Cyclin T1 is a regulatory subunit of a general RNA polymerase II elongation factor known as P-TEFb. Cyclin T1 is also required for Tat transactivation of HIV-1 LTR-directed gene expression. Translation of Cyclin T1 mRNA has been shown to be repressed in human monocytes, and this repression is relieved when cells differentiate to macrophages. We identified miR-198 as a microRNA (miRNA) that is strongly down-regulated when monocytes are induced to differentiate. Ectopic expression of miR-198 in tissue culture cells reduced Cyclin T1 protein expression, and plasmid reporter assays verified miR-198 target sequences in the 3′ untranslated region (3′UTR) of Cyclin T1 mRNA. Cyclin T1 protein levels increased when an inhibitor of miR-198 was transfected into primary monocytes, and overexpression of miR-198 in primary monocytes repressed the normal up-regulation of Cyclin T1 during differentiation. Expression of an HIV-1 proviral plasmid and HIV-1 replication were repressed in a monocytic cell line upon overexpression of miR-198. Our data indicate that miR-198 functions to restrict HIV-1 replication in monocytes, and its mechanism of action appears to involve repression of Cyclin T1 expression.

The replication of integrated human immunodeficiency virus type 1 (HIV-1) is dependent on the cellular cofactor cyclin T1, which binds the viral Tat protein and activates the RNA polymerase II transcription of the integrated provirus. The activation of resting CD4؉ T cells upregulates cyclin T1 protein levels independently of an increase in cyclin T1 mRNA levels, suggesting a translational repression of cyclin T1 in resting CD4 ؉ T cells. Hypothesizing that microRNAs (miRNAs) repress cyclin T1 translation in resting CD4؉ T cells and that this inhibition is lifted upon cell activation, we used microarray expression analysis to identify miRNAs miR-27b, miR-29b, miR-150, and miR-223 as being significantly downregulated upon CD4 ؉ T cell activation. The overexpression of these miRNAs decreased endogenous cyclin T1 protein levels, while treatment with the corresponding antagomiRs increased cyclin T1 protein levels. An miR-27b binding site within the cyclin T1 3= untranslated region (3=UTR) was identified and confirmed to be functional after the mutation of key resides abrogated the ability of miR-27b to decrease the expression of a luciferase reporter upstream of the cyclin T1 3=UTR. Ago2 immunoprecipitation revealed an association with cyclin T1 mRNA that was decreased following treatment with miR-27b and miR-29b antagomiRs. Cells overexpressing miR-27b showed decreased viral gene expression levels of the HIV-1 reporter virus and a decreased replication of strain NL4.3; a partial rescue of viral transcription could be seen following the transfection of cyclin T1. These results implicate miR-27b as a novel regulator of cyclin T1 protein levels and HIV-1 replication, while miR-29b, miR-223, and miR-150 may regulate cyclin T1 indirectly.

Extrachromosomal telomere repeat (ECTR) DNA is unique to cancer cells that maintain telomeres through the alternative lengthening of telomeres (ALT) pathway, but the role of ECTRs in ALT development remains elusive. We found that induction of ECTRs in normal human fibroblasts activated the cGAS-STING-TBK1-IRF3 signaling axis to trigger IFNβ production and a type I interferon response, resulting in cell-proliferation defects. In contrast, ALT cancer cells are commonly defective in sensing cytosolic DNA. We found that STING expression was inhibited in ALT cancer cell lines and transformed ALT cells. Notably, the ALT suppressors histone H3.3 and the ATRX-Daxx histone chaperone complex were also required to activate the DNA-sensing pathway. Collectively, our data suggest that the loss of the cGAS-STING pathway may be required to evade ECTR-induced anti-proliferation effects and permit ALT development, and this requirement may be exploited for treatments specific to cancers utilizing the ALT pathway.

Background: The latent reservoir of human immunodeficiency virus type 1 (HIV-1) in resting CD4 + T cells is a major obstacle to the clearance of infection by highly active antiretroviral therapy (HAART). Recent studies have focused on searches for adjuvant therapies to activate this reservoir under conditions of HAART. Prostratin, a non tumor-promoting phorbol ester, is a candidate for such a strategy. Prostratin has been shown to reactivate latent HIV-1 and Tat-mediated transactivation may play an important role in this process. We examined resting CD4 + T cells from healthy donors to determine if prostratin induces Cyclin T1/P-TEFb, a cellular kinase composed of Cyclin T1 and Cyclin-dependent kinase-9 (CDK9) that mediates Tat function. We also examined effects of prostratin on Cyclin T2a, an alternative regulatory subunit for CDK9, and 7SK snRNA and the HEXIM1 protein, two factors that associate with P-TEFb and repress its kinase activity.

While dengue virus is thought to replicate in mononuclear phagocytic cells in vivo, attempts to detect it in peripheral blood mononuclear cells (PBMC) by virus isolation or antigen detection have had variable and generally low rates. In this study, we developed a reverse transcription (RT)-real-time PCR assay to quantify positive-and negative-sense RNA of dengue virus type 2 within the cells. The assay includes an RT step using either sense or antisense primer followed by a real-time PCR step using the designed primers and probe, which target a capsid region highly conserved in dengue virus type 2 strains. It can be used to monitor the dynamic change of intracellular dengue virus RNA species during the course of infection. When this assay is employed in quantification of dengue virus RNA species in PBMC from 10 patients infected with dengue virus type 2, both positive-and negative-sense dengue RNA can be detected, indicating that dengue virus is actively replicating in PBMC in vivo. Moreover, the amounts of negative-sense dengue virus RNA in PBMC correlate very well with the viral load of dengue virus in plasma, suggesting that quantification of negative-sense dengue virus RNA in PBMC may provide another indicator of dengue virus replication in vivo. Use of this convenient, sensitive, and accurate method of quantification in clinical samples from patients with different disease severity would further our understanding of the pathogenesis of dengue.Dengue virus belongs to the genus Flavivirus of the family Flaviviridae. It contains a positive-sense single-stranded RNA genome of approximately 11 kb (9,22). Flanked by the two nontranslated regions at both ends, the single open reading frame consists of three structural genes at the 5Ј one-fourth and seven nonstructural genes at the 3Ј three-fourths. Among the 80 or so arthropod-borne flaviviruses, epidemics of infection by the four serotypes of dengue virus (DEN-1, DEN-2, DEN-3, and DEN-4) continue to be a major public health problem in tropical and subtropical areas (4,9,14,25). It has been estimated that approximately 100 million dengue infections occur annually worldwide (9,11,25).The clinical presentations of dengue virus infection range from asymptomatic, or a mild self-limited illness, dengue fever (DF), to a severe and potentially life-threatening disease, dengue hemorrhage fever/dengue shock syndrome (DHF/DSS) (9, 14, 37). Following an incubation period of 3 to 14 days, fever and a variety of symptoms occur, coinciding with the appearance of dengue virus in blood (9,14). Based on the pathological findings in experimentally infected rhesus monkeys and in humans with fatal infections, dengue virus is thought to replicate in mononuclear phagocytic cells in vivo (1,2,9,10,11,14,23,26). Several studies have attempted to detect dengue virus in peripheral blood mononuclear cells (PBMC). Scott et al. (15). Overall, the detection rates of dengue virus in PBMC through either virus isolation or antigen detection method were variable and generally low (3,15,30,36).Rec...