Biology subjects

Spearman, P.

Publications and source records attributed to Spearman, P..

5 recordsLinked to original sources

bioRxiv2025

HIV-1 infection of human microglia activates inflammatory pathways associated with HIV-associated neurocognitive disorders (HAND)

HIV-associated neurocognitive disorders (HAND) cause significant dysfunction among people living with HIV. Microglia are the primary immune cells of the central nervous system and are readily infected by HIV. Microglia are thought to contribute to neuroinflammation and cognitive dysfunction in neurodegenerative diseases such as Alzheimers Disease and are likely to play an important role in the pathogenesis of HAND. In order to identify pathways that may contribute to neuropathogenesis in HAND, we infected induced pluripotent stem cell-derived microglia (iMG) with HIV and defined gene expression changes over an 8-day period. Monocyte-derived macrophages (MDMs) were studied in parallel to identify common pathways stimulated in myeloid cells versus the unique aspects of microglia infection. Infection of iMG led to the induction of a robust early inflammatory response triggered within hours of infection, a pattern that differed significantly from that seen in MDMs. Remarkably, gene expression changes in iMG reproduced many of the characteristic genetic signatures previously identified in brain tissues obtained from individuals clinically diagnosed with HAND. Inflammatory activation representing interferon-mediated signaling, TNF/NF-{kappa}B, and IL-6/JAK/STAT pathways were particularly prominent over the time course of infection. Interferon-mediated signaling led to enhanced expression of multiple HIV restriction factors, yet viral replication in iMG remained robust. These findings suggest that HIV infection of microglia is the key cellular driver of neuroinflammation in the CNS of HIV-infected individuals. Further studies of infected microglia are likely to aid in understanding the pathogenesis of HAND and in evaluating therapeutic strategies to limit or eliminate HIV-induced neuropathogenesis. Author SummaryPersons living with HIV frequently develop debilitating problems with brain function that are collectively known as HIV-associated neurocognitive disorders (HAND). Treatment with antiretroviral drugs to control HIV has largely eliminated the most severe form of HIV-related brain dysfunction, HIV encephalitis, but a significant proportion of HIV-infected individuals receiving antiviral therapy still develop significant problems with cognitive function. Microglia are the major macrophage-like cells of the brain and are highly susceptible to HIV infection. Inflammation in the brain following infection contributes to damage to neurons and is the likely source of decline of brain function. This study used microglia that were derived from induced pluripotent stem cells, termed iMG, to study the kinetics of gene expression changes in HIV-infected microglia and compared those changes to those seen in monocyte-derived macrophages (MDMs). iMG were found to be easily infected with a macrophage-tropic HIV strain and exhibited a rapid increase in gene expression associated with inflammatory signaling through interferon- and tumor necrosis factor/NF-kappa B-mediated pathways. Remarkably, the patterns of gene expression in microglia strongly overlapped with gene expression signatures derived from brain tissue samples of HIV patients with HAND. The rapid onset and magnitude of inflammation as well as induction of specific HIV restriction factors differed from that seen in infected macrophages/MDMs. These studies reinforce the central role of microglia in the pathogenesis of HAND and provide insights into HIV-microglia interactions that can help direct interventions to reduce inflammation and preserve brain function.

microbiology↗

bioRxiv2024

The CLIC/GEEC pathway regulates particle endocytosis and formation of the virus-containing compartment (VCC) in HIV-1-infected macrophages

HIV-1 particles are captured by the immunoglobulin superfamily member Siglec-1 on the surface of macrophages and dendritic cells, leading to particle internalization and facilitating trans-infection of CD4+ T cells. HIV-1-infected macrophages develop a unique intracellular compartment termed the virus-containing compartment (VCC) that exhibits characteristic markers of the late endosome and is enriched in components of the plasma membrane (PM). The VCC has been proposed as the major site of particle assembly in macrophages. Depleting Siglec-1 from macrophages significantly reduces VCC formation, implying a link between the capture and endocytosis of external HIV-1 particles and the development of VCCs within HIV-infected cells. We found that endocytosis of particles to the VCC was independent of clathrin, but required dynamin-2. CD98 and CD44, classical markers of the CLIC/GEEC pathway, colocalized with Siglec-1 and HIV-1 particles within the VCC. Inhibition of CLIC/GEEC-mediated endocytosis by chemical or genetic means resulted in the arrest of captured HIV-1 particles on the macrophage cell surface and prevented VCC formation. Virus-like particles (VLPs) were taken up within CD98 and Siglec-1-enriched tubular membranes that migrated centripetally over time to form VCC-like structures. These findings indicate that following capture of virus by Siglec-1, particles follow an endocytic route to the VCC that requires both the CLIC/GEEC pathway and dynamin-2. We propose a model in which internalization of HIV-1 particles together with CLIC/GEEC membranes leads to the formation of the VCC in HIV-infected macrophages, creating an intracellular platform that facilitates further particle assembly and budding. Author SummaryThe major cell types infected by HIV are CD4+ T cells and macrophages. Infection of macrophages is of great interest because this cell type can contribute to transmission of virus within tissues, and because infected macrophages contribute to HIV-related complications that include neurologic disorders, endocrine disorders, and cardiovascular disease. HIV infection of macrophages may also create a latent reservoir that persists in infected individuals despite administration of antiretroviral therapy. Here we focused on a compartment that forms in HIV-infected macrophages termed the virus-containing compartment or VCC. The VCC is an intracellular compartment that has been described as a site of assembly and as a holding compartment for viruses, and contains components of both intracellular organelles and the plasma membrane. We identified an endocytosis pathway that helps to explain the origins of the VCC, the CLIC/GEEC pathway. Inhibition of the CLIC/GEEC pathway prevented virus-like particle delivery to the VCC and prevented the VCC from forming in HIV-infected macrophages. The GTPase dynamin-2 was also required for delivery of HIV particles to the VCC. This study identifies a new facet of how HIV interacts with macrophages, suggesting that disruption of this pathway could be a therapeutic strategy with implications for HIV cure efforts.

microbiology↗

bioRxiv2023

Incorporation of the HIV-1 envelope glycoprotein into viral particles is regulated by the tubular recycling endosome in a cell type-specific manner

The HIV-1 envelope glycoprotein (Env) is incorporated into particles during assembly on the plasma membrane (PM). Env initially reaches the PM through the secretory pathway, after which it is rapidly endocytosed via an AP-2- and clathrin-dependent mechanism. Here we show that endocytosed cell surface Env enters the tubular recycling endosome compartment (TRE). Trafficking to the TRE was dependent upon motifs within the CT previously implicated in Env recycling and particle incorporation. Depletion of TRE components MICAL-L1 or EHD1 led to defects in Env incorporation, particle infectivity, and viral replication. Remarkably, defects were limited to cell types defined as nonpermissive for incorporation of CT-deleted Env, including monocyte-derived macrophages, and not observed in 293T, HeLa, or MT-4 cells. This work identifies the TRE as an essential component of Env trafficking and particle incorporation, and provides evidence that the cell type-dependent incorporation of Env is defined by interactions with components of the TRE.

microbiology↗

bioRxiv2023

Tryptophan-based motifs in the LLP3 Region of the HIV-1 envelope glycoprotein cytoplasmic tail direct trafficking to the endosomal recycling compartment and mediate particle incorporation

The HIV-1 envelope glycoprotein complex (Env) is incorporated into developing particles at the plasma membrane (PM). The cytoplasmic tail (CT) of Env is known to play an essential role in particle incorporation, while the exact mechanisms underlying this function of the CT remain uncertain. Upon reaching the PM, trafficking signals in the CT interact with host cell endocytic machinery, directing Env into endosomal compartments within the cell. Prior studies have suggested that Env must traffic through the endosomal recycling compartment (ERC) in order for Env to return to the plasma membrane (PM) site of particle assembly. Expression of a truncated form of the ERC-resident trafficking adaptor Rab11-Family Interacting Proteins C (FIP1C) resulted in CT-dependent sequestration of Env in the condensed ERC, preventing recycling of Env to the PM. In this work, the motifs within the CT responsible for ERC localization of Env were systematically mapped. A small deletion encompassing the N-terminal portion of LLP3 eliminated ERC localization. Site-directed mutagenesis identified two tryptophan-based motifs (WE790-791 and WW796-797) within the N-terminus of LLP3 that were essential for ERC localization of Env. Mutant viruses bearing substitutions in these motifs were deficient in Env incorporation, with a corresponding loss of particle infectivity and a significant defect in replication in a spreading infection assay. These results identify two tryptophan-based motifs at the N-terminal portion of LLP3 that mediate ERC localization and Env incorporation, providing additional supporting evidence for the importance of cellular recycling pathways in HIV-1 particle assembly. IMPORTANCEThe HIV-1 envelope glycoprotein (Env) is an essential component of the virus, and has an exceedingly long cytoplasmic tail (CT). Previous studies have suggested that trafficking signals in the CT interact with host factors to regulate the incorporation of Env into particles. One particular area of interest is termed lentiviral lytic peptide 3 (LLP3), as small deletions in this region have been shown to disrupt Env incorporation. In this study, we identify a small region within LLP3 that regulates how Env associates with cellular recycling compartments. Mutants that reduced or eliminated Env from the recycling compartment also reduced Env incorporation into particles. These findings emphasize the importance of two tryptophan motifs in LLP3 to the incorporation of Env into particles, and provide additional support for the idea that the CT interacts with host recycling pathways to determine particle incorporation.

microbiology↗

bioRxiv2023

KIF16B mediates anterograde transport and modulates lysosomal degradation of the HIV-1 envelope glycoprotein

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) is incorporated into virions at the site of particle assembly on the plasma membrane (PM). The route taken by Env to reach the site of assembly and particle incorporation remains incompletely understood. Following initial delivery to the PM through the secretory pathway, Env is rapidly endocytosed, suggesting that recycling is required for particle incorporation. Endosomes marked by the small GTPase Rab14 have been previously shown to play a role in Env trafficking. Here we examined the role of KIF16B, the molecular motor protein that directs outward movement of Rab14-dependent cargo, in Env trafficking. Env colocalized extensively with KIF16B+ endosomes at the cellular periphery, while expression of a motor-deficient mutant of KIF16B redistributed Env to a perinuclear location. The half-life of Env labeled at the cell surface was markedly reduced in the absence of KIF16B, while a normal half-life was restored through inhibition of lysosomal degradation. In the absence of KIF16B, Env expression on the surface of cells was reduced, leading to a reduction in Env incorporation into particles and a corresponding reduction in particle infectivity. HIV-1 replication in KIF16B knockout cells was substantially reduced as compared to wildtype cells. These results indicate that KIF16B regulates an outward sorting step involved in Env trafficking, thereby limiting lysosomal degradation and enhancing particle incorporation. IMPORTANCEThe HIV-1 envelope glycoprotein is an essential component of HIV-1 particles. The cellular pathways that contribute to incorporation of envelope into particles are not fully understood. Here we identify KIF16B, a motor protein that directs movement from internal compartments toward the plasma membrane, as a host factor that prevents envelope degradation and enhances particle incorporation. This is the first host motor protein identified that contributes to HIV-1 envelope incorporation and replication.

microbiology↗