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Biomedical subjects

N Frenkel

Publications and source records attributed to N Frenkel.

At least 37 records · Page 2Linked to original sources
PubMed1991

Human herpesvirus 7: antigenic properties and prevalence in children and adults.

The recent isolation of human herpesvirus 7 (HHV-7) from activated CD4+ T lymphocytes of a healthy individual raises questions regarding the prevalence of this virus in humans and its immunological relationship to previously characterized human herpesviruses. We report that HHV-7 is a ubiquitous virus which is immunologically distinct from the highly prevalent T-lymphotropic HHV-6. Thus, (i) only two of six monoclonal antibodies to HHV-6 cross-reacted with HHV-7-infected cells, (ii) Western immunoblot analyses of viral proteins revealed different patterns for HHV-6- and HHV-7-infected cells, (iii) tests of sequential serum samples from children revealed seroconversion to HHV-6 without concomitant seroconversion to HHV-7, and (iv) in some instances HHV-7 infection occurred in the presence of high titers of HHV-6 antibodies, suggesting the lack of apparent protection of children seropositive for HHV-6 against subsequent infection with HHV-7. On the basis of the analyses of sera from children and adults it can be concluded that HHV-7 is a prevalent human herpesvirus which, like other human herpesviruses, infects during childhood. The age of infection appears to be somewhat later than the very early age documented for HHV-6.

Adult↗
PubMed1990

The replication of viral and cellular DNA in human herpesvirus 6-infected cells.

Human herpesvirus 6 (HHV-6) is a newly identified lymphotropic herpesvirus. We have analyzed viral and host DNA replication in peripheral blood lymphocytes infected in the absence of drugs or infected in the presence of phosphonoacetic acid (PAA) or acyclovir (ACV). The results revealed the following: (i) Infection with HHV-6 resulted in the shutoff of host DNA replication. (ii) PAA at concentrations of 100 and 300 micrograms/ml significantly reduced virus replication. The drug inhibited viral DNA replication, whereas host cell DNA replication was not affected. This strongly suggests that HHV-6 encodes a PAA sensitive viral DNA polymerase. (iii) ACV at 20 microM did not interfere with virus production and virus spread. ACV at 100 microM only partly interfered with virus replication, whereas at 400 microM the block was more complete. Viral DNA replication was not affected by ACV at 20 microM. However, approximately 60 and 85% inhibition in viral DNA replication was observed in the presence of 100 and 400 microM of ACV. (iv) Assays for viral thymidine kinase (TK) revealed no significant increase in TK activity, whereas increased TK activity was noted following infection of the same peripheral blood lymphocytes with herpes simplex virus. Thus, either HHV-6 does not encode a tk enzyme which can phosphorylate ACV or the inefficient block may reflect lower sensitivity of the HHV-6 DNA polymerase to the drug.

Acyclovir↗
PubMed1990

Interleukin-2 inhibits the replication of human herpesvirus-6 in mature thymocytes.

Human herpesvirus-6 (HHV-6) is a recently identified T lymphotropic virus. We have examined the ability of HHV-6 to replicate in mature and immature human thymocytes. Infection of both cell populations revealed that only mitogen-activated mature thymocytes could support efficient virus replication. Because interleukin-2 (IL-2) plays a central role in T cell activation we investigated its effect on HHV-6 replication. Unexpectedly, addition of recombinant IL-2 at concentrations-exceeding 10 U/ml strongly inhibited the virus-induced cytopathic effect. Electron microscopic examinations and immunofluorescence assays revealed a threefold reduction in the fraction of infected cells, and almost total absence of extracellular virions in the IL-2-treated cultures. It will therefore be of interest to determine whether the IL-2-mediated inhibitory effect plays some role in the establishment of HHV-6 latency in the human host.

Cells, Cultured↗
PubMed1990

Isolation of a new herpesvirus from human CD4+ T cells.

A new human herpesvirus has been isolated from CD4+ T cells purified from peripheral blood mononuclear cells of a healthy individual (RK), following incubation of the cells under conditions promoting T-cell activation. The virus could not be recovered from nonactivated cells. Cultures of lymphocytes infected with the RK virus exhibited a cytopathic effect, and electron microscopic analyses revealed a characteristic herpesvirus structure. RK virus DNA did not hybridize with large probes derived from herpes simplex virus, Epstein-Barr virus, varicella-zoster virus, and human cytomegalovirus. The genetic relatedness of the RK virus to the recently identified T-lymphotropic human herpesvirus 6 (HHV-6) was investigated by restriction enzyme analyses using 21 different enzymes and by blot hybridization analyses using 11 probes derived from two strains of HHV-6 (Z29 and U1102). Whereas the two HHV-6 strains exhibited only limited restriction enzyme polymorphism, cleavage of the RK virus DNA yielded distinct patterns. Of the 11 HHV-6 DNA probes tested, only 6 cross-hybridized with DNA fragments derived from the RK virus. Taken together, the maximal homology amounted to 31 kilobases of the 75 kilobases tested. We conclude that the RK virus is distinct from previously characterized human herpesviruses. We propose to designate it as the prototype of a new herpesvirus, the seventh human herpesvirus identified to date.

CD4 Antigens↗
PubMed1990

Variations in the replication and antigenic properties of human herpesvirus 6 strains.

The Z29 and U1102 strains of human herpesvirus 6 (HHV-6) were compared for their ability to replicate in fresh peripheral blood lymphocytes (PBL) and in continuous T cell lines. The replication of both strains in PBL was enhanced by mitogenic activation of cell growth. U1102 replicated in the continuous T cell lines, J JHAN and HSB-2, whereas no Z29 replication was observed in these cell lines as judged by infectious virus yields, the presence of viral antigens, and viral DNA replication. The two strains were compared with respect to their ability to react in immunofluorescence assays with monoclonal antibodies (MAbs) prepared against the GS strain of HHV-6. These MAbs are directed against six different polypeptides including three glycoproteins. All MAbs reacted with cells infected with the U1102 strain. The Z29-infected cells reacted with four MAbs but failed to react with MAbs specific for an 82- to 105-kDa major surface glycoprotein and with one MAb reactive with a nonglycosylated 180-kDa protein. Taken together, the two strains of HHV-6 exhibit variations with regard to their growth and antigenic properties.

Antibodies, Monoclonal↗
PubMed1990

Putative site for the acquisition of human herpesvirus 6 virion tegument.

The virion of human herpesvirus 6 (HHV-6) contains a very distinct tegument layer, occupying the space between the nucleocapsid and the virion envelope. Ultrastructural analyses of thymocytes infected with HHV-6 revealed the presence of intranuclear spherical compartments, approximately 1.5 microns in diameter, in which tegumentation seems to take place. These compartments, termed tegusomes, were bounded by two membranes and contained ribosomes, consistent with their derivation by cytoplasmic invagination into the nucleus. Capsids located within the nucleus outside the tegusomes were all naked, while those located in the cytoplasm were uniformly tegumented. In contrast, capsids present inside the tegusomes contains teguments of variable thicknesses. In addition, nucleocapsids were documented in the process of budding into the tegusomes. We thus suggest that the tegusomes represent a cellular site in which HHV-6 virions acquire their tegument.

Cell Nucleus↗
PubMed1990

T-cell activation is required for efficient replication of human herpesvirus 6.

We have investigated whether T-cell activation is required for the replication of the T-lymphotropic human herpesvirus 6. The virus did not replicate in quiescent peripheral blood lymphocytes but replicated efficiently following exposure of the cells to the polyclonal mitogen phytohemagglutinin (PHA). When purified T cells were treated with PHA in the absence of accessory cells, no virus replication was observed unless exogenous interleukin-2 (IL-2) was added to the medium, promoting cell division. Incubation of peripheral blood lymphocytes in the absence of PHA but in the presence of IL-2 resulted in delayed cell blastogenesis and virus replication. Cell blastogenesis and virus replication did not occur in the purified T-cell cultures incubated with IL-2 alone. Taken together, the results show that human herpesvirus 6 replication requires full progression of the cell cycle. This finding might have implications for the pathogenicity of the virus in the human host.

Cell Count↗
PubMed1989

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.

Animals↗
PubMed1988

Herpes simplex virus virion host shutoff function.

Herpes simplex virus (HSV) virions contain one or more functions which mediate the shutoff of host protein synthesis and the degradation of host mRNA. HSV type 1 (HSV-1) mutants deficient in the virion shutoff of host protein synthesis (vhs mutants) were isolated and were found to be defective in their ability to degrade host mRNA. Furthermore, it was found that viral mRNAs in cells infected with the vhs 1 mutant have a significantly longer functional half-life than viral mRNAs in wild-type virus-infected cells. In the present study we have mapped the vhs1 mutation affecting the virion shutoff of host protein synthesis to a 265-base-pair NruI-XmaIII fragment spanning map coordinates 0.604 to 0.606 of the HSV-1 genome. The mutation(s) affecting the functional half-lives of host mRNA as well as the alpha (immediate-early), beta (early), and gamma (late) viral mRNAs were also mapped within this 265-base-pair fragment. Thus, the shutoff of host protein synthesis is most likely mediated by the same function which decreases the half-life of viral mRNA. The shorter half-life of infected-cell mRNAs may allow a more rapid modulation of viral gene expression in response to changes in the transcription of viral genes. Interestingly, the vhs1 mutation of HSV-1 maps within a region which overlaps the Bg/II-N sequences of HSV-2 DNA shown previously to transform cells in culture. The possible relationship between the transformation and host shutoff functions are discussed.

Animals↗
PubMed1988

Herpes simplex virus induces the replication of foreign DNA.

Plasmids containing the simian virus 40 (SV40) DNA replication origin and the large T gene are replicated efficiently in Vero monkey cells but not in rabbit skin cells. Efficient replication of the plasmids was observed in rabbit skin cells infected with herpes simplex virus type 1 (HSV-1) and HSV-2. The HSV-induced replication required the large T antigen and the SV40 replication origin. However, it produced concatemeric molecules resembling replicative intermediates of HSV DNA and was sensitive to phosphonoacetate at concentrations known to inhibit the HSV DNA polymerase. Therefore, it involved the HSV DNA polymerase itself or a viral gene product(s) which was expressed following the replication of HSV DNA. Analyses of test plasmids lacking SV40 or HSV DNA sequences showed that, under some conditions, HSV also induced low-level replication of test plasmids containing no known eucaryotic replication origins. Together, these results show that HSV induces a DNA replicative activity which amplifies foreign DNA. The relevance of these findings to the putative transforming potential of HSV is discussed.

Animals↗
PubMed1987

Herpes simplex virus-infected cells contain a function(s) that destabilizes both host and viral mRNAs.

The herpes simplex virus virion contains a function that mediates the shutoff of host-protein synthesis and the degradation of host mRNA. Viral mutants affected in this function (vhs mutants) have previously been derived. Cells infected with these mutants exhibit a more stable synthesis of host as well as the immediate early (alpha)-viral proteins. We now show that a function associated with purified virions of the wild-type virus reduces the half-life of host and alpha mRNAs, whereas purified vhs-1 mutant virions lack this activity. The functional half-life of many early (beta)- and late (gamma)-viral mRNAs is also prolonged in mutant virus infections. These studies suggest that the wild-type virion brings into cells a function that indiscriminately reduces the half-life of both host and viral transcripts and that the early translational shutoff of the host is a consequence of this function. This function may facilitate rapid transitions in the expression of groups of genes that are transcriptionally turned on at different times after infection.

Animals↗
PubMed1987

Effects of herpes simplex virus on mRNA stability.

Herpes simplex virus virions contain one or more functions which mediate shutoff of host protein synthesis, disaggregation of host polyribosomes, and degradation of host mRNA. We studied aspects of the host shutoff mechanism by using herpes simplex virus type 1 mutants deficient in virion-induced shutoff of host protein synthesis (G. S. Read and N. Frenkel, J. Virol. 46:498-512, 1983). Shutoff of host protein synthesis by the wild-type virus was associated with degradation of host mRNAs, including beta-actin, alpha-tubulin, and heat shock protein 70. In contrast, the virion host shutoff (vhs) mutants were deficient to various degrees in their ability to induce host mRNA degradation; the extent of mRNA degradation correlated well with the extent of inhibition of host protein synthesis. This finding suggests that inhibition of host protein synthesis and degradation of host mRNA were mediated by the same virion-associated function. Virion-induced degradation of host mRNA was not prevented by inhibitors of ribosome translocation, nor could it be augmented, for mutant vhs-1, by drugs which disaggregate polyribosomes. This suggests that mRNA in polyribosomes, as well as nonpolyribosomal mRNA, is susceptible to virion-induced degradation. Finally, the half-life of viral transcripts was also prolonged in cells infected with the vhs-1 mutant virus, suggesting that the vhs function indiscriminately decreased the half-lives of both host and viral mRNAs. The vhs function may thus play a dual role in virus infection. (i) It inhibits host gene expression, and (ii) it enables rapid transitions in the expression of viral genes which are sequentially transcribed as infection progresses.

Actins↗
PubMed1986

Failure to induce cervical cancer in mice by long-term frequent vaginal exposure to live or inactivated herpes simplex viruses.

C57 mice aged 8-10 weeks in groups of 50 each received vaginal cotton pellets soaked in lysates of HEp-2 cells, either mock-infected or infected with herpes simplex virus I, herpes simplex virus 2, and highly attenuated recombinant viruses 5 times a week for 89 to 114 weeks. An untreated group was also included. The mock-infected and some of the infected cell lysates were exposed to ultraviolet light at a dose sufficient to inactivate virus. Smears of exfoliated vaginal cells collected once a month and histopathologic sections of genital organs removed at autopsy were coded and examined blind for the presence of abnormal cells indicative of malignant changes and cervical cancer, respectively. Sera collected before termination of the study were tested blind for the presence of antibody to infected cell lysates and to purified herpes simplex virus glycoprotein B. The results were as follows: Over 74% of 826 mice examined at autopsy contained tumors at non-genital sites. The tumors were randomly distributed among the various groups. Gross genital abnormalities were less common in untreated animals than in mice receiving vaginal implants. The fraction of mice which developed cervical cancer diagnosed by histopathologic examination was small (7.2%) and not significantly different among various groups. There was no correlation between the presence of abnormal exfoliated cells indicative of early invasive or invasive cancer lesions and the histopathologically proven diagnosis of micro-invasive or invasive cervical cancer. The incidence and levels of antibody were highest in animals exposed to live virus; some mice exposed to inactivated virus also developed weak or moderately high antibody levels. The presence of antibodies did not correlate with the presence of histopathologically proven cervical cancer. The results do not support the ability of herpes simplex viruses to cause genital neoplasia in mice.

Animals↗
PubMed1986

Analyses of transplanted murine tumors for HSV DNA sequences.

Meignier et al. (1986) report the results of exposure of C57BL/6NCr mice to vaginal plugs containing live or inactivated herpes simplex virus 1 or 2 (HSV-1 or HSV-2) or recombinant viruses 5 times a week for up to 114 weeks. Genital organs showing abnormalities were transplanted into nude mice. Of 33 transplants, 13 produced subcutaneous tumors in nude mice and 12 were subsequently transplanted into C57BL/6NCr mice. We report that the DNA extracted from coded tumor tissues of nude mice and from normal viscera of the same rodents did not hybridize with HSV-1 and HSV-2 DNA probes representing the viral genomic regions shown previously to be capable of morphologically transforming cells in culture. The sensitivity of the assays was such that we could detect 0.5 copies of the HSV sequences of complexity equal to or greater than 1 Kbp per cell DNA equivalent. To control for the sensitivity of the assays in the actual hybridizations, the tumor-cell DNA was also hybridized with a beta-globin mouse DNA probe. A striking feature of these control hybridizations was the detection of beta-globin polymorphism in some nude mouse tumors. The beta-globin polymorphism allowed us to conclude that the analyzed tissues contained significant amounts of the tumor cells occurring in the C57BL/6NCr mice.

Animals↗
PubMed1986

Sequences homologous to two separate transforming regions of herpes simplex virus DNA are linked in two human genital tumors.

Ten human genital invasive squamous cell carcinomas and five human premalignant tissues were analyzed for the presence of selected sets of herpes simplex virus 2 (HSV-2) DNA sequences. Two vulvar tumors and one vulvar dysplastic tissue were found to contain DNA sequences homologous to the BglII O fragment (coordinates 0.38-0.42) and the BglII N fragment (coordinates 0.58-0.63) of HSV-2 DNA. These two fragments overlap the subsets of HSV-1 and HSV-2 DNA sequences (respectively) shown previously to transform cells in culture. Sequences homologous to an additional HSV-2 DNA probe (BglII G) were not detected in the same tumors. Surprisingly, in each of the two positive vulvar tumors, the BglII N and BglII O sequences appeared to be linked, whereas in the standard HSV-2 genome the two fragments are separated by approximately 26 kb. This finding suggested that the two sets of sequences may have rearranged prior to or following the association of the HSV DNA sequences with the tumor cells. The same set of 10 tumors were analyzed for the presence of sequences complementary to human papillomavirus 16 (HPV16) DNA. The HPV16 DNA probe hybridized to three of six cervical tumors, whereas no hybridization was detected with the two vulvar tumors which contained the HSV DNA sequences.

Cell Transformation, Viral↗
PubMed1986

Herpes simplex virus amplicon: cleavage of concatemeric DNA is linked to packaging and involves amplification of the terminally reiterated a sequence.

Herpes simplex virus-infected cells contain large concatemeric DNA molecules arising from replication of the viral genome. The large concatemers are cleaved to generate unit-length molecules terminating at both ends with the a sequence. We have used constructed defective virus vectors (amplicons) derived from herpes simplex virus to study the mechanism of cleavage of viral DNA concatemers and the packaging of viral DNA into nucleocapsids. These studies revealed that (i) a 248-base-pair a sequence contained the signal(s) required for cleavage-packaging, (ii) the cleavage of viral DNA concatemers was coupled to packaging, (iii) the a sequence contained the information required for its own amplification, and (iv) cleavage-packaging occurred by a novel process involving the amplification of the a sequence.

Animals↗
PubMed1986

Functional domains within the a sequence involved in the cleavage-packaging of herpes simplex virus DNA.

Newly replicated herpes simplex virus (HSV) DNA consists of head-to-tail concatemers which are cleaved to generate unit-length genomes bounded by the terminally reiterated a sequence. Constructed defective HSV vectors (amplicons) containing a viral DNA replication origin and the a sequence are similarly replicated into large concatemers which are cleaved at a sequences punctuating the junctions between adjacent repeat units, concurrent with the packaging of viral DNA into nucleocapsids. In the present study we tested the ability of seed amplicons containing specific deletions in the a sequence to become cleaved and packaged and hence be propagated in virus stocks. These studies revealed that two separate signals, located within the Ub and Uc elements of the a sequence, were essential for amplicon propagation. No derivative defective genomes were recovered from seed constructs which lacked the Uc signal. In contrast, propagation of seed constructs lacking the Ub signal resulted in the selection of defective genomes with novel junctions, containing specific insertions of a sequences derived from the helper virus DNA. Comparison of published sequences of concatemeric junctions of several herpesviruses supported a uniform mechanism for the cleavage-packaging process, involving the measurement from two highly conserved blocks of sequences (pac-1 and pac-2) which were homologous to the required Uc and Ub sequences. These results form the basis for general models for the mechanism of cleavage-packaging of herpesvirus DNA.

Animals↗