Gene-specific MicroRNA antagonism protects against HIV Tat and TGF-ß-mediated suppression of CFTR mRNA and function.

BACKGROUND: MicroRNAs play an important role in health and disease. TGF-ß signaling, upregulated by HIV Tat, and in chronic airway diseases and smokers upregulates miR-145-5p to suppress cystic fibrosis transmembrane conductance regulator (CFTR). CFTR suppression in chronic airway diseases like Cystic Fibrosis, COPD and smokers has been associated with suppressed MCC and recurrent lung infections and inflammation. This can explain the emergence of recurrent lung infections and inflammation in people living with HIV. METHODS: Tat-induced aberrant microRNAome was identified by miRNA expression analysis. microRNA mimics and antagomirs were used to validate the identified miRNAs involved in Tat mediated CFTR mRNA suppression. CRISPR-based editing of the miRNA target sites in CFTR 3'UTR was used to determine rescue of CFTR mRNA and function in airway epithelial cell lines and in primary human bronchial epithelial cells exposed to TGF-ß and Tat. FINDINGS: HIV Tat upregulates miR-145-5p and miR-509-3p. The two miRNAs demonstrate co-operative effects in suppressing CFTR. CRISPR-based editing of the miRNA target site preserves CFTR mRNA and function in airway epithelial cells INTERPRETATION: Given the important roles of TGF-ß signaling and the multitude of genes regulated by miRNAs, we demonstrate that CRISPR-based gene-specific microRNA antagonism approach can preserve CFTR mRNA and function in the context of HIV Tat and TGF-ß signaling without suppressing expression of other genes regulated by miR-145-5p.

Novel Approaches of CRISPR-Cas Technology in Airway Diseases.

Clustered regularly interspaced palindromic repeats (CRISPR) technique plays a vital role in preclinical modelling of many respiratory diseases. Diseases such as chronic obstructive pulmonary disease (COPD), asthma, acute tracheal bronchitis, pneumonia, tuberculosis, lung cancer, and influenza infection continue to significantly impact human health. CRISPR associated (Cas) proteins, isolated from the immune system of prokaryotes, are one component of a very useful technique to manipulate gene sequences or editing and gene expression with significant implications for respiratory research in the field of molecular biology. CRISPR technology is a promising tool that is easily adaptable for specific editing of DNA sequences of interest with a goal towards modifying or eliminating gene function. Among its many potential applications, CRISPR can be applied to correcting genetic defects as well as for therapeutic approaches for treatment. This review elucidates recent advances in CRISPR-Cas technology in airway diseases.

TGF-ß1 increases viral burden and promotes HIV-1 latency in primary differentiated human bronchial epithelial cells.

Combination antiretroviral therapy (cART) has increased the life expectancy of HIV patients. However, the incidence of non-AIDS associated lung comorbidities, such as COPD and asthma, and that of opportunistic lung infections have become more common among this population. HIV proteins secreted by the anatomical HIV reservoirs can have both autocrine and paracrine effects contributing to the HIV-associated comorbidities. HIV has been recovered from cell-free bronchoalveolar lavage fluid, alveolar macrophages, and intrapulmonary lymphocytes. We have recently shown that ex-vivo cultured primary bronchial epithelial cells and the bronchial brushings from human subjects express canonical HIV receptors CD4, CCR5 and CXCR4 and can be infected with HIV. Together these studies suggest that the lung tissue can serve as an important reservoir for HIV. In this report, we show that TGF-ß1 promotes HIV latency by upregulating a transcriptional repressor BLIMP-1. Furthermore, we identify miR-9-5p as an important intermediate in TGF-ß-mediated BLIMP-1 upregulation and consequent HIV latency. The transcriptionally suppressed HIV can be reactivated by common latency reactivating agents. Together our data suggest that in patients with chronic airway diseases, TGF-ß can elevate the HIV viral reservoir load that could further exacerbate the HIV associated lung comorbidities.

Cigarette smoke promotes HIV infection of primary bronchial epithelium and additively suppresses CFTR function.

Recurrent lung infections are a common cause of morbidity and mortality in people living with HIV and this is exacerbated in smokers even when administered combination antiretroviral therapy (cART). The incidence of pneumonia is increased with smoking and treatment interruption and is directly dependent on viral load in patients when adjusted for CD4 counts. CFTR dysfunction plays an important role in aberrant airway innate immunity as it is pivotal in regulating mucociliary clearance (MCC) rates and other antibacterial mechanisms of the airway. In our earlier work, we have demonstrated that bronchial epithelium expresses canonical HIV receptors CD4, CCR5 and CXCR4 and can be infected with HIV. HIV Tat suppresses CFTR mRNA and function via TGF-ß signaling. In the present study, we demonstrate that cigarette smoke (CS) potentiates HIV infection of bronchial epithelial cells by upregulating CD4 and CCR5 expression. HIV and CS individually and additively suppress CFTR biogenesis and function, possibly explaining the increased incidence of lung infections in HIV patients and its exacerbation in HIV smokers.

HIV Infects Bronchial Epithelium and Suppresses Components of the Mucociliary Clearance Apparatus.

Recurrent lung infections and pneumonia are emerging as significant comorbidities in the HIV-infected population in the era of combination antiretroviral therapy (cART). HIV infection has been reported to suppress nasal mucociliary clearance (MCC). Since the primary components driving nasal MCC and bronchial MCC are identical, it is possible that bronchial MCC is affected as well. Effective MCC requires optimal ciliary beating which depends on the maintenance of the airway surface liquid (ASL), a function of cystic fibrosis transmembrane conductance regulator (CFTR) activity and the integrity of the signaling mechanism that regulates ciliary beating and fluid secretion. Impairment of either component of the MCC apparatus can compromise its efficacy and promote microbial colonization. We demonstrate that primary bronchial epithelium expresses HIV receptor CD4 and co-receptors CCR5 and CXCR4 and can be infected by both R5 and X4 tropic strains of HIV. We show that HIV Tat suppresses CFTR biogenesis and function in primary bronchial epithelial cells by a pathway involving TGF-ß signaling. HIV infection also interferes with bronchial epithelial cell differentiation and suppresses ciliogenesis. These findings suggest that HIV infection suppresses tracheobronchial mucociliary clearance and this may predispose HIV-infected patients to recurrent lung infections, pneumonia and chronic bronchitis.

Genetic analyses of cis-acting sequences controlling expression of human immunodeficiency virus type 1 coreceptor-CCR5 gene in rabbits and CXCR4 gene in monkeys.

INTRODUCTION: Human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus all use chemokine receptors (CCR5, CXCR4, and minor receptors) to gain entry into a susceptible cell and establish infection successfully by way of membrane fusion. Many such chemokine receptors that can act as entry cofactors under in vitro conditions have been identified, but the roles of CCR5 and CXCR4 chemokine receptors in infection, tropism, and pathogenesis have been studied in greater detail. The promoter region of CCR5 gene is quite polymorphic in humans, and mutations that affect the progression of HIV-1 have been identified. STUDY DESIGN/METHODS: We studied the nature of mutations in the CCR5 promoter region in rabbits. Large number of mutations, deletions, substitutions, and point mutations were observed all along the 400 base pair region of the promoter. RESULTS: We show that rabbit CCR5 promoter possesses features common to both humans and monkeys and lacks the second highly polymorphic region B in the CCR5 promoter that was previously identified in monkeys. Besides providing important evolutionary information, our findings can directly make an impact on the known expression levels of CCR5 protein that can modulate the progression of HIV-1 in rabbits. The CXCR4 promoter of monkeys showed polymorphisms that were largely caused by single nucleotide changes when compared with humans. CONCLUSIONS: This distinctly different evolutionary pattern suggests a more important role for chemokine receptor-CCR5 in the host defense.

Genetic analyses of the promoter region of interleukin-10 gene in different species of monkeys: implications for HIV/AIDS progression.

Recently a number of promoter alleles of interleukin-10 (IL-10) have been described in humans that affect the progression of HIV-1. Since infection of different species of monkeys with HIV-1, HIV-2 and SIV result in a widely varying outcome of the disease, we sought to study the nature of IL-10 promoter mutations in three different species of monkeys, namely rhesus, baboon and marmoset. We have identified three novel types of polymorphisms in monkeys that are characterized by insertions, deletions and substitutions besides several single nucleotide polymorphisms. Most interesting results were obtained with rhesus monkeys that lacked the 10-base long deletion in the polymorphic region B and the 12-base substitution in the polymorphic region C identified in marmoset monkeys. It is obvious that mutations of this nature involving insertions, deletions or substitutions of large sequences may potentially change the structure of the chromosome that are likely to affect binding of various transcription factors and consequently levels of transcription. The promoter region studied is highly polymorphic when compared to humans and at several places the changes are unique for a particular species of monkey. These observations may directly impact upon the progression of HIV-1 disease including other diseases/disorders where IL-10 levels have been implicated.

Inhibition of HIV-1 gene expression by novel macrophage-tropic DNA enzymes targeted to cleave HIV-1 TAT/Rev RNA.

Many regions of the HIV-1 genome have been targeted in earlier studies by RNA-cleaving DNA enzymes possessing the 10-23 catalytic motif, and efficient inhibition of HIV-1 gene expression was reported. All these studies employed charged synthetic lipids to introduce the catalytic DNA into the mammalian cells, which severely limits its practical application and usefulness in vivo. Taking advantage of the ability of G residues to interact directly with the scavenger receptors on the macrophages, we synthesized a DNA enzyme 5970 that contained 10 G residues at the 3' end. With the aim of improving the intracellular stability of the DNA enzyme 5970, we added two short stretches of stem-loop structures that were 12 bases long on either side of the DNA enzyme 5970. DNA enzyme 5970 without the poly-G tracts cleaved the synthetic RNA of HIV-1 TAT/Rev, two important regulatory proteins of HIV, very efficiently in a sequence-specific manner. Addition of 10 G residues at the 3' end of the DNA enzyme affected the cleavage efficiency only marginally whereas the same DNA enzyme with stem-loop structures on either end was significantly less efficient. The DNA enzyme with the poly-G tract at its 3' end was taken up specifically by a human macrophage-specific cell line directly in the absence of Lipofectin and was also able to inhibit HIV-1 gene expression in a transient-expression system as well as when challenged with the virus. The potential applications of these novel macrophage-tropic DNA enzymes are discussed.

Novel mono- and di-DNA-enzymes targeted to cleave TAT or TAT-REV RNA inhibit HIV-1 gene expression.

The regulatory proteins TAT and REV play a very important role in the transcription and replication of HIV-1. In order to seHIV-01lectively down regulate the expression of these genes we synthesized several mono- and one di-DNA-enzyme against the TAT or TAT-REV RNA. Several mono-DNA-enzymes possessing the 10-23 catalytic motif were assembled that were targeted to the predicted loop region of TAT or TAT/REV RNA. The cleavage efficiency of each mono-DNA-enzyme was variable and independent of the size of the predicted loop structure of the target RNA. DNA-enzyme targeted against the largest loop region cleaved the substrate RNA poorly. Mono-DNA-enzyme-5944 that targets only the TAT region cleaved the substrate poorly but the DNA-enzyme-5970 that overlaps TAT and REV showed potent cleavage activity. The two DNA-enzymes, when placed in tandem, cleaved the target RNA at multiple sites that were specific for the two mono-DNA-enzymes. Only Dz-5970 retained the ability to cleave the target RNA specifically at simulated physiological conditions. They were able to inhibit HIV-1 specific genes efficiently when introduced into a mammalian cell. The extent of inhibition correlated with their cleavage efficiency obtained at standard conditions of cleavage. Although DNA-enzyme-5970 showed the highest reduction (approximately 90%), other DNA-enzymes (mono-DNA-enzyme-5944 and the di-DNA-enzyme) also showed reduction to an extent of 60 and 80% respectively. The inhibitory effect of the DNA-enzyme could be overcome by providing HIV-1 TAT to the cells.

Targeted cleavage of HIV-1 envelope gene by a DNA enzyme and inhibition of HIV-1 envelope-CD4 mediated cell fusion.

With the ultimate aim of developing an effective antiviral strategy against HIV-1, a mono-DNA enzyme possessing the 10-23 catalytic motif [Santoro and Joyce (1997) Proc. Natl. Acad. Sci. USA 94, 4264-4266] was synthesized against the HIV-1 envelope gene. We tested the in vitro cleavage efficiency of the 178 bp long truncated HIV-1 Env transcript by DNA enzyme 6339. Protein independent and Mg2+ dependent specific cleavage products were obtained. As soon as 5 min after mixing equimolar concentrations of DNA enzyme and substrate RNA, more than 50% cleavage was observed which increased steadily over a period of 4 h. Very little cleavage was obtained at 1 mM MgCl2 concentration which improved significantly when the concentration of MgCl2 was increased up to 20 mM. Specific inhibition of cell membrane fusion caused by the interaction of gp160 and CD4 in HeLa cells was observed when the above DNA enzyme was used. Thus, these chemically synthesized DNA enzymes could prove to be very useful for in vivo application.