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S J Silverstein

Publications and source records attributed to S J Silverstein.

At least 19 recordsLinked to original sources
PubMed1991

The interaction of ICP4 with cell/infected-cell factors and its state of phosphorylation modulate differential recognition of leader sequences in herpes simplex virus DNA.

Regulation of herpes simplex virus (HSV) gene expression requires the synthesis of functional ICP4, a phosphoprotein that binds to several specific sites in virus DNA and acts in trans either to activate or to repress transcription of the three major kinetic classes of virus genes. Binding of ICP4 to specific sites in alpha genes (which are the first to be transcribed) causes repression of alpha-gene expression. ICP4 also indirectly participates in the formation of DNA-protein complexes with sequences present in the promoter/regulatory and leader regions of the beta and gamma genes that are sequentially activated later in infection. Here we demonstrate that the extent of phosphorylation of ICP4 contributes to its ability to participate differentially in complex formation with cis-acting elements present in beta and gamma genes. Dephosphorylated ICP4 retains its binding properties for the high affinity sites present in alpha promoters, whereas only phosphorylated forms of the protein are able to participate in complex formation with model beta and gamma sequences. These studies also reveal a requirement for cell and infected-cell factors to recognize the beta and gamma sequences. Our data suggest that the state of phosphorylation and concentration of ICP4 within the nucleus of infected cells determine the extent to which ICP4 interacts with these other factors.

Acid Phosphatase↗
PubMed1990

Regulation of herpes simplex virus immediate-early gene promoters in mouse neuroblastoma cells.

The non-permissivity of C1300 mouse neuroblastoma cells for herpes simplex virus (HSV) infection is due to a failure of such cells to transcribe the immediate-early (IE) genes following viral infection. We have transfected both C1300 cells and permissive cells with constructs in which each of the 5 IE promoters drives expression of the readily assayable chloramphenicol acetyl transferase (CAT) gene. These experiments show that the lack of IE gene transcription in C1300 cells is due to the weak activity of the five IE promoters in these cells compared to that observed in a range of permissive cell types. This effect is mediated both by up-stream elements and by sequences present in the minimal promoter. The different effects of DNA concentration on the activities of the minimal and complete promoters suggests that the up-stream sequences act by binding a repressor factor present in C1300 cells whilst the weak activity of the minimal promoter results from the absence of a positive factor in such cells.

Animals↗
PubMed1990

Renaturation of denatured lambda repressor requires heat shock proteins.

The temperature-sensitive bacteriophage lambda cI857 repressor protein rapidly renatures after thermal inactivation. E. coli mutants in the heat shock protein genes dnaK, dnaJ, and grpE do not efficiently reactivate heat-denatured repressor. Our results suggest that protein refolding is promoted by heat shock proteins and that such a process is the basis of the homeostatic role played by these proteins in the heat shock response.

Bacteriophage lambda↗
PubMed1990

Interaction of cell and virus proteins with DNA sequences encompassing the promoter/regulatory and leader regions of the herpes simplex virus thymidine kinase gene.

During the course of a productive infection with herpes simplex virus (HSV), gene expression is coordinately regulated in a cascade fashion. Three major kinetic classes of genes, termed alpha, beta, and gamma, are sequentially activated. The mechanism responsible for repression and subsequent activation of beta and gamma genes is not known. A mobility-shift electrophoresis assay was used to examine DNA fragments containing the promoter/regulatory and the mRNA leader regions of the thymidine kinase gene (TK, a model beta gene) for their ability to bind proteins present in nuclear extracts prepared from uninfected and infected cells. Specific complexes unique to each extract were formed. Using a monoclonal antibody specific for ICP4 (the major regulatory protein of HSV) we demonstrated that this protein is present in the complexes formed between probes encompassing either the promoter/regulatory or leader sequence DNAs and proteins in infected-cell extracts. These complexes formed despite the lack of a high affinity binding site for ICP4 in either of these regions. The stability of complexes formed in infected-cell extracts with DNA probes containing the promoter/regulatory, leader region, and a high affinity ICP4-binding site were compared by dissociation analysis. The relative kd(obs) for these DNA-protein complexes was in the order: TK-leader region much greater than TK-promoter/regulatory region greater than or equal to high affinity ICP4-binding site. Cu+/1,10-phenanthroline footprinting revealed that infected-cell complexes which form on a probe containing a high affinity ICP4-binding site generate a protection pattern, whereas those formed on a probe containing the TK-leader sequence do not. In contrast, complexes formed with the latter probe in extracts from uninfected cells are kinetically stable and refractile to cleavage. A model for activation of the TK gene which incorporates these results is presented.

Binding Sites↗
PubMed1990

Characterization of DNA-protein complex formation in nuclear extracts with a sequence from the herpes simplex virus thymidine kinase gene.

The biochemical characteristics of complex formation in nuclear extracts from mock-infected and herpes simplex virus (HSV)-infected Vero and HeLa cells with a sequence downstream of and adjacent to the promoter for the HSV thymidine kinase gene were studied using the mobility shift electrophoresis assay. This region is bound by host cell proteins, as evidenced by the formation of complexes after incubation in extracts from mock-infected cells. Unique virus-specific complexes form in extracts prepared from infected cells, and these complexes contain ICP4, the major regulatory protein of HSV. Examination of the salt requirements for assembly and the stability of preformed DNA-protein complexes to added salt demonstrate the distinct nature of the complexes that form in each extract. This finding is supported by analyses of the relative association and dissociation rates of these complexes which show that complexes formed in extracts prepared from infected cells are kinetically labile. After depletion with chelators, the divalent cation requirements for complex formation were assayed by supplementation with various metal salts. Addition of Mn2+ restored binding activity in extracts from both mock-infected and infected HeLa cells. Finally, footprinting assays revealed that sequences on each strand throughout this region of the thymidine kinase gene were involved in complex formation only in extracts from mock-infected cells. These experiments suggest that one consequence of virus gene expression is to alter the interaction of cell proteins with virus DNA.

Animals↗
PubMed1990

Occurrence of human papillomavirus type 16 DNA in cutaneous squamous and basal cell neoplasms.

Sixty-eight cutaneous squamous cell neoplasms (in situ and invasive) and 26 basal cell carcinomas from 89 patients were analyzed for DNA sequences homologous to the human papillomavirus (HPV) types found predominantly in the genital tract. Thirty-six (53%) of the squamous cell neoplasms contained HPV DNA as detected by filter or in situ hybridization analysis. The frequency of detection of HPV DNA was dependent on the site of the lesion. Of 40 genital squamous cell neoplasms (penile, vulvar, and perianal), 27 (68%) had detectable HPV DNA. In 25 of these, the HPV type was 16 or HPV-16-related, which was similar to the results for the squamous cell neoplasms of the finger (HPV DNA in 9 of 11 tumors with HPV-16 in seven). None of 16 squamous cell neoplasms from sites other than the genital tract or the finger had detectable HPV DNA. HPV DNA was detected in one of the 26 basal cell carcinomas (4%). We conclude that, for cutaneous epithelial malignancies, HPV-16 is restricted to squamous cell neoplasms of the genital tract and finger. These data are consistent with venereal transmission of HPV-16 to the periungual region and suggests a role for this virus in the evolution of squamous cell carcinoma at this site.

Adult↗
PubMed1990

Murine cytotoxic T lymphocytes specific for herpes simplex virus type 1 recognize the immediate early protein ICP4 but not ICP0.

Vaccinia virus recombinants expressing the herpes simplex virus type 1 (HSV-1) genes encoding ICP0 or ICP4 were used to identify the precise target antigen(s) of murine anti-viral cytotoxic T lymphocytes (CTL) specific for the non-structural immediate early proteins. These studies revealed that HSV-1-specific CTL, restricted to class I major histocompatibility complex genes of the H-2k haplotype but not the H-2d or H-2b haplotypes, would lyse autologous cells expressing ICP4. HSV-1-specific CTL derived from various mice strains failed to lyse target cells expressing ICP0. Calculation of the frequencies of H-2k-restricted virus-specific CTL demonstrated that approximately a third of the total HSV-1-specific response was directed against ICP4. Immunization of mice with either recombinant vaccinia virus or transfected L cells expressing ICP4 induced HSV-1-specific lymphoproliferation and delayed hypersensitivity but CTLs were not induced. More importantly, such immunized animals were unable to resist or control a subsequent challenge with virulent HSV-1.

Animals↗
PubMed1989

Cell proteins bind to multiple sites within the 5' untranslated region of poliovirus RNA.

The 5' noncoding region of poliovirus RNA contains sequences necessary for translation and replication. These functions are probably carried out by recognition of poliovirus RNA by cellular and/or viral proteins. Using a mobility-shift electrophoresis assay and 1,10-phenanthroline/Cu+ footprinting, we demonstrate specific binding of cytoplasmic factors with a sequence from nucleotides 510-629 within the 5' untranslated region (UTR). Complex formation was also observed with a second sequence (nucleotides 97-182) within the 5' UTR. These two regions of the 5' UTR appear to be recognized by distinct cell factors as determined by competition analysis and the effects of ionic strength on complex formation. However, both complexes contain eukaryotic initiation factor 2 alpha, as revealed by their reaction with specific antibody.

HeLa Cells↗
PubMed1989

Correlation of histology and human papillomavirus DNA detection in condyloma acuminatum and condyloma-like vulvar lesions.

The diagnosis of a vulvar condyloma is made when perinuclear halos are seen with nuclear atypia and binucleate forms (koilocytotic atypia). These changes are most prominent in the granular layer and are associated with the presence of human papillomavirus (HPV). However, these changes may be absent or minimal in patients with papillary vulvar lesions; this situation can thus present diagnostic difficulties. We analyzed the histologic features of 53 biopsies from 48 patients who had vulvar lesions suggestive of condylomata. Of the 26 biopsy specimens with koilocytotic atypia, 20 (77%) had sequences homologous to HPV DNA as detected by Southern blot hybridization analysis using a probe of HPV s 6/11, 16, 18, 31, 35, and 51. In cases where the histologic features were suggestive but not diagnostic of condylomata, because unequivocal koilocytotic atypia was not noted, five of 27 (19%) had detectable HPV DNA. In this latter group, we found no histologic feature to distinguish the cases that had detectable HPV DNA from those that did not. Analysis for HPV DNA by in situ hybridization in the cases that were histologically equivocal for condyloma was uniformly negative. We conclude that there is a marked decrease in the detection rate of the HPV types associated with genital tract neoplasms in vulvar lesions that lack koilocytotic atypia. Southern blot hybridization analysis was the only reliable way to distinguish the "equivocal for condyloma" cases that had HPV from those where HPV DNA was not detected.

Adult↗
PubMed1988

Histological correlates of clinically occult human papillomavirus infection of the uterine cervix.

Occult infection of the uterine cervix by human papillomavirus (HPV) is assumed when viral DNA sequences are detected from cervical swabs but no lesion is detectable and the Papanicolaou smear is negative. In an attempt to identify what histological changes correlate with occult infections, DNA was extracted from 200 cervical swabs taken from hysterectomy specimens. The DNA was analyzed by Southern blot hybridization for the presence of HPV sequences. Eleven cases (5.5%) were positive. The entire cervix from each case as well as from 28 negative cases was processed for histological analysis. One of the positive cases contained a CIN 2 lesion. The other 10 showed parakeratosis, papillomatosis, acanthosis, as well as focal nuclear pleomorphism and perinuclear halos (borderline koilocytotic atypia) in proportions equal to the negative controls. In situ hybridization analysis of the cases that showed borderline koilocytotic atypia were negative. These findings confirm that clinically and cytologically occult HPV infection of the uterine cervix is not associated with diagnostic histological changes. This underscores the need for caution when interpreting cervical biopsies that show changes suggestive, but not absolutely diagnostic, of HPV infection. Further, the precise epithelial location of the virus remains unclear.

Adult↗
PubMed1988

Accumulation of RNA homologous to human papillomavirus type 16 open reading frames in genital precancers.

The accumulation of human papillomavirus type 16 (HPV-16)-specific RNAs in tissue sections from biopsies of patients with genital precancers was studied by in situ hybridization with single-stranded 35S-labeled RNA. These analyses revealed that the most abundant early-region RNAs were derived from the E4 and E5 open reading frames (ORFs). RNAs homologous to the E6/E7 ORFs were also detected, whereas RNAs homologous to the intervening E1 ORF were not. This suggests that the E4 and E5 mRNAs are derived by splicing to the upstream E6/E7 ORFs, consistent with studies of HPV-11 in condylomata (L. T. Chow et al., Cancer Cells (Cold Spring Harbor) 5:55-72, 1987). Abundant RNAs homologous to the 5' portion of L1 were also detected. These RNAs were localized to the apical strata of the epithelium. HPV-16 RNAs accumulated in discrete regions of these lesions, and when present were most abundant in the upper cell layers of the precancerous epithelium. RNAs homologous to early ORFs were also detected in some germinal cells within the basal layer of the epithelium.

Epithelium↗
PubMed1988

Isolation of a novel human papillomavirus (type 51) from a cervical condyloma.

We cloned the DNA from a novel human papillomavirus (HPV) present in a cervical condyloma. When DNA from this isolate was hybridized at high stringency with HPV types 1 through 50 (HPV-1 through HPV-50), it showed weak homology with HPV-6 and -16 and stronger homology with HPV-26. A detailed restriction endonuclease map was prepared which showed marked differences from the maps for other HPVs that have been isolated from the female genital tract. Reassociation kinetic analysis revealed that HPV-26 and this new isolate were less than 10% homologous; hence, the new isolate is a novel strain of HPV. The approximate positions of the open reading frames of the new strain were surmised by hybridization with probes derived from individual open reading frames of HPV-16. In an analysis of 175 genital biopsies from patients with abnormal Papanicolaou smears, sequences hybridizing under highly stringent conditions to probes from this novel HPV type were found in 4.2, 6.1, and 2.4% of biopsies containing normal squamous epithelium, condylomata, and intraepithelial neoplasia, respectively. In addition, sequences homologous to probes from this novel isolate were detected in one of five cervical carcinomas examined.

Adult↗
PubMed1988

A comparison of biotin and isotope-labeled ribonucleic acid probes for in situ detection of HPV-16 ribonucleic acid in genital precancers.

Genital precancers were analyzed by in situ hybridization for the presence of HPV RNA using single-stranded RNA probes. To compare the sensitivity of biotin- and 35S-labeled probes, serial sections from three biopsies containing variable amounts of HPV 16 RNA were analyzed with probes containing each label. The probe template was a cloned fragment of the HPV 16 genome spanning portions of the E2, E5 and L2 orf's. When a focus contained a strong hybridization signal with 35S-labeled probes (i.e., target/background ratio greater than 40) serial sections of the same area usually demonstrated a strongly positive signal with biotin-labeled probes. When the signal with 35S in a given focus was weak (target/background ratio less than 20), serial sections contained either a very weak or no detectable signal with the biotinylated probe. From this study it appears than 35S-labeled RNA probes exceed the sensitivity of biotin-labeled probes by nearly an order of magnitude with short exposures (4 days), and with longer exposures (1 to 2 weeks), approach the sensitivity of that reported for tritiated probes and exposures of 1 month or longer.

Biotin↗
PubMed1988

Histologic correlates of papillomavirus infection of the vagina.

Vaginal warts (condylomata) secondary to human papillomavirus (HPV) infection are most easily identified when perinuclear halos are seen with clear-cut nuclear atypia (koilocytotic atypia) in the superficial cells of the lesion. However, vaginal lesions clinically resembling condylomata may exhibit lesser degrees of nuclear atypia with findings suggestive of, but not typical for, the presence of HPV. The diagnosis and management of such lesions are unclear. We correlated histologic findings with the detection of HPV DNA sequences in 39 vaginal lesions that contained a spectrum of clinical and histologic features suggestive of, or diagnostic for, condyloma. We detected HPV DNA in 63% (N = 22) of lesions with koilocytotic atypia and in 11.7% (N = 17) of lesions that showed some histologic features suggesting condyloma but that lacked koilocytotic atypia. These findings imply that vaginal lesions lacking koilocytotic atypia are not always caused by HPV, emphasizing the need for a conservative approach when the diagnosis of vaginal condyloma is not clear-cut.

Adult↗
PubMed1988

Comparison of formalin, buffered formalin, and Bouin's fixation on the detection of human papillomavirus deoxyribonucleic acid from genital lesions.

Detection of nucleic acid sequences homologous to human papillomavirus (HPV) relies primarily on their extraction from unfixed tissue. We detected HPV sequences in DNA extracted from paraffin-embedded tissue fixed in formalin (buffered and unbuffered) and Bouin's solution by dot blot hybridization. A detectable hybridization signal was noted in 32% of these fixed tissues which were chosen from cases where HPV DNA was detected in the unfixed tissue. When using a homologous 32P-labeled probe and a high stringency wash, the hybridization signal was lost if DNA was extracted after Bouin's fixation and diminished after formalin fixation, more so with unbuffered formalin. Similar differences in the hybridization signals among the different fixatives after high stringency wash were noted with in situ hybridization. Southern blot analysis showed that DNA extracted from tissues fixed in Bouin's was degraded and ranged in size from 100 to 500 base pairs as compared with 100 to 900 base pairs for DNA extracted from tissue fixed with unbuffered formalin. In contrast, no degradation was noted after fixation with buffered formalin. These results demonstrate that HPV sequences can be identified in DNA extracted from paraffin-embedded, fixed tissue. However, use of some fixatives may preclude identification of HPV type, by either dot blot or in situ hybridization.

Buffers↗
PubMed1986

Human papillomavirus type 16 infection: a morphological spectrum with evidence for late gene expression.

Human papillomavirus (HPV) type 16 is a unique strain found almost exclusively in squamous precancerous lesions and invasive carcinomas of the genital tract. A histological and immunohistochemical analysis of 34 cervical biopsies from which HPV 16 was isolated was performed to determine: the morphological spectrum of HPV 16 "infection" and if HPV late genes were expressed in lesions containing this virus. Twenty-eight of thirty-four (82%) biopsy specimens contained areas fulfilling the histological criteria for cervical intraepithelial neoplasia (CIN) (defined as the presence of abnormal mitoses and/or diffuse nuclear atypia in a portion of the biopsy). However, 19 of 28 (68%) CIN lesions also contained focal areas of epithelium indistinguishable from condylomata. Three biopsy specimens each contained condyloma only and normal-appearing squamous epithelium. Three of thirty-one lesions (10%) showed evidence of HPV capsid antigens. The presence of areas of condyloma, as well as capsid antigens, indicates that lesions containing HPV 16 share certain similarities with conventional warts associated with other HPVs. Furthermore, the wide range of morphology in lesions containing HPV 16 suggests that histological or cytological recognition of HPV 16-associated CIN lesions may not always be possible.

Carcinoma in Situ↗
PubMed1985

Latent papillomavirus and recurring genital warts.

After anogenital condylomata and intraepithelial neoplasms are removed, they frequently recur. Since these lesions are related to papillomaviruses, it has been suggested that latent papillomavirus infection is responsible for recurrence. We studied 20 cases of anogenital lesions that were treated by laser therapy and analyzed biopsy specimens of margins of normal skin adjacent to the lesions for papillomavirus sequences by Southern blot hybridization. In nine cases (45 per cent), papillomavirus sequences were detected in the normal skin margin; lesions recurred in 6 of the 9 patients (67 per cent), in contrast to only 1 of 11 patients (9 per cent) whose margins were negative for the presence of papillomavirus sequences. All but one recurrence developed within 15 mm of the treatment area. Our results demonstrate that clinically and histologically latent papillomavirus exists beyond the treatment area and that its presence influences subsequent recurrences.

Animals↗