A soluble transcription system derived from purified vaccinia virions.

A soluble extract from purified vaccinia virus particles has been developed which displays site-specific initiation of transcription on exogenous DNA templates that carry cloned vaccinia virus early gene sequences. Bacterial plasmid vectors with segments of a strongly expressed early region of the vaccinia virus genome were active templates, whether in supercoiled or linear, truncated forms. Correct initiation, corresponding to that found in vivo, was observed for all early genes tested. The involvement of other factors besides the viral RNA polymerase was demonstrated by the loss of specific initiation upon partial purification of the enzyme. Initiation activity was restored by reconstitution of the system with factors lacking polymerase activity. The soluble system retained properties of transcription characteristic of intact viral cores, including (i) similar relative rates of initiation of various genes, (ii) multiple requirement for ATP, (iii) methylation and polyadenylation of transcripts, and (iv) inhibition by a topoisomerase antagonist.

Transcriptional and translational analysis of a strongly expressed early region of the vaccinia virus genome.

A transcriptional and translational map was obtained for a 7-kilobase pair EcoRI fragment of vaccinia virus DNA containing 4% of the viral genome. This particular region contains a cluster of early genes which are transcribed in viral cores in vitro and in infected cells before uncoating of the viral DNA. Expression of this region was characterized by using vaccinia virus DNA sequences cloned in phage and plasmid vectors. Polypeptides encoded on the 7-kilobase pair fragment were identified by cell-free translation of viral mRNA selected by hybridization to immobilized DNA fragments. Early RNA programmed the synthesis of six proteins having apparent molecular weights of 63,000, 38,000, 37,000, 25,000, 15,000, and 13,000. The same result was obtained with RNA synthesized in vitro. A new species of 40 kilodaltons was synthesized in response to late RNA. Of the six "early" polypeptides, only the 13-kilodalton component was synthesized from late mRNA. RNA derived from the 7-kilobase pair region was analyzed by a variety of methods including hybridization blot, in vitro recapping, and S1 nuclease techniques. A surprisingly complex pattern of early transcription was found, indicating the existence of families of overlapping RNA species which share common 5'-proximal sequences. In addition, larger RNAs were identified spanning two or more adjacent genes. These RNAs appear to possess a common 5' terminus with transcripts derived from the first gene and are coterminal at the 3' end with RNAs from the downstream gene. Late RNA encoding the 40-kilodalton protein was shown to be heterogeneous in size. A single 5' terminus but no unique 3' terminus was identified for this class of transcripts. RNA species synthesized by cores in vitro were of similar size to authentic transcripts isolated from infected cells at early times.

Cellular differences in the molecular mechanisms of vaccinia virus host range restriction.

The biochemistry of vaccinia virus replication in two permissive (BSC-40, L-929), and two non-permissive (CHO, MDBK) cell lines has been compared. While CHO and MDBK cells differentially allowed expression of the various stages in the vaccinia developmental programme, neither cell supported production of any infectious progeny virions.

Identification of a virus-specified protein in the nucleus of vaccinia virus-infected cells.

A new protein has been detected in the nuclei of vaccinia virus-infected cells. This protein has an apparent mol. wt. of 28000 (VP28) on SDS--polyacrylamide gels and has been detected in Triton X-100-treated nuclei of infected BSC-40, L-929 and CVC cells. Within the infected cells, VP 28 was synthesized maximally at 1 to 2 h p.i. in the cytoplasm and accumulated in the nuclei at 4 to 5 h p.i. The appearance of VP28 was not affected by cytosine arabinoside (25 microgram/ml), an inhibitor of virus DNA synthesis, or rifampicin (100 microgram/ml), an inhibitor of vaccinia assembly, but was inhibited by irradiation of the infecting virions; thus classifying it as an early vaccinia virus gene product. Nuclear--cytoplasmic mixing experiments suggested that the nuclear location of VP28 was not an artefact of the cell fractionation techniques employed. VP28 did not appear to be phosphorylated.

Purification and characterization of regenerating mouse L929 karyoplasts.

Within 72-96 hr after preparation, about 10% of the karyoplasts made from mouse L929 cells regenerated to reform whole viable cells. As soon as 30 hr after preparation, however, nearly all of the remaining 90% of karyoplasts were dead. By separating living and dead karyoplasts at 30 hr, therefore, that fraction destined to complete regeneration was effectively purified. Complete separation was accomplished by sedimentation through Ficoll-paque (Pharmacia), a patented preparation originally developed for the separation of monocytes from whole blood. With the addition of this technique to the previously reported purification scheme for karyoplasts, various biochemical and morphological studies were attempted. Of particular importance are results indicating that karyoplasts that regenerate do not initially contain any more cytoplasm than the average karyoplasts in a preparation--that is, about 10% of the cytoplasm within a whole cell. Electron microscopy of karyoplasts immediately after preparation indicated an unequal partitioning of cytoplasmic organelles at the time of enucleation. For example, karyoplasts initially contain about 11.4% of the mitochondrial volume of whole cells, but only 2.9% of the Golgi apparatus. The size of the karyoplasts and the volume occupied by a variety of organelles was followed throughout the process of regeneration. Although there was an approximately linear increase in the diameter of regenerating karyoplasts, there appeared not to be a simple concordant increase in the volume occupied by all cellular organelles. An extensive investigation was performed to determine whether or not karyoplasts contained centrioles. Immediately after enucleation, 15,000 random thin sections through karyoplasts, which represented about 100 complete bodies, were examined for the presence or absence of centrioles. No centrioles were observed. Examination of the cytoplasts revealed that they contained a sufficient number of centrioles to account for all of the centrioles that were present in the whole cells before enucleation. Centrioles were first detected in karyoplasts in 24 hr after preparation, about the same time that karyoplasts regained the ability to adhere to the surface of tissue culture dishes. At this time, however, the average karyoplast had less than one centriole. By 72 hr, the regenerated karyoplasts had approximately the same number of centrioles as whole cells.

Vaccinia virus replication requires active participation of the host cell transcriptional apparatus.

The ability of vaccinia virus to replicate in BSC-40 monkey cells whose nuclei have been functionally inactivated was examined. Exposure of cell monolayers to ultraviolet radiation at doses that did not alter the cells' capacity to support a subsequent infection by a cytoplasmic virus (vesicular stomatitis virus) caused a reduction to less than 10% in the observed yield of infectious progeny from vaccinia virus and herpes simplex virus (type 1) infections. Similarly, replication of vaccinia virus was reduced to 5% by treatment of BCS-40 cells with alpha-amanitin (10 microgram/ml), a potent inhibitor of nuclear mRNA synthesis. In both situations, ultraviolet irradiation and alpha-amanitin treatment, early and late vaccinia viral genes were expressed at high levels, but the newly synthesized virion components were not assembled into mature infectious particles. Taken together, these data suggest that the active involvement of the host cell nuclear transcriptive system is obligatory in the vaccinia virus replicative cycle.

Separation of cytoplasts and whole cells using density gradients of renografin.

Cytoplasts prepared from L929 or Hepa-2 cells were separated from whole cells using density gradients of renografin. Using this technique, cytoplasts can be isolated from cell lines which cannot be routinely enucleated with an efficiency of 100%. The purified cytoplasts excluded the vital dye trypan blue and were utilized in nuclear transplantation experiments to reconstruct whole viable cells capable of division. In addition, the renografin gradient technique was used to separate the newly reconstructed cells from any contaminating "non-renucleated" cytoplasts. This will permit immediate biochemical characterization of cytoplasmic-nuclear hybrid cells without interference from contaminating cytoplasts.

Vaccinia virus replication. I. Requirement for the host-cell nucleus.

Using cytochalasin B-induced enucleation techniques, we examined the ability of vaccinia virus to replicate in the absence of the host-cell nucleus in several mammalian cell lines. It was found that virus-infected enucleated cells (cytoplasts) prepared from BSC-40, CVC, and L cells were incapable of producing infectious progeny virus. The nature of this apparent nuclear involvement was studied in detail in BSC-40 cells. Modulations designed to maximize cytoplast integrity and longevity, such as reduction of the growth temperature and initial multiplicity of infection, did not improve virus growth in cytoplasts. Sodium dodecyl sulfate-polyacrylamide gel analysis of the [(35)S]methionine pulse-labeled proteins synthesized in vaccinia virus-infected cytoplasts demonstrated that both early and late viral gene products were being expressed at high levels and with the proper temporal sequence. Vaccinia virus cytoplasmic DNA synthesis, as measured by [(3)H]thymidine incorporation, peaked at 3 h postinfection and was 70 to 90% of control levels in cytoplasts. However, in the cytoplasts this DNA was not converted to a DNase-resistant form late in infection, which was consistent with the failure to isolate physical particles from infected cytoplasts. Treatment of vaccinia virus-infected cells with 100 mug of rifampin/ml from 0 to 8 h to increase the pools of viral precursors, followed by subsequent removal of the drug, resulted in a threefold increase virus yield. This treatment had no effect on virus-infected cytoplasts. Finally, vaccinia virus morphogenesis was studied under an electron microscope in thin sections of virus-infected cells and cytoplasts which had been prepared at various times during a single-step growth cycle. It was apparent that, although early virus morphogenetic forms appeared, there was no subsequent DNA condensation or particle maturation in the cytoplasts. These results suggest that vaccinia virus requires some factor or function from the host-cell nucleus in order to mature properly and produce infectious progeny virus.

Recent developments with karyoplast regeneration and nuclear transplantation.

Karyoplasts and cytoplasts prepared by cytochalasin-induced enucleation techniques were used in the development of systems for large-scale nuclear transplantation and the regeneration of karyoplasts to reform whole viable cells. Ten to 20% of a preparation of mouse L929 karyoplasts remained viable. These bodies, purified from whole cells, cytoplasts, and nonviable karyoplats, contained less than 10% of the cytoplasm in whole cells. By the technique of mass nuclear transplantation, up to 40% of a culture of cytoplasts could be renucleated by fusion of karyoplasts to a monolayer of enucleated cells. More than 5 x 10(5) renucleated or true cytoplasmic-nuclear hybrid cells could be prepared from a single cytoplast monolayer culture.

The construction of viable nuclear-cytoplasmic hybrid cells by nuclear transplantation.

Using the mouse L-cell line as a model system, a generalized approach is presented for nuclear transplantation in cultured cells resulting in the construction of cytoplasmic-nuclear hybrid cells. Techniques were developed for the preparation of cytoplast and karyoplasts having minimum contamination by parent whole cells. Sendai virusmediated fusion was performed in a manner which maximized the formation of the desired fusion products-cells having one cell equivalent of cytoplasm from one parent and a nucleus from a second parent. The viability of the fusion products was established by examination of photographic records of the developing cultures. Using these techniques, we found that nuclei could be introduced routinely into 10-30% of a cytoplast culture. From determinations of the increase in cell number with time, it was estimated that at least 30% of the reconstructed cells were capable of division. The approach was next applied to the formation of hybrid cells from L-cell cytoplasts and A9 cell karyoplasts. The A9 cell line is an azaguanine-resistant derivative of L cells. Thus any whole cells remaining in the culture of fused cells were readily eliminated by treatment with the purine analogue. The culture of remaining cytoplasmic-nuclear hybrid cells grew to confluence in the presence of azaguanine. The applicability of the approach to the construction of hybrid cells using parent lines from different organisms is briefly discussed.

The regeneration and division of mouse L-cell karyoplasts.

A technique for efficient cytochalasin-induced enucleation was used to prepare "karyoplasts"--nuclei surrounded by a thin shell of cytoplasm and an outer cell membrane. Methods for estimating the quantity of cytoplasm remaining in karyoplasts indicated that they contained less than 10% of the amount found in whole cells. Procedures for separating karyoplasts from contaminating cytoplasmic fragments and whole cells are also described. Freshly prepared L-cell karyoplasts were unable to adhere to and spread upon a surface. However, after incubation for several days, about 30% of the karyoplasts regained these abilities to some degree. A portion of the regenerating karyoplasts were then observed to divide. These events were confirmed and recorded by time-lapse cinematography. In addition, by culturing karyoplasts under appropriate conditions, clones were isolated.

Poly(A) polymerase from vaccinia virus-infected cells. I. Partial purification and characterization.

Poly(A) polymerase activity is induced during vaccinia virus infection of HeLa cells. The enzyme is maximally induced at 3.5 h postinfection. Partial purification frees the preparation of RNase activity and RNA polymerase activity. ATP is the substrate for poly(A) synthesis. A small amount of poly(A) is produced from added adenosine diphosphate due to the production of ATP by an adenylate kinase present in the preparation. The incorporation of ATP into poly(A) is dependent on divalent cations (Mg(2+) or Mn(2+)) and is not inhibited by UTP, CTP, or GTP. Poly(U) stimulates ATP incorporation; poly(A) and poly(C) have little effect on ATP incorporation, and poly(dT) is extremely inhibitory. RNA prepared from HeLa cells and from the partially purified poly(A) polymerase (the enzyme preparation contains endogenous RNA [Brakel and Kates]) stimulates ATP incorporation by poly(A) polymerase which was subjected to DEAE-cellulose chromatography. RNase's, pancreatic and T(1), inhibit the production of poly(A). DNase has little effect. Poly(U) is able to stimulate poly(A) production in the presence of T(1) RNase.

Poly(A) polymerase from vaccinia virus-infected cells. II. Product and primer characterization.

The product of the in vitro reaction of a vaccinia virus-induced poly(A) polymerase (see preceding paper) with ATP is shown to be poly(A) by nuclease resistance and by annealing with poly(U). Polyacrylamide gel electrophoresis indicates that the in vitro synthesized poly(A) is associated with large RNA which is sensitive to RNase. RNA which co-purifies with the virus-induced enzyme is similar to vaccinia virus-specific RNA with respect to size and poly(A) content. Double labeling studies indicate that the RNA which co-purifies with the enzyme becomes associated with the poly(A) synthesized in vitro. The poly(A) formed in vitro is located on the 3'-OH terminus of this RNA. During in vitro poly(A) synthesis (32)P from alpha-[(32)P]ATP is transferred to nucleosides other than 2',3'-AMP, primarily to CMP. Inclusion of poly(U) in the in vitro reactions results in an increase in the transfer of (32)P to UMP.

Establishment and maintenance of the interferon-induced antiviral state: studies in enucleated cells.

Requirements for the physical presence of the cell's nucleus for the establishment and maintenance of the interferon-induced antiviral state were investigated. Enucleated chicken embryo fibroblasts were obtained by cytochalasin B treatment during centrifugation. The inhibition of vaccinia virus cytoplasmic DNA synthesis, monitored by autoradiography, was used to measure the antiviral activity resulting from interferon treatment. The antiviral state is not established in cells treated with interferon after removal of their nuclei. On the other hand, cells first treated with interferon for 6 or 12 h and then enucleated express the antiviral state. Furthermore, the antiviral state is maintained in enucleated cells for 16 h after enucleation. The antiviral state appears to be more stable in enucleates than in the residual nucleated cells found in the same cultures. Single cells of antiviral populations are found to be either fully permissive or fully restrictive to vaccinia DNA synthesis. The effect of an increasing intracellular multiplicity of infectious virus is to overcome the antiviral cell's block against viral DNA synthesis.

Mechanism of inhibition of vaccinia virus replication in adenovirus-infected HeLa cells.

The ability of vaccinia virus to replicate in HeLa cells which had been previously infected with adenovirus type 2 (Ad2) was studied in order to gain insight into the mechanism by which adenovirus inhibits the expression of host cell functions. Vaccinia virus was employed in these studies because it replicates in the cytoplasm, whereas Ad2 replicates in the nucleus of the cell. It was found that vaccinia deoxyribonucleic acid (DNA) synthesis is greatly inhibited in adeno-preinfected HeLa cells provided that vaccinia superinfection does not occur before 18 hr after adeno infection. The inhibition of vaccinia DNA synthesis can be traced to an inhibition of vaccinia protein synthesis and viral uncoating. Vaccinia ribonucleic acid (RNA) synthesis is not inhibited in adeno-preinfected cells, but the vaccinia RNA does not become associated with polysomes.

Mechanism of synthesis of vaccinia virus double-stranded ribonucleic acid in vivo and in vitro.

The synthesis of vaccinia virus double-stranded ribonucleic acid (RNA) in infected HeLa cells was sensitive to actinomycin D, suggesting that a deoxyribonucleic acid dependent reaction is involved. Some double-stranded RNA was made in the presence of cytosine arabinoside in infected cells. Double-stranded and complementary RNA were synthesized in vitro by using vaccinia cores. These two observations indicate that some of the double-stranded RNA is read from "early" genes. The double-stranded RNA synthesized in vitro had the same properties as that made in vivo. At least 70% of the double-stranded RNA made in vivo was in ribonuclease-resistant form prior to sodium dodecyl sulfate-phenol extraction. In addition, there was a complementary RNA in infected cells which could be converted to double-stranded RNA by annealing.