Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa sublines and recovery of a HeLa clonal line persistently infected with incomplete virus

Rustigian, Robert (Tufts University School of Medicine, Boston, Mass.). Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa clonal line persistently infected with incomplete virus. J. Bacteriol. 92:1805-1811. 1966.-The effect of viral antibody on persistent infection of HeLa cells by the Edmonston strain of measles virus was investigated by culturing cells from three persistently infected clones in medium supplemented with human immune globulin. The three infected HeLa clones were isolated from a persistently infected parent line. Two sublines which were grown in the presence of measles antibody developed a nonyielder state, wherein there is no detectable virus infectious for normal HeLa cultures. There is, however, continued synthesis of intracellular viral antigen and formation of viral intracytoplasmic inclusion bodies. The development of a nonyielder state was associated with a marked decrease in the degree of hemadsorption in cultures of both sublines. Further studies of the viral properties of non-yielder HeLa cell populations were made with a clone obtained from one of these sublines by plating under antibody. Persistent infection in this line was characterized by synthesis of incomplete virus even when the cells were cultured thereafter in anti-body-free medium. This was evidenced by (i) failure to recover infectious virus from the clonal population despite continued formation of intracellular viral antigen and viral intracytoplasmic inclusion bodies in a majority of the cells, (ii) the presence of only a few cells with surface viral antigen(s) including hemagglutinin, and (iii) the relatively weak antibody response to viral envelope antigen(s) after injection of cells into guinea pigs.     

Induction of Simian Virus 40 Antigen in BSC1 Transformed Cells

Heating to 45 C induced in virus-free clones of simian virus 40 (SV40) transformed BSC₁ cells the synthesis of SV40 viral antigen, as evidenced by immunofluorescence. Up to 3.8% of the cells exhibited viral antigen 72 hr after heating to 45 C for 30 min. Depletion of arginine from the medium of the heated cells enhanced and increased the percentage of cells synthesizing viral antigen to 11%. Cytosine arabinoside completely inhibited the induction of the viral antigen. No infectious virus was recovered from the cells in which synthesis of viral antigen was induced. However, small amounts of infectious SV40 virus were rescued from the BSC₁ transformed cells by fusion with rabbit kidney cells or by treatment with mitomycin C.     

Persistent infection of BHK-21 cells with pneumonia virus of mice.

Trypsinization of BHK-21 cells 72 h after primary infection with pneumonia virus of mice yielded clones of persistently infected cells which specifically adsorbed murine erythrocytes. We describe one clone of cells, the progeny of which, after more than 100 passages, still bore viral antigen demonstrable by immunofluorescence and immune electron microscopy, but produced little or no detectable infectious virus.     

Characterization of flat revertant cells isolated from simian virus 40-transformed mouse and rat cells which contain multiple copies of viral genomes.

Eight clones of flat revertants were isolated by negative selection from simian virus 40 (SV40)-transformed mouse and rat cell lines in which two and six viral genome equivalents per cell were integrated, respectively. These revertants showed either a normal cell phenotype or a phenotype intermediate between normal and transformed cells as to cellular morphology and saturation density and were unable to grow in soft agar medium. One revertant derived from SV40-transformed mouse cells was T antigen positive, whereas the other seven revertants were T antigen negative. SV40 could be rescued only from the T-antigen-positive revertant by fusion with permissive monkey cells. The susceptibility of the revertants to retransformation by wild-type SV40 was variable among these revertants. T-antigen-negative revertants from SV40-transformed mouse cells were retransformed at a frequency of 3 to 10 times higher than their grandparental untransformed cells. In contrast, T-antigen-negative revertants from SV40-transformed rat cells could not be retransformed. The arrangement of viral genomes was analyzed by digestion of cellular DNA with restriction enzymes of different specificity, followed by detection of DNA fragments containing a viral sequence and rat cells were serially arranged within the length of about 30 kilobases, with at least two intervening cellular sequences. A head-to-tail tandem array of unit length viral genomes was present in at least one insertion site in the transformed rat cells. All of the revertants had undergone a deletion(s), and only a part of the viral genome was retained in T-antigen-negative revertants. A relatively high frequency of reversion in the transformed rat cells suggests that reversion occurs by homologous recombination between the integrated viral genomes.     

A replication-defective variant of Moloney murine leukemia virus. I. Biological characterization.

We have studied the virus produced by a clone, termed 8A, that was isolated from a culture of murine sarcoma virus-transformed mouse cells after superinfection with Moloney murine leukemia virus (MuLV-M). Clone 8A produced high levels of type C virus particles, but only a low titer of infectious murine sarcoma virus and almost no infectious MuLV. When fresh cultures of mouse cells were infected with undiluted clone 8A culture fluids, they released no detectable pogeny virus for several weeks after infection. Fully infectious MuLV was then produced in these cultures. This virus was indistinguishable from MuLV-M by nucleic acid hybridization tests and in its insensitivity to Fv-1 restriction. It also induced thymic lymphomas in BALB/c mice. To explain these results, we propose that cone 8A is infected with a replication-defective variant of MuLV-M. Particles produced by clone 8A, containing this defective genome, can establish an infection in fresh cells but cannot produce progency virus at detectable levels. Several weeks after infection, the defect in the viral genome is corrected by back-mutation or by recombination with endogenous viral genomes, resulting in the formation of fully infectious progeny MuLV. The progeny MuLV'S that arose in two different experiments were found to be genetically different from each other. This is consistent with the hypothesis that, in each experiment, the progeny virus is formed clone 8A cells and assayed for infectivity by the calcium phosphate transfection technique. No detectable MuLV was produced by cells treated with this DNA. This finding, along with positive results obtained in control experiments, indicates that clone 8A cells do not contain a normal MuLV provirus.     

Reversion in Polyoma-transformed Cells: Retransformation, Induced Antigens and Tumorigenicity

We studied the properties of two morphologically reverted cell clones isolated as chromosomal segregants from a "hybrid" clone of BHK 21/13 hamster fibroblasts, transformed with polyoma virus. Both clones were less tumorigenic than control transformed cells. They contained no detectable polyoma-specific complement-fixing antigen. Induced transplantation antigen also appeared to be lost or reduced. Both clones could be retransformed with polyoma virus, suggesting that their reversion is due to the loss of viral genes from the transformed cell.     

Epstein-Barr virus DNA is amplified in transformed lymphocytes.

Leukocytes isolated from two adult donors who lacked detectable antibodies to antigens associated with Epstein-Barr virus were exposed to an average of 0.02 to 0.1 DNA-containing particles of Epstein-Barr virus per cell and immediately clones in agarose. Within about 30 generations all transformed cell clones contained between 5 and 800 copies of viral DNA per cell. Only 1 in 10(4) to less than 1 in 10(5) of the cells of each clone release virus, and the frequency of release did not correlate with the average number of copies of viral DNA in the cells of each clone. One clone that had an average of five copies of viral DNA per cell was recloned, and the average number of copies in four of six subclones increased 15-to 50-fold while the subclones were being propagated sufficiently to study them. These results indicate that Epstein-Barr virus DNA can undergo amplification relative to cell DNA at different times after it transforms cells.     

cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA.     

Properties of T antigens induced by wild-type SV40 and tsA mutants in lytic infection

T antigen in extracts of cells infected with tsA mutants is 2 to 6 times more labile at 32°C or 41°C than the antigen in extracts of cells infected with wild-type SV40, as assayed by complement fixation. The stabilities of wild-type and mutant antigens are not altered by mixing the extracts, and thus the stability is an intrinsic property of each antigen and is not determined by another component of the extract. This observation indicates that T antigen is probably the virus-coded product of the A gene. In cells infected at the permissive temperature of 32°C with a high multiplicity of either wild-type or tsA mutant virus, the amounts of T antigen are approximately equivalent and increase logarithmically during the entire period of infection, up to 96 hr. Cells infected at 32°C for 96 hr with mixtures of wild-type and tsA virus produce T antigen with the stability of wild-type, even when the infection is carried out with up to a 5 fold excess of the mutant. The more stable wild-type antigen may repress, directly or indirectly, the synthesis of the more labile mutant antigen.     

Properties of Simian Virus 40 Rescued from Cell Lines Transformed by Ultraviolet-Irradiated Simian Virus 40

Simian virus 40 (SV40) strains have been rescued from various clonal lines of mouse kidney cells that had been transformed by ultraviolet (UV)-irradiated SV40. To learn whether some of the rescued SV40 strains were mutants, monkey kidney (CV-1) cells were infected with the rescued virus strains at 37 C and at 41 C. The SV40 strains studied included strains rescued from transformed cell lines classified as "good," "average," "poor," and "rare" yielders on the basis of total virus yield, frequency of induction, and incidence of successful rescue trials. Four small plaque mutants isolated from "poor" yielder lines and fuzzy and small plaque strains isolated from an "average" and a "good" yielder line, respectively, were among the SV40 strains tested. Virus strains rescued from all classes of transformed cells were capable of inducing the transplantation antigen, and they induced the intranuclear SV40-T-antigen, thymidine kinase, deoxyribonucleic acid (DNA) polymerase, and cellular DNA synthesis at 37 C and at 41 C. With the exception of four small plaque strains rescued from "poor" yielders, the rescued SV40 strains replicated their DNA and formed infectious virus with kinetics similar to parental SV40 at either 37 or 41 C. The four exceptional strains did replicate at 37 C, but replication was very poor at 41 C. Thus, only a few of the rescued virus strains exhibited defective SV40 functions in CV-1 cells. All of the virus strains rescued from the "rare" yielder lines were similar to parental SV40. Several hypotheses consistent with the properties of the rescued virus strains are discussed, which may account for the significant variations in virus yield and frequency of induction of the transformed cell lines.     

Selective inhibition of translation of the mRNA coding for measles virus membrane protein at elevated temperatures.

The elevation of culture temperatures of C6 cells that were persistently infected with the Lec strain of the subacute sclerosing panencephalitis (SSPE) virus (C6/SSPE) resulted in immediate selective inhibition of membrane (M) protein synthesis. This phenomenon was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cytoplasmic lysates and immunoprecipitation with monoclonal antibody against the M protein in short-time labeling experiments. The synthesis of various viral mRNAs in the presence of actinomycin D decreased gradually at similar rates after a shift to 39 degrees C. No specific disappearance of the mRNA coding for the M protein was observed when viral RNAs isolated from the infected cells were compared before and after a shift up by Northern blot analysis. Results of pulse-chase experiments did not show any significant difference in M protein stability between 35 and 39 degrees C. This rapid block of M protein synthesis was observed not only in Vero cells that were lytically infected with plaque-purified clones from the Lec strain, clones isolated from C6/SSPE cells and the standard Edmonston strain of measles virus but also in CV1, MA160, and HeLa cells that were lytically infected with the Edmonston strain. Poly(A)+ RNAs that were extracted from C6/SSPE cells before and after a shift to 39 degrees C produced detectable phospho, nucleocapsid, and M proteins in cell-free translation systems at 32 degrees C. Even higher incubation temperatures did not demonstrate the selective depression of M protein synthesis described above in vitro. All these data indicate that M protein synthesis of measles virus is selectively suppressed at elevated temperatures because of an inability of the translation apparatus to interact with the M protein-encoded mRNA.     

Clonal analysis of mammalian cell cultures persistently infected with Japanese encephalitis virus.

More than 200 cells were cloned from populations of mammalian cells persistently infected with Japanese encephalitis virus. Only four cloned cultures contained cells that had viral antigen measurable by immunofluorescence and that released infectious virus, yet all clones harbored virus-specific RNA. Superinfection of cloned cells with wild-type Japanese encephalitis virus did not produce cytopathic effects, but resulted in production of viral antigen and infectious virus in formerly nonproducing clones. Cocultivation of nonproducer clone cells with normally permissive cells did not induce virus production, nor did treatment of nonproducer clones with various inhibitors of DNA, RNA, or protein synthesis. It is suggested that the cloning procedure may have selected for a particular subpopulation of cells and that defective virus is also involved in establishment and maintenance of persistent infection.     

Polyomavirus growth and persistence in Friend erythroleukemic cells

Infection of Friend erythroleukemic (FL) cells by polyomavirus (Py) invariably results in the selection of persistently infected FL-Py cell lines and clones. Anti-Py serum treatment of FL-Py lines and clones leads to the loss of Py genome and consequent cell cure. Conversely, cure has not been obtained in FL-PytsA cell lines (isolated after infection by a Py thermosensitive early mutant) and their derivative clones cultivated for a long time at nonpermissive temperature (39 degrees C), where viral large-T protein is inactive. Rescue of viral particles has always been obtained after shifting cells to 32 degrees C. Integrated viral genomes were detected by blot hybridization in an FL-PytsA clone at 39 degrees C. Long-term observation of FL-Py cell lines and their derivative clones reveals a reciprocal selection mechanism (coevolution) between the viral and the cellular populations, resulting in either a completely virus-free Py-resistant FL cell line (cure) or in a continuously Py-shedding line bearing Py genome variants. Structural analysis of these viral populations has been carried out, and some viral variants have been isolated and characterized. On the basis of the results obtained, the possible mechanisms of Py persistence in FL cells will be discussed.     

Cloning and structural analysis of integrated woodchuck hepatitis virus sequences from a chronically infected liver

We have isolated and determined the structure of a recombinant clone in lambda phage Charon 30 which contains woodchuck hepatitis virus sequences integrated in woodchuck genomic DNA sequences. This clone, in contrast to previously reported clones (Ogston et al., Cell 29:385-394, 1982), was isolated from a chronically infected liver which never developed hepatocellular carcinoma. Southern blot analysis of viral sequences in the clone in conjunction with electron microscope heteroduplex analysis showed that the integrated viral sequences did not contain internal rearrangements, as have those from hepatomas, but were colinear with the cloned viral genome except for the deletion of approximately 500 base pairs of viral sequences (between positions 1,000 and 1,550 on the viral map). Therefore, the integration was probably a defective genome incapable of supporting viral replication. However, the complete open reading frames coding for the viral X, core, presurface , and surface antigen genes were present, indicating that the viral sequences could code for viral antigens. Southern blot analysis of the normal cellular flanking sequences, using flanking sequence probes from the clone, showed that no detectable rearrangements of cellular DNA (less than 50 base pairs) had occurred at the site of viral integration.     

Temperature-sensitive virus derived from BHK cells persistently infected with HVJ (Sendai virus).

BHK-HVJ cells, a cell line of baby hamster kidney cells persistantly infected with HVJ (Sendai virus), started to produce infectious virus by shifting down the incubation temperature from 38 to 32 C. The virus derived from BHK-HVJ cells, designated as HJV-pB, was effectively neutralized with antibody against wild-type virus (HVJ-W) which was used for the establishment of BHK-HVJ cells. HVJ-pB replicated in eggs at 32 C, but not at 38 C, while HVJ-W grew equally well at both temperatures. When BHK cells infected with HVJ-PB were incubated at 38 C, production of infectious virus, hemagglutinin, and neuraminidase was markedly restrained, whereas a considerable amount of viral nucleocapisid and envelope antigens was detected in the cells by complement fixation tests. These viral activities became detectable immediately after temperature shift-down from 38 to 32 C even at the later stage of infection. HVJ-pB was indistinguishable from HJV-W with respect to particle size, density, and morphological characteristics, but appeared to possess a higher neuraminidase activity and was inactivated more rapidly at 50 C than HVJ-W. HVJ-pB was less cytocidal and could easily cause latent infection in BHK and mouse L cells.     

Spontaneous Conversion of Nontransformed Avian Sarcoma Virus-Infected Rat Cells to the Transformed Phenotype

Normal rat kidney (NRK) fibroblasts were infected with the Schmidt-Ruppin strain (SR-D) of avian sarcoma virus (ASV) and cloned 20 h after infection without selection for the transformed phenotype. Most infected clones initially exhibited the flat, nontransformed morphology that is characteristic of uninfected NRK cells. In long-term culture, however, the majority of the SR-D NRK clones began segregating typical ASV-transformed cells. Transforming ASV could be rescued by fusion with chicken embryo fibroblasts from most of the infected clones tested. Three predominantly flat, independently infected clones were further analyzed by subcloning 8 to 10 weeks after infection. Most flat progeny subclones derived at random from two of these "parental" SR-D NRK clonal lines did not yield virus upon fusion with chicken embryo fibroblasts, although a nondefective transforming ASV was repeatedly recovered from the parental clones. This observation suggested that most, but not all, daughter cells in these SR-D NRK clones lost the ASV provirus after cloning. The progeny of the third independent parental cell clone, c17, gave rise to both flat and transformed subclones that carried ASV. In this case, ASV recovery by fusion and transfection from the progeny subclones was equally efficient regardless of the transformation phenotype of the cells. The 60,000-dalton phosphoprotein product of the ASV src gene was, however, expressed at high level only in the transformed variants. The results of a Luria-Delbruck fluctuation analysis and of Newcombe's respreading test indicated that the event leading to the spontaneous conversion to the transformed state occurred at random in dividing cultures of these flat ASV NRK cells at a rate predicted for somatic mutation.