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S Silverstein

Publications and source records attributed to S Silverstein.

At least 37 records · Page 2Linked to original sources
PubMed1993

Identification of a promoter mapping within the reiterated sequences that flank the herpes simplex virus type 1 UL region.

Analysis of the promoter for the herpes simplex virus (HSV) immediate-early (alpha) gene alpha 0 in a short-term transient expression assay revealed that a SacI-to-NcoI fragment from -786 to +148 relative to the cap site directed the synthesis of chloramphenicol acetyltransferase when the fragment was present in either orientation. Although the constitutive levels of promoter activity were similar with either orientation, the reverse-orientation promoter was not induced in response to infection with HSV. Analysis of sequences composing the putative promoter in the opposite orientation revealed the presence of important regulatory elements associated with alpha promoters. These include an alpha-trans-inducing factor (alpha-TIF)-like response element, a high-affinity ICP4-binding site, numerous Sp1-binding sites, and a TATA box. Sequences contained within this region formed specific DNA-protein complexes in extracts from mock-infected and HSV-infected HeLa cells. Transient expression assays revealed that this sequence was positively regulated by the alpha 0 and alpha-TIF genes but negatively regulated by alpha 4. Finally, nuclear run-on transcription assays revealed that this promoter is active in its correct genomic context during the course of virus infection. We suggest that the promoter is a hybrid between an alpha and beta promoter because it exhibits maximal expression at 8 h postinfection and is expressed in the presence of cycloheximide.

Base Sequence↗
PubMed1993

A PCR strategy that differentiates two alleles for a single base insertion within a T repeat.

PCR amplification with modified primers, in combination with BcgI restriction fragment length polymorphism analysis, allowed us to differentiate a string of 7 Ts present in exon 7 of the 21-hydroxylase gene of man from the 8 Ts present in exon 7 of its pseudogene. This approach can substitute for hybridization with oligonucleotides, or for sequence analysis, when an allele containing a multiple repeated nucleotide sequence is to be differentiated from one with a different number of the identical nucleotide.

Alleles↗
PubMed1992

Relationship of human papillomavirus type to grade of cervical intraepithelial neoplasia.

OBJECTIVE--To determine the relationship of human papillomavirus (HPV) type to grade of cervical intraepithelial neoplasia (CIN) in a large series of cases. DESIGN--A survey of HPV types in CIN lesions detected using a new, highly accurate method for typing HPV that is based on restriction fragment length polymorphism analysis of amplimers produced during polymerase chain amplification of the conserved L1 region of HPV using consensus primers. SETTING--Private gynecologists' offices and inner-city colposcopy clinics. PATIENTS--A convenience sample of 276 HPV DNA-positive cervical biopsy specimens or samples from patients undergoing colposcopy for abnormal Papanicolaou smears. INTERVENTION--None. MAIN OUTCOME MEASURE(S)--Human papillomavirus type(s). RESULTS--Cervical intraepithelial neoplasia 1 lesions were relatively heterogeneous with regard to associated HPV types. Nineteen percent of CIN 1 lesions were associated with HPV types 6 or 11; 29% contained HPV types 16, 18, or 33; and 19% were associated with "novel types" of HPV. It was also found that 22% of CIN 1 lesions were associated with more than one HPV type. In contrast to CIN 1, both CIN 2 and CIN 3 were relatively homogeneous with regard to associated HPV types. Eighty-eight percent of CIN 2 and 3 lesions contained HPV types 16, 18, or 33. Unlike CIN 1 lesions, which often contained multiple types of HPV, only 7% of CIN 2 and 3 lesions were associated with multiple HPV types. CONCLUSIONS--Cervical intraepithelial neoplasia should be classified into two separate categories--low-grade and high-grade CIN. Since only 29% of low-grade lesions are associated with HPV types 16, 18, or 33, HPV type could potentially play a role in determining the most appropriate clinical management of patients with low-grade CIN. However, prospective follow-up studies of lesional behavior based on HPV type are required before clinical recommendations can be made.

DNA, Viral↗
PubMed1992

Typing of human papillomaviruses by polymerase chain reaction amplification with L1 consensus primers and RFLP analysis.

Human papillomaviruses (HPV) cause benign and malignant lesions of the epithelial and mucosal surfaces. Certain virus types are associated with cervical carcinomas, while others are associated with benign condylomata. We have developed a rapid method for determining HPV type that is based on restriction fragment length polymorphism (RFLP) analysis within the L1 region of HPVs that is amplified by PCR using the consensus primers described by Manos et al. Analysis of the product generated by PCR amplification of plasmids containing cloned HPV genomes and of 88 clinical specimens, known to contain HPV viral DNA by previous hybridization analysis, revealed that this method is useful for typing HPV sequences amplified from a variety of sources including cervical lavages, fresh tissue, and paraffin-embedded formalin-fixed biopsy material. The method can differentiate between most known types of HPV and discriminate between infections with single, multiple or novel HPV types. A high correlation (86%) was obtained when this method was compared with PCR amplification and Southern blot hybridization analysis of PCR product, or Southern blot hybridization analysis of total genomic DNA. Differences in typing occurred mostly for specimens that contained multiple or new/unknown HPV types. However, RFLP typing easily identified repeated patterns for new HPV types that were not detected by the other methods. In summary, PCR-RFLP typing is a sensitive and specific method to identify and characterize rapidly HPV DNA in clinical specimens from a variety of sources.

Blotting, Southern↗
PubMed1992

Varicella vaccine: the American experience.

Live attenuated varicella vaccine is safe and effective in preventing chickenpox. The best immune responses occur in healthy children. Leukemic children have a 50% incidence of mild-to-moderate adverse effects but have a high degree of protection once immune reactions to varicella-zoster virus (VZV) have developed. Adult vaccinees have a lower degree of protection (70%) than children. Vaccinees who develop breakthrough varicella usually have a modified infection. Another significant advantage of vaccination is that in leukemic children it leads to a lower incidence of zoster than after natural chickenpox. It is possible to differentiate between vaccine-type and wild-type VZV using a combination of polymerase chain reaction and restriction endonuclease digestion. A new assay for antibodies to VZV measured by latex agglutination reveals that 8-10 years after vaccination antibodies are detectable in greater than 90% of leukemic children who have not had breakthrough varicella.

Adult↗
PubMed1992

Restriction fragment length polymorphism of polymerase chain reaction products from vaccine and wild-type varicella-zoster virus isolates.

The nucleotide changes that result in two restriction endonuclease polymorphisms that differentiate wild-type varicella-zoster virus (VZV) from the vaccine strain were determined. Oligonucleotide primers that flank these sites were used to amplify the intervening sequences with the polymerase chain reaction to identify VZV DNA in clinical isolates. Restriction enzyme digestion of the amplification products distinguished vaccine and wild-type genomes from one another. This study confirms the feasibility of amplifying VZV sequences so that they may be detected in clinical specimens and provides a molecular epidemiological approach to strain identification of VZV in vesicular lesions.

Base Sequence↗
PubMed1992

Herpes simplex viruses with mutations in the gene encoding ICP0 are defective in gene expression.

Herpes simplex virus type 1 (HSV-1) mutants with codon insertions and deletions in IE-0, the gene encoding ICP0, were constructed. The HSV-1 deletion mutant dl1403 (N. D. Stow and E. C. Stow, J. Gen. Virol. 67:2571-2585, 1986) and an IE-0:lacZ transplacement vector isolated in this study were used to facilitate the construction of mutant viruses. Mutant viruses, all of which produced stable ICP0, were examined for their ability to plaque and grow on both Vero and HeLa cells because previous results showed that HSV-1 immediate-early (IE) gene promoters and their products are differentially expressed in these cells (J. Chen, X. Zhu, and S. Silverstein, Virology 180:207-220, 1991; I. H. Gelman and S. Silverstein, J. Virol. 61:2286-2296, 1987). Viruses with IE-0 genes that only poorly activated reporter genes in transient expression assays plaqued less efficiently on Vero cells and consistently accumulated decreased levels of late proteins. These mutants were also examined in single-step growth curve experiments and for the dependence of virus yield on multiplicity of infection (MOI). At low MOIs, their yields were less in Vero cells than in HeLa cells; by contrast, at high MOIs, there was no apparent difference in yield in either cell type, although each virus produced considerably fewer progeny than wild-type virus. Analysis of steady-state levels of RNA from genes representing each of the three major kinetic classes demonstrated that lower levels of RNAs accumulate in these mutants. We conclude from these studies that while ICP0 is not essential for virus growth in tissue culture, defects in this gene result in impairment of virus replication and delay the expression of early and late gene transcripts.

Animals↗
PubMed1992

Multimerization of ICP0, a herpes simplex virus immediate-early protein.

ICP0, a herpes simplex virus immediate-early gene product, is a highly phosphorylated nuclear protein that is a potent activator of virus and host genes. Using biochemical and genetic assays employing plasmids encoding mutant forms of ICP0 and a recombinant adenovirus that expresses ICP0, we mutant forms of ICP0 and a recombinant adenovirus that expresses ICP0, we provide evidence that the protein multimerizes. Some mutant forms of ICP0 were transdominant and interfered with activation of a target reporter gene or with complementation of an ICP0-minus virus.

Adenoviridae↗
PubMed1991

Mutational analysis of the sequence encoding ICP0 from herpes simplex virus type 1.

In-frame codon insertion and deletion mutants were constructed in a plasmid containing the sequence that encodes ICP0, a transcriptional activator of herpes simplex virus type 1 (HSV-1). The effect of these mutations was analyzed in a transient expression assay using the promoters for, the IE-0 gene (an immediate early (alpha) gene), the thymidine kinase gene (an early (beta) gene), and the glycoprotein C gene (a late (gamma) gene) fused to reporter cassettes that encoded either beta-galactosidase or chloramphenicol acetyl transferase. Assays were performed in the presence or absence of a plasmid encoding ICP4, the major regulatory protein of HSV-1. Our results demonstrate that ICP0-mediated transactivation varied depending on the position of the insertion in the gene. One region of this protein was consistently shown to be required for full activation of each promoter examined either in the presence or in the absence of ICP4. This region overlaps with a cysteine-rich region and coincides with a transactivator domain identified in another extensive mutational analysis of this sequence. Analysis of the deletion mutants generated in this study demonstrated that the carboxy-terminal regions were required for activation in certain circumstances and that this varied depending on the promoter being assayed and the cell type in which the analysis was performed.

Amino Acid Sequence↗
PubMed1991

Transactivation by herpes simplex virus proteins ICP4 and ICP0 in vaccinia virus infected cells.

Vaccinia virus recombinants containing the sequences from herpes simplex virus type 1 (HSV-1) encoding the immediate early (IE)(alpha) proteins ICP4 and ICP0, under the control of a mutated vaccinia virus 11K late promoter, were constructed. A cDNA copy of the gene encoding ICPO and an ICP4-encoding genomic segment were each inserted into the vaccinia virus genome at the thymidine kinase (TK) locus by homologous recombination. Steady-state analyses revealed that RNAs homologous to the IE-0 and IE-4 sequences accumulated in cells infected by recombinants with the kinetics of a typical vaccinia late mRNA. Western blot analyses demonstrated that the expression level of both ICPO and ICP4, produced by the recombinant viruses, was comparable to that in HSV-1-infected cells at late times postinfection. Both proteins synthesized in cells infected by the recombinants were located in the nucleus as revealed by immunofluorescence. Although in vitro studies reveal that extracts from vaccinia-virus-infected cells lose the ability to transcribe genes that contain RNA polymerase II promoters (Puckett and Moss (1983), Cell 35, 441-448) both ICPO and ICP4 expressed by the recombinant viruses can transactivate plasmids containing a reporter gene driven by the promoters for the HSV-1 TK and glycoprotein C genes. Nuclear extracts prepared from cells infected with the vaccinia virus vector expressing ICP4 exhibited sequence-specific DNA-binding activity.

Animals↗
PubMed1991

Analysis of the herpes simplex virus type 1 promoter controlling the expression of UL38, a true late gene involved in capsid assembly.

The cistrons encoding the herpes simplex virus type 1 (HSV-1) UL37 and UL38 genes are adjacent to one another but are transcribed from opposite strands of the viral DNA. The UL37 gene encodes a 1,123-amino-acid protein of unknown function, while the 465-amino-acid UL38 protein is involved in capsid assembly. Previous work from our laboratory indicated that the transcripts encoding these proteins are expressed with significantly different kinetics in productive infection. In the present communication we confirm the kinetic classes and precisely map the cap sites of the UL37 and UL38 mRNAs. A bifunctional reporter gene vector was used to demonstrate that divergent promoters control the expression of these reporter genes in trans-activation assays. The UL38 promoter is functionally separable from that controlling UL37 in a recombinant virus. We used deletion analysis to demonstrate that as few as 29 bases 5' of the mRNA cap site are adequate for full activity of the UL38 promoter in trans-activation assays. Finally, we analyzed the protein-binding properties of the UL38 promoter; several sites that form complexes containing ICP4, with clear homology to those identified in the HSV-1 gamma 42 promoter, are present. Thus, in general, the properties of this promoter are quite similar to those of other gamma promoters.

Animals↗
PubMed1991

Isolation and characterization of a functional cDNA encoding ICP0 from herpes simplex virus type 1.

The IE-0 gene of herpes simplex virus type 1 (HSV-1) contains two introns and encodes ICP0, a powerful transcriptional activator. We have isolated a cDNA clone that encodes ICP0 from a lambda gt10 cDNA library constructed from RNAs made from HSV-1-infected HeLa cells. DNA sequence analysis of this clone confirmed the predicted intron/exon boundaries (L. J. Perry, F. J. Rixon, R. D. Everett, M. C. Frame, and D. J. McGeoch, J. Gen. Virol. 67:2365-2380, 1986). Following transfection, a plasmid containing the cDNA copy of IE-0 directed the synthesis of ICP0, which was appropriately compartmentalized and distributed in the nucleus, as revealed by immunofluorescence. A transient expression assay was used to demonstrate that this cDNA copy retained the ability to transactivate the HSV-1 promoters for the IE-0 gene (an immediate-early gene), the thymidine kinase gene (an early gene), and the glycoprotein C gene (a late gene). The product of this cDNA clone cooperated with ICP4 to activate expression from the thymidine kinase gene promoter in a synergistic manner. The availability of a functional cDNA copy encoding ICP0 provides the opportunity to express this protein in vector systems that do not recognize eucaryotic donor and acceptor splicing signals to overexpress ICP0.

Base Sequence↗
PubMed1991

Biologic properties and nucleotide sequence analysis of human papillomavirus type 51.

Human papillomaviruses (HPVs) may be grouped according to the site from which they are isolated and the disease with which they are associated. We recently identified and cloned HPV type 51 (HPV-51) from a low-grade precancerous lesion (G. Nuovo, E. DeVilliers, R. Levine, S. Silverstein, and C. Crum. J. Virol. 62:1452-1455, 1988). Molecular epidemiologic analysis of cervical lesions, including condylomata and low- and high-grade precancers, revealed that HPV-51 was present in about 5% of the samples we examined. We have now determined the complete nucleotide sequence of this virus and compared it with other sequenced HPVs. Our analysis reveals that the 7,808-bp genome is composed of eight open reading frames which are encoded on the same strand and that this virus is most closely related to HPV-31. Sequence comparisons place this virus in the group of high-risk viruses (those with an increased risk of progressing to malignancy) along with HPV-16, -18, -31, and -33. Morphologic transformation experiments demonstrated that HPV-51 had transformation potential and that transformed cells contained RNAs homologous to E6 and E7.

Amino Acid Sequence↗
PubMed1990

Reactivation of latent herpes simplex virus by adenovirus recombinants encoding mutant IE-0 gene products.

We have previously shown that adenovirus recombinants expressing functional ICP0 reactivate latent herpes simplex virus type 2 (HSV-2) in an in vitro latency system. This study demonstrated that ICP0, independent of other HSV gene products, is sufficient to reactivate latent HSV-2 in this in vitro system. To assess the effects of defined mutations in the sequence encoding ICP0 (IE-0) on reactivation, seven in-frame insertion and three in-frame deletion mutants were moved into an adenovirus expression vector. Each recombinant directed the synthesis of stable ICP0 of the correct size. The transactivation activity of the mutated sequences in these recombinants was similar to that when they were tested in plasmids. When these recombinants were examined for their ability to reactivate in the in vitro latency system, mutants with dramatic defects in transactivation (Ad-0/125, Ad-0/89, Ad-0/2/7, and Ad-0/88/93) were unable to reactivate latent HSV-2 independent of the multiplicity of infection. An exception to this correlation was the finding that Ad-0/89, which transactivated poorly, was able to reactivate latent virus after prolonged incubation whereas other transactivation-deficient mutants could not. Moreover, the presence of ICP4 did not compensate for the inability of any of the recombinants tested to reactivate HSV-2. These results show that (i) the transactivation domains of ICP0 are also used in reactivation, (ii) the presence of another essential HSV regulatory protein ICP4 does not alter the pattern of reactivation by ICP0, and (iii) mutations in some regions of IE-0 previously shown to affect viral growth and plaque formation did not alter its ability to reactivate in this in vitro system.

Adenoviruses, Human↗
PubMed1989

Human papillomavirus type 16 DNA in periungual squamous cell carcinomas.

Ten squamous cell carcinomas (in situ or invasive) of the fingernail region were analyzed for the presence of DNA sequences homologous to human papillomavirus (HPV) by dot blot hybridization. In most patients, the lesions were verrucae of long-term duration that were refractory to conventional treatment methods. Eight of the lesions contained HPV DNA sequences, and in six of these the sequences were related to HPV 16 as deduced from low-stringency nucleic acid hybridization followed by low- and high-stringency washes. Furthermore, the restriction endonuclease digestion pattern of DNA isolated from four of these lesions was diagnostic of episomal HPV 16. The high-frequency association of HPV 16 with periungual squamous cell carcinoma is similar to that reported for HPV 16 with squamous cell carcinomas on mucous membranes at other sites, notably the genital tract. Our findings suggest that HPV 16 may play an important role in the development of squamous cell carcinomas of the finger, most notably those lesions that are chronic and located in the periungual area.

Adult↗
PubMed1989

Herpes simplex virus type 1 immediate-early protein Vmw110 reactivates latent herpes simplex virus type 2 in an in vitro latency system.

Reactivation of latent herpes simplex virus type 2 (HSV-2) by the immediate-early protein Vmw110 was studied by using an in vitro latency system. Adenovirus recombinants that express Vmw110 reactivated latent HSV-2. An HSV-1 mutant possessing a deletion in a carboxy-terminal region of Vmw110 reactivated latent HSV-2, whereas mutant FXE, which has a deletion in the second exon, did not. Therefore, Vmw110 alone is required to reactivate latent HSV-2 in vitro, and the region of Vmw110 defined by the deletion in FXE is important for this process.

Adenoviridae↗