Richard A. Consigli

DNA replication in chinese hamster cells: Evidence for a single replication fork per replicon☆

Chinese hamster cells were labelled with [3H]thymidine after fluorodeoxyuridine treatment. Autoradiograms of this DNA indicate that in each replicon, replication proceeds via a single replication fork.

Characterization of a protein kinase activity associated with purified capsids of the granulosis virus infecting Plodia interpunctella

Abstract A cyclic-nucleotide independent protein kinase activity has been demonstrated in highly purified preparations of the granulosis virus infecting the Indian meal moth, Plodia interpunctella . A divalent cation was required for activity. Manganese was the preferred cation and a pH of 8.0 resulted in optimal incorporation of 32 P radiolabel into acid-precipitable protein. Although both ATP and GTP could serve as phosphate donors, ATP was utilized more efficiently by the enzyme. The kinase activity was localized to purified capsids; and the basic, internal core protein, VP12, was found to be the predominant viral acceptor. Histones and protamine sulfate could also serve as acceptors for the capsid-associated kinase activity. Using acid hydrolysis and phosphoamino acid analysis of phosphorylated nucleocapsid protein and nuclear magnetic resonance of phosphorylated VP12, it was determined that the enzyme catalyzes the transfer of phosphate to both serine and arginine residues of acceptor proteins. We believe this kinase activity may play a significant role in the viral replication cycle.

Use of the baculovirus system to assemble polyomavirus capsid-like particles with different polyomavirus structural proteins: analysis of the recombinant assembled capsid-like particles.

The genes encoding the structural proteins (VP1, VP2 and VP3) of murine polyomavirus were cloned into the p2Bac dual multiple cloning site vector, individually or jointly, and the corresponding proteins were expressed in Spodoptera frugiperda (Sf9) insect cells by cotransfecting Sf9 cells with the constructed vector and the linear DNA of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Recombinant capsid-like particles could be purified 5 days post-infection from Sf9 cells infected with AcMNPV-VP1, with or without the involvement of minor protein (VP2 or VP3). Although VP2 and VP3 alone could not generate recombinant particles, they became incorporated into these particles when expressed with VP1 in Sf9 cells. Recombinant particles with different polyomavirus structural protein(s) were obtained by using different combined expression of these proteins in Sf9 cells. Cellular DNA of 5 kbp in size was packaged in all of the recombinant particles, which showed the same diameter as that of native virions. Agarose gel electrophoresis indicated that DNA packaged in these recombinant particles had a different pattern than that of native virions. Two-dimensional gel electrophoresis of the VP1 species of recombinant particles showed more VP1 species than those of the native virions from mouse cells, and an additional species of VP1 when VP2 was co-expressed with VP1. The recombinant particles were also compared for their ability to compete for polyomavirus infection. The competition assay indicated that the recombinant particles containing VP2 were the most efficient in inhibiting the native polyomavirus infection of 3T6 cells.

Early events in polyomavirus infection: fusion of monopinocytotic vesicles containing virions with mouse kidney cell nuclei☆

The entry of polyomavirus enclosed in monopinocytotic vesicles into mouse kidney cell nuclei was studied and evidence for a fusion mechanism was obtained. In vivo studies using the fluorescent lipophilic dye diI-C16(3) as a plasma membrane label showed that polyomavirus-infected nuclei accumulate plasma membrane, while uninfected or polyoma capsid-infected nuclei do not. Further evidence for fusion was obtained with electron microscopy of thin sections of infected mouse kidney cells. These specimens showed accumulation of plasma membrane in the outer nuclear membrane as well as evidence of recent fusion events. The polyoma virions (capsid proteins) were seen to accumulate on the inner nuclear membrane and in the nucleus and were identified by immunogold staining of the thin sections. The combined results of the in vivo dye studies and thin section immunoelectron microscopy studies provide evidence for a fusion mechanism for polyomavirus entry into mouse kidney cell nuclei.

A silver-staining technique for detecting minute quantities of proteins on nitrocellulose paper: retention of antigenicity of stained proteins.

Abstract A method using a silver-staining procedure to detect minute quantities of proteins on nitrocellulose paper is described. The technique is sensitive enough to detect nanogram quantities of proteins resolved on sodium dodecyl sulfate-polyacrylamide gels and then transferred to nitrocellulose paper by the electrotransfer technique. After the staining procedures, the proteins are shown to retain their antigenic properties.

Efficient transfer of proteins from acetic acid-urea and isoelectric-focusing gels to nitrocellulose membrane filters with retention of protein antigenicity

A method which facilitates the rapid and quantitative electrophoretic transfer of proteins from gels not containing sodium dodecyl sulfate (SDS) to nitrocellulose membranes is described. The equilibration of non-SDS-polyacrylamide gel electrophoretic gels in a buffer containing SDS confers a net negative charge to the proteins present, presumably as a result of the formation of SDS-protein complexes. Proteins from gels equilibrated in the SDS buffer and then electroblotted in a Tris-glycine buffer at pH 8.3 are transferred with much greater efficiency than are proteins from untreated gels. The method has been shown to significantly enhance the electrophoretic transfer of polyoma viral proteins resolved in either acetic acid-urea or isoelectric-focusing gels to nitrocellulose membranes, and it is suggested that the method should have universal applicability to all gel electrophoresis systems currently employed. The proteins from isoelectric-focusing gels treated with SDS and transferred to nitrocellulose membranes were found to retain antigenicity to antisera prepared against either denatured or native viral proteins.

Restriction enzyme analysis of the genomes of Plodia interpunctella and Pieris rapae granulosis viruses

The DNAs from the granulosis viruses of Plodia interpunctella and Pieris rapae were clearly distinguishable based on restriction endonuclease fragment patterns. By sizing single- and double-digestion products approximate molecular weights of 72 x 10(2) and 75 x 10(6), respectively, were estimated for the viral genomes.

Structural characterization of the hemocytes of Plodia interpunctella

Six types of hemocytes were identified in fifth instars of the Indian meal moth, Plodia interpunctella. The morphology of these cells was characterized by phase contrast and electron microscopy, with Sudan black B, Giemsa, Janus green B, and periodic acid‐Schiff staining. Reaction of the hemocytes with seven fluorescing lectin conjugates revealed distinctive binding patterns by their plasma and nuclear membranes and cytoplasmic inclusions. A direct line of descent from prohemocytes to plasmatocytes to granulocytes is suggested from these morphological observations.

Identification and protein analysis of polyomavirus assembly intermediates from infected primary mouse embryo cells.

A method is described for the isolation of polyoma virus assembly intermediates from infected mouse embryo cells. Sucrose gradient profiles revealed the presence of 90 S, 200 S, and 240 S intermediates. These intermediates were shown to be sensitive to a number of factors: ionic condition of the isolation buffer, presence of chelating agents and nonionic detergents during isolation, and sonication of nuclei during extraction of intermediates. Pulse-chase experiments demonstrated that the order of formation of the intermediates to be 90 S----240 S, with the 200 S particles as a possible intermediate form linking the 90 S and 240 S particles. Viral structural proteins VP1, VP2, and VP3 were shown to be present on all three intermediates, but the ratio of each protein varied on each intermediate species. Two-dimensional gel electrophoresis demonstrated that the distribution of the VP1 isoelectric focusing species were different among the three intermediates. Histone H1 was found exclusively with the 90 S species.

Avian polyomavirus major capsid protein VP1 interacts with the minor capsid proteins and is transported into the cell nucleus but does not assemble into capsid-like particles when expressed in the baculovirus system

The baculovirus system was used to construct and isolate AcMNPV-VP1, AcMNPV-VP2 and AcMNPV-VP3 recombinant viruses which express the respective avian polyomavirus (APV) structural proteins in Sf9 insect cells. These recombinant AcMNPVs containing APV structural protein genes were utilized to investigate protein-protein interactions between the structural proteins. Immunofluorescence studies utilizing Sf9 cells infected with the AcMNPV-VP1 revealed that the VP1 protein was expressed and localized in the cytoplasm and not transported into the nucleus. When the cells were co-infected with the VP1 and either VP2 or VP3 recombinant viruses, immunofluorescence of the VP1 protein was localized in the nucleus, indicating that the VP1 protein was transported to the nucleus by both the VP2 and VP3 minor proteins. This observation was suggestive of a protein-protein interaction between the expressed proteins. This protein-protein interaction was substantiated by laser scanning confocal microscopy of Sf9 cells that were co-infected with VP1, VP2 and VP3 recombinant viruses. However, the minor proteins could not be co-isolated with VP1 protein by immunoaffinity chromatography using a monoclonal anti-VP1 serum. In addition, capsid-like particles could not be purified either by CsC1 density gradient centrifugation or by immunoaffinity chromatography. VP1 capsomeres were isolated by immunoaffinity chromatography from Sf9 cells infected with AcMNPV-VP1, with or without the minor protein(s), and these capsomeres could assemble in vitro into capsid-like particles. Electron microscopic observation of thin-sectioned Sf9 cells, which were co-infected with VP1, VP2 and VP3 recombinant viruses, demonstrated capsomere-like structures in the nucleus, but capsid-like particles were not observed.