Ultrastructural characterization of an early, nonstructural polypeptide of herpes simplex virus type 1.

An immunoperoxidase procedure was employed to study the expression of a large-molecular-weight, virus-induced polypeptide (VP175; molecular weight, 175,000) at the light and electron microscopic levels in Vero cells infected with herpes simplex virus type 1 or with tsB2, a DNA-negative, temperature-sensitive mutant of herpes simplex virus type 1. In cells infected with herpes simplex virus type 1 and in cells infected with tsB2 at the permissive temperature (34 degrees C), VP175 was found within the nucleus. The protein was detected as early as 2 h postinfection and, by 3 h postinfection, was generally distributed in a marginated pattern contiguous with, and extending from, the inner lamella of the nuclear membrane. At 6 h postinfection, protein accumulations were dispersed throughout the nucleus, and, by 9 h postinfection, these accumulations tended to be localized in a marginated pattern near the nuclear membrane. It was also noted that, at 9 h postinfection, under permissive conditions, VP175 was not found in association with nucleocapsids or enveloped particles. In contrast, in cells infected with tsB2 at the nonpermissive temperature (39 degrees C) and harvested at 6 or 9 h postinfection, accumulations of VP175 were identified not only within the nucleus, but also within the cytoplasm in the form of annular or globular aggregates. These aggregates consisted of a granular matrix and were not bound by membranes.     

High-frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion.

The protoplast fusion technique of Schaffner (W. Schaffner, Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980) has been adapted to introduce cloned herpes simplex virus genes into cultured mammalian cells. The technique involves digesting bacterial cell walls with lysozyme to produce protoplasts and then fusing the protoplasts to mammalian cells by treatment with polyethylene glycol. For monitoring transfer, protoplasts were labeled with the fluorescent dye fluorescein isothiocyanate before fusion. After fusion, greater than 50% of the mammalian cells were fluorescent, demonstrating that bacterial material was transferred with high frequency. Transfer of plasmid pBR325 occurred at frequencies of 1 to 2%, as measured by in situ hybridization. Fusion transfer of a chimeric plasmid consisting of the herpes simplex virus type 1 (strain KOS) EcoRI fragment F in pBR325 resulted in expression of some viral genomic sequences in about 5% of the mammalian cells, as detected by indirect immunofluorescence. One Ltk- cell in 300 to 500 was transformed to the TK+ phenotype after fusion with protoplasts carrying the chimeric plasmid pX1, which consists of pBR322 and the BamHI fragment coding for the herpes simplex virus type 1 thymidine kinase gene.     

Short-term, high-efficiency expression of transfected DNA.

We have achieved high-efficiency uptake and expression of foreign DNA in mouse Ltk- cells by modifying the DEAE-dextran-mediated transfection method of McCutchan and Pagano (J. Natl. Cancer Inst. 42:351-357, 1968) to include an initial incubation at elevated pH followed by a shock treatment with dimethyl sulfoxide. Up to 80% of mouse Ltk- cells transfected with the herpes simplex virus thymidine kinase gene expressed thymidine kinase as measured by autoradiography.     

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.     

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.     

Isolation of cis-acting vaccinia virus DNA fragments promoting the expression of herpes simplex virus thymidine kinase by recombinant viruses.

Recombinant TK- vaccinia viruses containing the pBR322 sequence inserted in either orientation within the coding sequence of the viral thymidine kinase gene were constructed. They were characterized by genomic analysis, hybridization studies, reversion to wild-type virus by in vivo recombination, and rescue from their genomes of plasmids which contained all or parts of the pBR322 sequence. TK- cells were infected with one of these recombinant viruses and then transfected with pools of chimeric plasmids composed of a cloned herpes simplex virus thymidine kinase gene which contained upstream inserts of different vaccinia DNA fragments prepared by restriction or sonication. Recombination between homologous pBR322 sequences within infected cells generated selectable recombinant viruses in which expression of the herpes simplex virus thymidine kinase gene was promoted by the upstream vaccinia insert. These viruses were characterized by genomic analysis, hybridization, and in vivo or in vitro phosphorylation of (5-[125I]deoxycytidine as a specific assay for the expressed herpes simplex virus thymidine kinase. Vaccinia DNA inserts were isolated conveniently for transfer to bacteria by rescuing appropriate plasmids from the genome of recombinant viruses. The sequence of 100 nucleotides adjacent to the upstream region of the herpes simplex virus gene was determined in nine different inserts measuring 0.17 to 1.07 kilobase pairs.     

Sequences of herpes simplex virus type 1 that inhibit formation of stable TK+ transformants.

We have identified two regions of the herpes simplex virus type 1 (HSV-1) genome that inhibit DNA-mediated transformation of thymidine kinase-less L (Ltk-) cells by the cloned HSV-1 tk gene. When plasmids containing the EcoRI fragments EK or JK were mixed at 30 fmol/ml with the tk gene and transfected into Ltk- cells, the frequency of transformation was inhibited 80 to more than 90% relative to the control. Of the remaining 10 EcoRI fragments of the HSV-1 genome, 8 were inactive and 2 were weakly active. A 6.1-kilobase PstI subclone between 0.743 and 0.782 map units was isolated from pEK. This clone, pEK-P3P4, exhibited antitransformation activity toward HSV-1 tk and also the bacterial genes gpt and neo. pEK-P3P4 contains the alpha 27 gene, and restriction endonuclease inactivation and subcloning studies established that alpha 27 alone did not inhibit transformation. However, alpha 27 plus sequences both upstream and downstream of alpha 27 did inhibit transformation. In addition, alpha 0 or alpha 4 could substitute for alpha 27 in effecting antitransformation with these sequences. Therefore, an alpha gene and two additional loci in pEK-P3P4 are required for antitransformation. A second antitransforming locus in the reiterated sequences common to EK and JK and distinct from those in pEK-P3P4 was also identified but not characterized in detail. How antitransformation may be an expression of regulation of viral and host cell gene expression is discussed.     

Transfer of herpes simplex virus thymidine kinase synthesized in bacteria by a high-expression plasmid to tissue culture cells by protoplast fusion.

The introduction of a protein into living tissue culture cells may permit the in vivo study of functions of the protein. We have previously described a high-efficiency-expression plasmid, pHETK2, containing the herpes simplex virus type 1 thymidine kinase (TK) gene which, upon temperature induction, causes TK to be synthesized as greater than 4% of the bacterial protein. In this report we show that enzymatically active TK was transferred to mouse Ltk- cells by polyethylene glycol-mediated fusion with protoplasts prepared from bacteria containing induced levels of TK. The presence of TK in the Ltk- cells was detected by the incorporation of [3H]thymidine into cell nuclei as measured by autoradiography.     

Novel rearrangements of herpes simplex virus DNA sequences resulting from duplication of a sequence within the unique region of the L component.

We constructed insertion mutants of herpes simplex virus type 1 that contained a duplication of DNA sequences from the BamHI-L fragment (map units 0.706 to 0.744), which is located in the unique region of the L component (UL) of the herpes simplex virus type 1 genome. The second copy of the BamHI-L sequence was inserted in inverted orientation into the viral thymidine kinase gene (map units 0.30 to 0.32), also located within UL. A significant fraction of the progeny produced by these insertion mutants had genomes with rearranged DNA sequences, presumably resulting from intramolecular or intermolecular recombination between the BamHI-L sequences at the two different genomic locations. The rearranged genomes either had an inversion of the DNA sequence flanked by the duplication or were recombinant molecules in which different regions of the genome had been duplicated and deleted. Genomic rearrangements similar to those described here have been reported previously but only for herpes simplex virus insertion mutants containing an extra copy of the repetitive a sequence. Such rearrangements have not been reported for insertion mutants that contain duplications of herpes simplex virus DNA sequences from largely unique regions of the genome. The implications of these results are discussed.     

Foreign DNA introduced by calcium phosphate is integrated into repetitive DNA elements of the mouse L cell genome.

We investigated the sites of integration of exogenous DNA fragments introduced by DNA-mediated gene transfer. Mouse Ltk- cells were transformed with the herpes simplex virus thymidine kinase gene and pBR322 DNA by the calcium phosphate precipitation method. Some of the integrated exogenous DNA sequences were recovered from the stable tk+ transformants in the form of plasmids that were capable of propagation in bacteria. Four plasmids derived from two cloned cell lines were analyzed in detail by nucleotide sequencing and hybridization techniques. These plasmids contained a total of seven cellular-exogenous DNA junctions. In all cases, there was no sequence homology between the exogenous and cellular DNA sequences adjacent to the joining sites, and no specific exogenous or cellular sequences occurred at the junctions. Rearrangement or deletion of Ltk- DNA was always associated with the integration of exogenous DNA. All of the assignable cellular sequences at the junctions were repetitive sequences. Two of these sequences were from the MIF-1 repetitive sequence family, and a third consisted of a 40-base pair simple copolymer of alternating deoxyadenosine-deoxythymidine. Our results suggest that repetitive sequences are relatively favorable sites for the integration of exogenous DNA.     

Regulation of persistent infection with herpes simplex virus in vitro by hydrocortisone.

About 1% of Raji cells showed sensitivity to herpes simplex virus type 2 (HSV-2) infection when tested by infectious center assays or immunofluorescence tests, and the percentage did not change during cell passage. The addition of hydrocortisone to Raji cells persistently infected with HSV-2 caused a marked increase in virus production and in the number of HSV-producing cells. In the case of HSV-1, it was shown that the addition of hydrocortisone was required to maintain persistent infection. These observations suggest that control of replication of HSV-1 and HSV-2 in these cells is regulated by different mechanisms.     

Alterations in Glycosphingolipid Patterns in a Line of African Green Monkey Kidney Cells Infected with Herpesvirus

The major glycosphingolipids (GSLs) of a line of African green monkey kidney cells (BGM) were characterized as glucosylceramide, lactosylceramide, galactosyl-galactosyl-glucosylceramide, and N-acetylgalactosaminyl-galactosyl-galactosyl-glucosylceramide. Neutral GSLs accounted for approximately 80% of the total GSLs isolated. The predominant gangliosides were N-acetylneuraminyl-galactosyl-glucosylceramide, N-acetylgalactosaminyl-N-acetylneuraminyl-galactosyl- glucosylceramide, and galactosyl-N-acetylgalactosaminyl-N-acetylneuraminyl -galactosyl-glucosylceramide. The incorporation of labeled galactose into GSLs was compared in mock-infected and herpes simplex virus type 1-infected BGM cells. Herpes simplex virus type 1 infection resulted in a three- to four-fold increase in galactose incorporation into glucosylceramide and a decrease in galactose incorporation into galactosyl-galactosyl-glucosylceramide and N-acetyl-galactosaminyl-galactosyl-galactosyl-glucosylceramide. The virus-induced alteration in the GSL labeling pattern occurred early in infection, before the release of infectious virus, and was not prevented by the presence of cytosine arabinoside. Treatment of uninfected BGM cells with cycloheximide resulted in alterations in the GSL pattern which were similar to those observed in herpes simplex virus type 1-infected cells. These observations suggest that an early virus function such as inhibition of host cell protein synthesis is responsible for the observed alterations of GSL metabolism. Experiments with a syncytium-producing strain of herpes simplex virus type 1, herpes simplex virus type 2, and pseudorabies virus indicated that other herpes viruses altered GSL metabolism in a manner similar to herpes simplex virus type 1.     

Isolation of a simian virus 40 T-antigen-positive, transformation-resistant cell line by indirect selection.

In an attempt to identify cellular genes that might be involved in simian virus 40 (SV40) transformation, we have set out to isolate cells which express T antigen but are not transformed. SV40 DNA and the herpes simplex virus thymidine kinase gene were cotransfected into tk- 3T3 fibroblasts. Of 72 colonies screened that were resistant to hypoxanthine-aminopterin-thymidine, 57 were T antigen positive as judged by immunofluorescence. One of these lines, A27, had a normal growth phenotype in monolayer overgrowth and soft agar assays. It contained intact SV40 sequences that could be rescued by fusion to permissive cells. This rescued virus was fully capable of transforming nonpermissive cells to the same extent as did wild-type virus. The A27 cells, however, were not transformable by infection with SV40 or by transfection of SV40 DNA. It is likely that these cells were altered in a cellular function required for the establishment of the transformed state.     

Mutant of herpes simplex virus type 1 conditionally able to transform thymidine kinaseless L cells to a tk+ phenotype.

After nitrous acid mutagenesis of herpes simplex virus type 1 (HSV-1), a mutant, 1093, was isolated which, during productive infection, induced very low levels of thymidine kinase (tk). The mutant virus was found, after UV irradiation, to be unable to transform L cells lacking tk (Ltk-) to a tk+ phenotype as chararcterized by growth of the cells in a modified HAT-selective medium containing 1.6 X 10(-5) M thymidine. Cells transformed by wild-type virus grew vigorously under the same conditions. The mutant was able to transform Ltk- cells if the medium contained 10(-3) M thymidine. These transformed cells maintained their conditional character and would not grow in low concentrations of thymidine in selective medium. Therefore, this mutant is conditional on the thymidine concentration in the selection medium in its ability to transform Ltk- cells to a tk+ phenotype. The conditionally transformed cells could be supertransformed with wild-type UV-irradiated HSV-1 to a phenotype which would grow in low-thymidine selective medium. The frequency of supertransformation closely approximated the frequency of transformation of Ltk- cells by wild-type virus. Supertransformation at high frequency could not be effected by mutant 1093 or the tk- mutant B2006. These results indicate that the presence of HSV-1 genetic information in HSV-1-transformed cells does not preclude the acquisition by these cells of at least one additional HSV-1 gene, that for tk.