Proteins Specified by Herpes Simplex Virus V. Purification and Structural Proteins of the Herpesvirion
- 1 January 1972
- journal article
- Published by American Society for Microbiology in Journal of Virology
- Vol. 9 (1), 143-159
- https://doi.org/10.1128/jvi.9.1.143-159.1972
Abstract
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.Keywords
This publication has 24 references indexed in Scilit:
- Electrophoretic analysis of the major polypeptides of the human erythrocyte membraneBiochemistry, 1971
- Structural Proteins of Herpes Simplex VirusJournal of General Virology, 1971
- Herpesvirus Antigens on Cell Membranes Detected by Centrifugation of Membrane-Antibody ComplexesScience, 1971
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970
- A model for the behavior of vesicles in density gradients: Implications for fractionationBiochimica et Biophysica Acta (BBA) - Biomembranes, 1970
- Characterization of Herpes Simplex Virus Strains Differing in their Effects on Social Behaviour of Infected CellsJournal of General Virology, 1968
- Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gelsBiochemical and Biophysical Research Communications, 1967
- Polysomes and Protein Synthesis in Cells Infected with a DNA VirusScience, 1966
- Analysis of C14-labeled proteins by disc electrophoresisBiochemical and Biophysical Research Communications, 1965
- DISC ELECTROPHORESIS – II METHOD AND APPLICATION TO HUMAN SERUM PROTEINS*Annals of the New York Academy of Sciences, 1964