Herpes simplex virus DNA replication: the UL9 gene encodes an origin-binding protein.

Olivo, Paul D.; Nelson, Nancy J.; Challberg, Mark D.

doi:10.1073/pnas.85.15.5414

Cited by 161 publications

(145 citation statements)

References 21 publications

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“…Heteropolymeric DNA is much less effective than homopolymeric DNA in stimulating the DNA-dependent ATPase activity of the HSV-1 origin-binding protein, which is also a DNA helicase and is encoded by the UL9 gene (21)(22)(23). Both poly(dT) and poly(dC) effectively activated the ATPase activity of the UL5/UL52 subassembly (Fig.…”

Section: Fig 1 Effect Of Ph Mgmentioning

confidence: 99%

Interactions of a Subassembly of the Herpes Simplex Virus Type 1 Helicase-Primase with DNA

Healy

You

Dodson

1997

Journal of Biological Chemistry

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The UL5, UL8, and UL52 genes of herpes simplex virus type 1 encode a multisubunit assembly that possesses primase, DNA helicase, and DNA-dependent nucleoside triphosphatase activities. A subassembly consisting of the UL5 and UL52 gene products retains these activities. The nucleoside triphosphatase activity of the UL5/UL52 subassembly is strongly stimulated by both homo-and heteropolymeric single-stranded DNA. Double-stranded DNA has little ability to stimulate the ATPase activity. The subassembly binds both double and single-stranded DNA. Nucleotides are not required for DNA-binding. The minimum length of single-stranded DNA that is bound and that stimulates enzymatic activity is about 12 nucleotides. The kinetic parameters of the ATPase activity of the subassembly are affected by the length of the oligonucleotide coeffector. The K m decreases as the coeffector length is increased up to a length of about 20 nucleotides and then remains independent of coeffector length. The first order rate constant for ATPase activity exhibits a quasihyperbolic dependence on the length of the DNA coeffector and is maximal for coeffectors of 20 nucleotides and longer.Herpes simplex virus type 1 (HSV-1) 1 encodes a heterotrimeric DNA helicase-primase whose subunits are encoded by the UL5, UL8, and UL52 genes of the virus (1, 2). The helicase activity of the enzyme is coupled to the hydrolysis of either ATP or GTP (1, 3). The UL5 gene product possesses a set of domains that correspond with several conserved motifs found in other DNA helicases (4). Mutagenesis of any one of these conserved domains in UL5 protein obliterates viral DNA synthesis (5). The UL52 gene is also essential for viral DNA synthesis and encodes a conserved domain that is found in other primases (6 -8). Mutagenesis of this conserved domain specifically obliterates the primase activity of the HSV-1 helicase-primase (7,8). Deletion of the UL8 gene from HSV-1 renders the virus incapable of DNA replication (9).The HSV-1 helicase-primase can be isolated from insect cells that have been simultaneously infected with recombinant baculoviruses that express each of the three subunits that compose the holoenzyme (10). A subassembly consisting of the UL5 and UL52 gene products also exhibits the DNA-dependent NTPase, DNA helicase, and primase activities that are associated with the holoenzyme (11, 12). The primase activity of this subassembly exhibits DNA sequence dependence and is stimulated by the UL8 protein (13-15). However, purified UL8 protein itself lacks any type of discernible enzymatic activity (11,12).Homologs of the genes encoding the HSV-1 helicase-primase are found in several other human herpesviruses (16). The complex array of activities possessed by the helicase-primases encoded by this group of viruses may be attractive targets for antiviral drug design. An understanding of the properties of the HSV-1 helicase-primase may lead to strategies for developing such compounds.In this work we further examine the interaction of the UL5/ UL52 subassembly with various...

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Section: Fig 1 Effect Of Ph Mgmentioning

confidence: 99%

Interactions of a Subassembly of the Herpes Simplex Virus Type 1 Helicase-Primase with DNA

Healy

You

Dodson

1997

Journal of Biological Chemistry

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“…DNase footprinting and gel retardation studies demonstrated that the UL9 protein binds to two sites within ori s which overlap the ends of the palindromic DNA sequence ( Fig. 1) (Elias & Lehman, 1988;Koff & Tegtmeyer, 1988;Olivo et al, 1988;Weir et al, 1989). The results of methylation interference and site-directed mutagenesis experiments indicate that the UL9 recognition sequence comprises, or is included within, YGYTCGCACT (where Y represents C or T) (Koff & Tegtmeyer, 1988;Deb & Deb, 1989).…”

Section: Introductionmentioning

confidence: 99%

“…One of these proteins, encoded by the UL9 gene, binds to specific sequences within the origin regions (Olivo et al, 1988;Weir et al, 1989). DNase footprinting and gel retardation studies demonstrated that the UL9 protein binds to two sites within ori s which overlap the ends of the palindromic DNA sequence ( Fig.…”

Section: Introductionmentioning

confidence: 99%

Two binding sites for the herpes simplex virus type 1 UL9 protein are required for efficient activity of the oriS replication origin

Weir

Stow²

1990

Journal of General Virology

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Two sites within the short region origin of DNA replication (oris) in herpes simplex virus type 1 (HSV-1) which bind the product of the UL9 gene have previously been identified. One of these sites (site I) contains an 11 bp sequence which is also present in oris of varicella-zoster virus, and the other (site II) includes a related element differing in two positions. A third sequence (motif III), which lies close to binding site I, differs from the site I element at only a single position. We have deleted specifically each of these three 11 bp sequences from within functional copies of HSV-1 oris and have examined the effects on origin activity and binding of the UL9 protein. Gel retardation analyses confirmed the important roles of the regions deleted from sites I and II in interacting with the UL9 protein.In transient replication assays, copies of oris lacking the site I or II elements exhibited undetectable or residual (4 to 8%) activity respectively. The UL9 protein did not bind to motif III even in the absence of site I sequences, although removal of the motif Ill sequence caused a small reduction in oris activity. A single base change which converted the sequence within binding site I to that of motif III was sufficient to abolish both the interaction of the UL9 gene product at this locus and the replicative ability of oris. Therefore, interaction of the UL9 protein with binding site I is essential for origin activity, but the presence of binding site II is also required for efficient replication.

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“…Firstly, UL9 is a sequence-specific DNAbinding protein which interacts with defined sites within the viral origins of replication. These sequences are recognized in double-stranded DNA with the C-terminal 317 amino acids being sufficient for binding (Olivo et al, 1988;Koff & Tegtmeyer, 1988;Weir et al, 1989). Secondly, UL9 exhibits DNA helicase and associated DNA-dependent ATPase activities (Fierer & Challberg, 1992;, consistent with the presence of several motifs characteristic of a superfamily of DNA and RNA helicases within the N-terminal 400 amino acids (Gorbalenya & Koonin, 1993).…”

mentioning

confidence: 99%

The origin-binding domain of the herpes simplex virus type 1 UL9 protein is not required for DNA helicase activity

Abbotts¹,

Stow²

1995

Journal of General Virology

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The UL9 protein of herpes simplex virus type 1 binds to specific sequences within the viral origins of DNA replication and also functions as a DNA helicase. The C-terminal 317 amino acids of the 851 residue protein specify sequence-specific binding to the viral origins and the N-terminal 400 contain several motifs characteristic of many DNA and RNA helicases. To investigate whether the origin-binding domain is required for helicase function we have expressed a truncated version comprising amino acids 1-535 of UL9 using a recombinant baculovirus. Extracts were prepared from cells infected with the recombinant virus and chromatographer over ATP-agarose. DNA helicase, DNAdependent ATPase and a novel single-stranded DNAbinding activity were present in fractions containing the truncated UL9 protein but not in corresponding gradient fractions from a control virus infection. These results indicate that the DNA helicase function of UL9 does not require the presence of the origin-binding domain and suggest that an interaction between the N-terminal domain and distorted or partially single-stranded regions of DNA may play a role in unwinding the origin region.

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Herpes simplex virus DNA replication: the UL9 gene encodes an origin-binding protein.

Cited by 161 publications

References 21 publications

Interactions of a Subassembly of the Herpes Simplex Virus Type 1 Helicase-Primase with DNA

Interactions of a Subassembly of the Herpes Simplex Virus Type 1 Helicase-Primase with DNA

Two binding sites for the herpes simplex virus type 1 UL9 protein are required for efficient activity of the oriS replication origin

The origin-binding domain of the herpes simplex virus type 1 UL9 protein is not required for DNA helicase activity

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