p21(WAF1) gene promoter is epigenetically silenced by CTIP2 and SUV39H1.

Mainly regulated at the transcriptional level, the cellular cyclin-dependent kinase inhibitor, CDKN1A/p21(WAF1) (p21), is a major cell cycle regulator of the response to DNA damage, senescence and tumor suppression. Here, we report that COUP-TF-interacting protein 2 (CTIP2), recruited to the p21 gene promoter, silenced p21 gene transcription through interactions with histone deacetylases and methyltransferases. Importantly, treatment with the specific SUV39H1 inhibitor, chaetocin, repressed histone H3 lysine 9 trimethylation at the p21 gene promoter, stimulated p21 gene expression and induced cell cycle arrest. In addition, CTIP2 and SUV39H1 were recruited to the silenced p21 gene promoter to cooperatively inhibit p21 gene transcription. Induction of p21(WAF1) gene upon human immunodeficiency virus 1 (HIV-1) infection benefits viral expression in macrophages. Here, we report that CTIP2 further abolishes Vpr-mediated stimulation of p21, thereby indirectly contributing to HIV-1 latency. Altogether, our results suggest that CTIP2 is a constitutive p21 gene suppressor that cooperates with SUV39H1 and histone methylation to silence the p21 gene transcription.

Burkitt's/Burkitt's-like lymphoma presenting as bacterial sinusitis in two HIV-infected children.

Two children (ages 12 and 13 years) with transfusion-acquired human immunodeficiency virus (HIV) infection presented with facial pain and rhinorrhea. Radiographic imaging showed extensive paranasal sinus disease, presumed to be bacterial sinusitis, and the patients were treated with broad-spectrum oral antibiotics. Both patients were unresponsive to oral agents and were switched to intravenous antibiotics. Despite aggressive antimicrobial therapy, one patient (case 1) developed increased periorbital swelling and proptosis, and the other patient (case 2) developed symptoms of nasopharyngeal obstruction. Repeat imaging showed progression of the infiltrative process extending from the paranasal sinuses into the orbit (case 1), and nasopharynx (case 2). Surgical exploration and tissue biopsies were performed on both patients and the histopathology was consistent with Burkitt's/Burkitt's-like lymphoma. Combination systemic and intrathecal chemotherapy resulted in a complete remission in both patients. These reports illustrate the fact that Burkitt's/Burkitt's-like lymphoma in the paranasal sinuses may initially masquerade as an acute bacterial sinusitis. The ability of the tumor to extend rapidly from the sinuses into the orbit and nasopharynx reinforces the importance of early diagnosis and treatment. Burkitt's/Burkitt's-like lymphoma in the paranasal sinuses has not been previously described in HIV-infected children.

Genome-wide expression changes induced by HTLV-1 Tax: evidence for MLK-3 mixed lineage kinase involvement in Tax-mediated NF-kappaB activation.

The Tax protein of human T-lymphotropic virus type 1 (HTLV-1), an oncoprotein that transactivates viral and cellular genes, plays a key role in HTLV-1 replication and pathogenesis. We used cDNA microarrays to examine Tax-mediated transcriptional changes in the human Jurkat T-cell lines JPX-9 and JPX-M which express Tax and Tax-mutant protein, respectively, under the control of an inducible promoter. Approximately 300 of the over 2000 genes examined were differentially expressed in the presence of Tax. These genes were grouped according to their function and are discussed in the context of existing findings in the literature. There was strong agreement between our results and genes previously reported as being Tax-responsive. Genes that were differentially expressed in the presence of Tax included those related to apoptosis, the cell cycle and DNA repair, signaling factors, immune modulators, cytokines and growth factors, and adhesion molecules. Functionally, we provide evidence that one of these genes, the mixed-lineage kinase MLK-3, is involved in Tax-mediated NF-kappa-B signaling. Our current results provide additional insights into Tax-mediated signaling.

Long-term virologic and immunologic responses in human immunodeficiency virus type 1-infected children treated with indinavir, zidovudine, and lamivudine.

Virologic and immunologic responses were examined for 33 human immunodeficiency virus (HIV)-infected children who participated for > or = 96 weeks in a phase 1/2 protocol of 16 weeks of indinavir monotherapy, followed by the addition of zidovudine and lamivudine. At week 96, a median increase of 199 CD4+ T cells/microL and a median decrease of 0.74 log(10) HIV RNA copies/mL were observed. The relationship between control of viral replication and CD4) T cell count was examined. Patients were categorized into 3 response groups on the basis of duration and extent of control of viral replication. Of 21 children with a transient decrease in virus load of > or = 0.7 log(10) HIV RNA copies/mL from baseline, 7 experienced sustained increases in CD4+, CD4+ CD45RA+, and CD4+ CD45RO+ T cell counts. CD4+ CD45RA+ (naive) T cells were the major contributor to CD4+ T cell expansion. Continued long-term immunologic benefit may be experienced by a subset of children, despite only transient virologic suppression.

Telomere dynamics in monkeys: increased cell turnover in macaques infected with chimeric simian-human immunodeficiency viruses.

To address the question of how cell turnover is affected by retroviral infections, we used the telomeric terminal restriction fragments (TRFs) as markers of cell replicative history and measured their length in macaques infected with chimeric simian-human immunodeficiency viruses (SHIVs). The TRF lengths of mononuclear cells in 104 samples, including longitudinal samples from nine cynomolgus and ten pig-tailed macaques infected with SHIV, and in samples from 26 uninfected macaques, were quantitated by an improved method, based on two-dimensional calibration of DNA sizes, pulsed field electrophoresis, and high-resolution Southern blot images. The average TRF lengths of peripheral blood mononuclear cells (PBMCs) from uninfected pig-tailed (14.9+/-1.6 kbp) and cynomolgus (14.1+/-1.8 kbp) macaques were about 3 and 5 kbp longer than those of human infants and 30-year-old adults, respectively. The rate of TRF length shortening in infected pig-tailed macaques was significantly (P = 0.035) higher (2.2-fold) than in uninfected monkeys. The TRFs in SHIV-infected cynomolgus monkeys, which, in general, had lower viral loads than pig-tailed macaques, shortened on average more rapidly (1.6-fold) than in uninfected animals, but the difference was not statistically significant. The TRFs of mononuclear cells from the lymph nodes of two rapidly progressing SHIV-infected macaques that developed AIDS and died also shortened in parallel but somewhat more rapidly than in the PBMCs. These results suggest that the rate of PBMC turnover in macaques could be increased several-fold during infections by immunodeficiency viruses, likely due to immune activation by SHIV antigens.

Rapid telomere shortening in children.

Telomere shortening may reflect the total number of divisions experienced by a somatic cell and is associated with replicative senescence. We found that the average rate of telomere shortening in peripheral blood mononuclear cells (PBMCs) obtained longitudinally from nine different infants during the first 3 years of life (270 bp per year) is more than fourfold higher than in adults and does not correlate with telomerase activity. These results show that the rate of telomere loss changes during ontogeny, suggesting the existence of periods of accelerated cell division. Because human immunodeficiency virus (HIV) preferentially infects actively dividing cells, our observation suggesting accelerated cell division in children may provide an explanation for some of the distinctive pathogenic features of the HIV disease in infants, including higher viral loads and more rapid progression to acquired immunodeficiency syndrome (AIDS).

Long-term telomere dynamics: modest increase of cell turnover in HIV-infected individuals followed for up to 14 years.

To quantify the long-term dynamics of telomere lengths and the effect of HIV infection on lymphocyte turnover rates, we measured in a blinded study longitudinal samples from 6 individuals using a highly accurate method based on two-dimensional calibration of DNA sizes. For two uninfected controls followed 8 and 10 years the average telomeric terminal restriction fragment (TRF) shortening rate in peripheral blood mononuclear cells (PBMCs) was 50 and 60 bp/year, respectively, in agreement with previous measurements of cross-sectional samples. The TRF lengths of PBMCs from two slow progressors followed for 14 years declined by a rate of 120 +/-10 bp/year, i.e. 2-fold higher than the rate of TRF shortening for uninfected individuals. The rate of TRF shortening was higher in CD8 (140 +/-10 bp/year) than in CD4 (100 +/-10 bp/year) cells. The CD8 cell TRFs of the two fast progressors shortened faster (240 +/-10 bp/year) and the rate of CD4 cell TRF shortening in one of the fast progressors was 160 bp/year. These data suggest that HIV infection causes only a modest increase in the lymphocyte turnover which we speculate could be due to chronic activation of the immune system, and may not result in the exhaustion of its regenerative capacity and immunopathogenesis.

Individual prognoses of long-term responses to antiretroviral treatment based on virological, immunological and pharmacological parameters measured during the first week under therapy.

OBJECTIVE: To predict long-term (12 weeks or longer) virological responses to antiretroviral treatment from measurements made during the first few days on therapy. METHODS: Forty-one HIV-1-infected children were treated with ritonavir for 12 weeks followed by triple drug combination treatment, and the kinetics of virus decay in plasma, ritonavir concentration and CD4 cell counts were measured. A robust multivariate pattern recognition method was used for prediction of the longterm virological responses. RESULTS: The virus decay rate constants calculated from measurements of plasma viral RNA concentrations on the first, second, third, fourth and seventh day on therapy, the drug concentrations in the plasma on day seven, and the pretreatment levels of viral RNA and CD4 cell counts, correlated with long-term levels of plasma HIV-1 RNA. The combination of these parameters contained sufficient information for correct and robust prediction of the long-term response in 88% of the treated children. The predictions of individual responses were stable as demonstrated by a cross-validation analysis, which was highly statistically significant (r=0.87) and specific. CONCLUSION: These results demonstrate that multiple parameters determine the response to antiretroviral therapy and offer a very early measure of individual long-term responses, suggesting that treatment could be optimized after few days of therapy.

The expression of the essential nuclear splicing factor SC35 is altered by human immunodeficiency virus infection.

In order to identify cellular genes differentially expressed during human immunodeficiency virus 1 (HIV-1) infection, we conducted a screen using differential display. The sequence of one of the clones, 0085, was identical to a sequence present in the RNA splicing factor SC35. Since splicing is an essential point of control during HIV gene expression, we carried out additional experiments to examine SC35 expression during HIV infection. RNA blots confirmed that SC35 RNA was induced following HIV infection; a 2-3-fold increase in expression of SC35 RNA was detected by day 2 of HIV infection. Fluorescence-activated cell-sorting revealed concomitant increases in SC35 protein and double staining studies demonstrated that increases in SC35 protein occurred specifically in the HIV-infected cells. Laser scanning confocal microscopy revealed SC35 was associated with 2 microm 'nuclear speckles' in both infected and uninfected cells, suggesting that increases in SC35 accumulated in these nuclear structures and that HIV infection did not alter the intracellular distribution of SC35. These findings indicate that an essential splicing factor is induced after HIV infection, suggesting that the consequences of HIV infection include alterations in relative levels of a splicing factor.

Telomere dynamics in HIV-1 infected and uninfected chimpanzees measured by an improved method based on high-resolution two-dimensional calibration of DNA sizes.

We developed an improved method for accurately measuring telomere lengths based on two-dimensional calibration of DNA sizes combined with pulsed field electrophoresis and quantitative analysis of high-resolution gel images. This method was used to quantify the length of telomeres in longitudinal samples of peripheral blood mononuclear cells (PBMCs) from five chimpanzees infected with human immunodeficiency virus type 1 (HIV-1) and three uninfected animals, 14 to 27 years of age. The average length of the telomere restriction fragments (TRF) of infected and uninfected chimpanzees were 11.7 +/- 0.25 kbp, and 11.6 +/- 0.61 kbp, respectively, and were about 1 kbp and 3 kbp longer than those of human infants and 30 year old adults, respectively. There was a trend of a slight decrease (30-60 bp per year) in the TRF of two HIV infected chimpanzees over 30-35 months, while the TRF of one naive chimpanzee slightly increased over 20 months. Although the number of chimpanzees in this study is small and no statistically significant linear dependencies on time were observed, it appears that in chimpanzees, rates of shortening of the TRF are comparable or smaller than in adult humans and are not significantly affected by HIV-1 infection, which may be related to the inability of HIV-1 to cause disease in these animals.

HIV-1 infection and the developing nervous system: lineage-specific regulation of viral gene expression and replication in distinct neuronal precursors.

Neurologic abnormalities are common in HIV-1 infected patients and often represent the dominant clinical manifestation of pediatric AIDS. Although the neurological dysfunction has been directly related to CNS invasion by HIV-1, the pathogenesis of neurologic disorders remains unclear. Microglia and macrophages are major HIV-1 targets in the brain, whereas HIV-1 infected neurons or glial cells have been rarely reported. This suggests that indirect mechanisms may account for the severe neuronal damage observed in these patients. Nevertheless, immature, mitotically active neuronal and glial cells, which are present during fetal development, are susceptible to HIV-1 infection and replication in vitro, suggesting that HIV-1 infection during organ development may present unique features. To better characterize virus-host cells interactions in the developing CNS, we have examined the susceptibility of embryologically and biochemically distinct neuronal cell lines to HIV-1 infection. Here we show that mitotically active, immature neurons of distinct lineages, have different susceptibilities to HIV-1 infection and replication and different abilities to support viral gene expression. Mutational analysis of HIV-1 LTR reveals that a region of the viral promoter between nucleotide -255 to -166 is responsible for most quantitative and qualitative differences in viral transactivation among different neuroblasts. This suggests that specific regions of the viral promoter and cellular factors, either lineage- or differentiation-dependent, which bind to those regions, may contribute to control the levels of virus replication and possibly restrict the viral tropism in the developing brain. This may contribute to the establishment of a virus reservoir in the immature CNS and participate by either direct or indirect mechanisms to the severity of the AIDS-related pediatric neurological dysfunction.

Umbilical Cord Blood Mononuclear Cell HIV-1 LTR Binding Activities.

Vertically transmitted HIV disease constitutes a significant problem in pediatrics. In order to characterize some of the possible host factors involved in HIV replication in fetuses and newborns, we surveyed the HIV-1 LTR binding factors present in nuclear extracts from cord blood mononuclear cells. A series of electrophoretic mobility shift assays (EMSAs) showed that protein extracts from cord blood interacted with several regions of the HIV LTR. The most prominent binding activities involved the NF-kB sites, but other regions of the LTR also showed factor binding with the cord blood extracts. Some of these cord blood extract binding activities displayed qualitative differences when compared to adult peripheral blood mononuclear cell extracts in EMSA and UV cross-linking studies. Transient transfection experiments indicated that the NF-kB and Sp1 sequences were important for wild type levels of expression in cord blood cells, but that additional sequences 5' to the NF-kB sites also contributed activity. Thus, factors that interact with many of the well-known HIV LTR regulatory sites are present in cord blood cells. However, certain qualitative differences distinguished cord blood and adult peripheral blood binding activities and these may contribute to pathogenesis of HIV infection in neonates.

Human immunodeficiency virus type 1 infection of H9 cells induces increased glucose transporter expression.

A clone obtained from a differential display screen for cellular genes with altered expression during human immunodeficiency virus (HIV) infection matched the sequence for the human GLUT3 facilitative glucose transporter, a high-velocity-high-affinity facilitative transporter commonly expressed in neurons of the central nervous system. Northern (RNA) analysis showed that GLUT3 expression increased during infection. Flow cytometry showed that GLUT3 protein expression increased specifically in the HIV-infected cells; this increase correlated with increased 2-deoxyglucose transport in the HIV-infected culture. HIV infection therefore leads to increased expression of a glucose transporter normally expressed at high levels in other cell types and a corresponding increase in glucose transport activity. If HIV infection places increased metabolic demands on the host cell, changes in the expression of a cellular gene that plays an important role in cellular metabolism might provide a more favorable environment for viral replication.

Kinetics of HIV-1 RNA concentration changes in pediatric patients.

Recent studies have used potent antiviral agents to investigate the kinetics of HIV infection in vivo. They provided estimates for important kinetic parameters, including the decay constants for circulating virus and infected CD4+cells. However, since all of these studies fundamentally rely on the use of antiviral agents, it would be useful to develop other approaches capable of independently verifying the values of the kinetic parameters through other means. Since CD4+ cells are known to exhibit diurnal variations and since there have been suggestions that circulating virus concentrations also vary in a diurnal fashion, as well as nonperiodically, we developed a mathematical model to describe those natural variations. The model predicted variations in viral RNA concentrations and produced estimates of the values of viral kinetic parameters without the use of antiviral agents. To compare the model with experimental data we measured the temporal dependence of the concentration of plasma viral RNA obtained from pediatric HIV-1 patients. The data analysis led to finding diurnal variation in the viral RNA and an estimate of the circulating virus half-life in the order of few hours, in reasonable agreement with the estimates obtained using antiviral agents. These results are the first demonstration of diurnal variations in AIDS patients and confirm the order of magnitude of the virus half-life found by using antiviral drugs. These findings may have implications for understanding HIV-1 pathogenesis and the development of therapeutic protocols.

The molecular biology of HIV. Insights into pathogenesis and targets for therapy.

In the past 10 years, a large number of investigators have produced an enormous amount of information concerning the molecular biology of HIV. These studies at the most basic biological level have provided essential insights into the pathogenesis of the disease. They have supplied the information necessary for the creation of the antiviral therapies now available and have indicated the direction for the development of new therapies now in clinical trials and under investigation. Although the relatively ineffective therapies currently available serve as a constant source of disappointment for those practitioners who care for HIV-infected patients, there is some comfort to be gained from the rapid pace of investigation into the basic biology of the virus and the certainty that any more effective therapy must build upon the basic biological knowledge already obtained. A detailed study of some of the unique features observed during pediatric and perinatal HIV infection, particularly the relatively shortened time from infection to symptoms and the relative importance of CNS disease, may suggest new therapeutic approaches that will benefit both adult and pediatric patients. Finally, a comprehensive knowledge of HIV biology is an essential requirement for therapeutic maneuvers designed to interrupt the transmission of HIV from mother to child.

Replication of type 1 human immunodeficiency viruses containing linker substitution mutations in the -201 to -130 region of the long terminal repeat.

In previous transfection analyses using the chloramphenicol acetyltransferase reporter gene system, we determined that linker substitution (LS) mutations between -201 and -130 (relative to the transcription start site) of the human immunodeficiency virus type 1 long terminal repeat (LTR) caused moderate decreases in LTR transcriptional activity in a T-cell line (S. L. Zeichner, J. Y. H. Kim, and J. C. Alwine, J. Virol. 65:2436-2444, 1991). In order to confirm the significance of this region in the context of viral replication, we constructed several of these LS mutations (-201 to -184, -183 to -166, -165 to -148, and -148 to -130) in proviruses and prepared viral stocks by cocultivation of transfected RD cells with CEMx174 cells. In addition, two mutations between -93 and -76 and between -75 and -58 were utilized, since they affect the nuclear factor kappa B (NF-kappa B)- and Sp1-binding sites and were expected to diminish viral replication. Our results suggest that while transfection analyses offer an adequate approximation of the effects of the LS mutations, the analysis of viral replication using a mutant viral stock presents a more accurate picture, which is sometimes at variance with the transfection results. Three mutants (-201/-184 NXS, -165/-148 NXS, and -147/-130 NXS) had effects on viral replication that were much more severe than the effects predicted from their performance in transfection analyses, and the effects of two LS mutations (-201/-184 NXS and -183/-166 NXS) were not predicted by their effects in transfection. In addition, we observed cell type-specific permissiveness to replication of some mutant viruses. In the cell types tested, the LS mutations indicated an apparent requirement not only for the intact NF-kappa B and SP1-binding sites but also for several regions between -201 and -130 not previously associated with viral infectivity.

Differentiation-dependent human immunodeficiency virus long terminal repeat regulatory elements active in human teratocarcinoma cells.

We have examined the transcriptional utilization of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) under differentiating conditions by using the embryonal carcinoma cell line NTERA-2. NTERA-2 cells undergo two distinct pathways of terminal differentiation, to a neuronal phenotype in response to retinoic acid and to a nonneuronal phenotype in response to hexamethylene bisacetamide. To identify LTR regulatory elements active in each cell type we used a set of HIV LTR linker substitution mutants, which contain mutations that progressively replace adjacent 18-bp segments across the U3 region and into the R region (between nucleotides -453 and +15 relative to the transcription start site). Although each differentiating cell type showed utilization of expected key elements (e.g., NF-kappa B, SP1, TATA) in the 3' portion of the LTR (+1 to -112), the data indicated differentiation-dependent differences in the utilization of these elements. In addition, regions showing dramatic differentiation-dependent effects were detected in the 5' portion of the LTR (-112 to -453), in positions where transcription control elements have not been described previously. The marked differences in the sets of LTR regulatory elements required by each cell type indicate that the LTR can function under a variety of differentiation conditions. Together with previous findings, the data suggest that the complexity of the HIV LTR for transcriptional control is much greater than was previously thought and that the LTR maintains elements which facilitate transcription in many cell types.