Biomedical subjects

M A Whitt

Publications and source records attributed to M A Whitt.

25 records · Page 2Linked to original sources

PubMed1995

Replication and amplification of novel vesicular stomatitis virus minigenomes encoding viral structural proteins.

We have developed a system in which vesicular stomatitis virus (VSV) minigenomes encoding viral structural proteins can be expressed from plasmids. These RNAs can be replicated, transcribed, and packaged into infectious particles when coexpressed with the other VSV proteins. The minigenomes contain either the glycoprotein (G protein) gene (GMG [stands for G minigenome]) or both the G and matrix (M) protein genes (GMMG [stands for G/M minigenome]) from the Indiana serotype of VSV flanked by the trailer and leader regions from the wild-type VSV genome. Northern (RNA) blot analysis showed that the minigenome RNAs were replicated and that a positive-sense replicative intermediate was synthesized when coexpressed with the nucleocapsid (N) protein and the two VSV polymerase proteins (phosphoprotein [P] and the large catalytic subunit [L]) in vivo. In addition, functional mRNAs were transcribed from the minigenome templates, and the appropriate encoded proteins were expressed. Expression of the G and M proteins from GMMG resulted in the assembly and release of infectious particles that could be passaged on cells expressing the N, P, and L proteins only. Amplification occurred during successive passages, and after four passages approximately 30% of the cells expressed both the G and M proteins. Analysis of the RNAs produced in the GMMG-infected cells also showed that the minigenomes accurately reproduced all of the replicative and transcriptional events that normally occur in a VSV-infected cell. GMMG is therefore a novel type of defective particle which encodes functional viral proteins critical to its own propagation.

Animals↗

PubMed1991

Membrane fusion activity, oligomerization, and assembly of the rabies virus glycoprotein.

The spike glycoprotein (G protein) of rabies virus (CVS strain) expressed in HeLa cells from cloned cDNA mediated membrane fusion after exposure to pHs of 6.1 or below. Chemical crosslinking showed that the rabies G protein, like the vesicular stomatitis virus (VSV) G protein, could be crosslinked to dimers and trimers, indicating that rabies G protein is a trimer. However, unlike the VSV G protein, rabies G protein trimers were not stable to sedimentation in sucrose gradients, even at a mildly acidic pH which stabilizes the VSV G protein trimers. In addition, we report that the expressed rabies virus G protein was functional because it could assemble into VSV particles (tsO45) lacking VSV G protein and rescue infectivity. These VSV (rabies) pseudotypes were neutralized only by an antibody to the rabies G protein. We also examined the properties of a hybrid protein containing the extracellular domain of the rabies virus glycoprotein and the transmembrane and cytoplasmic domains of the VSV G protein. This protein was transported to the cell surface and could be crosslinked to form dimers and trimers, but had little or no detectable membrane fusion activity. The lack of fusion activity was paradoxical because the hybrid protein could rescue VSV infectivity, although the titers were lower than those obtained with the wild-type rabies G protein.

Animals↗

PubMed1991

Fatty acid acylation is not required for membrane fusion activity or glycoprotein assembly into VSV virions.

We have investigated the role of fatty acid acylation on two properties of the glycoprotein (G protein) from the Indiana serotype of vesicular stomatitis virus (VSV). Using a mutated G protein described previously (CS-2) that is not palmitylated, we found that fatty acid acylation was not required for the low pH-induced membrane fusion activity of VSV G protein. Transient expression of CS in HeLa cells resulted in syncytia formation that was indistinguishable from that induced by wild-type G protein. In addition, we found that expression of CS complemented a temperature-sensitive mutant of VSV (tsO45) as well as the wild-type protein. These results indicate that the presence of palmitate on the cytoplasmic domain of VSV G protein is not required for any step in the life cycle of the virus.

Acylation↗

PubMed1991

A new cationic liposome reagent mediating nearly quantitative transfection of animal cells.

One of the most efficient systems for the expression of genes in the cytoplasm of animal cells utilizes a recombinant vaccinia virus encoding the bacteriophage T7 RNA polymerase. Cells infected with this virus are transfected with plasmid DNAs containing the gene to be expressed under T7 promoter control. The major limitation of this system is the efficiency with which DNA is introduced into the cell. Recently, a cationic liposome-mediated transfection reagent has yielded transfection frequencies of greater than 80%. To determine if commercially available cationic lipids could form liposomes that would yield similar transfection efficiencies, we tested liposomes prepared with five different cationic lipids. When used at appropriate concentrations in liposomes that also contained a neutral lipid, four of the five cationic lipids were effective in the transfection of HeLa cells. However, liposomes formed with the neutral lipid and one of the cationic lipids, dimethyldioctadecylammonium bromide (DDAB), gave transfection frequencies of greater than 95% and had a broad spectrum of effectiveness on a variety of cell lines. Liposomes containing DDAB are an inexpensive, highly efficient and reproducible alternative for the transfection of animal cells and are well suited for use with the vaccinia virus/T7 expression system.

Animals↗

PubMed1990

A fusion-defective mutant of the vesicular stomatitis virus glycoprotein.

We have recently described an assay in which a temperature-sensitive mutant of vesicular stomatitis virus (VSV; mutant tsO45), encoding a glycoprotein that is not transported to the cell surface, can be rescued by expression of wild-type VSV glycoproteins from cDNA (M. Whitt, L. Chong, and J. Rose, J. Virol. 63:3569-3578, 1989). Here we examined the ability of mutant G proteins to rescue tsO45. We found that one mutant protein (QN-1) having an additional N-linked oligosaccharide at amino acid 117 in the extracellular domain was incorporated into VSV virions but that the virions containing this glycoprotein were not infectious. Further analysis showed that virus particles containing the mutant protein would bind to cells and were endocytosed with kinetics identical to those of virions rescued with wild-type G protein. We also found that QN-1 lacked the normal membrane fusion activity characteristic of wild-type G protein. The absence of fusion activity appears to explain lack of particle infectivity. The proximity of the new glycosylation site to a sequence of 19 uncharged amino acids (residues 118 to 136) that is conserved in the glycoproteins of the two VSV serotypes suggests that this region may be involved in membrane fusion. The mutant glycoprotein also interferes strongly with rescue of virus by wild-type G protein. The strong interference may result from formation of heterotrimers that lack fusion activity.

Animals↗

PubMed1989

Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant.

We have used transient expression of the wild-type vesicular stomatitis virus (VSV) glycoprotein (G protein) from cloned cDNA to rescue a temperature-sensitive G protein mutant of VSV in cells at the nonpermissive temperature. Using cDNAs encoding G proteins with deletions in the normal 29-amino-acid cytoplasmic domain, we determined that the presence of either the membrane-proximal 9 amino acids or the membrane-distal 12 amino acids was sufficient for rescue of the temperature-sensitive mutant. G proteins with cytoplasmic domains derived from other cellular or viral G proteins did not rescue the mutant, nor did G proteins with one or three amino acids of the normal cytoplasmic domain. Rescue correlated directly with the ability of the G proteins to be incorporated into virus particles. This was shown by analysis of radiolabeled particles separated on sucrose gradients as well as by electron microscopy of rescued virus after immunogold labeling. Quantitation of surface expression showed that all of the mutated G proteins were expressed less efficiently on the cell surface than was wild-type G protein. However, we were able to correct for differences in rescue efficiency resulting from differences in the level of surface expression by reducing wild-type G protein expression to levels equivalent to those observed for the mutated G proteins. Our results provide evidence that at least a portion of the cytoplasmic domain is required for efficient assembly of the VSV G protein into virions during virus budding.

Amino Acid Sequence↗

PubMed1988

A phosphorylated 34-kDa protein and a subpopulation of polyhedrin are thiol linked to the carbohydrate layer surrounding a baculovirus occlusion body.

Surrounding baculovirus occlusion bodies is an electron-dense layer reported to be composed of carbohydrate which we term calyx. Incubation of Autographa californica nuclear polyhedrosis virus occlusion bodies (AcMNPV OBs) with dilute alkaline saline (DAS) followed by centrifugation at 12,000 g resulted in the sedimentation of calyx material which contained pp34, residual polyhedrin (p32), and entrapped occluded virions (DAS P-12 fraction). Incubation of the DAS P-12 fraction with sodium dodecyl sulfate (SDS) resulted in solubilization of the entrapped virions and the majority of p32, while calyx material, pp34, and some p32 remained sedimentable at 12,000 g. Immunofluorescence microscopy of DAS-solubilized OBs using monoclonal antibody to pp34 and p32 revealed that both pp34 and p32 are closely associated with the calyx. When DAS P-12 fractions were resuspended in SDS and reducing agent, not only were the entrapped virions solubilized, but pp34 and the remaining p32 were also liberated, indicating that pp34 and a subpopulation of p32 are associated with the calyx via thiol linkages. Immunoblot analysis and peptide mapping demonstrated that pp34 is neither immunologically nor structurally related to p32. The kinetics of pp34 synthesis were also examined by immunoprecipitation of infected cell polypeptides using pp34-specific monoclonal antibody. pp34 was detected initially 15 hr postinfection (p.i.) and continued to be phosphorylated until 60-70 hr p.i. This study demonstrates that the AcMNPV calyx has a proteinaceous component and we propose that other occluded baculoviruses may also have a calyx-associated protein analogous to pp34.

Insect Viruses↗