SV40-infected Cultures Research Articles

Negative regulation of mitotic promoting factor by the checkpoint kinase chk1 in simian virus 40 lytic infection.

Lytic infection of African green monkey kidney (CV-1) cells by simian virus 40 (SV40) is characterized by stimulation of DNA synthesis leading to bypass of mitosis and replication of cellular and viral DNA beyond a 4C DNA content. To define mechanisms underlying the absence of mitosis, the expression levels of upstream regulatory molecules of mitosis-promoting factor (MPF) were compared in parallel synchronized cultures of SV40-infected and uninfected CV-1 cells. The DNA replication/damage checkpoint kinase Chk1 was phosphorylated in both uninfected and SV40-infected cultures arrested at G(1)/S by mimosine, consistent with checkpoint activation. Following release of uninfected cultures from G(1)/S, Chk1 phosphorylation was lost even though Chk1 protein levels were retained. In contrast, G(1)/S-released SV40-infected cultures exhibited dephosphorylation of Chk1 in S phase, followed by an increase in Chk1 phosphorylation coinciding with entry of infected cells into >G(2). Inhibitors of Chk1, UCN-01 and caffeine, induced mitosis and abnormal nuclear condensation and increased the protein kinase activity of MPF in SV40-infected CV-1 cells. These results demonstrate that SV40 lytic infection triggers components of a DNA damage checkpoint pathway. In addition, chemical inhibition of Chk1 activity suggests that Chk1 contributes to the absence of mitosis during SV40 lytic infection.

The susceptibility of Werner's syndrome and other human skin fibroblasts to SV40-induced transformation and immortalization.

In attempts to transform and immortalize human cell cultures, skin fibroblasts from normal donors of different ages, from patients with the premature ageing diseases Werner's syndrome (WS) and progeria (PR), and from donors with the cancer-prone diseases ataxia telangiectasia (AT), Bloom's syndrome (BS) and Fanconi's anaemia (FA), were infected with SV40 virus and their growth monitored thereafter. Lesch-Nyhan (LN) fibroblasts were also infected. SV40-infected cultures from two normal and from WS, AT and LN donors attained a spectrum of transformed properties, high mitotic activity at confluence, presence of T-antigen, anchorage independence and altered morphology. Most of these pretransformed cultures died in the crisis period. However, two cultures from the WS and LN patients survived the crisis period and have now been grown to more than 200 passages. For the LN culture the crisis period was at least 200 days. Both permanent lines retain the properties of pretransformed cells, but differ in their modal chromosome number and ability to grow in methionine-free medium. It can be concluded from these experiments that transformation by SV40 to permanent lines is a rare event in human skin fibroblasts, even when these cells were taken from patients predisposed to form cancers.

Effect of simian virus 40 infection on albumin production by hepatocytes cultured in chemically defined medium and plated on collagen and non-collagen attachment surfaces.

Simian virus 40 (SV40)-infected rat hepatocytes were tested for albumin production (a marker of differentiated hepatocyte function) by growth in serum-free medium for 30-40 days. We observed a biphasic peak of albumin production, with the first peak at 4-6 and the second at 10-20 days after plating. A 2- to 3-fold increase in the second peak was observed when albumin production by SV40-infected cultures was compared with that by uninfected cultures. No increase in cell number in SV40-infected cultures was observed during this 20-day period. By 25-35 days, albumin production by uninfected cultures ceased but was detected in 50-60% of SV40-infected cultures. SV40-induced stimulation and persistence occurred regardless of the attachment surface and did not require collagen. Transformation occurred regardless of whether stimulation or persistence of albumin production was seen; however, stimulation and persistence were necessary to yield transformed cultures that produced albumin.

Effect of azacytidine in SV40-infected BSC-1 cells.

5-Azacytidine (5-AzaCR) reduced significantly the levels of ribosomal 28S and 18S RNAs in the cytoplasm of uninfected and simian virus 40 (SV40)-infected BSC-1 cells. The percent inhibition of ribosomal RNAs was correlated to both the dose of AzaCR and the time of exposure. In SV40-infected cultures treated with 200 microgram/ml AzaCR, a portion of the total cytoplasmic radioactivity was observed to sediment as 19S and 16S by sucrose gradient analysis. The 19S and 16S peaks represented in part the viral-specific RNA species. This result was due to the drastic reduction (70--75%) of ribosomal RNAs in the cytoplasm of infected cultures.

Analysis of the molecular forms of simian virus 40 deoxyribonucleic acid synthesized in cycloheximide-treated cell cultures.

Cycloheximide addition at various times from 24 to 36 hr after virus infection markedly inhibits the rate of simian virus 40 (SV40) deoxyribonucleic acid (DNA) synthesis in monkey kidney (CV-1) cultures. To determine whether superhelical (form I) SV40 DNA was synthesized in the cycloheximide-inhibited cultures, extracts were prepared by the method of Hirt from cultures labeled with (3)H-thymidine ((3)H-dT) and were analyzed by cesium chloride-ethidium bromide (CsCl-EtBr) equilibrium centrifugation and by velocity sedimentation in neutral sucrose gradients. When control or cycloheximide-treated cultures were labeled for 2 or 4 hr with (3)H-dT at 36 or 37 hr after infection, 71 to 83% of the radioactivity soluble in 1 m NaCl was detected in closed-circular SV40 DNA (form I). Cycloheximide treatment did not generate an increase of higher multiple circular forms of SV40 DNA. In pulse-chase experiments with or without cycloheximide treatment, radioactivity first appeared in nicked molecular forms sedimenting faster than open-circular SV40 DNA (form II), and then was chased into superhelical form I SV40 DNA. These results suggest that in cycloheximide-treated SV40-infected cultures: (i) polynucleotide ligase concentrations are adequate, and (ii) duplication errors causing formation of circular oligomers of SV40 DNA are not enhanced.

Increased resistance of SV40 transformed human amnion cells to poliovirus infection.

The increased resistance to poliovirus of SV40 transformed human amnion cells compared with primary cells has been shown to involve reduced cytopathic effect (CPE), delayed and reduced production of virus, inefficiency of infectious center formation, and the failure of transformed cell monolayers to support poliovirus plaque formation. At multiplicities of exposure (plaque-forming units/ cell) less than 10, CPE was negligible in the poliovirus infected transformed cell cultures which could be maintained as living cultures yielding poliovirus up to three months. Infectious center formation at different multiplicities showed that both cell types could be infected with single infectious units, but in cultures of transformed cells, the probability of initiating infection was reduced about 25-fold. Resistance was not observed in primary amnion cultures infected with SV40 in the absence of transformation. However, cellular resistance developed as morphologic transformed cells appeared in SV40 infected cultures. Transformed cells maintained resistance when SV40 multiplication in transformed cultures was suppressed with metabolic inhibitors or eliminated by isolating virus-free clones of transformed cells in the presence of SV40 antiserum.

Enzyme induction in SV40-infected green monkey kidney cultures

After infection of primary cultures of green monkey kidney cells or of an established green monkey kidney cell line (CV-1) with SV40, thymidine kinase activity was induced. Increased enzyme activity was first detectable 12–16 hours after inoculation and was 4–15 times greater at 28–48 hours in infected than in noninfected cultures. Thymidine kinase has been partially purified from noninfected and SV40-infected cells. With deoxyuridine (UdR) as substrate, the Michaelis constant of the enzyme from infected cells was 2–3 times greater than that from noninfected cells. However, the kinetics of inactivation of the enzymes were about the same at 38 °. 1-β- d-Arabinofuranosylcytosine (ara-C) inhibited the incorporation of tritiated thymidine (H 3-TdR) into the deoxyribonucleic acid (DNA) of noninfected or SV40-infected monkey kidney cultures by 95% or more. Ara-C treatment increased greatly the thymidine kinase activity of noninfected monkey kidney cultures. The Michaelis constant of thymidine kinase purified from ara-C treated CV-1 cultures was identical to that from untreated cultures. Ara-C did not prevent the induction of thymidine kinase by SV40. Indeed, the effects of ara-C and SV40 on enzyme induction were approximately additive in drug treated and infected cultures. Bromodeoxyuridine (BUdR) treatment did not prevent thymidine kinase induction by SV40, although virus growth was inhibited by 99.9%. Unlike ara-C, BUdR did not have a stimulating effect on thymidine kinase activity in noninfected cells. Cycloheximide inhibited the incorporation of radioactive amino acids into proteins of monkey kidney cells. Cycloheximide prevented thymidine kinase induction when added to cultures early after SV40 infection and arrested further enzyme induction when added after thymidine kinase induction had begun. A gradual twofold increase in thymidylate synthetase activity occurred in SV40-infected cultures but thymidine 5′-monophosphate (TMP) kinase and uridine kinase activities increased by less than 50%. Radioautographic and biochemical experiments on the incorporation of radioactive precursors into DNA demonstrated that the percentage of cells with labeled nuclei and the uptake of either tritiated deoxyadenosine or H 3-TdR into DNA was sharply increased beginning at about 16–20 hours after infection. The total DNA content of SV40-infected cultures was also increased.