Long non-coding RNAs (lncRNAs) play critical roles in diverse cellular processes; however, their involvement in many critical aspects of the immune response including the interferon (IFN) response remains poorly understood. To address this gap, we compared the global gene expression pattern of primary human hepatocytes before and at three time points after treatment with IFN-α. Among ∼200 IFN-induced lncRNAs, one transcript showed ∼100-fold induction. This RNA, which we named lncRNA-CMPK2, was a spliced, polyadenylated nuclear transcript that was induced by IFN in diverse cell types from human and mouse. Similar to protein-coding IFN-stimulated genes (ISGs), its induction was dependent on JAK-STAT signaling. Intriguingly, knockdown of lncRNA-CMPK2 resulted in a marked reduction in HCV replication in IFN-stimulated hepatocytes, suggesting that it could affect the antiviral role of IFN. We could show that lncRNA-CMPK2 knockdown resulted in upregulation of several protein-coding antiviral ISGs. The observed upregulation was caused by an increase in both basal and IFN-stimulated transcription, consistent with loss of transcriptional inhibition in knockdown cells. These results indicate that the IFN response involves a lncRNA-mediated negative regulatory mechanism. lncRNA-CMPK2 was strongly upregulated in a subset of HCV-infected human livers, suggesting a role in modulation of the IFN response in vivo.

The major reservoirs for HIV in the CNS are in the microglia, perivascular macrophages, and to a lesser extent, astrocytes. To study the molecular events controlling HIV expression in the microglia, we developed a reliable and robust method to immortalize microglial cells from primary glia from fresh CNS tissues and commercially available frozen glial cells. Primary human cells, including cells obtained from adult brain tissue, were transformed with lentiviral vectors expressing SV40 T antigen or a combination of SVR40 T antigen and hTERT. The immortalized cells have microglia-like morphology and express key microglial surface markers including CD11b, TGFβR, and P2RY12. Importantly, these cells were confirmed to be of human origin by sequencing. The RNA expression profiles identified by RNA-seq are also characteristic of microglial cells. Furthermore, the cells demonstrate the expected migratory and phagocytic activity, and the capacity to mount an inflammatory response characteristic of primary microglia. The immortalization method has also been successfully applied to a wide range of microglia from other species (macaque, rat, and mouse). To investigate different aspects of HIV molecular regulation in CNS, the cells have been superinfected with HIV reporter viruses and latently infected clones have been selected that reactive HIV in response to inflammatory signals. The cell lines we have developed and rigorously characterized will provide an invaluable resource for the study of HIV infection in microglial cells as well as studies of microglial cell function.

Recent transcriptome analyses have indicated that a large part of mammalian genomes are transcribed into long non-protein-coding RNAs (lncRNAs). However, only a very small fraction of them have been individually studied, and whether the majority of lncRNAs found in large-scale studies have a cellular role is debated. To gain insight into the sequence features and genomic architecture of the subset of lncRNAs that have been proven to be functional, we created a database containing studied lncRNAs manually culled from the literature along with a parallel database containing all annotated protein-coding human RNAs. The Functional lncRNA Database, which contains 204 lncRNAs and their splicing variants, is available at valadkhanlab.org/database. Analysis of the lncRNAs and their comparison to protein-coding transcripts revealed sequence features including paucity of introns and low GC content in lncRNAs, which could explain several biological characteristics of these transcripts, such as their nuclear localization and low expression level. The predicted ORFs in lncRNAs have poor start codon and ORF contexts, which would lead to activation of the nonsense-mediated decay pathways and thus make it unlikely for most lncRNAs to code for even short peptides. Interestingly, our analyses revealed significant similarities between the lncRNAs and the 3′ untranslated regions (3′ UTRs) in protein-coding RNAs in structural features and sequence composition. The presence of these intriguing parallels between the lncRNAs and 3′ UTRs, which constitute the two main components of the RNA-mediated cellular regulatory system, indicates that highly similar evolutionary constraints govern the function of regulatory RNA sequences in the cell.

Significance: The molecular mechanisms leading to the creation and maintenance of the latent HIV reservoir remain incompletely understood. Unbiased shRNA screens showed that the estrogen receptor acts as a potent repressor of proviral reactivation in T cells. Antagonists of ESR-1 activate latent HIV-1 proviruses while agonists, including β-estradiol, potently block HIV reactivation. Using a well-matched set of male and female donors, we found that ESR-1 plays an important role in regulating HIV transcription in both sexes. However, women are much more responsive to estrogen and appear to harbor smaller inducible RNA reservoirs. Accounting for the impact of estrogen on HIV viral reservoirs will therefore be critical for devising curative therapies for women, a group representing 51% of global HIV infections.

The ubiquitous presence of long noncoding RNAs (lncRNAs) in eukaryotes points to the importance of understanding how their sequences impact function. As many lncRNAs regulate nuclear events and thus must localize to nuclei, we analyzed the sequence requirements for nuclear localization in an intergenic lncRNA named BORG (BMP2-OP1-responsive gene), which is both spliced and polyadenylated but is strictly localized in nuclei. Subcellular localization of BORG was not dependent on the context or level of its expression or decay but rather depended on the sequence of the mature, spliced transcript. Mutational analyses indicated that nuclear localization of BORG was mediated through a novel RNA motif consisting of the pentamer sequence AGCCC with sequence restrictions at positions −8 (T or A) and −3 (G or C) relative to the first nucleotide of the pentamer. Mutation of the motif to a scrambled sequence resulted in complete loss of nuclear localization, while addition of even a single copy of the motif to a cytoplasmically localized RNA was sufficient to impart nuclear localization. Further, the presence of this motif in other cellular RNAs showed a direct correlation with nuclear localization, suggesting that the motif may act as a general nuclear localization signal for cellular RNAs.