Here we demonstrate that an inducible anti-HIV short hairpin RNA (shRNA) expressed from a Pol II promoter inhibits HIV-1 gene expression in mammalian cells. Our strategy is based on a promoter system in which the HIV-1 LTR is fused to the Drosophila hsp70 minimal heat shock promoter. This system is inducible by HIV-1 TAT, which functions in a negative feedback loop to activate transcription of an shRNA directed against HIV-1 rev. Upon induction the shRNA is processed to an siRNA that guides inhibition of HIV replication in cultured T-lymphocytes and hematopoietic stem cell-derived monocytes. The fusion promoter system may be safer than drug-inducible systems for shRNA-mediated gene therapy against HIV as the shRNAs are only expressed following HIV infection.

We demonstrate a novel approach for coexpression of a short hairpin RNA (shRNA) with an open reading frame which exploits transcriptional read-through of a minimal polyadenylation signal from a Pol II promoter. We first observed efficient inducible expression of enhanced green fluorescent protein along with an anti-rev shRNA. We took advantage of this observation to test coexpression of the transdominant negative mutant (humanized) of human immunodeficiency type 1 (HIV-1) Rev (huRevM10) along with an anti-rev shRNA via an HIV-1-inducible fusion promoter. The coexpression of the shRNA and transdominant protein resulted in potent, long-term inhibition of HIV-1 gene expression and suppression of shRNA-resistant mutants. This dual expression system has broad-based potential for other shRNA applications, such as cases where simultaneous knockdown of mutant and wild-type transcripts must be accompanied by replacement of the wild-type protein.

Chronic bronchitis, caused by cigarette smoke exposure, is characterized by mucus hypersecretion and reduced mucociliary clearance (MCC). Effective MCC depends, in part, on adequate airway surface liquid. Cystic fibrosis transmembrane conductance regulator (CFTR) provides the necessary osmotic gradient for serosal to mucosal fluid transport through its ability to both secrete Cl 2 and regulate paracellular permeability, but CFTR activity is attenuated in chronic bronchitis and in smokers. b 2 -adrenergic receptor (b 2 -AR) agonists are widely used for managing chronic obstructive pulmonary disease, and can activate CFTR, stimulate ciliary beat frequency, and increase epithelial permeability, thereby stimulating MCC. Patients with chronic airway diseases and cigarette smokers demonstrate increased transforming growth factor (TGF)-b1 signaling, which suppresses b 2 -agonist-mediated CFTR activation and epithelial permeability increases. Restoring CFTR function in these diseases can restore the ability of b 2 -agonists to enhance epithelial permeability. Human bronchial epithelial cells, fully redifferentiated at the air-liquid interface, were used for 14 C mannitol flux measurements, Ussing chamber experiments, and quantitative RT-PCR. b 2 -agonists enhance epithelial permeability by activating CFTR via the b 2 -AR/adenylyl cyclase/cAMP/ protein kinase A pathway. TGF-b1 inhibits b 2 -agonist-mediated CFTR activation and epithelial permeability enhancement. Although TGF-b1 down-regulates both b 2 -AR and CFTR mRNA, functionally it only decreases CFTR activity. Cigarette smoke exposure inhibits b 2 -agonist-mediated epithelial permeability increases, an effect reversed by blocking TGF-b signaling. b 2 -agonists enhance epithelial permeability via CFTR activation. TGF-b1 signaling inhibits b 2 -agonist-mediated CFTR activation and subsequent increased epithelial permeability, potentially limiting the ability of b 2 -agonists to facilitate paracellular transport in disease states unless TGF-b1 signaling is inhibited.

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-β 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.