The herpes simplex virus type-1 single-strand DNA-binding protein (ICP8) promotes strand invasion

ICP8, the herpes simplex virus type-1 single-strand DNA-binding protein, was recently shown to promote strand exchange in conjunction with the viral replicative helicase (Nimonkar, A. V., and Boehmer, P. E. (2002) J. Biol. Chem. 277, 15182-15189). Here we show that ICP8 also catalyzes strand invasion in an ATP-independent manner. Thus, ICP8 promotes the assimilation of a single-stranded donor molecule into a homologous plasmid, resulting in the formation of a displacement loop. Invasion of a homologous duplex by single-stranded DNA requires homology at either 3' or 5' end of the invading strand. The reaction is dependent on the free energy of supercoiling and alters the topology of the acceptor plasmid. Hence, strand invasion products formed by ICP8 are resistant to the action of restriction endonucleases that cleave outside of the area of pairing. The ability to catalyze strand invasion is a novel activity of ICP8 and the first demonstration of a eukaryotic viral single-strand DNA-binding protein to promote this reaction. In this regard ICP8 is functionally similar to the prototypical prokaryotic recombinase RecA and its eukaryotic homologs. This strand invasion activity of ICP8 coupled with DNA synthesis may explain the high prevalence of branched DNA structures during viral replication.     

On the mechanism of strand assimilation by the herpes simplex virus type-1 single-strand DNA-binding protein (ICP8)

ICP8, the herpes simplex virus type-1 encoded single-strand DNA (ssDNA)-binding protein, promotes the assimilation of a single-stranded DNA molecule into a homologous duplex plasmid resulting in the formation of a displacement loop. Here we examine the mechanism of this process. In contrast to the RecA-type recombinases that catalyze strand invasion via an active search for homology, ICP8 acts by a salt-dependent strand annealing mechanism. The active species in this reaction is a ssDNA:ICP8 nucleoprotein filament. There appears to be no requirement for ICP8 to interact with the acceptor DNA. At higher concentrations, ICP8 promotes the reverse reaction, presumably owing to its helix destabilizing activity. ICP8-mediated strand assimilation imparts single-stranded character onto the acceptor DNA, consistent with the formation of a displacement loop. These data suggest that the recombination activity of ICP8 is similar to the mechanism of eukaryotic Rad52.     

Modulation of the herpes simplex virus type-1 UL9 DNA helicase by its cognate single-strand DNA-binding protein, ICP8

The mechanism of stimulation of a DNA helicase by its cognate single-strand DNA-binding protein was examined using herpes simplex virus type-1 UL9 DNA helicase and ICP8. UL9 and ICP8 are two essential components of the viral replisome that associate into a complex to unwind the origins of replication. The helicase and DNA-stimulated ATPase activities of UL9 are greatly elevated as a consequence of this association. Given that ICP8 acts as a single-strand DNA-binding protein, the simplest model that can account for its stimulatory effect predicts that it tethers UL9 to the DNA template, thereby increasing its processivity. In contrast to the prediction, data presented here show that the stimulatory activity of ICP8 does not depend on its single-strand DNA binding activity. Our data support an alternative hypothesis in which ICP8 modulates the activity of UL9. Accordingly, the data show that the ICP8-binding site of UL9 constitutes an inhibitory region that maintains the helicase in an inefficient ground state. ICP8 acts as a positive regulator by neutralizing this region. ICP8 does not affect substrate binding, ATP hydrolysis, or the efficiency of translocation/DNA unwinding. Rather, we propose that ICP8 increases the efficiency with which substrate binding and ATP hydrolysis are coupled to translocation/DNA unwinding.     

Intragenic complementation of herpes simplex virus ICP8 DNA-binding protein mutants.

The major DNA-binding protein, or infected-cell protein 8 (ICP8), of herpes simplex virus is required for viral DNA synthesis and normal regulation of viral gene expression. Previous genetic analysis has indicated that the carboxyl-terminal 28 residues are the only portion of ICP8 capable of acting independently as a nuclear localization signal. In this study, we constructed a mutant virus (n11SV) in which the carboxyl-terminal 28 residues of ICP8 were replaced by the simian virus 40 large-T-antigen nuclear localization signal. The n11SV ICP8 localized into the nucleus and bound to single-stranded DNA in vitro as tightly as wild-type ICP8 did but was defective for viral DNA synthesis and viral growth in Vero cells. Two mutant ICP8 proteins (TL4 and TL5) containing amino-terminal alterations could complement the n11SV mutant but not ICP8 gene deletion mutants. Cell lines expressing TL4 and TL5 ICP8 were isolated, and in these cells, complementation of n11SV was observed at the levels of both viral DNA replication and viral growth. Therefore, complementation between n11SV ICP8 and TL4 or TL5 ICP8 reconstituted wild-type ICP8 functions. Our results demonstrate that (i) the carboxyl-terminal 28 residues of ICP8 are required for a function(s) involved in viral DNA replication, (ii) this function can be supplied in trans by another mutant ICP8, and (iii) ICP8 has multiple domains possessing different functions, and at least some of these functions can complement in trans.     

In vitro strand exchange promoted by the herpes simplex virus type-1 single strand DNA-binding protein (ICP8) and DNA helicase-primase

The genome of herpes simplex virus type-1 undergoes a high frequency of homologous recombination in the absence of a virus-encoded RecA-type protein. We hypothesized that viral homologous recombination is mediated by the combined action of the viral single strand DNA-binding protein (ICP8) and helicase-primase. Our results show that ICP8 catalyzes the formation of recombination intermediates (joint molecules) between circular single-stranded acceptor and linear duplex donor DNA. Joint molecules formed by invasion of a 3'-terminal strand displaces the non-complementary 5'-terminal strand, thereby creating a loading site for the helicase-primase. Helicase-primase acts on these joint molecules to promote ATP-dependent branch migration. Finally, we have reconstituted strand exchange by the synchronous action of ICP8 and helicase-primase. Based on these data, we present a recombination mechanism for a eukaryotic DNA virus in which a single strand DNA-binding protein and helicase cooperate to promote homologous pairing and branch migration.     

Characterization of a major DNA-binding domain in the herpes simplex virus type 1 DNA-binding protein (ICP8).

We have studied the major DNA-binding protein (ICP8) from herpes simplex virus type 1 to identify its DNA-binding site. Since we obtained our protein from a cell line carrying multiple chromosomally located copies of the ICP8 gene, we first analyzed this protein to assess its similarity to the corresponding viral protein. Our protein resembled the viral protein by molecular weight, response to antibody, preference for binding single-stranded DNA, and ability to lower the melting temperature of poly(dA-dT). To define the DNA-binding domain, we subjected the protein to limited trypsin digestion and separated the peptide products on a sodium dodecyl sulfate-polyacrylamide gel. These fragments were then transferred to a nitrocellulose membrane, renatured in situ, and tested for their ability to bind DNA. From this assay, we identified four fragments which both bound DNA and exhibited the expected binding preference for single-stranded DNA. The sequence of the smallest of these fragments was determined and corresponds to a polypeptide spanning residues 300 to 849 in the intact protein. This peptide contains several regions which may be important for DNA binding based on sequence similarities in single-stranded DNA-binding proteins from other herpesviruses and, in one case, on a conserved sequence found in more distant procaryotic and eucaryotic proteins.     

An immunoassay for the study of DNA-binding activities of herpes simplex virus protein ICP8.

An immunoassay was used to examine the interaction between a herpes simplex virus protein, ICP8, and various types of DNA. The advantage of this assay is that the protein is not subjected to harsh purification procedures. We characterized the binding of ICP8 to both single-stranded (ss) and double-stranded (ds) DNA. ICP8 bound ss DNA fivefold more efficiently than ds DNA, and both binding activities were most efficient in 150 mM NaCl. Two lines of evidence indicate that the binding activities were not identical: (i) ds DNA failed to complete with ss DNA binding even with a large excess of ds DNA; (ii) Scatchard plots of DNA binding with various amounts of DNA were fundamentally different for ss DNA and ds DNA. However, the two activities were related in that ss DNA efficiently competed with the binding of ds DNA. We conclude that the ds DNA-binding activity of ICP8 is probably distinct from the ss DNA-binding activity. No evidence for sequence-specific ds DNA binding was obtained for either the entire herpes simplex virus genome or cloned viral sequences.     

Genetic evidence for multiple nuclear functions of the herpes simplex virus ICP8 DNA-binding protein

We have isolated several mutant herpes simplex viruses, specifically mutated in the infected cell protein 8 (ICP8) gene, to define the functional domains of ICP8, the major viral DNA-binding protein. To facilitate the isolation of these mutants, we first isolated a mutant virus, HD-2, with the lacZ gene fused to the ICP8 gene so that an ICP8-beta-galactosidase fusion protein was expressed. This virus formed blue plaques on ICP8-expressing cell lines in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Mutated ICP8 gene plasmids cotransfected with HD-2 DNA yielded recombinant viruses with the mutant ICP8 gene incorporated into the viral genome. These recombinants were identified by formation of white plaques. Four classes of mutants were defined: (i) some expressed ICP8 that could bind to DNA but could not localize to the cell nucleus; (ii) some expressed ICP8 that did not bind to DNA but localized to the nucleus; (iii) some expressed ICP8 that neither bound to DNA nor localized to the nucleus; and (iv) one expressed ICP8 that localized to the cell nucleus and bound to DNA in vitro, but the mutant virus did not replicate its DNA. These classes of mutants provide genetic evidence that DNA binding and nuclear localization are distinct functions of ICP8 and that ICP8 has nuclear functions other than binding to DNA. Furthermore, the portion of ICP8 needed for a nuclear function(s) distinct from DNA binding is the part of ICP8 showing sequence similarity to that of the cellular protein cyclin or proliferating cell nuclear antigen.     

The major herpes simplex virus type-1 DNA-binding protein is a zinc metalloprotein.

The primary amino acid sequence of the major herpes simplex virus type 1 (HSV-1)-infected cell polypeptide 8 (ICP8) deduced from the DNA sequence of the unique long open reading frame 29 (UL29 ORF) contains a potential metal-binding domain of the form Cys-X2-5-Cys-X2-15-A-X2-4-A where A may be either histidine or cysteine and X is any amino acid. The putative metal-binding sequence in ICP8 encompasses residues 499-512 as follows: C-N-L-C-T-F-D-T-R-H-A-C-V-H-. Atomic absorption analysis of several preparations of ICP8 indicates the presence of 1 mol of zinc/mol of protein. The zinc is resistant to removal by dialysis against concentrations of EDTA which deplete zinc from alcohol dehydrogenase. The bound zinc can be removed by reaction with the reversible sulfhydryl reagent p-hydroxymercurimethylsulfonate and the zinc-depleted protein transiently retains DNA binding activity. Digestion of both native and zinc-depleted ICP8 with V8 protease indicates that the bound zinc is required for the structural integrity of the protein.     

Correct intranuclear localization of herpes simplex virus DNA polymerase requires the viral ICP8 DNA-binding protein.

We used indirect immunofluorescence to examine the factors determining the intranuclear location of herpes simplex virus (HSV) DNA polymerase (Pol) in infected cells. In the absence of viral DNA replication, HSV Pol colocalized with the HSV DNA-binding protein ICP8 in nuclear framework-associated structures called prereplicative sites. In the presence of viral DNA replication, HSV Pol colocalized with ICP8 in globular intranuclear structures called replication compartments. In cells infected with mutant viruses encoding defective ICP8 molecules, Pol localized within the cell nucleus but showed a general diffuse intranuclear distribution. In uninfected cells transfected with a plasmid expressing Pol, Pol similarly showed a diffuse intranuclear distribution. Therefore, Pol can localize to the cell nucleus without other viral proteins, but functional ICP8 is required for Pol to localize to prereplicative sites. In cells infected with mutant viruses encoding defective Pol molecules, ICP8 localized to prereplicative sites. Thus, Pol or the portions of Pol not expressed by the mutant viruses are not essential for the formation of prereplicative sites or the localization of ICP8 to these structures. These results demonstrate that a specific nuclear protein can influence the intranuclear location of another nuclear protein.     

Equilibrium binding of single-stranded DNA with herpes simplex virus type I-coded single-stranded DNA-binding protein, ICP8

We have carried out solution equilibrium binding studies of ICP8, the major single-stranded DNA (ssDNA)-binding protein of herpes simplex virus type I, in order to determine the thermodynamic parameters for its interaction with ssDNA. Fluorescence anisotropy measurements of a 5'-fluorescein-labeled 32-mer oligonucleotide revealed that ICP8 formed a nucleoprotein filament on ssDNA with a binding site size of 10 nucleotides/ICP8 monomer, an association constant at 25 degrees C, K = 0.55 +/- 0.05 x 10(6) M(-1), and a cooperativity parameter, omega = 15 +/- 3. The equilibrium constant was largely independent of salt, deltalog(Komega)/deltalog([NaCl]) = -2.4 +/- 0.4. Comparison of these parameters with other ssDNA-binding proteins showed that ICP8 reacted with an unusual mechanism characterized by low cooperativity and weak binding. In addition, the reaction product was more stable at high salt concentrations, and fluorescence enhancement of etheno-ssDNA by ICP8 was higher than for other ssDNA-binding proteins. These last two characteristics are also found for protein-DNA complexes formed by recombinases in their active conformation. Given the proposed role of ICP8 in promoting strand transfer reactions, they suggest that ICP8 and recombinase proteins may catalyze homologous recombination by a similar mechanism.     

Conformational changes in the herpes simplex virus ICP8 DNA-binding protein coincident with assembly in viral replication structures

The herpes simplex virus (HSV) single-stranded DNA-binding protein, ICP8, is required for viral DNA synthesis. Before viral DNA replication, ICP8 colocalizes with other replication proteins at small punctate foci called prereplicative sites. With the onset of viral genome amplification, these proteins become redistributed into large globular replication compartments. Here we present the results of immunocytochemical and biochemical analysis of ICP8 showing that various antibodies recognize distinct forms of ICP8. Using these ICP8-specific antibodies as probes for ICP8 structure, we detected a time-dependent appearance and disappearance of ICP8 epitopes in immunoprecipitation assays. Immunofluorescence staining of ICP8 in cells infected with different HSV mutant viruses as well as cells transfected with a limited number of viral genes demonstrated that these and other antigenic changes occur coincident with ICP8 assembly at intranuclear replication structures. Genetic analysis has revealed a correlation between the ability of various ICP8 mutant proteins to form the 39S epitope and their ability to bind to DNA. These results support the hypothesis that ICP8 undergoes a conformational change upon binding to other HSV proteins and/or to DNA coincident with assembly into viral DNA replication structures.     

ATP-dependent unwinding of a minimal origin of DNA replication by the origin-binding protein and the single-strand DNA-binding protein ICP8 from herpes simplex virus type I

The Herpes simplex virus type I origin-binding protein, OBP, is encoded by the UL9 gene. OBP binds the origin of DNA replication, oriS, in a cooperative and sequence-specific manner. OBP is also an ATP-dependent DNA helicase. We have recently shown that single-stranded oriS folds into a unique and evolutionarily conserved conformation, oriS*, which is stably bound by OBP. OriS* contains a stable hairpin formed by complementary base pairing between box I and box III in oriS. Here we show that OBP, in the presence of the single-stranded DNA-binding protein ICP8, can convert an 80-base pair double-stranded minimal oriS fragment to oriS* and form an OBP-oriS* complex. The formation of an OBP-oriS* complex requires hydrolysable ATP. We also demonstrate that OBP in the presence of ICP8 and ATP promotes slow but specific and complete unwinding of duplex minimal oriS. The possibility that the OBP-oriS* complex may serve as an assembly site for the herpes virus replisome is discussed.     

Circular dichroism analysis of insect cell expressed herpes simplex virus type I single-stranded DNA-binding protein ICP8.

We have developed a baculovirus expression system for the rapid and efficient production of large quantities (>5 mg/10(8) cells) of ICP8. The recombinant ICP8 is fully functional and binds to single-stranded DNA. Secondary structure calculations from circular dichroism measurements indicate a content of 34.5% alpha-helix and 15.4% beta-sheet. This is the first structural report for ICP8 using CD analysis, which will be very useful for high-throughput assay development and mechanistic studies.     

Two-dimensional crystallization of herpes simplex virus type 1 single-stranded DNA-binding protein, ICP8, on a lipid monolayer

Herpes simplex virus type 1 single-stranded DNA-binding protein (ICP8) has been crystallized on a positively charged lipid monolayer. The crystals belong to the planar group p2 with a=39 nm, b=23.2 nm and gamma=87.2 degrees. The projected map of ICP8 crystals calculated at a resolution of 3.9 nm shows four ICP8 monomers per unit cell with the crystals formed by a parallel arrangement of 16.2 nm helical ICP8 filaments. This novel filamentous form has not been reported before. The ICP8 monomers show different appearances in projection, suggesting that they may adopt different orientations, probably reflecting the strong intermolecular and lipid-filament interactions in the crystal. When the 23 nm diameter filaments formed by ICP8 in solution at low temperature in the presence of magnesium were generated and then layered on the phospholipid monolayer, highly ordered arrays of an 8.5 nm filament with a shallow 31.2 nm pitch were observed and reconstruction revealed a double-helical structure.     

DNA-binding protein associated with herpes simplex virus DNA polymerase.

Purified preparations of herpes simplex virus type 2 DNA polymerase made by many different laboratories always contain at least two polypeptides. The major one, of about 150,000 molecular weight, has been associated with the polymerase activity. The second protein, of about 54,000 molecular weight, which we previously designated ICSP 34, 35, has now been purified. The purified protein has been used to prepare antisera (both polyclonal rabbit serum and monoclonal antibodies). These reagents have been used to characterize the protein, to demonstrate its quite distinct map location from that of the DNA polymerase on the herpes simplex virus genome, and to demonstrate the close association between the two polypeptides.     

Interaction between the DNA polymerase and single-stranded DNA-binding protein (infected cell protein 8) of herpes simplex virus 1

The herpes virus-encoded DNA replication protein, infected cell protein 8 (ICP8), binds specifically to single-stranded DNA with a stoichiometry of one ICP8 molecule/12 nucleotides. In the absence of single-stranded DNA, it assembles into long filamentous structures. Binding of ICP8 inhibits DNA synthesis by the herpes-induced DNA polymerase on singly primed single-stranded DNA circles. In contrast, ICP8 greatly stimulates replication of circular duplex DNA by the polymerase. Stimulation occurs only in the presence of a nuclear extract from herpes-infected cells. Appearance of the stimulatory activity in nuclear extracts coincides closely with the time of appearance of herpes-induced DNA replication proteins including ICP8 and DNA polymerase. A viral factor(s) may therefore be required to mediate ICP8 function in DNA replication.