Long-term persistence of transcriptionally active 'defective' HIV-1 proviruses: implications for persistent immune activation during antiretroviral therapy.

OBJECTIVES: People with HIV-1 (PWH) on effective antiretroviral therapy (ART) continue to exhibit chronic systemic inflammation, immune activation, and persistent elevations in markers of HIV-1 infection [including HIV-DNA, cell-associated HIV-RNA (CA HIV-RNA), and antibodies to HIV-1 proteins] despite prolonged suppression of plasma HIV-RNA levels less than 50 copies/ml. Here, we investigated the hypothesis that nonreplicating but transcriptionally and translationally competent 'defective' HIV-1 proviruses may be one of drivers of these phenomena. DESIGN: A combined cohort of 23 viremic and virologically suppressed individuals on ART were studied. METHODS: HIV-DNA, CA HIV-RNA, western blot score (measure of anti-HIV-1 antibodies as a surrogate for viral protein expression in vivo ), and key biomarkers of inflammation and coagulation (IL-6, hsCRP, TNF-alpha, tissue factor, and D-dimer) were measured in peripheral blood and analyzed using a combined cross-sectional and longitudinal approaches. Sequences of HIV-DNA and CA HIV-RNA obtained via 5'-LTR-to-3'-LTR PCR and single-genome sequencing were also analyzed. RESULTS: We observed similar long-term persistence of multiple, unique, transcriptionally active 'defective' HIV-1 provirus clones (average: 11 years., range: 4-20 years) and antibody responses against HIV-1 viral proteins among all ART-treated participants evaluated. A direct correlation was observed between the magnitude of HIV-1 western blot score and the levels of transcription of 'defective' HIV-1 proviruses ( r  = 0.73, P  < 0.01). Additional correlations were noted between total CD8 + T-cell counts and HIV-DNA ( r  = 0.52, P  = 0.01) or CA HIV-RNA ( r  = 0.65, P  < 0.01). CONCLUSION: These findings suggest a novel interplay between transcription and translation of 'defective' HIV-1 proviruses and the persistent immune activation seen in the setting of treated chronic HIV-1 infection.

The two sRNAs OmrA and OmrB indirectly repress transcription from the LEE1 promoter of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic bacterium that predominantly infects infants in developing countries. EPEC forms attaching and effacing (A/E) lesions on the apical surface of the small intestine, leading to diarrhea. The locus of enterocyte effacement (LEE) is both necessary and sufficient for A/E lesion morphogenesis by EPEC. Gene expression from this virulence determinant is controlled by an elaborate regulatory web that extends beyond protein-based transcriptional regulators and includes small regulatory RNA (sRNA) that exert their effects posttranscriptionally. To date, only 4 Hfq-dependent sRNAs-MgrR, RyhB, McaS, and Spot42-have been identified that affect the LEE of EPEC by diverse mechanisms and elicit varying regulatory outcomes. In this study, we demonstrate that the paralogous Hfq-dependent sRNAs OmrA and OmrB globally silence the LEE to diminish the ability of EPEC to form A/E lesions. Interestingly, OmrA and OmrB do not appear to directly target a LEE-encoded gene; rather, they repress transcription from the LEE1 promoter indirectly, by means of an as-yet-unidentified transcriptional factor that binds within 200 base pairs upstream of the transcription start site to reduce the expression of the LEE master regulator Ler, which, in turn, leads to reduced morphogenesis of A/E lesions. Additionally, OmrA and OmrB also repress motility in EPEC by targeting the 5' UTR of the flagellar master regulator, flhD.

Interneurons Regulate Locomotion Quiescence via Cyclic Adenosine Monophosphate Signaling During Stress-Induced Sleep in Caenorhabditis elegans.

Sleep is evolutionarily conserved, thus studying simple invertebrates such as Caenorhabditis elegans can provide mechanistic insight into sleep with single cell resolution. A conserved pathway regulating sleep across phylogeny involves cyclic adenosine monophosphate (cAMP), a ubiquitous second messenger that functions in neurons by activating protein kinase A. C. elegans sleep in response to cellular stress caused by environmental insults [stress-induced sleep (SIS)], a model for studying sleep during sickness. SIS is controlled by simple neural circuitry, thus allowing for cellular dissection of cAMP signaling during sleep. We employed a red-light activated adenylyl cyclase, IlaC22, to identify cells involved in SIS regulation. We found that pan-neuronal activation of IlaC22 disrupts SIS through mechanisms independent of the cAMP response element binding protein. Activating IlaC22 in the single DVA interneuron, the paired RIF interneurons, and in the CEPsh glia identified these cells as wake-promoting. Using a cAMP biosensor, epac1-camps, we found that cAMP is decreased in the RIF and DVA interneurons by neuropeptidergic signaling from the ALA neuron. Ectopic overexpression of sleep-promoting neuropeptides coded by flp-13 and flp-24, released from the ALA, reduced cAMP in the DVA and RIFs, respectively. Overexpression of the wake-promoting neuropeptides coded by pdf-1 increased cAMP levels in the RIFs. Using a combination of optogenetic manipulation and in vivo imaging of cAMP we have identified wake-promoting neurons downstream of the neuropeptidergic output of the ALA. Our data suggest that sleep- and wake-promoting neuropeptides signal to reduce and heighten cAMP levels during sleep, respectively.

Hfq and three Hfq-dependent small regulatory RNAs-MgrR, RyhB and McaS-coregulate the locus of enterocyte effacement in enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) is a significant cause of infantile diarrhea and death in developing countries. The pathogenicity island locus of enterocyte effacement (LEE) is essential for EPEC to cause diarrhea. Besides EPEC, the LEE is also present in other gastrointestinal pathogens, most notably enterohemorrhagic E. coli (EHEC). Whereas transcriptional control of the LEE has been meticulously examined, posttranscriptional regulation, including the role of Hfq-dependent small RNAs, remains undercharacterized. However, the past few years have witnessed a surge in the identification of riboregulators of the LEE in EHEC. Contrastingly, the posttranscriptional regulatory landscape of EPEC remains cryptic. Here we demonstrate that the RNA-chaperone Hfq represses the LEE of EPEC by targeting the 5' untranslated leader region of grlR in the grlRA mRNA. Three conserved small regulatory RNAs (sRNAs)-MgrR, RyhB and McaS-are involved in the Hfq-dependent regulation of grlRA MgrR and RyhB exert their effects by directly base-pairing to the 5' region of grlR Whereas MgrR selectively represses grlR but activates grlA, RyhB represses gene expression from the entire grlRA transcript. Meanwhile, McaS appears to target the grlRA mRNA indirectly. Thus, our results provide the first definitive evidence that implicates multiple sRNAs in regulating the LEE and the resulting virulence of EPEC.