Mechanism of Degradation of Duplex DNA by the DNase Induced by Herpes Simplex Virus

Herpes simplex virus type 1 (HSV-1) replication produces large intracellular DNA molecules that appear to be in a head-to-tail concatemeric arrangement. We have previously suggested (A. Severini, A. R. Morgan, D. R. Tovell, and D. L. J. Tyrrell, Virology 200:428-435, 1994) that these DNA species may have a complex branched structure. We now provide direct evidence for the presence of branches in the high-molecular-weight DNA produced during HSV-1 replication. On neutral agarose two-dimensional gel electrophoresis, a technique that allows separation of branched restriction fragments from linear fragments, intracellular HSV-1 DNA produces arches characteristic of Y junctions (such as replication forks) and X junctions (such as merging replication forks or recombination intermediates). Branched structures were resolved by T7 phage endonuclease I (gene 3 endonuclease), an enzyme that specifically linearizes Y and X structures. Resolution was detected by the disappearance of the arches on two-dimensional gel electrophoresis. Branched structures were also visualized by electron microscopy. Molecules with a single Y junction were observed, as well as large tangles containing two or more consecutive Y junctions. We had previously shown that a restriction enzyme which cuts the HSV-1 genome once does not resolve the large structure of HSV-1 intracellular DNA on pulsed-field gel electrophoresis. We have confirmed that result by using sucrose gradient sedimentation, in which both undigested and digested replicative intermediates sediment to the bottom of the gradient. Taken together, our experiments show that the intracellular HSV-1 DNA is held together in a large complex by frequent branches that create a network of replicating molecules. The fact that most of these branches are Y structures suggests that the network is held together by frequent replication forks and that it resembles the replicative intermediates of bacteriophage T4. Our findings add complexity to the simple model of rolling-circle DNA replication, and they pose interesting questions as to how the network is formed and how it is resolved for packaging into progeny virions.

Section: Discussionmentioning

confidence: 99%

Branched structures in the intracellular DNA of herpes simplex virus type 1

Severini

Scraba

Tyrrell

1996

“…Intracellular activity of HSV exonuclease. It has been speculated that the exonuclease activity found in HSV-infected cells may not repre- sent the true function of the polypeptide, since the optimal assay conditions are unlike an intracellular environment (9,28). Degradation of 3'end-labeled [32P]DNA after microinjection into oocytes was therefore investigated, and Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The enzyme synthesized in oocytes was found to act as an exonuclease in vivo. Two plasmids containing HSV DNA fragments directed the synthesis of exonuclease when microinjected into oocyte nuclei, and this finding enabled the coding and control sequences for this gene to be localized to 0.155 to 0.185 genome map units.Infection of cells with herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) results in the production of an alkaline exonuclease (9,10,21,28). At least one essential component of this enzyme is virus coded, since the activity induced by the HSV-2 mutant ts13 is thermolabile (7, 23).…”

mentioning

confidence: 99%

See 1 more Smart Citation

mRNA- and DNA-directed synthesis of herpes simplex virus-coded exonuclease in Xenopus laevis oocytes

Preston¹,

Cordingley²

1982

Microinjection of herpes simplex virus (HSV)-infected cell mRNA into Xenopus laevis oocytes resulted in the production of a new exonuclease activity. This enzyme strongly resembled the HSV alkaline exonuclease in many biochemical properties, and hybrid-arrested translation studies showed that it was virus coded, mapping at 0.080 to 0.185 genome map units. Exonuclease mRNA had a size and genome location equivalent to the mRNA encoding V185 in reticulocyte lysates, suggesting that VI85 is the exonuclease. The enzyme synthesized in oocytes was found to act as an exonuclease in vivo. Two plasmids containing HSV DNA fragments directed the synthesis of exonuclease when microinjected into oocyte nuclei, and this finding enabled the coding and control sequences for this gene to be localized to 0.155 to 0.185 genome map units.Infection of cells with herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) results in the production of an alkaline exonuclease (9,10,21,28). At least one essential component of this enzyme is virus coded, since the activity induced by the HSV-2 mutant ts13 is thermolabile (7, 23). The role of the exonuclease in the virus replication cycle is unclear at present.It has been shown previously that mRNAdirected synthesis of HSV-specified thymidine (pynmidine deoxyribonucleoside) kinase (TK) can be detected in the reticulocyte lysate cellfree system, provided appropriate assay conditions are used (5,25,29). These methods have facilitated studies on the size and genome map location of TK mRNA (5, 27), and have enabled the transcriptional control of the TK gene to be investigated (26). We report here that microinjection of HSV-infected cell mRNA into Xenopus laevis oocytes results in the production of active exonuclease, and therefore that similar studies are possible with this enzyme. We further show that synthesis of HSV exonuclease occurs after microinjection of suitable plasmid DNAs into oocyte nuclei. MATERIALS AND METHODSPlasmid DNAs. The plasmid DNAs used in these studies were as follows: (i) pGX41, which consists of the HSV-1 EcoRI D fragment cloned into the EcoRI site of pACYC184 (prepared by B. Matz); (ii) pGX24, which consists of the HSV-1 BamHI A fragment cloned into the BamHI site of pAT153 (prepared by V. Preston); and (iii) pGZ65, which consists of the HSV-2 BamHI F fragment cloned into the BamHI site of pAT153 (prepared by A. Davison). For microinjection into X. laevis oocytes, plasmid DNAs were purified by sucrose density gradient centrifugation.RNA preparations. Total cytoplasmic RNA was extracted from BHK cells at 5 h after infection with 20 PFU of HSV-1 or HSV-2 per cell at 31°C, as described previously (24). Polyadenylated RNA [poly(A)+ RNA] was selected from total cytoplasmic RNA by oligodeoxythymidylic acid-cellulose chromatography.For size fractionation, RNA was denatured by incubation in 50%o formamide-50 mM PIPES [piperazine-N-N'-bis(2-ethanesulfonic acid)] buffer (pH 7.4) at 60°C for 10 min, diluted twofold, and loaded onto a 13ml 15 to 40% sucrose density gradient prepared in ...

“…Infection of cells with herpes simplex virus type 1 (HSV-1) or HSV-2 results in the induction of many enzymes associated with DNA synthesis, one of which is an alkaline exonuclease (5,6,10,17). This enzyme is virus encoded since it can be synthesized in Xenopus laevis oocytes after microinjection of specific HSV-1 DNA fragments (14).…”

mentioning

confidence: 99%

Physical mapping of the herpes simplex virus type 2 nuc- lesion affecting alkaline exonuclease activity by using herpes simplex virus type 1 deletion clones

Wathen¹,

Hay²

1984

The nuc-lesion affecting alkaline exonuclease activity in the herpes simplex virus type 2 (HSV-2) mutant ts1348 had previously been mapped to the EcoRI-D restriction enzyme fragment of HSV-1. Eight clones with deletions representing most of HSV-1 EcoRI fragment D were selected with lambda gtWES hybrids. These clones were tested for their ability to rescue the alkaline exonuclease activity of HSV-2 nuc-ts1348 virus. The sequences colinear with the HSV-2 nuc-lesion were found to map between 0.169 and 0.174 map units on the HSV-1 Patton genome, representing an 0.8-kilobase-pair region that is 12.9 to 13.7 kilobase pairs from the left end of HSV-1 EcoRI fragment D.