Extracellular vesicles (EVs) serve as pivotal mediators of intercellular communication in both health and disease, delivering biologically active molecules from vesicle-producing cells to recipient cells. In the context of HIV infection, EVs have been shown to carry the viral protein Nef, a key pathogenic factor associated with HIV-related co-morbidities. Despite this recognition, the specific localisation of Nef within the vesicles has remained elusive. This study addresses this critical knowledge gap by investigating Nef-containing EVs. Less than 1% of the total released Nef was associated with EVs; most Nef existed as free protein released by damaged cells. Nevertheless, activity of EV-associated Nef in downregulating the major cholesterol transporter ABCA1, a critical aspect linked to the pathogenic effects of Nef, was comparable to that of free Nef present in the supernatant. Through a series of biochemical and microscopic assays, we demonstrate that the majority of EV-associated Nef molecules are localised on the external surface of the vesicles. This distinctive distribution prompts the consideration of Nef-containing EVs as potential targets for immunotherapeutic interventions aimed at preventing or treating HIV-associated co-morbidities. In conclusion, our results shed light on the localisation and functional activity of Nef within EVs, providing valuable insights for the development of targeted immunotherapies to mitigate the impact of HIV-associated co-morbidities.

The current study analyzed the intersecting biophysical, biochemical, and functional properties of extracellular particles (EPs) with the human immunodeficiency virus type-1 (HIV-1) beyond the currently accepted size range for HIV-1. We isolated five fractions (Frac-A through Frac-E) from HIV-infected cells by sequential differential ultracentrifugation (DUC). All fractions showed a heterogeneous size distribution with median particle sizes greater than 100 nm for Frac-A through Frac-D but not for Frac-E, which contained small EPs with an average size well below 50 nm. Synchronized and released cultures contained large infectious EPs in Frac-A, with markers of amphisomes and viral components. Additionally, Frac-E uniquely contained EPs positive for CD63, HSP70, and HIV-1 proteins. Despite its small average size, Frac-E contained membrane-protected viral integrase, detectable only after SDS treatment, indicating that it is enclosed in vesicles. Single particle analysis with dSTORM further supported these findings as CD63, HIV-1 integrase, and the viral surface envelope (Env) glycoprotein (gp) colocalized on the same Frac-E particles. Surprisingly, Frac-E EPs were infectious, and infectivity was significantly reduced by immunodepleting Frac-E with anti-CD63, indicating the presence of this protein on the surface of infectious small EPs in Frac-E. To our knowledge, this is the first time that extracellular vesicle (EV) isolation methods have identified infectious small HIV-1 particles (smHIV-1) that are under 50 nm. Collectively, our data indicate that the crossroads between EPs and HIV-1 potentially extend beyond the currently accepted biophysical properties of HIV-1, which may have further implications for viral pathogenesis.

The Gag-Pol polyprotein in human immunodeficiency virus type I (HIV-1) encodes enzymes that are essential for virus replication: protease (PR), reverse transcriptase (RT), and integrase (IN). The mature forms of PR, RT and IN are homodimer, heterodimer and tetramer, respectively. The precise mechanism underlying the formation of dimer or tetramer is not yet understood. Here, to gain insight into the dimerization of PR and RT in the precursor, we prepared a model precursor, PR-RT, incorporating an inactivating mutation at the PR active site, D25A, and including two residues in the p6* region, fused to a SUMO-tag, at the N-terminus of the PR region. We also prepared two mutants of PR-RT containing a dimer dissociation mutation either in the PR region, PR(T26A)-RT, or in the RT region, PR-RT(W401A). Size exclusion chromatography showed both monomer and dimer fractions in PR-RT and PR(T26A)-RT, but only monomer in PR-RT(W401A). SEC experiments of PR-RT in the presence of protease inhibitor, darunavir, significantly enhanced the dimerization. Additionally, SEC results suggest an estimated PR-RT dimer dissociation constant that is higher than that of the mature RT heterodimer, p66/p51, but slightly lower than the premature RT homodimer, p66/p66. Reverse transcriptase assays and RT maturation assays were performed as tools to assess the effects of the PR dimer-interface on these functions. Our results consistently indicate that the RT dimer-interface plays a crucial role in the dimerization in PR-RT, whereas the PR dimer-interface has a lesser role.

Broadly neutralizing antibodies are proposed as therapeutic and prophylactic agents against HIV-1, but their potency and breadth are less than optimal. This study describes the immunization of a llama with the prefusion-stabilized HIV-1 envelope (Env) trimer, BG505 DS-SOSIP, and the identification and improvement of potent neutralizing nanobodies recognizing the CD4-binding site (CD4bs) of vulnerability. Two of the vaccine-elicited CD4bs-targeting nanobodies, G36 and R27, when engineered into a triple tandem format with llama IgG2a-hinge region and human IgG1-constant region (G36×3-IgG2a and R27×3-IgG2a), neutralized 96% of a multiclade 208-strain panel at geometric mean IC80s of 0.314 and 0.033 µg mL-1, respectively. Cryo-EM structures of these nanobodies in complex with Env trimer revealed the two nanobodies to neutralize HIV-1 by mimicking the recognition of the CD4 receptor. To enhance their neutralizing potency and breadth, nanobodies are linked to the light chain of the V2-apex-targeting broadly neutralizing antibody, CAP256V2LS. The resultant human-llama bispecific antibody CAP256L-R27×3LS exhibited ultrapotent neutralization and breadth exceeding other published HIV-1 broadly neutralizing antibodies, with pharmacokinetics determined in FcRn-Fc mice similar to the parent CAP256V2LS. Vaccine-elicited llama nanobodies, when combined with V2-apex broadly neutralizing antibodies, may therefore be able to fulfill anti-HIV-1 therapeutic and prophylactic clinical goals.

Nucleic acid amplification, the bedrock of biotechnology and molecular diagnostics, surges in applications-especially isothermal approaches-heightening the demand for advanced and precisely engineered methods. Here, a novel approach for amplifying DNA with multiarm priming and looping optimization of nucleic acid (AMPLON) is presented. AMPLON relies on a novel polymeric material with unique set of multiarm polyethylene glycol-DNA primers for efficient DNA amplification under isothermal conditions. Each arm carries single-stranded DNA complementing the sense or antisense sequence of the target DNA. The amplification reaction begins with antisense arms binding to the target DNA, forming a template for sense-carrying arms to direct multiarm large DNA amplicon synthesis through successive DNA looping and unlooping steps. Using human immunodeficiency virus type 1 (HIV-1) as a model clinical target, AMPLON exhibits high sensitivity, detecting target concentrations as low as 100 copies mL-1. Compared to a quantitative real-time polymerase chain reaction assay using sensitive primers, AMPLON reliably identifies HIV-1 RNA in plasma samples (n = 20) with a significant agreement rate of 95%. With its ability to achieve highly specific and sensitive target amplification within 30 min, AMPLON holds immense potential to transform the field of nucleic acid research and unleashing new possibilities in medicine and biotechnology.

Due to its central role in cell biology, the cytoskeleton is a key regulator of viral infection, influencing nearly every step of the viral life cycle. In this review, we will discuss the role of two key components of the cytoskeleton, namely the actin and microtubule networks in early HIV-1 infection. We will discuss key contributions to processes ranging from the attachment and entry of viral particles at the cell surface to their arrival and import into the nucleus and identify areas where further research into this complex relationship may yield new insights into HIV-1 pathogenesis.

UNASSIGNED: During chronic human immunodeficiency virus (HIV)-1 infection, inhibitory molecules upregulated on lymphocytes contribute to effector cell dysfunction and immune exhaustion. People living with HIV (PLWH) are at greater risk for age-related morbidities, an issue magnified by human cytomegalovirus (CMV) coinfection. As CMV infection modifies natural killer (NK) cell properties and NK cells contribute to protection against HIV-1 infection, we considered the role of T-cell immunoreceptor with immunoglobulin and intracellular tyrosine inhibitory motif domains (TIGIT) in NK cell-based HIV-1 immunotherapy and elimination strategies.
UNASSIGNED: We measured TIGIT expression on immune cell subsets of 95 PLWH and assessed its impact on NK cell function, including elimination of autologous CD4+ T cells infected through reactivation of endogenous HIV-1.
UNASSIGNED: TIGIT was expressed on CD4+ T cells, CD8+ T cells and NK cells from PLWH. Although TIGIT levels on T cells correlated with HIV-1 disease progression, the extent of TIGIT expression on NK cells more closely paralleled adaptation to CMV. TIGIT interacts with its predominant ligand, poliovirus receptor (PVR), to inhibit effector cell functions. Circulating CD4+ T cells from PLWH more frequently expressed PVR than HIV-seronegative controls, and PVR expression was enriched in CD4+ T cells replicating HIV-1 ex vivo. Treatment with anti-TIGIT monoclonal antibodies increased NK cell HIV-1-specific antibody-dependent cytotoxicity in vitro and ex vivo.
UNASSIGNED: Blocking TIGIT may be an effective strategy to invigorate antibody-dependent NK cell activity against HIV-1 activated in cellular reservoirs for cure or treatment strategies.

OBJECTIVE: It is postulated that molecular methods along with mathematical modeling can provide critical inference regarding epidemiological parameters, transmission dynamics, spatiotemporal characteristics, and intervention efficacy. Hence, studying molecular epidemiology of human immunodeficiency virus (HIV)-1 infection, especially in resource-limited settings and with a large diaspora of the migrant population such as that of Bangladesh, is of paramount importance. The purpose of this systematic review was to concisely present and discuss the findings from previous studies conducted in Bangladesh regarding HIV-1 subtype prevalence.
METHODS: Articles were retrieved from six publicly available databases regarding HIV-1 molecular epidemiology using keywords HIV, HIV-1, subtype(s), Bangladesh, and any combination of aforementioned keywords using Boolean operators. A total of 14 articles were downloaded and screened for suitability. Finally, five studies, containing pooled sequences from 317 individuals, were included in this systematic review.
RESULTS: Results revealed a preponderance of subtype C among HIV-1 infected population (51.10%), followed by circulating recombinant form (CRF)_07BC (15.46%), CRF_01AE (5.68%), A1 (4.73%), CRF_02AG (3.47%), G (3.15%), CRF_62BC (2.84%), B (2.21%), and other subtypes and recombinant forms in small percentages. Subtype C was largely predominant in intravenous drug users as well as female sex workers, whereas the migrant population exhibited a diverse subtype including rare recombinant forms, largely due to their travel in the Middle East and other South East Asian countries.
CONCLUSIONS: With the number of HIV-1 infections increasing among the general population and a steady increase in the migrant population, molecular epidemiological data are required to curb the progression of the HIV-1 epidemic in Bangladesh.

Hexavalent sulfoglycodendrimers (SGDs) are synthesized as mimics of host cell heparan sulfate proteoglycans (HSPGs) to inhibit the early stages in viral binding/entry of HIV-1 and SARS-CoV-2. Using an HIV neutralization assay, the most promising of the seven candidates are found to have sub-micromolar anti-HIV activities. Molecular dynamics simulations are separately implemented to investigate how/where the SGDs interacted with both pathogens. The simulations revealed that the SGDs: 1) develop multivalent binding with polybasic regions within and outside of the V3 loop on glycoprotein 120 (gp120) for HIV-1, and consecutively bind with multiple gp120 subunits, and 2) interact with basic amino acids in both the angiotensin-converting enzyme 2 (ACE2) and HSPG binding regions of the Receptor Binding Domain (RBD) from SARS-CoV-2. These results illustrate the considerable potential of SGDs as inhibitors in viral binding/entry of both HIV-1 and SARS-CoV-2 pathogens, leading the way for further development of this class of molecules as broad-spectrum antiviral agents.

HIV-1 entry requires the redistribution of envelope glycoproteins (Env) into a cluster and the presence of cholesterol (chol) in the viral membrane. However, the molecular mechanisms underlying the specific role of chol in infectivity and the driving force behind Env clustering remain unknown. Here, gp41 is demonstrated to directly interact with chol in the viral membrane via residues 751-854 in the cytoplasmic tail (CT751-854). Super-resolution stimulated emission depletion (STED) nanoscopy analysis of Env distribution further demonstrates that both truncation of gp41 CT751-854 and depletion of chol leads to dispersion of Env clusters in the viral membrane and inhibition of virus entry. This work reveals a direct interaction of gp41 CT with chol and indicates that this interaction is an important orchestrator of Env clustering.