Transformation by Polyoma Virus affects Adhesion of Fibroblasts

WHEN freshly trypsinized hamster fibroblasts (BHK 21, Clone 13¹) are shaken in suspension, intercellular adhesions are rapidly formed and there is some support for the view that this process is the expression of an adhesive property of the cells in culture which has survived the procedures used to separate them and is not simply an artefact of the use of trypsin². Transformation of animal cells in culture by tumour viruses alters the cell surfaces, as various techniques have shown^3–9 and we have investigated whether intercellular adhesiveness, as defined by the particular assay we have been using, is also affected.     

Effect of the Inhibition of Protein Synthesis on the Establishment of Transformation by Polyoma Virus

Puromycin was used to study the effect of the inhibition of protein synthesis on transformation of hamster cells (BHK21) by polyoma virus. The drug was used at a concentration (10(-4)m) which caused in these cells a drastic but fully reversible inhibition of protein synthesis. A two- to threefold enhancement of transformation rate was obtained when the cells were exposed to puromycin for a period of 5 hr that started at the end of the virus adsorption period. No further enhancement was produced by prolonging puromycin treatment up to 13 hr after infection. The possibility that the observed effect on transformation rate could be mainly attributed to cell selection by puromycin was excluded. In addition, the relevance of a number of possible secondary effects of puromycin (inhibition of cell division, inhibition of deoxyribonucleic acid synthesis, etc.) was also ruled out. The effect of puromycin on transformation appeared to be dependent on the time (relative to infection) of addition of the drug. In fact, no transformation enhancement was observed when the cells were exposed to puromycin prior to infection or beyond the 10th hr after infection. Since another drug known to affect protein synthesis (p-fluorophenylalanine) was also shown to produce similar effects, it is suggested that transformation enhancement results from the inhibition of protein synthesis during a sensitive period closely following adsorption of the virus.     

Effect of Phleomycin on Polyoma Virus Synthesis in Mouse Embryo Cells

The addition of phleomycin (25 mug) to primary mouse embryo cells infected with polyoma virus was found to cause 96% inhibition of the synthesis of infectious virus. When ribonucleic acid and protein synthesis was investigated in these cells by use of isotope incorporation, it was found that neither was inhibited drastically. Immunofluorescent staining studies with the use of antibody directed to the viral structural proteins showed that proteins were synthesized in the presence of the antibiotic. However, when deoxyribonucleic acid (DNA) synthesis was investigated, it was found that DNA synthesis in uninfected cells was completely inhibited within the initial 10 hr of phleomycin addition, whereas DNA synthesis in infected cells proceeded at a reduced rate. Selective DNA extraction (Hirt method) of phleomycin-treated infected cells demonstrated that synthesized viral DNA was salt-extractable, similar to that in infected control cells lacking phleomycin. This extracted DNA was further fractionated by ethidium bromide-cesium chloride density gradient equilibrium centrifugation. The phleomycin-treated preparations revealed twice as much component II (circular nicked and linear) as component I (supercoiled) DNA, whereas the DNA from normally infected control cells showed the reverse picture. It was also demonstrated that viral particles synthesized in the presence of phleomycin did not contain component I DNA. This packaged DNA was found to consist of fragments of both the host and viral types. Cells that were prelabeled with (3)H-thymidine and then treated with phleomycin demonstrated host DNA degradation. However, fragments formed from prelabeled host DNA were not encapsidated into viral particles.     

Effect of β-Propiolactone Inactivation of Polyoma Virus on Viral Functions

Polyoma virus was inactivated by treatment with beta-propiolactone. T-antigen production, polyoma-RNA synthesis, induction of host DNA synthesis (measured by incorporation of labeled thymidine into the cell culture), and in vitro transforming ability were inactivated to a similar degree by various beta-propiolactone concentrations (0.25% beta-propiolactone reduced these functions approximately 96%), whereas plaque-forming ability and the ability of the virus to replicate its DNA and to synthesize capsid antigen were inactivated by a given concentration of beta-propiolactone to a much greater degree (0.25% beta-propiolactone led to a reduction of plaque-forming ability of over 8 logs). The significance of these data and their relationship to previously published experiments are discussed.     

多瘤病毒癌基因产物对PDGF刺激应答的影响

多瘤病毒癌基因产物对ＰＤＧＦ刺激应答的影响于爱鸣冈野幸雄＊于秉治（中国医科大学基础医学院生化教研室,沈阳１１０００１）＊（日本岐阜大学医学部生化学教室,日本岐阜５００）多瘤病毒癌基因主要表达三种转化蛋白．其中,中等分子肿瘤抗原（ｍｉｄｄｌｅｓｉｚｅｄ...     

Structural Roles of Polyoma Virus Proteins

The superhelical, closed circular form of polyoma deoxyribonucleic acid (DNA) (Co 1) is bound in a 25S DNA-protein complex to the viral histone-like proteins after alkaline disruption of the virion. Nicked viral DNA or linear DNA are largely free of protein. Most of the viral protein disruption is in the form of capsomeres, sedimenting principally at 10S and 7S. Despite the relatively constant ratio of 10S to 7S material in many preparations, (1:5.5 to 1:6.0, respectively), the two classes of capsomeres are indistinguishable by electron microscopy and contain only P(2), P(3), and P(4) in molar ratios of approximately 5:1:1 or 6:1:1, respectively. Material with sedimentation rates of approximately 1 to 3S is enriched for P(5) and contains small amounts of P(2), P(3), and P(4). During the in vitro reassembly of DNA-free, shell-like particles from disrupted virus, proteins P(1), P(2), P(3), P(4), and P(7) are reincorporated efficiently, whereas P(5) and P(6) are not. The presence in empty reassembled particles of histone-like protein, expecially P(7), implies that at least this one of the minor protein components of the virion may participate in protein-protein interactions with other components of the capsid.     

Molecular Aspects of Interferon Induction by Viruses

Virus-induced interferon formation depends on the presence within the cell of a viral ribonucleic acid. This RNA may either be double stranded or, in certain cases, single stranded. The double-stranded RNA can be derived from a virus, such as reovirus, which contains this type of RNA, or it may be synthesized within the cell using viral single-stranded RNA as a template. Single-stranded RNA must possess a stable configuration in solution to be active, and certain viral RNA molecules appear to be active for this reason. The presence of this RNA triggers a derepression event, which is probably nuclear, by an unknown mechanism, and this is followed by the production of an interferon messenger RNA and its translation. Little is known of the derepression event or the events that follow it.     

Gene with Quantitative Effect on Circulating Interferon Induced by Newcastle Disease Virus

Circulating interferon production, induced by Newcastle disease virus, is about seven times higher in C(57) Black mice than i Balb/c/Gif mice. A Mendelian analysis was carried out and circulating interferon production was measured in reciprocal F(1) hybrids, in the F(2) generation, in progeny of backcrosses of F(1) hybrids to either parent strain, and in second backcross progeny. The results indicate that a single, partly dominant, autosomal factor is responsible for the difference in circulating interferon production between both parent strains.     

Further Studies on the Effect of 5-Iododeoxyuridine on Polyoma Virus Multiplication in Mouse Cells

5-Iododeoxyuridine (IUDR) inhibited production of infectious polyoma virus in mouse embryo cells and mouse kidney cells in culture. Deoxythymidine reversed its effect. IUDR did not inactivate infectivity of free virus particles. IUDR did not prevent adsorption and penetration of polyoma virus to cells. The events sensitive to IUDR treatment occurred at around 20 hours after infection. The cytopathic effects of polyoma virus, including emergence of DNA containing-inclusions in the nucleus, were observable in infected cells in which viral replication was completely arrested by IUDR. It was shown by fluorescent antibody technique in infected mouse embryo cells and by complement fixation test in infected mouse kidney cells that IUDR inhibited completely the synthesis of viral antigen. No virus-like particles were demonstrated in the IUDR-treated infected-mouse kidney cells by electron microscope examinations.     

Effect of Ultraviolet Irradiation and Actinomycin D on Polyoma Virus Replication in Mouse Embryo Cell Cultures

Ultraviolet irradiation and actinomycin D impair the capacity of mouse embryo (ME) cells to support the replication of polyoma virus, but not of encephalomyocarditis (EMC) virus. The loss in capacity for polyoma virus synthesis was an “all-or-none” effect and followed closely upon the loss in cellular capacity for clone formation. Cells treated with either agent produced polyoma “T” antigen, but did not synthesize polyoma structural protein. Infection of untreated ME cells with polyoma virus produced marked stimulation of both deoxyribonucleic acid (DNA) synthesis and ribonucleic acid (RNA) synthesis. ME cell cultures irradiated with ultraviolet for 30 sec at 60 μw/cm² or treated with actinomycin D at 0.1 μg/ml for 6 hr prior to infection were incapable of synthesizing DNA or RNA, even after infection with polyoma virus. Irradiation of cells during infection produced cessation of synthesis of both RNA and DNA. Addition of actinomycin D during infection did not inhibit DNA synthesis but abolished RNA synthesis and reduced the yield of polyoma virus to 10% of that in untreated infected cultures. Both agents lost the ability to prevent replication of a full yield of polyoma virus when administered 30 hr after infection or later. The period after which neither agent inhibited polyoma replication corresponded with the period at which maximal RNA synthesis in untreated infected cultures had subsided. It can be concluded on the basis of the data presented that the functional integrity of the mouse embryo cell genome is required for the replication of polyoma virus, but not for EMC virus. Whereas the requirement for cellular DNA-dependent RNA synthesis for polyoma virus replication has been demonstrated, the exact nature of the host-cell function remains to be elucidated.     

Transient Inhibition of Polyoma Virus Synthesis by Sendai Virus (Parainfluenza I) II. Mechanism of the Interference by Inactivated Virus

The mechanism of the transient inhibition of polyoma virus synthesis by betapropiolactone-inactivated Sendai virus was studied. Polyoma virus early functions did not appear to be affected, although deoxyribonucleic acid (DNA) and structural protein synthesis were inhibited 60 and 35% respectively. The inhibition of macromolecular synthesis was not sufficient to account for the 90% inhibition of infectious progeny formation. Encapsidation of polyoma DNA into mature virions appears to be completely inhibited after superinfection by beta-propiolactone-inactivated Sendai virus. Ultraviolet irradiation of live or beta-propiolactone-inactivated Sendai virus preparations abolishes the interfering capacity, indicating that a functional Sendai virus ribonucleic acid molecule is the interfering component.     

Origin of the Thymidine Kinase Induced by Polyoma Virus in Productively Infected Cells

Cells of the 3T3 mouse line efficiently supported the multiplication of polyoma virus, and the infectious process was accompanied by a marked increase in thymidine kinase (TK) activity. Two lines of 5-bromodeoxyuridine-resistant 3T3 cells have been isolated. As expected, these cells incorporated practically no exogenous thymidine into their deoxyribonucleic acid (DNA) and contained negligible TK activity. Like the parental 3T3 cells, TK(-) lines were susceptible to productive infection by polyoma virus, but infection did not lead to an increase in TK activity. Since kinase activity did appear after infection with another virus (vaccinia) known to contain the gene(s) for that enzyme, it is concluded that TK is not one of the gene products of polyoma virus. As induction of cellular DNA synthesis by polyoma virus occurs normally when the TK(-) cells are infected in the stationary phase, TK cannot play a role in the determination of this phenomenon.     

Plaque Assay for Polyoma Virus on Primary Mouse Kidney Cell Cultures

A plaque assay for polyoma virus using primary baby mouse kidney cells is reported.     

Polyoma Virus Infection of Retinoic Acid-Induced Differentiated Teratocarcinoma Cells

The mouse teratocarcinoma stem cell line, F9, becomes permissive for productive polyoma infection upon treatment with retinoic acid. Through the use of M13-polyoma recombinant single-stranded DNA probes, spliced and unspliced early viral RNA were detected after polyoma infection of retinoic acid-treated and untreated F9 cultures.     

Rac is Essential in the Transformation of Endothelial Cells by Polyoma Middle T

Expression of the Polyoma Middle T (PyMT) antigen in endothelial cells results in single-step transformation to hemangioma producing malignant cells. To study the mechanism of PyMT transformation, we used the PyMT induced mouse brain endothelial cell line, bEND.3, expressing constitutively active and dominant negative mutants of the small GTPase Rac. The bEND.3 cell phenotype of tumorigenesis, loss of normal growth control and formation of cysts rather than capillary tubes in fibrin gels was reversed by expression of dominant negative Rac. The mechanism of N17 Rac action in blocking the endothelial cell transformant, PyMT, did not involve effects of Rac on the actin cytoskeleton since this component of the bEND.3 cell phenotype was not affected. Furthermore, the PyMT induced activation of the plasminogen activator (PA)/plasmin system was not affected by Rac inhibition. Inhibition of the downstream effectors of Rae, phosphatidylinositol 3-kinase (P13-K) and p70^S6k, which are known to be constitutively activated by PyMT transformation, inhibited bEND. cell proliferation and cyst formation in fibrin gels even in cells expressing V12 constitutively active Rac, but they did not restore capillary tube formation. These results demonstrate that middle T antigen induced endothelial cell transformation requires signal transduction by Rac. The downstream Rac effectors, P13-K and p70^S6k, mediate PyMT/Rac effects on cell proliferation and cyst formation, but other unknown effectors of PyMT are required for the cytoskeletal changes and activation of the PA/plasmin system.     

Marburg Virus Evades Interferon Responses by a Mechanism Distinct from Ebola Virus

Previous studies have demonstrated that Marburg viruses (MARV) and Ebola viruses (EBOV) inhibit interferon (IFN)-α/β signaling but utilize different mechanisms. EBOV inhibits IFN signaling via its VP24 protein which blocks the nuclear accumulation of tyrosine phosphorylated STAT1. In contrast, MARV infection inhibits IFNα/β induced tyrosine phosphorylation of STAT1 and STAT2. MARV infection is now demonstrated to inhibit not only IFNα/β but also IFNγ-induced STAT phosphorylation and to inhibit the IFNα/β and IFNγ-induced tyrosine phosphorylation of upstream Janus (Jak) family kinases. Surprisingly, the MARV matrix protein VP40, not the MARV VP24 protein, has been identified to antagonize Jak and STAT tyrosine phosphorylation, to inhibit IFNα/β or IFNγ-induced gene expression and to inhibit the induction of an antiviral state by IFNα/β. Global loss of STAT and Jak tyrosine phosphorylation in response to both IFNα/β and IFNγ is reminiscent of the phenotype seen in Jak1-null cells. Consistent with this model, MARV infection and MARV VP40 expression also inhibit the Jak1-dependent, IL-6-induced tyrosine phosphorylation of STAT1 and STAT3. Finally, expression of MARV VP40 is able to prevent the tyrosine phosphorylation of Jak1, STAT1, STAT2 or STAT3 which occurs following over-expression of the Jak1 kinase. In contrast, MARV VP40 does not detectably inhibit the tyrosine phosphorylation of STAT2 or Tyk2 when Tyk2 is over-expressed. Mutation of the VP40 late domain, essential for efficient VP40 budding, has no detectable impact on inhibition of IFN signaling. This study shows that MARV inhibits IFN signaling by a mechanism different from that employed by the related EBOV. It identifies a novel function for the MARV VP40 protein and suggests that MARV may globally inhibit Jak1-dependent cytokine signaling.