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Biomedical subjects

B Wolanski

Publications and source records attributed to B Wolanski.

9 recordsLinked to original sources
PubMed2003

An in vitro Flaviviridae replicase system capable of authentic RNA replication.

We have established an in vitro replication system for bovine viral diarrhea virus (BVDV), a surrogate for the closely-related hepatitis C virus. In an in vitro reaction, BVDV replication complexes synthesize vRNA and replicative form (RF) and replicative intermediate (RI) RNAs. Kinetic and heparin trapping experiments demonstrate the recycling of RF and RI products and the initiation of vRNA synthesis in this system. Consistent with this, quantitative hybridization reveals the asymmetric synthesis of positive and negative strand RNA products. These findings support the notion that RF serves as a template and RI as a precursor in the synthesis of vRNA. Furthermore, the antiviral activity of an NS5B inhibitor was similar in BVDV replicase and infectivity assays. Together, these results indicate that the in vitro activity of BVDV replicase complexes recapitulates RNA replication that occurs in infected cells, providing a system in which to study both mechanisms and inhibitors of Flaviviridae replication.

Animals↗
PubMed1997

Size and conformational stability of the hepatitis A virus used to prepare VAQTA, a highly purified inactivated vaccine.

A variety of biophysical techniques have been employed to examine the size and conformational integrity of highly purified hepatitis A virus (HAV) in solution (purified HAV particles are subsequently formalin-inactivated and adsorbed to aluminum salts for use as the vaccine VAQTA). The size of HAV particles was assessed by a combination of electron microscopy, sedimentation velocity, and dynamic light scattering. The effect of ionic strength and temperature on the overall conformational stability of HAV was determined by a combination of intrinsic HAV protein fluorescence, fluorescent probes of both RNA and protein, and UV-visible spectroscopy. A major structural change in HAV occurs near 60 degrees C with the addition of 0.2 M magnesium chloride enhancing the thermal stability of HAV by approximately 10 degrees C. Salt concentrations above 0.2 M, however, decrease the solubility of HAV. The effect of pH on the physical properties of HAV particles was monitored by dynamic light scattering, analytical size exclusion HPLC, and interaction with fluorescent dyes. HAV particles undergo a substantially reversible association/aggregation at pH values below 6 with the concomitant exposure of previously buried hydrophobic surfaces below pH 4. These results are in good agreement with previous studies of HAV thermal stability under extreme conditions in which the irreversible inactivation of the viral particles was measured primarily by the loss of viral infectivity. The wide variety of biophysical measurements described in this work, however, directly monitor structural changes as they occur, thus providing a molecular basis with which to monitor HAV stability during purification and storage.

Chromatography, High Pressure Liquid↗
PubMed1996

Anti-influenza virus activities of 4-substituted 2,4-dioxobutanoic acid inhibitors.

We previously identified a series of compounds which specifically inhibited the transcription of influenza A and B viruses (J. Tomassini, H. Selnick, M.E. Davies, M.E. Armstrong, J. Baldwin, M. Bourgeois, J. Hastings, D. Hazuda, J. Lewis, W. McClements, G. Ponticello, E. Radzilowski, G. Smith, A. Tebben, and A. Wolfe, Antimicrob. Agents Chemother. 38:2827-2837, 1994). The compounds, 4-substituted 2,4-dioxobutanoic acids, selectively targeted the cap-dependent endonuclease activity of the transcriptase complex. Additionally, several of these compounds effectively inhibited the replication of influenza virus but not other viruses in cell culture assays. Here, we report on the anti-influenza virus activities of other potent derivatives of the series evaluated in both in vitro and in vivo infectivity assays. These compounds inhibited the replication of influenza virus in yield reduction assays, with 50% inhibitory concentrations ranging from 0.18 to 0.71 microM. These 50% inhibitory concentrations were similar to those observed for inhibition of in vitro transcription (0.32 to 0.54 microM). One selected compound also elicited a dose-dependent inhibition of influenza virus replication in mice following an upper respiratory tract challenge. These studies demonstrate the antiviral efficacy of this inhibitor class and thereby establish the utility of influenza virus endonuclease as a chemotherapeutic target.

Animals↗
PubMed1994

Peptide substrate cleavage specificity of the human cytomegalovirus protease.

The human cytomegalovirus UL80 gene encodes an 80-kDa precursor polyprotein whose N-terminal 256-amino acid domain is a protease. This enzyme cleaves a specific peptide bond that results in its own release from the precursor, as well as a peptide bond near the C terminus of the viral assembly protein. The latter cleavage is apparently required for encapsidation of the viral genomic DNA and maturation of the viral capsid. A series of peptide substrates, representing the assembly protein cleavage site, was used to study the enzyme's substrate requirements and specificity. It was found that efficient cleavage minimally required the amino acid residues spanning the P4 to P4' positions. Substitution at any of these residues adversely affected the reaction. Conservation of the hydrophobic residues at P3 and P4 was essential. In addition, cleavage of a peptide representing the protease domain release site was reduced almost 100-fold relative to cleavage of the assembly protein maturation site peptide substrate.

Amino Acid Sequence↗
PubMed1991

Expression of active human immunodeficiency virus type 1 protease by noninfectious chimeric virus particles.

To generate nonpathogenic viral particles which express active human immunodeficiency virus type 1 (HIV-1) protease (PR), plasmids containing sequences from the genomes of HIV-1 and Moloney murine leukemia virus (M-MuLV) were constructed. Either the PR coding region alone; the gag, PR, and reverse transcriptase protein-coding regions; or the complete gag and pol protein-coding regions from HIV-1 were substituted for the corresponding regions of a full-length M-MuLV clone to yield the chimeric plasmids pMoHIV-I, pMoHIV-III, and pMoHIV-IV, respectively. Cell lines which express the viral gag polyprotein were isolated for hybrids pMoHIV-I and pMoHIV-III. These cells produced viral particles which contained processed core proteins. Cleavage of the gag polyprotein in the viral particles was inhibited by the HIV-1 PR inhibitor L-687908, indicating that the viral PR is responsible for the observed processing. The hybrid virions were not infectious; analyses indicated that the viral particles contained little or no reverse transcriptase activity. In addition, particles produced by pMoHIV-III transfectants failed to package the viral genomic RNA. The cell line which expresses and processes the HIV-1 gag polyprotein is a safe and effective reagent for the in vivo evaluation of potential inhibitors of the HIV-1 PR.

Animals↗
PubMed1985

Isolation and characterization of the Streptomyces cattleya temperate phage TG1.

A temperate actinophage, TG1, was isolated from soil by growth on Streptomyces cattleya and has been shown to be potentially useful for the cloning of DNA in this organism and other streptomycetes. It forms stable lysogens by integration at a unique site on the chromosome. The phage genome consists of 41 kb of double-stranded DNA with cohesive ends. It has unique sites for ClaI, NdeI, PstI, SmaI, and XbaI. The PstI site has been shown to be in a dispensable region of the phage genome. Deletions (2 kb in length) were obtained which retain this site and should be useful for the cloning of DNA.

Attachment Sites, Microbiological↗
PubMed1979

Viral etiology of age-dependent polioencephalomyelitis in C58 mice.

The etiology of immune polioencephalomyelitis (IPE) and the mechanisms of resistance to IPE induction were investigated in C58 mice. IPE was found to be induced by a lipid-solvent-sensitive, filterable replicating agent present in line Ib leukemic cell suspensions. IPE was serially transmitted in immunosuppressed mice with filtered extracts of spleens from diseased animals. The IPE-inducing activity of Ib cell extracts was abolished by chloroform or deoxycholate. Gel filtration of Ib cell extracts showed that the IPE agent has a molecular weight of at least 10(7). Electron microscopy of the active fractions from columns and of spinal cord extracts from mice with IPE revealed a virus-like particle, 40 nm in diameter, which is probably the IPE revealed a virus-like particle, 40 nm in diameter, which is probably the IPE agent. Administration of cyclophosphamide at various times after challenge increased the incidence of IPE in mice, suggesting that IPE is not autoimmune mediated. Immunosuppression resulted in maintenance of high levels of IPE agent in the central nervous system tissue, while immunization resulted in low levels. Moreover, immunized mice produced neutralizing antibodies. These data suggest that antibodies help restrict the amount of IPE agent in the nervous tissue, and that this restriction is required for resistance to IPE induction in C58 mice.

Aging↗