Differential and selective inhibition of cellular and herpes simplex virus DNA synthesis by arabinofuranosyladenine.

The influence of 9-beta-D-arabinofuranosyladenine (beta araAdo) and of its anomer 9-alpha-D-arabinofuranosyladenine (alpha araAdo) was studied in non-infected cells and cells infected with herpes simplex virus type 1 (HSV-1) and HSV type 2 (HSV-2). alpha AraAdo is a strong inhibitor of proliferation of non-infected cells. Multiplication of HSV-1 and HSV-2 is not affected at all by alpha araAdo, while their growth is strongly inhibited by beta araAdo. alpha AraAdo exerts no effect on the incorporation of dThd into HSV DNA, but blocks the incorporation into host cell DNA. Its anomer, beta araAdo, affects the incorporation rate of both the viral DNA system and the host cell DNA system (the latter one to a lesser extent). alpha AraAMP is incorporated into newly synthesized cellular DNA but not into HSV DNA. Enzymic studies relevant that alpha araATP has no effect on the HSV DNA polymerase system but a high inhibitory potency in the host cell DNA polymerase alpha system. The anomeric form, beta araATP, is a sensitive inhibitor of HSV DNA polymerase while the cellular DNA polymerases alpha and beta are more refractory.     

Anatomy of herpes simplex virus DNA. V. Terminally repetitive sequences.

Native DNA from four strains of herpes simplex virus 1 (HSV-1) circularized after digestion with the lambda exonuclease, indicating that the molecules were terminally repetitious. In two strains, the terminal repetition was evident in nearly 50% of the DNA molecules. Maximal circularization was observed when only 0.25 to 0.5% of the DNA was depolymerized by the exonuclease, suggesting that the minimal size of the terminally repetitious regions is in the range of 400 to 800 bases pairs. More extensive exonuclease treatment resulted in a reduction in the frequency of circularization. To determine whether the terminally repetitive regions themselves contained self-annealing sequences that were precluding circularization of more extensively digested DNA, the terminal fragments from HinIII restriction endonuclease digests were isolated, denatured, and tested for their ability to self-anneal. The results of hydroxyapatite column chromatography and electron microscope examination of the terminal regions are consistent with this hypothesis.     

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.     

Virion component of herpes simplex virus type 1 KOS interferes with early shutoff of host protein synthesis induced by herpes simplex virus type 2 186.

Herpes simplex virus (HSV) strains HSV type 1 (HSV-1) KOS and HSV-2 186 are representative of delayed and early shutoff strains, respectively, with regard to their ability to inhibit protein synthesis in Friend erythroleukemia cells. When these cells were simultaneously infected with HSV-1 KOS and HSV-2 186, HSV-1 KOS interfered with the rapid suppression of globin synthesis induced by HSV-2 186. The observed interference was competitive and not due to exclusion of HSV-2 by HSV-1 at the level of adsorption. Furthermore, UV-irradiated HSV-1 KOS was also effective at interfering with the early shutoff function of HSV-2 186, indicating that a virion component is responsible for the observed interference.     

Isolation and characterization of herpes simplex virus type 1 host range mutants defective in viral DNA synthesis.

Cell lines were generated by cotransfection of Vero cells with pSV2neo and a plasmid containing the herpes simplex virus type 1 (HSV-1) EcoRI D fragment (coordinates 0.086 to 0.194). One such cell line (S22) contained the genes for alkaline exonuclease and several uncharacterized functions. Three mutant isolates of HSV-1 strain KOS which grew on S22 cells but not on normal Vero cells were isolated and characterized. All three mutants (hr27, hr48, and hr156) were defective in the synthesis of viral DNA and late proteins when grown in nonpermissive Vero cells. Early gene expression in cells infected with these host range mutants appeared to be normal at the nonpermissive condition. The mutations were mapped by marker rescue to a 1.5-kilobase fragment (coordinates 0.145 to 0.155). The mutation of one of these mutants, hr27, was more finely mapped to an 800-base-pair region (coordinates 0.145 to 0.151). This position of these mutations is consistent with the map location of a putative 94-kilodalton polypeptide as determined by sequence analysis (D. McGeoch, personal communication). Complementation studies demonstrated that these mutants formed a new complementation group, designated 1-36. The results presented in this report indicate that the 94-kilodalton gene product affected by these mutations may have a direct role in viral DNA synthesis.     

Anatomy of herpes simplex virus DNA. VI. Defective DNA originates from the S component.

We previously reported that serial propagation of the Justin strain of herpes simplex virus 1 [HSV-1 (Justin)] results in the generation of defective DNA molecules consisting of tandem repetitions of sequences of limited complexity. In the present study, HSV-1 DNA was cleaved with the restriction endonucleases BglII and EcoRI. The fragments were electrophoretically separated on agarose gels, transferred to nitrocellulose strips, and then hybridized with 32P-labeled HSV-1 (Justin) defective DNA. The data allow us to conclude that DNA sequences contained in the repeat unit of defective DNA originate from the S segment of the wild-type viral DNA molecule.     

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.     

The herpes simplex virus virion host shutoff function.

The virion host shutoff (vhs) function of herpes simplex virus (HSV) limits the expression of genes in the infected cells by destabilizing both host and viral mRNAs. vhs function mutants have been isolated which are defective in their ability to degrade host mRNA. Furthermore, the half-life of viral mRNAs is significantly longer in cells infected with the vhs-1 mutant virus than in cells infected with the wild-type (wt) virus. Recent data have shown that the vhs-1 mutation resides within the open reading frame UL41. We have analyzed the shutoff of host protein synthesis in cells infected with a mixture of the wt HSV-1 (KOS) and the vhs-1 mutant virus. The results of these experiments revealed that (i) the wt virus shutoff activity requires a threshold level of input virions per cell and (ii) the mutant vhs-1 virus protein can irreversibly block the wt virus shutoff activity. These results are consistent with a stoichiometric model in which the wt vhs protein interacts with a cellular factor which controls the half-life of cell mRNA. This wt virus interaction results in the destabilization of both host and viral mRNAs. In contrast, the mutant vhs function interacts with the cellular factor irreversibly, resulting in the increased half-life of both host and viral mRNAs.     

Cell-free synthesis of herpes simplex virus proteins.

Polyribosomes isolated from herpes simplex virus type I (HSV-1)-infected cells have been used to program a eucaryotic cell-free translation system. At least 10 HSV-specific polypeptides, with apparent molecular weights of 25,000 to 160,000, are synthesized by wild-type HSV-infected polyribosomes. Polyribosomes prepared from thymidine kinase-negative mutants of HSV direct the synthesis of three putative nonsense termination polypeptides. HSV-specific polypeptides synthesized in vitro are precipitated with antiserum to HSV-infected cell proteins.     

Anatomy of herpes simplex virus DNA. III. Characterization of defective DNA molecules and biological properties of virus populations containing them.

We have characterized the virus progeny and its DNA from plaque-purified and undiluted passages of herpes simplex virus 1 in HEp-2 cells. Secifically, (i) infectious virus yields declined progressively in passages 1 through 10 and gradually increased at passages 11 through 14. The yields correlated with PFU/particle ratios. (ii) In cells infected with virus from passages 6 through 10, there was an overproduction of an early viral polypeptide (no. 4) and a delay in the synthesis of late viral proteins. In addition, the virus in these passages interfered with the replication of a nondefective marker virus. Cells infected with passage 14 virus produced normal amounts of polypeptide 4 and, moreover, this virus showed minimal interfering capacity. (iii) In addition to DNA of density 1.726 g/cm-3, which was the sole component present in viral progeny of passage 0, passages 6 through 14 contained one additional species (p 1.732) and in some instances (passages 6 and 10) also DNA of an intermediate buoyant density. The ratio of p 1.732 to p 1.726 DNA increased to a maximum of 4 in passages 6 through 9 and gradually decreased to 1 in passages 10 through 14. (iv) p 1.732 DNA cannot be differentiated from p 1.726 DNA with respect to size; however, it has no Hin III restriction enzyme cleavage sites and yields only predominantly two kinds of fragments with molecular weights of 5.1 x 10-6 and 5.4 x 10-6 upon digestion with EcoRI enzyme. (v) Partial denaturation profiles of purified p 1.732 DNA from passage 14 revealed the presence of two types of tandemly repeated units corresponding roughly in size to the EcoRI fragments and situated in different molecules. (vi) In addition to the two kinds of p 1.732 molecules consisting of tandem repaeat units of different sizes, other evidence for the diversity of defective DNA molecules emerged from comparisons of specific infectivity and interfering capacity of the progeny from various passages. The data suggest that some of the particles with DNA of normal buoyant density (1.726) must also be defective since the capacity to interfere and to produce an excess of polypeptide 4 did not appear to be proportional to the amount of high-buoyant-density defective DNA. The data suggest that defective interfering particles are replaced by defective particles with diminished capacity to interfere and that more than one species of defective DNA molecules evolves on serial preparation of HSV.     

Inhibition of human immunodeficiency virus replication by the herpes simplex virus virion host shutoff protein.

The herpes simplex virus (HSV) virion host shutoff gene (vhs) encodes a protein which nonspecifically accelerates the degradation of mRNA molecules, leading to inhibition of protein synthesis. This ability to inhibit a critical cellular function suggested that vhs could be used as a suicide gene in certain gene therapy applications. To investigate whether vhs might be useful for treatment of AIDS, we tested the ability of both HSV type 1 (HSV-1) and HSV-2 vhs to inhibit replication of human immunodeficiency virus (HIV). Replication of HIV was substantially inhibited when an infectious HIV proviral clone was cotransfected into HeLa cells together with vhs under the control of the cytomegalovirus (CMV) immediate-early promoter. HSV-2 vhs was more active than HSV-1 vhs in these experiments, consistent with previously published studies on these genes. Since expression of vhs from the CMV promoter is essentially unregulated, we also tested the ability of vhs expressed from the HIV long terminal repeat (LTR) promoter to inhibit HIV replication. Wild-type HSV-1 vhs inhibited HIV replication more than 44,000-fold in comparison to a mutant vhs gene encoding a nonfunctional form of the Vhs protein. Production of Vhs in transfected cells was verified by Western blot assays. A larger amount of Vhs was observed in cells transfected with plasmids expressing vhs from the HIV LTR than from the CMV promoter, consistent with the greater inhibition of HIV replication observed with these constructs. Mutant forms of Vhs were expressed at higher levels than wild-type Vhs, most likely due to the ability of wild-type Vhs to degrade its own mRNA. The strong inhibitory activity of the vhs gene and its unique biological properties make vhs an interesting candidate for use as a suicide gene for HIV gene therapy.     

Structure of replicating herpes simplex virus DNA.

We have investigated the molecular anatomy of the herpes simplex virus replicative intermediates by cleavage with the restriction endonuclease BglII. We find that in populations of multiply infected cells, pulse-labeled replicating herpes simplex virus DNA contains at least two and probably all four sequence isomers. Also, it contains no detectable termini. In pulse-chase experiments, we show that endless replicative intermediates are the precursors to virion DNA and that maturation is a relatively slow process. The results are discussed in terms of their significance to possible models of herpes simplex virus DNA replication.     

Inhibition of host protein synthesis and degradation of cellular mRNAs during infection by influenza and herpes simplex virus

Cloned DNA copies of two cellular genes were used to monitor, by blot hybridization, the stability of particular cell mRNAs after infection by influenza virus and herpesvirus. The results indicated that the inhibition of host cell protein synthesis that accompanied infection by each virus could be explained by a reduction in the amounts of cellular mRNAs in the cytoplasm, and they suggested that this decrease was due to virus-mediated mRNA degradation.     

Cell-free synthesis of herpes simplex virus DNA: the influence of polyamines.

The effect of polyamines on cell-free DNA synthesis of herpes simplex virus DNA in two different systems is investigated. Purified nuclei from infected cells are devoid of measurable amounts of putrescine, spermidine, and spermine, while an unfractionated lysate contains the polyamines at close to their respective cellular concentrations. Spermine, 0.3 mM, and 0.5 mM spermidine, when added to the nuclear system, decrease the extent of viral DNA synthesis to the level found in the lysate system, the size of the cell-free viral DNA product is increased, and a specific inhibition of repair-type DNA synthesis is observed. These effects of the polyamines occur only in the presence of ATP and not the other three ribonucleoside triphosphates.     

Involvement of DNA polymerase alpha in host cell reactivation of UV-irradiated herpes simplex virus.

Aphidicolin is a potent inhibitor of both host cell DNA polymerase alpha and herpes simplex virus (HSV)-induced DNA polymerase but has no effect on DNA polymerases beta and gamma of host cells. By using an aphidicolin-resistant mutant (Aphr) of HSV, a possible involvement of DNA polymerase alpha in host cell reactivation of UV-damaged HSV was studied. Plaque formation by UV-irradiated Aphr was markedly inhibited by 1 microgram of aphidicolin per ml, which did not affect the plating efficiency of nonirradiated Aphr. Aphidicolin added before 12 h postinfection inhibited plaque formation by irradiated Aphr, which became aphidicolin insensitive after 36 h postinfection. The results strongly suggest that host cell DNA polymerase alpha is involved in the repair of UV-irradiated HSV DNA.     

Induction of human cell DNA synthesis by herpes simplex virus type 2.

The effect of herpes simplex virus type 2 (HSV-2) infection on the synthesis of DNA in human embryonic fibroblast cells was determined at temperatures permissive (37 C) and nonpermissive (42 C) for virus multiplication. During incubation of HSV-2 infected cultures at 42 C for 2 to 4 days or after shift-down from 42 to 37 C, incorporation of (3H)TdR into total DNA was increased 2-to 30-fold as compared with mock-infected cultures. Analysis of the (3H)DNA suggested that host cell DNA synthesis was induced by HSV-2 infection. Induction of host cell DNA synthesis by HSV-2 also occurred in cells arrested in DNA replication by low serum concentration. The three strains of HSV-2 tested were capable of stimulating cellular DNA synthesis. Virus inactivated by UV irradiation, heat, or neutral red dye and light did not induce cellular DNA synthesis, suggesting that an active viral genome is necessary for induction.