Induction of the WAF1/CIP1 protein and apoptosis in human T-cell leukemia virus type I-transformed lymphocytes after treatment with adriamycin by using a p53-independent pathway.

The WAF1/CIP1 protein has been identified as a downstream mediator of the tumor suppressor p53 in regulating cell cycle progression through a G1-phase check-point. Recent work has implicated the functional status of p53 as a critical determinant in the apoptotic response of certain cell lines to DNA damaging agents. By using human T-cell leukemia virus type I-transformed lymphoid cell lines that differ in their level and function of wild-type p53, we investigated the induction of WAF1/CIP1 and apoptosis after exposure to Adriamycin, a genotoxic agent. We found that regardless of the p53 status in these cell lines, WAF1/CIP1 RNA was rapidly induced in response to Adriamycin treatment. An elevated level of WAF1/CIP1 protein was observed as well. Additionally, we demonstrated that apoptosis was induced in all cell lines analyzed despite some having functionally inactive p53 protein. Our data suggest that a p53-independent pathway may play a role in the apoptotic response observed in some cell lines after exposure to DNA damaging agents.

Phosphorylation-dependent human immunodeficiency virus type 1 infection and nuclear targeting of viral DNA.

In the replication of human immunodeficiency virus type 1 (HIV-1), gag MA (matrix), a major structural protein of the virus, carries out opposing targeting functions. During virus assembly, gag MA is cotranslationally myristoylated, a modification required for membrane targeting of gag polyproteins. During virus infection, however, gag MA, by virtue of a nuclear targeting signal at its N terminus, facilitates the nuclear localization of viral DNA and establishment of the provirus. We now show that phosphorylation of gag MA on tyrosine and serine prior to and during virus infection facilitates its dissociation from the membrane, thus allowing it to translocate to the nucleus. Inhibition of gag MA phosphorylation either on tyrosine or on serine prevents gag MA-mediated nuclear targeting of viral nucleic acids and impairs virus infectivity. The requirement for gag MA phosphorylation in virus infection is underscored by our finding that a serine/threonine kinase is associated with virions of HIV-1. These results reveal a novel level of regulation of primate lentivirus infectivity.

Inhibition of human immunodeficiency virus type 1 and vaccinia virus infection by a dominant negative factor of the interferon regulatory factor family expressed in monocytic cells.

ICSBP is a member of the interferon (IFN) regulatory factor (IRF) family that regulates expression of type I interferon (IFN) and IFN-regulated genes. To study the role of the IRF family in viral infection, a cDNA for the DNA-binding domain (DBD) of ICSBP was stably transfected into U937 human monocytic cells. Clones that expressed DBD exhibited a dominant negative phenotype and did not elicit antiviral activity against vesicular stomatitis virus (VSV) infection upon IFN treatment. Most notably, cells expressing DBD were refractory to infection by vaccinia virus (VV) and human immunodeficiency virus type 1 (HIV-1). The inhibition of VV infection was attributed to defective virion assembly, and that of HIV-1 to low CD4 expression and inhibition of viral transcription in DBD clones. HIV-1 and VV were found to have sequences in their regulatory regions similar to the IFN-stimulated response element (ISRE) to which IRF family proteins bind. Accordingly, these viral sequences and a cellular ISRE bound a shared factor(s) expressed in U937 cells. These observations suggest a novel host-virus relationship in which the productive infection of some viruses is regulated by the IRF-dependent transcription pathway through the ISRE.

Sleep electroencephalogram delta-frequency amplitude, night plasma levels of tumor necrosis factor alpha, and human immunodeficiency virus infection.

We tested the hypothesis that increases in tumor necrosis factor alpha (TNF-alpha) induced by human immunodeficiency virus (HIV) are associated with the increases in slow-wave sleep seen in early HIV infection and the decrease with sleep fragmentation seen in advanced HIV infection. Nocturnal sleep disturbances and associated fatigue contribute to the disability of HIV infection. TNF-alpha causes fatigue in clinical use and promotes slow-wave sleep in animal models. With slow progress toward a vaccine and weak effects from current therapies, efforts are directed toward extending productive life of HIV-infected individuals and shortening the duration of disability in terminal illness. We describe previously unrecognized nocturnal cyclic variations in plasma levels of TNF-alpha in all subjects. In 6 of 10 subjects (1 control subject, 3 HIV-seropositive patients with CD4+ cell number > 400 cells per microliters, and 2 HIV-positive patients with CD4+ cell number < 400 cells per microliters), these fluctuations in TNF-alpha were coupled to the known rhythm of electroencephalogram delta amplitude (square root of power) during sleep. This coupling was not present in 3 HIV-positive subjects with CD4+ cell number < 400 cells per microliters and 1 control subject. In 5 HIV subjects with abnormally low CD4+ cell counts ( < 400 cells per microliters), the number of days since seroconversion correlated significantly with low correlation between TNF-alpha and delta amplitude. We conclude that a previously unrecognized normal, physiological coupling exists between TNF-alpha and delta amplitude during sleep and that the lessened likelihood of this coupling in progressive HIV infection may be important in understanding fatigue-related symptoms and disabilities.

Maternal plasma human immunodeficiency virus type 1 RNA level: a determinant and projected threshold for mother-to-child transmission.

To prevent mother-to-child human immunodeficiency virus type 1 (HIV-1) transmission, it is important to identify its determinants. Because HIV-1 RNA levels can be reduced by antiviral therapy, we examined the role of maternal plasma HIV-1 RNA level in mother-to-child transmission. We used quantitative competitive PCR to measure HIV-RNA in 30 infected pregnant women and then followed their infants prospectively; 27% of the women transmitted HIV-1 to their infants and maternal plasma HIV-1 RNA level correlated strikingly with transmission. Eight of the 10 women with the highest HIV-1 RNA levels at delivery (190,400-1,664,100 copies per ml of plasma) transmitted, while none of the 20 women with lower levels (500-155,800 copies per ml) did (P = 0.0002). Statistical analysis of the distribution of HIV-1 RNA loads in these 30 women projected a threshold for mother-to-child transmission in a larger population; the probability of a woman with a viral RNA level of < or = 100,000 copies per ml not transmitting is predicted to be 97%. Examination of serial HIV-1 RNA levels during pregnancy showed that viral load was stable in women who did not initiate or change antiviral therapy. These data identify maternal plasma HIV-1-RNA level as a major determinant of mother-to-child transmission and suggest that quantitation of HIV-1 RNA may predict the risk of transmission.

Using in vitro selection to direct the covalent attachment of human immunodeficiency virus type 1 Rev protein to high-affinity RNA ligands.

We have used an in vitro selection procedure called crosslinking SELEX (SELEX = systematic evolution of ligands by exponential enrichment) to identify RNA sequences that bind with high affinity and crosslink to the Rev protein from human immunodeficiency virus type 1 (HIV-1). A randomized RNA library substituted with the photoreactive chromophore 5-iodouracil was irradiated with monochromatic UV light in the presence of Rev. Those sequences with the ability to photocrosslink to Rev were partitioned from the rest of the RNA pool, amplified, and used for the next round of selection. Rounds of photocrosslinking selection were alternated with rounds of selection for RNA sequences with high affinity to Rev. This iterative, dual-selection method yielded RNA molecules with subnanomolar dissociation constants and high efficiency photocrosslinking to Rev. Some of the RNA molecules isolated by this procedure form a stable complex with Rev that is resistant to denaturing gel electrophoresis in the absence of UV irradiation. In vitro selection of nucleic acids by using modified nucleotides allows the isolation of nucleic acid molecules with potentially limitless chemical capacities to covalently attack a target molecule.

Major histocompatibility complex class II DR alleles DRB1*1501 and those encoding HLA-DR13 are preferentially associated with a diminution in maternally transmitted human immunodeficiency virus 1 infection in different ethnic groups: determination by an automated sequence-based typing method.

Transmission of human immunodeficiency virus 1 (HIV-1) from an infected women to her offspring during gestation and delivery was found to be influenced by the infant's major histocompatibility complex class II DRB1 alleles. Forty-six HIV-infected infants and 63 seroreverting infants, born with passively acquired anti-HIV antibodies but not becoming detectably infected, were typed by an automated nucleotide-sequence-based technique that uses low-resolution PCR to select either the simpler Taq or the more demanding T7 sequencing chemistry. One or more DR13 alleles, including DRB1*1301, 1302, and 1303, were found in 31.7% of seroreverting infants and 15.2% of those becoming HIV-infected [OR (odds ratio) = 2.6 (95% confidence interval 1.0-6.8); P = 0.048]. This association was influenced by ethnicity, being seen more strongly among the 80 Black and Hispanic children [OR = 4.3 (1.2-16.4); P = 0.023], with the most pronounced effect among Black infants where 7 of 24 seroreverters inherited these alleles with none among 12 HIV-infected infants (Haldane OR = 12.3; P = 0.037). The previously recognized association of DR13 alleles with some situations of long-term nonprogression of HIV suggests that similar mechanisms may regulate both the occurrence of infection and disease progression after infection. Upon examining for residual associations, only only the DR2 allele DRB1*1501 was associated with seroreversion in Caucasoid infants (OR = 24; P = 0.004). Among Caucasoids the DRB1*03011 allele was positively associated with the occurrence of HIV infection (P = 0.03).

Parvovirus B19 promoter at map unit 6 confers autonomous replication competence and erythroid specificity to adeno-associated virus 2 in primary human hematopoietic progenitor cells.

The pathogenic human parvovirus B19 is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells. Although the target cell specificity for B19 infection has been suggested to be mediated by the erythrocyte P-antigen receptor (globoside), a number of nonerythroid cells that express this receptor are nonpermissive for B19 replication. To directly test the role of expression from the B19 promoter at map unit 6 (B19p6) in the erythroid cell specificity of B19, we constructed a recombinant adeno-associated virus 2 (AAV), in which the authentic AAV promoter at map unit 5 (AAVp5) was replaced by the B19p6 promoter. Although the wild-type (wt) AAV requires a helper virus for its optimal replication, we hypothesized that inserting the B19p6 promoter in a recombinant AAV would permit autonomous viral replication, but only in erythroid progenitor cells. In this report, we provide evidence that the B19p6 promoter is necessary and sufficient to impart autonomous replication competence and erythroid specificity to AAV in primary human hematopoietic progenitor cells. Thus, expression from the B19p6 promoter plays an important role in post-P-antigen receptor erythroid-cell specificity of parvovirus B19. The AAV-B19 hybrid vector system may also prove to be useful in potential gene therapy of human hemoglobinopathies.

Identification of a hexapeptide inhibitor of the human immunodeficiency virus integrase protein by using a combinatorial chemical library.

Integration of human immunodeficiency virus (HIV) DNA into the human genome requires the virus-encoded integrase (IN) protein, and therefore the IN protein is a suitable target for antiviral strategies. To find a potent HIV IN inhibitor, we screened a "synthetic peptide combinatorial library." We identified a hexapeptide with the sequence HCKFWW that inhibits IN-mediated 3'-processing and integration with an IC50 of 2 microM. The peptide is active on IN proteins from other retroviruses such as HIV-2, feline immunodeficiency virus, and Moloney murine leukemia virus, supporting the notion that a conserved region of IN is targeted. The hexapeptide was also tested in the disintegration reaction. This phosphoryl-transfer reaction can be carried out by the catalytic core of IN alone, and the peptide HCKFWW was found to inhibit this reaction, suggesting that the hexapeptide acts at or near the catalytic site of IN. Identification of an IN hexapeptide inhibitor provides proof of concept for the approach, and, moreover, this peptide may be useful for structure-function analysis of IN.

Human immunodeficiency virus 1 envelope-initiated G2-phase programmed cell death.

Despite intensive investigation, no clearly defined mechanism explaining human immunodeficiency virus (HIV)-induced cell killing has emerged. HIV-1 infection is initiated through a high-affinity interaction between the HIV-1 external envelope glycoprotein (gp120) and the CD4 receptor on T cells. Cell killing is a later event intimately linked by in vitro genetic analyses with the fusogenic properties of the HIV envelope glycoprotein gp120 and transmembrane glycoprotein gp41. In this report, we describe aberrancies in cell cycle regulatory proteins initiated by cell-cell contact between T cells expressing HIV-1 envelope glycoproteins and other T cells expressing CD4 receptors. Cells rapidly accumulate cyclin B protein and tyrosine-hyperphosphorylated p34cdc2 (cdk1) kinase, indicative of cell cycle arrest at G2 phase. Moreover, these cells continue to synthesize cyclin B protein, enlarge and display an abnormal ballooned morphology, and disappear from the cultures in a pattern previously described for cytotoxicity induced by DNA synthesis (S phase) inhibitors. Similar changes are observed in peripheral blood mononuclear cells infected in vitro with pathogenic primary isolates of HIV-1.

Production of avian leukosis virus particles in mammalian cells can be mediated by the interaction of the human immunodeficiency virus protein Rev and the Rev-responsive element.

In human immunodeficiency virus type 1-infected cells, the efficient expression of viral proteins from unspliced and singly spliced RNAs is dependent on two factors: the presence in the cell of the viral protein Rev and the presence in the viral RNA of the Rev-responsive element (RRE). We show here that the HIV-1 Rev/RRE system can increase the expression of avian leukosis virus (ALV) structural proteins in mammalian cells (D-17 canine osteosarcoma) and promote the release of mature ALV virions from these cells. In this system, the Rev/RRE interaction appears to facilitate the export of full-length unspliced ALV RNA from the nucleus to the cytoplasm, allowing increased production of the ALV structural proteins. Gag protein is produced in the cytoplasm of the ALV-transfected cells even in the absence of a Rev/RRE interaction. However, a functional Rev/RRE interaction increases the amount of Gag present intracellularly and, more strikingly, results in the release of mature ALV particles into the supernatant. RCAS virus containing an RRE is replication-competent in chicken embryo fibroblasts; however, we have been unable to determine whether the particles produced in D-17 cells are as infectious as the particles produced in chicken embryo fibroblasts.

Coexpression of NF-kappa B/Rel and Sp1 transcription factors in human immunodeficiency virus 1-induced, dendritic cell-T-cell syncytia.

Productive infection of T cells with human immunodeficiency virus 1 (HIV-1) typically requires that the T cells be stimulated with antigens or mitogens. This requirement has been attributed to the activation of the transcription factor NF-kappa B, which synergizes with the constitutive transcription factor Sp1 to drive the HIV-1 promoter. Recently, we have found that vigorous replication of HIV-1 takes place in nonactivated memory T cells after syncytium formation with dendritic cells (DCs). These syncytia lack activated cells as determined by an absence of staining for Ki-67 cell cycle antigen. The expression and activity of NF-kappa B and Sp1 were, therefore, analyzed in isolated T cells and DCs from humans and mice. We have used immunolabeling, Western blot analysis, and electrophoretic mobility shift and supershift assays. T cells lack active NF-kappa B but express Sp1 as expected. DCs express high levels of all known NF-kappa B and Rel proteins, with activity residing primarily within RelB, p50, and p65. However, DCs lack Sp1, which may explain the failure of HIV-1 to replicate in purified DCs. Coexpression of NF-kappa B and Sp1 occurs in the heterologous DC-T-cell syncytia that are induced by HIV-1. Therefore, HIV-1-induced cell fusion brings together factors that upregulate virus transcription. Since DCs and memory T cells frequently traffic together in situ, these unusual heterologous syncytia could develop in infected individuals and lead to chronic HIV-1 replication without ostensible immune stimulation.

Reactivated and latent varicella-zoster virus in human dorsal root ganglia.

Ganglia obtained at autopsy were examined by in situ hybridization from one patient with zoster (also called herpes zoster or shingles), two varicella-zoster virus (VZV)-seropositive patients with clinical evidence of zoster, one VZV-seronegative child, and one fetus. Ganglia positive for VZV had a hybridization signal in both neuronal and nonneuronal satellite cells. Ganglia obtained from the fetus and from the seronegative infant were consistently negative for VZV. Two striking observations were evident regarding the presence of VZV DNA in ganglia obtained from the individual with zoster at the time of death. First, ganglia innervating the sites of reactivation and ganglia innervating adjacent sites yielded strongly positive signals in neurons and satellite cells, whereas ganglia from distant sites were rarely positive. Second, VZV DNA was found in both the nuclei and the cytoplasm of neurons innervating areas of zoster. However, in neurons innervating zoster-free areas, VZV DNA was found only in the nucleus of neurons and their supporting satellite cells. Immunohistochemistry with a fluorescent monoclonal antibody to the VZV glycoprotein gpI, a late virus protein, revealed a positive signal in the cytoplasm of ganglia with clinical evidence of reactivation. These results illustrate that both neuronal and satellite cells become latently infected following primary VZV infection. The presence of VZV DNA and gpI in the cytoplasm of neurons demonstrates productive infection following reactivation at the site of latency.

Interleukin 2 induces CD8+ T cell-mediated suppression of human immunodeficiency virus replication in CD4+ T cells and this effect overrides its ability to stimulate virus expression.

The nonlytic suppression of human immunodeficiency virus (HIV) production from infected CD4+ T cells by CD8+ lymphocytes from HIV-infected individuals is one of the most potent host-mediated antiviral activities observed in vitro. We demonstrate that the pleiotropic cytokine interleukin 2 (IL-2), but not IL-12, is a potent inducer of the CD8+ HIV suppressor phenomenon. IL-2 induces HIV expression in peripheral blood or lymph node mononuclear cells from HIV-infected individuals in the absence of CD8+ T cells. However, IL-2 induces CD8+ T cells to suppress HIV expression when added back to these cultures, and this effect dramatically supersedes the ability to IL-2 to induce HIV expression. Five to 25 times fewer CD8+ cells were required to obtain comparable levels of inhibition of viral production if they were activated in the presence of IL-2 as compared with IL-12 or no exogenous cytokine. Furthermore, IL-2 appeared either to induce a qualitative increase in HIV suppressor cell activity or to increase the relative frequency of suppressor cells in the activated (CD25+) CD8+ populations. Analyses of proviral levels in peripheral blood mononuclear cells suggest that CD8+ T cell-mediated lysis of in vivo infected cells is not induced by IL-2. These results have implications for our understanding of the effects of impaired IL-2 production during HIV disease as well as the overall effects of IL-2-based immunotherapy on HIV replication in vivo.

Effects of TH1 and TH2 cytokines on CD8+ cell response against human immunodeficiency virus: implications for long-term survival.

CD8+ cells from long-term survivors [LTS; infected with human immunodeficiency virus (HIV) for 10 or more years and having CD4+ cell counts of > or = 500 cells per microliters] have a 3-fold greater ability to suppress HIV replication than do CD8+ cells from patients who have progressed to disease (progressors) during the same time period. A change in the pattern of cytokines produced in the host from those that typically favor cell-mediated immunity (T helper 1, TH1 or type 1) to those that down-regulate it (T helper 2, TH2 or type 2) was investigated as a cause of this reduced CD8+ cell anti-HIV function. Treatment of CD8+ cells from LTS with the TH1 cytokine interleukin (IL)-2 enhanced their anti-HIV activity, whereas exposure of these cells to TH2 cytokines IL-4 or IL-10 reduced their ability to suppress HIV replication and to produce IL-2. IL-2 could prevent and reverse the inhibitory effects of IL-4 and IL-10. Moreover, prolonged exposure of CD8+ cells from some progressors to IL-2 improved the ability of these cells to suppress HIV replication. These observations support previous findings suggesting that strong CD8+ cell responses play an important role in maintaining an asymptomatic state in HIV infection. The data suggest that the loss of CD8+ cell suppression of HIV replication associated with disease progression results from a shift in cytokine production within the infected host from a TH1 to a TH2 pattern. Modulation of these cytokines could provide benefit to HIV-infected individuals by improving their CD8+ cell anti-HIV activity.