A Method for Identifying the Viral Genes Required for Herpesvirus DNA Replication
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A Method for Identifying the Viral Genes Required for Herpesvirus DNA ReplicationAuthor(s): Mark D. ChallbergSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 83, No. 23 (Dec. 1, 1986), pp. 9094-9098Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/28895 .Accessed: 07/05/2014 19:09
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Proc. Natl. Acad. Sci. USA Vol. 83, pp. 9094-9098, December 1986 Genetics
A method for identifying the viral genes required for herpesvirus DNA replication
(plasmid transfection/transient assay/herpes simplex virus oriL and oris)
MARK D. CHALLBERG
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, 9000 Rockville Pike, Bethesda, MD 20894
Communicated by Daniel Nathans, August 13, 1986
ABSTRACT Several laboratories have shown that transfected plasmid DNAs containing either of the two known origins of herpes simplex virus (HSV) DNA replication, oris or oriL, are replicated in HSV-1-infected cells or in cells cotrans- fected with virion DNA. I have found that HSV-1 (KOS) DNA digested to completion with the restriction enzyme Xba I is as efficient as intact viral DNA in supporting the in vivo replica- tion of cotransfected plasmids containing oris. On the basis of this result, several of the Xba I restriction fragments of HSV-1 DNA were cloned into the plasmid vector pUC19, and combi- nations of cloned DNAs were tested for their ability to supply the trans-acting functions required for HSV origin-dependent replication. A combination of fi"ve cloned fragments of HSV-1 can supply all of the necessary functions: Xba I C (coordinates 0.074-0.294), Xba I F (coordinates 0.294-0.453), Xba I E (coordinates 0.453-0.641), Xba I D (coordinates 0.641-0.830), and EcoRI JK (coordinates 0.0-0.086; 0.830-0.865). Tran- sient plasmid replication in this system is dependent on the presence of either oris or oriL in cis. The plasmid containing Xba I F can be replaced by two smaller plasmids, one of which contains only the gene for the HSV-encoded DNA polymerase, and the other of which contains only the gene for the major DNA binding protein (ICP8). Thus, plasmid DNA replication in this system depends on two of the genes known from genetic studies to be essential for viral DNA replication in infected cells. This system defines a simple complementation assay for cloned fragments of HSV DNA that contain other genes involved in viral DNA replication and should lead to the rapid identifica- tion of all such genes.
Herpes simplex virus (HSV) is a potentially useful model for studying DNA replication in eukaryotic cells. The viral genome is a linear duplex DNA molecule of about 150 kilobases (kb) (1). Replication takes place within the nucleus of the infected cell, and the available genetic and biochemical evidence suggests that many, if not all, of the proteins involved in viral DNA replication are virus-encoded. Thus, in principle, HSV DNA replication should be amenable to the combined biochemical-genetical approach that has proven so powerful in the dissection of the process of DNA replication in prokaryotic systems.
Although the overall mechanism of HSV DNA replication is not yet well-understood, several important features have emerged. Three cis-acting elements that are thought to function as origins of replication have been identified (2-6). The existence of these origins was inferred from the structure of defective viral genomes (2, 3), and several laboratories have shown that plasmids containing these sequences are replicated when they are introduced into HSV-infected cells (2-6). Two of these origins, both named on's, are identical, since they are located within the innverted repeat sequence
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flanking the short component of the genome (2-5). The other origin, named oriL, is located within the unique sequences of the long component (2, 3, -6). Following initiation, DNA synthesis proceeds by way of intermediates in which the genomic termini are joined (7-9). The structure of these intermediates has not been precisely defined; they may consist of circular or concatemeric molecules, or both (7-9). Unit length linear genomes are subsequently formed during the packagirng of DNA into virions.
There are several viral gene products that have been shown by genetic means to be essential for DNA replication in cultured cells. These include a DNA polymerase (Pol; refs. 10-14), a single-stranded DNA binding protein (DBP; refs. 15-18), a ribonucleotide reductase (RR; ref. 19), and an exonuclease (Exo; ref. 20). These four proteins, which are thought to be directly involved in DNA replication, are the products of early genes. Another viral gene product, the immediate early protein IE175, is also required for DNA replication (21, 22); however, since the transcription of all early genes depends on the prior expression of one or more immediate early genes (23, 24), including IE175, the role of IE175 in]DNA replication may be indirect. In addition to these known proteins, it is likely that other viral gene products are also involved in viral DNA replication since other complementation groups of temperature-sensitive mu- tants that are unable to carry out DNA replication at the nonpermissive temperature have been reported (25, 26).
In this paper, I describe the development of a system for identifying segments of the HSV genome that encode pro- teins necessary for viral DNA replication. I show that a combination of several cloned restriction fragments of HSV DNA, collectively containing the majority of the viral DNA sequence, will supply all of the functions required for the replication of plasmids containing HSV replication origins when cotransfected into Vero cells. This approach defines a simple complementation assay for trans-acting HSV genes required for viral DNA replication as well as any additional genes necessary for their expression. In view of the relatively simple organization of HSV genes (27, 28), this assay should now make it possible to systematically identify all of the viral genes involved in HSV DNA replication.
MATERIALS AND METHODS Cells and Virus. Vero cells were propagated in Eagle's
minimal essential medium containing 10% fetal calf serum. The KOS strain of HSV-1 was used in this study and virus stocks were grown and assayed in Vero cells.
DNA Isolation. HSV-1 DNA was isolated by the procedure of Denniston et al. (29) from Vero cells infected at a multiplicity of infection of 0.001 plaque-forming unit per cell. Bacterial plasmid DNAs were isolated by the method of
Abbreviations: HSV, herpes simplex virus; Pol, DNA polymerase; DBP, DNA binding protein; RR, ribonucleotide reductase; Exo, exonuclease; kb, kilobase(s); bp, base pair(s).
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Genetics: Challberg Proc. Natl. Acad. Sci. USA 83 (1986) 9095
Birnboim and Doly (30) and were further purified by banding in cesium chloride/ethidium bromide gradients.
Construction of Recombinant Plasmids. All recombinant plasmids containing HSV DNA were derived from the KOS strain of HSV-1, and plasmids were introduced into Esche- richia coli strain DH5 by the procedure of Hanahan (31).
pMC110. pKOS-BX (obtained from S. Weller), containing the BamHI X fragment of HSV-1 inserted into the BamHI site of pBR325, was digested with Sma I and a 230-base-pair (bp) fragment containing oris was purified by agarose gel electro- phoresis. This fragment was' inserted into the Sma I site of pUC19 (32). The structure of the recombinant clone was verified by sequencing the 230-bp insertion.
pMC121, -122, -123, and -124. HSV-1 DNA was incubated with the Klenow fragment of Pol I in the presence of the four dNTPs to form blunt ends at the genomic termini. This DNA' was then ligated to a 10-fold molar excess of Xba I linker oligonucleotide (New England Biolabs) and digested with Xba I. The digested DNA was fractioned by agarose gel electrophoresis using low-melting-temperature agarose (Bethesda Research Laboratories). DNA fragments =20-30 kb in size were excised from the gel and purified by extraction of the melted gel slice with phenol and chloroform, followed by ethanol precipitation. This DNA was then joined, using T4 DNA ligase, to Xba I-cleaved pUC19 DNA that had been treated with calf intestine phosphatase, and the ligation mixture was used to transform E. coli strain DH5. Trans- formed colonies were screened by hybridization with one of four nick-translated fragments of HSV DNA: EcoRI D, EcoRI F, EcoRI A, and EcoRI EK. Plasmid DNA was isolated from, several colonies which hybridized to the individual probes, and plasmid structure was analyzed by digestion with several different restriction enzymes, includ- ing BamHI, EcoRI, Hindlll, and Hpa I. Approximately 50% of the hybridizing colonies had plasmid' DNA with the predicted restriction enzyme patterns; the remaining 50% had large deletions or other rearrangements. Purified prepara- tions of pMC122 (containing Xba I F) are heterogeneous; -50% of the pMC122 DNA in the preparation used in this work contained small deletions in the oriL region.
pNNI and pNN3. Subclones of the Xba I F region were constructed starting with plasmid pMC119, which contains the Hpa I B fragment of HSV-I DNA cloned into the Sma I site of pUC19. Plasmid DNA from single colonies of DH5 containing pMC119 was digested with BamHI to determine the structure of the DNA in the intragenic region between dbp and pol (6). Several colonies contained a single species of plasmid DNA with a 150-bp deletion in that region, and DNA from one of these clones was used as the starting material in the construction of pNN1 and pNN3. Detailed restriction mapping suggested that the end points of the 150-bp deletion are identical to those of a similarly sized deletion described by Weller et al. (6). This is the most common stable deletion in the oriL region of plasmids derived from the KOS strain of HSV-1 (S. Weller, personal communication). pNN1 was constructed by isolating the 6.5-kb Sac I fragment containing the dbp gene from pMC119 and ligating this fragment to Sac I-digested pUC19. pNN3 was constructed as follows: pMC119 was digested with Pvu I and Xba I. The DNA was incubated with the Klenow fragment of PolI and dNTPs to form blunt ends, and a 9-kb fragment containing the pol gene was isolated by gel electrophoresis. This fragment was ligated into the Sma I site of pUC19 to form pNN2. pNN2 was partially digested with Kpn I and completely digested with HindIII, and a 5.5-kb fragment containing the pol gene was cloned between the Kpn I and HindIII sites of pUC19. The locations of restriction sites within the genes for Pol and DBP were deduced from the published DNA sequences (33, 34).
Assay for Plasmid DNA Replication. Vero cells were plated in 28-cm2 dishes at a density of about 3 x 106 cells per dish
and incubated for 14-16 hr at 37?C, at which time they were nearly confluent. Cells were transfected using the calcium phosphate coprecipitation technique (35, 36) as follows. Three to 4 hr before transfection, the medium was replaced. Combinations of HSV DNA and/or plasmid DNAs (0.5 Ag each) were mixed with sonicated calf thymus DNA and 2.0 M CaCl2 so that the final concentrations were 20 ,ug/ml of DNA and 0.25 M CaCl2 in a volume of 0.25 ml. This mixture was added dropwise to 0.25 ml of 2x concentrated HBS (HBS = 25 mM Hepes/140 mM NaCl/1.4 mM Na2PO4) and incubated at room temperature for 20-30 min, at which point it was added directly to the medium of the cells. After 4 hr at 37?C, the medium was removed and 0.5 ml of 15% glycerol in HBS was added. After 3-4 min, the cells were washed with isotonic buffer and the medium was replaced. After 14-16 hr at 37?C the medium was removed and the cells were lysed by the addition of 2.0 ml of 10 mM Tris HCl, pH 8.0/10 mM EDTA/2% NaDodSO4/100 ,Ag of proteinase K per ml. The cell lysate was incubated at 37?C for 2-8 hr, after which sodium acetate was added to a concentration of 0.3 M, and the resulting solution was extracted once each with phenol and chloroform/isoamyl alcohol (24:1).- RNase A was added at a concentration of 50 Ag/ml. Following incubation at 37?C for 30 min, DNA was precipitated with ethanol and redis- solved in 10 mM Tris-HCl, pH 8.0/1 mM EDTA (TE) containing 50 Ag of RNase A per ml. One-fourth of the DNA from a dish was digested with EcoRI, Dpn I, and, in some experiments, HindIII, in a reaction mixture (0.1 ml) contain- ing 10 mM Tris HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 6 mM 2-mercaptoethanol, and 10 units of each restriction enzyme for 4-14 hr at 37?C. The DNA was precipitated with ethanol, dissolved in TE, and fractionated by gel electropho- resis in a 1% agarose gel. The DNA in the gel was transferred to nitrocellulose and hybridized with 32P-labeled pUC19 DNA (1-3 x 108 dpm/,g) as described by Maniatis et al. (37). DNA fragments on the filter that hybridized to the probe were located by autoradiography for 5-24 hr using Kodak XAR film and an intensifying screen.
Xba I Does Not Cleave Any HSV-1 Gene That Is Essential for DNA Replication. As mentioned above, it has been shown previously that cells transfected with purified HSV virion DNA will support the replication of cotransfected plasmids that contain oris or oriL (2-6). It seems likely that plasmid replication in such a system depends on the expression of only a limited subset of viral genes-namely, those genes directly involved in replication of viral DNA and any regu- latory genes necessary for their expression. As a first step in locating essential replication genes,...