Epitranscriptomic RNA modifications, including methylation of adenine and cytidine residues, are now recognized as key regulators of both cellular and viral mRNA function. Moreover, acetylation of ...the N4 position of cytidine (ac4C) was recently reported to increase the translation and stability of cellular mRNAs. Here, we show that ac4C and N-acetyltransferase 10 (NAT10), the enzyme that adds ac4C to RNAs, have been subverted by human immunodeficiency virus 1 (HIV-1) to increase viral gene expression. HIV-1 transcripts are modified with ac4C at multiple discrete sites, and silent mutagenesis of these ac4C sites led to decreased HIV-1 gene expression. Similarly, loss of ac4C from viral transcripts due to depletion of NAT10 inhibited HIV-1 replication by reducing viral RNA stability. Interestingly, the NAT10 inhibitor remodelin could inhibit HIV-1 replication at concentrations that have no effect on cell viability, thus identifying ac4C addition as a potential target for antiviral drug development.
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•Cytidines on HIV-1 RNAs are acetylated to ac4C by NAT10•Deposition of ac4C residues enhances viral RNA stability•Silent mutagenesis of ac4C sites causes a NAT10-dependent drop in HIV-1 replication•Remodelin, a small molecule NAT10 inhibitor, can inhibit HIV-1 gene expression
Tsai et al. report that multiple cytidines on HIV-1 RNAs are acetylated to ac4C by cellular NAT10. Depletion of ac4C by mutagenesis or drug treatment reduces the stability of HIV-1 transcripts, causing reduced viral replication. Thus, HIV-1 has evolved to highjack a host epitranscriptomic modification enzyme to enhance viral replication.
The purpose of the present study was to test the applicability of self-determination theory (Deci and Ryan in J Res Pers 19:109–134. doi:
10.1016/0092-6566(85)90023-6
,
1985
; Can Psychol 49:182–185. ...doi:
10.1037/a0012801
,
2008
) across developmental periods by differentiating children and adolescents on the importance of individual needs (i.e., autonomy, competence, relatedness) and the role of balance across contexts (i.e., home, school, peers) in predicting depressive symptoms. Participants completed the Children’s Intrinsic Need Satisfaction Scale (Koestner and Veronneau in The Children’s Intrinsic Needs Satisfaction Scale. McGill University, Montreal,
2001
) and the Children’s Depression Inventory (Kovacs in Children’s depression inventory manual. Multi-Health Systems, North Tonawanda,
1992
). Results indicated that only the need for competence was significantly related to depressive symptoms in the child sample (
n
= 149) whereas, the satisfaction of autonomy and relatedness were significant predictors in the adolescent sample (
n
= 153). In both samples, need balance across contexts was a significant predictor over and above the level of satisfaction of each individual need. Implications for clinical practice and for theory will be presented.
The present study applied self-determination theory to examine the onset, maintenance, and cessation of non-suicidal self-injury (NSSI) in adolescents. Specifically, the study examined the ...relationship between the basic psychological needs of autonomy, competence, and relatedness, and NSSI status. Participants were classified into the NSSI Maintain (n = 30), NSSI Start (n = 44), NSSI Stop (n = 21), or Control (n = 98) groups based on NSSI status over 2 time points within a 12-month period. Repeated measures multiple analysis of variance was employed. Satisfaction of the need for competence decreased over time in all adolescents. Adolescents who maintained NSSI behavior reported significantly lower levels of need satisfaction compared to adolescents reporting no history of NSSI engagement, and adolescents who began NSSI over the course of the study reported significantly lower levels of need satisfaction compared to those reporting no history of NSSI engagement. The findings suggest that need satisfaction varies as a function of NSSI status.
Objective: We applied self-determination theory to examine a model whereby perceived parental autonomy support directly and indirectly affects nonsuicidal self-injury (NSSI) through difficulties in ...emotion regulation. Method: 639 participants (53% female) with a mean age of 13.38 years (SD = 0.51) completed the How I Deal with Stress Questionnaire as a screener for NSSI, the Perceptions of Parents Scale, and the Difficulties in emotion Regulation Scale. Participants who indicated having ever hurt themselves on purpose without the intent to die (n = 116, 66% female) were classified in the NSSI lifetime group. Results: A mediation analysis with bootstrapping procedure revealed that adolescents who reported their parents as being less supportive of their need for autonomy were more likely to have engaged in NSSI. Further, this relationship was partially mediated by emotion regulation. Conclusion: Adolescents who do not perceive autonomy support from their parents, have more difficulties regulating their emotions, and may turn to NSSI as a means to cope. Clinical implications of the findings suggest involving the family, and specifically, targeting parental autonomy support may be beneficial when working with young adolescents who self-injure.
Impact and Implications
High school students in the present study report that a lack of parental autonomy support leads to difficulties in emotion regulation and increased risk for engagement in nonsuicidal self-injury. Considering this pathway, school psychologists may find it helpful to involve parents in interventions and to target specific parenting practices that can increase autonomy support.
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•The HIV splice site A3 regulatory locus intrinsically adopts multiple structures in solution, which are phylogenetically conserved across different viral strains.•The major structure ...observed in vitro folds to completely sequester the PPyT in base pairs with the ESS2p element and a downstream Linker.•The 3D structure of A3SL1 reveals that the PPyT is completely engaged in tertiary interactions including with the A3 cleavage site, which undergoes slow conformational exchange.•Mutations designed to destabilize the PPyT:ESS2p helix correlate with increasing splicing to acceptor site A3 within HIV infected cells, validating the functional significance of this interaction.•Biophysical characterizations provide evidence that hnRNP A1 but not hnRNP H binds with high affinity and specificity to A3SL1.
Alternative splicing of the HIV transcriptome is controlled through cis regulatory elements functioning as enhancers or silencers depending on their context and the type of host RNA binding proteins they recruit. Splice site acceptor A3 (ssA3) is one of the least used acceptor sites in the HIV transcriptome and its activity determines the levels of tat mRNA. Splice acceptor 3 is regulated by a combination of cis regulatory sequences, auxiliary splicing factors, and presumably RNA structure. The mechanisms by which these multiple regulatory components coordinate to determine the frequency in which ssA3 is utilized is poorly understood. By NMR spectroscopy and phylogenetic analysis, we show that the ssA3 regulatory locus is conformationally heterogeneous and that the sequences that encompass the locus are conserved across most HIV isolates. Despite the conformational heterogeneity, the major stem loop (A3SL1) observed in vitro folds to base pair the Polypyrimdine Tract (PPyT) to the Exon Splicing Silencer 2p (ESS2p) element and to a conserved downstream linker. The 3D structure as determined by NMR spectroscopy further reveals that the A3 consensus cleavage site is embedded within a unique stereochemical environment within the apical loop, where it is surrounded by alternating base–base interactions. Despite being described as a receptor for hnRNP H, the ESS2p element is sequestered by base pairing to the 3′ end of the PPyT and within this context it cannot form a stable complex with hnRNP H. By comparison, hnRNP A1 directly binds to the A3 consensus cleavage site located within the apical loop, suggesting that it can directly modulate U2AF assembly. Sequence mutations designed to destabilize the PPyT:ESS2p helix results in an increase usage of ssA3 within HIV-infected cells, consistent with the PPyT becoming more accessible for U2AF recognition. Additional mutations introduced into the downstream ESS2 element synergize with ESS2p to cause further increases in ssA3 usage. When taken together, our work provides a unifying picture by which cis regulatory sequences, splicing auxiliary factors and RNA structure cooperate to provide stringent control over ssA3. We describe this as the pair-and-lock mechanism to restrict access of the PPyT, and posit that it operates to regulate a subset of the heterogenous structures encompassing the ssA3 regulatory locus.
Between October 1977 and February 2002, a total of 343 patients (mean age 62 +/- 13 years; range: 19-91 years) underwent double valve replacement (DVR) with the St. Jude Medical (SJM) heart valve. ...Among the replacements, 337 (98%) were aortic and mitral in nature. Concomitant coronary artery bypass was performed in 73 patients (21%).
Cardiac Surgical Associates has maintained an independent database of patients undergoing valve replacement with the SJM prosthesis since the valve's first implantation in October 1977. Patients were contacted by questionnaire and/or telephone (94% complete) between November 2002 and June 2003. The patients' hospital course and valve-related events were verified by patient chart review and/or physician contact.
Operative mortality was 8% (n = 29); mortality was valve-related in two cases. The mean follow up was 6.5 +/- 6.0 years (range: 1 month to 24 years); total follow up was 2,226 patient-years. Over 25 years, patient freedom from late mortality was 62%, and from valve-related mortality 78%. Freedom from thromboembolic events was 82% (93% from permanent defect), from bleeding events 76%, from endocarditis 98%, from valve thrombosis 99.9%, and from reoperation 98%. Six reoperations were carried out in five patients (2%), valve repair or replacement in five (2%), and suture closure of paravalvular leak in one patient (0.3%). There were no valve structural failures reported.
The SJM valve has proven to be an effective and durable heart valve prosthesis. Over the long-term, the event rate is low and there is excellent freedom from reoperation in the double valve configuration.
Previous work has demonstrated that the epitranscriptomic addition of m
A to viral transcripts can promote the replication and pathogenicity of a wide range of DNA and RNA viruses, including HIV-1, ...yet the underlying mechanisms responsible for this effect have remained unclear. It is known that m
A function is largely mediated by cellular m
A binding proteins or readers, yet how these regulate viral gene expression in general, and HIV-1 gene expression in particular, has been controversial. Here, we confirm that m
A addition indeed regulates HIV-1 RNA expression and demonstrate that this effect is largely mediated by the nuclear m
A reader YTHDC1 and the cytoplasmic m
A reader YTHDF2. Both YTHDC1 and YTHDF2 bind to multiple distinct and overlapping sites on the HIV-1 RNA genome, with YTHDC1 recruitment serving to regulate the alternative splicing of HIV-1 RNAs. Unexpectedly, while YTHDF2 binding to m
A residues present on cellular mRNAs resulted in their destabilization as previously reported, YTHDF2 binding to m
A sites on HIV-1 transcripts resulted in a marked increase in the stability of these viral RNAs. Thus, YTHDF2 binding can exert diametrically opposite effects on RNA stability, depending on RNA sequence context.
Viral protein R (Vpr) is an HIV-1 accessory protein whose function remains poorly understood. In this report, we sought to determine the requirement of Vpr for facilitating HIV-1 infection of ...monocyte-derived dendritic cells (MDDCs), one of the first cell types to encounter virus in the peripheral mucosal tissues. In this report, we characterize a significant restriction of Vpr-deficient virus replication and spread in MDDCs alone and in cell-to-cell spread in MDDC-CD4
T cell cocultures. This restriction of HIV-1 replication in MDDCs was observed in a single round of virus replication and was rescued by the expression of Vpr in
in the incoming virion. Interestingly, infections of MDDCs with viruses that encode Vpr mutants unable to interact with either the DCAF1/DDB1 E3 ubiquitin ligase complex or a host factor hypothesized to be targeted for degradation by Vpr also displayed a significant replication defect. While the extent of proviral integration in HIV-1-infected MDDCs was unaffected by the absence of Vpr, the transcriptional activity of the viral long terminal repeat (LTR) from Vpr-deficient proviruses was significantly reduced. Together, these results characterize a novel postintegration restriction of HIV-1 replication in MDDCs and show that the interaction of Vpr with the DCAF1/DDB1 E3 ubiquitin ligase complex and the yet-to-be-identified host factor might alleviate this restriction by inducing transcription from the viral LTR. Taken together, these findings identify a robust
cell culture system that is amenable to addressing mechanisms underlying Vpr-mediated enhancement of HIV-1 replication.
Despite decades of work, the function of the HIV-1 protein Vpr remains poorly understood, primarily due to the lack of an
cell culture system that demonstrates a deficit in replication upon infection with viruses in the absence of Vpr. In this report, we describe a novel cell infection system that utilizes primary human dendritic cells, which display a robust decrease in viral replication upon infection with Vpr-deficient HIV-1. We show that this replication difference occurs in a single round of infection and is due to decreased transcriptional output from the integrated viral genome. Viral transcription could be rescued by virion-associated Vpr. Using mutational analysis, we show that domains of Vpr involved in binding to the DCAF1/DDB1/E3 ubiquitin ligase complex and prevention of cell cycle progression into mitosis are required for LTR-mediated viral expression, suggesting that the evolutionarily conserved G
cell cycle arrest function of Vpr is essential for HIV-1 replication.
To investigate the role of bone morphogenetic protein (BMP) signaling in osteoclastogenesis in vivo, we eliminated BMPRII in osteoclasts by creating a BMPRIIfl/fl;lysM-Cre mouse strain. Conditional ...knock-out (cKO) mice are osteopetrotic when compared with WT controls due to a decrease in osteoclast activity. Bone marrow macrophages (BMMs) isolated from cKO mice are severely inhibited in their capacity to differentiate into mature osteoclasts in the presence of M-CSF and receptor activator of NF-κB (RANK) ligand. We also show that BMP noncanonical (MAPK) and canonical (SMAD) pathways are utilized at different stages of osteoclast differentiation. BMP2 induces p38 phosphorylation in pre-fusion osteoclasts and increases SMAD phosphorylation around osteoclast precursor fusion. Phosphorylation of MAPKs was decreased in differentiated BMMs from cKO animals. Treating BMMs with the SMAD inhibitor dorsomorphin confirms the requirement for the canonical pathway around the time of fusion. These results demonstrate the requirement for BMP signaling in osteoclasts for proper bone homeostasis and also explore the complex signaling mechanisms employed by BMP signaling during osteoclast differentiation.
Background: BMPs affect osteoclastogenesis in vitro, but the effects of BMP signaling on osteoclastogenesis in vivo are not well understood.
Results: Conditional deletion of BMPRII in osteoclasts results in reduced osteoclastogenesis, resulting in increased bone.
Conclusion: BMP signaling is required for proper bone remodeling in vivo.
Significance: Identifying factors affecting osteoclast differentiation increase understanding of bone remodeling regulation in vivo.
Latency reveRsaI strategies for HIV cure using inhibitor of apoptosis protein (IAP) antagonists (IAPi) induce unprecedented levels of latent reservoir expression without immunotoxicity during ...suppressive antiretroviral therapy (ART). However, full targeting of the reservoir may require combinatorial approaches. A Jurkat latency model screen for IAPi combination partners demonstrated synergistic latency reveRsaI with bromodomain (BD) and extraterminal domain protein inhibitors (BETi). Mechanistic investigations using CRISPR-CAS9 and single-cell RNA-Seq informed comprehensive ex vivo evaluations of IAPi plus pan-BET, bD-selective BET, or selective BET isoform targeting in CD4.sup.+ T cells from ART-suppressed donors. IAPi+BETi treatment resulted in striking induction of cell-associated HIV gag RNA, but lesser induction of fully elongated and tat-rev RNA compared with T cell activation-positive controls. IAPi+BETi resulted in HIV protein induction in bulk cultures of CD4.sup.+ T cells using an ultrasensitive p24 assay, but did not result in enhanced viral outgrowth frequency using a standard quantitative viral outgrowth assay. This study defines HIV transcriptional elongation and splicing as important barriers to latent HIV protein expression following latency reveRsaI, delineates the roles of BET proteins and their BDs in HIV latency, and provides a rationale for exploration of IAPi+BETi in animal models of HIV latency.
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