Characterization of the continuous, differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia

We are reporting a procedure for the purification of herpes simplex enveloped nucleocapsids (virions), an evaluation of the purification procedure and the results of analyses of the virion proteins by high-resolution acrylamide gel electrophoresis. The data may be summarized as follows. (i) The procedure for the purification of virions consists of careful extraction of cytoplasm to prevent nuclear breakage, separation of enveloped nucleocapsids from soluble proteins and membrane vesicles by rate zonal centrifugation of cytoplasmic extracts through dextran 10 gradients, treatment with urea to dissociate virus-debris aggregates, and, lastly, separation of virions from naked nucleocapsids and free membranes by isopycnic flotation in discontinuous sucrose gradients. (ii) Purity was evaluated in three ways, i.e., electron microscopic examination, analysis of purified virions produced in cells labeled with amino acids before infection, and analysis of purified virions from artificial mixtures of infected and labeled, uninfected cells. The extent of purification was 120-to 200-fold with respect to host proteins. Residual contaminants were identified as host and viral constituents of membrane vesicles. Residual host proteins are very likely contaminants and not structural components of the virion. (iii) Analyses by staining and autoradiography of structural proteins of purified virions in 6, 7, 8.5, 9, and 14% acrylamide gels revealed 24 bands of proteins and glycoproteins made and labeled after infection. Co-electrophoresis of viral proteins with six known standards ranging from 25,700 to 220,000 daltons in molecular weight in 6, 7, 8.5, and 9% acrylamide gels indicate that viral proteins range from 25,000 to 275,000 daltons. The sum of the molecular weights of viral proteins is 2,580,000 daltons. Assuming that messenger transcription is asymmetric and noncomplementary, this corresponds to 47% of the genetic information of the virus. (iv) The nonionic detergent NP-40 removes from purified virions some nonglycosylated proteins and a large fraction of the glycosylated proteins. It leaves behind traces of the envelope visible in the electron microscope as well as some glycoproteins thought to be in the envelope.

Proteins Specified by Herpes Simplex Virus V. Purification and Structural Proteins of the Herpesvirion

Nitric oxide (NO) has antimicrobial activity against a wide spectrum of infectious pathogens, but an antiviral effect has not been reported. The impact of NO, from endogenous and exogenous sources, on herpes simplex virus type 1 (HSV 1) replication was studied in vitro. HSV 1 replication in RAW 264.7 macrophages was reduced 1,806-fold in monolayers induced to make NO by activation with gamma IFN and LPS. A competitive and a noncompetitive inhibitor of nitric oxide synthetase substantially reduced the antiviral effect of activated RAW macrophages. S-nitroso-L-acetyl penicillamine (SNAP) is a donor of NO and was added to the media of infected monolayers to assess the antiviral properties of NO in the absence of gamma IFN and LPS. A single dose of S-nitroso-L-acetyl penicillamine 3 h after infection inhibited HSV 1 replication in Vero, HEp2, and RAW 264.7 cells in a dose-dependent manner. Neither virucidal nor cytocidal effects of NO were observed under conditions that inhibited HSV 1 replication. Nitric oxide had inhibitory effects, comparable to that of gamma IFN/LPS, on protein and DNA synthesis as well as on cell replication. This report demonstrates that, among its diverse properties, NO has an antiviral effect.

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Evidence for antiviral effect of nitric oxide. Inhibition of herpes simplex virus type 1 replication.

Two classes of herpesvirus capsids, designated A and B, were isolated from the nuclei of human cells infected with herpes simplex virus (HSV) A and B capsids share in common four structural proteins, ie, no 5, 19, 23, and 24 B capsids contain 77 to 97 times more deoxyribonucleic acid than A capsids; moreover, they contain proteins no 21 and 22a in addition All of the proteins contained in the capsid except no 22a are present in the enveloped nucleocapsids (virions) in approximately the same molar ratios The capsid proteins of HSV-1 cannot be differentiated from their HSV-2 counterparts with respect to electrophoretic mobility A third class of capsids, designated C capsids, was isolated from virions contained in the cytoplasm of infected cells by the same procedure used to obtain A and B capsids The C capsids contain all of the proteins present in A capsids plus proteins 1 to 3 and 21 Images

Proteins Specified by Herpes Simplex Virus VIII. Characterization and Composition of Multiple Capsid Forms of Subtypes 1 and 2

Experiments done with a temperature"sensitive mutant of herpes simplex virus type 1 (HSV-1) have revealed that one of the virisn glycoproteins, designated VP7(B2), is apparently not required for the production of enveloped virus particles, whereas it does play a critical role in virion infectivity The mutant, designated HSV-1[HFEM]tsB5, fails to accumulate VP7(B2) at nonpermissive temperature and produces virions that lack detectable quantities of this glycoprotein and that have very low specific infectivity The poor infectivity of the virions is most readily explained by failure of penetration into the host cell rather than by failure of adsorption to cells because it was shown that the VP7(B2)-deficient virions can bind to cells and that polyethylene glycol, an agent known to promote membrane fusion, can significantly enhance infectivity of the adsorbed virions

Membrane proteins specified by herpes simplex viruses. III. Role of glycoprotein VP7(B2) in virion infectivity.

Summary
Arginine is essential for the replication of herpes simplex virus. Omission of arginine from the culture medium did not affect the synthesis of viral DNA but prevented the formation of virions. Addition of arginine immediately stimulated protein synthesis which was followed by virus formation.

The role of arginine in the replication of herpes simplex virus.

Herpes simplex virus structural proteins

Studies on the synthesis of herpes simplex virions.

STUDIES on human leukaemic cells reveal the presence in the cytoplasmic fraction of an RNase sensitive DNA polymerase1 complexed with 70S RNA2 and virus specific polysomal RNA3. The DNA product of this enzymatic reaction specifically hybridized to leukaemic nuclear DNA and not to nuclear DNA from normal leukocytes4. This does not establish a viral aetiology for human leukaemia, but suggests that such a possibility may exist. The isolation and characterization of virus-like particles may therefore be possible in conditions which induce the leukaemic cells to produce virions. Our approach was based on the observation5 that arginine deprivation of Rous sarcoma virus (RSV) transformed rat cells (which do not synthesize virions) induced the synthesis and release of C type virions. We have found that leukaemic cells, obtained from patients with acute lymphoblastic leukaemia (ALL) and chronic lymphoblastic leukaemia (CLL) and myeloblastic leukaemia, when incubated in an arginine deficient medium, released particles labelled with 3H-uridine which banded at a density of 1.17 g ml−1. These particles were found to contain an RNase sensitive DNA polymerase activity which transcribed DNA from 70S RNA molecules and formed 70S RNA : DNA hybrid molecules.

Udy Olshevsky

Papers

The role of arginine in the replication of herpes simplex virus.

Herpes simplex virus structural proteins

Studies on the synthesis of herpes simplex virions.

Particles released from arginine deprived human leukaemic cells.

Studies on Herpes Simplex virus DNA: Denaturation properties