerase active sites appear to be largely independent, such that the two activities appear noncoordinated. Finally, the presence or absence of UL42 did not impact the coordination of the polymerase and exonuclease activities. In addition to providing fundamental insights into how the polymerase and exonuclease function together, these activities provide a rationale for understanding why the exonuclease minimally impacts accumulation of mismatches by the purified polymerase and raise the question of how these two activities function together in vivo.