Shirley A. Harmon

Detection of hepatitis A virus RNA and capsid antigen in individual cells

The replication of hepatitis A virus (HAV) RNA and the production of HAV VP1 protein were examined in cultures of BS-C-1 cells under one-step growth conditions by in situ hybridization and immunofluorescence. Individual cells that had undergone active viral RNA replication were detectable at 24 h post-infection. During subsequent days, increasing numbers of cells began replicating viral RNA, so that by seven days post-infection, all cells had accumulated significant amounts of viral RNA. The results show that the protracted replication cycle of HAV in cultured cells represents a slow recruitment of infected cells into a replication mode, rather than an inherently slow virus reproduction in all cells. With the reagents utilized in this study, nucleic acid hybridization was more sensitive than antigen detection by immunofluorescence or immunoblot analysis.

Expression of hepatitis A virus capsid sequences in insect cells.

A cDNA coding for hepatitis A virus (HAV) VP1 and portions of the flanking VP3 and P2 sequences was inserted into the genome of Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter and translational start codon. Cells infected with recombinant virus produced high levels of a 55 kDa protein, identified as containing HAV VP1 by reactivity with anti-VP1 serum. The expressed protein also reacted on immunoblots with human HAV convalescent sera as well as sera from rabbits immunized with intact HAV. This protein was found predominantly in the cytoplasm of infected insect cells, probably as an insoluble aggregate.

Characterization of monospecific antisera against all five vesicular stomatitis virus-specific proteins: Anti-L and Anti-NS inhibit transcription in vitro

Abstract Antisera against each of the five proteins of vesicular stomatitis virus (VSV) were produced in rabbits, and the IgG fractions were shown to be specific for the individual proteins of purified VSV or VSV-infected cytoplasm by immunodiffusion and by immunoprecipitation. All of the IgG fractions inhibited transcription in vitro by ribonucleocapsids (RNPs) isolated from detergent-lysed virions. However, when RNPs were isolated from VSV-infected cytoplasm, only anti-L and anti-NS IgG fractions inhibited transcription, providing direct evidence that both L and NS proteins are necessary for transcription. None of the IgG fractions inhibited protein kinase activity by RNPs isolated from virions or from VSV-infected cytoplasm.

Ultrastructural localization of L and NS enzyme subunits on vesicular stomatitis virus RNPs using gold sphere-staphylococcal protein A-monospecific IgG conjugates

Colloidal gold spheres were coated with staphylococcal protein A and were used to determine the location of NS and L proteins on vesicular stomatitis virus (VSV) ribonucleoprotein (RNP) complexes using monospecific anti-NS and anti-L IgG preparations. Conjugates using either anti-NS or anti-L demonstrated that these enzyme subunits were uniformly distributed along the entire length of the RNP complex. Under saturating conditions of IgG concentrations, it was observed that there were at least 60-70 molecules of NS protein and 30-35 molecules of L protein labeled per RNP complex.

Transformation-Defective Virus Generation from Cloned Nondefective Avian Sarcoma Virus

An avian sarcoma virus, twice cloned by soft agar colony assay without intervening passage of the virus and apparently freed of transformation-defective ( td ) viruses, has been found

Candidate Products of Avian Myeloblastosis Virus Oncogene Region Produced by in vitro Translation of Genomic RNA

Avian myeloblastosis virus (AMV) genomic RNA was translated in vitro to identify the AMV transforming gene (myb) product(s). Full-length RNA yielded, in addition to the expected Pr76gag, a gag-related product of 92,000 daltons which did not appear to be a gag-oncogene fusion protein. Subgenomic AMV RNA in vitro translation, nonstructural products (specific for AMV) were of 54,000, 49,000 and 34,000 daltons. Based on their sizes and similar peptide maps for the smaller two proteins, it is proposed that they derive from the myb region of AMV.

PRODUCTS OF CELL-FREE RNA SYNTHESIS USING CONDITIONS PREVENTING INITIATION AND PROCESSING

ABSTRACT Nuclei prepared by a non-aqueous method, in which lyophilized cells were fractionated in glycerol, were rehydrated in the presence of nucleotide triphosphates and incubated at 35°. Sodium heparin was added to 1 to 3 mg/ml to suppress ribonuclease activities. Heparin incidentally disrupted nuclear structure somewhat and presumably prevented reinitiation of RNA synthesis. Overall rates of RNA synthesis in the presence or absence of heparin were 0.05-0.10 p moles UMP incorporated per μg DNA per min for 20 min, with decreasing rates of synthesis beyond 40 min. The products of 20 min of synthesis, analyzed by non-denaturing sedimentation and by α-amanitin sensitivity, were 45s RNA as polymerase I product (no added KCl) and a heterogenous population of polymerase II products (0.3 M KCl plus 1 mM MnCl2) with a distinct peak near 9-13s. Polymerase II products became larger with incubation beyond 20 min.