Human Immunodeficiency Virus Type 1 Transcription Research Articles

Generation of HIV-1 and Internal Control Transcripts as Standards for an In-House Quantitative Competitive RT-PCR Assay to Determine HIV-1 Viral Load

Human immunodeficiency virus type-1 (HIV-1) viral load is useful for monitoring disease progression in HIV-infected individuals. We generated RNA standards of HIV-1 and internal control (IC) by in vitro transcription and evaluated its performance in a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. HIV-1 and IC standards were obtained at high RNA concentrations, without DNA contamination. When these transcripts were included as standards in a qRT-PCR assay, it was obtained a good accuracy (±0.5 log10 unit of the expected results) in the quantification of the HIV-1 RNA international standard and controls. The lower limit detection achieved using these standards was 511.0 IU/mL. A high correlation (r = 0.925) was obtained between the in-house qRT-PCR assay and the NucliSens easyQ HIV-1 test (bioMerieux) for HIV-1 RNA quantitation with clinical samples (N = 14). HIV-1 and IC RNA transcripts, generated in this study, proved to be useful as standards in an in-house qRT-PCR assay for determination of HIV-1 viral load.

The Role of Estradiol and Progesterone in Mucosal HIV-1 Infection, Transmission and Pathogenesis

Heterosexual transmission of the human immunodeficiency virus type-1 (HIV-1) to women occurs after exposure of the mucosal surfaces of the female reproductive tract (FRT) to cell-free virus or to HIV-1 infected cells present in the seminal fluid of an infected male partner. However, exposure to virus at this site does not always result in transmission of HIV-1, indicating that the local microenvironment in the FRT at the time of viral exposure may influence a womans susceptibility to infection. Cleary, the sex steroid hormones, estradiol and progesterone, play an important role in the FRT. Although the primary function of these hormones is to regulate the reproductive status of a woman, they also exert profound effects on the subsets of immune cells in the FRT as well as on their phenotype and functional activity. In addition, estradiol and progesterone regulate the local cytokine and chemokine milieu by influencing the activation state of immune cells. Fluctuations in estradiol and progesterone levels in the FRT regulate expression of HIV-1 receptors CD4 and galactosyl ceramide (Gal-Cer), as well as HIV-1 co-receptors CCR5 and CXCR4. Moreover, these hormones control the development of leukocyte aggregates in the FRT that contain T cells and macrophages, resulting in foci of potential target cells for HIV-1 infection. Estradiol has also been shown to enhance HIV-1 transcription from infected cells, thereby increasing levels of viral shedding from the genital tract. In sum, these steroid hormones serve to alter a womans susceptibility to HIV-1 infection after exposure to virus in the genital tract. Keywords: HIV-1, ectocervix, estradiol, progesterone, steroid hormone, mucosal immunology, Mucosal HIV-1 Infection, Heterosexual transmission, human immunodeficiency virus type-1, female reproductive tract, HIV-1 infected cells, sex steroid hormones, cytokine, chemokine milieu, progesterone levels, HIV-1 receptors CD4, galactosyl ceramide, HIV-1 co-receptors, CXCR4, T cells, HIV-1 transcription, steroid hormones, heterosexual intercourse, highly active antiretroviral therapy, cervicovaginal fluid, genital mucosa, chronic inflammation, lamina propria, GalC, endometrial glands, R5 tropic strains, urethra, epididymal epithelium, columnar epithelium, cytokine Interleukin-6, pro-inflammatory cytokine, IL-1, IL-6, blood mononuclear cells, cervical biopsies, Toll-like receptors, pathogen associated molecular patterns, secretory leukocyte peptidase, macrophage inflammatory, uterine-derived natural killer, chemokine ligand-12

Interaction of human dipeptidyl peptidase IV and human immunodeficiency virus type-1 transcription transactivator in Sf9 cells

BackgroundDipeptidyl peptidase IV (DPPIV) also known as the T cell activation marker CD26 is a multifunctional protein which is involved in various biological processes. The association of human-DPPIV with components of the human immunodeficiency virus type-1 (HIV1) is well documented and raised some discussions. Several reports implicated the interaction of human-DPPIV with the HIV1 transcription transactivator protein (HIV1-Tat) and the inhibition of the dipeptidyl peptidase activity of DPPIV by the HIV1-Tat protein. Furthermore, enzyme kinetic data implied another binding site for the HIV1-Tat other than the active centre of DPPIV. However, the biological significance of this interaction of the HIV1-Tat protein and human-DPPIV has not been studied, yet. Therefore, we focused on the interaction of HIV1-Tat protein with DPPIV and investigated the subsequent biological consequences of this interaction in Spodoptera frugiperda cells, using the BAC-TO-BAC baculovirus system.ResultsThe HIV1-Tat protein (Tat-BRU) co-localized and co-immunoprecipitated with human-DPPIV protein, following co-expression in the baculovirus-driven Sf9 cell expression system. Furthermore, tyrosine phosphorylation of DPPIV protein was up-regulated in Tat/DPPIV-co-expressing cells after 72 h culturing and also in DPPIV-expressing Sf9 cells after application of purified recombinant Tat protein. As opposed to the expression of Tat alone, serine phosphorylation of the Tat protein was decreased when co-expressed with human-DPPIV protein.ConclusionsWe show for the first time that human-DPPIV and HIV1-Tat co-immunoprecipitate. Furthermore, our findings indicate that the interaction of HIV1-Tat and human-DPPIV may be involved in signalling platforms that regulate the biological function of both human-DPPIV and HIV1-Tat.

DNA-dependent activator of IFN-regulatory factors enhances the transcription of HIV-1 through NF-κB

Pattern recognition receptors (PRRs) play a pivotal role in host innate immune responses against microbial infection. Viruses are primarily recognized by PRRs such as Toll-like receptor 3, 7, 8 and 9, and RIG-I-like receptors. Recent studies have demonstrated that DNA-dependent activator of IFN-regulatory factors (DAI) is a cytosolic sensor molecule for dsDNA, and is implicated in antiviral responses to some DNA viruses. Soon after infection, human immunodeficiency virus type-1 (HIV-1) synthesizes viral dsDNA in the cytoplasm by reverse transcriptase. In addition, an immune compromised state due to chronic HIV-1 infection results in opportunistic infection with some microbes that potentially activate DAI. However, it has not been elucidated whether DAI affects HIV-1 replication, or its possible mechanisms. Here, we showed that forced expression of DAI markedly enhanced HIV-1 replication, which was largely impaired by mutations at κB sites in HIV-1 LTR or by suppressing activation of NF-κB. Moreover, intact structure around the D3 region (174–232 aa) and two RIP homotypic interaction motifs (198–214 aa and 256–272 aa) within DAI were critical for its activity. These results suggest that activation of DAI might contribute to augment HIV-1 replication through DAI- NF-κB pathway.

HIV‐1 Tat is unconventionally secreted through the plasma membrane

The Tat protein is required for efficient HIV-1 (human immunodeficiency virus type 1) transcription. Moreover, Tat is secreted by infected cells, and circulating Tat can affect several cell types, thereby contributing to HIV-1 pathogenesis. We monitored Tat secretion by transfected CD4+ T-cells. A Tat chimaera carrying an N-glycosylation site did not become glycosylated when expressed in cells, while the chimaera was glycosylated when mechanically introduced into purified microsomes. These data indicate that secreted Tat does not transit through the endoplasmic reticulum. The use of pharmacological inhibitors indicated that the Tat secretion pathway is unusual compared with previously identified unconventional secretion routes and does not involve intracellular organelles. Moreover, cell incubation at 16 degrees C inhibited Tat secretion and caused its accumulation at the plasma membrane, suggesting that secretion takes place at this level.

A novel method for promoting heterologous protein expression in Escherichia coli by fusion with the HIV-1 TAT core domain

The human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (TAT) protein, a member of the protein transduction domain (PTD) superfamily, can deliver heterologous proteins across most biomembranes without losing bioactivity. However, there is no report on whether the TAT core domain containing the sequence 'YGRKKRRQRRR' has other functions. As the TAT core domain is most basic (pI=12.8) and has biomembrane crossing ability, we hypothesized it might probably influence the protein expression level due to subcellular redistribution of target proteins in the cells. To address this issue, we constructed the prokaryotic expression vector pET28b-TAT-EGFP (using the vector pET28b-EGFP for control) containing the core domain coding region, and transformed the vector into E. coli BL21 (DE3) cells for expression of the enhanced green fluorescent protein (EGFP) with the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). Equal amount of the total proteins were fractionated using 15% SDS-PAGE and identified by western blot, and the plasmid copy number was assayed by Southern blot. In order to further study the subcellular localization of heterologous proteins in E. coli cells, the cytoplasmic and periplasmic components were extracted by chloroform and osmotic shock techniques. Interestingly, our data showed that the TAT core domain was not only able to promote the heterologous protein expression in E. coli, but also improve the yields and the solubility of heterologous proteins, while the plasmid copy number of TAT-containing clones and TAT-free clones was not affected by the TAT core domain. In addition, the TAT-tagged protein was mainly localized in the cytoplasm and also accumulated in the periplasmic space along with the time for protein expression, while in contrast, the TAT-free protein was mainly expressed in the periplasm and only a few in cytoplasm. A further examination on the distribution of the expressed proteins in cytoplasm and periplasm suggested that the TAT core domain might promote protein expression in the cytoplasm initially and then partially deliver them across the cytomembrane to the periplasmic space in a concentration-dependent manner. Taken together, our current data have provided a novel method for improving heterologous protein expression in prokaryotic cells by fusion with the TAT core domain, which will promote expression efficiency of bioactive proteins for protein engineering.

M2 pyruvate kinase enhances HIV-1 transcription from its long terminal repeat

Both thymocytes and tumor cells express M2 type isoenzyme of pyruvate kinase (M2PK), which is different from R type isoenzyme of pyruvate kinase (RPK) that is expressed in erythrocytes. In this report, the effect of RPK and M2PK on the transcription of human immunodeficiency virus type 1 (HIV-1) was tested. The results indicated that M2PK could enhance HIV-1 transcription from its long terminal repeat (LTR) promoter, while RPK did not have such an effect. Specific down-regulation of M2PK could inhibit HIV-1 transcription from its LTR region. Furthermore, it was found that the C terminal region of M2PK is responsible for this effect. Collectively, the cellular factor M2PK that is expressed in thymocytes could facilitate the transcription of HIV-1.

Regulation of HIV-1 transcription in cells of the monocyte-macrophage lineage.

Human immunodeficiency virus type 1 (HIV-1) has been shown to replicate productively in cells of the monocyte-macrophage lineage, although replication occurs to a lesser extent than in infected T cells. As cells of the monocyte-macrophage lineage become differentiated and activated and subsequently travel to a variety of end organs, they become a source of infectious virus and secreted viral proteins and cellular products that likely initiate pathological consequences in a number of organ systems. During this process, alterations in a number of signaling pathways, including the level and functional properties of many cellular transcription factors, alter the course of HIV-1 long terminal repeat (LTR)-directed gene expression. This process ultimately results in events that contribute to the pathogenesis of HIV-1 infection. First, increased transcription leads to the upregulation of infectious virus production, and the increased production of viral proteins (gp120, Tat, Nef, and Vpr), which have additional activities as extracellular proteins. Increased viral production and the presence of toxic proteins lead to enhanced deregulation of cellular functions increasing the production of toxic cellular proteins and metabolites and the resulting organ-specific pathologic consequences such as neuroAIDS. This article reviews the structural and functional features of the cis-acting elements upstream and downstream of the transcriptional start site in the retroviral LTR. It also includes a discussion of the regulation of the retroviral LTR in the monocyte-macrophage lineage during virus infection of the bone marrow, the peripheral blood, the lymphoid tissues, and end organs such as the brain. The impact of genetic variation on LTR-directed transcription during the course of retrovirus disease is also reviewed.

Subcellular localization of the interaction between the human immunodeficiency virus transactivator Tat and the nucleosome assembly protein 1

The histone chaperone nucleosome assembly protein, hNAP-1, is a host cofactor for the activity of the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. The interaction between these two proteins has been shown to be important for Tat-mediated transcriptional activation and for efficient viral infection. Visualization of HIV-1 transcription and fluorescence resonance energy transfer experiments performed in this work demonstrate that hNAP-1 is not recruited to the site of Tat activity but the two proteins interact at the nuclear rim. These data are consistent with a mechanism that requires hNAP-1 for the transport of Tat within the nucleus rather than for the remodeling of nucleosomes on the provirus. Protein-protein docking and molecular modeling of the complex suggest that this interaction occurs between the basic domain of Tat and the histone-binding domain. The combination of theoretical and whole cell studies provided new insights into the functional significance of the Tat:hNAP-1 recognition.

Short Communication: RNA Interference Directed against Axin1 Upregulates Human Immunodeficiency Virus Type 1 Gene Expression by Activating the Wnt Signaling Pathway in HeLa-Derived J111 Cells

Axin1, a regulator of Wnt signaling, was previously identified as playing a negative role in the late phase of human immunodeficiency virus type 1 (HIV-1) replication in HeLa-derived J111 cells. In this report, we studied the molecular mechanism of how Axin1 regulates HIV-1 replication. HIV-1 transactivator, Tat-dependent viral reporter gene expression was enhanced in J111 cells transfected with small interfering RNA (siRNA) against Axin1. In addition, viral transcription was upregulated in J111 cells transfected with siRNA against Axin1. In contrast, HIV-1 gene expression was not enhanced by transfecting HeLa cells with siRNA against Axin1. The expression levels of T cell factor-4 (TCF4) and beta-catenin were higher in J111 than HeLa cells. In addition, siRNAs against TCF4 and beta-catenin inhibited the Axin1 siRNA-dependent enhancement of HIV-1 gene expression in J111 cells. These results suggest that Axin1 plays a negative role in HIV-1 transcription through the Wnt signaling pathway in J111 cells under normal cell culture conditions.

HIV Type 1 Infection in Women: Increased Transcription of HIV Type 1 in Ectocervical Tissue Explants

Mucosal surfaces of the female reproductive tract are the main routes of heterosexual transmission of human immunodeficiency virus type 1 (HIV-1), but the contribution of each of the reproductive sites to mucosal transmission is unknown. We compared levels of HIV-1 transcription between ectocervical and endometrial tissue explants infected ex vivo with HIV-1. We detected higher levels of HIV-1 transcription in the ectocervix. Although CD45 expression was also increased at this site, higher levels of HIV-1 transcription could not be accounted for exclusively by differences in CD45 expression. This suggests that factors other than CD45 levels regulate HIV-1 transcription within the ectocervix. We detected higher levels of interleukin (IL)-6 at this site. Furthermore, addition of recombinant IL-6 to tissue explants enhanced HIV-1 transcription to a much greater degree in the ectocervix than in the endometrium. This is, to our knowledge, the first study to compare ectocervix and endometrium in a tissue explant model of HIV-1 infection and to demonstrate greater HIV-1 transcription in the ectocervix. Our results suggest that the ectocervix is more conducive to HIV-1 replication than is the endometrium and that IL-6 enhances HIV-1 transcription at this site. Thus, the ectocervix is an important site to be considered in heterosexual transmission of HIV-1.

The ups and downs of HIV-1 gene expression

Following infection, Human Immunodeficiency Virus type 1 (HIV-1) becomes stably integrated into the genome of the host cell. It depends, therefore, on host cell factors to regulate its transcriptional output, both positively and negatively. HIV-1 transcriptional silencing requires chromatin remodelling and transcriptional repressor complexes, as well as heterochromatin formation at the integrated provirus. An important feature of silenced proviruses is their ability to be re-activated by either the viral transcription factor, Tat, or environmental stimuli. Activation of transcription is also controlled by numerous host cell factors. We have identified novel mechanisms of transcriptional activation and repression that depend on cellular macromolecular complexes such as proteasome and nuclear exosome. I will discuss recent developments in understanding the mechanisms by which these complexes control HIV-1 transcription, and the implications for viral latency.

Resveratrol inhibited Tat-induced HIV-1 LTR transactivation via NAD +-dependent SIRT1 activity

Aims Tat protein plays a pivotal role in both the human immunodeficiency virus type 1 (HIV-1) replication cycle and the pathogenesis of HIV-1 infection. Sirtuins 1 (SIRT1) is a possible candidate for redox modulation because its activity is regulated by nicotinamide adenine dinucleotide (NAD +) or NAD +/NADH ratio. The aim of the present study was to determine whether the redox status and SIRT1 expression are related to HIV-1 Tat protein-induced transactivation. Main methods HeLa-CD4-long terminal repeat (LTR)-β-gal (MAGI) cells were transfected with Tat plasmid. Tat-induced HIV-1 LTR transactivation was determined by MAGI cell assay. The NAD + or NADH levels and SIRT1 activity were measured. In addition, the protein expression of SIRT1 was assayed by western blotting. Key findings Pretreatment with resveratrol increased intracellular NAD + levels and SIRT1 protein expression after Tat plasmid transfection in a concentration-dependent manner. Pretreatment with resveratrol attenuated Tat-induced HIV-1 transactivation in MAGI cells. These effects of resveratrol were largely abolished by knockdown of SIRT1 by short interfering RNA (siRNA). Pretreatment with nicotinamide, a SIRT1 inhibitor, potentiated Tat-induced HIV-1 transactivation in MAGI cells, and overexpression of SIRT1 attenuated Tat-induced HIV-1 transcription in MAGI cells. Significance Inhibition of SIRT1 activity by Tat is considered a critical step of Tat transactivation. Resveratrol and related compounds represent potential candidates for novel anti-HIV therapeutics.

Epigenetic Regulation of HIV-1 Latency by Cytosine Methylation

Human immunodeficiency virus type 1 (HIV-1) persists in a latent state within resting CD4+ T cells of infected persons treated with highly active antiretroviral therapy (HAART). This reservoir must be eliminated for the clearance of infection. Using a cDNA library screen, we have identified methyl-CpG binding domain protein 2 (MBD2) as a regulator of HIV-1 latency. Two CpG islands flank the HIV-1 transcription start site and are methylated in latently infected Jurkat cells and primary CD4+ T cells. MBD2 and histone deacetylase 2 (HDAC2) are found at one of these CpG islands during latency. Inhibition of cytosine methylation with 5-aza-2′deoxycytidine (aza-CdR) abrogates recruitment of MBD2 and HDAC2. Furthermore, aza-CdR potently synergizes with the NF-κB activators prostratin or TNF-α to reactivate latent HIV-1. These observations confirm that cytosine methylation and MBD2 are epigenetic regulators of HIV-1 latency. Clearance of HIV-1 from infected persons may be enhanced by inclusion of DNA methylation inhibitors, such as aza-CdR, and NF-κB activators into current antiviral therapies.

A Limited Group of Class I Histone Deacetylases Acts To Repress Human Immunodeficiency Virus Type 1 Expression

Silencing of the integrated human immunodeficiency virus type 1 (HIV-1) genome in resting CD4(+) T cells is a significant contributor to the persistence of infection, allowing the virus to evade both immune detection and pharmaceutical attack. Nonselective histone deacetylase (HDAC) inhibitors are capable of inducing expression of quiescent HIV-1 in latently infected cells. However, potent global HDAC inhibition can induce host toxicity. To determine the specific HDACs that regulate HIV-1 transcription, we evaluated HDAC1 to HDAC11 RNA expression and protein expression and compartmentalization in the resting CD4(+) T cells of HIV-1-positive, aviremic patients. HDAC1, -3, and -7 had the highest mRNA expression levels in these cells. Although all HDACs were detected in resting CD4(+) T cells by Western blot analysis, HDAC5, -8, and -11 were primarily sequestered in the cytoplasm. Using chromatin immunoprecipitation assays, we detected HDAC1, -2, and -3 at the HIV-1 promoter in Jurkat J89GFP cells. Targeted inhibition of HDACs by small interfering RNA demonstrated that HDAC2 and HDAC3 contribute to repression of HIV-1 long terminal repeat expression in the HeLa P4/R5 cell line model of latency. Together, these results suggest that HDAC inhibitors specific for a limited number of class I HDACs may offer a targeted approach to the disruption of persistent HIV-1 infection.

Transcriptional Restriction of Human Immunodeficiency Virus Type 1 Gene Expression in Undifferentiated Primary Monocytes

Monocytes are critical precursors of dendritic cells and macrophages, which play an important role in the pathogenesis of human immunodeficiency virus type 1 (HIV-1). HIV-1 postentry infection is blocked in undifferentiated monocytes in vitro, while the underlying mechanisms are not fully understood. HIV-1 Tat-mediated transactivation of the viral long terminal repeat (LTR) promoter is essential for HIV-1 transcription. Two critical cellular cofactors of HIV-1 Tat, cyclin T1 (CycT1) and cyclin-dependent kinase 9 (CDK9), are required for LTR-directed HIV-1 transcription. In addition to the previously identified restrictions in early viral life cycle, we find that HIV-1 gene expression is impaired in undifferentiated primary monocytes. Transfection of monocytes by nucleofection with HIV-1 proviral DNA could not produce infectious HIV-1. The lack of Tat transactivation of the LTR promoter correlated with the impaired HIV-1 gene expression in monocytes. Interestingly, heterokaryons between primary monocytes and a human embryonic kidney cell line restored Tat transactivation of LTR, suggesting that monocytes lack cellular factors required for Tat transactivation. CycT1 protein was undetectable in freshly isolated monocytes and induced in monocyte-differentiated macrophages, while the expression of CDK9 remained constant. Transient expression of CycT1 in undifferentiated monocytes could not rescue Tat transactivation, suggesting that CycT1 is not the only limiting factor of HIV-1 infection in monocytes. Furthermore, monocyte differentiation into macrophages appeared to enhance the phosphorylation of CDK9, which correlated with significantly increased HIV-1 infection in macrophages. Our results provide new insights into HIV-1 infection and regulation in primary monocytes and viral pathogenesis.

Noncytotoxic Suppression of Human Immunodeficiency Virus Type 1 Transcription by Exosomes Secreted from CD8+T Cells

CD8(+) T cells display a noncytotoxic activity that suppresses transcription of human immunodeficiency virus type 1 (HIV-1) in an antigen-independent and major histocompatibility complex-unrestricted manner. To date, the precise cellular and molecular factors mediating this CD8(+) T-cell effector function remain unsolved. Despite evidence indicating the dependence of the activity on cell-cell contact, the possibility of a membrane-mediated activity that represses transcription from the viral promoter remains unexplored. We therefore investigated whether this inhibition of HIV-1 transcription might be elicited by a membrane-bound determinant. Using a CD8(+) T-cell line displaying potent noncytotoxic HIV-1 suppression activity, we have identified a membrane-localized HIV-1-suppressing activity that is concomitantly secreted as 30- to 100-nm endosome-derived tetraspanin-rich vesicles known as exosomes. Purified exosomes from CD8(+) T-cell culture supernatant noncytotoxically suppressed CCR5-tropic (R5) and CXCR4-tropic (X4) replication of HIV-1 in vitro through a protein moiety. Similar antiviral activity was also found in exosomes isolated from two HIV-1-infected subjects. The antiviral exosomes specifically inhibited HIV-1 transcription in both acute and chronic models of infection. Our results, for the first time, indicate the existence of an antiviral membrane-bound factor consistent with the hallmarks defining noncytotoxic CD8(+) T-cell suppression of HIV-1.

CDK13, a New Potential Human Immunodeficiency Virus Type 1 Inhibitory Factor Regulating Viral mRNA Splicing

The human immunodeficiency virus type 1 (HIV-1) Tat is a 14-kDa viral protein that acts as a potent transactivator by binding to the transactivation-responsive region, a structured RNA element located at the 5' end of all HIV-1 transcripts. Tat transactivates viral gene expression by inducing the phosphorylation of the C-terminal domain of RNA polymerase II through several Tat-activated kinases and by recruiting chromatin-remodeling complexes and histone-modifying enzymes to the HIV-1 long terminal repeat. Histone acetyltransferases, including p300 and hGCN5, not only acetylate histones but also acetylate Tat at lysine positions 50 and 51 in the arginine-rich motif. Acetylated Tat at positions 50 and 51 interacts with a specialized protein module, the bromodomain, and recruits novel factors having this particular domain, such as P/CAF and SWI/SNF. In addition to having its effect on transcription, Tat has been shown to be involved in splicing. In this study, we demonstrate that Tat interacts with cyclin-dependent kinase 13 (CDK13) both in vivo and in vitro. We also found that CDK13 increases HIV-1 mRNA splicing and favors the production of the doubly spliced protein Nef. In addition, we demonstrate that CDK13 acts as a possible restriction factor, in that its overexpression decreases the production of the viral proteins Gag and Env and subsequently suppresses virus production. Using small interfering RNA against CDK13, we show that silencing of CDK13 leads to a significant increase in virus production. Finally, we demonstrate that CDK13 mediates its effect on splicing through the phosphorylation of ASF/SF2.

Identification of functional microRNAs released through asymmetrical processing of HIV-1 TAR element

The interaction between human immunodeficiency virus type 1 (HIV-1) and RNA silencing pathways is complex and multifaceted. Essential for efficient viral transcription and supporting Tat-mediated transactivation of viral gene expression, the trans-activation responsive (TAR) element is a structured RNA located at the 5′ end of all transcripts derived from HIV-1. Here, we report that this element is a source of microRNAs (miRNAs) in cultured HIV-1-infected cell lines and in HIV-1-infected human CD4+ T lymphocytes. Using primer extension and ribonuclease (RNase) protection assays, we delineated both strands of the TAR miRNA duplex deriving from a model HIV-1 transcript, namely miR-TAR-5p and miR-TAR-3p. In vitro RNase assays indicate that the lack of a free 3′ extremity at the base of TAR may contribute to its low processing reactivity in vivo. Both miR-TAR-5p and miR-TAR-3p down-regulated TAR miRNA sensor activity in a process that required an integral miRNA-guided RNA silencing machinery. miR-TAR-3p exerted superior gene downregulatory effects, probably due to its preferential release from HIV-1 TAR RNA by the RNase III Dicer. Our study suggests that the TAR element of HIV-1 transcripts releases functionally competent miRNAs upon asymmetrical processing by Dicer, thereby providing novel insights into viral miRNA biogenesis.

Proximity and Branch Migration Mechanisms in HIV-1 Minus Strand Strong Stop DNA Transfer

Human immunodeficiency virus type 1 minus strand transfer was measured using a genomic donor-acceptor template system in vitro. Donor RNA D199, having the minimum region required for minus strong stop DNA synthesis, was previously shown to transfer with 35% efficiency to an acceptor RNA representing the 3' repeat region. Donor D520, having an additional 321-nucleotide segment extending into gag, transferred at 75% efficiency. In this study each transfer step was analyzed to account for the difference. Measurement of terminal transfer indicated that the 3' terminus of the cDNA generated using D520 is more accessible for transfer than that of D199. Nevertheless, acceptor competition experiments demonstrated that D520 has a greater preference for invasion-driven versus terminal transfer than D199. Competition mapping showed that the base of the transactivation response element is the primary invasion site for D520, important for efficient acceptor invasion. Acceptors complementary to the invasion and terminal transfer sites, but not the region between, allowed assessment of the significance of hybrid propagation by branch migration. These bipartite acceptors showed that with D520, invasion raises the local concentration of the acceptor for efficient terminal transfer by a proximity effect. However, with D199, invasion is relatively inefficient, and the cDNA 3' terminus is not very accessible. For most transfers that occurred, the acceptor accessed the cDNA 3' end by branch migration. Results suggest that both proximity and branch migration mechanisms contribute to transfers, with the proportion determined by donor-cDNA structure. D520 transfers better because it has greater accessibility for both invasion and terminus transfer.