Lawrence A. Caliguiri

Influenza virus proteins: I. Analysis of polypeptides of the virion and identification of spike glycoproteins☆

Abstract The polypeptides of influenza virions have been analyzed by polyacrylamide gel electrophoresis. Seven polypeptides were detected in purified virions grown in three different cell types, and four of these polypeptides appear to be covalently linked with carbohydrate. Virions grown in different cell types were compared by coelectrophoresis, and host-dependent differences were detected in the electrophoretic mobility of some glycoproteins, but not of nonglycoproteins, suggesting that the carbohydrate moiety of such glycoproteins is specified by the host cell. The molecular weights of the seven proteins, estimated by coelectrophoresis with marker proteins, ranged from 83,500 to 26,500, with a total molecular weight of 380,500. Treatment of virions with the protease bromelain degraded the viral spikes resulting in particles which were bounded by a smooth-surfaced membrane, and which could be purified in a potassium tartrate gradient. Such particles were lacking three of the four glycoproteins; the four remaining viral proteins were unaltered by the enzyme. The particles devoid of spikes were noninfective, and they lacked hemagglutinin and neuraminidase activities, indicating that these properties are associated with the spike glycoproteins.

Isolation of paramyxovirus glycoproteins. Association of both hemagglutinating and neuraminidase activities with the larger SV5 glycoprotein

A method has been developed for the isolation of the glycoproteins of the parainfluenza virus SV5 using the nonionic detergent Triton X-100. Full recovery of hemagglutinating and neuraminidase activities was obtained. By rate zonal centrifugation in sucrose gradients containing 1% Triton X-100 and 0.5 M or 1 M potassium chloride, it was possible to separate the two glycoproteins. Under these conditions, the sedimentation coefficient of the larger glycoprotein, virus protein 2, was 8.9 S and that of the smaller glycoprotein, virus protein 4, was 6.7 S. Each of the proteins aggregated when the detergent and KCl were removed, and the appearance of the aggregates differed with the two proteins. Both hemagglutinating and neuraminidase activities were found to be associated with protein 2; protein 4 exhibited neither activity. The results suggest that in this paramyxovirus both hemagglutinating and neuraminidase activities reside on a single glycoprotein. The biological function of the smaller SV5 glycoprotein remains to be determined.

The role of cytoplasmic membranes in poliovirus biosynthesis

Abstract Chemical analysis and electron microscopy show that isopycnic centrifugation in discontinuous sucrose density gradients separates smooth and rough cytoplasmic membranes of HeLa cells. By 3.25 hours after poliovirus infection, there is evidence of marked proliferation of cytoplasmic membranes in the smooth microsomal fraction. Sixty-five percent of labeled virus-specific RNA is found in this fraction after a 2.5-min pulse of uridine-3H 3.25 hours after infection; however, after a 10-min pulse, most of the labeled RNA is found in the rough microsomal fraction. Over 50% of newly synthesized viral protein is found in the rough microsomal fraction throughout a 30-min chase period following a 3-min pulse of leucine-3H. This pattern of distribution of viral RNA and protein is dependent on the presence of cytoplasmic membranes. The major site of translation of viral RNA is associated with rough membranes, whereas replication of viral RNA takes place in association with smooth membranes. Results after addition of guanidine to infected cells support the hypothesis that viral RNA synthesis is initiated in a complex associated with smooth membranes.

Membranous Structures Associated with Translation and Transcription of Poliovirus RNA

Poliovirus RNA and proteins are synthesized in association with distinct membranous structures that were separated by means of Isopycnic centrifugation of cytoplasmic extracts in discontinuous sucrose-density gradients. Viral RNA is replicated in a structure that contains rapidly labeled replicative intermediate RNA and viral RNA polymerase associated with the smooth membrane fraction. In sucrose gradients this viral RNA replication complex is distributed at densities in the range of 1.12 to 1.18 grams per cubic centimeter. Viral proteins are synthesized on polyribosomes bound to membranes and sediment with polyribosomes at densities of less than 1.25 grams per cubic centimeter.

Inhibition of uncoating of poliovirus by arildone, a new antiviral drug

Abstract The antiviral effects of a new drug, arildone (4-[6-(2-chloro-4-methoxyphenoxy)hexyl]-3,5-heptanedione, on poliovirus type 2 replication and host cell functions are described. Arildone inhibits poliovirus replication at a minimal inhibitory concentration (MIC) of 0.2 μM, while transport of radioactively labeled precursors and synthesis of DNA, RNA, and protein in uninfected HeLa cells are not inhibited. This drug is not virucidal and does not interfere with adsorption or penetration. Arildone inhibits uncoating of poliovirus and thereby prevents virus-induced shutoff of host cell protein synthesis. The possible mechanisms by which arildone interacts with the poliovirus icosahedral capsid to prevent uncoating are discussed.

Characterization of poliovirus-specific structures associated with cytoplasmic membranes

Abstract The hydrodynamic properties and the components of membrane-associated virus-specific structures isolated from the cytoplasm of poliovirus-infected HeLa cells have been determined. Fraction 2, a smooth microsomal fraction, contains a structure that sediments at approximately 130 S after deoxycholate lysis of membranes. This structure contains replicative intermediate RNA, double-stranded replicative form, and ribosomal subunits, but no completed single-stranded viral RNA or ribosomes. The structure in Fraction 3, another smooth microsomal fraction, sediments at approximately 230 S after lysis of membranes, and it contains 74 S ribosomes. The major species of virus-specific RNA in this structure is completed single-stranded viral RNA, but replicative intermediate RNA and replicative form are also present. Fraction 5, the rough microsomal fraction, contains a structure that sediments at about 320 S after lysis of membranes. This structure has the properties of viral polyribosomes and contains completed single-stranded viral RNA but no replicative intermediate RNA. These results suggest a model for the membrane-associated replication and translation of viral RNA.

The proteins of the parainfluenza virus SV5. II. The carbohydrate content and glycoproteins of the virion.

Abstract The carbohydrate composition and glycoproteins of the parainfluenza virus SV5 grown in primary rhesus monkey kidney cells and in the MDBK line of bovine kidney cells have been examined. Galactose, mannose, glucosamine, and fucose are linked to viral proteins. Glucose, galactosamine, and galactose are present in the virion as constituents of glycolipids. Neuraminic acid has not been found in SV5 virions, although it is present in other enveloped viruses which do not possess the enzyme neuraminidase as a viral component. Two glycoproteins have been identified in the SV5 virion; both are associated with the viral envelope. The other viral envelope proteins and the nucleocapsid protein have no detectable carbohydrate.

Action of guanidine on the replication of poliovirus RNA

Abstract Guanidine (1.5m M ) inhibits viral RNA synthesis in cells that are actively producing virus. It was found that synthesis of viral RNA becomes progressively less inhibitable by guanidine after the fourth hour in the viral growth cycle when the rate of viral RNA synthesis diminishes. Guanidine added at 3 hours inhibited the synthesis of single-stranded RNA faster than synthesis of double-stranded RNA; however, 35 S single-stranded RNA was still the major RNA moiety formed. While there was a net reduction in the cumulative radioactivity of ribonuclease-resistant RNA in the replicative intermediate, there was an increase in the amount of radioactivity in the double-stranded replicative form after guanidine was added. These results indicate that guanidine does not inhibit growth and completion of viral RNA chains, but blocks the initiation of new chains.

The proteins of the parainfluenza virus SV5: I. Separation of virion polypeptides by polyacrylamide gel electrophresis

Abstract The polypeptides of isolated, purified SV5 nucleocapsid and of SV5 virions dissociated with sodium dodecyl sulfate have been separated by polyacrylamide gel electrophoresis. The nucleocapsid contains a single polypeptide. A total of six polypeptides, 2 major and 4 minor bands, have been found in the SV5 virion. The nucleocapsid protein represents one of the major polypeptides, and the other five are presumably associated with the viral envelope. Similar results have been obtained with SV5 virions grown in four different cell types. Estimation of the molecular weights of the viral proteins based on coelectrophoresis with marker proteins indicate that the nucleocapsid polypeptide has the lowest molecular weight, ∼43,000. The molecular weights of the other five viral polypeptides range from approximately 50,000 to 76,000.

Proteins associated with the poliovirus RNA replication complex

Abstract The poliovirus RNA replication complex is a heterogeneous structure associated with smooth-surfaced cytoplasmic membranes. The membrane-bound complex has an average buoyant density of 1.08 g/cm 3 in potassium tartrate. After lysis of membranes the replication complex sediments heterogeneously in the 75 to 265 S region of linear sucrose gradients. The peaks of pulse-labeled virus-specific RNA and viral RNA polymerase activity coincide and sediment at 155 S. Virus-specific protein sediments as a peak at 110 S with a faster sedimenting shoulder. The proteins in the 155 S region are digested by trypsin; however, proteins in the 110 S peak are trypsin-resistant. SDS-acrylamide gel electrophoresis reveals a relative increase of NCVP6 in the 110 S peak. A polypeptide with an estimated molecular weight of 33,500 is clearly distinct in the 155 S region and lower in sucrose gradients. NCVP3 and NCVP7 are enriched in the lysed membranes that float near the top of sucrose gradients. These results suggest that nascent viral RNA and procapsids may interact in the viral RNA replication complex and thereby initiate viral maturation. One or more of the virus-specific proteins found in this complex may represent the enzyme, viral RNA polymerase, or a subunit of a modified cellular enzyme.