Growth of measles virus in a human macrophagelike cell line: U937

Measles virus infection was established in U937, a continuous human macrophagelike cell line. Unlike cultured human peripheral macrophages, infection resulted in prominent giant cell formation, indicating that these cells are susceptible to viral-induced fusion. Although a high proportion of cells in culture contained measles viral antigen by immunofluorescent assay a relatively small amount of infectious virus was produced. In contrast to continuously cultured human lymphoblastoid cell lines, infection of U937 was lytic, and persistent infection could not be established. The U937 cell line may be useful for further studies of viral interaction with macrophages, including those related to the induction of cell fusion by measles or other syncytium-forming viruses.     

Replication of human adenoviruses in guinea pig embryo cells: an ultrastructural study

Summary Guinea pig embryo (GPE) cells showed different degrees of susceptibility to human adenovirus types as determined by virus infectivity assay and electron microscopic examination. Adenovirus 2 and 5 induced extensive cellular changes and produced high titers of infectious virus in GPE cells as in human cells. Mature progeny virus and protein crystals were observed in both cell types. Adenovirus 7 induced some cellular changes in GPE cells but only a small number of cells yielded progeny virus as determined by electron microscopy. Adenovirus 3, 8 and 31 induced some cellular changes but no progeny virus was found under electron microscopic examination. Characteristic fibers were observed in nuclei of adenovirus 31 infected cells. The ability of human adenovirus 2 and 5 to replicate in GPE cells is an example of an unusual cross-species biological property of certain adenovirus types. This property may be useful as a biological marker for these virus types.With 8 Figures     

Construction of human cell lines which contain and express the adenovirus DNA binding protein gene by cotransformation with the HSV-1 tk gene

We have introduced the DNA binding protein (DBP) gene of human adenovirus type 5 (Ad5) into high molecular weight DNA of permissive human cells by cotransformation of tk- cells with the cloned DBP and HSV-1 thymidine kinase genes. 110 tk+ cell lines were isolated after selection in HAT medium. The amount and arrangement of adenovirus sequences in the tk+ cell lines were analyzed by restriction endonuclease digestion and filter hybridization. Twelve of the 110 lines carry at least a segment of the DBP gene while only three of these contain the entire DBP gene at approximately one copy per cell. Cytoplasmic, polyadenylated DBP mRNA is made in all three cell lines though the amount is very low compared to that present in infected HeLa cells. The cell line U13-2 which contains approximately 1/30 the steady-state level of DBP mRNA found in infected HeLa cells produces a few percent of the amount of DBP made during the peak period of DBP synthesis in infected cells. The other two lines contain lower levels of DBP mRNA and do not synthesize detectable levels of the protein. When these DBP-tk+ cell lines are infected with adenovirus mutants containing temperature-sensitive (ts) mutations in the DBP gene, only U13-2 permits some viral DNA replication (and hence late gene expression) at the nonpermissive temperature, indicating that sufficient quantities of DBP from the integrated gene are produced to allow complementation of the ts mutation in this cell line. However, growth of these ts mutants (as measured by virus production) is only partially complemented in U13-2 at the nonpermissive temperature.     

Human immunodeficiency virus infection of monocytic and T-lymphocytic cells: Receptor modulation and differentiation induced by phorbol ester

The monocytic leukemic cell line U937 can be infected with human immunodeficiency virus type 1 (HIV-1) to become permanently infected virus producers. Uninfected U937 cells express T4 (CD4) antigen and form syncytia when mixed with HIV-1 producing cells. Anti-T4 monoclonal antibodies block syncytium formation indicating that the HIV-1 receptors on U937 cells include T4 antigen. The promyelocytic leukemic cell line HL60, while expressing only low amounts of surface T4 and not forming syncytia on exposure to HIV-1, can be infected by HIV-1 at lower efficiency than U937 and T-cell lines. 12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment induces the differentiation of U937 cells into macrophages. HIV-infected U937 cells retain the ability to differentiate, though less efficiently, as shown by the appearance of monocyte/macrophage surface markers. T4 antigen on both U937 and T-cell lines is down regulated by TPA treatment. Functional receptors for HIV-1, assayed by syncytium induction and pseudotype plating, are lost concomitantly with T4 antigen following TPA treatment of U937 cells and T cells.     

Restriction of HIV-1 replication in promonocytic cells: a role for IFN-alpha

A comparative study of the replication kinetics of human immunodeficiency virus type 1 (HIV-1) was performed in the promonocytic U937 cells and in the T lymphoblastoid H9 cells. If a productive HIV-1 infection of both cell types could be established, the time which elapses before most of the cells could express viral proteins is always proportionally longer for U937 cells than for H9 cells. Indeed, when U937 cells are infected with HIV-1, this nonproductive phase is followed by a lag phase during which the percentage of virus-producing cells is slowly increasing when compared to H9 cells. The restriction of HIV-1 replication in U937 cells might be consecutive to the lower adsorption of viral particles to these cells, even though the same percentage of U937 and H9 cells was expressing the CD4 molecule. Furthermore, we demonstrate that HIV-1 replication in U937 cells is mainly restricted by endogenous IFN-alpha. Indeed, addition of anti-IFN-alpha antibodies at the time of infection, during the nonproductive phase of the viral replication cycle, or during the lag phase leads to an earlier expression of viral proteins and/or to a rapid increase in the percentage of virus-producing cells. Likewise, the treatment of cultures of HIV-1 chronically infected U937 cells with the same antibodies induces an increased production of viral particles. Thus, IFN-alpha appears to be involved in the persistence of HIV-1 in the monocytes/macrophages of infected individuals.     

Difference in the capacity to repair DNA damage in human cells chronically infected with the rubella virus

Centrifugation of cell lysates in alkaline sucrose gradients and chromatography on hydroxyapatite columns were used to demonstrate inhibition of reparation of mitomycin C-induced DNA damages at the stage of reunification of single-strand breaks of DNA in human HEp-2 cell cultures chronically infected with rubella virus. At the same time, reparation of single-strand breaks of DNA caused by bleomycin occurs with similar intensity both in chronically infected and noninfected HEp-2 cultures. The experimental results suggest that the chronic course of infection in human cells leads to disorders in reparative synthesis of cellular DNA and/or is due to disconnected effect of reparation enzymes in this system.     

Comparison of replication of adenovirus type 2 and type 4 in human lymphocyte cultures.

Adenovirus type 2 was capable of replicating in purified lymphocyte cultures from human adenoid specimens. Phytohemagglutinin stimulation enhanced the replication of virus. Viral titers of 103 to 104 50% tissue culture infective doses per ml were reached after 4 to 8 days. Only 1 to 3 per 106 cells were found to produce virus. In contrast, there was no evidence that lymphocyte cultures could support the replication of adenovirus type 4. The life span of cultures infected with type 2 or 4 was not reduced. The possibility that lymphocytes infected with virus play a role in initiating natural, persisting adenovirus infections of human adenoids is discussed.     

Multiplicity-dependent host range restriction of human adenovirus in human embryonal carcinoma cells

The ability of human adenovirus type 5 to replicate and produce infectious virus was examined in two different cell lines, HeLa cells and Tera I, a human embryonal carcinoma cell line. High titers of infectious adenovirus were produced (10⁹ PFU/ml) by growth in HeLa cell cultures independent of the input multiplicity of infection (by 96 hr postinfection). On the other hand, 3–4 logs less infectious virus were synthesized in Tera I cells when low (0.01–0.1 PFU/cell) input multiplicities of infection were used. At input multiplicities of 1, 10, or 100 PFU/cell, $\text{1?1}\text{1}\text{2}$ logs less infectious virus was produced by Tera I cells than HeLa cell cultures at late times (96–144 hr) postinfection. These results demonstrate an adenovirus input multiplicity-dependent, host range restriction in human embryonal carcinoma cells. These observations may provide a means of examining the differences between cells with distinct developmental potentials.     

Role of mycoplasma infection in the cytopathic effect induced by human immunodeficiency virus type 1 in infected cell lines.

In addition to previously reported tetracycline analogs, other antibiotics known for antimycoplasmal activities inhibited the cytopathic effect in CEM cl13 cells infected with human immunodeficiency virus type 1 (HIV-1) or HIV-2 but were unable to block virus replication. A contaminating mycoplasma was isolated from our CEM cl13 cells and identified as a strain of Mycoplasma fermentans. Following infection of lymphoblastoid (CEM) or promonocytic (U937 and THP1) cell lines with HIV-1, cytopathic effect was observed only in association with mycoplasmal contamination. Moreover, HIV-1 infection of U937 cells after experimental inoculation with a human isolate of M. fermentans led to pronounced cell killing. We have verified that this effect is not merely an artifact caused by arginine and/or glucose depletion in the cell culture medium. These results confirm that mollicutes, in particular M. fermentans, are able to act synergistically with HIV-1 to kill infected cells in some in vitro systems.     

Chronically HIV-1 infected human monocyte culture U937 as a model for assessing the efficacy of anti-HIV agents]

Cell culture U937 chronically infected with HIV-1 is suggested as a model for adequate evaluation of antiviral activity of HIV inhibitors. Azidothimidine (AZT) notable decreased HIV-1 reproduction in chronically infected U937 cells to passages 15-18. Glycirrhizic acid (GA) effectively inhibited the virus production during the first four passages, while in subsequent passages (up to passage 20) decreased the virus production by only 60% in comparison with the control. If a combination of AZT and GA (1:1000) was used, p24 was not detected in the culture fluid by passage 20. Culturing of U937 cells with AZT led to a 10-fold decrease in the amount of DNA 2-LTR in comparison with the total content of proviral DNA, the content of HIV-1 DNA 1-LTR remaining virtually unchanged. Culturing of U937 with a combination of AZT and GA resulted in a notable decrease in the content of proviral DNA 2-LTR after passage 3, while after passage 9 this form of HIV-1 DNA was not detected at all.     

Biological characterization of infectious molecular clones derived from a human immunodeficiency virus type-1 isolate with rapid/high replicative capacity.

In order to molecularly characterize rapidly and slowly replicating HIV-1 variants, molecular clones were obtained from a rapid/high virus isolate. This isolate, 4803, had only been passaged in peripheral blood mononuclear cells (PBMC) prior to cloning. Molecular cloning was done in bacteriophage lambda-dash using high molecular weight DNA of isolate 4803 infected PBMC. Seven recombinant phages were identified. The clones were found to be related to each other and differed only at 1 or 2 restriction sites (out of 28). The molecular clones were transfected into various cell types by electroporation. The phenotype of progeny viruses was found to be dependent on the cell type used for transfection. Progeny viruses produced by PBMC cultures differed from the parental isolate in that they did not form syncytia and lacked the capacity to replicate in cell lines. Since transfection of PBMC yielded progeny viruses within 1 week, this phenotype is considered to be the true phenotype of the clones. Transfection of the T-lymphoid HUT-78 cell line and of the monocytoid U937-2 cell line yielded progeny viruses after considerable delay (more than 1 month). Progeny viruses from HUT-78 cells were similar to the parental isolate in that they formed syncytia in PBMC and replicated in all cell lines tested. Progeny viruses from U937-2 cells showed an intermediate phenotype in that they replicated in U937-2 but not in T-lymphoid cell lines. These results indicate that molecular clones of a rapid/high virus may have a restricted replicative capacity compared to the parental, genetically heterogenous virus isolate.     

Enhancement of adenovirus infection in WI-38 and AGMK cells by pretreatment of cells with 5-iododeoxyuridine.

Adenovirus replicates inefficiently in WI-38 and AGMK cells. Treatment of these cells with 5-iododeoxyuridine (IdU) prior to infection enhances the yield of adenovirus by 10- to 1000-fold. IdU-pretreated cultures demonstrate an increased percentage of adenovirus T-antigen-positive cells in comparison to untreated controls. IdU treatment has no effect on virus adsorption, and in AGMK cells IdU enhancement is additive to SV40 enhancement of adenovirus yield. Thus, IdU treatment relieves a restriction to virus growth that occurs prior to T-antigen synthesis. IdU pretreatment has no effect on virus growth in HEK, a permissive cell type. Incorporation of IdU into cellular DNA is required for enhancement.     

Comparative study of variants of the Venezuelan equine encephalomyelitis virus isolated from chronically infected cells by transfection in different cells

Comparative data with regard to the properties of Venezuelan equine encephalomyelitis (VEE) virus isolated from HeLa carrier cultures by transfection in different cell cultures have been obtained. Introduction of DNA extracted from the carrier cultures into BHK-21 cell cultures resulted in production of an actively multiplying medium-plaque virus, and parallel addition of the same DNA preparations in chick fibroblast or monkey kidney cultures led to production of small-plaque virus with a low reproduction potential. The virus produced by transfection of BHK-21 cells differed from that produced in chick fibroblast and monkey kidney cultures in electrophoretic mobility of virion envelope proteins. The infection of these cultures with virions as well as infection with genome RNA did not result in production of differing virus variants. The importance of the experimental genetic data for the problem of the nature of the infectious principle of cellular DNA preparations and of the form of existence of viral genome in chronically infected cultures with infection of the integrative type is discussed.     

Differentiation of the U937 macrophage cell line removes an early block of HSV-1 infection.

In the human macrophage-like cell line U937, which is resistant to infection with herpes simplex virus type 1 (HSV-1), it was previously shown that resistance can be overcome by inducing differentiation of the cells by treatment with phorbol 12-myristate 13-acetate (PMA). The present data show that differentiation, and not PMA treatment alone, enabled HSV-1 replication, because vitamin D3 and mezerein were also able to cause U937 cells to differentiate to a state permissive for HSV-1 infection. Additionally, a portion of the undifferentiated cells underwent a productive infection when treated with PMA 2 days after infection, suggesting persistence of HSV-1 in these cells. The nonpermissiveness of the undifferentiated cells was further defined. Resistance did not involve differences in virus uptake, because the amounts of viral DNA in the infected cells and nuclei of differentiated and undifferentiated U937 cells were not significantly different early after infection. However, only very low levels of RNA for HSV-1 immediate-early, early, and late genes could be detected in the undifferentiated U937 cells by Northern blot analysis compared with the differentiated U937 cells. These data suggest that the primary block in HSV-1 replication in undifferentiated U937 cells occurred after transport of the viral DNA to the cell nucleus but prior to steady-state accumulation of viral RNA for immediate-early genes.     

Dual role of TNF-alpha in NK/LAK cell-mediated lysis of chronically HIV-infected U1 cells. Concomitant enhancement of HIV expression and sensitization of cell-mediated lysis

The U937-derived chronically HIV-infected U1 cell line and uninfected U937 cell clones were efficiently lysed by both unstimulated (NK) and IL-2-stimulated (lymphokine-activated killer; LAK) peripheral blood mononuclear cells (PBMC) of healthy HIV-seronegative donors. Pretreatment of target cells with IFN-gamma down-modulated killing of both U1 cells and two U937 cell clones, and up-regulated MHC class I expression. In contrast, TNF-alpha enhanced the sensitivity of infected U1 cells, but not of U937 cell clones to NK / LAK cell lysis. Co-cultivation of IL-2-stimulated PBMC with U1 cells triggered expression and replication of HIV by cell-cell contact, and this effect was inhibited by anti-TNF-alpha antibodies (Ab); virus production was partially inhibited by zidovudine. Of interest, anti-TNF-alpha Ab protected U1 cells from LAK cell activity. Thus, TNF-alpha can induce HIV expression from chronically infected U1 cells, but also plays an important role in sensitizing these cells to lysis.     

Impairment of Brucella growth in human macrophagic cells that produce nitric oxide

In mice, nitric oxide (NO) production by inducible NO synthase (iNOS), is a component of the control of Brucella infection. In humans, the involvement of iNOS in infection is still a matter of debate. Based on in vitro experiments, it was recently postulated that in humans, Brucella infection tends to become chronic because NO cannot exert its deleterious effect. In fact, conditions allowing NO production by human macrophages in culture are poorly defined, rendering the in vitro study of NO function difficult. Using DFGiNOS U937 macrophagic cells engineered to produce NO and U937 cells activated by ligation of IgE receptors, we showed that the intracellular development of Brucella was impaired in human macrophages, which produced NO. Although Brucella-infected human macrophagic phagocytes did not release NO in commonly used models of infection, the machinery required to produce NO was expressed in these cells and could be triggered by cell membrane receptors present on the infected cells. Therefore, the lack of NO production in isolated human macrophages infected by Brucella under in vitro conditions did not exclude a possible involvement of NO in the control of human brucellosis.     

Differentiation of a human monocytic cell line associated with increased production of Rift Valley fever virus by infected cells

Rift Valley fever (RVF) virus is a cause of significant human and animal disease in many parts of Africa. In some cases, it causes a hemorrhagic fever, which is frequently fatal. Prior studies have shown that RVF virus productively infects peritoneal macrophages from susceptible rat strains. The U937 human monocytic cell line was used to determine the effect of monocytic cell differentiation on the degree of viral production by cell cultures infected with RVF virus. Differentiation of U937 cells to more mature monocytic cells by phorbol ester resulted in production of 10 times more infectious virions in comparison with undifferentiated cells. These studies imply that monocytic cell differentiation increases permissiveness for RVF virus production.     

Adenovirus infection enhances in vitro adherence of Streptococcus pneumoniae.

Viruses are thought to facilitate bacterial infections of the respiratory tract, but the mechanisms are poorly understood. The present study analyzed the effect of adenovirus on bacterial adherence to human respiratory tract epithelial cells. The human lung carcinoma cell line A549 was infected with adenovirus of types 1, 2, 3, 4, 5, and 9. At a multiplicity of infection of 75 particles per cell, cytopathic effects occurred in 75 to 100% of the cells within 48 h. The virus-infected cells were harvested at various times after infection and analyzed for the ability to bind strains of Haemophilus influenzae and Streptococcus pneumoniae. Adenovirus (types 1, 2, 3, and 5) commonly causing respiratory tract infections increased the binding of adherent S. pneumoniae strains to the cells. This effect was not seen for other adenovirus types. Adenovirus infection did not change the adherence of cells of poorly adhering strains of S. pneumoniae or H. influenzae. The increase in adherence of S. pneumoniae could be inhibited by the DNA synthesis inhibitor cytosine arabinofuranoside, which is known to block the late phase of the adenovirus infection. When electron microscopy was used, there was no evidence that virus particles bound directly to bacteria. Adherence was not affected by pretreatment of the cells with virus particles or viral proteins. This suggested that adenovirus infection upregulated receptors for S. pneumoniae. The increased attachment may be one mechanism by which viruses precondition the respiratory mucosa for bacterial infection.