Miriam Margalith

Differential inhibition of DNA polymerase and RNase H activities of the reverse transcriptase by phosphonoformate

SummaryThree potential inhibitors of reverse transcriptase activities, phosphonoformate (PF), phosphonoacetate (PAA), and ethyl-diethyl phosphonoformate (Et-PF), were compared in this study. Only PF was found to inhibit the DNA polymerase activity of the purified reverse transcriptase of Moloney murine leukemia virus (M-MuLV) and avian myeloblastosis virus (AMV). The degree of DNA polymerase inhibition was linear with PF concentration; 50% inhibition was achieved at 10 µM. Whereas PF inhibited both the RNA and DNA dependent DNA polymerase activities, the RNase H activity of the reverse transcriptase was unaffected. Both the endogenous DNA polymerase activity in detergent disrupted virus and the activity of the purified enzyme with the isolated virus genome 70S RNA were inhibited by PF However, higher concentrations of PF were needed to inhibit the endogenous reaction. The inhibition by PF appeared to be reversible and noncompetitive with respect to the substrate deoxythymidine triphosphate (dTTP). Addition of PF after the initiation of DNA synthesis immediately arrested the reaction.

Transformation of BSC1 Cells following Chronic Infection with SV40

Summary The BSC/SV 40 virus carrier state was established by infecting monolayers of BSC1 cells with high concentrations of SV40. In these carrier cultures, only 4% of the cells contained intranuclear tumour (T) antigen as determined by the immunofluorescent method, but every cell produced infectious virus. During passage of the BSC/SV40 cultures, a cell line was selected in which all the cells exhibited T antigen and only 0.2–1% of the cells produced infectious virus (transformed state). The BSC1 transformed cells were like the BSC1 parent cells sensitive to challenge with heterologous viruses (attenuated polio 1, herpes simplex and vaccinia), but were resistant to superinfection by the homologous SV40. Cytosine arabinoside, actinomycin D and antiserum to SV40 inhibited virus and T antigens in the BSC/SV40 carrier cultures. In the transformed cells, infectious virus and virus antigen only were inhibited but synthesis of the T antigen remained unaffected. By cell cloning, two virus-free clones were obtained.

Phosphonoformate inhibits synthesis of epstein-barr virus (EBV) capsid antigen and transformation of human cord blood lymphocytes by EBV☆

Abstract Phosphonoformic acid trisodium salt (PF) and ethyl diethylphosphonoformate (Et-PF) inhibited Epstein—Barr virus (EBV) viral capsid antigen (VCA) synthesis in B.95-8 cells, at concentrations which were nontoxic to cells. PF inhibited 96.7% of VCA synthesis when used at 500 μM whereas Et-PF inhibited only 68.9% of VCA synthesis when used at 2000 μM. The synthesis of EBV nuclear antigen (EBNA) was not inhibited by PF or Et-PF. PF inhibited the transformation of human cord blood lymphocytes (CBL) by EBV as detected by [ 3 H]thymidine uptake. The inhibition of transformation was a function of the dose of PF used. A concentration of 2000 μM PF completely inhibited the transformation; the same concentration of ET-PF had almost no effect.

Segregation of mouse virulent and avirulent ECHO virus type 9 strains and the correlation of mouse-virulence with the “temperature” (rct/40) marker

A quantitative study of mouse virulence of ECHO virus type 9 strains, isolated from cases of aseptic meningitis was carried out using the plaque technique. Plaques obtained directly from ECHO virus type 9 isolates exhibited marked variation in mouse virulence; the majority of plaques was of high or low virulence, and only 3 out of 80 plaques tested were completely mouse avirulent. Subplating of these mouse avirulent plaques yielded strains which were predominantly mouse avirulent (45 out of 47 plaques). However, the passage in undiluted form of the avirulent plaque progeny in MKCC and especially in infant mice was selective for mouse virulence. The rct/40 marker was employed for the study of mouse virulence of the ECHO virus type 9 strains. An excellent correlation was found between the rct/40 marker and mouse virulence; rct/40+ strains were mouse virulent whereas rct/40− strains were avirulent. The rct/40 marker is suggested for use in the determination of mouse virulence of ECHO virus type 9 isolates.

Genetic characteristics of echovirus type 9 strains: association of mouse virulence with other genetic markers.

Summary In naturally occurring strains and laboratory selected variants of echovirus type 9, the association between virulence for infant mice (v) and the reproductive capacity at 40° (t) was of the v+t+ and v-t- types. By different selection methods the two other marker combinations, v+t- and v-t+, were obtained. The frequency of t+ in a t- echovirus type 9 population was 10-5. Of the four possible combinations between the v and c (cytopathic effect for human epithelial cell lines) markers, only three were found. The v+c+ covariation could not be obtained in spite of appropriate methods of selection.

Genetic Characteristics of Echovirus Type 9 Strains: Selection and Characterization of Variants

Summary Using different methods of selection, three main types of echovirus type 9 variants were obtained from a single isolate derived from a naturally occurring strain. The three variants pl 88, pl 43 and pl 29 differed in a number of genetic characters as follows: virulence for infant mice (v), capacity to grow at 40° as compared to 36.5° (t), cytopathic effect on epithelial cell lines of human origin (c), inhibition by rhesus monkey serum (m) and protection against heat inactivation by Al ions (a). They also differed in the size and time of appearance of plaques on monkey kidney cell monolayers. The pl 88 variant was mouse-virulent, grew at 40° to the same degree as at 36.5°, was not cytopathogenic on epithelial cell lines of human origin, was not inhibited by normal rhesus monkey serum, not protected by Alions from heat inactivation and produced early large plaques on baboon kidney cell monolayers. Accordingly its genetic markers are v + t + c - m - a -. The genetic markers of the pl 43 variant are v - t - c - m - a -; pl 43 differs from pl 88 only in the v and t markers. Though avirulent for mice, mouse-virulent variants could be selected from it by repeated passage in mice. pl 29 had the characters v -s t - c +. It was distinctly different from both pl 88 and pl 43 by its c marker and produced late and small plaques on baboon kidney cell monolayers. pl 29 was stable in its mouse avirulence and no virulent mutants could be obtained from it by repeated passage in mice. Progeny of pl 29 occurred in two ‘subforms’, m + a + and m - a -. The v -s t - c + m + a + form was identical in these characters with both the quigley and the prototype echovirus type 9, hill strains.

On the continuous culturing of B.95-8 cells en the presence of phosphonoacetic acid

Lymphoblastoid B.95-8 cells were cultured for four months and three weeks in the presence of increasing concentrations (50--200 microgram/ml) of phosphonoacetic acid (PAA). Several weeks after removal of the PAA, the cultures, in parallel with untreated B.95-8 cells, were tested for the presence of: 1) Epstein-Barr virus (EBV) viral capsid antigen (VCA), and b) transformation of human cord blood lymphocytes. There was no difference in the percentage of cells exhibiting VCA in the B.95-8 PAA treated and untreated cells. However, transformation assays indicated 10 times less transforming virus in culture supernatant harvested from B.95-8 cultures treated with PAA, as compared with the control cultures. Electron microscopic studies indicated the presence of virus particles in B.95-8 control cells and their almost complete absence in the PAA-treated cells.