The incorporation of cellular proteins into viral particles is well-documented for numerous enveloped viruses, including the human immunodeficiency virus type 1 (HIV-1). This phenomenon represents a unique host-pathogen interplay whereby HIV-1 can exploit the functions of non-virally encoded proteins to interact with the susceptible host. Given that HIV-1 can infect multiple cell-types, we hypothesized that progeny virions incorporate a protein repertoire reflective of the producer cells from which they emerged. These virion-incorporated proteins in turn can facilitate interactions between HIV-1 and immune cells, with implications for pathogenesis.

Herein, we produced HIV-1(BaL) viral stocks in donor-matched peripheral-blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs), along with donor-matched microglia and bone-marrow derived macrophages (BMDMs). Virus-containing supernatants were then analyzed by mass spectrometry to identify the broad repertoire of virion-incorporated proteins, with curated lists of top protein hits extensively validated with orthogonal assays, like flow virometry and immunoprecipitation assays. With donor variation controlled, we observed differential virion-incorporation of numerous human proteins in the PBMC/MDM and Microglia/BMDM datasets, reinforced by hierarchical clustering. Enrichment analyses also revealed differential protein functionalities, allowing for unique cell-virus interactions driven by the repertoire of incorporated proteins. StringDB was used to model physical protein interactions between various leukocytes, revealing predicted cell-type interactions enabled by the virion-incorporated proteins identified in this study. Finally, select targets were validated on plasma-derived HIV-1 particles from people living with HIV (PLWH) to determine in vivo relevance of the identified protein fingerprints.

Collectively, these results reinforce our hypothesis that virions emerge from infected cells with a unique protein fingerprint, allowing for interactions with various immune cells. Ongoing work aims to validate these interactions to identify their contributions to viral pathogenesis and potential as new therapeutic targets.

The HIV-1 accessory protein Nef is expressed early during infection and contributes to evasion of the host immune response. Nef modulates cellular trafficking pathways to downregulate various cell surface immune effectors, including the HIV-1 entry receptor CD4. Another protein downregulated by Nef is Serine incorporator 5 (SERINC5), a multi-pass transmembrane protein restriction factor that incorporates into progeny virions during egress and reduces infectivity by inhibiting fusion with target cells. Nef counteracts SERINC5 by recruiting cellular adaptor protein 2 (AP-2) to internalize SERINC5 via clathrin-mediated endocytosis. An endocytic dileucine sorting motif ([D/E]xxxL[L/I]₁₆₅) in the Nef C-terminal region establishes associations with adaptor proteins (APs) necessary for antagonism of SERINC5 and CD4, suggesting both proteins are downregulated through equivalent trafficking pathways.

We have identified a Nef DN₁₆₄ND polymorphism within the dileucine motif that decreases SERINC5 downregulation, while having minimal effects on CD4 downregulation, rendering antagonism of SERINC5 and CD4 functionally uncoupled. We next hypothesized that the DN₁₆₄ND polymorphism alters interactions between Nef and APs during endolysosomal trafficking to uncouple the two downregulatory pathways. We used the protein-protein interaction reporter assay, bimolecular fluorescence complementation (BiFC), with confocal microscopy to detect Nef and SERINC5 interactions within the endocytic pathway of transfected cells. Our analysis revealed a pronounced decrease in Rab7-positive late endosome localized BiFC associated with the Nef DN₁₆₄ND polymorphism, while no significant differences were observed in Rab5-positive early endosomes and the trans-Golgi network, suggesting that the DN₁₆₄ND polymorphism is causing increased recycling of SERINC5 to the plasma membrane or compromising entry into the degradative pathway. We aim to elucidate the pathway of Nef-mediated SERINC5 downregulation by further characterizing the subcellular compartments and APs involved in the functional uncoupling. This investigation may inform the development of novel therapeutics that inhibit Nef and improve health outcomes in people living with HIV-1.

The HIV-1 accessory protein Nef contributes to HIV-mediated immune evasion by downregulating major histocompatibility complex class I (MHC-I). Nef-mediated MHC-I downregulation results from interaction between Nef and Src family kinases (SFKs) and prevents recognition of HIV-infected cells by cytotoxic T lymphocytes (CTLs). Inhibiting the Nef:SFK interaction has been shown to prevent MHC-I downregulation. Using an in silico docking screen, previous work in our lab identified a dipeptide inhibitor (H3-1) of the Nef:SFK interaction. However, H3-1 was determined to be unstable as it was undetectable via mass spectrometry in mouse plasma 60 minutes post-intraperitoneal injection. Thus, analogues of H3-1 were designed by replacing the C-terminal carboxyl group with various functional groups to decrease susceptibility to proteolysis. The stability of these analogues was analyzed similarly via mass spectrometry and was increased compared to that of H3-1. Results of a NanoBiT assay based on luciferase readout of protein-protein interactions suggests that two of the analogues are potent inhibitors of the interaction between Nef and hematopoietic cell kinase (Hck), a specific SFK. Characterization of the analogues via mass spectrometry suggests that at least three are more stable than H3-1, as they were detectable in mouse plasma for at least one hour. Preliminary characterization done by treating HIV-infected primary CD4+ T cells with the H3-1 analogues and analyzing surface MHC-I via flow cytometry determined that H3-1 and its analogues restore MHC-I on the surface of infected cells. Analyzing surface MHC-I levels on splenocytes of transgenic mice expressing HIV-1 Nef in immune cells also suggested the inhibitors are able to increase antigen presentation. We will also assess whether increased CTL activation results from changes in MHC-I levels using humanized C3H mice. Characterization of these inhibitors will determine their utility as part of an immune-directed HIV therapeutic strategy.

Background: Combination antiretroviral therapy (ART) remains a cornerstone of HIV treatment, with over one million pregnant people receiving ART, reducing perinatal transmission. Children who are HIV-exposed but uninfected (CHEU) have higher rates of adverse birth outcomes, after in utero exposure to integrase strand transfer inhibitors (INSTIs). The 2018 Botswana Tsepamo study reported increased incidence of neural tube defects (NTDs) with dolutegravir exposure, and folate deficiency is linked to NTDs. The mechanisms behind these risks and long-term ART effects remain not fully understood. Placental transporters and enzymes regulate antiretroviral drug (ARV) disposition and fetal delivery. We investigated whether ART-mediated dysregulation of these pathways affects fetal development.
Methodology: HTR8/SVneo and BeWo human placental cell lines, representing first and third trimesters, were treated with clinically relevant concentrations of INSTIs, dolutegravir, bictegravir, cabotegravir, or DMSO control. mRNA, protein expression, and function of key placental nutrient and drug transporters, including FR-alpha (FRα), RFC (SLC19A1), PCFT (SLC46A1), P-gp (ABCB1), BCRP (ABCG2), and MRPs 1- 5 (ABCC1-5), along with metabolic enzymes, UGT1A1, CYP3A4 were assessed by qPCR, immunoblotting, and radiolabeled uptake assays. Complementary in vivo studies in pregnant mice evaluated placental and fetal nutrient/drug transporter and metabolic enzymes expression following oral INSTIs treatment.
Results: Dolutegravir reduced FRα and PCFT mRNA and protein expression as well as function by 50% and increased BCRP (25%), MRP1 (50%), and MRP3 (30%) in HTR8 cells. Bictegravir decreased FRα, RFC, and PCFT by 20% and downregulated CYP3A4 by 60% in BeWo cells. Cabotegravir produced modest effects on FRα and RFC but increased P-gp by 50% in HTR8 cells.
Conclusion: These findings suggest that INSTI exposure may dysregulate key SLC, ABC placental transporters and metabolic enzymes, potentially altering transplacental transfer of ARVs and essential nutrients. This could affect fetal drug exposure and contribute to adverse developmental outcomes. (Supported by OHTN and CIHR).

Background: HIV-associated neurocognitive impairment (NCI) affects nearly half of people living with HIV and is primarily characterized by chronic neuroinflammation. Peroxisome proliferator-activated receptor gamma (PPARγ), which exhibits anti-inflammatory and neuroprotective effects, represents a potential therapeutic target for HIV-related neurological complications. We investigated whether activation of PPARγ with INT131, a selective partial agonist, could mitigate HIV-induced neuroinflammation and cognitive deficits in an EcoHIV mouse model. In parallel, postmortem human brain tissues were analyzed to assess neuroinflammatory responses.

Methods: Male and female C57BL/6 mice were assigned to four groups: Saline, Saline+INT131, EcoHIV (4×10⁶ pg/mL p24 IP), and EcoHIV+INT131. Two weeks later, osmotic pumps delivered INT131 (20 mg/kg/day) or saline for 28 days. Behavioral assessments evaluated locomotion, learning, memory, and anxiety. Brain and spleen tissues were analyzed by qPCR, immunohistochemistry, and western blot for viral genes/proteins, inflammatory and oxidative stress markers, BBB tight junction proteins, and PPARγ/NF-κB signaling. Postmortem brain tissues from HIV– controls, HIV+ without NCI, and HIV+ with NCI obtained from the National NeuroAids Tissue Consortium were examined for PPARγ, NF-κB, IL-6, IBA-1, GFAP, and CASP-3.

Results: INT131 reduced EcoHIV Vif, Tat, and p24, decreased inflammatory (Tnf-α, Il-1β, C3, Ccl2, Ifn-γ) and oxidative stress markers (Nos2, Hmox) in brain and spleen of both sexes, restored PPARγ, and normalized BBB protein expression (Cldn5, Ocln, Tjp1). In behavioral studies, it reversed locomotor, memory, and anxiety deficits in the EcoHIV group. In post-mortem human brain tissues from HIV+ individuals with NCI, we observed an increased NF-κB activation, IL-6 upregulation, microgliosis (Iba-1), astrocytosis, and increased caspase-3, with reduced PPARγ versus HIV– and HIV+ without NCI, providing translational insight for PPARγ-targeted interventions.

Conclusion: These findings identify PPARγ as a relevant target for HIV-associated neuroinflammation, BBB dysfunction, and cognitive impairments supporting further investigation in PPARγ-targeted therapies for HIV-associated NCI. (Supported by the Canadian Institutes of Health Research.)