Limits...
Opposing regulation of endolysosomal pathways by long-acting nanoformulated antiretroviral therapy and HIV-1 in human macrophages.

Araínga M, Guo D, Wiederin J, Ciborowski P, McMillan J, Gendelman HE - Retrovirology (2015)

Bottom Line: NanoART and native ATV treated uninfected cells showed limited effects.The data was confirmed by Western blot.DAVID and KEGG bioinformatics analyses of proteomic data showed relationships between secretory, mobility and phagocytic cell functions and virus and particle trafficking.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, 985880 Nebraska Medical Center, Omaha 68198-5880, NE, USA. hegendel@unmc.edu.

ABSTRACT

Background: Long-acting nanoformulated antiretroviral therapy (nanoART) is designed to improve patient regimen adherence, reduce systemic drug toxicities, and facilitate clearance of human immunodeficiency virus type one (HIV-1) infection. While nanoART establishes drug depots within recycling and late monocyte-macrophage endosomes, whether or not this provides a strategic advantage towards viral elimination has not been elucidated.

Results: We applied quantitative SWATH-MS proteomics and cell profiling to nanoparticle atazanavir (nanoATV)-treated and HIV-1 infected human monocyte-derived macrophages (MDM). Native ATV and uninfected cells served as controls. Both HIV-1 and nanoATV engaged endolysosomal trafficking for assembly and depot formation, respectively. Notably, the pathways were deregulated in opposing manners by the virus and the nanoATV, likely by viral clearance. Paired-sample z-scores, of the proteomic data sets, showed up- and down- regulation of Rab-linked endolysosomal proteins. NanoART and native ATV treated uninfected cells showed limited effects. The data was confirmed by Western blot. DAVID and KEGG bioinformatics analyses of proteomic data showed relationships between secretory, mobility and phagocytic cell functions and virus and particle trafficking.

Conclusions: We posit that modulation of endolysosomal pathways by antiretroviral nanoparticles provides a strategic path to combat HIV infection.

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Related in: MedlinePlus

Schematic representation of the MDM phagosome network identified in HIV-1-infected (A) and HIV-1-infected and nanoATV treated cells (B). Proteins identified were compared against control uninfected MDM cultures (p < 0.05). The acquired profiles were analyzed through the bioinformatics program using a comprehensive set of functional annotation tools to uncover biological data sets behind the uncovered list of genes. Data for Annotation, Visualization and Integrated Discovery (DAVID) facilitated the linked sets of enriched functional-related protein groups. This tool was employed to identify enriched biological processes among the expressed proteins. Gene Ontology terms were used to identify related pathways with the assistance of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The KEGG database facilitated the elucidation of the functions for the MDM as derived from the proteomic datasets. Statistical significance was determined using a p-value < 0.05. Proteins in red and blue, display up- and down- regulation, respectively. Proteins in green belong to the phagosome network and not deregulated by ATV treatment. The differences in protein up- and down-regulation between HIV infection alone and HIV infection with nanoATV treatment are circled in red.
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Fig1: Schematic representation of the MDM phagosome network identified in HIV-1-infected (A) and HIV-1-infected and nanoATV treated cells (B). Proteins identified were compared against control uninfected MDM cultures (p < 0.05). The acquired profiles were analyzed through the bioinformatics program using a comprehensive set of functional annotation tools to uncover biological data sets behind the uncovered list of genes. Data for Annotation, Visualization and Integrated Discovery (DAVID) facilitated the linked sets of enriched functional-related protein groups. This tool was employed to identify enriched biological processes among the expressed proteins. Gene Ontology terms were used to identify related pathways with the assistance of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The KEGG database facilitated the elucidation of the functions for the MDM as derived from the proteomic datasets. Statistical significance was determined using a p-value < 0.05. Proteins in red and blue, display up- and down- regulation, respectively. Proteins in green belong to the phagosome network and not deregulated by ATV treatment. The differences in protein up- and down-regulation between HIV infection alone and HIV infection with nanoATV treatment are circled in red.

Mentions: HIV-1 infection engages a spectrum of cellular proteins seen in specialized cell populations that support its replication [20-23]. The effect of nanoART on cell protein expression has not yet been defined, in its target macrophage. To such ends, we applied quantitative SWATH-MS proteomics followed by bioinformatics to uncover proteins deregulated by native ATV or nanoATV with or without HIV-1 infection. For these experiments MDM were first infected with HIV-1ADA and four hours later medium was removed and cells were treated with 100 μM P407-ATV. Following 16 hours of drug treatment, the media was replaced with drug-free fluids and cells were harvested for proteomic tests after an additional seven days. This experimental paradigm was followed to assess the role that the antiretroviral delivery system had on the macrophage proteome during spreading viral infection. To separate the effects of the antiretroviral drug, the nanoparticle and the viral infection, separate and combined analyses of each of these were required. MDM were infected with HIV-1 at a MOI of 0.1 then treated with 100 μM native- or nanoATV. After seven days, cells were harvested and SWATH-MS was performed on whole cell lysates [15]. Because of the expansive proteomic data sets and analyses based on the biological response variables amongst the nanoATV, ATV and HIV-1 treatments the data are presented in 7 additional files and two figures. Figure 1 illustrates differences in protein expression between HIV-1 infected cells with or without nanoART as compared to controls (uninfected-untreated MDM) and Figure 2 illustrates differences in protein expression between uninfected cells treated with native- and nanoATV compared to controls. Quantitative profiling identified 527 significantly changed MDM proteins following HIV-1 infection. These were up- or down-regulated (p < 0.05) and were assessed by paired-samples z-scores (Additional file 1). The numbers of proteins exhibiting changed expression in HIV-1-infected cells were greater than in replicate infected cells treated with nanoATV, 527 versus 376 respectively (Additional file 2A). Up- and down- regulated proteins in HIV-1 infected cells were 41 and 59% of total (n = 216 and 311, respectively). In contrast for nanoATV-treated HIV-1-infected MDM, up- and down-regulated proteins were 59 and 41% of total (n = 222 and 154, respectively). Uninfected cells treated with nanoATV had fewer deregulated proteins (n = 195) compared to the other groups (Additional file 1). The proteins uncovered engaged the PANTHER database which sorted the deregulated proteins by classes. This illustrated the relative numbers of proteins in each class for the HIV-1-infected and infected and nanoATV-treated cells (Additional file 2B). This showed that the number of deregulated proteins in HIV-1-infected versus infected and nanoATV-treated cells was greater for each of the classes (nucleic acid binding, hydrolase, transferase, protease, signalling molecule, transporter, transcription factors and ligases). These results demonstrate that the deregulation of cellular proteins by HIV-1 infection can be altered by nanoATV treatment. To uncover the function of the classes of proteins deregulated by HIV infection we examined the functional categories by PANTHER classification. These data are based on Gene Ontology annotations (GO molecular function, GO biological processes and GO cellular component). Additional file 3A shows the proteins sorted according to molecular function with the percentages of total deregulated proteins classified for subgroups. Common proteins were separated based on binding (28 to 31%), enzyme regulator (5%) and transporter (3-4%) activities between the nanoATV, HIV-1 and HIV-1 and nanoATV treated MDM. Proteins included Rab5, −7 (GDP/GTP and protein binding) and LAMP1 (enzyme and protein binding). The classification revealed enrichment for metabolic, cellular, localization, regulation and cellular organizational cellular processes. These were the principal categories or protein sets (Additional file 3B). The relative ratios for the proteins were similar amongst groups. GO for cellular component showed that deregulated proteins sorted by cell organelle and macromolecular complex (Additional file 3C). Note that such groupings were common to HIV-1 and HIV-1 and nanoATV treated cells. The data demonstrated that HIV-1 and HIV-1 and nanoATV, as compared to control, affect similar cellular processes. However, the numbers of proteins in each were reduced following infection and nanoATV treatment.Figure 1


Opposing regulation of endolysosomal pathways by long-acting nanoformulated antiretroviral therapy and HIV-1 in human macrophages.

Araínga M, Guo D, Wiederin J, Ciborowski P, McMillan J, Gendelman HE - Retrovirology (2015)

Schematic representation of the MDM phagosome network identified in HIV-1-infected (A) and HIV-1-infected and nanoATV treated cells (B). Proteins identified were compared against control uninfected MDM cultures (p < 0.05). The acquired profiles were analyzed through the bioinformatics program using a comprehensive set of functional annotation tools to uncover biological data sets behind the uncovered list of genes. Data for Annotation, Visualization and Integrated Discovery (DAVID) facilitated the linked sets of enriched functional-related protein groups. This tool was employed to identify enriched biological processes among the expressed proteins. Gene Ontology terms were used to identify related pathways with the assistance of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The KEGG database facilitated the elucidation of the functions for the MDM as derived from the proteomic datasets. Statistical significance was determined using a p-value < 0.05. Proteins in red and blue, display up- and down- regulation, respectively. Proteins in green belong to the phagosome network and not deregulated by ATV treatment. The differences in protein up- and down-regulation between HIV infection alone and HIV infection with nanoATV treatment are circled in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4307176&req=5

Fig1: Schematic representation of the MDM phagosome network identified in HIV-1-infected (A) and HIV-1-infected and nanoATV treated cells (B). Proteins identified were compared against control uninfected MDM cultures (p < 0.05). The acquired profiles were analyzed through the bioinformatics program using a comprehensive set of functional annotation tools to uncover biological data sets behind the uncovered list of genes. Data for Annotation, Visualization and Integrated Discovery (DAVID) facilitated the linked sets of enriched functional-related protein groups. This tool was employed to identify enriched biological processes among the expressed proteins. Gene Ontology terms were used to identify related pathways with the assistance of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The KEGG database facilitated the elucidation of the functions for the MDM as derived from the proteomic datasets. Statistical significance was determined using a p-value < 0.05. Proteins in red and blue, display up- and down- regulation, respectively. Proteins in green belong to the phagosome network and not deregulated by ATV treatment. The differences in protein up- and down-regulation between HIV infection alone and HIV infection with nanoATV treatment are circled in red.
Mentions: HIV-1 infection engages a spectrum of cellular proteins seen in specialized cell populations that support its replication [20-23]. The effect of nanoART on cell protein expression has not yet been defined, in its target macrophage. To such ends, we applied quantitative SWATH-MS proteomics followed by bioinformatics to uncover proteins deregulated by native ATV or nanoATV with or without HIV-1 infection. For these experiments MDM were first infected with HIV-1ADA and four hours later medium was removed and cells were treated with 100 μM P407-ATV. Following 16 hours of drug treatment, the media was replaced with drug-free fluids and cells were harvested for proteomic tests after an additional seven days. This experimental paradigm was followed to assess the role that the antiretroviral delivery system had on the macrophage proteome during spreading viral infection. To separate the effects of the antiretroviral drug, the nanoparticle and the viral infection, separate and combined analyses of each of these were required. MDM were infected with HIV-1 at a MOI of 0.1 then treated with 100 μM native- or nanoATV. After seven days, cells were harvested and SWATH-MS was performed on whole cell lysates [15]. Because of the expansive proteomic data sets and analyses based on the biological response variables amongst the nanoATV, ATV and HIV-1 treatments the data are presented in 7 additional files and two figures. Figure 1 illustrates differences in protein expression between HIV-1 infected cells with or without nanoART as compared to controls (uninfected-untreated MDM) and Figure 2 illustrates differences in protein expression between uninfected cells treated with native- and nanoATV compared to controls. Quantitative profiling identified 527 significantly changed MDM proteins following HIV-1 infection. These were up- or down-regulated (p < 0.05) and were assessed by paired-samples z-scores (Additional file 1). The numbers of proteins exhibiting changed expression in HIV-1-infected cells were greater than in replicate infected cells treated with nanoATV, 527 versus 376 respectively (Additional file 2A). Up- and down- regulated proteins in HIV-1 infected cells were 41 and 59% of total (n = 216 and 311, respectively). In contrast for nanoATV-treated HIV-1-infected MDM, up- and down-regulated proteins were 59 and 41% of total (n = 222 and 154, respectively). Uninfected cells treated with nanoATV had fewer deregulated proteins (n = 195) compared to the other groups (Additional file 1). The proteins uncovered engaged the PANTHER database which sorted the deregulated proteins by classes. This illustrated the relative numbers of proteins in each class for the HIV-1-infected and infected and nanoATV-treated cells (Additional file 2B). This showed that the number of deregulated proteins in HIV-1-infected versus infected and nanoATV-treated cells was greater for each of the classes (nucleic acid binding, hydrolase, transferase, protease, signalling molecule, transporter, transcription factors and ligases). These results demonstrate that the deregulation of cellular proteins by HIV-1 infection can be altered by nanoATV treatment. To uncover the function of the classes of proteins deregulated by HIV infection we examined the functional categories by PANTHER classification. These data are based on Gene Ontology annotations (GO molecular function, GO biological processes and GO cellular component). Additional file 3A shows the proteins sorted according to molecular function with the percentages of total deregulated proteins classified for subgroups. Common proteins were separated based on binding (28 to 31%), enzyme regulator (5%) and transporter (3-4%) activities between the nanoATV, HIV-1 and HIV-1 and nanoATV treated MDM. Proteins included Rab5, −7 (GDP/GTP and protein binding) and LAMP1 (enzyme and protein binding). The classification revealed enrichment for metabolic, cellular, localization, regulation and cellular organizational cellular processes. These were the principal categories or protein sets (Additional file 3B). The relative ratios for the proteins were similar amongst groups. GO for cellular component showed that deregulated proteins sorted by cell organelle and macromolecular complex (Additional file 3C). Note that such groupings were common to HIV-1 and HIV-1 and nanoATV treated cells. The data demonstrated that HIV-1 and HIV-1 and nanoATV, as compared to control, affect similar cellular processes. However, the numbers of proteins in each were reduced following infection and nanoATV treatment.Figure 1

Bottom Line: NanoART and native ATV treated uninfected cells showed limited effects.The data was confirmed by Western blot.DAVID and KEGG bioinformatics analyses of proteomic data showed relationships between secretory, mobility and phagocytic cell functions and virus and particle trafficking.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, 985880 Nebraska Medical Center, Omaha 68198-5880, NE, USA. hegendel@unmc.edu.

ABSTRACT

Background: Long-acting nanoformulated antiretroviral therapy (nanoART) is designed to improve patient regimen adherence, reduce systemic drug toxicities, and facilitate clearance of human immunodeficiency virus type one (HIV-1) infection. While nanoART establishes drug depots within recycling and late monocyte-macrophage endosomes, whether or not this provides a strategic advantage towards viral elimination has not been elucidated.

Results: We applied quantitative SWATH-MS proteomics and cell profiling to nanoparticle atazanavir (nanoATV)-treated and HIV-1 infected human monocyte-derived macrophages (MDM). Native ATV and uninfected cells served as controls. Both HIV-1 and nanoATV engaged endolysosomal trafficking for assembly and depot formation, respectively. Notably, the pathways were deregulated in opposing manners by the virus and the nanoATV, likely by viral clearance. Paired-sample z-scores, of the proteomic data sets, showed up- and down- regulation of Rab-linked endolysosomal proteins. NanoART and native ATV treated uninfected cells showed limited effects. The data was confirmed by Western blot. DAVID and KEGG bioinformatics analyses of proteomic data showed relationships between secretory, mobility and phagocytic cell functions and virus and particle trafficking.

Conclusions: We posit that modulation of endolysosomal pathways by antiretroviral nanoparticles provides a strategic path to combat HIV infection.

Show MeSH
Related in: MedlinePlus