bafilomycin-a1 and HIV-Infections

Low vitamin D levels in human immunodeficiency virus type-1 (HIV) infected persons are associated with more rapid disease progression and increased risk for Mycobacterium tuberculosis infection. We have previously shown that 1α,25-dihydroxycholecalciferol (1,25D3), the active form of vitamin D, inhibits HIV replication in human macrophages through the induction of autophagy. In this study, we report that physiological concentrations of 1,25D3 induce the production of the human cathelicidin microbial peptide (CAMP) and autophagic flux in HIV and M. tuberculosis co-infected human macrophages which inhibits mycobacterial growth and the replication of HIV. Using RNA interference for Beclin-1 and the autophagy-related 5 homologue, combined with the chemical inhibitors of autophagic flux, bafilomycin A₁, an inhibitor of autophagosome-lysosome fusion and subsequent acidification, and SID 26681509 an inhibitor of the lysosome hydrolase cathepsin L, we show that the 1,25D3-mediated inhibition of HIV replication and mycobacterial growth during single infection or dual infection is dependent not only upon the induction of autophagy, but also through phagosomal maturation. Moreover, through the use of RNA interference for CAMP, we demonstrate that cathelicidin is essential for the 1,25D3 induced autophagic flux and inhibition of HIV replication and mycobacterial growth. The present findings provide a biological explanation for the benefits and importance of vitamin D sufficiency in HIV and M. tuberculosis-infected persons, and provide new insights into novel approaches to prevent and treat HIV infection and related opportunistic infections.

Topics: Antimicrobial Cationic Peptides; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 5; Beclin-1; Calcifediol; Cathelicidins; Cells, Cultured; HIV Infections; HIV-1; Humans; Hydrazines; Macrolides; Macrophages; Membrane Proteins; Microtubule-Associated Proteins; Mycobacterium tuberculosis; Phagosomes; RNA Interference; RNA, Small Interfering; Thiourea; Tuberculosis

During a comparison of the infectivity of mNDK, a CD4-independent human immunodeficiency virus type 1 (HIV-1) strain, to various cell lines, we found that HeLa cells were much less susceptible than 293T and TE671 cells. Hybridoma cells between HeLa and 293T cells were as susceptible as 293T cells, suggesting that cellular factors enhance the mNDK infection in 293T cells. By screening a cDNA expression library in HeLa cells, cystatin C was isolated as an enhancer of the mNDK infection. Because cathepsin B protease, a natural ligand of cystatin C, was upregulated in HeLa cells, we speculated that the high levels of cathepsin B activities were inhibitory to the CD4-independent infection and that cystatin C enhanced the infection by impairing the excessive cathepsin B activity. Consistent with this idea, pretreatment of HeLa cells with 125 µM of CA-074Me, a cathepsin B inhibitor, resulted in an 8-fold enhancement of the mNDK infectivity. Because cathepsin B is activated by low pH in acidic endosomes, we further examined the potential roles of endosomes in the CD4-independent infection. Suppression of endosome acidification or endocytosis by inhibitors or by an Eps15 dominant negative mutant reduced the infectivity of mNDK in which CD4-dependent infections were not significantly impaired. Taken together, these results suggest that endocytosis, endosomal acidification, and cathepsin B activity are involved in the CD4-independent entry of HIV-1.

Topics: Acids; Cathepsin B; CD4 Antigens; Chloroquine; Cystatin C; Endocytosis; Endosomes; Genes, Dominant; Genetic Vectors; HEK293 Cells; HeLa Cells; HIV Infections; HIV-1; Humans; Hydrazones; Immunity, Innate; Macrolides; Protease Inhibitors; Signal Transduction; Toll-Like Receptor 4

Autophagy is critical to maintaining cell homeostasis and is implicated in neurodegenerative diseases. This research examined the role of autophagy in human immunodeficiency virus type 1 (HIV-1)-associated encephalitis, the pathologic hallmark of neuroAIDS.. The frontal cortex from 32 HIV-infected persons (12 without evidence HIV-1 encephalitis or clinical signs of central nervous system impairment and 20 with histopathological findings of HIV-1 encephalitis) and 8 persons without HIV infection and any neuropathology were examined postmortem. Green fluorescent protein-labeled (GFP) light chain 3 (LC3)-expressing neuroblastoma SK-N-SH cells treated with gp120 from CXCR4 and CCR5 viruses were also examined. Autophagic markers were assessed by means of Western blot analysis, transmission electron microscopy (TEM), and confocal microscopy.. Autophagic proteins Beclin 1, Autophagy-related gene (Atg)-5, Atg-7, and LC3-II were significantly increased in brains with HIV-1 encephalitis (P < .05). These findings were confirmed by TEM and immunostaining of brain tissue. Additionally, levels of autophagic proteins and autophagosomes were increased in neuronal cells treated with both CXCR4- or CCR5-tropic HIV-1 gp120. No increase in the level of autophagy was observed in the brains of HIV-infected persons without HIV-1 encephalitis compared with the level in brains of HIV-uninfected persons.. Postmortem brains with HIV-1 encephalitis exhibit increased markers of autophagy compared with brains from HIV-infected persons without HIV-1 encephalitis or HIV-uninfected control brains, which suggests that dysregulation of autophagy may be important in the pathogenesis of neuroAIDS.

Topics: Adult; Aged; Autophagy; Autopsy; Blotting, Western; Case-Control Studies; Cell Line, Tumor; Encephalitis, Viral; Female; Frontal Lobe; HIV Envelope Protein gp120; HIV Infections; Humans; Immunohistochemistry; Lysosomal Membrane Proteins; Lysosomes; Macrolides; Male; Microtubule-Associated Proteins; Middle Aged; Phagosomes

Plasmacytoid dendritic cells (pDCs) play a major role in anti-viral immunity by virtue of their ability to produce high amounts of type I interferons (IFNs) and a variety of inflammatory cytokines and chemokines in response to viral infections. Since recent studies have established that pDCs accumulate at the site of virus entry in the mucosa, here we analyzed whether epithelial cells were able to modulate the function of pDCs. We found that the epithelial cell lines HT-29 and Caco-2, as well as a primary culture of human renal tubular epithelial cells (HRTEC), induced the phenotypic maturation of pDCs stimulating the production of inflammatory cytokines. By contrast, epithelial cells did not induce any change in the phenotype of conventional or myeloid DCs (cDCs) while significantly stimulated the production of the anti-inflammatory cytokine IL-10. Activation of pDCs by epithelial cells was prevented by Bafilomycin A1, an inhibitor of endosomal acidification as well as by the addition of RNase to the culture medium, suggesting the participation of endosomal TLRs. Interestingly, the cross-talk between both cell populations was shown to be associated to an increased expression of TLR7 and TLR9 by pDCs and the production of LL37 by epithelial cells, an antimicrobial peptide able to bind and transport extracellular nucleic acids into the endosomal compartments. Interestingly, epithelium-activated pDCs impaired the establishment of a productive HIV infection in two susceptible target cells through the stimulation of the production of type I IFNs, highlighting the anti-viral efficiency of this novel activation pathway.

Topics: Caco-2 Cells; Cell Line, Tumor; Chemokines; Culture Media; Cytokines; Dendritic Cells; Endosomes; Epithelial Cells; HIV; HIV Infections; Humans; Inflammation; Interferons; Macrolides; Phenotype

We previously reported that inhibition of endosomal/lysosomal function can dramatically enhance human immunodeficiency virus type 1 (HIV-1) infectivity, suggesting that under these conditions productive HIV-1 infection can occur via the endocytic pathway. Here we further examined this effect with bafilomycin A1 (BFLA-1) and show that this enhancement of infectivity extends to all HIV-1 isolates tested regardless of coreceptor usage. However, isolate-specific differences were observed in the magnitude of the effect. This was particularly evident in the case of the weakly infectious HIV-1(SF2), for which we observed the greatest enhancement. Using reciprocal chimeric viruses, we were able to determine that both the disproportionate increase in the infectivity of HIV-1(SF2) in response to BFLA-1 and its weak infectivity in the absence of BFLA-1 mapped to its envelope gene. Further, we found HIV-1(SF2) to have lower fusion activity and to be 12-fold more sensitive to the fusion inhibitor T-20 than HIV-1(NL4-3). Proteasomal inhibitors also enhance HIV-1 infectivity, and we report that the combination of a lysosomal and a proteasomal inhibitor greatly enhanced infectivity of all isolates tested. Again, HIV-1(SF2) was unique in exhibiting a synergistic 400-fold increase in infectivity. We also determined that inhibition of proteasomal function increased the infectivity of HIV-1 pseudotyped with vesicular stomatitis virus G protein. The evidence presented here highlights the important role of the lysosomes/proteasomes in the destruction of infectious HIV-1(SF2) and could have implications for the development of novel antiviral agents that might take advantage of these innate defenses.

Topics: Cell Line; Enzyme Inhibitors; HIV Infections; HIV-1; Humans; Lysosomes; Macrolides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Recombination, Genetic; Species Specificity; Virus Replication