Despite receiving antiretroviral therapy, most patients with HIV still have latent reservoirs of the virus; here, these reservoirs are shown to be dominated by viruses with cytotoxic T lymphocyte escape mutations, with potential implications for the development of therapeutic vaccines. Antiretroviral therapy does not cure HIV-1 infection: despite drug treatment, most patients still have latent reservoirs of the virus. This study of immune cells isolated from 30 HIV-1-infected patients who had been on antiretroviral therapy for at least two years and had maintained undetectable plasma HIV-1 RNA levels, shows that these viral reservoirs are dominated by viruses with cytotoxic T lymphocyte escape mutations. This finding implies that future directions in therapeutic vaccine design may need to focus on boosting broad cytotoxic T lymphocyte responses. Despite antiretroviral therapy (ART), human immunodeficiency virus (HIV)-1 persists in a stable latent reservoir1,2, primarily in resting memory CD4+ T cells3,4. This reservoir presents a major barrier to the cure of HIV-1 infection. To purge the reservoir, pharmacological reactivation of latent HIV-1 has been proposed5 and tested both in vitro and in vivo6,7,8. A key remaining question is whether virus-specific immune mechanisms, including cytotoxic T lymphocytes (CTLs), can clear infected cells in ART-treated patients after latency is reversed. Here we show that there is a striking all or none pattern for CTL escape mutations in HIV-1 Gag epitopes. Unless ART is started early, the vast majority (>98%) of latent viruses carry CTL escape mutations that render infected cells insensitive to CTLs directed at common epitopes. To solve this problem, we identified CTLs that could recognize epitopes from latent HIV-1 that were unmutated in every chronically infected patient tested. Upon stimulation, these CTLs eliminated target cells infected with autologous virus derived from the latent reservoir, both in vitro and in patient-derived humanized mice. The predominance of CTL-resistant viruses in the latent reservoir poses a major challenge to viral eradication. Our results demonstrate that chronically infected patients retain a broad-spectrum viral-specific CTL response and that appropriate boosting of this response may be required for the elimination of the latent reservoir.

Genes expressed under circadian-clock control are found in organisms ranging from prokaryotes to humans. In Drosophila melanogaster, the period (per) gene, which is required for clock function, is transcribed in a circadian manner. We have identified a circadian transcriptional enhancer within a 69-bp DNA fragment upstream of the per gene. This enhancer drives high-amplitude mRNA cycling under light-dark-cycling or constant-dark conditions, and this activity is per protein (PER) dependent. An E-box sequence within this 69-bp fragment is necessary for high-level expression, but not for rhythmic expression, indicating that PER mediates circadian transcription through other sequences in this fragment.

Objective: To determine the feasibility of next-generation sequencing (NGS) microbiome approaches in the diagnosis of infectious disorders in brain or spinal cord biopsies in patients with suspected CNS infections. Methods: In a prospective pilot study, we applied NGS in combination with a new computational analysis pipeline to detect the presence of pathogenic microbes in brain or spinal cord biopsies from 10 patients with neurologic problems indicating possible infection but for whom conventional clinical and microbiology studies yielded negative or inconclusive results. Results: Direct DNA and RNA sequencing of brain tissue biopsies generated 8.3 million to 29.1 million sequence reads per sample, which successfully identified with high confidence the infectious agent in 3 patients for whom validation techniques confirmed the pathogens identified by NGS. Although NGS was unable to identify with precision infectious agents in the remaining cases, it contributed to the understanding of neuropathologic processes in 5 others, demonstrating the power of large-scale unbiased sequencing as a novel diagnostic tool. Clinical outcomes were consistent with the findings yielded by NGS on the presence or absence of an infectious pathogenic process in 8 of 10 cases, and were noncontributory in the remaining 2. Conclusions: NGS-guided metagenomic studies of brain, spinal cord, or meningeal biopsies offer the possibility for dramatic improvements in our ability to detect (or rule out) a wide range of CNS pathogens, with potential benefits in speed, sensitivity, and cost. NGS-based microbiome approaches present a major new opportunity to investigate the potential role of infectious pathogens in the pathogenesis of neuroinflammatory disorders.

Astrocytes are known to be critical regulators of neuronal function. However, relatively few mediators of astrocyte to neuron communication have been identified. Recent advancements in the biology of extracellular vesicles have begun to implicate astrocyte derived extracellular vesicles (ADEV) as mediators of astrocyte to neuron communication, suggesting that alterations in the release and/or composition of ADEVs could influence gliotransmission. TNFα and IL-1β are key mediators of glial activation and neuronal damage, but the effects of these cytokines on the release or molecular composition of ADEVs is unknown. We found that ADEVs released in response to IL-1β (ADEV-IL-1β) and TNFα (ADEV-TNFα) were enriched with miRNAs that target proteins involved in neurotrophin signaling. We confirmed that miR-125a-5p and miR-16-5p (both enriched in ADEV-IL-1β and ADEV-TNFα) targeted NTKR3 and its downstream effector Bcl2. Downregulation of these targets in neurons was associated with reductions in dendritic growth, dendritic complexity, reduced spike rates, and burst activity. Molecular interference of miR-125a-5p and miR-16-5p prevented ADEV-IL-1β from reducing dendritic complexity, spike, and burst rates. These findings suggest that astrocytes respond to inflammatory challenge by modifying the miRNA cargo of ADEVs to diminish the activity of target neurons by regulating the translational expression of proteins controlling programs essential for synaptic stability and neuronal excitability.

RNA polymerase II synthesizes a diverse set of transcripts including both protein-coding and non-coding RNAs. One major difference between these two classes of transcripts is the mechanism of termination. Messenger RNA transcripts terminate downstream of the coding region in a process that is coupled to cleavage and polyadenylation reactions. Non-coding transcripts like Saccharomyces cerevisiae snoRNAs terminate in a process that requires the RNA–binding proteins Nrd1, Nab3, and Sen1. We report here the transcriptome-wide distribution of these termination factors. These data sets derived from in vivo protein–RNA cross-linking provide high-resolution definition of non-poly(A) terminators, identify novel genes regulated by attenuation of nascent transcripts close to the promoter, and demonstrate the widespread occurrence of Nrd1-bound 3′ antisense transcripts on genes that are poorly expressed. In addition, we show that Sen1 does not cross-link efficiently to many expected non-coding RNAs but does cross-link to the 3′ end of most pre–mRNA transcripts, suggesting an extensive role in mRNA 3′ end formation and/or termination. Transcription in eukaryotes is widespread including both protein-coding transcripts and an increasing number of non-coding RNAs. Here we present the results of transcriptome-wide mapping of a set of yeast RNA–binding proteins that control expression of some protein-coding genes and a number of novel non-coding RNAs. The yeast Nrd1-Nab3-Sen1 pathway is required for termination and exosome-mediated processing of non-coding RNA polymerase II transcripts. Our data show that these components bind unexpected targets including a large number of antisense transcripts originating from the 3′ end of genes that are poorly expressed in the sense direction. We also show that Sen1 helicase, involved in termination of non-coding RNAs, is also present at the 3′ end of mRNAs, suggesting a more fundamental role in transcription termination. Mis-regulation of transcription is the underlying cause of many disease states. For example, mutation of the human Sen1 gene, senataxin, causes a range of neurodegenerative disorders. Understanding the roles of yeast RNA–binding proteins in controlling termination of coding and non-coding RNAs will be useful in deciphering the mechanism of these proteins in human cells.