Semipermissive Cells Research Articles

Indiscriminate activity from the B19 parvovirus p6 promoter in nonpermissive cells

B19 parvovirus is absolutely tropic for human erythroid progenitor cells. Among the untested mechanisms underlying this tropism is the possibility of cell-specific positive regulation of the promoter in permissive cells. Using the bacterial chloramphenicol acetyltransferase and firefly luciferase reporter genes, we detected strong activity from the B19 P6 promoter in transfected nonpermissive cells. Very high-level expression was seen in a T lymphoblastoid cell line, CEM. No transcriptional enhancement occurred in an erythropoietin-dependent semipermissive cell line. A putative second B19 promoter at map unit 44 (1344) was nonfunctional and unable to confer tissue specificity. Thus, tropism is unlikely to be regulated at the level of transcriptional initiation from either the P6 or P44 promoter.

Adenovirus 41 growth in semi-permissive cells shows multiple-hit kinetics

Adenovirus type 41 infection of semi-permissive Chang conjunctival cells, monitored by fluorescent focus assay, followed multiple-hit kinetics. In non-permissive human cells, type 41 showed infectivity with two-hit kinetics in the presence of type 2. Type 41 infectivity was seen to be directly proportional to input concentration (one-hit) only in 293 cells, a continuous human line expressing Ad 5 E1 products.

Simian virus 40 DNA replication correlates with expression of a particular subclass of T antigen in a human glial cell line

Immunocytochemistry and in situ hybridization were used to identify simian virus 40 (SV40) large T-antigen expression and viral DNA replication in individual cells of infected semipermissive human cell lines. SV40 infection aborts before T-antigen expression in many cells of each of the human cell lines examined. In all but one of the human cell lines, most of the T-antigen-producing cells replicated viral DNA. However, in the A172 line of human glial cells only a small percentage of the T-antigen-expressing cells replicated viral DNA. Since different structural and functional classes of T antigen can be recognized with anti-T monoclonal antibodies, we examined infected A172 cells with a panel of 10 anti-T monoclonal antibodies to determine whether viral DNA replication might correlate with the expression of a particular epitope of T antigen. One anti-T monoclonal antibody, PAb 100, did specifically recognize that subset of A172 cells which replicated SV40 DNA. The percentage of PAb 100-reactive A172 cells was dramatically increased by the DNA synthesis inhibitors hydroxyurea and aphidicolin. Removal of the hydroxyurea was followed by an increase in the percentage of cells replicating viral DNA corresponding to the increased percentage reactive with PAb 100. The pattern of SV40 infection in A172 cells was not altered by infection with viable viral mutants containing lesions in the small t protein, the agnoprotein, or the enhancer region. Finally, in situ hybridization was used to show that the percentage of human cells expressing T antigen was similar to the percentage transcribing early SV40 mRNA. Thus, the block to T-antigen expression in human cells is at a stage prior to transcription of early SV40 mRNA.

Infection by enteric adenoviruses, rotaviruses, and other agents in a rural african environment

From February 1985 to January 1986, 432 stool samples, 310 from rural African children with diarrhea and 122 from controls, were analysed for the presence of enteric viruses known to be associated with diarrhea. Group A rotavirus ELISA indicated 12.9% positivity among patients and 2.5% positivity among controls. Only 23 of the 43 rotavirus ELISA‐positive stools were also positive by electron microscopy. Nine children, three of whom were controls, were found to be shedding coronavirus‐like particles, detected by electron microscopy. Stools from all but one of the nine children had been taken within 1 month of each other. Dot‐blot hybridization tests for the presence of Ad40 or Ad41 DNA revealed 44 positive stools, 41 of which were from patients (13.2% positivity). Only three of the Ad40‐ or Ad41‐positive stools by DNA hybridization were positive by electron microscopy, and only these three strains could be grown in semipermissive Chang conjunctival cells and their identity checked by restriction enzyme analysis. Further attempts to rescue the other strains using a helper virus failed, but nine of the stools proved positive by ELISA using a subgroup F‐specific monoclonal antibody. On the basis of the DNA hybridization results alone, subgroup F adenoviruses were encountered as frequently as rotavirus in the study and were significantly associated with diarrhea, although the viability and intactness of virus particles by the time of laboratory analysis appeared to be very low.

Relationship between methylation status and expression of an epstein-barr virus (EBV) capsid antigen gene

The methylation status of the 160 kD viral capsid antigen ( VCA ) gene promoter was determined by hybridization analysis. The semi-permissive marmoset cell line FF41-1 lacked cytosine methylation in approximately three quarters of the VCA promoter CpG dinucleotide residues. In the stringently infected HH514CL16 cell line the same CpG residues were methylated in three quarters of the genomes. 5′deoxy-5′-S-isobutyladenosine (SIBA), a DNA methylase inhibitor, was utilized to disrupt the EBV latent state. As determined by flow cytometry, SIBA treatment significantly increased expression of VCA. The VCA promoter was hypomethylated in VCA-positive FF41-1 cells sorted by flow cytometry. While hypomethylation alone was not sufficient for VCA transcriptional activity, the absence of methylation of VCA promoter CpG dinucleotide residues was associated with expression of VCA.

The nucleotide sequence of the region ofsrcgene deletion in transformation-defective Rous sarcoma virus adapted to semi-permissive host cells

The nucleotide sequence of the region of<i>src</i>gene deletion in transformation-defective Rous sarcoma virus adapted to semi-permissive host cells

The nucleotide sequence of the region of sre gene deletion in transformation-defective Rous sarcoma virus adapted to semi-permissive host cells

The nucleotide sequence of the region of sre gene deletion in transformation-defective Rous sarcoma virus adapted to semi-permissive host cells

Helper function of adenovirus 2 for adenovirus 41 antigen synthesis in semi-permissive and non-permissive cells.

Using a fluorescent focus assay, complementation and interference effects of Ad2 and Ad41 on each other in mixed infection were investigated. Ad2 provided a helper function for Ad41 late antigen synthesis in cells normally non-permissive for Ad41 growth (HEF cells), and enhanced Ad41 late antigen synthesis in semi-permissive Chang conjunctival cells. The degree of complementation by Ad2 was dependent on its input concentration. In addition, interference by Ad41 on Ad2 replication was seen in HEF cells. The degree of interference by Ad41 was dependent on the relative time of infection by each serotype. The complementation results in HEF cells suggest an absolute dependency of Ad41 on an adenovirus helper function in these cells. The results presented are consistent with the postulated helper function provided in trans by 293 cells, which are transformed by Ad5 early region 1.

Early events of importance in determining host cell permissiveness to mouse hepatitis virus infection.

Three categories of cell lines are described which differ with respect to their permissiveness to mouse hepatitis virus (MHV), strain A59. Fully permissive L-2 cells gave rise to 100- to 1000-fold higher numbers of infectious centres than did semi-permissive LM, LM-K or C-1300 cells, whereas non-permissive Vero or C-6 cells were refractory to MHV infection. On an infected cell basis, semi-permissive cells (LM, LM-K or C-1300) were as efficient in replicating viral RNA, protein and progeny virions as fully permissive L-2 cells. This result suggested that LM, LM-K and C-1300 cells were deficient in their ability to permit full expression (as compared to L-2 cells) of an early event in MHV infection. Assays of radiolabelled MHV binding to cells of all three categories (L-2, LM, LM-K and C-6) and of infectious MHV binding to L-2 and LM-K cells showed no correlation between virion binding and degree of permissiveness to MHV infection. Internalization of MHV virions into L-2 and LM-K cells, as assayed by proteinase K-resistant infectious centres, showed that, in both cases, maximum virion uptake was complete by approximately 40 min post-inoculation. Direct assays of infectious virion uptake showed similar numbers of internalized viruses (only a threefold difference between L-2 and LM-K cells, as compared to a 500-fold difference in infectious centres). Attempts to enhance MHV uptake into LM-K cells relative to L-2 cells, with DEAE-dextran or the cytoskeleton-disrupting drugs colchicine and cytochalasin B, were unsuccessful, further suggesting that the ability of LM-K cells to internalize the virus was not lacking. The results suggest that MHV infection of at least some semi-permissive cells, such as the LM-K line, is limited by a process which chronologically correlates with virion uncoating. Since LM-K cells have been shown previously to be resistant to membrane fusion in MHV infection, it is postulated that they may also restrict uncoating of MHV by limiting the degree of normal endosomal membrane fusion with the viral envelope.

Enhanced Production of a Human Spumavirus (Retroviridae) in Semi-permissive Cell Cultures after Treatment with 5-Azacytidine

Infection by a human spumavirus of human foetal diploid lung (HFDL) cells was found to be productive with virus titres ranging from 10(3) to 10(5) p.f.u./ml. In contrast, infection of recovered amnion (RA) aneuploid cells resulted in a persistent infection with less than 100 p.f.u./ml infectious virus produced. The decreased sensitivity of RA cells to the spumavirus was not due to the failure of virus to penetrate into the cell since infectious virus was not produced even after transfection of infectious proviral DNA. The effect of 5-azacytidine, an inhibitor of DNA methylation, on virus replication was examined. Whereas virus production in HFDL cells was not affected, there was a 100-fold increase in virus yield in RA cells treated with the drug for at least 48 h and maximum virus yields were obtained 4 days post-infection.

The role of protease-dependent cell membrane fusion in persistent and lytic infections of murine hepatitis virus.

We have defined three categories of cultured cell lines on the basis of their permissiveness (susceptibility to initial infection) to mouse hepatitis virus (MHV). Fully permissive L-2 cells gave rise to 100-1000-fold higher numbers of infectious centers than did semi-permissive LM, LM-K or C-1300 cells, whereas non-permissive Vero or C-6 cells were refractory to MHV infection. On an infected cell basis, there was no deficiency on the part of semi-permissive cell lines to replicate total viral RNA, viral polypeptides or progeny virions. Two of the semi-permissive cell lines (LM and LM-K) supported persistent MHV infection, while a third (C-1300) succumbed to lytic infection. LM and LM-K cells, but not C-1300 cells showed resistance to MHV-induced membrane fusion, even when placed in contact with fusion-active MHV-infected L-2 cells. The ability of a given cell to undergo fusion did not correlate with membrane lipid characteristics (unsaturated fatty acid and sterol content) which contribute to membrane "fluidity". In order to more closely study the parameters of MHV-induced cell fusion, membranes were prepared from MHV-infected L-2 cells and monitored for their fusogenic potential with permissive L-2 cells, semi-permissive LM cells and non-permissive vero cells. Fusion was only observed with the permissive L-2 cells, and only when exogenous protease (trypsin or chymotrypsin) was added. When the membranes were prepared from 35S-methionine-labeled MHV-infected L-2 cells and subjected to protease treatment, the radiolabeled 180,000 dalton form of the E2-glycoprotein underwent proteolytic cleavage to yield a major product of approximately 90,000 daltons. Both trypsin and chymotrypsin were effective in this proteolytic cleavage and in activating membrane fusion. In a normally permissive, fusogenic infection of MHV in L-2 cells, the protease inhibitors TPCK and ZPCK, but not TLCK, were found to inhibit cell fusion. In MHV-infected L-2 cells, E2 was found almost exclusively as the 180,000 dalton form but turned over rapidly as shown by pulse-chase studies. TPCK and ZPCK but not TLCK inhibited turnover. The results suggest that L-2 cells contain a protease which cleaves at aromatic amino acids such as phenylalanine, and that this protease cleaves the 180,000 dalton form of the E2 to peptide fragments, one or more of which may activate cell fusion.

Enhanced induction of SV40 replication from transformed rat cells by fusion with UV-irradiated normal and repair-deficient human fibroblasts

Fusion of SV40-transformed rat (BRKSV) cells which do not spontaneously produce infectious virus, with permissive monkey cells resulted in a low level of production of infectious virus in the heterokaryons. UV-Irradiation of the BRKSV cells prior to fusion did not result in increased virus production, but irradiation of the monkey cells prior to fusion did result in enhanced induction (EI) of SV40, as compared to control experiments in which neither cell type was irradiated. This indicated that rat cells lack the ability to initiate replication of integrated SV40 upon UV-irradiation and do not contain “permissiveness” factors that are required to support SV40 replication. In contrast, monkey cells do contain such permissiveness factors which seem to be temporally enhanced by UV-irradiation, and thus may be responsible for the EI phenomenon. Expression of EI was dose-dependent and reached maximum values approximately 24 h after UV-irradiation. The kinetics of EI resembled that of EI previously established for SV40 induction in semi-permissive cells, and of enhanced reactivation (ER) and enhanced mutagenesis (EM) of SV40 in monkey cells. Similar kinetics of EI were obtained when human diploid fibroblasts were used for fusion with BRKSV cells. Similar levels of EI were found with normal human cells and repair-deficient xeroderma pigmentosum (XP) cells of complementation groups A and C, and XP variant cells. This suggests thatt expression of EI is not related to excision repair. Since EI is also normally expressed in XP cells which display an abnormal ER of HSV and in XP variant cells which show a delayed EM of HSV, we conclude that EI may occur independently of ER and EM. Finally it was shown that treatment of human cells with N-ethyl- N-nitrosourea results in similar induction of EI as irradiation with UV-light, and that addition of TPA in fusion experiments has no effect on EI.

Semipermissive Replication of a Nuclear Polyhedrosis Virus of Autographa californica in a Gypsy Moth Cell Line

Several gypsy moth cell lines have been previously described as nonpermissive for the multiple-embedded nuclear polyhedrosis virus of Autographa californica (AcMNPV). In this report, we demonstrate the semipermissive infection of a gypsy moth cell line, IPLB-LD-652Y, with AcMNPV. IPLB-LD-652Y cells infected with AcMNPV produced classic cytopathic effects but failed to yield infectious progeny virus. Results of experiments employing DNA-DNA dot hybridization suggested that AcMNPV DNA synthesis was initiated from 8 to 12 h postinfection (p.i.), continued at a maximum rate from 12 to 20 h p.i., and declined from 20 to 36 h p.i. The rate of AcMNPV DNA synthesis approximated that observed in the permissive TN-368 cell line. AcMNPV-infected IPLB-LD-652Y cells, pulse-labeled with [(35)S]methionine at various time intervals p.i. and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed four virus-induced proteins, one novel to the semipermissive system and three early alpha proteins, synthesized from 1 to 20 h p.i. Thereafter, both host and viral protein synthesis was completely suppressed. These results suggest that AcMNPV adsorbed, penetrated, and initiated limited macromolecular synthesis in the semipermissive gypsy moth cell line. However, the infection cycle was restricted during the early phase of AcMNPV replication.

Adenovirus Type 2 Activates Cell Cycle-Dependent Genes That Are a Subset of Those Activated by Serum

We have studied a panel of 10 genes and cDNA sequences that are expressed in a cell cycle-dependent manner in different types of cells from different species and that are inducible by different mitogens. These include five sequences (c-myc, 4F1, 2F1, 2A9, and KC-1) that are preferentially expressed in the early part of the G1 phase, three genes (ornithine decarboxylase, p53, and c-rasHa) preferentially expressed in middle or late G1, and two genes (thymidine kinase and histone H3) preferentially expressed in the S phase of the cell cycle. We have studied the expression of these genes in nonpermissive (tsAF8) and semipermissive (Swiss 3T3) cells infected with adenovirus type 2. Under the conditions of these experiments, adenovirus type 2 infection stimulates cellular DNA synthesis in both tsAF8 and 3T3 cells. However, four of the five early G1 genes (c-myc, 4F1, KC-1, and 2A9) and one of the late G1 genes (c-ras) are not induced by adenovirus infection, although they are strongly induced by serum. The other sequences (2F1, ornithine decarboxylase, p53, thymidine kinase, and histone H3) are activated by both adenovirus and serum. We conclude that the cell cycle-dependent genes activated by adenovirus 2 are a subset of the cell cycle-dependent genes activated by serum. The data suggest that the mechanisms by which serum and adenovirus induce cellular DNA synthesis are not identical.

Adenovirus type 2 activates cell cycle-dependent genes that are a subset of those activated by serum.

We have studied a panel of 10 genes and cDNA sequences that are expressed in a cell cycle-dependent manner in different types of cells from different species and that are inducible by different mitogens. These include five sequences (c-myc, 4F1, 2F1, 2A9, and KC-1) that are preferentially expressed in the early part of the G1 phase, three genes (ornithine decarboxylase, p53, and c-rasHa) preferentially expressed in middle or late G1, and two genes (thymidine kinase and histone H3) preferentially expressed in the S phase of the cell cycle. We have studied the expression of these genes in nonpermissive (tsAF8) and semipermissive (Swiss 3T3) cells infected with adenovirus type 2. Under the conditions of these experiments, adenovirus type 2 infection stimulates cellular DNA synthesis in both tsAF8 and 3T3 cells. However, four of the five early G1 genes (c-myc, 4F1, KC-1, and 2A9) and one of the late G1 genes (c-ras) are not induced by adenovirus infection, although they are strongly induced by serum. The other sequences (2F1, ornithine decarboxylase, p53, thymidine kinase, and histone H3) are activated by both adenovirus and serum. We conclude that the cell cycle-dependent genes activated by adenovirus 2 are a subset of the cell cycle-dependent genes activated by serum. The data suggest that the mechanisms by which serum and adenovirus induce cellular DNA synthesis are not identical.

Establishment and characterization of a permanent pSV ori −-transformed ataxia-telangiectasia cell line

A permanent ataxia-telangiectasia (A-T) cell line has been established from the fibroblast strain AT2SF after transfection with the bacterial plasmid pSV ori −, which contains replication origin-defective SV40 sequences. The original transfection frequency, as measured by transformed foci, was markedly reduced in two A-T strains when compared with either normal or xeroderma pigmentosum fibroblasts. As with SV40 virion-transformed fibroblasts, pSV ori −-transformed cells entered a crisis phase, from which about one-fourth of the original clones from A-T and normal fibroblasts recovered. Both the pSV ori −-transformed TAT2SF cell line and an SV40 virion-transformed AT5BI (GM5489) cell line retained their characteristic sensitivity to the lethal effects of ionizing radiation, as well as their X ray-resistant DNA synthesis. Southern blot analysis of cellular SV40 sequences demonstrated a single major integration site of pSV ori − in the AT2SF cells. In contrast, AT5BI cells transformed with SV40 virions demonstrated a high degree of heterogeneity of integrated viral sequences. Neither the TAT2SF nor the GM5489 transformed cell line contains any detectable freely replicating SV40 viral sequences, which are seen in many other semipermissive SV40-transformed cells.

Definition of the simian virus 40 early promoter region and demonstration of a host range bias in the enhancement effect of the simian virus 40 72-base-pair repeat.

The simian virus 40 (SV40) origin region includes the viral replication origin and the early and late promoters and consists of a few palindromes, a 17-base-pair (bp) A + T-rich sequence, three copies of a G + C-rich 21-bp repeat, and two copies of a 72-bp repeat. We have made sequential deletions in the SV40 origin region and determined the early promoter efficiencies of these truncated DNA segments by connecting them in the correct orientation with the coding regions of selectable marker genes and assaying the expression of the chimeric marker genes in vivo in different host cell lines. A truncated SV40 early promoter segment containing only the TATA box and the major in vivo mRNA initiation sites has essentially no promoter efficiency. We have located the major component of the SV40 early promoter within the 21-bp repeated sequences, which consist of an alternating and mutually overlapping array of two C-rich oligonucleotides having the consensus sequences Y-Y-C-C-G-C-C-C (Y = pyrimidine nucleoside) and G-C-C-C-(C)-TA-AT-A(T)-C-T. Between one and two copies of the 21-bp repeat were adequate for gene expression under conditions in which the enhancement effect of the 72-bp repeat was minimal. We also find that the SV40 72-bp repeat exhibits a pronounced host range in its enhancement of gene expression; the enhancement is only 2-fold in the nonpermissive mouse cells but amounts to 10- or 20-fold in the permissive monkey cells or the semipermissive human cells, respectively.

Effect of butyrate on adenovirus infection in semipermissive cells

Adenovirus 2 stimulated cellular DNA synthesis in quiescent cultures of semipermissive tsAF8 cells and 3T3 cells. Such stimulation was inhibited by Na-butyrate, which also inhibited viral DNA replication in tsAF8 cells. In addition, butyrate inhibited the expression of early regions ElA and E2 of adenovirus 2 in both tsAF8 and 3T3 cells, while it had little effect on permissive HeLa cells.

Characterization of a Cellular Protein that Promotes SV40 Infection in Human Cells

Publisher Summary This chapter discusses the characterization of a cellular protein that promotes SV40 infection in human cells. Studies show that a cellular protein promotes SV40 infection of cultured primate cells. The protein is released into the medium by uninfected cells that are susceptible (permissive) to productive SV40 infection. This protein, designated as enhancer, facilitates the infection of human cells that exhibit low susceptibility to SV40. Addition of the SV40 enhancer before or with virus to cultured semipermissive host cells results in approximately 100-fold increases in the number of infected cells as determined by the plaquing efficiency of infectious centers and/or by immunofluorescence of nuclear SV40 T-antigen utilizing specific, fluorescein-conjugated antisera. A direct relationship exists between the susceptibility of SV40 infection and presence of active enhancer in uninfected cell cultures. Simian cells (CV-1) are very susceptible to SV40 and release easily detectable amounts of SV40 enhancer activity (10 unit/ml). In contrast, semipermissive human fibroblasts release minimal amounts of enhancer activity, and nonpermissive WISH and chick embryo fibroblast cells do not release any detectable SV40 enhancer activity. Thus, the amount of SV40 enhancer activity recovered from cell cultures coincided with the ability of SV40 to infect these cells.

Host Antigens on Avian Oncoviruses: Presence on Viruses Produced by Quail, Duck and Rat Cells of Antigens Related to Membrane Antigens of Chick Embryo Fibroblasts and Chicken Erythrocytes

Avian sarcoma viruses (ASV) produced by Japanese quail embryo fibroblasts (QEF) were inactivated to the same degree as ASV produced by chick embryo fibroblasts (CEF), with or without complement, by rabbit antisera to CEF and to membrane antigens of chick embryo and adult chicken erythrocytes, in particular their unique age-specific antigens. ASV produced by duck embryo fibroblasts (DEF) were only inactivated by the antisera to the chicken erythrocyte antigens. Hence, viruses produced by QEF and DEF bear on their envelope antigens related to the chicken antigens picked up by ASV in CEF. These antigens are presumably coded by the quail and duck cells, since antigens related to the chicken antigens were found on QEF and on quail and duck erythrocytes. Antigens related to the chicken antigens have also been found on ASV shed in low amounts by semi-permissive rat sarcoma cells (line 17RBI77) and on the surface of these cells, as well as on that of another semi-permissive cell line (RBH) originating from a hamster sarcoma produced by inoculation of the rat cells. The origin of these antigens, which may play a role in semi-permissiveness, remains to be explained since they do not appear to be normally expressed by rat or hamster cells. It was also found that, contrary to an earlier conclusion and in agreement with a recent report, uninfected CEF bear on their membrane an antigen that is related or identical to the specific antigen of chick embryo erythrocytes. Therefore, only the antigen related to the adult-specific chicken erythrocyte antigen does not pre-exist on CEF.