Purified Herpes Simplex Virus Type Research Articles

Herpesviruses Enhance Fibrinogen Clot Lysis.

AbstractAbstract 1153 Background:Members of the Herpesvirus family have been implicated in vascular disease. To explain the correlation on a molecular basis we have shown that the virus envelope contains anionic phospholipid derived from host cells, and proteins encoded by the host (tissue factor) as well as the viral (glycoprotein C) genomes, which initiate blood coagulation. This suggests that virus infection should be a strong independent predictor of vascular disease. Nevertheless, the clinical correlation is relatively weak, becoming more significant in combination with other risk factors. To explain this discrepancy, the current work is based on our additional report that at least one Herpesvirus (cytomegalovirus (CMV)) has host-genome-encoded annexin II on its surface. Annexin II is known to accelerate tissue plasminogen activator (tPA)-mediated activation of plasminogen to plasmin because of C-terminal lysines that interact with both plasminogen and tPA. Plasmin is the primary fibrinolytic protease, and is necessary for physiological clot dissolution. Thus, these viruses may facilitate clearance of the fibrin they generate. Hypothesis:We hypothesize that Herpesviruses enhance tPA-mediated plasmin generation and this mechanism correlates to the presence of annexin II on the virus. Methods:Purified herpes simplex virus type 1 (HSV1) and 2 (HSV2) and CMV were quantified by electron microscopy. Annexin II expression varies between cell types, therefore HSV1 was propagated in several cell lines. Virus-dependent plasmin generation was followed in the presence of purified plasminogen and tPA using a chromogenic assay. The contribution of viruses to fibrin clot lysis using purified proteins was investigated by light scattering. Plasminogen-conjugated horse radish peroxidase (plasminogen-HRP) and western blots were used to identify plasminogen-binding species and annexin II associated with the virus, respectively. The effect of plasmin-mediated signalling on virus infection was determined using cytopathic plaque assays. Results:Chromogenic experiments demonstrated that HSV1, HSV2 and CMV enhanced plasminogen activation in a dose-dependent manner by up to 5-fold, regardless of the parental cell line. Prolonged incubation confirmed the requirement for exogenous tPA. Plasminogen-HRP bound to a number of virus-associated proteins and was shown to be C-terminal lysine-dependent by complete inhibition with epsilon-aminocaproic acid (EACA). Annexin II was demonstrated to be associated with purified HSV1 cultured in different cells except when propagated in a melanoma (A7) line that did not express annexin II. An annexin II antibody inhibited binding of plasminogen-HRP to viral annexin II. HSV1, HSV2 and CMV accelerated fibrin clot lysis, which was inhibited in the presence of EACA and aprotinin, a plasmin inhibitor. However, in contrast to the chromogenic experiment for plasmin generation, each virus also exhibited a clot lysis mechanism independent of added tPA. As we have previously identified for thrombin, incubation of host cells with purified plasmin during inoculation enhanced virus infection by over 3-fold. Conclusion:Cumulatively these data demonstrate that HSV1, HSV2 and CMV accelerate tPA- mediated plasmin generation in the absence of fibrin and identify annexin II as one of several plasminogen binding partners. These viruses furthermore enhance the rate of fibrin clot lysis. However, in the presence of fibrin, purified HSV1, HSV2 and CMV are capable of facilitating clot dissolution in the absence of exogenous tPA. The molecular basis for this novel mechanism is not yet known, but requires plasminogen activation. The finding that purified plasmin enhances infection suggests these viruses may have evolved to initiate plasmin generation. Hence, the virus-mediated activation of fibrinolysis may compensate for its ability to trigger coagulation and attenuate potential contributions as an independent predictor of vascular disease. Disclosures:No relevant conflicts of interest to declare.

Thrombin enhances herpes simplex virus infection of cells involving protease-activated receptor 1.

We have previously shown that the surface of purified herpes family viruses can initiate thrombin production by expressing host-encoded and virus-encoded procoagulant factors. These enable the virus to bypass the normal cell-regulated mechanisms for initiating coagulation, and provide a link between infection and vascular disease. In the current study we investigated why these viruses may have evolved to generate thrombin. Using cytolytic viral plaque assays, the current study examines the effect of thrombin on human umbilical vein endothelial cell (HUVEC) or human foreskin fibroblast (HFF) infection by purified herpes simplex virus type 1 (HSV1) and type 2 (HSV2). Demonstrating that the availability of thrombin is an advantage to the virus, purified thrombin added to serum-free inoculation media resulted in up to a 3-fold enhancement of infection depending on the virus strain and cell type. The effect of thrombin on HUVEC infection was generally greater than its effect on HFF. To illustrate the involvement of thrombin produced during inoculation, hirudin was shown to inhibit the infection of each HSV strain, but only when serum containing clotting factors for thrombin production was present in media. The involvement of protease-activated receptor 1 (PAR1) was supported using PAR1-activating peptides in place of thrombin and PAR1-specific antibodies to inhibit the effects of thrombin. These data show that HSV1 and HSV2 initiate thrombin production to increase the susceptibility of cells to infection through a mechanism involving PAR1-mediated cell modulation.

Role of Protein-Protein Interactions during Herpes Simplex Virus Type 1 Recombination-dependent Replication

Recombination-dependent replication is an integral part of the process by which double-strand DNA breaks are repaired to maintain genome integrity. It also serves as a means to replicate genomic termini. We reported previously on the reconstitution of a recombination-dependent replication system using purified herpes simplex virus type 1 proteins (Nimonkar A. V., and Boehmer, P. E. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10201-10206). In this system, homologous pairing by the viral single-strand DNA-binding protein (ICP8) is coupled to DNA synthesis by the viral DNA polymerase and helicase-primase in the presence of a DNA-relaxing enzyme. Here we show that DNA synthesis in this system is dependent on the viral polymerase processivity factor (UL42). Moreover, although DNA synthesis is strictly dependent on topoisomerase I, it is only stimulated by the viral helicase in a manner that requires the helicase-loading protein (UL8). Furthermore, we have examined the dependence of DNA synthesis in the viral system on species-specific protein-protein interactions. Optimal DNA synthesis was observed with the herpes simplex virus type 1 replication proteins, ICP8, DNA polymerase (UL30/UL42), and helicase-primase (UL5/UL52/UL8). Interestingly, substitution of each component with functional homologues from other systems for the most part did not drastically impede DNA synthesis. In contrast, recombination-dependent replication promoted by the bacteriophage T7 replisome was disrupted by substitution with the replication proteins from herpes simplex virus type 1. These results show that although DNA synthesis performed by the T7 replisome is dependent on cognate protein-protein interactions, such interactions are less important in the herpes simplex virus replisome.

Rep-dependent initiation of adeno-associated virus type 2 DNA replication by a herpes simplex virus type 1 replication complex in a reconstituted system.

Productive infection by adeno-associated virus type 2 (AAV) requires coinfection with a helper virus, e.g., adenovirus or herpesviruses. In the case of adenovirus coinfection, the replication machinery of the host cell performs AAV DNA replication. In contrast, it has been proposed that the herpesvirus replication machinery might replicate AAV DNA. To investigate this question, we have attempted to reconstitute AAV DNA replication in vitro using purified herpes simplex virus type 1 (HSV-1) replication proteins. We show that the HSV-1 UL5, UL8, UL29, UL30, UL42, and UL52 gene products along with the AAV Rep68 protein are sufficient to initiate replication on duplex DNA containing the AAV origins of replication, resulting in products several hundred nucleotides in length. Initiation can occur also on templates containing only a Rep binding site and a terminal resolution site. We further demonstrate that initiation of DNA synthesis can take place with a subset of these factors: Rep68 and the UL29, UL30, and UL42 gene products. Since the HSV polymerase and its accessory factor (the products of the UL30 and UL42 genes) are unable to efficiently perform synthesis by strand displacement, it is likely that in addition to creating a hairpin primer, the AAV Rep protein also acts as a helicase for DNA synthesis. The single-strand DNA binding protein (the UL29 gene product) presumably prevents reannealing of complementary strands. These results suggest that AAV can use the HSV replication apparatus to replicate its DNA. In addition, they may provide a first step for the development of a fully reconstituted AAV replication assay.

Synthesis of a Photoaffinity Analog of 3′-Azidothymidine, 5-Azido-3′-azido-2′,3′-dideoxyuridine.: INTERACTIONS WITH HERPESVIRUS THYMIDINE KINASE AND CELLULAR ENZYMES

Long term administration of 3'-azidothymidine (AZT) for the treatment of AIDS has led to detrimental clinical side effects in some patients, the biochemical causes of which are still being delineated. Base-substituted, azido-nucleotide photoaffinity analogs have routinely proven to be effective tools for identifying and characterizing nucleotide-utilizing enzymes. Therefore, we have synthesized 5-azido-3'-azido-2',3'-dideoxyuridine, which is a potential photoaffinity analog of two human immunodeficiency virus drugs, AZT and 3'azido-2',3'-dideoxyuridine. A partially purified herpes simplex virus type 1 thymidine kinase and [gamma-32P]ATP were used to make an AZT monophosphate analog, [32P]5-azido-3'-azido-2',3'-dideoxyuridine monophosphate. The photoaffinity properties of this analog were initially tested with herpes simplex virus type 1 thymidine kinase. Photoaffinity labeling of this enzyme was saturable (half-maximal, 30 microM) and could be specifically inhibited by AZT, AZT monophosphate, thymidine, and thymidine monophosphate. Photolabeling of rat liver microsomal membranes was also done, and several membrane proteins that interact with AZT monophosphate were identified. The antiviral and cytotoxic activities of 5-azido-3'-azido-2',3'-dideoxyuridine were determined using human immunodeficiency virus, type 1 strain IIIB and an AZT drug-resistant strain in human T lymphocyte H9 cells.

Mechanism of cytostatic action of novel 5-(thien-2-yl)- and 5-(furan-2-yl)-substituted pyrimidine nucleoside analogues against tumor cells transfected by the thymidine kinase gene of herpes simplex virus.

Several novel 5-substituted 2'-deoxyuridine (dUrd) analogues were evaluated as substrates for highly purified herpes simplex virus type 1 (HSV-1)-encoded thymidine kinase (TK) derived from HSV-1 TK gene-transfected murine mammary carcinoma FM3A cells, and human platelet thymidine (dThd) phosphorylase. The Ki of 5-(furan-2-yl)-dUrd, 5-(thien-2-yl)-dUrd and 5-(thien-2-yl)-dCyd for HSV-1 TK was 0.94, 0.71, and 1.32 microM, respectively. Inhibition was competitive with respect to the natural substrate dThd. With dCyd as substrate, the Ki of 5-(thien-2-yl)-dCyd for HSV-1 TK was 4.5 microM (Ki/Km = 0.17). In striking contrast with (E)-5-(2-bromovinyl)-dUrd, the 5-(thien-2-yl)- and 5-(furan-2-yl)-dUrd derivatives were not substrates for human dThd phosphorylase. 5-(Thien-2-yl)-dUrd, 5-(furan-2-yl)-dUrd and 5-(thien-2-yl)-dCyd were at least 100-fold more cytostatic to the HSV TK gene-transfected FM3A tumor cells than wild-type FM3A/0 cells. The viral TK expressed in the HSV-1 TK gene-transfected tumor cells merely acts as an activating enzyme, whereas thymidylate synthase serves as the target enzyme for the cytostatic action of the compounds. The novel 5-substituted dUrd analogues should be further pursued as candidate drugs in the treatment of HSV TK gene-transfected tumors.

Deletions of the carboxy terminus of herpes simplex virus type 1 UL42 define a conserved amino-terminal functional domain

The herpes simplex virus type 1 UL42 protein was synthesized in reticulocyte lysates and assayed for activity in vitro. Three functional assays were used to examine the properties of in vitro-synthesized UL42: (i) coimmunoprecipitation to detect stable complex formation with purified herpes simplex virus type 1 DNA polymerase (Pol), (ii) a simple gel-based assay for DNA binding, and (iii) a sensitive assay for the stimulation of Pol activity. UL42 synthesized in reticulocyte lysates formed a stable coimmunoprecipitable complex with Pol, bound to double-stranded DNA, and stimulated the activity of Pol in vitro. Carboxy-terminal truncations of the UL42 protein were synthesized from restriction enzyme-digested UL42 gene templates and gene templates made by polymerase chain reaction and assayed for in vitro activity. Truncations of the 488-amino-acid (aa) UL42 protein to aa 315 did not abolish its ability to bind to Pol and DNA or to stimulate Pol activity. Proteins terminating at aas 314 and 313 showed reduced levels of binding to Pol, but these and shorter proteins were unable to bind to DNA or to stimulate Pol activity. These results suggest that all three of the biochemical functions of UL42 colocalize entirely within the N-terminal 315 aas of the UL42 protein. Amino acid sequence alignment of alpha herpesvirus UL42 homologs revealed that the N-terminal functional domain corresponds to the most highly conserved region of the protein, while the dispensable C terminus is not conserved. Conservative aa changes at the C terminus of the 315-aa truncated protein were used to show that conserved residues were important for activity. These results suggest that 173 aa of UL42 can be deleted without a loss of activity and that DNA-binding and Pol-binding activities are correlated with the ability of UL42 to stimulate Pol activity.

Identification of a new glycoprotein of herpes simplex virus type 1 and genetic mapping of the gene that codes for it.

A type-specific monoclonal antibody, LP10, precipitated a glycoprotein with a molecular weight of approximately 59,000 from purified herpes simplex virus type 1. Although this glycoprotein was similar in size to glycoprotein D (gD), it was shown to be less abundant in both virions and infected cells, to migrate more rapidly in its precursor form, to incorporate glucosamine but not mannose, and to have a more stable precursor in tunicamycin-treated cells than the gD precursor (pgD). Immunoassays of cells infected with insertion recombinants and intertypic recombinants localized the gene coding for the target antigen of LP10 to the unique short (Us) region at map units 0.892 to 0.924 excluding gD. The target antigen of LP10 was then definitively mapped to the Us4 open reading frame by immunoprecipitation of a polypeptide synthesized by in vitro translation of a Us4-specific transcript prepared by using an SP6 cloning This newly identified glycoprotein product of the Us4 gene of herpes simplex virus type 1 is distinct from the previously identified gB1, gC1, gE1, and gH1.

Metabolism and mode of action of (R)-9-(3,4-dihydroxybutyl)guanine in herpes simplex virus-infected vero cells.

The metabolism and mode of action of the anti-herpes compound buciclovir [R)-9-(3,4-dihydroxybutyl)-guanine, BCV) has been studied in herpes simplex virus-infected and uninfected Vero cells. In uninfected cells, a low and constant concentration of intracellular BCV was found, while in herpes simplex virus-infected cells, an increasing concentration of BCV phosphates was found due to metabolic trapping. The major phosphorylation product was BCV triphosphate (BCVTP) which was 92% of the total amount of BCV phosphates. BCV phosphates were accumulated to the same extent in cells infected with either a herpes simplex virus type 1 or a herpes simplex virus type 2 strain while thymidine kinase-deficient mutants of herpes simplex virus type 1 were 10 times less efficient in accumulating BCV phosphates. In uninfected Vero cells, the concentration of the phosphorylated forms of BCV was less than 1% of that found in herpes simplex virus-infected cells. The BCVTP formed in herpes simplex virus-infected cells was highly stable, as 80% of the amount of BCVTP was still present even 17 h after removal of extracellular BCV. BCV was a good substrate for herpes simplex virus type 1- and type 2-induced thymidine kinases but not for the cellular cytosol or mitochondrial thymidine kinases. BCV monophosphate could be phosphorylated by cellular guanylate kinase to BCV diphosphate. BCVTP was a selective and competitive inhibitor to deoxyguanosine triphosphate of the purified herpes simplex virus type 1- and type 2-induced DNA polymerases. BCVTP could neither act as an alternative substrate in the herpes simplex virus type 2 or cellular DNA polymerase reactions, nor could [3H]BCV monophosphate be detected in DNA formed by herpes simplex virus type 2 DNA polymerase, or be detected in nucleic acids extracted from herpes simplex virus type 1-infected cells. These data indicate that BCVTP may inhibit the herpes simplex virus-induced DNA polymerase without being incorporated into DNA.

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.

Binding to cells of virosomes containing herpes simplex virus type 1 glycoproteins and evidence for fusion.

Envelope proteins and lipids were extracted from purified herpes simplex virus type 1 virions with octyl glucoside and mixed with phosphatidylcholine for preparation of virosomes by removal of the detergent. Greater than 85% of the extracted envelope proteins, including all the glycoproteins and the nonglycosylated protein designated VP16, were associated with virosomes, which ranged in density from ca. 1.07 to 1.13 g/cm3. All the glycoproteins except gC were as susceptible to degradation by added protease in virosomes as in virions, indicating similar orientations in both. Approximately 30 to 40% of radiolabel incorporated into virosomes bound to HEp-2 cells within 1.5 h at either 4 or 37 degrees C. The cell-bound virosomes were enriched for gB and deficient in other glycoproteins, in comparison with unbound or total virosomes. Binding of virosomes to HEp-2 cells could be inhibited by purified virus, heparin, and monospecific antiviral antibodies. Polyclonal and monoclonal anti-gB antibodies were more effective at inhibiting virosome binding than were anti-gD or anti-gC antibodies. Virosomes depleted of gB or gD did not bind to cells as efficiently as did virosomes containing all the extracted enveloped components; this loss of binding activity was especially pronounced on depletion of gB. The binding of herpes simplex virus type 1 virosomes to cells is discussed in relation to possible heterogeneity of the virosomes and comparisons with binding of virions to cells. We also present electron microscopic evidence that bound virosomes can fuse with the cell surface.

Phosphorylation of four acyclic guanosine analogs by herpes simplex virus type 2 thymidine kinase.

Four acyclic guanosine analogs, the (R) and (S) enantiomers of 9-(3,4-dihydroxybutyl)guanine, 9-(4-hydroxybutyl)guanine, and acyclovir were compared in enzyme kinetic experiments, using purified herpes simplex virus type 2 thymidine kinase. All four analogs showed competitive patterns of inhibition in the phosphorylation of thymidine by the viral thymidine kinase, but different affinities and relative rates of phosphorylation were observed.

Direct demonstration that the abundant 6-kilobase herpes simplex virus type 1 mRNA mapping between 0.23 and 0.27 map units encodes the major capsid protein VP5.

The two partially colinear 6-kilobase (kb) and 1.5-kb mRNAs mapping between 0.23 and 0.27 map units on the herpes simplex virus type 1 genome were precisely located. The 5' end of the 6-kb mRNA was located 28 bases downstream of the sequence ATATATT and was 10 bases to the left of the BamHI site at 0.268. This position is ca. 90 bases to the left of our earlier reported sequence (R. J. Frink, K. G. Draper, and E. K. Wagner, Proc. Natl. Acad. Sci. U.S.A. 78:6139-6143, 1981). We used a polyclonal antibody made against purified herpes simplex virus type 1 VP5 to demonstrate that the 155,000-dalton translation product of the 6-kb mRNA is this capsid protein. The antibody did not react with the 35,000-dalton translation product of the 1.5-kb mRNA. We also confirmed our identification of VP5 as the translation product of the 6-kb mRNA by comparison of tryptic peptides of the in vitro-translated protein and authentic VP5.

Inactivation of herpes simplex virus by a pregnene derivative (TX 3047).

1. Two strains of independently purified Herpes simplex virus type 1 were irreversibly inactivated after incubation with the pregnene derivative methyl-p-toluene sulphonate of diethylamino-3-beta-ethoxy-20-hydroxy-pregn-5-ene (TX 3047) in vitro. 2. RNA and DNA synthesis (both cellular and viral) were studied in the presence of TX 3047. Viral and cellular DNA synthesis but not viral RNA synthesis was reduced. 3. Formation of infectious particles was not affected by TX 3047 if the parental virus was absorbed to the cell before it was incubated with TX 3047. This suggests that TX 3047 acts at the level of the membrane.

Correlation of herpes simplex virus antibody titers and specific lymphocyte stimulation in adult blood donors.

Antibody titers to herpes simplex virus type 1 in sera from healthy adult donors were assayed by complement fixation, microneutralization, and an enzyme immunoassay (ELISA). This last test proved to be the most sensitive method for antibody detection. It was estimated that ELISA antibody titers were up to 40-fold higher than neutralizing antibody titers and up to 100-fold higher than complement fixation antibody titers. Due to the higher sensitivity of ELISA, only 3 of 36 blood donors tested in this assay were shown to be seronegative, whereas 6 additional persons of the same group were termed seronegative by the microneutralization assay. Furthermore, four of the latter also did not respond in the complement fixation test. In vitro stimulation of peripheral lymphocytes by using a partially purified herpes simplex virus type 1 particle antigen was achieved for all seropositive blood donors. Only those three donors who were ELISA negative reacted negatively in this stimulation assay. From these results it may be concluded that ELISA is an appropriate method not only for rapid and sensitive antibody determination but also for selecting herpes simplex virus-negative patients.

5'-Amino-5'-deoxythymidine: synthesis, specific phosphorylation by herpesvirus thymidine kinase, and stability to pH of the enzymically formed diphosphate derivative.

Radioactive 5'-amino-5'-deoxythymidine (5'-AdThd), an inhibitor of herpes simplex virus, was synthesized by direct amination of monotosylated thymidine with concentrated ammonium hydroxide. Incubation of 5'-AdThd with purified herpes simplex virus type 1-encoded pyrimidine deoxyribonucleoside kinase produced the diphosphate derivative. The monphosphate derivative of 5'-AdThd was not detected as a reaction product of this enzymatic phosphorylation. A purified mixture of nonviral thymidine kinase and thymidylate kinase derived from uninfected Vero cells was unable to phosphorylate 5'-AdThd under identical conditions. The rate of hydrolysis of 5'-AdThd diphosphate increased as the pH of the reaction mixture decreased, with a shoulder region appearing between pH 3 and 5. The rate of hydrolysis was markedly increased below pH 3. 5'-AdThd, but not the monophosphate derivative, was detected in the hydrolysis mixture. The hydrolysis of 5'-AdThd diphosphate pH 3 also yielded some thymine in addition to 5'-AdThd.

Serum antibodies to herpes simplex virus type 1 during active oral herpes infection.

Subjects with oral herpes lesions at the time of serum sampling had higher-efficiency antibody (higher proportion of neutralizing antibody as determined by plaque reduction, compared with total antibody as detected by radioimmunoassay) to herpes simplex virus type 1 (HSV-1) than did subjects with no lesions at the time of serum sampling. These higher-efficiency sera also had higher antibody titers to structural components of herpes simplex virus type 1 than did the low-efficiency sera. Absorption of high- and low-efficiency sera with purified herpes simplex virus type 1 particles removed all neutralizing antibody but not all antibody detected by radioimmunoassay. High-efficiency serum was depleted of more antibody to particulate antigen that was the low-efficiency serum, indicating that the high-efficiency serum contained a higher proportion of antibody to the virus particle.

Deoxythymidine kinase induced in HeLa TK- cells by herpes simplex virus type I and type II. II. Purification and characterization.

Deoxythymidine kinase activities were induced in HeLa TK- (deoxythymidine kinase-deficient) cells infected with either herpes simplex virus type I or herpes simplex virus type II. The herpes simplex virus type I-induced enzyme was found in the cytoplasmic and nuclear fractions of the infected cells, whereas the herpes simplex type II-induced deoxythymidine kinase could only be found in the cytoplasm. Herpes simplex virus type I and II specific deoxythymidine kinases were purified by affinity column chromatography. Both purified deoxythymidine kinases retained the deoxycytidine kinase activity present in the crude preparation. The purified herpes simplex virus type I deoxythymidine kinase had a different mobility on electrophoresis, but the same sedimentation rate on a glycerol gradient as the corresponding unpurified enzyme, whereas the purified herpes simplex virus type II deoxythymidine kinase had the same mobility and sedimentation rate as the corresponding unpurified enzyme. In the presence of Mg2+ATP and dithiothreitol, herpes simplex virus type II deoxythymidine kinase was more stable than herpes simplex virus type I deoxythymidine kinase at both 45 degrees and 4 degrees. The deoxycytidine kinase activity present in the purified preparations was inactivated at the same rate as the deoxythymidine kinase activity. In the presence of the other substrate, deoxythymidine, herpes simplex virus type I deoxythymidine kinase was more stable than herpes simplex virus type II kinase. The purified herpes simplex virus type I and II deoxythymidine kinase had different activation energies when Mg2+ATP and deoxythymidine were used as substrates, but showed the same sensitivity toward ammonium sulfate inhibition.

Comparison of the virion proteins specified by herpes simplex virus types 1 and 2

Purified herpes simplex virus type 2 (HSV-2) virions were found to contain approximately the same number of polypeptides as HSV type 1 (HSV-1) virions. Comparisons of the structural proteins specified by five independent HSV-2 isolates revealed some minor differences in their electrophoretic profiles on sodium dodecyl sulfate-acrylamide gels; certain invariant features of the electrophoretic profiles, however, allowed clear differentiation between all the HSV-2 isolates and HSV-1.

Defective Virions of Herpes Simplex Viruses

Serial undiluted passage of clonally purified herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in 4 different types of cells resulted in (i) partial loss (84–98%) of infectivity and (ii) the app