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N Frenkel

Publications and source records attributed to N Frenkel.

At least 55 records · Page 3Linked to original sources
PubMed1985

The herpes simplex virus amplicon. IV. Efficient expression of a chimeric chicken ovalbumin gene amplified within defective virus genomes.

cDNA sequences of the chicken ovalbumin gene were fused to an alpha (immediate early) promoter of herpes simplex virus and to genomic ovalbumin 3'-flanking sequences. The chimeric alpha-ovalbumin gene was introduced into defective virus genomes which were stably propagated in serially passaged virus stocks in the presence of helper virus. Analyses of polypeptides synthesized in cells infected with the resultant defective virus stocks revealed the abundant expression of the chimeric alpha-ovalbumin gene. The presence of introns was not essential for this expression.

Carcinoma, Squamous Cell↗
PubMed1985

The herpes simplex virus amplicon: analyses of cis-acting replication functions.

Previous studies have shown that defective virus vectors (amplicons) derived from herpes simplex viruses could be efficiently propagated in virus stocks in the presence of trans-acting helper virus functions. The present study established that two separate cis-acting functions--a DNA replication origin and a cleavage/packaging signal--are required for amplicon propagation. Using deleted derivatives of cloned amplicons, we mapped one of the viral DNA replication origins (ori-2 or oriL) at coordinate 0.422 of the standard HSV-1 genome and at an equivalent position within the HSV-2 genome.

Base Sequence↗
PubMed1985

Nucleotide sequence and structural features of a novel US-a junction present in a defective herpes simplex virus genome.

Defective genomes generated during serial propagation of herpes simplex virus type 1 (Justin) consist of tandem reiterations of sequences that are colinear with a portion of the S component of the standard viral genome. We determined the structure of the novel US-a junction, at which the US sequences of one repeat unit join the a sequences of the adjacent repeat unit. Comparison of the nucleotide sequence at this junction with the nucleotide sequence of the corresponding US region of the standard virus genome indicated that the defective genome repeat unit arose by a single recombinational event between an L-S junction a sequence and the US region. The recombinational process might have been mediated by limited sequence homology. The sequences retained within the US-a junction further define the signal for cleavage and packaging of viral DNA.

Base Sequence↗
PubMed1984

Herpes simplex virus amplicon: effect of size on replication of constructed defective genomes containing eucaryotic DNA sequences.

Previous studies (R. R. Spaete and N. Frenkel, Cell 30:295-304, 1982) have documented the potential use of defective virus vectors (amplicons) derived from herpes simplex virus for the efficient introduction of foreign DNA sequences into eucaryotic cells. Specifically, cotransfection of cells with helper virus DNA and cloned amplicons (8 to 10 kilobases [kb]) containing bacterial plasmid DNA sequences linked to a set of herpes simplex virus cis-acting propagation signals (a replication origin and a cleavage-packaging signal) resulted in the generation of virus stocks containing packaged defective genomes that consisted of uniform head-to-tail reiterations of the chimeric seed amplicon sequences. The chimeric defective genomes could be stably propagated in virus stocks and could thus be used to efficiently infect cells. We now report on additional studies designed to propagate relatively large sets of eucaryotic DNA sequences within chimeric packaged defective genomes. These studies have utilized a 12-kb chicken DNA sequence encoding the chicken ovalbumin gene and cloned by Lai et al. (Proc. Natl. Acad. Sci. U.S.A. 77:244-248, 1980) in the plasmid pOV12. Virus stocks derived from cells cotransfected with helper virus DNA and chimeric amplicons (overall size of 19.8 kb, of which 12 kb corresponded to the chicken DNA) contained defective genomes composed of reiterations of the 19.8-kb seed amplicon sequences. However, in addition to the authentically sized repeat units, defective genomes in the derivative virus stocks contained smaller repeat units representing deleted versions of the seed 19.8-kb amplicons. The recombinational events leading to the formation of deleted repeats did not appear to occur at unique sites, as shown by comparative analyses of multiple, independently generated virus series propagated from separate transfections. In contast, seed amplicons ranging in size from 11 to 15 kb and containing subsets of the 12-kb chicken DNA sequences replicated efficiently and could be stably propagated in virus stocks. The results of these studies suggest the existence of size restrictions (up to 15 kb) on the efficient replication of seed herpes simplex virus amplicons.

Animals↗
PubMed1983

Herpes simplex virus mutants defective in the virion-associated shutoff of host polypeptide synthesis and exhibiting abnormal synthesis of alpha (immediate early) viral polypeptides.

Six mutants isolated from herpes simplex virus type 1 were judged to be defective with respect to the virion-associated function acting to rapidly shut off host polypeptide synthesis in herpes simplex virus-infected cells. The mutants were capable of proper entry into the cells, but, unlike the parent wild-type virus, they failed to shut off host polypeptide syntehsis in the presence of actinomycin D. They were consequently designated as virion-associated host shutoff (vhs) mutants. In the presence of actinomycin D, three of the mutants, vhs1, -2, and -3, failed to shut off the host at both 34 and 39 degrees C, whereas vhs4, -5, and -6 exhibited a temperature-dependent vhs phenotype. Since the mutants were capable of growth at 34 degrees C, it appeared that the vhs function was not essential for virus replication in cultured cells. Temperature-shift experiments performed with the vhs4 mutant showed that an active vhs function was required throughout the shutoff process and that, once established, the translational shutoff could not be reversed. In the absence of actinomycin D, the mutants induced a generalized, secondary shutoff of host translation, which required the synthesis of beta (early) or gamma (late) viral polypeptide(s). The vhs mutants appeared to be defective also with respect to post-transcriptional shutoff of alpha (immediate early) viral gene expression, since (i) cells infected with mutant viruses overproduced alpha viral polypeptides, (ii) there was an increased functional stability of alpha mRNA in the vhs1 mutant virus-infected cells, and (iii) superinfection of vhs1-infected cells with wild-type virus, in the presence of actinomycin D, resulted in a more pronounced shutoff of alpha polypeptide synthesis from preformed alpha mRNA than equivalent superinfection with vhs1 virus. The data suggest that the synthesis of alpha polypeptides in wild-type virus infections is subject to a negative post-transcriptional control involving viral gene product(s) present in infected cell lysates constituting virus stocks. The vhs1 mutant and possibly other vhs mutants contain a mutation in the gene encoding this function.

Animals↗
PubMed1982

The herpes simplex virus amplicon: a new eucaryotic defective-virus cloning-amplifying vector.

We have employed repeat units of herpes simplex virus (HSV) defective genomes to derive a cloning-amplifying vector (amplicon) that can replicate in eucaryotic cells in the presence of standard HSV helper virus. The design of the HSV amplicon system is based on the previous observation that cotransfection of cells with helper virus DNA and seed monomeric repeat units of HSV defective genomes results in the regeneration of concatemeric defective genomes composed of multiple reiterations of the seed repeats. Cotransfection of cells with helper virus DNA and chimeric repeat units containing bacterial plasmid pKC7 DNA resulted in the generation of defective genomes composed of reiterations of the seed HSV-pKC7 repeats. These chimeric defective genomes were packaged into virus particles and could be propagated in virus stocks, with the most enriched passages containing more than 90% chimeric defective genomes. Furthermore, monomeric chimeric repeat units could be transferred back and forth between bacteria and eucaryotic cells. A derivative vector constructed so as to contain several unique restriction enzyme sites could be potentially employed in the introduction of additional viral or eucaryotic DNA sequences into eucaryotic cells.

Animals↗
PubMed1982

Site-specific cleavage/packaging of herpes simplex virus DNA and the selective maturation of nucleocapsids containing full-length viral DNA.

Defective genomes present in serially passaged herpes simplex virus (HSV) stocks have been shown to consist of tandemly arranged repeat units containing limited sets of the standard virus DNA sequences. Invariably, the HSV defective genomes terminate with the right (S component) terminus of HSV DNA. Because the oligomeric forms can arise from a single repeat unit, it has been concluded that the defective genomes arise by a rolling circle mechanism of replication. We now report on our studies of defective genomes packaged in viral capsids accumulating in the nuclei and in mature virions (enveloped capsids) translocated into the cytoplasm of cells infected with serially passaged virus. These studies have revealed that, upon electrophoresis in agarose gels, the defective genomes prepared from cytoplasmic virions comigrated with nondefective standard virus DNA (M(r) 100 x 10(6)). In contrast, DNA prepared from capsids accumulating in nuclei consisted of both full-length defective virus DNA molecules and smaller DNA molecules of discrete sizes, ranging in M(r) from 5.5 to 100 x 10(6). These smaller DNA species were shown to consist of different integral numbers (from 1 to approximately 18) of defective genome repeat units and to terminate with sequences corresponding to the right terminal sequences of HSV DNA. We conclude on the basis of these studies that (i) sequences from the right end of standard virus DNA contain a recognition signal for the cleavage and packaging of concatemeric viral DNA, (ii) the sequence-specific cleavage is either a prerequisite for or occurs during the entry of viral DNA into capsid structures, and (iii) DNA molecules significantly shorter than full-length standard viral DNA can become encapsidated within nuclear capsids provided they contain the cleavage/packaging signal. However, capsids containing DNA molecules significantly shorter than standard virus DNA are not translocated into the cytoplasm.

Capsid↗
PubMed1982

Structure and expression of class II defective herpes simplex virus genomes encoding infected cell polypeptide number 8.

Defective genomes present in serially passaged virus stocks derived from the tsLB2 mutant of herpes simplex virus type 1 were found to consist of repeat units in which sequences from the U(L) region, within map coordinates 0.356 and 0.429 of standard herpes simplex virus DNA, were covalently linked to sequences from the end of the S component. The major defective genome species consisted of repeat units which were 4.9 x 10(6) in molecular weight and contained a specific deletion within the U(L) segment. These tsLB2 defective genomes were stable through more than 35 sequential virus passages. The ratios of defective virus genomes to helper virus genomes present in different passages fluctuated in synchrony with the capacity of the passages to interfere with standard virus replication. Cells infected with passages enriched for defective genomes overproduced the infected cell polypeptide number 8, which had previously been mapped within the U(L) sequences present in the tsLB2 defective genomes. In contrast, the synthesis of most other infected cell polypeptides was delayed and reduced. The abundant synthesis of infected cell polypeptide number 8 followed the beta regulatory pattern, as evident from kinetic studies and from experiments in which cycloheximide, canavanine, and phosphonoacetate were used. However, in contrast to many beta (early) and gamma (late) viral polypeptides, the synthesis of infected cell polypeptide number 8 was only minimally reduced when cells infected with serially passaged tsLB2 were incubated at 39 degrees C. The tsLB2 mutation had previously been mapped within the domains of the gene encoding infected cell polypeptide number 4, the function of which was shown to be required for beta and gamma viral gene expression. It is thus possible that the tsLB2 mutation affects the synthesis of only a subset of the beta and gamma viral polypeptides. An additional polypeptide, 74.5 x 10(3) in molecular weight, was abundantly produced in cells infected with a number of tsLB2 passages. This polypeptide was most likely expressed from truncated gene templates within the most abundant, deleted repeats of tsLB2 defective virus DNA.

Cell Line↗
PubMed1981

Replication of herpes simplex virus DNA: localization of replication recognition signals within defective virus genomes.

Serially passaged herpes simplex virus type 1 (HSV-1) strain Justin was previously shown to contain defective virus genomes consisting of head-to-tail reiterations of sequences derived from the end of the S component of the standard virus DNA. Cotransfection of purified monomeric defective genome repeat units with foster helper virus DNAs onto rabbit skin cells resulted in regeneration and replication of concatemeric defective DNA molecules which were successfully encapsidated. Thus, defective HSV-1 (Justin) genomes contain, within their limited DNA sequences, a sufficient set of recognition sites required for HSV DNA replication and packaging. The arrangement of repeat units within the regenerated defective virus genomes was consistent with their replication by a rolling circle mechanism in which a single repeat unit served as the circularized template. This replication occurred most actively late after infection and could be shown to be inhibited by low concentrations of phosphonoacetate known to inhibit the HSV-specified viral DNA polymerase selectively. The resultant concatemers were shown to be cleaved to Mr 100 X 10(6) DNA molecules which were terminated at one end with the proper ac end sequence of the parental standard virus DNA.

Animals↗
PubMed1980

Mapping of the herpes simplex virus DNA sequences present in herpes simplex virus type-1 thymidine kinase-transformed cells.

Analyses of the herpes simplex virus (HSV) DNA sequences which are present in three HSV thymidine kinase-transformed (HSVtk+) mouse cell lines have revealed that these cells contain relatively large and variable portions of the viral genome. Two of these cell lines do not contain the viral DNA sequences known to encode the early viral genes normally responsible for regulating tk gene expression during lytic HSV infections. This finding suggests that cell-associated viral tk gene expression may be regulated by cellular rather than viral control mechanisms. In addition, we have compared the viral DNA sequences present in one unstable HSVtk+ cell line to those present in tk- revertant and tk+ rerevertant cell lines sequentially derived from it. Our results have shown that within the limits of sensitivity of our mapping approach, these three related cell lines contain the same set of viral DNA sequences. Thus, gross changes in viral DNA content do not appear to be responsible for the different tk phenotypes of these cells.

Animals↗
PubMed1980

Identification of the herpes simplex virus DNA sequences present in six herpes simplex virus thymidine kinase-transformed mouse cell lines.

We have used a novel filter hybridization approach to detect and map the herpes simplex virus (HSV) DNA sequences which are present in four HSV thymidine kinase (HSVtk+)-transformed cell lines which were derived by exposure of thymidine kinase negative (tk-) mouse cells to UV light-irradiated HSV type 2 (HSV-2). In addition, we have mapped the HSV-1 DNA sequences which are present in two HSV-1tk+-transformed cell lines produced by transfection of tk- mouse cells with sheared HSV-1 DNA. The results of these studies can be summarized as follows. (i) The only HSV DNA sequences which were common to all HSVtk+-transformed cells were those located between map coordinates 0.28 and 0.32. Thus, this region contains all of the viral DNA sequences which are necessary for the expression of HSV-mediated tk transformation. (ii) Many of the cell lines also contained variable amounts of non-tk gene viral DNA sequences located between map coordinates 0.11 to 0.57 and 0.82 to 1.00, suggesting that incorporation of the viral DNA sequences located between these map coordinates is a relatively random event. (iii) The viral DNA sequences located between map coordinates 0 to 0.11 and 0.57 to 0.82 were uniformly absent from all of the HSVtk+ cell lines tested, suggesting that there is a strong negative selective pressure against incorporation of these viral DNA sequences.

Animals↗
PubMed1979

Structure and origin of defective genomes contained in serially passaged herpes simplex virus type 1 (Justin).

Restriction enzyme and hybridization analyses have revealed that high-density DNA prepared from passage 15 of serially passaged herpes simplex virus type 1 (Justin) contains three major classes of modified viral DNA molecules, each composed of distinct but closely related types of repeate units. The DNA sequences within the three types of repeat units are colinear with the DNA sequences located at the right end (between coordinates 0.94 and 1.0) of the parental herpes simplex virus type 1 genome. Thus, the three types of repeat units each contain the entire repeat sequence (ac) (which brackets the unique sequences of the small [S] component of herpes simplex virus type 1 DNA) and differ only with respect to the amount of unique S sequences which they contain. The three classes of high-density DNA molecules were found to be stably propagated between passages 6 and 15 of this series.

Base Sequence↗
PubMed1979

BamI, KpnI, and SalI restriction enzyme maps of the DNAs of herpes simplex virus strains Justin and F: occurrence of heterogeneities in defined regions of the viral DNA.

We present the locations of the cleavage sites for the BamI, KpnI, and SalI restriction endonucleases within the DNA molecules of herpes simplex virus type 1 (HSV-1) strains Justin and F. These restriction enzymes cleave the HSV-1 DNA at many sites, producing relatively small fragments which should prove useful in future studies of HSV-1 gene structure and function. The mapping data revealed the occurrence of heterogeneity within three regions of the viral genome including (i) the region spanning map coordinates 0.74--0.76, (ii) the ends of the large (L) DNA component, and (iii) the junction between the large (L) and the small (S) components. The heterogeneity in the ends of L and the S-L junctions of HSV-1 (Justin) and HSV-1 (F) DNAs was grossly similar to that previously reported to occur in the ends of L and the S-L junctions of the HSV-1 (KOS) DNA (M. J. Wagner and W. C. Summers, J. Virol. 27:374--387, 1978). Thus, cleavage of these regions with restriction endonucleases yielded sets of minor fragments differing in size by constant increments. However, the various strains of HSV-1 differed with respect to the numbers, size increments, and relative molarities of the various minor fragments, suggesting that the parameters of the heterogeneity are inherited in the structural makeup of the HSV-1 genome. The strain dependence of the pattern of heterogeneity can be most easily explained in terms of variable sizes of the terminally reiterated a sequence, contained in the DNA molecules of these three strains of HSV-1.

Base Sequence↗
PubMed1978

Mapping of the herpes simplex virus DNA sequences in three herpes simplex virus thymidine kinase-transformed cell lines.

We have made use of a novel filter hybridization approach in order to map the herpes simplex virus (HSV) DNA sequences which are present in three HSV thymidine kinase (TK)-transformed cell lines. The cell line 33A+ which was produced by infection of 3T3 TK- cells with UV-irradiated HSV-2 (333) was found to contain one contiguous stretch of viral DNA sequences which maps between 0.15 and 0.57 on the HSV-2 genome. The sequences mapping from 0.31 to 0.37 were present in 3--4-fold higher abundance than the rest of the viral DNA sequences in this cell line. Cell lines 5A and 8N were produced by transfection of mouse CL1D cells with sheared HSV-1 (1023) DNA. The cell line 5A was found to contain a contiguous set of viral DNA sequences mapping between 0.26 and 0.41 on the HSV-1 genome. The cell line 8N was found to contain three non-contiguous sets of viral DNA sequences, mapping between 0.09 and 0.41, 0.53 and 0.58, and 0.94 and 1.0 on the HSV-1 genome. These results seem to indicate that many different sets of viral DNA sequences can be incorporated into the cell during HSV-mediated biochemical transformation.

Animals↗
PubMed1978

The DNA of serially passaged herpes simplex virus: organization, origin, and homology to viral RNA.

High-density DNA prepared from serially passaged herpes simplex virus contains three major classes of modified viral DNA molecules. The altered DNA molecules are composed of multiple repetitions of sequences derived from the right-hand side of the S region of the parental plaque-purified viral DNA. The repeat units contained in the three types of high-density DNA share most of their DNA sequences but differ with respect to a small region derived from the unique sequences of the S component of HSV-1 DNA. Hybridization of the defective DNA to HSV-infected cell RNA shows that the high-density DNA contains sequences complementary to both early and late viral transcripts.

Base Sequence↗
PubMed1976

Herpes simplex virus DNA in transformed cells: sequence complexity in five hamster cell lines and one derived hamster tumor.

Analyses of the hybridization kinetics of labeled herpes simplex virus 2 (HSV-2) DNA with DNA from five hamster cell lines transformed by UV light-irradiated HSV-2 revealed the following. (i) Viral DNA sequences were detected in all five cell lines tested. (ii) None of the cell lines contained the full complement of HSV-2 DNA. (iii) The amount of viral DNA present in the cells varied in different transformed cell lines and ranged from 8 to 32% of the HSV-2 DNA genome in 1 to 3 copies/cell. (iv) Two parallel passages of the same cell line (333-2-29) differed in the amount of viral DNA they contained. We also compared the viral DNA sequences present in (i) one transformed cell line (333-8-9) propagated serially in culture for 80 passages, (ii) a tumor produced by inoculation of a newborn hamster with the 333-8-9 cells, and (iii) a cell line derived from a hamster tumor as above and propagated in culture for 32 passages. The results show that viral DNA present in the hamster tumor and in the cells derived from the tumor had a lower sequence complexity than that present in the original serially passaged 333-8-9 cell line.

Animals↗
PubMed1975

Anatomy of herpes simplex virus DNA: strain differences and heterogeneity in the locations of restriction endonuclease cleavage sites.

Digestion of herpes simplex virus DNA by the HinIII or Eco RI restriction endonucleases yielded 11 to 15 fragments with molecular weights between 1 x 10(6) and 28 x10(6). The electrophoretic profiles obtained in 0.3% agarose gels with DNA fragments from none different strains of herpes simplex virus type 1 could be readily differentiated from the patterns exhibited by the corresponding fragments from four separate strains of type 2 virus; however, with each serotype, the laboratory strains differed significantly among themselves and also from isolates passaged a minimum number of times outside the human host. Digestion of all DNAs of herpes simples virus with either enzyme reproducibly generated two classes of fragments (major and minor) which differed in molar ocncentration. Moreover, although the molecular weight of an intact herpes simplex 1(F1) DNA molecule is approximately 98 x 10(6), the summed molecular weights of all major and minor HinIII fragments totalled 160 x 10(6), and the seven major fragments alone accounted for only 60 x 10(6). These unusual features indicate the existence of limited heterogeneity in the positions of cleavage sitet along individual molecules. We have eliminated the possibility that minor fragments arose from contamination with the defective DNA of high byoyant density which appears on serial undiluted passage of the virus. In fact, this latter type of DNA was resistant to cleavage by HinIII and gave large amounts of only two species of EcoRI fragments; suggesting that the defective molecules consist of many tandem repeats of a small segment of viral DNA. The heterogeneity in the viral DNA of normal density appears to be related to the structural organization of the molecules and does not necessarily imply differences in genetic content.

Carcinoma, Squamous Cell↗