Marc S. Collett

Bovine viral diarrhea virus genomic organization

In previous work, we developed a preliminary description of the genetic organization of the prototypic pestivirus bovine viral diarrhea virus (BVDV). In order to refine this genetic map and to further elucidate the gene products and expression strategy of this virus, we have generated a broad panel of sequence-specific antibody reagents. Use of these reagents not only allowed the identification of several previously undescribed viral polypeptides, but when used in in vivo pulse-chase experiments, they identified precursor polyproteins and processing intermediates. Data generated from these studies provide a more accurate and complete view of viral gene organization, as well as insight into several aspects of protein processing and the gene expression strategy employed by this pestivirus. These experiments also revealed varying stability and turnover rates for the mature BVDV proteins. These latter results have implications for the functional roles of certain gene products.

Porcine parvovirus DNA: characterization of the genomic and replicative form DNA of two virus isolates.

The genomic and replicative form (RF) DNA of porcine parvovirus (PPV) have been characterized. PPV isolate NADL-8 was found to have a 5000-base single-stranded genome, and a unique strand was encapsidated in virus particles. The RF DNA of isolate NADL-8 was found to be an infectious 5000-base pair (bp) molecule. Select restriction endonuclease sites were mapped along the RF DNA of PPV (NADL-8), and oriented with respect to the viral genomic DNA. The RF DNA of a second isolate of PPV, the less pathogenic, cell culture-adapted NADL-2 virus, was also analyzed. DNA preparations isolated from NADL-2 virus-infected cells contained two viral RF DNA species: a 5000-bp infectious molecule very similar if not identical to the NADL-8 virus RF DNA, and a noninfectious 4700-bp DNA molecule. The 4700-bp RF DNA molecule found in NADL-2 DNA preparations was similar in all respects to the 5000-bp RF DNA, except for a 300-bp deletion in the region encoding the viral capsid proteins. The presence of this defective variant in PPV (NADL-2) virus preparations is discussed in relation to the reduced pathogenic potential of the NADL-2 virus as compared to the NADL-8 virus.

Initiation of DNA synthesis by the avian oncornavirus RNA-directed DNA polymerase: Structural and functional localization of the major species of primer RNA on the oncornavirus genome

Abstract We have both structurally and functionally localized the site on the avian oncornavirus RNA genome at which the major species of primer RNA is bound. Localization of the primer site involved either determining the minimum size class of poly(A)-containing fragments of the viral RNA genome to which radiolabeled primer RNA would anneal, or determining the minimum size class of poly(A)-containing fragments that could be reconstituted as an active template primer complex for the RNA-directed DNA polymerase subsequent to reannealing to unlabeled purified primer RNA. Both approaches indicate that the primer site appears to be very close to (within 10%), or at the 5′ end of the 35 S RNA genome. The implications of these findings on the transcription of the oncornavirus genome in vitro and in vivo are discussed.

Physical modification of purified Rous sarcoma virus pp60v-src protein after incubation with ATP/Mg2+.

Using partially purified enzyme preparations, we show that incubation of the Rous sarcoma virus transforming protein kinase, pp60v-src, with Mg2+ and ATP at concentrations near or above the enzymes Km for ATP resulted in a physical modification of the pp60v-src polypeptide. Under such conditions, a portion of pp60v-src was converted to a form that migrated more slowly in SDS-polyacrylamide gels than enzyme incubated without ATP or with low concentrations of ATP. Comparative tryptic peptide mapping of pp60v-src incubated with low and high levels of ATP revealed that more extensive tyrosine phosphorylation of the pp60v-src polypeptide occurred at the higher concentrations of ATP. This more extensive phosphorylation was characterized by the appearance of several new phosphorylated tyrosine residues on both the amino-terminal and carboxy-terminal portions of the pp60v-src molecule. The possible consequences of these modifications on the protein kinase activity of pp60v-src, and the functional regulation of retrovirus transforming proteins in general, are discussed.

Avian retrovirus RNA-directed DNA synthesis: Transcription at the 5′ terminus of the viral genome and the functional role for the viral terminal redundancy

Abstract The size and structure of DNA synthesized on 35 S RNA·tRNAtrp template primer complexes in reconstructed reactions by the avian retrovirus RNA-directed DNA polymerase in vitro have been elucidated. Under standard enzymatic reaction conditions three major species of DNA are initiated on tRNAtrp at the 5′ end of the viral genome. Two of these are single stranded in nature and exhibit lengths of 70 and 100 nucleotides. The third is hairpin in structure and is 185 nucleotides in length. A precursor-product relationship has been demonstrated between DNA70, DNA100, and the hairpin DNA185 species. Furthermore, it appears that hairpin DNA185 is formed during DNA synthesis rather than after DNA synthesis as a consequence of the intramolecular folding of complementary sequences present in the DNA185 transcript. Finally, we present data indicating that, in addition to conversion of DNA100 to hairpin DNA, the DNA100 species can attach to the 3′ end of the viral genome presumably by base pairing with the terminally redundant sequences at the 3′ terminus to provide a primer for the continuation of DNA transcription at this end of the viral RNA. These data provide a functional role in proviral DNA synthesis for the terminally repeated genomic sequences.

Distribution of the globin gene in active and inactive chromatin fractions from friend erythroleukemia cells

SUMMARY Stimulation of the T3C12 clone of Friend erythroleukemia cells with 1.2% dimethyl sulfoxide (DMSO) results in progressive increase in the concentration of globin mRNA sequences in the total cellular RNA of treated cells, as measured by nucleic acid hybridization employing a globin cDNA probe. The greatest increment in the content of globin RNA occurs between 30 and 40 h after addition of DMSO. Globin cDNA was also used to measure the concentration of globin-specific sequences in the DNA and RNA of transcriptionally active and inactive chromatin fractions prepared from these cells by the DNase II - MgCl, procedure of Gottesfeld et al. [ 161. Essentially equal concentrations of globin sequences are present in the DNA isolated from active and inactive chromatin fractions of cells grown in the presence of 1.2 % DMSO for 50 h (the time of initiation of hemoglobin synthesis). Furthermore, there are no significant differences in the globin gene concentrations between the active chromatin fractions from DMSO-treated and control cultures at either 50 or 120 h after initiation of DMSO treatment. However, chromatin-associated RNA isolated from the active chromatin of cells synthesizing maximum amounts of hemoglobin (120 h) contains a higher concentration of globin sequences than RNA from the active chromatin of control cells. Chromatin fractions from untreated cells also contain a significant amount of RNA which hybridizes to the globin cDNA probe. These observations suggest that both transcriptional and post-transcriptional control mechanisms are involved in hemoglobin gene expression in T3C12 erythroleukemia cells.

Elimination of residual RNase activity from purified preparations of RNA-directed DNA polymerase

Abstract Preparations of RNA-directed DNA polymerase purified from RNA tumor viruses by standard methods generally contain trace amounts of single-stranded RNA endonucleolytic activity detectable only by relatively sensitive methods. This contaminating RNase activity has been found to be completely inhibited when RNA-directed DNA polymerase reactions are carried out in the presence of low concentrations of bentonite. Under these conditions, only minimal inhibition of the DNA polymerase and RNase H activities of the RNA-directed DNA polymerase was observed.

In vitro transcription of reconstituted 35s RNA.tRNAtrp template.primer complexes by the avian oncornavirus DNA polymerase. Effect of temperature on the size of the DNA transcripts.

Abstract The synthesis of long DNA transcripts from avian sarcoma virus 35S RNA·tRNA trp template·primer complexes in reconstituted reactions in vitro is temperature dependent. At 15° DNA transcripts accumulate that are 100 nucleotides in length (DNA 100 ), whereas at 30° DNA transcripts ranging to 3000 nucleotides in length (DNA >100 ) are synthesized from these template·primer complexes. By hybridization analysis we have demonstrated that DNA 100 synthesized at 15° contains nucleotide sequences complementary to the 5′ end of the viral RNA genome whereas longer DNA transcripts synthesized at 30° contain nucleotide sequences complementary to the 3′ terminus of the viral genome. Pulse-chase experiments indicate that the 5′-located DNA 100 transcripts are precursors of the larger DNA transcripts. These data are consistent with the elongation of 5′ terminally located DNA transcripts at the 3′ end of the viral genome.