Excision of DNA fragments corresponding to the unit-length a sequence of herpes simplex virus type 1 and terminus variation predominate on one side of the excised fragment.

ABSTRACT The a sequence of herpes simplex virus type 1 (HSV-1) is a region bracketed by two direct repeats named DR1. Concatemeric HSV-1 DNA, the product of DNA replication, is cleaved at a specific site on the second DR1 distal from the S component (authentic cleavage) to yield unit-length linear HSV-1 DNA prior to or during packaging of HSV-1 DNA. The presence of two DNA bands, of 0.25 kb (shorter band) and 0.5 kb (longer band), the lengths of which correspond to one and two units of the a sequence, was identified using acrylamide gel electrophoresis of HSV-1 DNA preparations extracted by the method of Hirt. Twelve DNA fragments from each band were molecularly cloned, and nucleotide sequences were determined. Both termini of eight (67%) DNA clones from the shorter band corresponded to the specific cleavage site on DR1. Five (41%) DNA clones from the longer band had a terminus corresponding to the specific cleavage site on DR1 on one side, but not on the opposite side. Thirteen (54%) of 24 termini of 12 analyzed DNA clones from the longer band were in and around DR1. Thus, cleavage events of DR1 can be classified into three categories: (i) authentic cleavage; (ii) site-specific cleavage on the third DR1 distal from the S component (secondary site-specific cleavage), which is related to the generation of the shorter DNA band in combination with authentic cleavage; and (iii) less-specific cleavage events in and around other DR1 elements which relate to the generation of the longer DNA band.

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In Vitro Processing of Herpes Simplex Virus Type 1 DNA Replication Intermediates by the Viral Alkaline Nuclease, UL12

Journal of Virology ◽

10.1128/jvi.72.11.8772-8781.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 8772-8781 ◽

Cited By ~ 32

Author(s):

Joshua N. Goldstein ◽

Sandra K. Weller

Keyword(s):

Herpes Simplex Virus ◽

Dna Replication ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Unit Length ◽

Simplex Virus ◽

Replication Intermediates ◽

Alkaline Nuclease

ABSTRACT Herpes simplex virus type 1 (HSV-1) DNA replication intermediates exist in a complex nonlinear structure that does not migrate into a pulsed-field gel. Genetic evidence suggests that the product of the UL12 gene, termed alkaline nuclease, plays a role in processing replication intermediates (R. Martinez, R. T. Sarisky, P. C. Weber, and S. K. Weller, J. Virol. 70:2075–2085, 1996). In this study we have tested the hypothesis that alkaline nuclease acts as a structure-specific resolvase. Cruciform structures generated with oligonucleotides were treated with purified alkaline nuclease; however, instead of being resolved into linear duplexes as would be expected of a resolvase activity, the artificial cruciforms were degraded. DNA replication intermediates were isolated from the well of a pulsed-field gel (“well DNA”) and treated with purified HSV-1 alkaline nuclease. Although alkaline nuclease can degrade virion DNA to completion, digestion of well DNA results in a smaller-than-unit-length product that migrates as a heterogeneous smear; this product is resistant to further digestion by alkaline nuclease. The smaller-than-unit-length products are representative of the entire HSV genome, indicating that alkaline nuclease is not inhibited at specific sequences. To further probe the structure of replicating DNA, well DNA was treated with various known nucleases; our results indicate that replicating DNA apparently contains no accessible double-stranded ends but does contain nicks and gaps. Our data suggest that UL12 functions at nicks and gaps in replicating DNA to correctly repair or process the replicating genome into a form suitable for encapsidation.

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Structural Characterization of the UL25 DNA-Packaging Protein from Herpes Simplex Virus Type 1

Journal of Virology ◽

10.1128/jvi.80.5.2309-2317.2006 ◽

2006 ◽

Vol 80 (5) ◽

pp. 2309-2317 ◽

Cited By ~ 33

Author(s):

Brian R. Bowman ◽

Robert L. Welschhans ◽

Hariharan Jayaram ◽

Nigel D. Stow ◽

Valerie G. Preston ◽

...

Keyword(s):

Amino Acids ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Unit Length ◽

Dna Packaging ◽

Crystallographic Structure ◽

Simplex Virus

ABSTRACT Herpesviruses replicate their double stranded DNA genomes as high-molecular-weight concatemers which are subsequently cleaved into unit-length genomes by a complex mechanism that is tightly coupled to DNA insertion into a preformed capsid structure, the procapsid. The herpes simplex virus type 1 UL25 protein is incorporated into the capsid during DNA packaging, and previous studies of a null mutant have demonstrated that its function is essential at the late stages of the head-filling process, either to allow packaging to proceed to completion or for retention of the viral genome within the capsid. We have expressed and purified an N-terminally truncated form of the 580-residue UL25 protein and have determined the crystallographic structure of the region corresponding to amino acids 134 to 580 at 2.1-Å resolution. This structure, the first for any herpesvirus protein involved in processing and packaging of viral DNA, reveals a novel fold, a distinctive electrostatic distribution, and a unique “flexible” architecture in which numerous flexible loops emanate from a stable core. Evolutionary trace analysis of UL25 and its homologues in other herpesviruses was used to locate potentially important amino acids on the surface of the protein, leading to the identification of four putative docking regions for protein partners.

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