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L Haarr

Publications and source records attributed to L Haarr.

At least 37 records · Page 2Linked to original sources
PubMed1988

Prominence of the herpes simplex virus latency-associated transcript in trigeminal ganglia from seropositive humans.

Although herpes simplex virus type 1 (HSV-1) is known to reside latently in trigeminal ganglia between episodes of reactivation, the mechanisms involved in restricting the virus to this state are not understood. Using in situ nucleic acid hybridization methods, we show that there is HSV-encoded RNA in ganglion cells from 10 of 12 seropositive and zero of three seronegative patients studied at autopsy. Transcripts mapping to the region encoding the immediate-early polypeptide ICPO and the latency-associated transcript (LAT) were detected in the nuclei of these neurons. No other region of the HSV-1 genome was found to be expressed. When carefully defined probes were used to identify the transcripts, RNA corresponding to the LAT and, rarely, to ICPO was found. These results suggest that HSV-1 latency is an active process and that the LAT may be involved in regulating viral genetic expression.

Herpes Simplex↗
PubMed1988

Interaction of polylysine with the cellular receptor for herpes simplex virus type 1.

We earlier reported that neomycin blocked reversibly the binding of herpes simplex virus type 1 (HSV-1) to the receptor of BHK cells, while the binding of HSV-2 to the receptor was unaffected. We could not determine whether the effect was on the virus particle, the receptor, or both. We have now tested several other cationic substances, and report that polylysine (and polyarginine) block the binding of HSV-1 to the receptor by interfering with the cellular receptor function; higher molecular weight polylysines were more potent than those of lower molecular weight. Polylysine and neomycin showed additive effects. In vitro, polylysine showed the same strong binding to the plasma membrane phosphoinositides as did neomycin. Together these data suggest that the drugs may have a common target in the cell membrane. The HSV-1 and HSV-2 virus particles were unaffected by the drugs, as was the cellular HSV-2 receptor.

Animals↗
PubMed1988

Identification of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1.

Hybrid arrest of in vitro translation was used to localize the region of the herpes simplex virus type 1 genome encoding the 65-kilodalton DNA-binding protein (65KDBP) to between genome coordinates 0.592 and 0.649. Knowledge of the DNA sequence of this region allowed us to identify three open reading frames as likely candidates for the gene encoding 65KDBP. Two independent approaches were used to determine which of these three open reading frames encoded the protein. For the first approach a monoclonal antibody, MAb 6898, which reacted specifically with 65KDBP, was isolated. This antibody was used, with the techniques of hybrid arrest of in vitro translation and in vitro translation of selected mRNA, to identify the gene encoding 65KDBP. The second approach involved preparation of antisera directed against oligopeptides corresponding to regions of the predicted amino acid sequence of this gene. These antisera reacted specifically with 65KDBP, thus confirming the gene assignment.

DNA-Binding Proteins↗
PubMed1987

DNA- and RNA-binding proteins of chromatin from Escherichia coli.

The different proteins present in chromatin of Escherichia coli have been analyzed by a variety of techniques. The chromatin was isolated using a previously published procedure (Sjåstad, K., Fadnes, P., Krüger, P.G. Lossius, I. and Kleppe, K. (1982) J. Gen. Microbiol. 128, 3037) and solubilized by the action of micrococcal nuclease or DNAase I. The DNA-protein and RNA-protein complexes thus obtained were purified by sucrose gradient centrifugation and isopycnic gradient centrifugation in metrizamide in low ionic strength. The protein: DNA ratio of the DNA-protein complexes was estimated from the latter method and found to be approx. 1.75. The protein components were analyzed further by one- and two-dimensional gel electrophoresis. Approx. 15 major polypeptides were detected in the DNA-protein complex, whereas 10 were present in the RNA-protein complex. The majority of the polypeptides in both complexes had acidic isoelectric pH. The polypeptides in the two complexes differed markedly and only two polypeptides, having molecular weights of 57,000 and 37,000, respectively, were found to be common in both complexes. In agreement with earlier studies, the basic protein HU was not present in the DNA-protein complex. Affinity studies of the proteins from chromatin using DNA- and RNA-Sepharose columns in general confirmed the above conclusions. The two-dimensional gel electrophoretic patterns of the proteins in the different complexes were compared with those of proteins in the inner and outer membranes. Only one of the major polypeptides present in the inner membrane, having a molecular weight of 57,000, was enriched in the DNA-protein complex.

Carrier Proteins↗
PubMed1987

Evidence that deletion of coding sequences in the 5' end of the thymidine kinase gene of herpes simplex virus type 1 affects the stability of the gene products.

The experiments described in the present work were designed to study the function of the N-terminal end of thymidine kinase (TK) encoded by herpes simplex virus type 1. Specifically we were interested to know whether this end was involved in binding of the enzyme to other molecules, had any influence on its subcellular localization or affected one or more of the activities associated with the enzyme. A parental enzyme and a deletion mutant, lacking the 45 N-terminal amino acids, derived from this strain, were used. Thymidine kinase from the parental virus bound to DNA-Sepharose, but the truncated enzyme did not. This was apparently not due to a specific ability to bind to DNA, since immunofluorescence studies indicated that both the normal and the deleted TK were mainly located in the cytoplasm, preferentially in the perinuclear region. Phosphorylation of thymidine as well as the amounts of TK polypeptides were markedly reduced at late times after infection with the mutant, but not to the same extent after infection with the wild-type. The deleted TK gene was efficiently transcribed as shown by hybridization of RNA to a probe specific for the gene, and this RNA directed the synthesis in vitro of TK polypeptides. Deletion of the 5' end of the gene seems to affect the stability of either the enzyme or TK-specific mRNA, or both. The TMP phosphorylating activity seems to be particularly destabilized relative to the thymidine phosphorylating activity.

Cell Compartmentation↗
PubMed1987

Evidence that neomycin inhibits binding of herpes simplex virus type 1 to the cellular receptor.

The effect of neomycin, a phosphoinositide-binding aminoglycoside, on herpes simplex virus type 1 (HSV-1) infection of BHK cells was studied. We showed earlier that it specifically inhibits HSV-1 production but not HSV-2 production (Langeland et al., Biochem Biophys. Res. Commun. 141:198-203, 1986). We now show that neomycin had no effect on cellular protein synthesis, as judged by the appearance of 35S-labeled polypeptides separated by polyacrylamide gel electrophoresis. Virus-induced polypeptides, however, were strongly inhibited at neomycin concentrations above 2 mM. Comparison among different aminoglycosides showed a variation in inhibition of HSV-1 production that paralleled the cationic charge of the aminoglycosides. HSV-1 receptor binding at 4 degrees C was completely inhibited by neomycin. At 37 degrees C both receptor binding and internalization, as measured by an indirect assay, appeared to be inhibited by more than 90%. The effect of neomycin on the infection was almost immediate upon the addition of the drug and preceded virus internalization. Possible mechanisms of the neomycin effect are discussed.

Animals↗
PubMed1987

The 65,000-Mr DNA-binding and virion trans-inducing proteins of herpes simplex virus type 1.

The possible identity of the herpes simplex virus type 1 (HSV-1) 65K (65,000-Mr) virion protein which stimulates transcription from immediate-early genes with the HSV-1 65K DNA-binding protein was investigated. The two proteins were found to be distinct by the three separate criteria of immunological reactivity, tryptic peptide fingerprinting, and mobility in two-dimensional gels. Using HSV-1/HSV-2 intertypic recombinants and a serotype-specific antiserum, we located the gene encoding the 65K DNA-binding protein between coordinates 0.574 and 0.682 on the HSV-1 genome. The protein is posttranslationally modified by phosphorylation. In crude extracts of HSV-1-infected cells the 65K trans-inducing protein did not detectably bind to double-stranded calf thymus DNA under the conditions of our assay.

Antigens, Viral↗
PubMed1986

Changes in polypeptide synthesis and glycosylation in mouse embryonic fibroblast C3H/10T1/2 Cl 8 cells caused by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate.

The mechanism of tumor promotion is not well understood. We have used the transformable, tumor promotable, mouse embryo fibroblast C3H/10T1/2 Cl 8 cells to study tumor promoter specific changes in protein synthesis and protein glycosylation. Two-dimensional gel electrophoresis showed that 12-O-tetradecanoylphorbol 13-acetate caused a significant increase in the synthesis of five cellular and 34 extracellular polypeptides. One of these polypeptides has tentatively been identified as ornithine decarboxylase. One new polypeptide (p 62, Mr 58,000) was found in the medium of 12-O-tetradecanoylphorbol 13-acetate-treated cells. The amounts of several excreted proteins were enhanced 5-10 fold by 12-O-tetradecanoylphorbol 13-acetate. 12-O-tetradecanoylphorbol 13-acetate interfered with glycosylation both by affecting protein synthesis and also directly with glycosylation. At least 15 polypeptides in the medium and two cellular polypeptides decreased after 12-O-tetradecanoylphorbol 13-acetate treatment. Two of the major polypeptides found in the medium (p 8 and 10, Mr approx. 200,000-220,000) have properties similar to fibronectin, while p 9 and 11 both found in the cellular preparations and in the medium (Mr 180,000 and 150,000) were collagenase sensitive and their synthesis was inhibited by 12-O-tetradecanoylphorbol 13-acetate.

Animals↗
PubMed1986

Evidence that neomycin inhibits HSV 1 infection of BHK cells.

The effect of neomycin on the Herpes Simplex Virus (HSV) type 1 and 2 infection of baby hamster kidney cells was studied. Neomycin concentrations of 3 mM or more caused a more than 90% inhibition of HSV 1 proliferation, while it had no effect on HSV 2 proliferation, measured as plaque-forming units. Furthermore, neomycin must be present at the time of infection in order to exert full effect, addition 1 hour postinfection was comparable to untreated cells. This indicates that neomycin may specifically interfere with very early stages of HSV 1 infection.

Animals↗
PubMed1986

Polyphosphoinositide metabolism in baby-hamster kidney cells infected with herpes simplex virus type 1.

The incorporation of [32P]Pi and [3H]inositol into the inositol lipids of baby-hamster kidney cells was studied in herpes-simplex-virus-type-1(HSV-1)-infected and mock-infected cells. The infection was conducted during incorporation of, as well as after prelabelling with, the precursors. These methods were used in order to study both synthesis de novo of, and steady-state changes in, the phosphoinositides. Both with infection during labelling, and after prelabelling, we found increased [32P]- and [3H]-phosphatidylinositol 4,5-bisphosphate (PIP2) and decreased [32P]- and [3H]-phosphatidylinositol 4-monophosphate in infected as compared with mock-infected cells, whereas no effect was observed on phosphatidylinositol. This altered inositol-lipid metabolism was (at least in the case of PIP2) not present until 3-6 h after infection and remained stable, or increased slightly, throughout the infection period. Polyphosphoinositide metabolism constitutes an important step in signal processing in many forms of cellular stimulation, and the results obtained suggest that HSV-1 infection may induce such events in our cell system.

Animals↗
PubMed1985

Utilization of internal AUG codons for initiation of protein synthesis directed by mRNAs from normal and mutant genes encoding herpes simplex virus-specified thymidine kinase.

Previous studies (H.S. Marsden, L. Haarr, and C.M. Preston, J. Virol. 46:434-445, 1983) have shown that at least three polypeptides, with molecular weights of 43,000, 39,000, and 38,000, are encoded by the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene. It has been suggested that the 39,000- and 38,000-molecular-weight polypeptides arise from preinitiation complexes bypassing the first and second AUG codons before commencement of translation since, according to previous work (M. Kozak, Nucleic Acids Res. 9:5233-5252, 1981), these codons are not of the most efficient structure for initiation. This possibility was investigated by using specific herpes simplex virus mutants with alterations in the TK gene. Mutant TK4 has an amber mutation between the first and second AUG codons, whereas mutant delta 1 has a deletion which removes the first AUG codon but leaves other AUG codons, as well as transcriptional promoter sequences, intact. Both mutants synthesized only the 39,000- and 38,000-molecular-weight polypeptides, and the amounts produced were normal in TK4-infected cells but increased in delta 1-infected cells. Furthermore, the levels of TK produced after infection with the mutant viruses correlated with the amounts of the 39,000- and 38,000-molecular-weight polypeptides synthesized. The 43,000-, 39,000-, and 38,000-molecular-weight polypeptides were shown to be related by their positive reaction with anti-TK serum in both immunoprecipitation and immunoblotting experiments. The production of the 39,000- and 38,000-molecular-weight polypeptides through bypassing of the first AUG codon was examined by hybrid arrest experiments with a DNA fragment complementary to only 50 bases at the 5' terminus of TK mRNA. This fragment arrested the synthesis of the 30,000- and 38,000-molecular-weight polypeptides when annealed to mRNA from wild-type HSV-1- or TK4-infected cells, showing that those polypeptides arise from an mRNA initiated upstream from the first AUG codon. mRNA from cells infected with mutant delta 1, which lacks DNA sequences upstream from the first AUG, was not affected by the 50-base-pair fragment. The data therefore confirm that three polypeptides encoded by the HSV-1 TK gene arise by differential use of in-phase AUG codons for the initiation of protein synthesis. This mechanism for the production of related but distinct polypeptides has not previously been demonstrated in a eucaryotic system, and the implications for the regulation of TK enzyme activities are discussed.

Cell Line↗
PubMed1984

Two-dimensional gel electrophoretic separation of the proteins present in chromatin of Escherichia coli.

The polypeptides present in 35S-labelled chromatin prepared from Escherichia coli cells, and polypeptides present in the DNA and RNA complexes obtained by micrococcal nuclease digestion of the chromatin, were analysed by two-dimensional non-equilibrium polyacrylamide gel electrophoresis. Three hundred and thirty-five 35S-labelled polypeptides were detected in the chromatin whereas the DNA- and RNA-containing fractions of the micrococcal nuclease digest contained 126 and 183 polypeptides respectively. The major basic low-molecular-weight polypeptides were found in the DNA-containing fractions.

Bacterial Proteins↗
PubMed1983

Characterization of the DNA-cellulose-binding proteins from Escherichia coli K 12.

The various [35S]DNA-binding proteins present in lysates of Escherichia coli K 12 cells have been analyzed by means of two-dimensional SDS-polyacrylamide gel electrophoresis. The proteins were isolated by the DNA-cellulose technique and eluted by increasing concentrations of NaCl (0.15, 0.4, 0.6 and 2 M). Only 2% of the total 35S radioactivity in the lysate became bound to the DNA-cellulose column. A total of 237 polypeptides were detected and the distribution among the salt eluates were 85, 83, 40 and 29 polypeptides, respectively. The 40 major polypeptides with regard to concentrations were also identified from gels stained with a protein-specific reagent. The polypeptides could be divided into two main groups according to pI values, namely, acidic polypeptides (total number, 174) and basic polypeptides (total number, 63). The ratio between acidic and basic polypeptides decreased with increasing salt concentrations in the eluates. The majority of the basic polypeptides had molecular weights in the range 10 000-30 000, whereas the acidic polypeptides had molecular weights from 10 000 to 165 000.

Cellulose↗
PubMed1983

Processing of herpes simplex virus type 1 glycoproteins: two-dimensional gel analysis using monoclonal antibodies.

The number of discrete species of glycoprotein induced by herpes simplex virus type 1 (HSV-1, strain 17 syn+) and their processing has been examined by a combination of immunoprecipitation with monoclonal antibodies and analysis of immune precipitates by two-dimensional (2D) gel electrophoresis. Seventeen different monoclonal antibodies directed against glycoproteins A/B, C, D and E were used. Polypeptides intermediate in the synthesis of gA/B, C and D were visualized and two early intermediates of glycoprotein A/B (pgA/B1 and pgA/B2), both mannose-containing, were identified. Comparison on 2D gels of polypeptides synthesized in vitro from HSV-1-infected cell mRNA with those synthesized in pulse-chase experiments in vivo has allowed identification of early precursors of pgA/B and pgD. Their apparent mol. wt. were 105000 and 47000 respectively. The 2D gel analysis of glycoproteins induced in HFL cells infected with 17 syn+ revealed a number of previously unreported glycoprotein species. One had mobility on both gradient and single concentration SDS-polyacrylamide gels similar to that of gC but was resolved from gC on non-equilibrium pH gradient gels. This glycoprotein was produced in relatively large amounts and was not precipitated by any of the monoclonal antibodies used in this study. The data suggest that this glycoprotein either has a polypeptide chain unrelated to those of glycoproteins A/B, C, D or E or alternatively is derived from one of them and modified in such a way as to mask or remove the antigenic sites with which the monoclonal antibodies interact. Sixteen other previously unreported glycoprotein species not obviously related, as judged by their electrophoretic mobility, to gA/B, C, D or E were also identified: two reacted with gA/B-specific monoclonal antibodies and five with glycoprotein C-specific monoclonal antibodies. The origin of the remaining nine new glycoproteins has still to be ascertained.

Antibodies, Monoclonal↗
PubMed1983

Processing of herpes simplex virus proteins and evidence that translation of thymidine kinase mRNA is initiated at three separate AUG codons.

The role which post-translational modification plays in the genesis of herpes simplex virus-induced polypeptides was investigated. Two-dimensional gel electrophoresis was used to identify those polypeptides (i) synthesized in vitro, (ii) labeled in vivo during a pulse, and (iii) labeled after a chase. Excluding glycoproteins, we detected 36 precursor or short-lived polypeptides, 8 polypeptides which were generated by post-translational modification, 46 polypeptides which were apparently not modified after synthesis, and 19 polypeptides which were either transient intermediates or not modified. Comparison of polypeptides synthesized in vitro and during an in vivo pulse showed that translation in vitro resembles quite closely translation in vivo and that amounts of protein synthesized in vivo are determined largely by the levels of mRNA. This analysis provided the basis for an investigation of the suggestion (C.M. Preston and D.J. McGeoch, J. Virol. 38:593-605, 1981) that the two polypeptides of apparent molecular weights of 43,000 (VI 43) and 39,000 (VI 39) encoded by the herpes simplex virus type 1 thymidine kinase gene are translated from a single mRNA by two in-phase initiation codons. Hybrid arrest was used to identify in vitro translation products encoded by the thymidine kinase gene. Two-dimensional gel electrophoresis showed that VI 39 was more acidic than VI 43, consistent with the predicted amino acid composition of a polypeptide whose synthesis was initiated at the second AUG codon, located 135 bases downstream from the first. Furthermore, two-dimensional gels revealed a third polypeptide whose synthesis was arrested by the same fragment. Its pI and apparent molecular weight (38,000) were compatible with initiation of translation at a third AUG codon an additional 42 bases downstream. Our findings provide strong evidence that downstream initiation codons within the thymidine kinase mRNA are used.

Codon↗
PubMed1981

Two-dimensional gel analysis of HSV type 1-induced polypeptides and glycoprotein processing.

Two hundred and thirty virus-induced polypeptides have been detected in BHK cells infected with herpes simplex virus type 1 (HSV-1; strain 17) by means of two-dimensional gel electrophoresis. The polypeptides have been characterized by both relative mobility following isoelectric focusing and apparent mol. wt. in SDS-polyacrylamide gels. Some polypeptides, visualized as a single band on a one-dimensional SDS-polyacrylamide gel, were resolved into several spots. Three were identified in Vmw43, the band thought to contain thymidine kinase activity. Not all the observed polypeptides are unique species: some appear to be related and have altered mobilities as a consequence of post-translational modification events. Pulse-chase experiments and treatment of infected cells with neuraminidase suggested that glycoproteins gB, gC and gD contain sialic acid and that synthesis of gB and gD occurs by at least 15 and 10 discrete steps respectively.

Animals↗
PubMed1981

Inhibition of T7 development at high concentrations of the phage.

Escherichia coli B exposed to high doses of bacteriophage T7 did not lyse. A similar effect was observed when the high dose was added within the first 7 min after primary infection. No viable phage was formed. DNA synthesis was inhibited rapidly and the nucleoid structure was absent. Protein synthesis was in general markedly reduced and so were the activities of the phage-specific enzymes endolysin and DNA polymerase. However, phage genes were transcribed both by the host RNA polymerase and by the phage-specific enzyme. We suggest that inhibition of phage development is due to structural alterations occurring in the cell wall/membrane such that replication is inhibited.

Bacteriolysis↗