Quantitative Proteomics by SWATH-MS Reveals Altered Expression of Nucleic Acid Binding and Regulatory Proteins in HIV-1-Infected Macrophages

Human immunodeficiency virus type 1 (HIV-1) infection remains a worldwide epidemic, and innovative therapies to combat the virus are needed. Developing a host-oriented antiviral strategy capable of targeting the biomolecules that are directly or indirectly required for viral replication may provide advantages over traditional virus-centric approaches. We used quantitative proteomics by SWATH-MS in conjunction with bioinformatic analyses to identify host proteins, with an emphasis on nucleic acid binding and regulatory proteins, which could serve as candidates in the development of host-oriented antiretroviral strategies. Using SWATH-MS, we identified and quantified the expression of 3608 proteins in uninfected and HIV-1-infected monocyte-derived macrophages. Of these 3608 proteins, 420 were significantly altered upon HIV-1 infection. Bioinformatic analyses revealed functional enrichment for RNA binding and processing as well as transcription regulation. Our findings highlight a novel subset of proteins and processes that are involved in the host response to HIV-1 infection. In addition, we provide an original and transparent methodology for the analysis of label-free quantitative proteomics data generated by SWATH-MS that can be readily adapted to other biological systems

Proteomic Analysis of Early HIV‑1 Nucleoprotein Complexes

After entry into the cell, the early steps of the human immunodeficiency virus type 1 (HIV-1) replication cycle are mediated by two functionally distinct nucleoprotein complexes, the reverse transcription complex (RTC) and preintegration complex (PIC). These two unique viral complexes are responsible for the conversion of the single-stranded RNA genome into double-stranded DNA, transport of the DNA into the nucleus, and integration of the viral DNA into the host cell chromosome. Prior biochemical analyses suggest that these complexes are large and contain multiple undiscovered host cell factors. In this study, functional HIV-1 RTCs and PICs were partially purified by velocity gradient centrifugation and fractionation, concentrated, trypsin digested, and analyzed by LC–MS/MS. A total of seven parallel infected and control biological replicates were completed. Database searches were performed with Proteome Discoverer and a comparison of the HIV-1 samples to parallel uninfected control samples was used to identify unique cellular factors. The analysis produced a total data set of 11055 proteins. Several previously characterized HIV-1 factors were identified, including XRCC6, TFRC, and HSP70. The presence of XRCC6 was confirmed in infected fractions and shown to be associated with HIV-1 DNA by immunoprecipitation-PCR experiments. Overall, the analysis identified 94 proteins unique in the infected fractions and 121 proteins unique to the control fractions with ≥2 protein assignments. An additional 54 and 52 were classified as enriched in the infected and control samples, respectively, based on a 3-fold difference in total Proteome Discoverer probability score. The differential expression of several candidate proteins was validated by Western blot analysis. This study contributes additional novel candidate proteins to the growing published bioinformatic data sets of proteins that contribute to HIV-1 replication

Pulsed Stable Isotope Labeling of Amino Acids in Cell Culture Uncovers the Dynamic Interactions between HIV-1 and the Monocyte-Derived Macrophage

Dynamic interactions between human immunodeficiency virus-1 (HIV-1) and the macrophage govern the tempo of viral dissemination and replication in its human host. HIV-1 affects macrophage phenotype, and the macrophage, in turn, can modulate the viral life cycle. While these processes are linked to host–cell function and survival, the precise intracellular pathways involved are incompletely understood. To elucidate such dynamic virus–cell events, we employed pulsed stable isotope labeling of amino acids in cell culture. Alterations in <i>de novo</i> protein synthesis of HIV-1 infected human monocyte-derived macrophages (MDM) were examined after 3, 5, and 7 days of viral infection. Synthesis rates of cellular metabolic, regulatory, and DNA packaging activities were decreased, whereas, those affecting antigen presentation (major histocompatibility complex I and II) and interferon-induced antiviral activities were increased. Interestingly, enrichment of proteins linked to chromatin assembly or disassembly, DNA packaging, and nucleosome assembly were identified that paralleled virus-induced cytopathology and replication. We conclude that HIV-1 regulates a range of host MDM proteins that affect its survival and abilities to contain infection

Pulsed Stable Isotope Labeling of Amino Acids in Cell Culture Uncovers the Dynamic Interactions between HIV-1 and the Monocyte-Derived Macrophage

Dynamic interactions between human immunodeficiency virus-1 (HIV-1) and the macrophage govern the tempo of viral dissemination and replication in its human host. HIV-1 affects macrophage phenotype, and the macrophage, in turn, can modulate the viral life cycle. While these processes are linked to host–cell function and survival, the precise intracellular pathways involved are incompletely understood. To elucidate such dynamic virus–cell events, we employed pulsed stable isotope labeling of amino acids in cell culture. Alterations in <i>de novo</i> protein synthesis of HIV-1 infected human monocyte-derived macrophages (MDM) were examined after 3, 5, and 7 days of viral infection. Synthesis rates of cellular metabolic, regulatory, and DNA packaging activities were decreased, whereas, those affecting antigen presentation (major histocompatibility complex I and II) and interferon-induced antiviral activities were increased. Interestingly, enrichment of proteins linked to chromatin assembly or disassembly, DNA packaging, and nucleosome assembly were identified that paralleled virus-induced cytopathology and replication. We conclude that HIV-1 regulates a range of host MDM proteins that affect its survival and abilities to contain infection

Pulsed Stable Isotope Labeling of Amino Acids in Cell Culture Uncovers the Dynamic Interactions between HIV-1 and the Monocyte-Derived Macrophage

Dynamic interactions between human immunodeficiency virus-1 (HIV-1) and the macrophage govern the tempo of viral dissemination and replication in its human host. HIV-1 affects macrophage phenotype, and the macrophage, in turn, can modulate the viral life cycle. While these processes are linked to host–cell function and survival, the precise intracellular pathways involved are incompletely understood. To elucidate such dynamic virus–cell events, we employed pulsed stable isotope labeling of amino acids in cell culture. Alterations in <i>de novo</i> protein synthesis of HIV-1 infected human monocyte-derived macrophages (MDM) were examined after 3, 5, and 7 days of viral infection. Synthesis rates of cellular metabolic, regulatory, and DNA packaging activities were decreased, whereas, those affecting antigen presentation (major histocompatibility complex I and II) and interferon-induced antiviral activities were increased. Interestingly, enrichment of proteins linked to chromatin assembly or disassembly, DNA packaging, and nucleosome assembly were identified that paralleled virus-induced cytopathology and replication. We conclude that HIV-1 regulates a range of host MDM proteins that affect its survival and abilities to contain infection

Pulsed Stable Isotope Labeling of Amino Acids in Cell Culture Uncovers the Dynamic Interactions between HIV-1 and the Monocyte-Derived Macrophage

Dynamic interactions between human immunodeficiency virus-1 (HIV-1) and the macrophage govern the tempo of viral dissemination and replication in its human host. HIV-1 affects macrophage phenotype, and the macrophage, in turn, can modulate the viral life cycle. While these processes are linked to host–cell function and survival, the precise intracellular pathways involved are incompletely understood. To elucidate such dynamic virus–cell events, we employed pulsed stable isotope labeling of amino acids in cell culture. Alterations in <i>de novo</i> protein synthesis of HIV-1 infected human monocyte-derived macrophages (MDM) were examined after 3, 5, and 7 days of viral infection. Synthesis rates of cellular metabolic, regulatory, and DNA packaging activities were decreased, whereas, those affecting antigen presentation (major histocompatibility complex I and II) and interferon-induced antiviral activities were increased. Interestingly, enrichment of proteins linked to chromatin assembly or disassembly, DNA packaging, and nucleosome assembly were identified that paralleled virus-induced cytopathology and replication. We conclude that HIV-1 regulates a range of host MDM proteins that affect its survival and abilities to contain infection

Astrocyte effect on the proteome of HIV-1/VSV infected microglia.

a<p>Compared with uninfected microglia.</p>b<p>Theoretical isoelectric point calculated by Swissprot database at <a href="http://ca.expasy.org/sprot/" target="_blank">http://ca.expasy.org/sprot/</a>.</p>c<p>Number of peptides detected by mass spectrometry for each identified protein.</p

BMM mobility after exposure to micoglia/astrocvte conditioned media.

<p>Migration of CMFDA-labeled BMM was assessed for 3 h using confocal microscopy live imaging in μ-slide chemotaxis assays comparing fresh media with conditioned media (CM) from microglia alone (MCG), astrocytes alone (AST) or microglia-astrocyte co-cultures (MCG+AST). Inhibitors wiskostatin (WISK, 20 µM) and latrunculin A (LatA, 0.2 µg/ml) were utilized to inhibit BMM migration. (A) Representative migrating BMM cultured in fresh medium (Medium) or CM fro microglia-astrocyte co-cultures (MCG+AST) were tracked (X's in different color tracings) and evaluated at 60 min, 120 min, and 180 min. Scale bar, 10 µm; original magnification, ×10. (B) Plots of each individual cell migration were generated from tracking data acquired for 3 h cultures in medium (Fresh Medium), CM from microglia (MCG), astrocytes (AST), or astrocyte-microglia co-cultures (CM), and in the presence of WISK and LatA inhibitors. Data were acquired by laser confocal microscopy and ImageJ software (NIH) interfaced with the ManulTrack plugin. (C) Migration velocities for each individual cell track were determined with the Chemotaxis and Tool software tool (ibidi). Significant differences in the mean±SEM migration velocities for n = 50 determinations/group were assessed by two-tail Student's t test where *p<0.05 was considered significant.</p

Immunohistochemical validation of proteomic profiling.

<p>Uninfected or HIV-1/VSV infected microglia were cultured for 24 h in the absence (MCG) or presence of astrocytes (MCG+AST). This arbitrary time point was chosen as it reflected dynamic changes in cell-cell interactions. Uninfected microglia cultured in the presence of 20 µg/ml TNF-α for 6 hours served as an activated microglia control. (A) Microglia were stained for the expression of the cytoskeletal proteins tubulin-α (green) and F-actin (red); the specific microglial marker ionized calcium binding adaptor molecule 1 (Iba-1) (red); and the activation indicator, vimentin (green). DAPI staining of nuclei shows total number of microglia. Arrowheads point to migratory morphologies. Scale bar, 20 µm; original magnification, ×63. Quantification of (A) tubulin-α, (B) F-actin, and (C) vimentin expression was performed via laser confocal microscopy and the ratio of overall fluorescence to cell numbers was calculated by digital image analysis using Image-Pro Plus version 5.1 software (Media Cybernetics, Inc.). Significant differences in mean fluorescence±SEM for n = 5 determinations/group was performed by one-way ANOVA and Tukey's post-hoc multiple comparisons where p<0.05 was considered significant.</p

Neurotoxic activities of culture fluids from HIV-1/VSV infected microglia attenuated by microglia-astrocyte co-cultivation.

<p>Mouse neurons were cultured for 10 d and exposed to the 20% conditioned media (CM) from uninfected or HIV-1/VSV infected microglia and/or astrocytes for 24 h. MAP-2 (green) and NeuN (red) expression by neurons was visualized by immunohistochemical staining. Characteristic neurotoxicity, including released nuclear materials and broken neurites, were observed in neurons treated with CM from HIV-1/VSV infected microglia alone. Percentages of apoptotic neurons were evaluated by the ratio of TUNEL⁺ (green) neurons to DAPI⁺ (blue) cells. The results are depicted as a mean percentage of apoptotic cells±SEM of three experiments. Significant reduction of percentage of apoptotic cells were observed in HIV-1/VSV infected co-cultures and astrocyte groups, compared with those of neurons cultured in microglial CM alone (n = 3 determines/group, p<0.01, determined by one-way ANOVA analysis and Tukey's multiple comparison post-hoc tests). Bars for MAP-2/NeuN, 20 µm; bars for DAPI and TUNEL, 50 µm.</p