Distinct modes of human immunodeficiency virus type 1 proviral latency revealed by superinfection of nonproductively infected cell lines with recombinant luciferase-encoding viruses.

To study the basis of cellular latency of human immunodeficiency virus (HIV), we have used a recombinant luciferase-encoding HIV (HXB-Luc) to superinfect nonproductively HIV-1-infected human leukemic cell lines. HXB-Luc contains the Photinus pyralis luciferase gene in place of the nef gene and provides a highly sensitive, simple assay for HIV infection and expression. To circumvent any superinfection block in latently infected cells, we also generated viruses pseudotyped with murine leukemia virus amphotropic envelope (HXB-Luc:ampho). The parental uninfected lines, U937 and A3.01, from which the latently infected cell lines U1 and ACH-2, respectively, were derived could be readily infected with pseudotyped or nonpseudotyped reporter viruses. However, superinfection of U1 cells with either HXB-Luc or HXB-Luc:ampho resulted in only low levels of luciferase activity. Like the endogenous provirus, HXB-Luc provirus could be efficiently activated by phorbol ester treatment of HXB-Luc:ampho-superinfected U1 cells. In contrast, superinfection of ACH-2 cells resulted in active expression of the secondarily introduced virus even in unstimulated cells and luciferase production higher than in the parental cell line A3.01. Thus, the proviral latency in U1 cells appears to result from a defect in the cellular environment (a trans effect), whereas the latency in ACH-2 is specific to the integrated provirus and is probably a cis effect due to the site of integration. These results demonstrate distinct modes of proviral latency in these two cell line models and may have implications in our understanding of the regulation and significance of cellular latency in HIV infection.     

Cells nonproductively infected with HIV-1 exhibit an aberrant pattern of viral RNA expression: a molecular model for latency

U1 and ACH-2 cells are subclones of HIV-1-infected monocyte/macrophage-like and T lymphocyte cell lines, respectively, which express the HIV-1 genome at very low levels. We have examined whether they might provide a model of HIV-1 latency. The patterns of HIV-1-specific RNA expressed in these cells consisted of singly and multiply spliced RNA species, with little or no full-length genomic RNA. Upon stimulation with agents that activate the HIV-1 long terminal repeat in these cells, a marked rise in the amount of small mRNAs, encoding the viral regulatory proteins, preceded the increase in the unspliced RNA. Thus, U1 and ACH-2 cells maintain HIV-1 in a state equivalent to the early phase of a lytic infection and, after stimulation, recapitulate the events of a single cycle infection of highly susceptible target cells.     

UV activation of human immunodeficiency virus gene expression in transgenic mice.

Human immunodeficiency virus (HIV) infection is associated with a clinical latency of as long as 10 years before the development of disease. One explanation for this delay is the requirement of cofactors such as other DNA or RNA viruses, cytokines critical for immune modulation, or environmental UV light. At least in tissue culture studies, these agents are capable of inducing HIV gene expression in cell lines which either harbor the entire viral genome or contain a reporter gene under the control of the viral long terminal repeat regulatory region. The role of these cofactors in terminating clinical latency and inducing disease has been difficult to ascertain because of the lack of an appropriate animal model. We now report that UV light can markedly induce HIV gene expression in transgenic mice carrying both the cis-acting (long terminal repeat) and trans-acting (the tat gene) elements which are essential for viral transactivation and replication in infected cells. Our finding may explain the clinical observations that cutaneous lesions in HIV-infected individuals are often seen in the sunlight exposed areas of the skin, including the face and neck.     

Effect of cellular differentiation on cytokine-induced expression of human immunodeficiency virus in chronically infected promonocytic cells: dissociation of cellular differentiation and viral expression.

Cellular differentiation is thought to play an important role in the susceptibility of monocytic lineage cells to human immunodeficiency virus (HIV) infection as well as in their ability to support virus replication. In addition, virus replication in monocytes/macrophages has been demonstrated in vitro to be strongly modulated by several cytokines such as tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor. The purpose of the present study was to investigate the interaction between cellular differentiation and cytokines in the regulation of HIV expression from chronically infected monocytic lineage cells. U1, a persistently HIV-infected promonocytic cell line, is characterized by low levels of virus expression which can be modulated by several cytokines. 1 alpha,-25-Dihydroxyvitamin D3 (Vit.D3), a well-known differentiating agent for myelomonocytic cells which has been previously reported to modulate HIV replication in other in vitro systems, induced maturation of U1 cells toward a macrophage-like phenotype, as demonstrated by the induction of the differentiation-associated cell surface markers CD14 and CD11b. Vit.D3-induced differentiation did not result in induction of HIV expression; however, when U1 cells were stimulated with tumor necrosis factor alpha in the presence of Vit.D3, a synergistic induction of cell differentiation and viral expression was demonstrated. In contrast, Vit.D3 suppressed the induction of HIV expression in U1 cells stimulated with gamma interferon, interleukin-6, and granulocyte-macrophage colony-stimulating factor, although synergy between Vit.D3 and these cytokines was observed in terms of cellular differentiation. These data suggest that differentiation of monocytic cells does not necessarily correlate with increased HIV expression.     

Different sensitivity to apoptosis in cells of monocytic or lymphocytic origin chronically infected with human immunodeficiency virus type-1

Apoptotic death of CD4+ T lymphocytes is a major cause of the immunodeficiency caused by human immunodeficiency virus (HIV), but it is still unclear how this process precisely occurs. To characterize a potentially useful cellular model, we have analyzed the tendency of chronically HIV-infected CD4+ human cell lines of different origin to undergo apoptosis. We studied ACH-2 and U1 lines, derived from the CD4+ T-cell A301 and the promonocytic U937 cell lines, respectively, and induced apoptosis via several stimuli that trigger different pathways. Their capacity to regulate plasma membrane CD95 expression and to produce soluble CD95 was also analyzed. Using staurosporine, TNF-alpha plus cycloheximide, and gamma-radiations, we observed that ACH-2 were more sensitive to programmed cell death than A301, while U1 were less sensitive than U937. Both infected cell types had a lower sensitivity to CD95-induced apoptosis; the analysis of changes in mitochondrial membrane potential corroborated these observations. Plasma membrane CD95 was similarly regulated in all cell types, which, however, presented a different capacity to produce soluble CD95 molecules. Our in vitro results may offer a new perspective for developing further studies on the pathogenesis of HIV infection. A chronically infected cell line of lymphocytic origin is more susceptible to apoptosis than its parental cell type, while infected monocytic cells are less sensitive than their uninfected counterpart. Thus, it is possible to hypothesize that one of the reasons by which circulating monocytes survive and represent a viral reservoir is the capacity of HIV to decrease the sensitivity to apoptosis of this cell type. However, further studies on ex-vivo collected fresh cells, as well as on other cell lines, are urgently needed to confirm such hypothesis.     

Cytokine-mediated induction of human immunodeficiency virus (HIV) expression and cell death in chronically infected U1 cells: do tumor necrosis factor alpha and gamma interferon selectively kill HIV-infected cells?

Infection with several DNA or RNA viruses induces a state of increased sensitivity to cell lysis mediated by tumor necrosis factor (TNF), particularly in the presence of gamma interferon (IFN-gamma). Infection of human cells with the human immunodeficiency virus (HIV) may induce a similar phenomenon. However, TNF and IFN-gamma are known upregulators of HIV replication, raising the question of the potential role of these cytokines in the selective elimination of cells infected with this virus. The present study demonstrates that chronically infected U1 cells were killed with much greater efficiency by costimulation with TNF-alpha and IFN-gamma than their uninfected parental cell line U937. However, synergistic induction of viral expression also occurred in U1 cells as a consequence of treatment with the two cytokines. Cell death in U1 cells was not caused by the massive production of virions, in that costimulation with glucocorticoid hormones and TNF-alpha or IFN-gamma resulted in high levels of virion production without cytopathicity. To investigate the nature of the selective cytotoxic effect observed in U1 cells costimulated with TNF-alpha plus IFN-gamma, a panel of uninfected cell clones was generated by limiting dilution of U937 cells and tested for response to TNF-alpha and/or IFN-gamma. In contrast to the uncloned bulk parental U937 cell line, most uninfected cell clones showed a very high susceptibility to being killed by TNF-alpha and IFN-gamma. Similar findings were obtained when both infected U1 cells and several uninfected U937 cell clones were costimulated with an anti-Fas monoclonal antibody in the presence of IFN-gamma, although, unlike cells stimulated with TNF-alpha, cells treated with anti-Fas antibody did not express virus. Therefore, the increased susceptibility to cytokine-mediated lysis observed in cell lines infected with HIV is likely due to the selection of preexisting cell clones rather than viral infection.     

Alpha interferon suppresses virion but not soluble human immunodeficiency virus antigen production in chronically infected T-lymphocytic cells.

Alpha interferon (IFN-alpha) is effective in preventing the release of human immunodeficiency virus (HIV) from chronically infected T-lymphocytic (ACH-2) and promonocytic (U1) cell lines stimulated with the phorbol ester phorbol-12-myristate-13 acetate (PMA). In the present study, we observed that together with particle production, shedding of HIV antigen (p24gag) occurs in the T-cell line ACH-2 both constitutively and after stimulation with PMA. IFN-alpha, although effective in suppressing the release of HIV particles, did not inhibit shedding of p24gag into the culture supernatants of either unstimulated or PMA-stimulated cells. These observations may be of relevance in the evaluation of the in vivo efficacy of IFN-alpha treatment of HIV-infected individuals as determined by levels of p24 antigen in plasma.     

Cell-specific differences in activation of NF-kappa B regulatory elements of human immunodeficiency virus and beta interferon promoters by tumor necrosis factor.

Three aspects of the involvement of tumor necrosis factor in human immunodeficiency virus (HIV) pathogenesis were examined. Tumor necrosis factor alpha (TNF-alpha) mRNA production was analyzed by polymerase chain reaction amplification in monocytic U937 cells and in a chronically HIV infected U937 cell line (U9-IIIB). TNF-alpha RNA was undetectable in U937 cells, whereas a low constitutive level was detected in U9-IIIB cells. Paramyxovirus infection induced a 5- to 10-fold increase in the steady-state level of TNF-alpha RNA in U9-IIIB cells compared with U937 cells, suggesting that HIV-infected monocytic cells produced higher levels of TNF-alpha than did normal cells after a secondary virus infection. The effects of TNF-alpha on gene expression were examined by transient expression assays using reporter chloramphenicol acetyltransferase plasmids linked to regulatory elements from the HIV long terminal repeat (LTR) and the beta interferon promoter. In U937 and Jurkat T lymphoid cells, the inducibility of the different hybrid promoters by TNF-alpha or phorbol ester varied in a cell type- and promoter context-specific manner; the levels of gene activity of NF-kappa B-containing plasmids correlated directly with induction of NF-kappa B DNA-binding activity. Although the intact beta interferon promoter was only weakly stimulated by phorbol ester or TNF-alpha, multimers of the PRDII NF-kappa B-binding domain were inducible by both agents. TNF-alpha was able to increase expression of the HIV LTR in T cells, but in monocytic cells, TNF-alpha did not induce the HIV LTR above a constitutive level of activity. This level of NF-kappa B-independent activity appears to be sufficient for virus multiplication, since TNF-alpha treatment had no effect on the kinetics of de novo HIV type 1 (HIV-1) infection and viral RNA production in U937 cells. However, in Jurkat cells, TNF-alpha dramatically enhanced the spread of HIV-1 through the cell population and increased viral RNA synthesis, indicating that in T cells HIV-1 multiplication was stimulated by TNF-alpha treatment.     

Recombinant gp120 specifically enhances tumor necrosis factor-alpha production and Ig secretion in B lymphocytes from HIV-infected individuals but not from seronegative donors

The effect of recombinant protein from the envelope (gp120) of the HIV on B lymphocytes purified from either HIV-infected individuals or healthy seronegative controls was examined. B cells from peripheral blood and lymph nodes of HIV-infected individuals spontaneously secreted TNF-alpha; this secretion was augmented by the presence of gp120, whereas B cells from healthy seronegative donors failed to secrete significant levels of TNF-alpha in the presence or absence of gp120. In a coculture system of B cells and chronically HIV-infected T cells (ACH-2), where viral expression is largely mediated by TNF-alpha, gp120 increased virus expression only if the B cells were obtained from HIV-infected individuals. The effects of gp120 on viral expression in this system were not mediated via CD4 receptor binding or FcR binding of anti gp120-gp120 immune complexes. Besides its effect on cytokine production, gp120 also stimulated Ig secretion in B cells from HIV-infected individuals, but not from normal donors. Finally, it was demonstrated by in situ hybridization that germinal centers of lymph nodes from HIV-infected individuals contain large amounts of HIV RNA that is in close proximity to germinal center B cells. These findings suggest that the hyperplastic germinal centers of lymph nodes provide an unique environment for virus expression and accumulation where gp120 stimulates B cells to secrete HIV inductive cytokines, such as IL-6 and TNF-alpha, and thereby further enhances virus expression in infected cells in a paracrine manner.     

Herpes simplex virus type 1-mediated induction of human immunodeficiency virus type 1 provirus correlates with binding of nuclear proteins to the NF-kappa B enhancer and leader sequence.

Herpes simplex virus type 1 (HSV-1) infection induces expression of the human immunodeficiency virus type 1 (HIV-1) provirus in the chronically infected T-cell line ACH-2. The HSV-1-mediated induction correlates with the appearance of two NF-kappa B-specific proteins of 55 and 85 kDa in the nucleus and with the binding of 50-kDa nuclear protein to the LBP-1 binding site of the untranslated leader sequence of the HIV-1 long terminal repeat. The HSV-1-induced LBP-1 binding protein, designated HLP-1, is present exclusively in HSV-1-infected, but not in phorbol-12-myristate-13-acetate- or tumor necrosis factor alpha-treated ACH-2 cells. Both the NF-kappa B and LBP-1 target sequences, when inserted either alone or together 5' of a heterologous minimal promoter (thymidine kinase), confer inducibility by HSV-1 infection in a transient transfection assay. Thus, it appears that the HSV-1-mediated activation of HIV-1 provirus is brought about by the binding of both NF-kappa B and HLP-1 specific proteins to two distinct regions of HIV-1 long terminal repeat.     

TAR independent activation of the human immunodeficiency virus in phorbol ester stimulated T lymphocytes.

Multiple regulatory elements in the human immunodeficiency virus long terminal repeat (HIV LTR) are required for activation of HIV gene expression. Previous transfection studies of HIV LTR constructs linked to the chloramphenicol acetyltransferase gene indicated that multiple regulatory regions including the enhancer, SP1, TATA and TAR regions were important for HIV gene expression. To characterize these regulatory elements further, mutations in these regions were inserted into both the 5' and 3' HIV LTRs and infectious proviral constructs were assembled. These constructs were transfected into either HeLa cells, Jurkat cells or U937 cells in both the presence and absence of phorbol esters which have previously been demonstrated to activate HIV gene expression. Viral gene expression was assayed by the level of p24 gag protein released from cultures transfected with the proviral constructs. Results in all cell lines indicated that mutations of the SP1, TATA and the TAR loop and stem secondary structure resulted in marked decreases in gene expression while mutations of the enhancer motif or TAR primary sequence resulted in only slight decreases. However, viruses containing mutations in either the TAR loop sequences or stem secondary structure which were very defective for gene expression in untreated Jurkat cells, gave nearly wild-type levels of gene expression in phorbol ester-treated Jurkat cells but not in phorbol ester-treated HeLa or U937 cells. High level gene expression of these TAR mutant constructs in phorbol ester-treated Jurkat cells was eliminated by second site mutations in the enhancer region or by disruption of the tat gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Failure of human immunodeficiency virus entry and infection in CD4-positive human brain and skin cells.

CD4 molecules on human cells function as a major receptor for human immunodeficiency virus (HIV); however, certain CD4-negative cell types may also be susceptible to infection. Therefore, we attempted to quantitate the relationship between HIV infection and CD4 expression on human cell lines before and after introduction of the CD4 gene by using a retrovirus vector. Prior to introduction of the CD4 expression vector, low levels of HIV infection were detected by a sensitive focal immunoassay on all three cell types studied. With several HIV strains in clones of human cervical carcinoma (HeLa) cells expressing different levels of CD4, HIV titer increased with increasing CD4 expression. In contrast, in squamous cell carcinoma cells (SCL1) and astroglial cells (U87MG), even high levels of CD4 expression failed to augment HIV infection. The CD4 protein expressed in these two cell lines had the expected molecular weight and was capable of binding HIV virions. However, in contrast to CD4-positive HeLa cells, CD4-positive U87MG and SCL1 cells were unable to form syncytia when cultured with cells expressing HIV envelope protein. Thus, the inability of HIV to infect these cells appeared to be due to lack of fusion between HIV virion envelope proteins and CD4-positive cell membranes. This block is infectivity was overcome when cells were infected with HIV which was pseudotyped with the envelope protein of amphotropic murine leukemia virus. Thus, in addition to CD4, other cell surface molecules appear to be required for successful HIV entry into and infection of these two human cell lines.     

c-Myc and Sp1 contribute to proviral latency by recruiting histone deacetylase 1 to the human immunodeficiency virus type 1 promoter

Histone deacetylase (HDAC) inhibitors such as valproic acid (VPA) induce the expression of quiescent proviral human immunodeficiency virus type 1 (HIV-1) and may deplete proviral infection in vivo. To uncover novel molecular mechanisms that maintain HIV latency, we sought cellular mRNAs whose expression was diminished in resting CD4(+) T cells of HIV-1-infected patients exposed to VPA. c-Myc was prominent among genes markedly downregulated upon exposure to VPA. c-Myc expression repressed HIV-1 expression in chronically infected cell lines. Chromatin immunoprecipitation (ChIP) assays revealed that c-Myc and HDAC1 are coordinately resident at the HIV-1 long terminal repeat (LTR) promoter and absent from the promoter after VPA treatment in concert with histone acetylation, RNA polymerase II recruitment, and LTR expression. Sequential ChIP assays demonstrated that c-Myc, Sp1, and HDAC1 coexist in the same DNA-protein complex at the HIV promoter. Short hairpin RNA inhibition of c-Myc reduces both c-Myc and HDAC1 occupancy, blocks c-Myc repression of Tat activation, and increases LTR expression. These results expand the understanding of mechanisms that recruit HDAC and maintain the latency of HIV-1, suggesting novel therapeutic approaches against latent proviral HIV infection.