The uptake of pan-ethnic expanded carrier screening is higher when offered during preconception or early prenatal genetic counseling.

OBJECTIVE: To examine factors that influence uptake of expanded carrier screening (ECS) among women undergoing preconception and prenatal genetic counseling. METHODS: We retrospectively reviewed 500 medical records from women with prenatal or preconception genetic counseling at a prenatal genetic counseling service. We tabulated acceptance of ECS by indication for genetic counseling along with demographic and pregnancy-related factors. RESULTS: ECS was offered to 483 of 500 women, and 192 (39.8%) accepted. Of the 67 women counseled preconceptionally, 46 (68.7%) accepted ECS. This was significantly more than for 416 women counseled during pregnancy, of whom 146 (35.1%) accepted (P ≤ 0.001). For pregnant patients, the mean gestational age of those accepting ECS (12 weeks 3 days; n = 146) was significantly lower than those declining (13 weeks 4 days; n = 270; P ≤ 0.001). The acceptance rates were 7 of 12 (58.3%, P = 0.195) for Ashkenazi Jewish women, 12 of 41 (29.3%; P = 0.186) for Asian women, and 7 of 25 (28.0%; P = 0.241) for women of mixed ethnicity. CONCLUSIONS: These results suggest that receiving genetic counseling prior to or earlier in the first trimester is associated with acceptance of ECS and support the importance of early genetic counseling about carrier screening options.

Altertoxins with potent anti-HIV activity from Alternaria tenuissima QUE1Se, a fungal endophyte of Quercus emoryi.

Screening of a small library of natural product extracts derived from endophytic fungi of the Sonoran desert plants in a cell-based anti-HIV assay involving T-cells infected with the HIV-1 virus identified the EtOAc extract of a fermentation broth of Alternaria tenuissima QUE1Se inhabiting the stem tissue of Quercus emoryi as a promising candidate for further investigation. Bioactivity-guided fractionation of this extract led to the isolation and identification of two new metabolites, altertoxins V (1) and VI (2) together with the known compounds, altertoxins I (3), II (4), and III (5). The structures of 1 and 2 were determined by detailed spectroscopic analysis and those of 3-5 were established by comparison with reported data. When tested in our cell-based assay at concentrations insignificantly toxic to T-cells, altertoxins V (1), I (3), II (4), and III (5) completely inhibited replication of the HIV-1 virus at concentrations of 0.50, 2.20, 0.30, and 1.50 µM, respectively. Our findings suggest that the epoxyperylene structural scaffold in altertoxins may be manipulated to produce potent anti-HIV therapeutics.

Cdk9 T-loop phosphorylation is regulated by the calcium signaling pathway.

Eukaryotic RNA polymerase II transcriptional elongation is a tightly regulated process and is dependent upon positive transcription elongation factor-b (P-TEFb). The core P-TEFb complex is composed of Cdk9 and Cyclin T and is essential for the expression of most protein coding genes. Cdk9 kinase function is dependent upon phosphorylation of Thr186 in its T-loop. In this study, we examined kinases and signaling pathways that influence Cdk9 T-loop phosphorylation. Using an RNAi screen in HeLa cells, we found that Cdk9 T-loop phosphorylation is regulated by Ca(2+)/calmodulin-dependent kinase 1D (CaMK1D). Using small molecules inhibitors in HeLa cells and primary CD4(+) T lymphocytes, we found that the Ca(2+) signaling pathway is required for Cdk9 T-loop phosphorylation. Inhibition of Ca(2+) signaling led to dephosphorylation of Thr186 on Cdk9. In reporter plasmid assays, inhibition of the Ca(2+) signaling pathway repressed the PCNA promoter and HIV-1 Tat transactivation of the HIV-1 LTR, but not HTLV-1 Tax transactivation of the HTLV-1 LTR, suggesting that perturbation of the Ca(2+) pathway and reduction of Cdk9 T-loop phosphorylation inhibits transcription units that have a rigorous requirement for P-TEFb function.

Phosphatase PPM1A negatively regulates P-TEFb function in resting CD4(+) T cells and inhibits HIV-1 gene expression.

BACKGROUND: Processive elongation of the integrated HIV-1 provirus is dependent on recruitment of P-TEFb by the viral Tat protein to the viral TAR RNA element. P-TEFb kinase activity requires phosphorylation of Thr186 in the T-loop of the CDK9 subunit. In resting CD4+T cells, low levels of T-loop phosphorylated CDK9 are found, which increase significantly upon activation. This suggests that the phosphorylation status of the T-loop is actively regulated through the concerted actions of cellular proteins such as Ser/Thr phosphatases. We investigated the role of phosphatase PPM1A in regulating CDK9 T-loop phosphorylation and its effect on HIV-1 proviral transcription. RESULTS: We found that overexpression of PPM1A inhibits HIV-1 gene expression during viral infection and this required PPM1A catalytic function. Using an artificial CDK tethering system, we further found that PPM1A inhibits CDK9, but not CDK8 mediated activation of the HIV-1 LTR. SiRNA depletion of PPM1A in resting CD4+T cells increased the level of CDK9 T-loop phosphorylation and enhanced HIV-1 gene expression. We also observed that PPM1A protein levels are relatively high in resting CD4+T cells and are not up-regulated upon T cell activation. CONCLUSIONS: Our results establish a functional link between HIV-1 replication and modulation of CDK9 T-loop phosphorylation by PPM1A. PPM1A represses HIV-1 gene expression by inhibiting CDK9 T-loop phosphorylation, thus reducing the amount of active P-TEFb available for recruitment to the viral LTR. We also infer that PPM1A enzymatic activity in resting and activated CD4+ T cells are likely regulated by as yet undefined factors.

Identification of novel CDK9 and Cyclin T1-associated protein complexes (CCAPs) whose siRNA depletion enhances HIV-1 Tat function.

BACKGROUND: HIV-1 Tat activates RNA Polymerase II (RNAP II) elongation of the integrated provirus by recruiting a protein kinase known as P-TEFb to TAR RNA at the 5' end of nascent viral transcripts. The catalytic core of P-TEFb contains CDK9 and Cyclin T1 (CCNT1). A human endogenous complexome has recently been described - the set of multi-protein complexes in HeLa cell nuclei. We mined this complexome data set and identified 12 distinct multi-protein complexes that contain both CDK9 and CCNT1. We have termed these complexes CCAPs for CDK9/CCNT1-associated protein complexes. Nine CCAPs are novel, while three were previously identified as Core P-TEFb, the 7SK snRNP, and the Super-Elongation Complex. We have investigated the role of five newly identified CCAPs in Tat function and viral gene expression. RESULTS: We examined five CCAPs that contain: 1) PPP1R10/TOX3/WDR82; 2) TTF2; 3) TPR; 4) WRNIP1; 5) FBXO11/CUL1/SKP1. SiRNA depletions of protein subunits of the five CCAPs enhanced Tat activation of an integrated HIV-1 LTR-Luciferase reporter in TZM-bl cells. Using plasmid transfection assays in HeLa cells, we also found that siRNA depletions of TTF2, FBXO11, PPP1R10, WDR82, and TOX3 enhanced Tat activation of an HIV-1 LTR-luciferase reporter, but the depletions did not enhance expression of an NF-κB reporter plasmid with the exception of PPP1R10. We found no evidence that depletion of CCAPs perturbed the level of CDK9/CCNT1 in the 7SK snRNP. We also found that the combination of siRNA depletions of both TTF2 and FBXO11 sensitized a latent provirus in Jurkat cells to reactivation by sub-optimal amounts of αCD3/CD28 antibodies. CONCLUSIONS: Our results identified five novel CDK9/CCNT1 complexes that are capable of negative regulation of HIV-1 Tat function and viral gene expression. Because siRNA depletions of CCAPs enhance Tat function, it is possible that these complexes reduce the level of CDK9 and CCNT1 available for Tat, similar to the negative regulation of Tat by the 7SK snRNP. Our results highlight the complexity in the biological functions of CDK9 and CCNT1.

Reproductive Outcomes from Maternal Loss of Nlrp2 Are Not Improved by IVF or Embryo Transfer Consistent with Oocyte-Specific Defect.

Nlrp2 encodes a protein of the oocyte subcortical maternal complex (SCMC), required for embryo development. We previously showed that loss of maternal Nlrp2 in mice causes subfertility, smaller litters with birth defects, and growth abnormalities in offspring, indicating that Nlrp2 is a maternal effect gene and that all embryos from Nlrp2-deficient females that were cultured in vitro arrested before the blastocysts stage. Here, we used time-lapse microscopy to examine the development of cultured embryos from superovulated Nlrp2-deficient and wild-type mice after in vivo and in vitro fertilization. Embryos from Nlrp2-deficient females had similar abnormal cleavage and fragmentation and arrested by blastocyst stage, irrespective of fertilization mode. This indicates that in vitro fertilization does not further perturb or improve the development of cultured embryos. We also transferred embryos from superovulated Nlrp2-deficient and wild-type females to wild-type recipients to investigate if the abnormal reproductive outcomes of Nlrp2-deficient females are primarily driven by oocyte dysfunction or if a suboptimal intra-uterine milieu is a necessary factor. Pregnancies with transferred embryos from Nlrp2-deficient females produced smaller litters, stillbirths, and offspring with birth defects and growth abnormalities. This indicates that the reproductive phenotype is oocyte-specific and is not rescued by development in a wild-type uterus. We further found abnormal DNA methylation at two maternally imprinted loci in the kidney of surviving young adult offspring, confirming persistent DNA methylation disturbances in surviving offspring. These findings have implications for fertility treatments for women with mutations in NLRP2 and other genes encoding SCMC proteins.

Maternal stress in Shank3ex4-9 mice increases pup-directed care and alters brain white matter in male offspring.

Gene-environment interactions contribute to the risk for Autism Spectrum Disorder (ASD). Among environmental factors, prenatal exposure to stress may increase the risk for ASD. To examine if there is an interaction between exposure to maternal stress and reduced dosage or loss of Shank3, wild-type (WT), heterozygous (HET) and homozygous (HOM) female mice carrying a deletion of exons four through nine of Shank3 (Shank3ex4-9) were exposed to chronic unpredictable mild stress (CUMS) from prior to conception throughout gestation. This study examined maternal care of these dams and the white matter microstructure in the brains of their adult male offspring. Overall, our findings suggest that maternal exposure to CUMS increased pup-directed care for dams of all three genotypes. Compared to WT and HET dams, HOM dams also exhibited increased maternal care behaviors with increased time spent in the nest and reduced cage exploration, regardless of exposure to CUMS. Diffusion tensor imaging showed higher mean fractional anisotropy in the hippocampal stratum radiatum of WT and HOM male offspring from dams exposed to CUMS and HOM offspring from unexposed dams, compared to WT male offspring from unexposed dams. These data support that CUMS in Shank3-mutant dams results in subtle maternal care alterations and long-lasting changes in the white matter of the hippocampus of their offspring.

Genetic characterization of HIV type 1 long terminal repeat following vertical transmission.

Human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) sequences were characterized from six mother-infant pairs following vertical transmission. The LTR sequences exhibited a low degree of heterogeneity within mothers, within infants, and between epidemiologically linked mother-infant pairs. However, LTR sequences were more heterogeneous between epidemiologically unlinked individuals compared with linked mother-infant pairs. These data were further supported by low estimates of genetic diversity and clustering of each mother-infant pair's sequences into a separate subtree as well as the presence of common signature sequences between mother-infant pairs. The functional domains essential for LTR (promoter) function, including the promoter (TATAA), enhancers (three Sp-I and two NF-kappaB), the modulatory regions (two AP-I sites, two NFAT, one NF-IL6 site, one Ets-1, and one USF-1), and the TAR region were generally conserved among mother-infant pairs. Taken together, limited heterogeneity and conservation of functional domains in the LTR following vertical transmission support the notion that a functional LTR is critical in viral replication and pathogenesis in HIV-1-infected mothers and their infected infants.

Correction: Chronic Maternal Low-Protein Diet in Mice Affects Anxiety, Night-Time Energy Expenditure and Sleep Patterns, but Not Circadian Rhythm in Male Offspring.

[This corrects the article DOI: 10.1371/journal.pone.0170127.].

Role of HIV-1 subtype C envelope V3 to V5 regions in viral entry, coreceptor utilization and replication efficiency in primary T-lymphocytes and monocyte-derived macrophages.

BACKGROUND: Several subtypes of HIV-1 circulate in infected people worldwide, including subtype B in the United States and subtype C in Africa and India. To understand the biological properties of HIV-1 subtype C, including cellular tropism, virus entry, replication efficiency and cytopathic effects, we reciprocally inserted our previously characterized envelope V3-V5 regions derived from 9 subtype C infected patients from India into a subtype B molecular clone, pNL4-3. Equal amounts of the chimeric viruses were used to infect T-lymphocyte cell lines (A3.01 and MT-2), coreceptor cell lines (U373-MAGI-CCR5/CXCR4), primary blood T-lymphocytes (PBL) and monocyte-derived macrophages (MDM). RESULTS: We found that subtype C envelope V3-V5 region chimeras failed to replicate in T-lymphocyte cell lines but replicated in PBL and MDM. In addition, these chimeras were able to infect U373MAGI-CD4+-CCR5+ but not U373MAGI-CD4+-CXCR4+ cell line, suggesting CCR5 coreceptor utilization and R5 phenotypes. These subtype C chimeras were unable to induce syncytia in MT-2 cells, indicative of non-syncytium inducing (NSI) phenotypes. More importantly, the subtype C envelope chimeras replicated at higher levels in PBL and MDM compared with subtype B chimeras and isolates. Furthermore, the higher levels subtype C chimeras replication in PBL and MDM correlated with increased virus entry in U373MAGI-CD4+-CCR5+. CONCLUSION: Taken together, these results suggest that the envelope V3 to V5 regions of subtype C contributed to higher levels of HIV-1 replication compared with subtype B chimeras, which may contribute to higher viral loads and faster disease progression in subtype C infected individuals than other subtypes as well as rapid HIV-1 subtype C spread in India.

Chronic Maternal Low-Protein Diet in Mice Affects Anxiety, Night-Time Energy Expenditure and Sleep Patterns, but Not Circadian Rhythm in Male Offspring.

Offspring of murine dams chronically fed a protein-restricted diet have an increased risk for metabolic and neurobehavioral disorders. Previously we showed that adult offspring, developmentally exposed to a chronic maternal low-protein (MLP) diet, had lower body and hind-leg muscle weights and decreased liver enzyme serum levels. We conducted energy expenditure, neurobehavioral and circadian rhythm assays in male offspring to examine mechanisms for the body-weight phenotype and assess neurodevelopmental implications of MLP exposure. C57BL/6J dams were fed a protein restricted (8%protein, MLP) or a control protein (20% protein, C) diet from four weeks before mating until weaning of offspring. Male offspring were weaned to standard rodent diet (20% protein) and single-housed until 8-12 weeks of age. We examined body composition, food intake, energy expenditure, spontaneous rearing activity and sleep patterns and performed behavioral assays for anxiety (open field activity, elevated plus maze [EPM], light/dark exploration), depression (tail suspension and forced swim test), sociability (three-chamber), repetitive (marble burying), learning and memory (fear conditioning), and circadian behavior (wheel-running activity during light-dark and constant dark cycles). We also measured circadian gene expression in hypothalamus and liver at different Zeitgeber times (ZT). Male offspring from separate MLP exposed dams had significantly greater body fat (P = 0.03), less energy expenditure (P = 0.004), less rearing activity (P = 0.04) and a greater number of night-time rest/sleep bouts (P = 0.03) compared to control. MLP offspring displayed greater anxiety-like behavior in the EPM (P<0.01) but had no learning and memory deficit in fear-conditioning assay (P = 0.02). There was an effect of time on Per1, Per 2 and Clock circadian gene expression in the hypothalamus but not on circadian behavior. Thus, transplacental and early developmental exposure of dams to chronic MLP reduces food intake and energy expenditure, increases anxiety like behavior and disturbs sleep patterns but not circadian rhythm in adult male offspring.

Molecular characterization of the HIV-1 gag nucleocapsid gene associated with vertical transmission.

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) plays a pivotal role in the viral lifecycle: including encapsulating the viral genome, aiding in strand transfer during reverse transcription, and packaging two copies of the viral genome into progeny virions. Another gag gene product, p6, plays an integral role in successful viral budding from the plasma membrane and inclusion of the accessory protein Vpr within newly budding virions. In this study, we have characterized the gag NC and p6 genes from six mother-infant pairs following vertical transmission by performing phylogenetic analysis and by analyzing the degree of genetic diversity, evolutionary dynamics, and conservation of functional domains. RESULTS: Phylogenetic analysis of 168 gag NC and p6 genes sequences revealed six separate subtrees that corresponded to each mother-infant pair, suggesting that epidemiologically linked individuals were closer to each other than epidemiologically unlinked individuals. A high frequency (92.8%) of intact open reading frames of NC and p6 with patient and pair specific sequence motifs were conserved in mother-infant pairs' sequences. Nucleotide and amino acid distances showed a lower degree of viral heterogeneity, and a low degree of estimates of genetic diversity was also found in NC and p6 sequences. The NC and p6 sequences from both mothers and infants were found to be under positive selection pressure. The two important functional motifs within NC, the zinc-finger motifs, were highly conserved in most of the sequences, as were the gag p6 Vpr binding, AIP1 and late binding domains. Several CTL recognition epitopes identified within the NC and p6 genes were found to be mostly conserved in 6 mother-infant pairs' sequences. CONCLUSION: These data suggest that the gag NC and p6 open reading frames and functional domains were conserved in mother-infant pairs' sequences following vertical transmission, which confirms the critical role of these gene products in the viral lifecycle.

Characterization of HIV-1 envelope gp41 genetic diversity and functional domains following perinatal transmission.

BACKGROUND: HIV-1 envelope gp41 is a transmembrane protein that promotes fusion of the virus with the plasma membrane of the host cells required for virus entry. In addition, gp41 is an important target for the immune response and development of antiviral and vaccine strategies, especially when targeting the highly variable envelope gp120 has not met with resounding success. Mutations in gp41 may affect HIV-1 entry, replication, pathogenesis, and transmission. We, therefore, characterized the molecular properties of gp41, including genetic diversity, functional motifs, and evolutionary dynamics from five mother-infant pairs following perinatal transmission. RESULTS: The gp41 open reading frame (ORF) was maintained with a frequency of 84.17% in five mother-infant pairs' sequences following perinatal transmission. There was a low degree of viral heterogeneity and estimates of genetic diversity in gp41 sequences. Both mother and infant gp41 sequences were under positive selection pressure, as determined by ratios of non-synonymous to synonymous substitutions. Phylogenetic analysis of 157 mother-infant gp41 sequences revealed distinct clusters for each mother-infant pair, suggesting that the epidemiologically linked mother-infant pairs were evolutionarily closer to each other as compared with epidemiologically unlinked sequences. The functional domains of gp41, including fusion peptide, heptad repeats, glycosylation sites and lentiviral lytic peptides were mostly conserved in gp41 sequences analyzed in this study. The CTL recognition epitopes and motifs recognized by fusion inhibitors were also conserved in the five mother-infant pairs. CONCLUSION: The maintenance of an intact envelope gp41 ORF with conserved functional domains and a low degree of genetic variability as well as positive selection pressure for adaptive evolution following perinatal transmission is consistent with an indispensable role of envelope gp41 in HIV-1 replication and pathogenesis.

Evaluations of HIV type 1 rev gene diversity and functional domains following perinatal transmission.

The human immunodeficiency virus type 1 (HIV-1) rev exons 1 and 2 sequences were analyzed from six mother-infant pairs following perinatal transmission. The rev open reading frame was maintained with a frequency of 93.96% in six mother-infant pairs' sequences. There was a low degree of viral heterogeneity and estimates of genetic diversity in mother-infant pairs' rev sequences. However, the distances of rev sequences between epidemiologically unlinked individuals were greater than in epidemiologically linked mother-infant pairs. Furthermore, phylogenetic parameters revealed that the epidemiologically linked mother-infant pairs were closer evolutionarily to each other as compared with epidemiologically unlinked mother-infant pairs. Both mothers and infants were under positive selection pressure as determined by the ratios of nonsynonymous to synonymous substitutions. The functional domains required for Rev activity, including nuclear export of RNA, RNA binding domain, and nuclear import signals, were conserved in all mother-infant pairs' sequences. The conservation of functional domains of rev and a low degree of heterogeneity following vertical transmission are consistent with an indispensable role of rev in the HIV-1 life cycle.

Short communication: SAHA (vorinostat) induces CDK9 Thr-186 (T-loop) phosphorylation in resting CD4+ T cells: implications for reactivation of latent HIV.

The histone deacetylase inhibitor (HDACi) suberoylanilide hydroxyamic acid (SAHA), also known as vorinostat, has recently been reported to activate latent HIV-1 in patients undergoing antiretroviral therapy. It is possible that SAHA reactivation of latent viruses may involve effects on cellular transcription factors such as positive transcription elongation factor b (P-TEFb), a protein kinase whose core is composed of CDK9 and Cyclin T1. P-TEFb is recruited by the HIV-1 Tat protein to activate productive RNA polymerase II elongation of the integrated provirus. We found that SAHA treatment of isolated resting CD4(+) T cells induced CDK9 Thr-186 (T-loop) phosphorylation in six of eight healthy donors and increased Cyclin T1 expression in one donor; Thr-186 phosphorylation is required for P-TEFb function. Disulfiram, another small molecule currently under evaluation in clinical trials for reactivation of latent HIV-1, was also found capable of inducing CDK9 Thr-186 phosphorylation and Cyclin T1 levels in resting CD4(+) T cells from healthy donors. In a Jurkat CD4(+) T cells HIV-1 latency system, disulfiram reactivated the latent provirus and induced CDK9 Thr-186 phosphorylation. Our findings suggest that small molecules capable of reactivating latent HIV-1 in resting CD4(+) T cells may function in part by increasing CDK9 Thr-186 phosphorylation and perhaps Cyclin T1 expression, thereby up-regulating P-TEFb function.

Inhibition of HIV-1 Replication by Secondary Metabolites From Endophytic Fungi of Desert Plants.

Most antiretroviral drugs currently in use to treat an HIV-1 infection are chemically synthesized and lead to the development of viral resistance, as well as cause severe toxicities. However, a largely unexplored source for HIV-1 drug discovery is endophytic fungi that live in a symbiotic relationship with plants. These fungi produce biologically active secondary metabolites, which are natural products that are beneficial to the host. We prepared several hundred extracts from endophytic fungi of desert plants and evaluated the inhibitory effects on HIV-1 replication of those extracts that showed less than 30% cytotoxicity in T-lymphocytes. Those extracts that inhibited viral replication were fractionated in order to isolate the compounds responsible for activity. Multiple rounds of fractionation and antiviral evaluation lead to the identification of four compounds, which almost completely impede HIV-1 replication. These studies demonstrate that metabolites from endophytic fungi of desert plants can serve as a viable source for identifying potent inhibitors of HIV-1 replication.

Making a Short Story Long: Regulation of P-TEFb and HIV-1 Transcriptional Elongation in CD4+ T Lymphocytes and Macrophages.

Productive transcription of the integrated HIV-1 provirus is restricted by cellular factors that inhibit RNA polymerase II elongation. The viral Tat protein overcomes this by recruiting a general elongation factor, P-TEFb, to the TAR RNA element that forms at the 5' end of nascent viral transcripts. P-TEFb exists in multiple complexes in cells, and its core consists of a kinase, Cdk9, and a regulatory subunit, either Cyclin T1 or Cyclin T2. Tat binds directly to Cyclin T1 and thereby targets the Cyclin T1/P-TEFb complex that phosphorylates the CTD of RNA polymerase II and the negative factors that inhibit elongation, resulting in efficient transcriptional elongation. P-TEFb is tightly regulated in cells infected by HIV-1-CD4+ T lymphocytes and monocytes/macrophages. A number of mechanisms have been identified that inhibit P-TEFb in resting CD4+ T lymphocytes and monocytes, including miRNAs that repress Cyclin T1 protein expression and dephosphorylation of residue Thr186 in the Cdk9 T-loop. These repressive mechanisms are overcome upon T cell activation and macrophage differentiation when the permissivity for HIV-1 replication is greatly increased. This review will summarize what is currently known about mechanisms that regulate P-TEFb and how this regulation impacts HIV-1 replication and latency.

Limited redundancy in genes regulated by Cyclin T2 and Cyclin T1.

BACKGROUND: The elongation phase, like other steps of transcription by RNA Polymerase II, is subject to regulation. The positive transcription elongation factor b (P-TEFb) complex allows for the transition of mRNA synthesis to the productive elongation phase. P-TEFb contains Cdk9 (Cyclin-dependent kinase 9) as its catalytic subunit and is regulated by its Cyclin partners, Cyclin T1 and Cyclin T2. The HIV-1 Tat transactivator protein enhances viral gene expression by exclusively recruiting the Cdk9-Cyclin T1 P-TEFb complex to a RNA element in nascent viral transcripts called TAR. The expression patterns of Cyclin T1 and Cyclin T2 in primary monocytes and CD4+ T cells suggests that Cyclin T2 may be generally involved in expression of constitutively expressed genes in quiescent cells, while Cyclin T1 may be involved in expression of genes up-regulated during macrophage differentiation, T cell activation, and conditions of increased metabolic activity To investigate this issue, we wished to identify the sets of genes whose levels are regulated by either Cyclin T2 or Cyclin T1. FINDINGS: We used shRNA lentiviral vectors to stably deplete either Cyclin T2 or Cyclin T1 in HeLa cells. Total RNA extracted from these cells was subjected to cDNA microarray analysis. We found that 292 genes were down- regulated by depletion of Cyclin T2 and 631 genes were down-regulated by depletion of Cyclin T1 compared to cells transduced with a control lentivirus. Expression of 100 genes was commonly reduced in either knockdown. Additionally, 111 and 287 genes were up-regulated when either Cyclin T2 or Cyclin T1 was depleted, respectively, with 45 genes in common. CONCLUSIONS: These results suggest that there is limited redundancy in genes regulated by Cyclin T1 or Cyclin T2.

Epstein-Barr virus BART9 miRNA modulates LMP1 levels and affects growth rate of nasal NK T cell lymphomas.

Nasal NK/T cell lymphomas (NKTCL) are a subset of aggressive Epstein-Barr virus (EBV)-associated non-Hodgkin's lymphomas. The role of EBV in pathogenesis of NKTCL is not clear. Intriguingly, EBV encodes more than 40 microRNAs (miRNA) that are differentially expressed and largely conserved in lymphocryptoviruses. While miRNAs play a critical role in the pathogenesis of cancer, especially lymphomas, the expression and function of EBV transcribed miRNAs in NKTCL are not known. To examine the role of EBV miRNAs in NKTCL, we used microarray profiling and qRT-PCR to identify and validate expression of viral miRNAs in SNK6 and SNT16 cells, which are two independently derived NKTCL cell lines that maintain the type II EBV latency program. All EBV BART miRNAs except BHRF-derived miRNAs were expressed and some of these miRNAs are expressed at higher levels than in nasopharyngeal carcinomas. Modulating the expression of BART9 with antisense RNAs consistently reduced SNK6 and SNT16 proliferation, while antisense RNAs to BARTs-7 and -17-5p affected proliferation only in SNK6 cells. Furthermore, the EBV LMP-1 oncoprotein and transcript levels were repressed when an inhibitor of BART9 miRNA was transfected into SNK6 cells, and overexpression of BART9 miRNA increased LMP-1 protein and mRNA expression. Our data indicate that BART9 is involved in NKTCL proliferation, and one of its mechanisms of action appears to be regulating LMP-1 levels. Our findings may have direct application for improving NKTCL diagnosis and for developing possible novel treatment approaches for this tumor, for which current chemotherapeutic drugs have limited effectiveness.

Characterization of Cdk9 T-loop phosphorylation in resting and activated CD4(+) T lymphocytes.

The cellular kinase complex P-TEFb is composed of Cdk9 and cyclin T, and it is required for expression of most protein-coding genes by RNAP II. Cdk9 has been shown recently to be activated in cis by autophosphorylation of Thr186 in its T-loop. Using a phosphospecific Cdk9 antibody, we examined the level of Cdk9 T-loop phosphorylation in resting and activated CD4(+) T lymphocytes. Cdk9 T-loop phosphorylation was found to be low-to-undetectable in resting CD4(+) T lymphocytes, and upon activation by distinct stimuli, there is a rapid (<1 h) increase in pCdk9 that does not require protein synthesis. The low level of Cdk9 T-loop phosphorylation was not to be a result of the absence of an associated regulatory cyclin partner. These observations suggest that autophosphorylation of the Cdk9 T-loop is repressed in resting CD4(+) T lymphocytes. The low level of T-loop phosphorylation in resting cells is also reflected in a low level of phosphorylation of Ser2 in the carboxyl terminal domain of RNAP II, suggesting that lack of Cdk9 T-loop autophosphorylation may limit RNAP II elongation in quiescent CD4(+) T lymphocytes.