Preparative method of isolating influenza virus glycoproteins

Two preparative methods for isolation of biologically active glycoproteins from influenza virus A - hemagglutinin and neuraminidase, were elaborated. A three-step procedure involves solubilization of glycoproteins with nonionic (Triton N-101, TN) or cationic (cetylpyridinium chloride, CPC) detergents, separation from degraded virions by centrifugation, and removal of detergents and lipids by precipitation of the glycoproteins with butanol (TN), or, alternatively, precipitation of CPC upon cooling. Using virion concentration of approximately 1 mg/ml and optimal weight ratio of detergent to virus (protein) of approximately 20:1 (for TN) and 1:1 (for CPC), the glycoproteins were obtained with the overall yield of 70-80%. The isolated glycoproteins exhibit the same immunological and enzymatic activities as intact virus A/Texas/77 and A/Leningrad/80.     

High performance liquid chromatography and characterization of structural proteins of the influenza virus

Constituent proteins of human influenza virus A have been separated by reverse-phase HPLC on Polysil ODS-500. Their homogeneity is confirmed by the data of amino acid composition, Edman analysis and gel electrophoresis.     

Preparative isolation and some properties of SV40 T-antigen]

SV40 T-antigen was isolated from hamster tumors and purified about 1600-fold by the procedure including successive ammonium sulfate precipitation, chromatography on DEAE-cellulose, preparative polyacrylamide gel electrophoresis and elimination of the bulk of contaminating cell proteins by the interaction with antibodies to the tissues of normal hamsters. The resulting preparation was not quite homogenous being contaminated with some of cell proteins left. T-antigen in the tumor extract was revealed at least in three distinct forms with molecular weight of 100 000, 200 000, and 400 000. It is proposed that these forms correspond to mono-, di-, and tetramers of the basal protein of T-antigen, although the alternative explanation, the existence of complex of T-antigen with cell proteins, cannot be ruled out.     

Densonucleosis virus structural proteins

The protein coats of two densonucleosis viruses (types 1 and 2) were examined by a variety of biophysical, biochemical, and serological techniques. The viruses were 24 nm in diameter, contained at least four polypeptides, were remarkably stable to extremes of pH and denaturing agents, and were serologically closely related. The two viruses could, however, be distinguished serologically and by differences in migration of their structural polypeptides. For each virus the “top component” (i.e., the protein coat minus DNA, found occurring naturally in infections) appeared to have a composition identical to that of the coat of the virus and was a more stable structure. Electrometric titration curves of the virus particles and top components demonstrated that the DNA phosphate in densonucleosis virus particles was neutralized by cations other than basic amino acid side chains of the protein coat. Circular dichroism studies showed that there was a conformational difference between the protein coats of top components and virus particles.     

Preparative isolation of gonadotropin and urokinase on carboxylic cation exchangers]

Low-pressure ion-exchange chromatography was used for sequential isolation of chorionic gonadotropin and urokinase (EC 3.4.99.26) from urine of pregnant women. Theoretical analysis of electrostatic interactions of protein macromolecules with the sorptive surface of a carboxylic cation exchanger was performed. This analysis allowed us to optimize the pH values for selective sorption and reversible desorption of the target component on a cation exchanger with a selected acidity of functional groups. The use of carboxylic cation exchangers KM-2p and Biokarb-GM allowed us to obtain the preparations with purity not inferior to commercially available preparations.     

Fate of influenza A virus proteins

In this review we summarize the current state, history, future, mutation tendency and species susceptibility of influenza A virus proteins based on our probabilistic analyses on amino acid pairs, and compare the current state of influenza A virus proteins with that of proteins which we have studied in the past.     

Preparative scale isolation of sphingosine

A high-yield method is described for the preparation of sphingenine from galactosylceramide. Two successive cleavage reactions are used, alkaline butanol to form galactosylsphingosine, then acidic acetonitrile to form the sphingenine. A column purification procedure utilizing silica gel is described. The method is superior to previously described methods in its rapidity and avoidance of isomerization and contamination by racemization and rearrangement products.     

Cellular proteins in influenza virus particles

Virions are thought to contain all the essential proteins that govern virus egress from the host cell and initiation of replication in the target cell. It has been known for some time that influenza virions contain nine viral proteins; however, analyses of other enveloped viruses have revealed that proteins from the host cell can also be detected in virions. To address whether the same is true for influenza virus, we used two complementary mass spectrometry approaches to perform a comprehensive proteomic analysis of purified influenza virus particles. In addition to the aforementioned nine virus-encoded proteins, we detected the presence of 36 host-encoded proteins. These include both cytoplasmic and membrane-bound proteins that can be grouped into several functional categories, such as cytoskeletal proteins, annexins, glycolytic enzymes, and tetraspanins. Interestingly, a significant number of these have also been reported to be present in virions of other virus families. Protease treatment of virions combined with immunoblot analysis was used to verify the presence of the cellular protein and also to determine whether it is located in the core of the influenza virus particle. Immunogold labeling confirmed the presence of membrane-bound host proteins on the influenza virus envelope. The identification of cellular constituents of influenza virions has important implications for understanding the interactions of influenza virus with its host and brings us a step closer to defining the cellular requirements for influenza virus replication. While not all of the host proteins are necessarily incorporated specifically, those that are and are found to have an essential role represent novel targets for antiviral drugs and for attenuation of viruses for vaccine purposes.     

Junin virus structural proteins.

Polyacrylamide gel electrophoresis of purified Junin virus revealed six distinct structural polypeptides, two major and four minor ones. Four of these polypeptides appeared to be covalently linked with carbohydrate. The molecular weights of the six proteins, estimated by coelectrophoresis with marker proteins, ranged from 25,000 to 91,000. One of the two major components (number 3) was identified as a nucleoprotein and had a molecular weight of 64,000. It was the most prominent protein and was nonglycosylated. The other major protein (number 5), with a molecular weight of 38,000, was a glucoprotein and a component of the viral envelope. The location on the virion of three additional glycopeptides with molecular weights of 91,000, 72,000, and 52,000, together with a protein with a molecular weight of 25,000, was not well defined.     

Preparative scale isolation of galactosylhydroxylysine.

A method is described for the isolation and purification of gram quantities of the hydroxylysine-monosaccharide from commercially available marine sponge. The procedure utilized alkaline hydrolysis followed by purification by ionexchange chromatography and gel filtration. Compositional analysis indicated that the final product contained only galactose, hydroxylysine, and HCl which were present in equimolar quantities and comprised 94% of the dry weight. This preparation has been utilized as a substrate for the assay of UDP-glucose:collagen glucosyltransferase (EC 2.4.1.66) of human platelets.     

Placement of the structural proteins in Sindbis virus

The structure of the lipid-enveloped Sindbis virus has been determined by fitting atomic resolution crystallographic structures of component proteins into an 11-A resolution cryoelectron microscopy map. The virus has T=4 quasisymmetry elements that are accurately maintained between the external glycoproteins, the transmembrane helical region, and the internal nucleocapsid core. The crystal structure of the E1 glycoprotein was fitted into the cryoelectron microscopy density, in part by using the known carbohydrate positions as restraints. A difference map showed that the E2 glycoprotein was shaped similarly to E1, suggesting a possible common evolutionary origin for these two glycoproteins. The structure shows that the E2 glycoprotein would have to move away from the center of the trimeric spike in order to expose enough viral membrane surface to permit fusion with the cellular membrane during the initial stages of host infection. The well-resolved E1-E2 transmembrane regions form alpha-helical coiled coils that were consistent with T=4 symmetry. The known structure of the capsid protein was fitted into the density corresponding to the nucleocapsid, revising the structure published earlier.     

Preparative isolation of serum a albumin

A simple method is suggested for the preparative isolation of native albumin in the thick (6 mm) block of the agar-agar gel on the veronal-medinal buffer solution (pH 8.6, ionic strength 0.1). The method is based on the analytical horizontal electrophoresis. 0.8-1 ml of 10% solution of proteins was introduced into each of two starting trenches. 5h after the beginning of distillation in the anode edge of the plate at a distance of 35 mm from the start two collector slits were cut out perpendiculary to the protein movement. Then they were filled with the NaCl physiological solution and protein portions were taken thrice each half an hour. The purity of albumins was checked immunologically. The method may be applied for obtaining other individual proteins.     

Preparative labeling of proteins with [35S]methionine.

The most common technique for preparative labeling of proteins with radioisotopes for experimental purposes utilizes 125I. This isotope has certain limitations, including the emission of gamma- and X-irradiation, the release of gaseous 125I2 from solutions of Na 125I, and the potential for concentration of 125I in thyroid glands. We have discovered a means for labeling proteins rapidly and simply with [35S]methionine. The technique is applicable to a wide variety of proteins. Antibodies labeled by our technique remain functional.