Herpes Simplex Virus Type 1 Helicase-Primase: DNA Binding and Consequent Protein Oligomerization and Primase Activation

Chen, Yan; Bai, Ping; Mackay, Shannon; Korza, George; Carson, John H.; Kuchta, Robert D.; Weller, Sandra K.

doi:10.1128/jvi.01690-10

Cited by 24 publications

(27 citation statements)

References 54 publications

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“…In this respect it might parallel the gene 59 protein in the T4 system which acts as a helicase loader and then remains bound at moving forks as seen in the EM using nanoscale tags (41). It has previously been shown that UL5/UL8/UL52 binds preferentially to the oligo-based fork-like structures (42). Whether this binding is dictated by UL8 is to be seen.…”

Section: Discussionmentioning

confidence: 99%

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O-GlcNAc Transferase Regulates Mitotic Chromatin Dynamics

Sakabe

Hart

2010

Journal of Biological Chemistry

121

115

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During lytic infection, herpes simplex virus (HSV) DNA is replicated by a mechanism involving DNA recombination. For instance, replication of the HSV-1 genome produces X-and Y-branched structures, reminiscent of recombination intermediates. HSV-1`s replication machinery includes a trimeric helicase/primase is composed of helicase (UL5) and primase (UL52) subunits and a third subunit, UL8. UL8 has been reported to stimulate the helicase and primase activities of the complex in the presence of ICP8, an HSV-1 protein that functions as an annealase, a protein that binds complementary single-stranded (ss)DNA and facilitates its annealing to duplex DNA. UL8 also influences the intracellular localization of the UL5/UL52 subunits, but UL8`s catalytic activities are not known. In this study, we used a combination of biochemical techniques and transmission electron microscopy. First, we report that UL8 alone forms protein filaments in solution. Moreover, we also found that, UL8 binds to ssDNAs >50 nt long and promotes the annealing of complementary ssDNA to generate highly branched duplex DNA structures. Finally, UL8 has a very high affinity for replication fork structures containing a gap in the lagging strand as short as 15 nt, suggesting that UL8 may aid in directing or loading the trimeric complex onto a replication fork. The properties of UL8 uncovered here suggest that UL8 may be involved in the generation of the X-and Y-branched structures that are the hallmarks of HSV replication.Herpes simplex virus type 1 (HSV-1) has a 152-kb linear double-stranded (ds) DNA genome. During HSV-1 infection, the viral genome is released from the capsid into the nucleus and is replicated to form concatemeric DNA that is recognized by the packaging machinery to produce infectious virus. Seven virus-encoded proteins are required for viral DNA synthesis including an origin binding protein (UL9) and six core replication proteins: a two subunit DNA polymerase (UL30 and UL42), a three subunit helicase/primase complex (UL5, UL8 and UL52) and a multifunctional ssDNA binding protein (ICP8) (reviewed in ref.1). The six purified core replication proteins can function to produce linear concatemers in vitro if provided a primed template (2). On the other hand, in vivo, late replicating viral DNA has been reported to adopt a mixture of complex structures such as X-and Y-shaped branches and tangled masses, suggesting that recombination may play a role in the replication of HSV-1 DNA (3-7). Additional support for this concept was provided by Blümel et al. who showed that when SV40 genomes were replicated by the six HSV-encoded replication factors and SV40 large T antigen, concatemers composed of X-shaped DNA structures were observed (8). Since SV40 replication normally produces two circular daughter molecules, the complex DNA generated in the presence of HSV replication proteins suggests that the HSV replication machinery is inherently recombinogenic. A better understanding of the functions and properties of the essential viral replicatio...

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Section: Discussionmentioning

confidence: 99%

“…The oligonucleotides (42,55,69, 82 and 90 nt) described in Table 1 were used in the binding assays. M13mp18 (7249 nt) and ΦX174 (5386 nt) circular ssDNAs were from New England Biolabs.…”

Section: Methodsmentioning

confidence: 99%

O-GlcNAc Transferase Regulates Mitotic Chromatin Dynamics

Sakabe

Hart

2010

Journal of Biological Chemistry

121

115

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“…Consistent with this, the HSV H/P behaves as a single heterotrimer by gel filtration [80]. However, in the presence of forked DNA and AMP–PNP or ATP, higher-order complexes can form [96]. Furthermore, two or more H/P heterotrimers are needed in order to observe efficient primase activity [96].…”

Section: Hsv H/pmentioning

confidence: 94%

“…However, in the presence of forked DNA and AMP–PNP or ATP, higher-order complexes can form [96]. Furthermore, two or more H/P heterotrimers are needed in order to observe efficient primase activity [96]. In these experiments, primase exhibited a cooperative dependence on protein concentration while helicase activity did not.…”

Section: Hsv H/pmentioning

confidence: 99%

The DNA helicase–primase complex as a target for herpes viral infection

Weller

Kuchta

2013

Expert Opinion on Therapeutic Targets

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Introduction The Herpesviridae are responsible for debilitating acute and chronic infections, and some members of this family are associated with human cancers. Conventional anti-herpesviral therapy targets the viral DNA polymerase and has been extremely successful; however, the emergence of drug-resistant virus strains, especially in neonates and immunocompromised patients, underscores the need for continued development of anti-herpes drugs. In this article, we explore an alternative target for antiviral therapy, the HSV helicase/primase complex. Areas covered This review addresses the current state of knowledge of HSV DNA replication and the important roles played by the herpesvirus helicase–primase complex. In the last 10 years several helicase/primase inhibitors (HPIs) have been described, and in this article, we discuss and contrast these new agents with established inhibitors. Expert opinion The outstanding safety profile of existing nucleoside analogues for a-herpesvirus infection make the development of new therapeutic agents a challenge. Currently used nucleoside analogues exhibit few side effects and have low occurrence of clinically relevant resistance. For HCMV, however, existing drugs have significant toxicity issues and the frequency of drug resistance is high, and no antiviral therapies are available for EBV and KSHV. The development of new anti-herpesvirus drugs is thus well worth pursuing especially for immunocompromised patients and those who develop drug-resistant infections. Although the HPIs are promising, limitations to their development into a successful drug strategy remain.

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“…This template was initially developed to study T7 replication (4) and was re-engineered by Falkenberg et al (12) to examine HSV-1 replication. The 70-base-long minicircle they employed was a partial dsDNA circle with a 40-base ss 5Ј-tail required for efficient UL5/UL8/UL52 binding and unwinding (13) and a 20-base leading strand gap at the fork, providing a site for assembly of the replisome. They observed leading strands up to 10,000 nt and Okazaki fragments of ϳ3,000 nt in length.…”

mentioning

confidence: 99%