Structure-function studies of the herpes simplex virus type 1 DNA polymerase.

The analysis of the deduced amino acid sequence of the herpes simplex virus type 1 (HSV-1) DNA polymerase reported here suggests that the polymerase structure consists of domains carrying separate biological functions. The HSV-1 enzyme is known to possess 5'-3'-exonuclease (RNase H), 3'-5'-exonuclease, and DNA polymerase catalytic activities. Sequence analysis suggests an arrangement of these activities into distinct domains resembling the organization of Escherichia coli polymerase I. In order to more precisely define the structure and C-terminal limits of a putative catalytic domain responsible for the DNA polymerization activity of the HSV-1 enzyme, we have undertaken in vitro mutagenesis and computer modeling studies of the HSV-1 DNA polymerase gene. Sequence analysis predicts that the major DNA polymerization domain of the HSV-1 enzyme will be contained between residues 690 and 1100, and we present a three-dimensional model of this region, on the basis of the X-ray crystallographic structure of the E. coli polymerase I. Consistent with these structural and modeling studies, deletion analysis by in vitro mutagenesis of the HSV-1 DNA polymerase gene expressed in Saccharomyces cerevisiae has confirmed that certain amino acids from the C terminus (residues 1073 to 1144 and 1177 to 1235) can be deleted without destroying HSV-1 DNA polymerase catalytic activity and that the extreme N-terminal 227 residues are also not required for this activity.     

A DNA helicase induced by herpes simplex virus type 1.

We have identified and partially purified a DNA-dependent ATPase that is present specifically in herpes simplex virus type 1-infected Vero cells. The enzyme which has a molecular weight of approximately 440,000 differs from the comparable host enzyme in its elution from phosphocellulose columns and in its nucleoside triphosphate specificity. The partially purified DNA-dependent ATPase is also a DNA helicase that couples ATP or GTP hydrolysis to the displacement of an oligonucleotide annealed to M13 single-stranded DNA. The enzyme requires a 3' single-stranded tail on the duplex substrate, suggesting that the polarity of unwinding is 5'----3' relative to the M13 DNA. The herpes specific DNA helicase may therefore translocate on the lagging strand in the semidiscontinuous replication of the herpes virus 1 genome.     

Comparison of DNA facilitators in the uptake and intracellular fate of infectious herpes simplex virus type 2 DNA.

The comparative efficiencies of polyornithine, CaCl2 and DEAE-dextran in enhancing the infectivity of exogenous herpes simplex virus (HSV) type 2 DNA were examined. CaCl2 was 12-times more effective in promoting genetic expression of viral DNA than DEAE-dextran, while polyornithine did not mediate any HSV DNA infectivity. A comparison of sedimentation profiles of DNA extracted from cells inoculated with viral DNA in the presence of each facilitator revealed that input 56 S HSV DNA underwent marked alteration with time. There was also extensive processing of a 20 S DNA species. The data indicated that the biological efficiency of each facilitator was related to the amount of 20 S DNA remaining at the final time periods. One possible explanation for the efficiency of CaCl2 as a facilitator was that it allowed for the reutilization of the 20 S DNA species during HSV replication. The persistence of the 20 S peak in cells treated with DEAE-dextran was a measure of the decreased efficacy of this facilitator. Finally, the absence of a 56 S peak and the greatly elevated levels of 20 S DNA at final time points in polyornithine-treated cells accounted for the failure of this compound to promote HSV DNA infectivity.     

Ribonucleotide reductase of herpes simplex virus type 2 resembles that of herpes simplex virus type 1.

The ribonucleotide reductase (ribonucleoside-diphosphate reductase; EC 1.17.4.1) induced by herpes simplex virus type 2 infection of serum-starved BHK-21 cells was purified to provide a preparation practically free of both eucaryotic ribonucleotide reductase and contaminating enzymes that could significantly deplete the substrates. Certain key properties of the herpes simplex virus type 2 ribonucleotide reductase were examined to define the extent to which it resembled the herpes simplex virus type 1 ribonucleotide reductase. The herpes simplex virus type 2 ribonucleotide reductase was inhibited by ATP and MgCl2 but only weakly inhibited by the ATP X Mg complex. Deoxynucleoside triphosphates were at best only weak inhibitors of this enzyme. ADP was a competitive inhibitor (K'i, 11 microM) of CDP reduction (K'm, 0.5 microM), and CDP was a competitive inhibitor (K'i, 0.4 microM) of ADP reduction (K'm, 8 microM). These key properties closely resemble those observed for similarly purified herpes simplex virus type 1 ribonucleotide reductase and serve to distinguish these virally induced enzymes from other ribonucleotide reductases.     

Physical map of the origin of defective DNA in herpes simplex virus type 1 DNA.

The origin of defective DNA (dDNA) of the Patton strain of herpes simplex virus type 1 (HSV-1) was physically mapped with BamHI in the parental DNA. The dDNA obtained from virus passaged at high multiplicities of infection was resistant to cleavage with HindIII, whereas digestion with EcoRI yielded a cluster of fragments 5.4 to 5.7 megadaltons (Mdal) in size. Cleavage with BamHI gave a cluster of fragments 2.6 to 3.2 Mdal in size, plus two homogeneous, comigrating 1-Mdal fragments. One of the latter fragments contained the single EcoRI site approximately 65 base pairs from one end. Hybridization of in vitro labeled dDNA probe to EcoRI, HindIII, BamHI, and Hpa I digests of nondefective HSV-1 DNA demonstrated that, in addition to the S-region terminal repeat, only one end of the S region was involved in the generation of this class of dDNA. Thus, the dDNA probe did not hybridize to either the S region 3.0-Mdal HindIIIN fragment or a 3.0-Mdal BamHI fragment of the adjacent 8.7-Mdal HindIIIG fragment, but did hybridize to four BamHI fragments of HindIII G (approximately 5.7 Mdal). The cluster of 2.6- to 3.2-Mdal fragments obtained with BamHI digestion of dDNA appears to represent a novel junction between the termination of dDNA adjacent to the 3.0-Mdal BamHI fragment in HindIII G and the 2.0- to 2.3-Mdal BamHI fragment terminal in HSV-1 DNA.     

Properties of herpes simplex virus type 1 and type 2 DNA polymerase

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) DNA polymerases were highly purified from infected HeLa BU cells by DEAE cellulose, phosphocellulose and DNA cellulose column chromatography. DNA exonuclease activity but not endonuclease activity was found associated with both types of DNA polymerase. Both DNA polymerase activities could be activated by salt in a similar fashion with the optimal activity in the range of ionic strength between 0.22 and 0.29 alpha. At an ionic strength of 0.14, spermidine and putrescine in the concentration range (0--5 mM) studied could mimic the action of KCI in stimulating DNA polymerase activity. Spermine, in the same concentration range, had a biphasic effect. At an ionic strength of 0.29 all three polyamines were inhibitory. HSV-1 and HSV-2 DNA polymerase are similar in their column chromatographic behavior, sedimentation rate in sucrose gradient centrifugation, and activation energy, but they differ in their heat stability at 45 degrees C with the HSV-2 enzyme more stable than the HSV-1 enzyme. Kinetic behavior of both enzymes is similar, with Km values for deoxyribonucleoside triphosphates in the range of 5 . 10(-7) to 1.8 . 10(-8) M. IdUTP and dUTP served as apparent competitive inhibitors with respect to dTTP, and AraATP acted as an apparent competitive inhibitor with respect to dATP. AraATP could not replace dATP in the DNA polymerization reaction; in contrast, IdUTP could replace TTP. Phosphonoformic acid behaved as an uncompetitive inhibitor with respect to DNA. The ID(50) value estimated was foind to be dependent on the purity of the DNA polymerase used and the ionic strength of the assay condition. Each DNA-polymerase associated DNA exonuclease had the same stability at 45 degrees C as its DNA polymerase. The associated DNAase activity was inhibited by phosphonoformic acid and high ionic strength of the assay condition.     

Induced extrusion of DNA from the capsid of herpes simplex virus type 1.

DNA-filled capsids (C capsids) of herpes simplex virus type 1 were treated in vitro with guanidine-HCl (GuHCl) and analyzed for DNA loss by sucrose density gradient ultracentrifugation and electron microscopy. DNA was found to be lost quantitatively from virtually all capsids treated with GuHCl at concentrations of 0.5 M or higher, while 0.1 M GuHCl had little or no effect. DNA removal from 0.5 M GuHCl-treated capsids was effected without significant change in the capsid protein composition, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or in its structure, as judged by electron microscopy. Electron microscopic examination of capsids in the process of emptying showed that DNA was extruded from multiple, discrete sites which appeared to coincide with capsid vertices. DNA exited the capsid in the form of thick strands or fibers that varied in diameter from approximately 4 to 13 nm with preferred diameters of 7 and 11 nm. The fibers most probably correspond to multiple, laterally aligned DNA segments, as their diameters are nearly all greater than that of a single DNA double helix. The results suggest that GuHCl treatment promotes an alteration in the capsid pentons which allows DNA to escape locally. Hexons must be more resistant to this change, since DNA loss appears to be restricted to the pentons. The ability of GuHCl to cause loss of DNA from C capsids with no accompanying change in capsid morphology or protein composition suggests that penton sites may open transiently to permit DNA exist and then return to their original state.     

Suppression of apoptotic DNA fragmentation in herpes simplex virus type 1-infected cells.

HEp-2 cells underwent apoptosis when the cells were incubated in medium containing sorbitol. Infection with herpes simplex virus type 1 (HSV-1) prior to the sorbitol treatment suppressed this apoptosis completely, indicating that HSV-1 carries an antiapoptosis gene. In addition, HSV-1 multiplication was restricted in these apoptotic HEp-2 cells.     

DNA synthesis and DNA polymerase activity of herpes simplex virus type 1 temperature-sensitive mutants.

Fifteen temperature-sensitive mutants of herpes simplex virus type 1 were studied with regard to the relationship between their ability to synthesize viral DNA and to induce viral DNA polymerase (DP) activity at permissive (34 C) and nonpermissive (39 C) temperatures. At 34 C, all mutants synthesized viral DNA, while at 39 C four mutants demonstrated a DNA+ phenotype, three were DNA+/-, and eight were DNA-. DNA+ mutants induced levels of DP activity similar to thhose of the wild-type virus at both temperatures, and DNA+/- mutants induced reduced levels of DP activity at 39 C but not at 34 C. Among the DNA- mutants three were DP+, two were DP+/-, and three showed reduced DP activity at 34 C with no DP activity at 39 C. DNA-, DP- mutants induced the synthesis of a temperature-sensitive DP as determined by in vivo studies.     

Characterization of nuclear structures in cells infected with herpes simplex virus type 1 in the absence of viral DNA replication.

Herpes simplex virus type 1 DNA replication occurs in nuclear domains termed replication compartments, which are areas of viral single-stranded DNA-binding protein (UL29) localization (M.P. Quinlan, L. B. Chen, and D. M. Knipe, Cell 36:857-868). In the presence of herpesvirus-specific polymerase inhibitors, UL29 localizes to punctate nuclear foci called prereplicative sites. Using versions of the helicase-primase complex proteins containing short peptide epitopes which can be detected in an immunofluorescence assay, we have found that the helicase-primase complex localizes to prereplicative sites and replication compartments. To determine if prereplicative site formation is dependent upon these and other essential viral replication proteins, we have studied UL29 localization in cells infected with replication-defective viruses. Cells infected with viruses that fail to express one of the three helicase-primase subunits or the origin-binding protein show a diffuse nuclear staining for UL29. However, in the presence of polymerase inhibitors, mutant-infected cells contain UL29 in prereplicative sites. Replication-defective viruses containing subtle mutations in the helicase or origin-binding proteins behaved identically to their null mutant counterparts. In contrast, cells infected with viral mutants which fail to express the polymerase protein contain prereplicative sites in the absence and presence of polymerase inhibitors. We propose that active viral polymerase prevents the formation of prereplicative sites. Models of the requirement of essential viral replication proteins in the assembly of prereplicative sites are presented.     

Renaturation of complementary DNA strands by herpes simplex virus type 1 ICP8.

ICP8, the major single-stranded DNA-binding protein of herpes simplex virus type 1, promotes renaturation of complementary single strands of DNA. This reaction is ATP independent but requires Mg2+. The activity is maximal at pH 7.6 and 80 mM NaCl. The major product of the reaction is double-stranded DNA, and no evidence of large DNA networks is seen. The reaction occurs at subsaturating concentrations of ICP8 but reaches maximal levels with saturating concentrations of ICP8. Finally, the renaturation reaction is second order with respect to DNA concentration. The ability of ICP8 to promote the renaturation of complementary single strands suggests a role for ICP8 in the high level of recombination seen in cells infected with herpes simplex virus type 1.     

Latent herpes simplex virus type 1 DNA contains two copies of the virion DNA joint region.

Southern blot analysis of latent herpes simplex virus DNA detected in mouse brain and digested with a restriction enzyme revealed two copies of the virion DNA joint fragment. Thus, the absence of free ends noted previously in latent herpes simplex virus type 1 DNA is due to joining of the termini.     

Identification of herpes simplex virus type 1 genes required for origin-dependent DNA synthesis.

The herpes simplex virus (HSV) genome contains both cis- and trans-acting elements which are important in viral DNA replication. The cis-acting elements consist of three origins of replication: two copies of oriS and one copy of oriL. It has previously been shown that five cloned restriction fragments of HSV-1 DNA together can supply all of the trans-acting functions required for the replication of plasmids containing oriS or oriL when cotransfected into Vero cells (M. D. Challberg, Proc. Natl. Acad. Sci. USA, 83:9094-9098, 1986). These observations provide the basis for a complementation assay with which to locate all of the HSV sequences which encode trans-acting functions necessary for origin-dependent DNA replication. Using this assay in combination with the data from large-scale sequence analysis of the HSV-1 genome, we have now identified seven HSV genes which are necessary for transient replication of plasmids containing either oriS or oriL. As shown previously, two of these genes encode the viral DNA polymerase and single-stranded DNA-binding protein, which are known from conventional genetic analysis to be essential for viral DNA replication in infected cells. The functions of the products of the remaining five genes are unknown. We propose that the seven genes essential for plasmid replication comprise a set of genes whose products are directly involved in viral DNA synthesis.     

Recombinogenic properties of herpes simplex virus type 1 DNA sequences resident in simian virus 40 minichromosomes.

In a previous work, it was demonstrated that the bacterial transposon Tn5 is capable of undergoing sequence inversion via recombination between its duplicated IS50 elements when replicated by the herpes simplex virus type 1 (HSV-1) origin oris but not by the simian virus 40 (SV40) origin orisv. Further analysis of the latter phenomenon indicated that this lack of recombination was the result of topological constraints imposed by the SV40 minichromosome, such that recombination events could be readily detected in Tn5 derivatives in which the IS50 elements were arranged in a direct rather than inverted orientation. With this information, a second set of experiments were carried out to examine how the highly recombinogenic sequences which mediate the inversion of the long (L) and short (S) components of the HSV-1 genome behave in an SV40 minichromosome. Tandem copies of the L-S junction of the HSV-1 genome were observed to promote deletions in an SV40 shuttle plasmid at a frequency that was considerably greater than that of duplicated bacterial plasmid vector DNA. However, the presence of superinfecting HSV-1 did not enhance the frequency of these recombination events. These results support our previous findings that HSV-1 genome isomerization is mediated by a homologous recombination mechanism which is intimately associated with the act of viral DNA synthesis. Moreover, they demonstrate that the sequences which comprise the L-S junction appear to be inherently recombinogenic and, therefore, do not contain specific signals required for HSV-1 genome isomerization.     

Functional expression of a cloned herpes simplex virus type 1 DNA polymerase gene.

A vector which expresses the herpes simplex virus type 1 (HSV-1) (strain 17) DNA polymerase gene was constructed by ligating two separately cloned HSV DNA restriction fragments into an intermediate plasmid and then mobilizing the intact polymerase gene-encoding sequence into a pSV2 derivative. The expression vector (pD7) contains a functional simian virus 40 replication origin and early enhancer-promoter upstream from the HSV DNA polymerase-encoding sequence. COS-1 cells transfected with pD7 contained an RNA species, shown by Northern blot analysis to hybridize specifically with an HSV DNA pol probe and to be the same size (4.3 kilobases) as the pol mRNA found in HSV-1-infected COS-1 cells. A genetic complementation test was used to establish that pD7 expresses a functional pol gene product. COS-1 cells transfected with pD7 were able to partially complement the growth defect of an HSV-1 (KOS) temperature-sensitive mutant, tsC7, in the DNA polymerase gene at the nonpermissive temperature.     

The DNA replication origins of herpes simplex virus type 1 strain Angelotti

The nucleotide sequences of the origins of DNA replication (ori) of the S- and L-component (oriS, oriL) of the herpes simplex virus type 1 (HSV-1) standard genome were determined from HSV-1 strain Angelotti (ANG). In contrast to other HSV-1 strains, the ANG oriS sequence revealed an insertion of an TA-dinucleotide in an otherwise very conserved but imperfect palindromic sequence of 47 bp. The oriL sequence of the standard ANG genome was found to be identical to that of an ANG class II defective genome which exhibits a duplication of a 144 bp palindrome. A model is presented to explain the origination of the amplified ANG oriL sequences from the parental genome with a single copy of oriL via illegitimate recombination. Alignment of the ori sequences of HSV, adeno- and papovaviruses unveiled that the HSV ori region can be subdivided into two distinct sites of homology to the DNA initiation signals of papova- and adenoviruses, suggesting that the HSV origins of replication comprise elements for DNA replication by both, cellular and virus-encoded DNA polymerases.     

Varicella-zoster virus complements herpes simplex virus type 1 temperature-sensitive mutants.

Varicella-zoster virus (VZV) can complement temperature-sensitive mutants of herpes simplex virus. Of seven mutants tested, two, carrying mutations in the immediate-early ICP4 and ICP27 proteins, were complemented. This complementation was not seen in coinfections with adenovirus type 5 or cytomegalovirus. Following transfection into CV-1 cells, a DNA fragment containing the VZV short repeat sequence complemented the ICP4 mutant. These data demonstrate a functional relationship between VZV and herpes simplex virus and have allowed localization of a putative VZV immediate-early gene.