Lewis I. Pizer

Entry of Herpes Simplex Virus Type 1 into Primary Sensory Neurons In Vitro Is Mediated by Nectin-1/HveC

ABSTRACT Primary cultures of rat and mouse sensory neurons were used to study the entry of herpes simplex virus type 1 (HSV-1). Soluble, truncated nectin-1 but not HveA prevented viral entry. Antibodies against nectin-1 also blocked infection of rat neurons. These results indicate that nectin-1 is the primary receptor for HSV-1 infection of sensory neurons.

Varicella-Zoster Virus DNA in Cells Isolated from Human Trigeminal Ganglia

ABSTRACT To determine the type of cell(s) that contain latent varicella-zoster virus (VZV) DNA, we prepared pure populations of neurons and satellite cells from trigeminal ganglia of 18 humans who had previously had a VZV infection. VZV DNA was present in 34 of 2,226 neurons (1.5%) and in none of 20,700 satellite cells. There was an average of 4.7 (range of 2 to 9) copies of VZV DNA per latently infected neuron. Latent VZV DNA was primarily present in large neurons, whereas the size distribution of herpes simplex virus DNA was markedly different.

The metabolism of serine and glycine in mutant lines of Chinese hamster ovary cells

Abstract This report describes studies of mutant lines of cultured Chinese hamster ovary cells that have different levels of serine transhydroxymethylase (EC 2.1.2.1). This enzyme, which splits serine to yield glycine and N5,N10-methylene tetrahydrofolic acid, is found in both the mitochondria and cytosol of these cells (see Chasin et al. (1974) Proc. Nat. Acad. Sci. USA71, 718–722). Our experiments with these mutant lines have established a correlation among the amount of mitochondrial serine transhydroxymethylase, the intracellular glycine concentration, and the extent that exogenous serine increases the glycine pool. Limited amino acid incorporation into protein occurred with all cell lines, but in contrast to the glycine-requiring mutant line 51-11, revertants that no longer required glycine for growth showed increased incorporation when the medium was supplemented with serine. These results indicate that normally the mitochondrial serine transhydroxymethylase together with the intracellular serine concentration regulate the supply of glycine and under certain conditions can control the rate of protein synthesis. Additional experiments with radioactive serine and glycine have shown that the mitochondrial serine transhydroxymethylase regulates the interconversion of these amino acids as well as serine oxidation. Calculations based on the 14CO2 produced from l -[14C]serine by the mutant and parental cell lines indicate that approximately 50% of the serine oxidized is initially converted to glycine and an oxidizable one-carbon unit.

Deoxyribonucleic acid-dependent ribonucleic acid synthesis in Escherichia coli infected with bacteriophage T2

Following infection of Escherichia coli by the bacteriophage T2, ribonucleic acid polymerase activity in unfractionated extracts is reduced to approximately twenty per cent of the activity in unfractionated extracts of uninfected cells. The residual enzyme activity is sensitive to deoxyribonuclease, to ribonuclease and to actinomycin D. RNA polymerase can be purified from extracts of T2-infected cells with a yield of more than 200%. The purified enzyme has properties similar to the enzyme purified from uninfected cells. Deoxyribonucleic acid isolated from T2-infected cells is a mixture of high molecular weight bacterial and viral DNA. Both components of this mixture are active as templates for RNA polymerase in vitro, although only the viral DNA is a template in vivo. As reported by Skold & Buchanan (1964) , material which inhibits purified RNA polymerase in vitro appears in cell extracts following T2 infection. The inhibitory material is destroyed by acid treatment and reduced in amount by digestion with alkali. Incubation overnight at 37°C and heating at 100°C increase the amount of inhibitory material. In control experiments with uninfected E. coli, inhibitory material is not present in the initial extract or after treatment with acid. Treatment with alkali, incubation overnight at 37°C or heating at 100°C result in levels of inhibitory material similar to that in extracts of infected cells subjected to the same treatment.

Postentry Events Are Responsible for Restriction of Productive Varicella-Zoster Virus Infection in Chinese Hamster Ovary Cells

ABSTRACT Productive infection of varicella-zoster virus (VZV) in vitro is restricted almost exclusively to cells derived from humans and other primates. We demonstrate that the restriction of productive VZV infection in CHO-K1 cells occurs downstream of virus entry. Entry of VZV into CHO-K1 cells was characterized by utilizing an ICP4/β-galactosidase reporter gene that has been used previously to study herpes simplex virus type 1 entry. Entry of VZV into CHO-K1 cells involved cell surface interactions with heparan sulfate glycosaminoglycans and a cation-independent mannose-6-phosphate receptor. Lysosomotropic agents inhibited the entry of VZV into CHO-K1 cells, consistent with a low-pH-dependent endocytic mechanism of entry. Infection of CHO-K1 cells by VZV resulted in the production of both immediate early and late gene products, indicating that a block to progeny virus production occurs after the initiation of virus gene expression.

[178] 3-phosphoglycerate dehydrogenase (Escherichia coli)

Publisher Summary This chapter discusses the methods of preparation of 3-Phosphoglycerate Dehydrogenase (Escherichia coli). Phosphoglycerate dehydrogenase catalyzes the oxidation of 3-phosphoglycerate (PGA) to hydroxypyruvate phosphate (HPAP). This is the initial reaction in serine biosynthesis, and the enzyme from E. coil is inhibited by L-serine. Most of the studies associated with the purification of the enzyme and its crystalline preparation, enzyme activity is most readily measured by following the disappearance of DPNH spectrophotometrically or fluorometrically. This assay is inaccurate in cell extracts that contain a DPNH oxidase. When this situation exists an assay based on the chromatographic separation of 14C-labeled serine phosphate produced from PGA-14C can be used. A unit of enzyme activity is defined as that amount of enzyme that catalyzes the disappearance of 1 millimicromole of DPNH per minute at 25 °. Recrystallization of the enzyme to constant specific activity provides the evidence that the preparation is free of contaminating proteins. Analytical ultracentrifugation and zone electrophoresis failed to detect inhomogeneity in the crystalline preparation.

The effect of herpes virus infection on mRNA in polyoma virus-transformed cells

Abstract Polyoma-transformed BHK cells are permissive for herpes simplex virus. Transcripts from the polyoma genome in these cells have been well characterized and provide an example of a specific class of mRNA. The effect of herpes simplex type I infection on the synthesis and amount of polyoma-specific RNA was investigated by hybridization to polyoma DNA. Hybridization of unlabeled cytoplasmic RNA to radioactive E-strand polyoma DNA (that is, the DNA strand complementary to RNA present in polyoma-infected cells prior to viral DNA synthesis) demonstrated that by 5 hr after infection with herpes virus the amount of polyoma-specific RNA was 20% of that found in uninfected controls. Hybridization of radioactive RNA to polyoma DNA immobilized on filters indicated that after herpes infection, the synthesis of polyoma-specific RNA rapidly declined. The reduced synthesis of this specific class of mRNA could account for the drop in cytoplasmic levels.

Characterization of a mutant of E.coli with elevated levels of seryl-tRNA synthetase

Abstract A mutant strain of E.coli K12 with an elevated level of seryl-tRNA synthetase activity has been described. The enzyme from the mutant was inactivated by antiserum prepared against the K12 enzyme and the inactivation curve demonstrated that the increased activity was a consequence of more enzyme protein rather than an enzyme with altered kinetic properties. The gene controlling the enzyme level was shown by transduction to be linked to the structural gene for the enzyme. Regulatory processes involving the supply of serine or charging of seryl-tRNA were not detected.

Abortive infection of Escherichia coli strain W by T2 bacteriophage

Abstract This paper describes the abortive infection of Escherichia coli strain W by phage T2 and attempts to relate the biology of the infection with the extent of viral synthesis and the fate of the parental DNA. When strain W cells were infected with T2, less than 1% of the cells produced phage; the phage produced were not modified, and only one out of two to three phage were effective killers of strain W. The infection was characterized by an abrupt, early cessation of all macromolecular syntheses. Early phage enzymes were produced at lower than normal levels. No DNA and no late proteins were synthesized. Isotope incorporation data indicated that protein synthesis stopped abruptly early after infection. RNA synthesis continued a few minutes longer than protein synthesis and then stopped. When the fate of 32 P-labeled infecting phage DNA was studied, two degradative processes were found. Initially there was a rapid degradation of 30 to 50% of the phage DNA molecules to acid-soluble fragments. When this activity was completed, the remaining acid-precipitable phage DNA was intact as measured by neutral and alkaline sucrose gradient centrifugation, thus indicating that the prior acid solubilization did not involve these molecules. The remaining intact DNA was subsequently slowly attacked by a double-stranded endonuclease which cleaved the DNA to smaller acid-precipitable pieces. It is postulated that these nucleolytic activities are related to the reduced efficiency of killing strain W by T2 infection, although they may not be the primary cause of the inability to produce phage.

Transcription of a bacteriophage DNA by mammalian RNA polymerase

Abstract T even bacteriophage DNA is a relatively inefficient template for mammalian RNA polymerase and transcription is not stimulated by bacterial sigma factor. The RNA that is transcribed corresponds primarily to that synthesized early after bacteriophage infection although some RNA corresponding to that synthesized late after infection and some anti-messenger are transcribed. The data suggest that the enzyme preferentially recognizes initiation sites for early RNA.