Biomedical subjects

D M Knipe

Publications and source records attributed to D M Knipe.

At least 73 records · Page 4Linked to original sources

PubMed1989

A deletion mutant of the latency-associated transcript of herpes simplex virus type 1 reactivates from the latent state with reduced frequency.

We have generated and characterized a deletion mutant of herpes simplex virus type-1, dlLAT1.8, which lacks the putative promoter region, transcriptional start site, and 1,015 base pairs of the DNA sequences specifying the latency-associated transcripts (LATs). When tested in a CD-1 mouse ocular model, dlLAT1.8 was replication competent in the eye and in ganglia during acute infection but reactivated from explant cultures of ganglia with reduced efficiency (49%) relative to those of wild-type and marker-rescued viruses (94 and 85%, respectively) despite the fact that levels of mutant viral DNA in ganglia during latent infection were comparable to wild-type levels. The neurovirulence of KOS was not significantly altered by the removal of sequences specifying the LATs, as judged by numbers of animals dying on or before 30 days postinfection. Examination of ganglia latently infected with dlLAT1.8 by in situ hybridization revealed no LAT expression. The genotype of reactivated virus was identical to that of input dlLAT1.8 virus as judged by Southern blot analysis. These studies suggest that although the LATs are not essential for the establishment and reactivation of latency in our model, they may play a role in determining the frequency of reactivation of virus from the latent state.

Animals↗

PubMed1989

Herpes simplex virus alpha protein ICP27 possesses separable positive and negative regulatory activities.

The HSV-1 alpha (immediate-early) protein ICP27 expressed in transfected cells can activate the expression of certain HSV-1 promoters as well as inhibit the transactivated expression of others. We constructed a set of plasmids encoding mutant ICP27 molecules truncated at their carboxyl termini and used transfection assays to determine the functional properties of the mutant proteins. A polypeptide containing the amino-terminal 263 amino acid residues of ICP27 retained partial ability to activate gene expression but was unable to inhibit transactivation. Mutant proteins possessing 406 or 504 amino acids of ICP27 were unable to activate gene expression but retained full ability to inhibit transactivation. These results define two separable regulatory activities of ICP27, one positive and one negative, which can modulate gene expression in transfected cells. Immunoblot and immunofluorescence experiments were used to study the immunological reactivities and intracellular localizations of the mutant proteins. All proteins possessing the amino-terminal 263 amino acids of ICP27 reacted with an ICP27-specific monoclonal antibody and were localized to the cell nucleus. The mutant proteins, however, exhibited a number of phenotypes with regard to intranuclear localization. A mutant possessing 504 residues of ICP27 was similar to the wild-type protein in apparently localizing to all regions of the nucleus. A mutant containing 406 residues of ICP27, on the other hand, was mostly excluded from the nucleolar regions, while a 263-residue mutant was localized predominantly in the nucleoli. Thus, some aspect of ICP27 structure or function can dramatically affect its intranuclear distribution.

Animals↗

PubMed1988

Formation of DNA replication structures in herpes virus-infected cells requires a viral DNA binding protein.

Eukaryotic DNA synthesis is thought to occur in multienzyme complexes present at numerous discrete sites throughout the nucleus. We demonstrate here that cellular DNA replication sites identified by bromodeoxyuridine labeling are relocated in cells infected with herpes simplex virus such that they correspond to viral prereplicative structures containing the HSV DNA replication protein, ICP8. Thus components of the cellular DNA replication apparatus are present at viral prereplicative sites. Mutant virus strains expressing defective ICP8 do not alter the pattern of host cell DNA replication sites, indicating that functional ICP8 is required for the redistribution of cellular DNA replication complexes. This demonstrates that a specific protein molecule can play a role in the organization of DNA replication proteins at discrete sites within the cell nucleus.

Animals↗

PubMed1988

Alternative mechanisms for activation of human immunodeficiency virus enhancer in T cells.

The expression of human immunodeficiency virus (HIV) after T cell activation is regulated by NF-kappa B, an inducible DNA-binding protein that stimulates transcription. Proteins encoded by a variety of DNA viruses are also able to activate expression from the HIV enhancer. To determine how this activation occurs, specific genes from herpes simplex virus type 1 and adenovirus that activate HIV in T lymphoma cells have been identified. The cis-acting regulatory sequences in the HIV enhancer that mediate their effect have also been characterized. The relevant genes are those for ICP0-an immediate-early product of herpes simplex virus type 1-and the form of E1A encoded by the 13S messenger RNA of adenovirus. Activation of HIV by adenovirus E1A was found to depend on the TATA box, whereas herpesvirus ICP0 did not work through a single defined cis-acting element. These findings suggest multiple pathways that can be used to bypass normal cellular activation of HIV, and they raise the possibility that infection by herpes simplex virus or adenovirus may directly contribute to the activation of HIV in acquired immunodeficiency syndrome by mechanisms independent of antigenic stimulation in T cells.

Adenoviruses, Human↗

PubMed1988

Genetic identification of a portion of the herpes simplex virus ICP8 protein required for DNA-binding.

The major DNA-binding protein or infected cell protein 8 (ICP8) encoded by herpes simplex virus exhibits multiple interactions with the cell nucleus in that it interacts with the host cell nuclear matrix and viral DNA molecules as sequential stages in its maturational process (M. P. Quinlan, L. B. Chen, and D. M. Knipe (1984), Cell 36, 857-868). To define the portion(s) of ICP8 required for DNA binding, we have fine-mapped and identified the sequence changes in mutant genes causing changes in the protein that affect DNA binding. These mutations lead to amino acid changes between residues 348 and 450 of ICP8. Construction of a mutant ICP8 gene specifically altered at residues 499 and 502 led to a gene product that was also defective in a nuclear function. Thus, at least part of the region of ICP8 from residues 348 to 450 is required for DNA binding by ICP8. This portion of the protein may be involved in binding to DNA or forming intermolecular contacts needed for cooperative DNA binding. If this region is directly involved in binding of the protein to DNA, the most likely structure predicted for this region involves folding of beta-strands to form a channel for binding to a nucleotide chain.

Amino Acid Sequence↗

PubMed1988

Gene-specific transactivation by herpes simplex virus type 1 alpha protein ICP27.

Herpes simplex virus type 1 (HSV-1) encodes several alpha (immediate-early) gene products that modulate gene expression during viral replication. We report here that the alpha protein ICP27 specifically stimulates expression of a later viral gene, that encoding glycoprotein B (gB). Using temperature-sensitive viral mutants, the effect of ICP27 on HSV-1 protein synthesis was examined at early times after infection or at later times when viral DNA replication was inhibited. Under these conditions, the expression of gB showed a marked dependence on the presence of functional ICP27, whereas several other beta and gamma 1 genes showed a lesser dependence. It was also noted that cells infected with ICP27 temperature sensitive mutants at the nonpermissive temperature showed a reduction in the electrophoretic mobility of the alpha protein ICP4. To examine the mechanism by which ICP27 stimulated gB expression, a plasmid was constructed in which the promoter-regulatory region of the gB gene was fused to the gene encoding chloramphenicol acetyltransferase (CAT). CAT expression from this plasmid was induced significantly by ICP27 expressed from a cotransfected plasmid. Induction of CAT activity by ICP27 correlated well with an increase in the amount of CAT transcripts initiated from the transcriptional start site of the gB gene. The transactivating activity of ICP27 was specific for the gB promoter-regulatory region, as expression from several other HSV-1 promoter-CAT chimeric genes was not stimulated by ICP27. The DNA sequences which conferred the response to ICP27 mapped within 175 base pairs upstream and 41 base pairs downstream of the gB transcriptional start site. Our results suggest that the full expression of gB and perhaps other viral genes during HSV-1 infection requires the combined action of multiple viral transactivators.

Acetyltransferases↗

PubMed1987

Stages in the nuclear association of the herpes simplex virus transcriptional activator protein ICP4.

The nuclear localization of the herpes simplex virus transcriptional activator protein ICP4 was studied by indirect immunofluorescence. At early times after viral infection, ICP4 quickly localized to a diffuse intranuclear distribution. ICP4 later concentrated in globular compartments within the nucleus. The redistribution to the compartments was dependent on viral DNA replication. Double staining for ICP4 and ICP8, the early major DNA-binding protein, revealed that both were found in the same intranuclear globular compartments at late times. These were previously named "replication compartments" (M. P. Quinlan, L. B. Chen, and D. M. Knipe, Cell 36:857-868, 1984). Because ICP4 and ICP8 are known to function in transcriptional activation and DNA replication, respectively, both DNA replication and late transcription may occur in these compartments. The association of ICP4 and ICP8 with the replication compartments appeared to be independent in that the retention of ICP8 in the compartments required ongoing viral DNA synthesis, while the association of ICP4 was independent of viral DNA synthesis once the compartments were formed. Because ICP4 shows a different distribution at early and late times, stimulation of transcription by ICP4 may involve different molecular events or contacts during these two periods of the replicative cycle.

Animals↗

PubMed1987

Mapping of the transcriptional initiation site of the herpes simplex virus type 1 ICP8 gene in infected and transfected cells.

The initiation site for transcription of the herpes simplex virus type 1 (HSV-1) gene encoding the major DNA-binding protein. ICP8, was mapped by nuclease S1 analysis of RNA-DNA hybrids. When RNA isolated from cells infected with HSV-1 was used, one major start site of ICP8 gene transcription was mapped at 89 base pairs to the right of the BstEII site at 0.409 map units. In cells transfected with a cloned ICP8 gene, the same major start site was detected either in the presence or absence of the immediate-early (alpha) genes encoding ICP4 or ICP0, which have been shown to stimulate ICP8 gene expression in transfected cells. Both ICP4 and ICP0 stimulated the accumulation of the ICP8 gene transcripts in the transient expression system, and their effects were synergistic. By comparison of the sequence of the putative promoter region of the ICP8 gene with the promoter of the HSV-1 TK gene, a significant similarity was detected between the three transcriptional regulatory signals of the TK gene and the upstream sequences of the ICP8 gene. Analysis of promoters of other delayed-early (beta) genes showed that they all contained regions of significant homology with the distal signals of the upstream sequences of the TK or ICP8 gene.

Animals↗

PubMed1986

Transcriptional control of herpesvirus gene expression: gene functions required for positive and negative regulation.

We have used an in vitro nuclear run-off assay to measure the levels of transcription of specific herpes simplex virus genes at different times during a lytic infection. We analyzed the effects of inhibition of DNA replication and of defects in two herpes simplex virus regulatory proteins on the transcription of these genes. We present evidence that the transcription of the alpha ICP4 gene is negatively regulated during a lytic infection. The regulation of ICP4 gene transcription requires the beta protein ICP8 (where ICP = infected cell polypeptide). Transcription of the beta ICP8, gamma 1 ICP5, and gamma 2 glycoprotein C (gC) genes was dependent on ICP4, and transcription of the gamma 2gC gene was strongly inhibited when DNA replication was blocked. Defects in ICP8 also resulted in increased levels of transcription of the ICP4, ICP8, ICP5, and gC genes from parental viral genomes. Our results suggest that ICP8 may be important in maintaining the highly ordered cascade of viral gene expression.

DNA Replication↗

PubMed1986

A mutant herpesvirus protein leads to a block in nuclear localization of other viral proteins.

The herpes simplex virus mutants KOS1.1 ts756 and HFEM tsLB2 express temperature-sensitive ICP4 proteins that are not localized properly to the cell nucleus at the nonpermissive temperature. In these infected cells at the nonpermissive temperature, nuclear localization of at least two other viral proteins, ICP0 and ICP8, is impaired. Replacement of the mutated sequences in the ICP4 gene of tsLB2 restored proper nuclear localization of all of the proteins. The ICP0 and ICP8 proteins expressed in cells transfected with their individual genes were localized to the cell nucleus. Therefore, in infected cells, the mutant ICP4 gene product appears to be the primary defect which leads to the block in nuclear localization of the other proteins. One viral protein, ICP27, was not inhibited for nuclear localization in these cells. These data indicate that there are at least two pathways for nuclear localization of HSV proteins, one of which is inhibited by the mutant ICP4 protein. The mutant ICP4 protein may define a probe for one of the pathways of nuclear localization of proteins.

Animals↗

PubMed1985

A genetic test for expression of a functional herpes simplex virus DNA-binding protein from a transfected plasmid.

The major DNA-binding protein, ICP8, encoded by herpes simplex virus is localized to the infected cell nucleus where it plays a role in viral DNA replication and control of viral gene expression. To identify the parts of the ICP8 protein that are important for its localization and functions, we have developed a system to test the ability of recombinant plasmids to express functional ICP8. A recombinant plasmid containing the wild-type ICP8 gene was transfected into cells. The cells were later infected with a temperature-sensitive ICP8 mutant virus at the nonpermissive temperature. Sufficient wild-type ICP8 was expressed from the transfected plasmid to complement the replication of the mutant virus. This provides a genetic system to test the properties of ICP8 expressed from mutagenized plasmids without the establishment of a stable cell line or the reintroduction of the ICP8 gene into the herpes simplex virus genome.

DNA Replication↗

PubMed1985

An immunoassay for the study of DNA-binding activities of herpes simplex virus protein ICP8.

An immunoassay was used to examine the interaction between a herpes simplex virus protein, ICP8, and various types of DNA. The advantage of this assay is that the protein is not subjected to harsh purification procedures. We characterized the binding of ICP8 to both single-stranded (ss) and double-stranded (ds) DNA. ICP8 bound ss DNA fivefold more efficiently than ds DNA, and both binding activities were most efficient in 150 mM NaCl. Two lines of evidence indicate that the binding activities were not identical: (i) ds DNA failed to complete with ss DNA binding even with a large excess of ds DNA; (ii) Scatchard plots of DNA binding with various amounts of DNA were fundamentally different for ss DNA and ds DNA. However, the two activities were related in that ss DNA efficiently competed with the binding of ds DNA. We conclude that the ds DNA-binding activity of ICP8 is probably distinct from the ss DNA-binding activity. No evidence for sequence-specific ds DNA binding was obtained for either the entire herpes simplex virus genome or cloned viral sequences.

Animals↗

PubMed1985

Identification of a herpes simplex virus function that represses late gene expression from parental viral genomes.

The expression of herpes simplex virus gamma 2 (late) genes is inhibited before the onset of viral DNA replication. We report that the block in the expression of certain gamma 2 genes is relieved, at least in part, by defects in the beta ICP8 protein. We have examined the expression of the gamma 2 gene encoding glycoprotein C (gC) in cells infected with a temperature-sensitive ICP8 mutant. Under conditions in which viral DNA replication is inhibited, cells infected with the ICP8 mutant overproduce the gC family of mRNAs relative to the level observed in cells infected with a wild-type virus. The gC mRNA synthesized in cells infected with the ICP8 mutant virus is correctly initiated and spliced and is translated with the same relative efficiency as in cells infected with a replicating wild-type virus. These results suggest that ICP8 is involved in the negative regulation of gamma 2 genes expressed from parental viral genomes. The level of gC expression was greatest in cells infected with a replicating wild-type virus. These data suggest that DNA replication and genome amplification are not absolute requirements for gamma 2 gene expression but may facilitate full-level expression of these genes.

Animals↗

PubMed1985

Stimulation of expression of a herpes simplex virus DNA-binding protein by two viral functions.

We examined the expression and localization of herpesvirus proteins in monkey cells transfected with recombinant plasmids containing herpes simplex virus (HSV) DNA sequences. Low levels of expression of the major HSV DNA-binding protein ICP8 were observed when ICP8-encoding plasmids were introduced into cells alone. ICP8 expression was greatly increased when a recombinant plasmid encoding the HSV alpha (immediate-early) ICP4 and ICP0 genes was transfected with the ICP8 gene. Deletion and subcloning analysis indicated that two separate functions capable of stimulating ICP8 expression were encoded on the alpha gene plasmid. One mapped in or near the ICP4 gene, and one mapped in or near the ICP0 gene. Their stimulatory effects were synergistic when introduced on two separate plasmids. Thus, two separate viral functions can activate herpesvirus early gene expression in transfected cells.

Cell Nucleus↗

PubMed1984

The intranuclear location of a herpes simplex virus DNA-binding protein is determined by the status of viral DNA replication.

The herpes simplex viral DNA-binding protein, ICP8, is targeted to two different locations in the cell nucleus as part of its maturation pathway. Prior to viral DNA synthesis ICP8 was found at discrete pre-replicative sites throughout the nucleus, where it exhibited a high salt-labile association with the nuclear matrix. During viral DNA replication ICP8 was localized in randomly distributed replication compartments, where it is bound to viral DNA. Initiation of viral DNA replication caused the protein to move from the prereplicative sites to the replication compartments, while inhibition of replication caused movement in the opposite direction. In cells where viral DNA synthesis was proceeding, a minor population of ICP8 may also have been associated with the prereplicative sites. The prereplicative sites may serve as a nuclear reservoir for ICP8 not bound to replicating or progeny DNA.

Animals↗

PubMed1984

Characterization of the major mRNAs transcribed from the genes for glycoprotein B and DNA-binding protein ICP8 of herpes simplex virus type 1.

The structural genes encoding the herpes simplex virus type 1 glycoprotein B and the major DNA-binding protein ICP8 have been mapped previously within the EcoRI-F restriction fragment (map coordinates 0.314 to 0.420) of the viral genome. In this study the mRNAs transcribed from these DNA sequences were identified by hybridization selection of 32P-labeled RNA and by Northern blot analysis of polyadenylated cytoplasmic RNA. A 3.4-kilobase RNA was the major mRNA homologous to the DNA sequences between coordinates 0.343 and 0.386 in which mutations in the glycoprotein B gene have been mapped. A 4.5-kilobase RNA was the major mRNA homologous to the viral DNA sequences between coordinates 0.361 and 0.417 in which mutations in the ICP8 gene have been mapped. Hybridization-selected mRNAs were translated in vitro to determine the primary translation products encoded in each region. The glycoprotein B- and ICP8-specific polypeptides were identified by immunoprecipitation with specific antisera. The translation products encoded by the glycoprotein B gene were 103,000 and 99,000 in molecular weight. The translation products encoded by the ICP8 gene were 125,000 and 122,000 in molecular weight.

Animals↗

PubMed1983

Characterization of herpes simplex virus 2 temperature-sensitive mutants whose lesions map in or near the coding sequences for the major DNA-binding protein.

By marker rescue with cloned herpes simplex virus 2 DNA fragments, we have mapped the temperature-sensitive mutations of a series of herpes simplex virus 2 mutants to a region of the herpes simplex virus 2 genome that lies within or near the coding sequences for the major DNA-binding protein, ICP8. In cells infected with certain of these mutants at the nonpermissive temperature, the association of the major DNA-binding protein with the cell nucleus was defective. In these cells, the DNA-binding protein accumulated in the cytoplasmic and the crude nuclear detergent wash fractions. At the permissive temperature, the maturation of the mutant ICP8 was similar to that of the wild-type viral protein. With the remainder of the mutants, the nuclear maturation of ICP8 was similar to that encoded by the wild-type virus at the nonpermissive and permissive temperatures as assayed by cell fractionation.

Animals↗

PubMed1983

Thermolabile in vivo DNA-binding activity associated with a protein encoded by mutants of herpes simplex virus type 1.

The major DNA-binding protein encoded by several temperature-sensitive mutants of herpes simplex virus type 1 was thermolabile for binding to intracellular viral DNA. The ability of DNase I to release this protein from isolated nuclei was used as a measure of the amount of protein bound to viral DNA. This assay was based upon our previous observation that the fraction of herpesviral DNA-binding protein which can be eluted from nuclei with DNase I represents proteins associated with progeny viral DNA (D. M. Knipe and A. E. Spang, J. Virol. 43:314-324, 1982). In this study, we found that several temperature-sensitive mutants encoded proteins which rapidly chased from a DNase I-sensitive to a DNase I-resistant nuclear form upon shift to the nonpermissive temperature. We interpret this change in DNase I sensitivity to represent the denaturation of the DNA-binding site at the nonpermissive temperature and the association with the nuclear framework via a second site on the protein. The DNA-binding activity measured by the DNase I sensitivity assay represents an important function of the protein in viral replication because three of five mutants tested were thermolabile for this activity. A fourth mutant encoded a protein which did not associate with the nucleus at the nonpermissive temperature and therefore would not be available for DNA binding in the nucleus. We also present supportive evidence for the binding of the wild-type protein to intracellular viral DNA by showing that a monoclonal antibody coprecipitated virus-specific DNA sequences with the major DNA-binding protein.

Cell Nucleus↗