Eva Margalith

Characterization and Mixed Infections of Three Strains of Vaccinia Virus: Wild Type, IBT-resistant and IBT-dependent Mutants

Summary The characteristics of IBT-dependent (IBT d ) and IBT-resistant (IBT r ) mutants of vaccinia virus were compared with those of the wild type (wt) strain. The mutants did not differ from the wild type strain by their sedimentation in sucrose gradients. Minor differences in the polypeptide composition of the virus particles and in the neutralization of the three virus strains by anti-vaccinia human immunoglobulin were observed. Mixed infections of the viruses in HeLa cells enabled the growth of the strains under their unfavourable conditions (wt in the presence of IBT and IBT d in the absence of IBT).

Inhibition of Vaccinia Virus Growth by the Nucleoside Analogue 1-beta-D-Ribofuranosyl-1,2,4-Triazole-3-Carboxamide (Virazole, Ribavirin)

Virazole or Ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) inhibits the growth of vaccinia virus at a concentration ode to a certain extent in the presence of Virazole, the DNA fails to acquire resistance to deoxyribonuclease and virus particles are not formed. Reversibility of the antiviral effect occurs when the drug is washed out from the infected cultures or when guanosine at an equimolar concentration is added.

Antiviral activity of tunicamycin on herpes simplex virus.

Tunicamycin (0.5 microgram/ml) significantly lowers (2 to 3 log10) the infectious yield of herpes simplex virus type 1 grown in chicken embryo fibroblasts and in BSC1 cells. Although virus particles are formed and the synthesis of the viral deoxyribonucleic acid is only partially affected by the antibiotic, the glycosylation of herpesvirus glycopeptides is amost completely inhibited. The morphology of virus particles made in the presence of tunicamycin is similar to that of intact virus particles, as demonstrated by electron microscopy. This suggests that the absence of the carbohydrate side-chain from the viral glycopeptides does not affect the overall integrity of the virion but decreases very significantly the infectivity of these particles. Images

Location of vaccinia virus structural polypeptides on the surface of the virus particle.

Vaccinia virus has a complex structure. Thin sections of virus particles show three structural components: the outer envelope, the lateral bodies and a central structure, known as the ‘core’, which contains the virus DNA (Dales, 1963). Easterbrook (1966) showed that treatment with the nonionic detergent NP40, in the presence of 2-mercaptoethanol (2 ME) leads to the dissociation of the outer membrane and release of the core. By polyacrylamide gel electrophoresis, Holowczak & Joklik (1967) identified at least 17 structural polypeptides, three of which were associated with the core. Katz & Moss (1970) showed that some of these polypeptide peaks could be further separated into two to three polypeptides, thus increasing the total number of resolved structural polypeptides to 22. Our knowledge concerning the arrangement of vaccinia virus polypeptides on the particle surface is very limited. The present paper attempts to determine which of the virus polypeptides is situated on the surface.

Synthesis of Vaccinia Virus Polypeptides in the Presence of Isatin-β-Thiosemicarbazone

Isatin-β-thiosemicarbazone (IBT) at a concentration of 14 μM inhibited the multiplication of vaccinia virus in HeLa cells. For the first 3 h after infection, viral deoxyribonucleic acid (DNA) was synthesized in the presence of IBT at the same rate as in the control culture; the replication rate declined at a later stage. The DNA failed to be coated with proteins and to become resistant to deoxyribonuclease unless IBT was removed. “Early” and “late” viral polypeptides were formed in the presence of IBT, as revealed by polyacrylamide gel electrophoresis. The formation from precursor of a core polypeptide, a reaction blocked by rifampin, was not affected by IBT. Therefore, it is suggested that a maturation step later than the one blocked by rifampin is involved in the inhibition of vaccinia virus by IBT. Images

Isolation and Characterization of an IBT-Dependent Mutant of Vaccinia Virus

The antiviral activity of thiosemicarbazones against poxviruses was reported by Hamre, Bernstein & Donovick (1950). Thiosemicarbazone derivatives are clinically used in India and Africa. Appearance of drug-resistant and drug-dependent mutants in the course of viral therapy is a potential danger. Appleyard & Way (1966) and Ghendon & Chernos (1972) observed the selection of thiosemicarbazone-derivatives resistant mutants, during growth of the wild-type strain in the presence of these drugs. We shall describe here the isolation and characterization of an isatin-β-thiosemicarbazone (IBT)-dependent mutant (IBTD) of vaccinia virus, which needs IBT for its growth. The characteristics of this mutant are compared with these of the wild-type (wt) strain and an IBT-resistant mutant (IBTR), isolated in our laboratory. The two vaccinia mutants IBTD and IBTR were isolated after treatment of cells infected with wt virus with the mutagenic agent iododeoxyuridine in the presence of IBT.

An isatin beta-thiosemicarbazone (IBT)-dependent mutant of vaccinia virus: the nature of the IBT-dependent step.

Summary The IBT-dependent mutant of vaccinia virus needs IBT for its growth. IBT is not essential for the synthesis of virus DNA, but the formed DNA does not become resistant to deoxyribonuclease in its absence. Both ‘early’ and ‘late’ virus proteins are synthesized in cells infected with the mutant, but in the absence of IBT one of the virus core structural polypeptides, which is normally formed from a higher mol. wt. precursor, is not made. These findings suggest that the IBT-dependent step of the mutant occurs earlier in the maturation process of the virus than does the block produced by IBT with the wild-type strain.

Inhibition of Herpesvirus Deoxyribonucleic Acid and Protein Synthesis by Tilorone Hydrochloride

Tilorone hydrochloride at a concentration of 10 μg/ml very efficiently inhibited herpes simplex virus growth in BSC1 cells when the virus is infected at a low multiplicity of infection. The adsorption of the virus was not affected by the drug, and the penetration of the deoxyribonucleic acid of the input virus into the cytoplasm and nuclei proceeded normally when tilorone hydrochloride was present. However, newly synthesized viral deoxyribonucleic acid was not detectable under these conditions, there was a remarkable decrease in the rate of viral polypeptide synthesis, and virus particles were not formed. The inhibition of herpesvirus growth by tilorone hydrochloride was absolutely dependent on the presence of the drug in the cultures. Pretreatment of the cells with the drug did not result in resistance to herpesvirus infection after the removal of the drug. Images

The Effect of Isatin beta Thiosemicarbazone (IBT)-related Compounds on IBT-resistant and on IBT-dependent Mutants of Vaccinia Virus

Summary IBT-related compounds were examined for their ability to inhibit wild-type and IBT-resistant mutants of vaccinia virus and for their capability to support the growth of an IBT-dependent mutant. Among thirteen compounds tested, six did not affect any one of the three virus strains and five behaved similarly to IBT, but two inhibited the growth of all three viruses and therefore differed from IBT. It was found that the last two compounds also differed from IBT in that they interfered with synthesis of the virus DNA. This difference is surprising in view of a substantial similarity in chemical structure.

The effect of tilorone hydrochloride on the growth of several animal viruses in tissue cultures.

Tilorone hydrochloride, at a concentration of 10 mug/ml inhibits the growth of herpes simplex virus type 1 in BS-C-1 cells. The growth of vaccinia virus in BS-C-1 cells is partially inhibited; however, six viruses containing RNA, including four members of the togavirus group grown in chick fibroblasts, are not affected by the drug. The inhibition of the growth of herpes virus by tilorone hydrochloride is greater when the multiplicity of infection is lower than 1 p.f.u/cell and when the drug is added early in the course of infection.