Slow Release of HIV-1 Protein Nef from Vesicle-like Structures Is Inhibited by Cytosolic Calcium Elevation in Single Human Microglia

Abstract

Once infected by HIV-1, microglia abundantly produce accessory protein Nef that enhances virus production and infectivity, but little is known about its intracellular compartmentalization, trafficking mode(s), and release from microglia. Here, we transfected immortalized human microglia with a plasmid encoding Nef tagged with green fluorescent protein (Nef.GFP) to biochemically and microscopically identify Nef.GFP-associated cellular compartments and examine their mobility and Nef release from cultured cells. Immunoblotting revealed that Nef.GFP confined to subcellular fractions with a buoyant density similar to organelles positive for lysosomal-associated membrane protein 1 (LAMP1) but structurally segregated from dextran-laden and LysoTracker-laden endo-/lysosomes in live cells. As revealed by confocal microscopy, Nef.GFP-positive vesicle-like structures were smaller than dextran-laden vesicles and displayed slow and non-directional mobility, in contrast to the faster and directional mobility of dextran-laden vesicles. Ionomycin-evoked elevation in intracellular free Ca concentration ([Ca] ) negligibly affected mobility of Nef.GFP structures but strongly and irrecoverably attenuated mobility of dextran-laden vesicles. A slow time-dependent decrease in the number of Nef.GFP-positive structures was observed in non-stimulated controls (5 ± 1 structures/min), but not in ionomycin-stimulated cells (0 ± 2 structures/min; P< 0.05), indicating that elevated [Ca] inhibits the release of Nef.GFP structures. The latter significantly co-localized with membrane sites immunopositive for the tetraspanins CD9 (36 ± 4%) and CD81 (22 ± 1%). This is the first report to demonstrate that microglial CD9- and CD81-positive plasma membrane-derived compartments are associated with biogenesis and Nef release.

Astroglia in Alzheimer’s Disease

“…In the nervous system, the HIV-1 virus primarily infects and propagates in microglial cells. Microglia contributes to neuronal death by releasing various neurotoxic factors [123,171], including Nef protein [267]. In HAD, astroglial cells develop both astrodegeneration and reactive astrogliosis.…”

Complexities of JC Polyomavirus Receptor-Dependent and -Independent Mechanisms of Infection

“…For instance, several ESCRT related proteins are implicated in HIV budding and cytomegalovirus maturation [162][163][164], tetraspanins play a role in HIV, herpes simplex virus-1, and influenza virus infections [113,165,166], and β-coronaviruses like SARS-CoV-2 were recently shown to use secretory autophagy pathways for cellular escape [115]. Importantly, there are instances of viral proteins, mRNA, and microRNAs disseminated in EVs to uninfected cells [110,116,[118][119][120][167][168][169][170][171]. Many research groups are pushing to understand which EV biogenesis pathways are exploited by viruses and how viral cargo is packaged into EVs.…”

Astrogliopathology in the infectious insults of the brain

“…Endocytosis appears to be common mechanism of neurotropic virus brain infection, since human immunodeficiency virus 1 (HIV-1) enters astroglia by endocytosis as well, albeit the fate of HIV-1 virus seems to be linked with its destruction in the endolysosomes [16]. Some of the HIV-1 virus parts, such as the protein Nef can be propagated to neighbouring cells via vesicle-like structures, and this process is inhibited by elevated cytosolic calcium levels, [Ca 2+ ]i [98], which contrasts to the case where elevated [Ca 2+ ]i stimulates vesicle-based secretion [47,60,95]. The internalisation of TBEV particles into astrocytes [78] is consistent with the clathrin-dependent endocytotic entry known for several members of Flaviviridae family including West Nile virus (WNV) [19], Dengue virus [3,69], Hepatitis C virus [10] and Bovine Viral Diarrhoea virus (BVDV) [38].…”