William A. Carter

Ribosomes: Effect of Interferon on Their Interacton with Rapidly Labeled Cellular and Viral RNA's

Rapidly labeled RNA of mouse L cells and labeled RNA of Mengo virus, unlike cellular RNA labeled under steady-state conditions, form detectable complexes with L-cell ribosomes. These ribosome-RNA complexes formed in vitro appear analogous to those assembled during polysome formation in vivo. When ribosomes are prepared from L cells exposed to homologous interferon, their capacity to associate with cell messenger is preserved, while their ability to interact with viral RNA is markedly reduced. The ribosomes from cells exposed to interferon are thus altered selectively to permit only certain messages to be bound and translated.

Fibroblast interferon treatment of a patient with chronic active hepatitis: Increased number of circulating T lymphocytes and elimination of rosette-inhibitory factor

A 23 year old woman with chronic active hepatitis documented by liver biopsy demonstrated persistent hepatitis B surface antigen, hepatitis B virus specific DNA polymerase hepatitis B core antigen (HBcAg), for approximately one year. The number of circulating T lymphocytes that rosetted with sheep erythrocytes was decreased, and a rosette-inhibitory factor was present in her peripheral blood. Interferon treatment (1 X 10(6) U/day intramuscularly for 82 days) resulted in a decrease of HBsAg and disappearance of HBcAg, (HBeAg) and specific DNA polymerase. In addition, the number of T lymphocytes increased to normal, and the rosette-inhibitory factor disappeared from the circulation. These findings suggest that the effect of interferon in chronic active hepatitis is mediated in part through its action on the immune system.

Effects of tunicamycin on the physical properties and antiviral activities of murine L cell interferon

Abstract In the present study, we have examined the physicochemical, antigenic, and biological properties of murine L cell interferons produced in the presence of the glycosylation inhibitor, tunicamycin. Interferon produced by L cells treated with tunicamycin exhibited fourfold less antiviral activity on murine cells, but similar heterologous activity on human cultures when compared to interferon synthesized in the absence of the antibiotic. The antiviral activities of the interferons produced in the presence of tunicamycin were neutralized by specific anti-interferon sera to the same degree as control L cell interferon. A 17,000 molecular weight interferon was synthesized in the presence of tunicamycin, whereas two larger (43,000 and 26,000) interferon species were synthesized by untreated cultures. Isoelectric focusing of a control L cell interferon revealed considerable molecular microheterogeneity, whereas L cell interferon produced in the presence of tunicamycin focused as two discrete peaks (designated 1 and 2); only peak 2 was neutralized by antisera raised against human leukocyte interferon serum. These results suggest that two 17,000 molecular weight nonglycosylated interferon species were produced by murine L cells treated with tunicamycin, one of which possesses a common antigenic determinant(s) with interferon produced by human leukocytes.

Purified human fibroblast interferon in vivo: skin reactions and effect on bone marrow precursor cells.

Human interferon from normal diploid fibroblasts, purified by sequential chromatography on concanavalin A-agarose and phenyl-sepharose, was administered parenterally in 4 subjects. Fever, marked skin hypersensitivity reactions and suppression of marrow stem cells (estimated by the count of myeloid colony-forming cells), side-effects common for less purified fibroblast and leukocyte interferons, were absent. Purified fibroblast interferon retained antiviral and immunomodulatory activity, evidenced by reduction of the blastogenic response of peripheral lymphocytes and decrease of hepatitis B virus markers in a patient with chronic hepatitis B infection treated with this substance.

The human interferon-albumin interaction: The influence of albumin conformation

Abstract When human interferon is chromatographed on bovine serum albumin immobilized via a molecular arm to agarose, there is a selective, transient retention of the interferon. When albumin is defatted prior to immobilization, its interaction with interferon becomes stronger, suggesting that hydrophobic binding is a major force in complex formation. The strength of the interferon-albumin interaction correlates with the extent of deformation of the albumin ligand. Human interferons, induced either by rI n ·rC n or Newcastle disease virus (NDV), are retained to the same extent, thus suggesting the existence of common structural features involved in their interactions with immobilized albumin.

Differential Susceptibility of Spleen Focus-Forming Virus and Murine Leukemia Viruses to Ansamycin Antibiotics

The streptovaricin complex (SvCx) and rifamycin SV derivatives display potent antiviral activity against the polycythemic strain of Friend leukemia virus (FV-P), as measured by a reduction in the number of spleen foci produced in mice. Such reductions may be explained by inactivation of functions of (i) the spleen focus-forming virus (SFFV), (ii) its “helper” murine leukemia virus (MuLV), or (iii) both viruses normally present in FV-P. We noted that preincubation of FV-P with fractionation products of SvCx, or derivatives of rifamycin SV, at low concentrations (3 to 5 μg/ml) reduces the number of spleen foci 80 to 97%, whereas titers of MuLV (from the same inoculum) remain unaffected (MuLV titers were measured by XC, S+L−, and “helper activity” assays). Our findings indicate a remarkable biological selectivity of ansamycins, as well as nonansamycin components of SvCx, against the transforming and defective spleen focus-forming virus as compared to MuLV. Thus, the drugs might be useful in distinguishing other types of oncornaviruses.

Activity of Pure Streptovaricins and Fractionated Streptovaricin Complex Against Friend Virus

Chromatographic fractionation of streptovaricin complex yields two stable components enriched (4- to 16-fold) in activity directed against the polycythemic strain of Friend virus; both components apparently contain no streptovaricins. When compared with their unfractionated parent streptovaricin complex, eight individual intact streptovaricins (A through G and J) show at least a 30-fold reduction in antiviral activity. These results further support the conclusion that the diversified biological properties of streptovaricin complex probably reside in different molecular structures.

Study of ansamycin inhibition of a ribonucleic acid-directed deoxyribonucleic acid polymerase by an immobilized template assay.

A series of structurally related ansamycins have been analyzed, in a new immobilized template assay, to determine the mechanism by which they inhibit a ribonucleic acid-directed deoxyribonucleic acid (DNA) polymerase from Moloney murine leukemia virus. By this assay, we can better correlate specific structures of these drugs with inhibitory mechanisms. Using an immobilized template, we were also able to observe drug effects on the stability of complexes formed between the polymerase, a template (polyadenylic acid-agarose), and a primer, as well as to monitor the synthesis of DNA in the presence of drug. For each drug, we determined the complex (intermediate in DNA synthesis) which was primarily affected and whether the effect was due to a destabilization process. Although the activity and specificity of the unsubstituted ansamycins (streptovaricins and rifamycin SV) were modulated by conformation of the molecule and electron density of the aromatic ring, the principal mode of inhibition is, apparently, drug binding to a polymerase-template complex; the drug binds in a manner which prevents subsequent formation of a polymerase-template-primer complex. However, some derivatives of rifamycin SV, when substituted at carbon-3 with bulky or hydrophobic side chains, displayed markedly different modes of action. For example, demethyl dimethyl rifampin prevented the formation of polymerase-template complexes, whereas rifazacyclo 16 acted by promoting the dissociation of polymerase-template-primer complexes.

Responses of the Murine Myeloid Colony-Forming Cell to Ansamycin Antibiotics

The in vitro susceptibility of murine myeloid colony-forming cells to the antiproliferative activities of three ansamycin antibiotics was determined. These cells were found to be 10- to 40-fold more susceptible than the corresponding human ones.

Antivirion Effects of Streptovaricin Complex Against Friend Virus

The in vitro antivirion activities of five different streptovaricin complex lots against the polycythemic strain of the Friend virus were evaluated. The assay system was based on the inhibition of the Friend virus-induced spleen foci. The virus inactivation process was shown to be susceptible to variation in temperature, pH, and time. The antivirion activity and the acute toxicity for mice, as well as the optical properties of these streptovaricin complexes, do not co-vary; this suggests that their biological activities are not associated with a single molecular structure. In addition, the antivirion activity of the five preparations of streptovaricin complex differs about 30-fold, indicating that this activity does not reside in a major component of the complex.