Edith N. Rosenblum

Protein cleavage and poxvirus morphogenesis: Tryptic peptide analysis of core precursors accumulated by blocking assembly with rifampicin

Abstract Rifampicin was used to block vaccinia virus assembly and accumulate structural proteins that have not undergone maturational processing. Two of these proteins were purified and shown by tryptic peptide analysis to be higher molecular weight precursors of the major core components. In contrast, rifampicin did not prevent the formation from its precursor of another virion protein located outside the virus core.

Infectious Mononucleosis: Complement-Fixing Antibodies to Herpes-Like Virus Associated with Burkitt Lymphoma

Complement-fixing antibodies to a herpes-like virus derived from a Burkitt tumor-cell line developed in each of 21 patients with infectious mononu-cleosis. These antibodies were absent in all serums before the patients became ill, appeared during the early phases of illness, and persisted for long periods of time. These antibodies are distinct from heterophile antibodies. None of the patients developed immune responses to herpes simplex, cytomegalo-, or reoviruses in the course of their illness. The data suggest that the development of complement-fixing antibodies to this herpes-like virus in these patients may be linked to infectious mononucleosis.

The incidence of complement-fixing antibodies to herpes simplex and herpes-like viruses in man and rhesus monkeys.

Summary The results of the comparative sero-epidemiology of herpes-like viruses (HLV) and herpes simplex virus (HSV) infections in man are presented. Sera from 573 subjects of various age groups were examined for the presence of complement-fixing antibodies to these viruses. Antibodies to HLV appeared at an earlier age than HSV antibodies and reached a frequency of 65% in the 3-5 year age group. Both antibodies were present in 85-90% of the adult population and were distributed on a worldwide basis. Both antibodies persisted for at least 10 years in sera of 26 normal individuals. Fifty percent of sera obtained from 99 rhesus monkeys within 1-4 days after time of capture contained antibodies reacting with HLV antigen.

Vaccinia virus directed RNA and protein synthesis in the presence of rifampicin

Abstract The effects of rifampicin, an inhibitor of bacterial DNA transcription, on the formation of infectious vaccinia virus and viral particles, RNA and proteins were studied under single step growth conditions. Although virion formation was inhibited by more than 99%, the synthesis of early and late viral directed RNA species, as characterized by their sedimentation properties, was unaffected. Rifampicin did not prevent the synthesis of early and late viral proteins identified by immunodiffusion, disc gel electrophoresis and disc gel immunoelectrophoresis. The drug also did not inhibit the in - vitro activity of the virion associated RNA polymerase.

Assembly of virus particles during mixed infection with wild-type vaccinia and a rifampicin-resistant mutant

Abstract A rifampicin-resistant vaccinia virus mutant was isolated from wild-type virus stocks. The mutant was purified by sucrose density gradient centrifugation and had an infectivity, electron microscopic appearance, and polypeptide composition that was similar to the wild-type virus. Growth of the mutant was inhibited only slightly at concentrations of rifampicin which completely prevented the growth of drug-sensitive virus, and at no concentration tested was there a detectable effect on viral envelope formation. The mutant virus was capable of rescuing wild-type during mixed infection in the presence of rifampicin; after simultaneous infection the ratio of genomes in the progeny reflected that of the original inoculum. Superinfection experiments demonstrated that beat-inactivated mutant was capable of efficiently rescuing wild-type virus and that extensive replication of the mutant genome was not required for this. Characteristic rifampicin blocked viral forms, normal maturing forms, and intermediate structures were seen in the same ultrathin cell sections after simultaneous infection with low multiplicities of mutant and wild-type virus. In cells simultaneously infected with high multiplicities of mutant and wild-type virus, rifampicin had no detectable effect on viral envelope formation. No difference was found in pulse-labeled polypeptides synthesized in rifampicin-treated cells infected with either mutant virus, wild-type virus, or both when analyzed by SDS-polyacrylamide gel electrophoresis. After a 4-hr chase, cleavage of the high molecular weight precursors of structural polypeptides was inhibited in cells infected with wild-type virus but occurred to similar extents in cells infected with mutant alone and cells simultaneously infected with mutant and wild-type virus.

Assembly of vaccinia virus particles from polypeptides made in the presence of rifampicin

Abstract Vaccinia viral polypeptides, labeled in the presence of rifampicin, were incorporated into virus particles after rifampicin was removed. All structural polypeptides, resolved by polyacrylamide gel electrophoresis, were labeled in the presence of rifampicin. The specific effect of rifampicin on the formation of the vaccinia viral envelope was reversed within 10 min even in the presence of NaF or inhibitors of protein or nucleic acid synthesis. Further maturation of some virus particles, which were characterized by their ultrastructure and isolated by sucrose gradient sedimentation, proceeded in the presence of inhibitors of protein synthesis. In contrast, addition of NaF or actinomycin D almost completely blocked the development of mature virus particles. Rifampicin acted specifically at the stage of envelope formation and did not prevent later steps in maturation.

Attempts to transmit infectious mononucleosis to rhesus monkeys and marmosets and to isolate herpes-like virus.

Summary Buffy coat cells obtained from the blood of 11 patients with infectious monucleosis were inoculated into selected rhesus monkeys and marmosets. These monkeys were free of detectable complement-fixing antibodies to a herpes-like virus (EBV) considered to be etiologically related to infectious mononucleosis. None of the inoculated animals developed detectable illness, or hematological and serological changes. Attempts to isolate a viral agent from the throats of the 11 patients in cultures of human leukocytes were unsuccessful.

Glycoprotein synthesis in cells infected with vaccinia virus: I. Non-virion glycoproteins

Abstract When vaccinia-virus infected HeLa cells were incubated in medium containing radioactively labeled glucosamine, the specific activity of the UDP- N -acetylhexosamine pool increased linearly at a rate similar to that of uninfected cells. Nevertheless, starting at 2 hr after infection, hexosamines were incorporated into glycoproteins at a progressively lower rate. Furthermore, from electrophoretic analysis it appeared as though synthesis of the major glycoproteins of uninfected cells was arrested and new glycoproteins were labeled. After equilibrium centrifugation the major vaccinia-induced glycoproteins did not sediment with infectious virus but a large portion was recovered from fractions rich in cell membranes and containing particulate material. Glycoproteins of similar electrophoretic mobilities were made in vaccinia-infected HeLa and chick cells. Both actinomycin D and cycloheximide inhibited the labeling of all virus-induced glycoproteins whereas rifamycin derivatives had a selective effect.

Glycoprotein synthesis in cells infected with vaccinia virus III. Purification and biosynthesis of the virion glycoprotein

A 2-step procedure was used for the purification of the vaccinia virion glycoprotein. The glycoprotein was purified according to molecular weight by SDS-polyacrylamide gel electrophoresis and then separated from three similar molecular weight proteins by SDS-hydroxylapatite chromatography. Peptide maps of tryptic digests indicated that the proteins separated by chromatography had different amino acid sequences. The molecular weight of the virion glycoprotein, determined by electrophoresis at several gel concentrations was approximately 38,500. Attempts were made to use trypsin as a probe to locate the glycoprotein within the virion. Although the glycoprotein was degraded or released by treatment of virions with trypsin under conditions in which lipid was not removed, DNA was not liberated and no loss in plaque-forming units was detected, the proteolytic activity was not selective enough for precise localization. Radioisotopic labeling experiments were carried out to determine the relative time of synthesis of the glycoprotein. These studies suggested that both the polypeptide and carbohydrate portions were formed primarily after the onset of viral DNA synthesis.

Rifamycins: Modulation of Specific Anti-Poxviral Activity by Small Substitutions on the Piperazinyliminomethyl Side Chain

Rifamycin derivatives differing in the substitutent at the 4 position of the piperazinyliminomethyl side chain were tested for anti-poxviral activity. The effects of each derivative on wild-type vaccinia virus and on a mutant selected for resistance to rifampin were determined. Antiviral activity was measured in tissue culture by plaque inhibition, reduction in virus yield, and specific interruption of virus morphogenesis. Rifamycin derivatives containing H, ethyl, or propyl groups at the 4 position of the piperazinyliminomethyl side chain were much less active than rifampin, which has a methyl group at this position. Thus, minimal shortening or lengthening of the methyl piperazinyliminomethyl side chain of rifampin led to loss of specific antiviral activity. In contrast, the derivative containing an amino group at the 4 position of the piperazinyliminomethyl side chain had enhanced anti-poxviral activity.