Zinc chloride (0.1 mM) inhibited by 96 The cytoplasmic fraction obtained after low-speed centrifugation was subjected to sonic vibrations for 30 s in a Bransonic 12 sonicator and then layered on a 25 to 40% sucrose gradient in 1 mM tris(hydroxymethyl)-aminomethane-hydrochloride, pH 9.0, and sedimented in an SW50.1 rotor at 13,000 rpm for 35 min at 4°C. Fractions (10 drops) were collected from the bottom of the tube. One part (100 pi) of each fraction was directly precipitated with trichloroacetic acid, and another part was first digested with deoxyribonuclease (50 ug/ml; Worthington Diagnostics, Freehold, N.J.) in the presence of 10 mM MgCl2 for 30 min at 37°C.Polyacrylamide gel analysis. A discontinuous buffer system was used for sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The separating 13-cm-long gel contained 7.5% acrylamide-0.2% N,N'-methylene-bisacrylamide-0.1 M sodium phosphate (pH 7.1)-0.1% sodium dodecyl sulfate. The samples for electrophoresis were solubilized by incubation in 2% sodium dodecyl sulfate-1% mercaptoethanol at 1000C for 1 min. Electrophoresis took place at a constant current of 2 mA per gel overnight, until the dye front reached the bottom of the gel. After the gels were stained for 5 h with 0.1% Coomassie brilliant blue in 10% trichloroacetic acid, they were destained with 7.5% acetic acid. Gels were sliced longitudinally, dried, and placed in contact with X-ray film.Chemicals

Tunicamycin (0.5 ,ig/ml) significantly lowers (2 to 3 log,o) 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 almost 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.Herpes simplex type 1 virus (HSV) is an enveloped deoxyribonucleic acid (DNA)-containing virus that assembles by budding from the cell nuclear membranes during its maturation process (13,14). The virion contains more than 30 structural polypeptides, to several of which carbohydrate side chains are covalently linked (7,21). The role of these glycopeptides in the overall integrity and stability of the virions is yet to be clarified.Tunicamycin is a glucosamine-containing antibiotic (23, 24) that specifically inhibits glycosylation of proteins (2,18,22). It blocks the transfer of N-acetylglucosamine-l-phosphate from uridine 5'-diphosphate-N-acetylglucosamine to the dolichyl monophosphate that serves as a lipid carrier (12,25) by inhibition of the transferase involved (6). Changes in the cell morphology and in the membrane properties of normal and virus-transformed fibroblasts occur in the presence of tunicamycin (3, 4). Tunicamycin significantly lowers the yield of several enveloped ribonucleic acid (RNA)-containing viruses (10,15,17,20). Whereas formation of virions of Semliki Forest, fowl plague (20), Sindbis, and vesicular stomatitis viruses (10) is greatly inhibited by tunicamycin, formation of Rous sarcoma and influenza virions occurs in the presence of the antibiotic; however, these virus particles show reduced infectivity and undetectable amounts of glycoproteins (15,20).In the present study, we examine the effect of tunicamycin on formation, structure, and infectivity of HSV particles to determine the involvement of the carbohydrate side chains of the glycopeptides in these viral processes and functions. MATERIALS

SUMMARYIBT-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 Iast 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.

SUMMARYThe 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).

A mutant of herpes simplex virus type 2, which induces low levels of thymidine-kinase activity in infected BSC1 cells and consequently able to grow in the presence of acycloguanosine, was isolated. This mutant has also been shown to cause fusion of BSC1 cells. In BSC1 cells, co-infected with the wild-type strain and the mutant, the yield of each of the two viruses was normal but the rounding and aggregation of cells observed, resembled that found in wild-type infected cultures. When the mixed infection was performed in the presence of acycloguanosine (100 micrometers), the growth of the two virus strains was inhibited, as well as the cytopathic effect in the cultures. It is suggested that under these conditions, the thymidine-kinase which was induced in the infected cells by the wild-type strain, phosphorylated acycloguanosine and the activated drug formed, inhibited the growth of the two viruses by interference in their DNA syntheses.