Publications

Nonhuman primates (NHPs), particularly macaques, are indispensable models for studying human infectious diseases due to their close immunological and physiological similarities. Understanding species-specific molecular differences is essential for maximizing the translational value of these models. Here we report that APOBEC3G (A3G) , a potent antiviral restriction factor and the major source of genetic variations in HIV, exhibits a widespread mRNA splicing defect in the Cercopithecinae subfamily, which includes the commonly used NHP models. Driven by intronic polymorphisms, this splicing defect substantially reduces A3G protein levels and consequently results in a markedly reduced A3G-mediated mutation signatures, fewer defective viral genomes, and greater viral diversification in SIV compared to HIV. This species-specific effect is not restricted to lentiviruses: reduced A3G signatures have also been reported in simian foamy virus and simian T-cell leukemia virus, suggesting broader effects across primate retroviruses. These findings reveal a lineage-specific alteration in a major antiviral restriction factor, with important implications for viral restriction, evolution, drug resistance, and immune evasion. They also highlight the importance of incorporating naturally occurring genetic variation into NHP model selection to improve the reproducibility, translational fidelity, and biological relevance of preclinical research.

The persistence of HIV latent reservoir is the major challenge to HIV cure because latent viruses serve as sources for viral rebound upon ART cessation. Mechanisms regulating viral persistence are not well understood; thus, there is a compelling need for research focusing on addressing the knowledge gap related to HIV persistence. The present study focuses on the effect of extracellular condensates (ECs) on latent HIV/SIV reactivation in the brain in the context of HIV infection using the SIV-infected rhesus macaque model. We used in vitro model systems of post-integration latency and primary peripheral blood mononuclear cells isolated from HIV-infected ART-suppressed donors to explore the role of basal ganglia (BG) isolated extracellular condensates (ECs) in reprogramming HIV latent cells. We found that BG ECs from uninfected macaques (VEH) and SIV infected macaques (VEH | SIV) activated latent HIV transcription in various model systems. VEH | SIV ECs significantly increased the expression and production of viral antigen in latently infected cells. Activation of viral transcription, antigen expression, and latency reactivation was inhibited by ECs from the brain of macaques treated with Delta-9-tetrahydrocannabinol (THC) and infected with SIV (THC | SIV). Virus produced by latently infected cells treated with VEH | SIV ECs potentiated cell-cell and cell-free HIV transmission. VEH | SIV ECs also reversed dexamethasone-mediated inhibition of HIV transcription while TNFα-mediated reactivation of latency was reversed by THC | SIV ECs. Transcriptome and secretome analyses of total RNA and supernatants from latently infected cells treated with ECs revealed significant alterations in gene expression and cytokine secretion. THC | SIV ECs increased secretion of Th2 and decreased secretion of proinflammatory cytokines. Most strikingly, while VEH/SIV ECs robustly induced expression of HIV RNA in latently HIV-infected cells, increased the frequency of HIV gag p24 expressing cells in HIV-infected CD4 + T cells within PBMCs, and production of extracellular HIV gag p24, long-term low-dose THC administration enriched ECs with anti-inflammatory cargo that significantly diminished their ability to reactivate latent HIV, an indication that ECs are endogenous host factors that may regulate HIV persistence.

Extracellular particles (EPs), an umbrella term encompassing membrane-enclosed extracellular vesicles (EVs) and non-vesicular extracellular particles ([NVEPs], previously described as extracellular condensates [ECs]) contain a complex cargo of biomolecules, including DNA, RNA, proteins, and lipids, reflecting the physiological state of their cell of origin. Identifying proteins associated with EPs that regulate host responses to physiological and pathophysiological processes is of critical importance. Here, we report the findings of our study to gain insight into the proteins associated with NVEPs. We used samples from human semen, the rat brain, and the rhesus macaque (RM) brain and blood to assess the physical properties and proteome profiles of NVEPs from these specimens. The results show significant differences in the zeta potential, concentration, and size of NVEPs across different species. We identified 938, 51, and 509 total proteins from NVEPs isolated from rat brain tissues, RM blood, and human seminal plasma, respectively. The species-specific protein networks show distinct biological themes, while the species-conserved protein interactome was identified with six proteins (ALB, CST3, FIBA/FGA, GSTP1, PLMN/PLG, PPIA) associated with NVEPs in all samples. The six NVEP-associated proteins are prone to aggregation and formation of wide, insoluble, unbranched filaments with a cross-beta sheet quaternary structure, such as amyloid fibrils. Protein-to-function analysis indicates that the six identified proteins are linked to the release of dopamine, immune-mediated inflammatory disease, replication of RNA viruses, HIV-HCV co-infection, and inflammation. These interesting findings have created an opportunity to evaluate NVEPs for their potential use as biomarkers of health and disease. Additional in-depth studies are needed to clarify when and how these proteins sustain their physiological role or transition to pathogenic roles.

Epitranscriptomic modifications [N6-methyladenosine (m6A)] regulate various diseases, including cancer and inflammation. Despite their functional relevance in neural development and differentiation, the role of m6A modifications in HIV neuropathogenesis is unknown. Using anti-N6-methyladenosine (m6A) antibody-immunoprecipitation and microarray profiling, we identified m6A modifications in miRNAs in basal ganglia (BG) of uninfected (VEH) and SIV-infected Rhesus macaques (RMs) on combination anti-retroviral therapy (ART) and either VEH-treated (VEH/SIV/ART) or THC:CBD-treated (THC:CBD/SIV/ART). HIV/SIV infection promoted an overall hypomethylated miRNA m6A profile. While THC:CBD did not significantly impact the overall hypomethylated m6A profile, specific miRNAs predicted to target proinflammatory genes showed marked m6A hypomethylation compared to VEH-treated RMs. Additionally, specific BG m6A-modified miRNAs were detected in BG-derived extracellular vesicles. Mechanistically, the DRACH motif in the miR-194-5p seed region was significantly m6A hypomethylated in THC:CBD/SIV/ART RMs. Unlike wild-type, in-vitro transfected m6A-modified miR-194-5p mimics failed to downregulate STAT1 protein expression. Further, compared to VEH/SIV/ART RMs, THC:CBD significantly reduced m6A methylation of 44 miRNAs directly involved in regulating CNS network genes. Our findings indicate that m6A epi-transcriptomic marks in the seed nucleotides can impair miRNA function and that cannabinoids may preserve it by reducing m6A methylation levels, thus providing a mechanistic explanation underlying their anti-neuroinflammatory effects in HIV/SIV infection.

With the advancement of combination antiretroviral therapy (ART), over 50% of people with HIV (PWH) in the United States are now over the age of 50. A hallmark of the aging immune system is a progressive dysfunction of both the innate and adaptive immune responses, often characterized by clonal expansion of memory T cells. However, the impact of age-related immune dysfunction on HIV/SIV reservoir dynamics remains understudied. We hypothesized that age-associated clonal expansion of memory T cells contributes to the increase of the HIV reservoirs in older PWH (OPWH). In this retrospective study, we utilized archived peripheral blood mononuclear cells (PBMCs) from young and older PWH with suppressed plasma viremia for at least 5 years and quantified both intact and total HIV proviral DNA from CD4+ T cells. Alongside the human study, we also analyzed samples from SIV-infected, ART-suppressed young and aged rhesus macaques, quantifying intact and total proviruses in CD4+ T cells. We observed a significantly higher level of intact and total proviral DNA in older compared to younger PWH. The frequency of intact provirus was positively correlated with activated CD4+ and CD8+ T cells. Consistently, in the non-human primate model, aged macaques exhibited significantly higher levels of intact and total SIV proviruses in CD4+ T cells than their younger counterparts. Collectively, these findings suggest that the HIV/SIV reservoir expands with age, potentially driven by immune activation. Future studies are warranted to elucidate the mechanisms underlying reservoir expansion in the aging population.

Despite effective antiretroviral therapy (ART), people with HIV (PWH) experience persistent inflammation and metabolic dysfunction, increasing their risk for non-AIDS comorbidities. Accordingly, we evaluated the effects of long-term/low-dose Δ9-tetrahydrocannabinol (THC) supplementation in simian immunodeficiency virus (SIV)-infected, ART-treated rhesus macaques (RMs). THC significantly increased plasma/jejunum serotonin and indole-3-propionate, enhancing gut-brain communication through up-regulation of serotonin receptors (HTR4/HTR7) and aryl hydrocarbon receptor (Ahr) signaling via a cannabinoid receptor (CBR)-2-mediated mechanism. Furthermore, THC enriched cholesterol-metabolizing Oscillibacter and reduced plasma cholesterol and toxic secondary bile acids (SBAs), thus improving cholesterol and SBA homeostasis. Furthermore, THC increased β-hydroxybutyrate (BHB) levels via a CBR1-mediated mechanism, suggesting enhanced hepatic fatty acid oxidation for metabolic and cardiovascular health. THC restored ART/SIV-induced elevation of pro-inflammatory and cardiotoxic long-chain acylcholines to preinfection levels. THC-treated RMs maintained viral suppression despite reduced plasma ART levels, suggesting diminished ART-related toxicity. Our findings demonstrate phytocannabinoids to be a safe adjunct therapy alongside ART to mitigate chronic inflammation and metabolic dysfunction in PWH.

The gut is a major reservoir in HIV-infected individuals on antiretroviral therapy (ART) and in long-term non-progressors (LTNPs). Whether ART reduces gut infection and reservoirs in LTNPs is unknown. Herein, SIV-infected LTNP Rhesus macaques were treated with short- or long-term ART, and SIV envelope gp120 sequences obtained from single genome amplification were analyzed before and after ART in peripheral blood and the intestine. Although ART does not eliminate SIV in these LTNPs, a longer ART period dramatically reduces SIV infection in the gut. This study highlights the importance of long-term ART in LTNPs to minimize gut infection and prolong remission.

Long noncoding RNAs (lncRNAs) are transcripts measuring >200 bp in length and devoid of protein-coding potential. LncRNAs exceed the number of protein-coding mRNAs and regulate cellular, developmental, and immune pathways through diverse molecular mechanisms. In recent years, lncRNAs have emerged as epigenetic regulators with prominent roles in health and disease. Many lncRNAs, either host or virus-encoded, have been implicated in critical cellular defense processes, such as cytokine and antiviral gene expression, the regulation of cell signaling pathways, and the activation of transcription factors. In addition, cellular and viral lncRNAs regulate virus gene expression. Viral infections and associated immune responses alter the expression of host lncRNAs regulating immune responses, host metabolism, and viral replication. The influence of lncRNAs on the pathogenesis and outcomes of viral infections is being widely explored because virus-induced lncRNAs can serve as diagnostic and therapeutic targets. Future studies should focus on thoroughly characterizing lncRNA expressions in virus-infected primary cells, investigating their role in disease prognosis, and developing biologically relevant animal or organoid models to determine their suitability for specific therapeutic targeting. Many cellular and viral lncRNAs localize in the nucleus and epigenetically modulate viral transcription, latency, and host responses to infection. In this review, we provide an overview of the role of nuclear lncRNAs in the pathogenesis and outcomes of viral infections, such as the Influenza A virus, Sendai Virus, Respiratory Syncytial Virus, Hepatitis C virus, Human Immunodeficiency Virus, and Herpes Simplex Virus. We also address significant advances and barriers in characterizing lncRNA function and explore the potential of lncRNAs as therapeutic targets.

Intestinal epithelial barrier dysfunction, a hallmark of HIV/SIV infection, persists despite viral suppression by combination antiretroviral therapy (cART). Emerging evidence suggests a critical role for long noncoding RNAs (lncRNAs) in maintaining epithelial homeostasis. We simultaneously profiled lncRNA/mRNA expression exclusively in colonic epithelium (CE) of SIV-infected rhesus macaques (RMs) administered vehicle (VEH) or Δ-9-tetrahydrocannabinol (THC). Relative to controls, fewer lncRNAs were up- or downregulated in CE of THC/SIV compared with VEH/SIV RMs. Importantly, reciprocal expression of the natural antisense lncRNA MMP25-AS1 (up 2.3-fold) and its associated protein-coding gene MMP25 (attracts neutrophils by inactivating alpha-1 anti-trypsin/SERPINA1) (down 2.2-fold) was detected in CE of THC/SIV RMs. Computational analysis verified 2 perfectly matched complementary regions and an energetically stable (normalized binding free energy = -0.2626) MMP25-AS1/MMP25 duplex structure. MMP25-AS1 overexpression blocked IFN-γ-induced MMP25 mRNA and protein expression in vitro. Elevated MMP25 protein expression in CE of VEH/SIV but not THC/SIV RMs was associated with increased infiltration by myeloperoxidase/CD11b++ neutrophils (transendothelial migration) and epithelial CD47 (transepithelial migration) expression. Interestingly, THC administered in combination with cART increased MMP25-AS1 and reduced MMP25 mRNA/protein expression in jejunal epithelium of SIV-infected RMs. Our findings demonstrate that MMP25-AS1 is a potentially unique epigenetic regulator of MMP25 and that low-dose THC can reduce neutrophil infiltration and intestinal epithelial injury potentially by downregulating MMP25 expression through modulation of MMP25-AS1.

Historically, cannabis has been valued for its pain-relieving, anti-inflammatory, and calming properties. Ancient civilizations like the Egyptians, Greeks, and Chinese medicines recognized their therapeutic potential. The discovery of the endocannabinoid system, which interacts with cannabis phytoconstituents, has scientifically explained how cannabis affects the human immune system, including the central nervous system (CNS). This review explores the evolving world of cannabis-based treatments, spotlighting its diverse applications. By researching current research and clinical studies, we probe into how cannabinoids like Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) help to manage conditions ranging from chronic pain, persistent inflammation, cancer, inflammatory bowel disease, and neurological disorders to even viral diseases such as Human Immunodeficiency virus (HIV), SARS-CoV-2. and the emerging monkeypox. The long-term recreational use of cannabis can develop into cannabis use disorder (CUD), and therefore, understanding the factors contributing to the development and maintenance of cannabis addiction, including genetic predisposition, neurobiological mechanisms, and environmental influences, will be timely. Shedding light on the adverse impacts of CUD underscores the importance of early intervention, effective treatment approaches, and public health initiatives to address this complex issue in an evolving landscape of cannabis policies and perceptions.