Transformation by a temperature sensitive mutant of Rous sarcoma virus in the absence of serum.

Cultures of chicken embryo fibroblasts infected with the temperature-sensitive transformation mutant of Rous sarcoma virus, tsLA24PR-A, were arrested between mitosis and S phase by exposure to serum-free medium at the non-permissive temperature (41degree C) for 2 days. On shifting to the permissive temperature (35degree C) the cells assumed a transformed morphology and increased uptake of [2minus 3H]-Deoxy-glucose. There was a concomitant increase in acid insoluble [3H]-thymidine. This suggests that the virus transforming function can cause stationary cells to enter their growth cycle. The level of release of infectious virus was shown to decrease on cell cycle arrest in serum-free medium and not to recover on a shift to 35 degrees, when cellular DNA synthesis and transformation was induced. Cultures rendered stationary in medium containing serum depleted of multiplication stimulating factor did not show this reduction in virus production.     

Characterization of the infection of equine fibroblasts by equine infectious anemia virus

Summary Equine dermal fibroblasts persistently infected with equine infectious anemia virus (EIAV) show no alterations in cell morphology or growth kinetics when compared to uninfected cells. The percentage of cells immunofluorescent positive for viral proteins fluctuated, depending upon the stage of the cell cycle, while production of extracellular virus was uniform throughout the cell cycle, increasing only as the cell number increased. This was shown in log versus stationary phase cultures as well as in cultures synchronized by serum starvation. The establishment of productive infection did not require host cell DNA synthesis. Normal levels of progeny virus were produced in cultures pretreated with mitomycin C and placed in serum-containing medium. Serum-starved cultures, however, did not support EIAV replication as well as other cultures, presumably because synthesis of provirus was inhibited.With 3 Figures     

Cell-cycle perturbation in Sf9 cells infected with Autographa californica nucleopolyhedrovirus.

Flow cytometry analysis of the cell-cycle progression was performed in Sf9 cells infected with Autographa californica nucleopolyhedrovirus (AcNPV) in the cultures partially synchronized by aphidicolin exposure and deprivation. Cells infected with AcNPV during the G1 phase progressed and were arrested in the S phase in the 4 h following the infection, whereas cells infected during the S phase did not progress past the S phase. Cells infected during the G2/M phase remained in the G2/M phase without mitosis during a period of 10 h. Such cell-cycle arrest was also observed in the cells infected with ts8, a temperature-sensitive mutant of AcNPV that is defective in both genomic DNA synthesis and late gene expression. Cells with >4 N DNA content accumulated in the cultures infected with wild-type AcNPV, whereas no such cells appeared in the cultures infected with ts8, suggesting that viral origin of the DNA overaccumulated in the cells with >4 N DNA content. This was confirmed by the slot blot hybridization experiments, which showed that viral DNA, but not cellular DNA, increased strikingly in Sf9 cells during the infection with AcNPV. These results indicate that AcNPV targets at least two different checkpoints to prevent normal cell-cycle progression of Sf9 cells and that neither viral DNA replication nor expression of viral late genes is a necessary prerequisite for such AcNPV-induced cell-cycle arrest. It is suggested that the cell-cycle arrest in AcNPV-infected Sf9 cells is an event triggered early in infection by specific interaction of viral gene products with cellular components that regulate cell-cycle progression.     

Effect of bovine parvovirus replication on DNA,RNA, and protein synthesis in S phase cells

The kinetics of replication of bovine parvovirus (BPV) and the effect of viral replication on cellular macromolecular synthesis were examined in hydroxyurea (HU)-synchronized fetal bovine spleen cells. Immediately after release of cells from HU block, 80–85% of the cells began to synthesize DNA. The production of infectious progeny BPV proceeded at a faster rate in synchronized cells infected at the beginning of S phase than in asynchronous cultures. In synchronized cells, titers of infectious virus increased at 8 hr p.i. and the maximum titer was achieved by 20 hr. BPV DNA synthesis preceded the production of progeny virus by 2 hr. Although the rates of RNA and protein synthesis in infected cells were severely reduced after 8 hr p.i., BPV replication did not affect the rate of progression of cells through S phase.     

Syntheses of virus-induced thymidine kinase and viral DNA in herpes simplex type 1 virus-infected chick embryo fibroblasts.

Herpes simplex virus type 1, strain Kupka, did not replicate in chick embryo fibroblasts (CEF), but the infection was followed by the development of cytopathic changes. This effect could be abolished by UV irradiation of the virus. Virus-induced thymidine kinase was synthesized in the infected cells reaching a maximum level at 24 hours post infection (p.i.). In the presence of cytosine arabinoside, thymidine kinase synthesis was enhanced. This suggested that the late (post-replicative) viral function, which turns off thymidine kinase synthesis, was expressed in the infected CEF untreated with the drug. HSV type 1 laboratory strains Kupka and KOS were capable of inducing the synthesis of virus-specific DNA in CEF. But in CEF infected with fresh type 1 virus isolates replication of viral DNA was not observed.     

Occurrence and role of an early antigen and evidence for transforming ability of porcine circovirus

Summary By means of indirect immunofluorescence assay (IFA) using natural swine immune serum and hyperimmune serum from rabbits infected with porcine circovirus (PCV), a PCV antigen was detected present prior to the onset of viral and cellular DNA synthesis in nucleoli of cells of synchronized and growth stimulated infected PS cell cultures grown for more than 12 h in the presence of hydroxyurea. The number of cells containing specifically fluorescing nucleoli increased with increasing time of growth in the presence of hydroxyurea. The concomitant increase in the number of cells containing virus structural (VS) antigen in the nuclei and the increase in the amount of replicative (RF) DNA and accompanying 5 S DNA after release from the hydroxyurea block suggest that EA is involved in induction of PCV DNA replication. Primary pig kidney cell cultures persistently infected with PCV survived mock-infected control cultures for 16 passages. They had lost contact inhibition and formed cell colonies in soft agar at a ratio of 0.1 to 0.4%. Cell lines derived from agar colonies showed properties of transformed cells e.g. low requirement for serum growth factors, ability to overgrow a continuous cell layer, anchorage independence of growth. In transformed cells stimulated to growth and grown in the presence of hydroxyurea, non-structural viral antigen visible by IFA in nucleoli and VS antigen located in the cytoplasm were expressed. Contrary to virus bound nuclear VS antigen in productive infection, accumulation of cytoplasmatic VS antigen was independent of DNA synthesis and caused cell destruction, thus limiting growth of cell layers and colonies in soft agar.     

Productive influenza virus infection of synchronized chick embryo fibroblast cells.

The effects of cell metabolic activity on the outcome of influenza virus infection were studied in partially synchronized chick embryo fibroblast cultures. There was no evidence to show that the time in the cell cycle at which cells were infected had any significant effect on the final virus yield. However, some differences were detected in the length of the latent period between infections established in synchronized or in stationary cells. Influenza virus could replicate in synchronized or normal cell cultures in which DNA synthesis was inhibited with 9-beta-D-arabinofuranosyladenine (ara-A).     

Evidence for interferon production and its correlation with YF 17DD vaccine virus yields in primary chick embryo cells

Early experiments have resulted in the establishment of an efficient methodology for the production of a yellow fever vaccine in chicken embryo fibroblasts (CEF) using the 17DD virus strain [Freire, M.S., Mann, G.F., Marchevsky, R.S., Yamamura, A.M., Almeida, L.F., Jabor, A.V., Malachias, J.M., Coutinho, E.S., Galler, R., 2005. Production of yellow fever 17DD vaccine virus in primary culture of chicken embryo fibroblasts: yields, thermo and genetic stability, attenuation and immunogenicity. Vaccine 23, 2501-2512]. To investigate the role of the interferon system in vaccine virus yields, CEF cultures seeded at high and low cell densities and infected with the yellow fever 17DD virus were used. The supernatants of these cultures were tested for the presence of interferon by an assay based on the reduction of cytopathic effect of a challenge virus (Sindbis), for the enzymatic activity of the interferon-induced 2',5'-oligoadenylate synthetase and for the expression of 2',5'-oligoadenylate synthetase mRNA. The presence of interferon and its influence in the replication of yellow fever 17DD virus in CEF cultures was clearly demonstrated.     

Kinetics of parvovirus replication,cytopathic effects,and mitotic arrest in synchronized bovine fetal spleen cells

Initiation of autonomous parvovirus replication depends on the S phase of host cells actively traversing the cell cycle. The parvoviruses Lu III and H-1 inhibit synchronized cells from entering mitosis, implying that parvoviruses rapidly shut down cell cycle traverse during G2 phase. Bovine parvovirus (BPV) did not inhibit the entry into mitosis of hydroxyurea synchronized bovine fetal spleen cells. Mitotic indexes of infected cultures were as much as 60-fold higher than those of mock-infected controls. Mitosis in control and infected cultures peaked at 10 hr after infection corresponding to the end of the BPV eclipse period. Cytopathic changes, including morphological alteration of mitotic chromosomes, were detected in mitotic cells from infected cultures by light and electron microscopy. Arrest of BPV-infected cells in mitosis may explain these results. Not all infected cells were killed in mitosis, since some developed intranuclear inclusions and became pycnotic as nucleated, interphase cells. Inclusion formation was coincident with viral morphogenesis in interphase nuclei at 16 to 18 hr postinfection, late in the viral replication cycle. The cell cycle stage at which parvovirus-infected cells are arrested and cytopathic events ensue may be determined by the cellular progression rate from S phase through G2 and M.     

Identification of self-complementary virus-specific ribonucleic acid in chick kidney cells infected with chicken embryo lethal orphan virus.

RNA was extracted from primary chicken embryo kidney (CEK) cells infected with chicken embryo lethal orphan (CELO) virus and exposed to a pulse of (5-3H)-uridine late in infection. When this RNA was self-annealed, 4.5% became resistant to pancreatic ribonuclease digestion. The ribonuclease-resistant RNA was isolated by chromatography on Sephadex G-100, and the RNA was found to have the characteristics of a double-stranded molecule of sedimentation coefficient 8S. Half of the column-isolated RNA hybridized to CELO DNA with equal amounts of virus RNA binding to the heavy or light stands of the CELO DNA, indicating the presence of complementary RNA species late in the infectious cycle of CELO.     

Virus Production and Release, Cell Longevity, and Cloning Efficiency of Chicken Embryo Fibroblasts Infected with Rous Sarcoma Virus

Continuous virus production is a characteristic of chicken embryo fibroblasts (CEF) infected and transformed by a nondefective Schmidt-Ruppin subgroup A Rous sarcoma virus. This virus production has been examined with particular attention to the amount of newly budded virus which remained cell-associated, and to the amount and degree of viral aggregation at the cell surface and in the fluid tissue culture medium. The total biologically active virus associated with a Schmidt-Ruppin subgroup A Rous sarcoma virus-infected CEF culture was divided almost equally between that portion of virus which was present in the fluid medium and that portion which was cell-associated. Various mechanical and enzymatic methods were used to remove cell-bound virus and to disperse aggregates of virus in the tissue culture medium to assess cell production of virus per hour accurately, which was determined as an average of 16.4 focus-forming units per cell per hour. With appropriate culture conditions, it was found that Schmidt-Ruppin subgroup A Rous sarcoma virus-infected and -transformed CEF replicated faster, could be passaged more times, and grew to higher cell densities than did normal CEF and CEF infected with a subgroup A Rous associated virus. Subgroup A Rous sárcoma virus-infected CEF cloned with much lower efficiency than did subgroup A Rous associated virus-infected CEF or normal CEF. Experiments employing a temperature-sensitive mutant of subgroup A Schmidt-Ruppin Rous sarcoma virus- and Rous associated virus-infected CEF indicated that the poor cloning efficiency of Schmidt-Ruppin subgroup A Rous sarcoma virus infected cells was not due to the constant production of virus but was probably related to some property associated with transformation of the cell by Rous sarcoma virus.     

Replication of porcine circovirus: induction by glucosamine and cell cycle dependence

Summary Multiplication of porcine circovirus (PCV) was found to be inducible by treatment of infected cell cultures with 300mm glucosamine. One day after glucosamine treatment and after growth in fresh medium, an increase in the number of cells containing virus antigen of up to 50 times as compared to mock-treated cultures was observed. Analysis of this phenomenon revealed that replication of PCV DNA was induced. Only aminohexoses but not hexoses and acetylated aminohexoses were efficacious. The course of PCV replication in synchronized cell cultures infected at different periods of the cell cycle showed that PCV DNA synthesis depends on cellular enzymes expressed during S phase growth of cells. However, whereas in cell cultures treated with glucosamine after infection in G₀ or during G₁, the start of PCV replication was observed during the first S phase after growth stimulation, the latent period in mock-treated cultures lasted until the second S phase. Also in cell cultures transfected with PCV DNA in G₀ or during G₁ using DEAE-dextran as mediator, PCV replication started during the first S phase after growth release of the cells. From these findings the conclusion is drawn that glucosamine and DEAE-dextran initiate PCV replication by enabling the PCV genome to get entry to the cell nucleus that normally can be achieved only by inclusion in the daughter nuclei at the end of mitosis.     

Studies on polyoma virus DNA replication in synchronized C3H2K cells.

In G1-arrested cells infected between 1 and 12 h after having been stimulated by fresh serum to progress to S phase, polyoma virus DNA synthesis proceeded in the first half of S phase, and virus and whole cellular DNA accumulated at about the same time. However, in cells infected later than 14 h after serum stimulation, virus DNA synthesis was shifted to the next S phase. Thus, a permissive cell attains competence for polyoma virus DNA replication at a precise moment during an S phase initiated by fresh serum, which can efficiently replace the early virus host DNA stimulation function. When cells were incubated in serum that had lost its capacity to stimulate host DNA synthesis by pre-absorption with growing cells, normal yields of polyoma DNA could nevertheless be observed, which shows that extensive replication of host DNA does not seem to be an obligatory condition for virus DNA replication.     

Alterations in nucleic acid metabolism induced by infection with an oncogenic virus

Summary Polyoma virus multiplies inin vitro cultures of mouse embryo fibroblasts, and the replicating cycle is accompanied by an increase in the incorporation of labelled precursors into the nucleic acids of the host cells particularly of DNA. From polyoma-infected mouse cells a virus-specific DNA can be isolated which, after purification by chromatography, retains its infectivity forin vitro cultures and oncogenic properties for suckling rodents. Fractionation on a column of methylated albumin allows separation of such virus type DNA from cellular DNA.Rat embryo cells infected with the oncogenic virus do not replicate the particles or form viral antigen, but undergo a malignant transformation. In this case no evident quantitative change or characteristic qualitative alteration in nucleic acids metabolism of the host cells was observed. Though these data do not offer absolute proof that the alternative pathways induced by polyoma virus, i. e., multiplication of viral particles and malignant transformation, involve two different metabolic pictures in the host cell, they suggest this possibility.     

Cell cycle dependence of synthesis of unintegrated viral DNA in mouse cells newly infected with murine leukemia virus

We have studied Moloney murine leukemia virus (MuLV) replication in newly infected NIH/3T3 cells brought to a stationary phase by serum depletion. Progeny viruses were markedly decreased under these conditions. Studies of the early phase of the virus cycle by the Southern blot hybridization procedure revealed that levels of unintegrated linear double-stranded and supercoiled viral DNAs were decreased in quiescent NIH/3T3 cells as compared to levels detected in serum-replenished cells. When serum was added to quiescent cells up to 48 hr postinfection, we could detect an increase of viral DNA, suggesting the presence of a stable intermediate encoding viral information. In order to characterize this intermediate, stationary cells were labeled with BrdU at the time of serum addition so that substituted viral DNA molecules made under serum stimulation could be separated on CsCl gradients from those made under serum depletion. The analysis of this experiment revealed that upon serum addition, the majority of viral DNA was fully substituted (HH), indicating that it must have been synthesized from an RNA template. Also, an important part of viral DNA made after serum addition had an intermediate density (HL), suggesting that incomplete molecules made in quiescent cells were completed after serum addition. Our results clearly show that host factors are required for synthesis of viral DNA in NIH/3T3 cells newly infected with MuLV.     

Induction of cellular DNA synthesis and increased mitotic activity in syrian hamster embryo cells abortively infected with human cytomegalovirus.

The effect of human cytomegalovirus (CMV) on cell DNA synthesis and mitotic activity in hamster embryo fibroblasts was examined. The results indicated that CMV infected cells had increased rates of cell DNA replication and mitotic activity. Detection of the effect of CMV on these two parameters necessitated arrest of cells prior to infection with low serum concentrations. This lowered the background levels of DNA synthesis and cell division so that the effect of virus infection could be detected. The data indicate that cells arrested prior to infection demonstrate increased susceptibility to virus infection. It was also observed that the effect of CMV on both DNA replication and mitotic activity could be enhanced by irradiation with ultraviolet light of the virus prior to infection.     

Relation of bovine leukosis virus production on cell growth cycle

Summary The cell cycle of fetal lamb kidney (FLK) cultures chronically infected with bovine leukosis virus was synchronized by double thymidine block. The synchronized FLK cells were examined for production of BLV antigen and virion release by cytoplasmic immunofluorescence and syncytia forming assay, respectively. The production of BLV antigens was increased during S and G2 phases and was decreased during M and G1 phases. BLV release was associated with the M phase of FLK cells. Short term lymphocyte cultures from BLV infected cattle were treated with hydroxyurea and mitomycin C. The expression of BLV antigen and DNA synthesis of PHA stimulated lymphocytes was inhibited by both drugs.With 2 Figures     

Influence of cell cycle on the efficiency of transfection with purified human cytomegalovirus DNA

The efficiency of transfection of purified HCMV genome was studied in synchronized human embryo fibroblasts. The data show that HCMV DNA infection performed in S phase is more efficient than in cells infected in G2, M and G1 phases of cell cycle, respectively. These findings stress the importance of the metabolic state of cell cultures in HCMV genome transfection.     

Avian adeno-associated virus (AAAV) and fowl adenoviruses (FAV): Studies of viral interactions in chicken cell cultures

As the growth kinetics of avian adeno-associated virus (AAAV) in chicken cells demonstrate, the three serotypes of fowl adenovirus (FAV), FAV -1, -5 and -8, provide complete helper activity for the production of infectious AAAV. Under one step conditions, the growth cycle of AAAV in primary chicken kidney cell (CKC) cultures is characterised by an eclipse phase of 8 hours and an exponential increase of the virus infectivity by 4 to 5 logs until 24 hours post-adsorption (p.a.). These growth characteristics do not depend on the serotype of FAV used as helper. In chicken embryo fibroblast (CEF) cultures the eclipse phase is prolonged to 12 hours p.a. and the virus infectivity increases only by 2 logs. In addition, the low efficiency of plating of FAV -1 in this cell system does not allow one step growth curves for AAAV. In CKC and CEF cultures coinfected with FAV and AAAV the multiplication of helper FAV is reduced. The degree of growth inhibition depends on the AAV multiplicity used. Sequential infection of CKC cultures with FAV -1 and AAAV modifies the AAAV growth cycle, i.e. there is a time reduction of the eclipse phase and a decrease of the virus yield. Infectious AAAV was determined by an indirect immunofluorescence assay and infectious FAV by a plaque assay.