HIV-1 and methamphetamine alter galectins -1, -3, and -9 in human monocyte-derived macrophages.

Macrophages are key elements of the innate immune system. Their HIV-1 infection is a complex process that involves multiple interacting factors and various steps and is further altered by exposure of infected cells to methamphetamine (Meth), a common drug of abuse in people living with HIV. This is reflected by dynamic changes in the intracellular and secreted proteomes of these cells. Quantification of these changes poses a challenge for experimental design and associated analytics. In this study, we measured the effect of Meth on expression of intracellular and secreted galectins-1, -3, and -9 in HIV-1 infected human monocyte-derived macrophages (hMDM) using SWATH-MS, which was further followed by MRM targeted mass spectrometry validation. Cells were exposed to Meth either prior to or after infection. Our results are the first to perform comprehensive quantifications of galectins in primary hMDM cells during HIV-1 infection and Meth exposure a building foundation for future studies on the molecular mechanisms underlying cellular pathology of hMDM resulting from viral infection and a drug of abuse-Meth.

HIV and Proteomics: What We Have Learned from High Throughput Studies.

The accelerated development of technology over the last three decades has driven biological sciences to high-throughput profiling experiments, now broadly referred to as systems biology. The unprecedented improvement of analytical instrumentation has opened new avenues for more complex experimental designs and expands the knowledge in genomics, proteomics, and other omics fields. Despite the collective efforts of hundreds of researchers, gleaning all the expected information from omics experiments is still quite far. This paper summarizes what has been learned from high-throughput proteomics studies thus far, and what is believed should be done to reveal even more valuable information from such studies. It is drawn from the background in using proteomics to study human immunodeficiency virus 1 infection of macrophages and/or T cells, but it is believed that some conclusions will be more broadly applicable.

Palmitoylated mNeonGreen Protein as a Tool for Visualization and Uptake Studies of Extracellular Vesicles.

Extracellular vesicles (EVs) are membranous nanoparticles released by cells as vital mediators of intercellular communication. As such, EVs have become an attractive target for pathogens and cancer cells, which can take control over their cargo composition, as well as their trafficking, shaping the pathogenesis. Despite almost four decades of research on EVs, the number of specific and efficient EV labeling methods is limited, and there is still no universal method for the visualization of their transport in living cells. Lipophilic dyes that non-specifically intercalate into the EVs membranes may diffuse to other membranes, leading to the misinterpretation of the results. Here, we propose a palmitoylated fluorescent mNeonGreen (palmNG) protein as an alternative to chemical dyes for EVs visualization. The Branchiostoma lanceolatum-derived mNeonGreen is a brighter, more stable, and less sensitive to laser-induced bleaching alternative to green fluorescent protein (GFP), which makes it a more potent tag in a variety of fluorescence-based techniques. A palmNG-expressing stable human melanoma cell line was generated using retrovirus gene transfer and cell sorting. This protein partially localizes to cellular membranes, and can be detected inside size-exclusion (SEC)-purified EVs. With the use of flow cytometry and fluorescent confocal microscopy, we performed qualitative and quantitative analyses of palmNG-EVs uptake in recipient human hepatoma cells, in comparison to PKH67-labeled vesicles. Our findings confirm that membrane-embedded mNeonGreen can be successfully applied as a tool in EVs transfer and uptake studies.

Alphaherpesvirus gB Homologs Are Targeted to Extracellular Vesicles, but They Differentially Affect MHC Class II Molecules.

Herpesvirus envelope glycoprotein B (gB) is one of the best-documented extracellular vesicle (EVs)-incorporated viral proteins. Regarding the sequence and structure conservation between gB homologs, we asked whether bovine herpesvirus-1 (BoHV-1) and pseudorabies virus (PRV)-encoded gB share the property of herpes simplex-1 (HSV-1) gB to be trafficked to EVs and affect major histocompatibility complex (MHC) class II. Our data highlight some conserved and differential features of the three gBs. We demonstrate that mature, fully processed BoHV-1 and PRV gBs localize to EVs isolated from constructed stable cell lines and EVs-enriched fractions from virus-infected cells. gB also shares the ability to co-localize with CD63 and MHC II in late endosomes. However, we report here a differential effect of the HSV-1, BoHV-1, and PRV glycoprotein on the surface MHC II levels, and MHC II loading to EVs in stable cell lines, which may result from their adverse ability to bind HLA-DR, with PRV gB being the most divergent. BoHV-1 and HSV-1 gB could retard HLA-DR exports to the plasma membrane. Our results confirm that the differential effect of gB on MHC II may require various mechanisms, either dependent on its complex formation or on inducing general alterations to the vesicular transport. EVs from virus-infected cells also contained other viral glycoproteins, like gD or gE, and they were enriched in MHC II. As shown for BoHV-1 gB- or BoHV-1-infected cell-derived vesicles, those EVs could bind anti-virus antibodies in ELISA, which supports the immunoregulatory potential of alphaherpesvirus gB.