High susceptibility of U937-derived subclones to human immunodeficiency virus type 1 infection correlates with accumulation of unintegrated circular viral DNA

Our previous report showed that U937-derived subclones were differentiated into at least three types (high, middle, and low types), even in the subclones expressing similar levels of surface CD4, in terms of the kinetics of the appearance of viral antigens and virus production after infection with human immunodeficiency virus type 1 (HIV-1). Here we showed the evidence that high susceptibility to HIV-1 infection, which was confirmed by the profound expression of viral messages and antigens, was exclusively associated with a high number of the unintegrated extrachromosomal form of viral DNA, but not with the amounts of adsorbed virus RNA nor those of integrated DNA form. The difference in the amounts of extrachromosomal form of viral DNA was also observed in the culture with 3-azido-3-deoxythymidine (AZT), indicating that the susceptibility is essentially unrelated to multiple infection events. Thus, the susceptibility of U937-derived subclones to HIV-1 infection seems to be affected by the occurrence of specific events involved in the accumulation of unintegrated viral DNA after viral adsorption.     

Establishment of novel cell lines latently infected with human immunodeficiency virus 1

Many human immunodeficiency virus 1 (HIV-1) researchers focus on the developing new anti-reservoir therapy to eradicate HIV-1 provirus from the HIV-1-infected patients. HIV-1 provirus is the major obstacle for effective HIV-1 treatment because it integrates into the host genome and can produce a virus progeny after stopping highly active antiretroviral therapy (HAART). We established two novel cell lines latently infected with HIV-1 by limiting dilution cloning of A3.01 cells infected with HIV-1. Analysis of the flanking sequence of HIV-1 proviral DNA integrated into chromosomal cellular DNA revealed that proviral DNA was inserted into different sites of different chromosomes in the two examined cell lines. In these lines, virus reactivation could be induced by a phorbol 12-myristate 13-acetate (PMA) treatment that resulted in a marked increase of the production HIV-1 p24 antigen and appearance of the infectious virus. The novel cell lines latently infected with HIV-1 represent further tool for the study of molecular mechanisms of viral latency and development of anti-reservoir therapy of HIV-1 infection.     

Analysis of the basis for persistence of herpes simplex virus type 1 in undifferentiated U937 cells.

Replication of herpes simplex type 1 (HSV-1) is inhibited in the human monocyte-like cell line, U937, when the cells are in the undifferentiated state, but when the cells are stimulated to differentiate by treatment with the phorbol ester, phorbol 12-myristate 13-acetate virus is replicated. Because HSV-1 has been shown to persist in these cells and in their in vitro counterparts freshly isolated human blood monocytes, we initiated an analysis of viral persistence in undifferentiated U937 cells. No appreciable HSV-1 DNA replication was observed in undifferentiated U937 cells compared with differentiated U937 cells and with fully permissive Vero cells. However, using in situ hybridization, we established that a significant percent of the undifferentiated U937 cells contained viral DNA sequences. Interestingly, when analyzed by Southern blot hybridization, this DNA was found to have assumed a nonlinear configuration similar to that found in latently infected neurons. Analysis of viral proteins in undifferentiated U937 cells revealed a marked absence of proteins of all three kinetic classes. However, in transient transfection assays, the major viral transactivating protein ICP4, functioned normally, whereas ICP0, a promiscuous transactivator of both viral and cellular genes, was unable to transactivate viral promoters in undifferentiated U937 cells. Thus, a subtle dysfunction in the activity of ICP0 may account, at least in part, for the inability of undifferentiated U937 cells to support replication of HSV-1.     

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.     

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.     

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.     

Two bases are deleted from the termini of HIV-1 linear DNA during integrative recombination

Proviral integration is a required step in the retrovirus life cycle. The mechanism of integration involves specific modification of the ends of linear viral DNA and subsequent recombination with host sequences. Integration results in the limited loss of sequence information at the termini of the viral genome. The composition of the intact linear DNA termini were inferred by sequencing the 2-long terminal repeat (2-LTR) circle junction that is formed when the linear molecule undergoes intramolecular, blunt-end ligation. The junction sequence contained the nucleotides GTAC that were not present at the ends of the integrated provirus. Comparison with the sequence of the LAV-1 strain of HIV-1 demonstrated that the GT dinucleotide derived from the right-hand terminus (U5) of the linear viral DNA and the AC dinucleotide came from the left-hand terminus (U3). Therefore, the corrected size of the LAV-1 LTR is 637 bp. This conclusion was confirmed independently by assessing the structure of linear viral DNA in acutely infected T cells. A portion of the population of linear HIV-1 DNA molecules were specifically deleted at their 3' ends; the extent of this deletion was 2 bases. This result is consistent with the activity of viral integrase protein on linear viral DNA and it accounts for the structure of integrated HIV-1 proviruses.     

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.     

Type I interferon is a powerful inhibitor of in vivo HIV-1 infection and preserves human CD4(+) T cells from virus-induced depletion in SCID mice transplanted with human cells.

Although several studies are available on the in vitro inhibitory activities of type I interferon (IFN) on HIV-1 replication, the role of these cytokines in the pathogenesis of AIDS is still matter of conjecture. Both beneficial and adverse effects have been envisaged and considered as a possible rationale for the development of either IFN or anti-IFN therapies in HIV-1-infected patients. In the present study, we have evaluated the efficacy of human type I IFN on HIV-1 infection and virus-induced depletion of human CD4 T cells in two models established in SCID mice. In SCID mice transplanted with human U937 cells (U937-SCID mouse model), continuous treatment with type I consensus IFN (CIFN) resulted in a total suppression of HIV-1 infection. This inhibitory effect was superior to that obtained after AZT treatments. Results from an ensemble of experiments in SCID mice transplanted with either control or genetically modified human U937 cells transduced with a Tat-inducible IFN-alpha gene (LTR-IFN-A2 U937) indicated that low levels of IFN-alpha, produced locally as a result of virus infection, were extremely effective in inhibiting acute HIV infection and virus replication. Of interest, LTR-IFN-A2 U937 cells conferred a strong anti-HIV-1 protection to coinjected bystander U937 cells. Notably, experiments with SCID mice reconstituted with human PBL (hu-PBL-SCID mouse model) showed that treatment with CIFN inhibited HIV-1 replication more effectively than AZT treatment. Remarkably, treatment with CIFN resulted in a clear-cut protection from the virus-induced depletion of human CD4 T cells, which was also associated with the generation of an antibody response toward HIV-1 antigens in 50% of the virus-injected xenografts. These results suggest that type I IFN efficiently preserves human CD4(+) cells from virus-induced damage in hu-PBL-SCID mice, not only by inducing an antiviral state in target cells but also by stimulating anti-HIV-1 human immune responses in vivo.     

Identification of autonomous replication sequences in genomic and mitochondrial DNA of Crithidia fasciculata

High frequency transformation of Saccharomyces cerevisiae was used as a functional assay to isolate autonomous replication sequences (ars) from the genomic and kinetoplast DNA of the insect trypanosomatid Crithidia fasciculata. Three independent cloned genomic sequences and one kinetoplast DNA sequence promoted high frequency transformation and extrachromosomal maintenance of the YIp5 plasmid DNA in yeast. The kinetoplast DNA clone was sub-cloned to further localize the DNA sequence essential for ars activity. This element was shown to be contained in a 2 kb HindIII-EcoRI fragment derived from a 8 kb HindIII fragment of the maxicircle component of the kinetoplast DNA. This 2 kb fragment is within a DNA sequence that has been shown to strongly hybridize to Trypanosoma brucei maxicircle DNA.