Multiplication Of Vaccinia Virus Research Articles

Reconstitution of carbonic anhydrase activity of the cell-surface-binding protein of vaccinia virus

The N-terminal region of a 32 kDa cell-surface-binding protein, encoded by the D8L gene of vaccinia virus, shows sequence homology to CAs (carbonic anhydrases; EC 4.2.1.1). The active CAs catalyse the reversible hydration of CO2 to bicarbonate participating in many physiological processes. The CA-like domain of vaccinia protein [vaccCA (vaccinia virus CA-like protein)] contains one of the three conserved histidine residues required for co-ordination to the catalytic zinc ion and for enzyme activity. In the present study, we report the engineering of catalytically active vaccCA mutants by introduction of the missing histidine residues into the wild-type protein. The wild-type vaccCA was inactive as a catalyst and does not bind sulfonamide CA inhibitors. Its position on a phylogram with other hCAs (human CAs) shows a relationship with the acatalytic isoforms CA X and XI, suggesting that the corresponding viral gene was acquired from the human genome by horizontal gene transfer. The single mutants (vaccCA N92H/Y69H) showed low enzyme activity and low affinity for acetazolamide, a classical sulfonamide CA inhibitor. The activity of the double mutant, vaccCA N92H/Y69H, was much higher, of the same order of magnitude as that of some human isoforms, namely CA VA and CA XII. Moreover, its affinity for acetazolamide was high, comparable with that of the most efficient human isoenzyme, CA II (in the low nanomolar range). Multiplication of vaccinia virus in HeLa cells transfected with the vaccCA N92H/Y69H double mutant was approx. 2-fold more efficient than in wild-type vaccCA transfectants, suggesting that the reconstitution of the enzyme activity improved the virus life cycle.

The vaccinia virus-stimulated mitogen-activated protein kinase (MAPK) pathway is required for virus multiplication.

Early events play a decisive role in virus multiplication. We have shown previously that activation of MAPK/ERK1/2 (mitogen-activated protein kinase/extracellular-signal-regulated kinase 1/2) and protein kinase A are pivotal for vaccinia virus (VV) multiplication [de Magalhães, Andrade, Silva, Sousa, Ropert, Ferreira, Kroon, Gazzinelli and Bonjardim (2001) J. Biol. Chem. 276, 38353-38360]. In the present study, we show that VV infection provoked a sustained activation of both ERK1/2 and RSK2 (ribosomal S6 kinase 2). Our results also provide evidence that this pattern of kinase activation depends on virus multiplication and ongoing protein synthesis and is maintained independently of virus DNA synthesis. It is noteworthy that the VGF (VV growth factor), although involved, is not essential for prolonged ERK1/2 activation. Furthermore, our findings suggest that the VV-stimulated ERK1/2 activation also seems to require actin dynamics, microtubule polymerization and tyrosine kinase phosphorylation. The VV-stimulated pathway MEK/ERK1/2/RSK2 (where MEK stands for MAPK/ERK kinase) leads to phosphorylation of the ternary complex factor Elk-1 and expression of the early growth response (egr-1) gene, which kinetically paralleled the kinase activation. The recruitment of this pathway is biologically relevant, since its disruption caused a profound effect on viral thymidine kinase gene expression, viral DNA replication and VV multiplication. This pattern of sustained kinase activation after VV infection is unique. In addition, by connecting upstream signals generated at the cytoskeleton and by tyrosine kinase, the MEK/ERK1/2/RSK2 cascade seems to play a decisive role not only at early stages of the infection, i.e. post-penetration, but is also crucial to define the fate of virus progeny.

Neplanocin A. A potent inhibitor of S-adenosylhomocysteine hydrolase and of vaccinia virus multiplication in mouse L929 cells.

Neplanocin A, a novel cyclopentenyl analog of adenosine, is a naturally occurring antibiotic which exhibits significant antitumor activity against L1210 leukemia in mice (Yaginuma, S., Muto, N., Tsujino, M., Sudate, Y., Hayashi, M., and Otani, M. (1981) J. Antibiot. 34, 359-366). In the present study we demonstrate that neplanocin A is also a potent inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1) having a Ki of 8.39 nM for the purified bovine liver enzyme. Analysis of the apparent irreversible inactivation of AdoHcy hydrolase by neplanocin A indicates that the drug is a tight binding inhibitor, exhibiting a stoichiometry of one molecule of inhibitor to one molecule (tetramer) of enzyme. In addition, we show that neplanocin A is a potent inhibitor of vaccinia virus (WR) multiplication in monolayer cultures of mouse L-cells. Concentrations of the drug as low as 0.5 and 1.0 microM in the culture medium produce 84 and 95% inhibition of plaque formation, respectively, while exhibiting little toxicity to the host cells. The inhibition of virus multiplication by neplanocin A coincides with a rapid inhibition of AdoHcy hydrolase activity in the infected cells and a subsequent 10-fold increase in the intracellular AdoHcy/S-adenosylmethionine ratio. These findings suggest that the antiviral actions of this compound may be related to an inhibition of S-adenosylmethionine-dependent macromolecular methylation reactions which are essential to the production of new virus particles (e.g. viral messenger RNA).

Über biologische Wirkungen von Koordinationsverbindungen der Übergangsmetalle 3. Zur antiviralen Wirkung von Cis-dichloro-diammin-platin (II): „Cis-Platin“

The coordination compound cis-dichlorodiammineplatinum(II) (cis-DDP) was shown by Rosenberg et al. (17) to exhibit antitumour activity. Several authors have indicated limited virustatic properties of cis-DDP against bacterial, oncogenic, avipox and paramyxo viruses.In our investigations, cis-DDP significantly showed an antiviral action in vitro against enveloped DNA and RNA viruses, such as vaccinia, pseudorabies, herpes simplex type 1, Newcastle disease, influenza A/fowl plague, influenza A/Victoria 3/75, influenza A/Jena 48/78, influenza B/Johannesburg and vesicular stomatitis viruses. Out of the group of non-enveloped viruses, adenovirus type 4 and 5 were inhibited, whereas no inhibition against naked cardiovirus Mengo could be estimated.The antiviral action was proved against extracellular virus by dialysis experiments with vaccinia virus and also during the replication cycles of enveloped viruses. In trials with cell-free viruses the plaque reduction of all sensitive viruses mentioned above amounted up to 100 per cent in comparison to the untreated controls caused by virus inactivation with loss of infectivity in contact with several concentrations of cis-DDP.On the other hand, the addition of the compound for one hour only immediately after infection or up to 8 hrs later produced a complete depression of further multiplication of vaccinia virus. Likewise, the replication of influenza virus A/FPV or VSV was inhibited, whereas the multiplication of adenoviruses was not influenced in a comparable manner.

The effect of viruses on megakaryocytes in vitro (author's transl)

Bone marrow suspensions from adolescent rats contain 0.3% megakaryocytes; this rate decreases to almost zero within 72--96 h cultivation in vitro in Leighton tubes because of thrombocytopoesis. Such cultures were inoculated immediately after seeding with different viruses in high multiplicity. After infection with herpes simplex virus type 1 or adenovirus type 2, no deviation of the number and morphology of the megakaryocytes was seen when compared with control cultures. However, after infection with vaccinia virus and, still more marked, with Newcastle disease virus, morphological alterations and interference with thrombocytopoesis were seen. Furthermore, a considerable portion of the altered megakaryocytes persisted for 72--96 h. Finally, the cytoplasm of the megakaryocytes after infection with vaccinia virus or NDV, but not with HSV 1 or adenovirus 2, showed a specific immunofluorescence with antiviral antisera, which indicates a direct cellular involvement and multiplication of vaccinia virus and NDV in megakaryocytes.

Combined inhibition of vaccinia virus multiplication by inhibitors of DNA synthesis.

Nine purine or pyrimidine analogues or other inhibitors of DNA synthesis were studied for their inhibition of vaccinia virus multiplication by a plaque inhibition test with a fluid overlay in Vero cell cultures, either as single substances or in combination. Experiments on combined inhibition were made with substance concentrations leading to 80-90% plaque inhibition; indifference, additive, or synergistic interaction were evaluated from normalized plaque counts. 12 substance pairs acted in a synergistic manner and 22 showed indifference. A comparison was made with the results of other DNA viruses (adenovirus, herpes simplex virus).

Process of cap formation of messenger RNA by vaccinia virus particles carrying an organized enzymes system.

Vaccinia virus mRNAs carry the cap structure m7G5'pppAm- or m7G5'pppGm- at the 5'-terminus, which is synthesized by a series of RNA polymerase and capping enzymes contained in the virus particle. The process of the cap formation at the 5'-terminus of mRNA was studied using an in vitro system under similar conditions to those of vaccinia virus multiplication in its host cell. After adding a methyl-group donor, methyl-3H-S-adenosylmethionine, the oligonucleotides, which were the de novo synthesized 5'-terminal part of mRNA, were isolated from the RNA-synthesizing virion at appropriate time intervals, and were analysed. The 5'-5' confronting nucleotides with 2'-O-methylation, G5'pppAm and G5'pppGm, were found with the completed cap structure, m7G5'pppAm and m7G5'pppGm. The confronting nucleotides with only 7-methyl guanine as a methylated component, m7G5'pppA and m7G5'pppG, were not detected at any incubation time, and it was concluded that methylation at the 2'-position of the 5'-terminal purine nucleoside of mRNA precedes methylation of the 7-position of the blocking guanosine. This result is different from that obtained using the enzymes isolated from vaccinia virus (Moss et al., 1976) and also from the results obtained using other kinds of virus particles, which carry RNA polymerase and capping enzymes. These differences may be due to the specific organization of a series of capping enzymes and RNA polymerase in each virus particle.

Mise en évidence de deux étapes arginino-dépendantes du cycle de multiplication du virus vaccinal: Rôle des polyamines dans la synthèse des ARNm tardifs

The multiplication of vaccinia virus in KB cells is dependent on arginine. In the absence of arginine, the synthesis of late mRNA does not occur. In order to characterize the mechanism by which the synthesis of late mRNA is dependent on arginine, it has been demonstrated that ornithine and the polyamines derived from it — putrescine, spermidine and spermine — can replace arginine and allow the synthesis of late mRNA. In contrast, arginine cannot be substituted for ornithine or the polyamines to allow the production of infectious particles. Thus, these are two arginine-dependent steps in the multiplication cycle of vaccinia virus: the synthesis of late mRNA in which polyamines are involved and a later step, viral maturation, which is presumably dependent on the synthesis of one or several arginine-rich proteins. The existence of two arginine-dependent steps has also been demonstrated by electron microscopic studies. The first step, which is the formation of immature nucleoid containing particles, requires the presence of polyamines. The second step is the formation of mature virions.

ChemInform Abstract: 3‐SUBSTITUTED ADENINES. IN VITRO ENZYME INHIBITION AND ANTIVIRAL ACTIVITY

AbstractDie direkte Alkylierung von Adenin ergibt die 3‐Substitutionsprodukte (II) sowie die isomeren Alkylderivate (III) und (IV).

Rifamycins as inhibitors of RNA and DNA polymerases

The rifamycins are a class of compounds obtained by chemical modification of one of the fermentation products of S t rep tomyces mediterranei (Sensi et al., 1966). This group of compounds was characterized in early reports to possess strong antimicrobial activity which led to their clinical application as antibiotics, particularly in the treatment of tuberculosis (Furesz et al., 1965). The interest of molecular biologists in these compounds was promoted by the finding of a specific anti-polymerase activity for bacterial RNA polymerase, which appears to be the mechanism by which the anti-bacterial activity is expressed. One of the compounds, rifampicin, was successfully used to elucidate steps of the complex process of RNA transcription. Although the low doses of drug required for inhibition and the stoichiometry of binding are indicative of specificity of the interaction of this complex molecule with the bacterial enzyme, other targets appear to be reached by the drug when high concentrations are used. For example, effects on mitochondrial RNA polymerase from N e u r o s p o r a crassa (Kuntzel and Schafer, 1971), on RNA synthesis in heart mitochondria (Gamble and McCluer, 1970), and on the multiplication of vaccinia virus (see Riva and Silvestri, 1972, for a review) have been reported, suggesting that a broader range of action involving polymerases from different sources might be inherent to this class of compounds. It seemed reasonable to assume that the limited effectiveness of rifampicin for polymerases from sources other than bacteria could be overcome by modification of the drug molecule. With this in mind, rifampicin and a few of its analogs were used as inhibitors of the RNA-dependent DNA polymerase of RNA tumor viruses at a time when the discovery of this enzyme was being utilized in studies designed to obtain an understanding of the mechanism of cell transformation and as a tool to explore the possible link between viruses and human cancer. In this article, the outcome of these studies, which indicated a common antipolymerase activity of rifamycin compounds, will be presented. An updated outline of the studies of rifampicin with the bacterial RNA polymerase, the subject of previous review articles (Wehrli and Staehelin, 1970; Riva and Silvestri, 1972), will be presented for parallel comparisons with the studies on the interaction of rifamycins with the viral reverse transcriptase and with eucaryotic RNA polymerases.

Antiviral activity of 5-thiocyanatopyrimidine nucleosides

The antiviral activity of the 5-thiocyanatopyrimidine nucleosides 5-NCSrU ∗ ∗ Abbreviations: 5-NCSrU, 5-thiocyanatouridine; 5-NCSdU, 5-thiocyanato-2'-deoxyuridine: 5-NCSaraU, 5-thiocyanato uracil arabinoside or 1-β- d-arabinofuranosyl-5-thiocyanatouracil; tri-O'-acetyl-5-NCSrU, 2',3',5'-tri- O-acetyl-5-thiocyanatouridine; ara-C, cytosine arabinoside, 1-β- d-arabinofuranosylcytosine; 5-ethyl-dU, 5-ethyl-2'-deoxyuridine; dT, 2'-deoxythymidine; 5-IdU, 5-iodo-2'-deoxyuridine; CCID 50, cell culture infecting dose 50 (dose infecting 50% of the cell cultures); PFU, plaque forming units. , 5-NCSdU, 5-NCSaraU and tri- O′-acetyl-5-NCSrU has been evaluated in primary rabbit kidney (PRK) cell cultures challenged with either DNA (vaccinia, herpes simplex) or RNA (vesicular stomatitis) viruses. 5-NCSdU inhibited vaccinia virus multiplication at 10 μg/ml, and vaccinia and herpes simplex virus induced cytopathogenicity at 4 μg/ml. Tri- O′-acetyl-5-NCSrU inhibited vesicular stomatitis virus-induced cytopathogenicity at 1–10 μg/ml. None of the compounds had profound effects on host cell RNA or DNA synthesis, even at 200 μg/ml, as monitored by [ 3H]uridine and [ 3H]thymidine incorporation respectively, except 5-NCSdU, which brought about a 10–30-fold increase of [ 3H]thymidine incorporation at 200 μg/ml. The inhibitory effect of 5-NCSdU on vaccinia virus replication and its stimulatory effect on [ 3H]thymidine incorporation were almost completely reversed by thymidine at concentrations 100 times lower than that of the thiocyanato derivative. When treated with dithiothreitol, the 5-thiocyanatopyrimidine nucleosides also lost a significant part of their biological activity, presumably due to reduction to the corresponding 5-mercapto analogs.

Antiviral activity of 5-ethyl pyrimidine deoxynucleosides

The antiviral activity of the α- and β-anomers of 5-ethyl-2′-deoxyuridine (EtUdR) and 5-ethyl-2′-deoxycytidine (EtCdR) has been assessed in primary rabbit kidney (PRK) cell cultures. Cytosine arabinoside (ara-C) and 5-iodo-2′-deoxyuridine (IUdR) were included as reference materials. When inhibition of vaccinia virus multiplication was measured the following order of (decreasing) activity was established: ara-C > IUdR > (β-)EtUdR > (β-)EtCdR. The α-anomers of EtUdR and EtCdR were completely inactive. In marked contrast with ara-C and IUdR which were found to inhibit PRK cell growth and thymidine incorporation into cell DNA at relatively low doses (0.08–0.4 μg/ml and 8–40 μg/ml respectively), EtUdR did not inhibit cell growth or thymidine incorporation into DNA unless very high doses were used (200–500 μg/ml). At 0.4–200 μg/ml EtUdR had a stimulatory effect on the subsequent incorporation of thymidine and deoxycytidine into DNA, most probably due to a starvation of the cells for these precursors during their contact period with EtUdR. EtCdR did not markedly alter thymidine incorporation into DNA. Antiviral indices were calculated for EtUdR, IUdR and ara-C. These were defined as the ratios of the minimum toxic doses (inhibiting cell growth or thymidine incorporation by 50 per cent) to the minimum effective doses (inhibiting vaccinia virus induced cytopathogenicity by 50 per cent). The antiviral indices of EtUdR and IUdR were almost identical but considerably greater than that of ara-C.

Der Einfluß von 4-Hydroxy-2.3-pentenal (HPE) auf die Vermehrung von Vaccinia-Virus in Hühnerfibroblastenkulturen / Influence of 4-Hydroxy-2,3-pentenale (HPE) on the Multiplication of Vaccinia-virus in Chick-embryo Fibroblast Cultures

Abstract The influence of 4-hydroxy-2,3-/rans-pentenale (HPE) on multiplication of Vaccinia virus in chick fibroblast cultures has been investigated. The cytotoxicity of HPE has been estimated by in ­ corporation of [3H] thymidine, [3H] uridine, [3H] leucine and by morphological tests. The greatest influence of H PE was seen after incorporation of [3H] thymidine. Chick-embryo cultures infected with Vaccinia virus showed a marked increase of DNA synthesis in contrast to the controls. This increase averaged 180% after 16 to 14 hours. HPE 0.05-10_ 3 M caused a reduction of 72% in [3H] thymidine incorporation in virus infected cultures. At the same time virus concentration decreased by 76%. HPE 0.1-10~3 M, a concentration which inhibits the normal cell DNA synthesis by 50%, caused about 90% inhibition in infected cultures; virus m ulti­ plication was 99% inhibited. These findings have been attributed to a selective inhibition of virus DNA synthesis by HPE. and possible mechanisms are discussed.

Synthesis of vaccinia virus polypeptides in the presence of isatin-beta-thiosemicarbazone.

Isatin-beta-thiosemicarbazone (IBT) at a concentration of 14 muM 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.

Inhibition of release of vaccinia virus by N1-isonicotinoly-N2-3-methyl-4-chlorobenzoylhydrazine.

N(1)-isonicotinoyl-N(2)-3-methyl-4-chlorobenzoylhydrazine (IMCBH) is a selective inhibitor of vaccinia virus multiplication. In concentrations up to 50 microg/ml, IMCBH causes neither toxic morphologic changes, nor does it inhibit the multiplication of cells. Viruses other than vaccinia are not affected by IMCBH. The virus-inhibitory effect of IMCBH is dependent on the type of host cell used, i.e., the compound is effective in chick embryo fibroblasts and monkey kidney cells but not in L cells. IMCBH does not exhibit any protecting effect on vaccinia virus-infected mice or rabbits. IMCBH interferes with virus release: in single cycle experiments in chick embryo fibroblasts, IMCBH strongly blocks the release of vaccinia virus at concentrations as low as 3 microg/ml, while intracellular virus synthesis is hardly affected. Viral cytopathic changes are completely suppressed by IMCBH within the span of a single cycle infection, although extensive changes eventually occur. By inhibiting virus release from initially infected cells, IMCBH markedly inhibits the multiplication of vaccinia virus in cell cultures infected at low virus/ cell multiplicities. IMCBH does not inhibit the early toxic cytopathic changes induced by large inocula of vaccinia virus in BHK21 cells.

Hybridization and sedimentation studies on “early” and “late” vaccinia messenger RNA

The vaccinia virus multiplication cycle can be divided into two portions: the early portion, which precedes viral DNA replication and lasts for about one-and-a-half hours; and the late portion which follows until maturation of progeny is complete. Vaccinia messenger RNA transcribed during these two periods, that is, early and late messenger RNA, has been studied by means of hybridization and density gradient sedimentation. The following results were obtained: 1. (1) Early vaccinia messenger RNA is distinctly smaller than late messenger RNA (10 to 12 s against 16 to 20 s). Late messenger RNA contains nucleotide sequences not present in early messenger RNA; however, all the sequences transcribed early are also transcribed late. If late messenger RNA is transcribed from the entire viral genome, one-half to two-thirds is transcribed early. 2. (2) Messenger RNA transcribed in the presence of cytosine arabinoside, which completely inhibits viral DNA replication, appears to be identical to early messenger RNA. The cytosine arabinoside RNA transcribed in HeLa and L cells contains the same base sequences. 3. (3) The pattern of transcription of early and late vaccinia messenger RNA in HeLa and L cells is quite different. In HeLa cells the total amount of late messenger RNA synthesized greatly exceeds the amount of early; in L cells the position is reversed. 4. (4) The effect of varying the multiplicity of infection on the pattern of early and late messenger RNA transcription has been studied. The rate of early mRNA synthesis is proportional to the multiplicity over a certain limited range. Once the maximum rate of mRNA transcription has been reached, which occurs the earlier the higher the multiplicity, the rate of transcription of messenger RNA falls to a steady low level equal to no more than 5% of the maximum value; this rate of transcription then persists until at least 19 hours after infection. 5. (5) The large messenger RNA molecules transcribed late contain nucleotide sequences also present in small early messenger RNA molecules. Some small messenger RNA molecules are also transcribed late: these contain at least some sequences characteristic of late messenger RNA. 6. (6) At five hours after infection the messenger RNA molecules present in polyribosomes contain all the sequences characteristic of early messenger RNA molecules. By eight hours after infection messenger RNA in polyribosomes is very significantly depleted with respect to sequences characteristic of early messenger RNA. 7. (7) The stability of early and late vaccinia messenger RNA in the presence of actinomycin D has been studied in HeLa and L cells. Early vaccinia messenger RNA is very stable in HeLa cells. Those late messenger RNA molecules which contain sequences characteristic of late messenger RNA are mostly unstable (half-life less than one hour); but those containing sequences characteristic of early messenger RNA are as stable as early messenger RNA itself. In L cells early and late vaccinia messenger RNA are equally stable (half-life two to three hours). mRNA is considerably more stable in extracts of HeLa cells than of L cells; and early mRNA is somewhat more stable in extracts of HeLa cells than is late mRNA. These results are discussed in relation to the pattern of macromolecular biosynthesis during the vaccinia virus multiplication cycle.

Dactinomycin: relative resistance of green monkey kidney cell cultures to its action.

SummaryThe action of dactinomycin on cellular RNA synthesis and vaccinia virus multiplication in HeLa, mouse embryo (ME), African green monkey kidney (GMK) cells in culture were compared. Approxima...

Effect of "cephaloridine" on vaccinia virus in vitro.

EXPERIMENTS in our laboratory have indicated that a new cephalosporin preparation, ‘Cephaloridine’ (CR), is capable of arresting the development of a trachoma agent at an early stage, when used in concentrations as low as 0.1 µg/ml, of culture medium. In the present communication, some findings on the effect of this antibiotic on the multiplication of vaccinia virus in cell culture will be described.

The inhibition of vaccinia virus multiplication by isatin-beta-thiosemicarbazone.

The inhibition of vaccinia virus multiplication by isatin-beta-thiosemicarbazone.

The cycle of multiplication of vaccinia virus in Earle's strain L cells II. Initiation of DNA synthesis and morphogenesis

Electron microscopic and autoradiographic methods were used to study the effects of 4 inhibitors of nucleic acid metabolism upon the synthesis of viral DNA and development of vaccinia particles. Multiplication of the virus was inhibited either by a pretreatment of the inoculum with ultraviolet light (UV) or, following its uptake and penetration, of virus-cell complexes with actinomycin D, fluorodeoxyuridine (FU-dR), or mitomycin C. Virus pretreated with UV penetrates normally, and cores are released into the cytoplasmic matrix. However, only a few cores break down and the majority remain intact, cytoplasmic DNA synthesis is absent, and foci of virus development fail to form. In cells treated with 1 μg of actinomycin per milliliter all steps in the penetration and release of the genome appear to be normal and viroplasmic matrices or factories are formed. Although in such cells viral DNA synthesis continues at about one-third the normal rate, virus morphogenesis is completely arrested. After FUdR treatment cytoplasmic factories are formed consisting presumably of protein since DNA synthesis is stopped. In these factories immature forms of vaccinia occur, generally devoid of nucleoids, low numbers of mature virus are observed corresponding to a low yield of infectious progeny, probably derived from parental genomes reincorporated into new coats. With mitomycin a proportional reduction of 90% in DNA synthesis and virus formation is obtained. Cells pretreated, but infected in the absence of the drug, continue to synthesize DNA in the cytoplasm but disintegrate before progeny virus is assembled. The significance of these findings in respect to initiation of genetic functions and virus development are discussed.