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HIV-associated cardiovascular diseases remain a leading cause of death in people living with HIV/AIDS (PLWHA). Although antiretroviral drugs suppress the viral load, they fail to remove the virus entirely. HIV-1 Nef protein is known to play a role in viral virulence and HIV latency. Expression of Nef protein can be detected in different organs, including cardiac tissue. Despite the established role of Nef protein in HIV-1 replication, its impact on organ function inside the human body is not clear. To understand the effect of Nef at the organ level, we created a new Nef-transgenic (Nef-TG) mouse that expresses Nef protein in the heart. Our study found that Nef expression caused inhibition of cardiac function and pathological changes in the heart with increased fibrosis, leading to heart failure and early mortality. Further, we found that cellular autophagy is significantly inhibited in the cardiac tissue of Nef-TG mice. Mechanistically, we found that Nef protein causes the accumulation of Bcl2 and Beclin-1 proteins in the tissue, which may affect the cellular autophagy system. Additionally, we found Nef expression causes upregulation of the cellular senescence marker p21 and senescence-associated β-galactosidase expression. Our findings suggest that the Nef-mediated inhibition of autophagy and induction of senescence markers may promote aging in PLWHA. Our mouse model could help us to understand the effect of Nef protein on organ function during latent HIV infection. [ABSTRACT FROM AUTHOR]

CPG makes a great contribution to the control of gait motions in hexapod robots. To manage the gait movements of hexapod robots more efficiently and with lower energy consumption, this work introduced a Spiking-CPG(SCPG) neural network as the bionic control system for these robots. The system integrated Hopf oscillators with biologically inspired LIF neurons, which communicated through sparse spiking signals, in a ring topology. It was composed of six groups, each containing 2 000 LIF neurons, interconnected to form the network. This SCPG control system could produce common hexapod robot gaits such as the wave, tetrapod, and tripod gaits. It facilitated rapid, smooth, and stable transitions between motion gaits by adjusting phase difference parameters, and could dynamically adjust the required frequency and amplitude, exhibiting excellent robustness by quickly recovering from external disturbances. It controlled a simple 3D hexapod robot model on the Webots platform. It developed the robot by transforming the SCPG signal output through joint mapping functions, validating the motion stability of the designed hexapod robot and the feasibility and effectiveness of the SCPG control scheme. Finally, it ported the SCPG neural network controller proposed to Intel's Loihi chip, showing that it has high execution speed and lower energy consumption, indicating a promising prospect for application in the motion control of hexapod robots. [ABSTRACT FROM AUTHOR]

Blood–brain barrier (BBB) injury and dysfunction following infection with the human immunodeficiency virus (HIV) enables viral entry into the brain, infection of resident brain cells, neuronal injury and subsequent neurodegeneration leading to HIV-associated neurocognitive disorders (HAND). Although combination antiretroviral therapy has significantly reduced the incidence and prevalence of acquired immunodeficiency syndrome and increased the life expectancy of people living with HIV, the prevalence of HAND remains high. With aging of people living with HIV associated with increased comorbidities, the prevalence of HIV-related central nervous system (CNS) complications is expected to remain high. Considering the principal role of the brain endothelium in HIV infection of the CNS and HAND, the purpose of this manuscript is to review the current literature on the pathobiology of the brain endothelium structural and functional dysregulation in HIV infection, including in the presence of HIV-1 and viral proteins (gp120, Tat, Nef, and Vpr). We summarize evidence from human and animal studies, in vitro studies, and associated mechanisms. We further summarize evidence of synergy or lack thereof between commonly abused substances (cocaine, methamphetamine, alcohol, tobacco, opioids, and cannabinoids) and HIV- or viral protein-induced BBB injury and dysfunction. [ABSTRACT FROM AUTHOR]

A split-voltage amplifier architecture is proposed which improves the power efficiency compared to a conventional implementation. The approach is verified with a prototype fabricated in 0.35 µm CMOS technology using lateral bipolar input transistors. It achieves a measured DC gain of 105 V/V, a differential AC gain of 40.3 dB with a bandwidth of 55 kHz, a CMRR of approximately 75 dB, and a PSRR of 55 dB. The input-referred noise is 7 nV/√Hz and 923 nVrms integrated from 100 Hz to 10 kHz, resulting in a Noise Efficiency Factor (NEF) of 2.84 and a Power Efficiency Factor (PEF) of 18.3. The split-voltage configuration improves power efficiency by nearly 25% compared to a full voltage supply and maintains a small area design. Action potentials of the medial and lateral giant fiber of an earthworm are recorded as an example application. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The host transmembrane protein SERINC5 is incorporated into retrovirus particles and inhibits HIV-1 infectivity. The lentiviral Nef protein counteracts SERINC5 by downregulating it from the cell surface and preventing its incorporation into virions. The ability of Nef to antagonize the host factor varies in magnitude between different HIV-1 isolates. After having identified a subtype H nef allele unable to promote HIV-1 infectivity in the presence of SERINC5, we investigated the molecular determinants responsible for the defective counteraction of the host factor. Chimeric molecules with a subtype C Nef highly active against SERINC5 were constructed to locate Nef residues crucial for the activity against SERINC5. An Asn at the base of the C-terminal loop of the defective nef allele was found in place of a highly conserved acidic residue (D/E 150). The conversion of Asn to Asp restored the ability of the defective Nef to downregulate SERINC5 and promote HIV-1 infectivity. The substitution was also found to be crucial for the ability of Nef to downregulate CD4, but not for Nef activities that do not rely on the internalization of receptors from the cell surface, suggesting a general implication in promoting clathrin-mediated endocytosis. Accordingly, bimolecular fluorescence complementation revealed that the conserved acidic residue contributes to the recruitment of AP2 by Nef. Altogether, our results confirm that Nef downregulates SERINC5 and CD4 by engaging a similar machinery and indicates that, in addition to the di-leucine motif, other residues in the C-terminal flexible loop are important for the ability of the protein to sustain clathrin-mediated endocytosis.

Abstract HIV-associated neurocognitive disorders (HAND) are a spectrum of cognitive impairments that continue to affect approximately half of all HIV-positive individuals despite effective viral suppression through antiretroviral therapy (ART). White matter pathologies have persisted in the ART era, and the degree of white matter damage correlates with the degree of neurocognitive impairment in patients with HAND. The HIV protein Nef has been implicated in HAND pathogenesis, but its effect on white matter damage has not been well characterized. Here, utilizing in vivo, ex vivo, and in vitro methods, we demonstrate that Nef-containing extracellular vesicles (Nef EVs) disrupt myelin sheaths and inflict damage upon oligodendrocytes within the murine central nervous system. Intracranial injection of Nef EVs leads to reduced myelin basic protein (MBP) staining and a decreased number of CC1 + oligodendrocytes in the corpus callosum. Moreover, cerebellar slice cultures treated with Nef EVs exhibit diminished MBP expression and increased presence of unmyelinated axons. Primary mixed brain cultures and enriched oligodendrocyte precursor cell cultures exposed to Nef EVs display a decreased number of O4 + cells, indicative of oligodendrocyte impairment. These findings underscore the potential contribution of Nef EV-mediated damage to oligodendrocytes and myelin maintenance in the pathogenesis of HAND.

People living with HIV (PLWH) face a growing burden of chronic diseases, owing to the combinations of aging, environmental triggers, lifestyle choices, and virus-induced chronic inflammation. The rising incidence of pulmonary vascular diseases represents a major concern for PLWH. The study of HIV-associated pulmonary vascular complications ideally requires a strong understanding of pulmonary vascular cell biology and HIV pathogenesis at the molecular level for effective applications in infectious diseases and vascular medicine. Active HIV infection and/or HIV proteins disturb the delicate balance between vascular tone and constriction, which is pivotal for maintaining pulmonary vascular health. One of the defining features of HIV is its high genetic diversity owing to several factors including its high mutation rate, recombination between viral strains, immune selective pressures, or even geographical factors. The intrinsic HIV genetic diversity has several important implications for pathogenic outcomes of infection and the overall battle to combat HIV. Challenges in the field present themselves from two sides of the same coin: those imposed by the virus itself and those stemming from the host. The field may be advanced by further developing in vivo and in vitro models that are well described for both pulmonary vascular diseases and HIV for mechanistic studies. In essence, the study of HIV-associated pulmonary vascular complications requires a multidisciplinary approach, drawing upon insights from both infectious diseases and vascular medicine. In this review article, we discuss the fundamentals of HIV virology and their impact on pulmonary disease, aiming to enhance the understanding of either area or both simultaneously. Bridging the gap between preclinical research findings and clinical practice is essential for improving patient care. Addressing these knowledge gaps requires interdisciplinary collaborations, innovative research approaches, and dedicated efforts to prioritize HIV-related pulmonary complications on the global research agenda.

HIV-associated neurocognitive disorders (HAND) are a spectrum of cognitive impairments that continue to affect approximately half of all HIV-positive individuals despite effective viral suppression through antiretroviral therapy (ART). White matter pathologies have persisted in the ART era, and the degree of white matter damage correlates with the degree of neurocognitive impairment in patients with HAND. The HIV protein Nef has been implicated in HAND pathogenesis, but its effect on white matter damage has not been well characterized. Here, utilizing in vivo, ex vivo, and in vitro methods, we demonstrate that Nef-containing extracellular vesicles (Nef EVs) disrupt myelin sheaths and inflict damage upon oligodendrocytes within the murine central nervous system. Intracranial injection of Nef EVs leads to reduced myelin basic protein (MBP) staining and a decreased number of CC1 + oligodendrocytes in the corpus callosum. Moreover, cerebellar slice cultures treated with Nef EVs exhibit diminished MBP expression and increased presence of unmyelinated axons. Primary mixed brain cultures and enriched oligodendrocyte precursor cell cultures exposed to Nef EVs display a decreased number of O4 + cells, indicative of oligodendrocyte impairment. These findings underscore the potential contribution of Nef EV-mediated damage to oligodendrocytes and myelin maintenance in the pathogenesis of HAND. [ABSTRACT FROM AUTHOR]

An application specific integrated circuit (ASIC) and a custom-made microelectromechanical system (MEMS) sensor are presented, designed to function together as a sensor system for measuring low amplitude low frequency magnetic fields. The MEMS system comprises several free-standing double-wing magnetoelectric resonators with a size of $900~\mu $ m x $150~\mu $ m to measure alternating magnetic fields in the sub-kilohertz regime. It utilizes piezolelectric (AlN) and magnetostrictive (FeCoSiB) layers to exploit the delta-E effect for magnetic field sensing. On the ASIC a three-channel current-reuse amplifier with lateral bipolar transistors in the input stage is implemented occupying a chip area of 0.0864 mm2. Measurements demonstrate a voltage gain of 40 dB with a 3-dB bandwidth of 75 kHz and an input referred noise floor of 8 nV/ $\surd $ Hz while consuming $199~\mu $ W per channel. The sensor system is capable of detecting magnetic fields with a limit of detection (LOD) of 16 nT/ $\surd $ Hz for single sensor elements. By operating three sensor elements in parallel, one on each amplifier channel, the LOD is further reduced to 10 nT/ $\surd $ Hz. Owing to the high reproducibility of the sensor elements, this improvement in the LOD is close to the ideal value of $\surd 3$ . The results imply that the system can be scaled to large numbers of sensor elements without principle obstacles.

Epidemic Kaposi's sarcoma (KS), defined by co-infection with Human Herpes Virus 8 (HHV-8) and the Human Immunodeficiency Virus (HIV), is a major cause of mortality in sub-Saharan Africa. Antiretroviral therapy (ART) significantly reduces the risk of developing KS, and for those with KS, tumors frequently resolve with ART alone. However, for unknown reasons, a significant number of KS cases do not resolve and can progress to death. To explore how HIV responds to ART in the KS tumor microenvironment, we sequenced HIV env-nef found in DNA and RNA isolated from plasma, peripheral blood mononuclear cells, and tumor biopsies, before and after ART, in four Ugandan study participants who had unresponsive or progressive KS after 180-250 days of ART. We performed immunohistochemistry experiments to detect viral proteins in matched formalin-fixed tumor biopsies. Our sequencing results showed that HIV diversity and RNA expression in KS tumors are maintained after ART, despite undetectable plasma viral loads. The presence of spliced HIV transcripts in KS tumors after ART was consistent with a transcriptionally active viral reservoir. Immunohistochemistry staining found colocalization of HIV Nef protein and tissue-resident macrophages in the KS tumors. Overall, our results demonstrated that even after ART reduced plasma HIV viral load to undetectable levels and restored immune function, HIV in KS tumors continues to be transcriptionally and translationally active, which could influence tumor maintenance and progression.

Species living in spatially structured populations require a network of interconnected habitat patches. Due to changes in forest management, this network of habitat patches has been lost for insect species inhabiting open spots within forests. We studied two of the last populations of the Southern White Admiral (Limenitis reducta) in Germany. The aim of our study was to provide information for the conservation of this species. We conducted a capture-mark-recapture study over three consecutive years and we estimated population sizes and demographic parameters using Jolly-Seber and Cormack-Jolly-Seber models. Furthermore, we used different dispersal kernels to study the dispersal of L. reducta. We found that apparent survival rates differed greatly between the sexes. The mean residence times were eight to nine days for males and only two to four days for females. Apparent survival rates of both sexes decreased with increasing wing deterioration. Total population sizes of L. reducta varied between sites and years and ranged between 61 and 123. Daily abundances were generally low, especially that of females. The mean dispersal distance of individual recaptured butterflies increased in years with higher population densities, suggesting density-dependent dispersal. The dispersal data in our study was clearly bimodal, probably reflecting ‘routine movement’ at short distances and ‘displacement movement’ between habitat patches at longer distances. Consequently, the processes generating the dispersal data were better represented by the lognormal mixture model than by the negative exponential and the inverse power function. The mixed kernel predicted that about 9 % of the population disperses over > 1 km but that long-distance dispersal is rare. Our study highlights the urgent need for conservation measures to protect L. reducta in Germany and, based on our data, we recommend to create new habitat patches at distances of 1 to 1.5 km from existing habitats.

HIV-1 encodes four accesory proteins in addition to its structural and regulatory genes. Uniquely amongst them, Vpr is abundantly present within virions, meaning it is poised to exert various biological effects on the host cell upon delivery. In this way, Vpr contributes towards the establishment of a successful infection, as evidenced by the extent to which HIV-1 depends on this factor to achieve full pathogenicity in vivo. Although HIV infects various cell types in the host organism, CD4+ T cells are preferentially targeted since they are highly permissive towards productive infection, concomitantly bringing about the hallmark immune dysfunction that accompanies HIV-1 spread. The last several decades have seen unprecedented progress in unraveling the activities Vpr possesses in the host cell at the molecular scale, increasingly underscoring the importance of this viral component. Nevertheless, it remains controversial whether some of these advances bear in vivo relevance, since commonly employed cellular models significantly differ from primary T lymphocytes. One prominent example is the "established" ability of Vpr to induce G2 cell cycle arrest, with enigmatic physiological relevance in infected primary T lymphocytes. The objective of this review is to present these discoveries in their biological context to illustrate the mechanisms whereby Vpr supports HIV-1 infection in CD4+ T cells, whilst identifying findings that require validation in physiologically relevant models. [ABSTRACT FROM AUTHOR]

This paper investigates spiking neural networks (SNN) for novel robotic controllers with the aim of improving accuracy in trajectory tracking. By emulating the operation of the human brain through the incorporation of temporal coding mechanisms, SNN offer greater adaptability and efficiency in information processing, providing significant advantages in the representation of temporal information in robotic arm control compared to conventional neural networks. Exploring specific implementations of SNN in robot control, this study analyzes neuron models and learning mechanisms inherent to SNN. Based on the principles of the Neural Engineering Framework (NEF), a novel spiking PID controller is designed and simulated for a 3-DoF robotic arm using Nengo and MATLAB R2022b. The controller demonstrated good accuracy and efficiency in following designated trajectories, showing minimal deviations, overshoots, or oscillations. A thorough quantitative assessment, utilizing performance metrics like root mean square error (RMSE) and the integral of the absolute value of the time-weighted error (ITAE), provides additional validation for the efficacy of the SNN-based controller. Competitive performance was observed, surpassing a fuzzy controller by 5% in terms of the ITAE index and a conventional PID controller by 6% in the ITAE index and 30% in RMSE performance. This work highlights the utility of NEF and SNN in developing effective robotic controllers, laying the groundwork for future research focused on SNN adaptability in dynamic environments and advanced robotic applications. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) infection causes myelodysplasia, anemia, and accumulation of inflammatory monocytes (CD14+ CD16+) through largely unknown cellular and molecular pathways. The mouse cells thought to be equivalent to human CD14+ CD16+ cells are CD11b+ Gr1+ myeloid-derived suppressor cells (MDSC). We used HIV transgenic (Tg) mouse models to study MDSC, namely, CD4C/Nef Tg mice expressing nef in dendritic cells (DC), pDC, CD4+ T, and other mature and immature myeloid cells and CD11c/Nef Tg mice with a more restricted expression, mainly in DC and pDC. Both Tg strains showed expansion of granulocytic and CD11b+ Gr1low/int cells with MDSC characteristics. Fetal liver cell transplantation revealed that this expansion was stroma-independent and abrogated in mixed Tg/non-Tg 50% chimera. Tg bone marrow (BM) erythroid progenitors were decreased and myeloid precursors increased, suggesting an aberrant differentiation likely driving CD11b+ Gr1+ cell expansion, apparently cell autonomously in CD4C/Nef Tg mice and likely through a bystander effect in CD11c/Nef Tg mice. Hck was activated in Tg spleen, and Nef-mediated CD11b+ Gr1+ cell expansion was abrogated in Hck/Lyn-deficient Nef Tg mice, indicating a requirement of Hck/Lyn for this Nef function. IL-17 and granulocyte colony-stimulating factor (G-CSF) were elevated in Nef Tg mice. Increased G-CSF levels were normalized in Tg mice treated with anti-IL-17 antibodies. Therefore, Nef expression in myeloid precursors causes severe BM failure, apparently cell autonomously. More cell-restricted expression of Nef in DC and pDC appears sufficient to induce BM differentiation impairment, granulopoiesis, and expansion of MDSC at the expense of erythroid maturation, with IL-17→G-CSF as one likely bystander contributor. IMPORTANCE HIV-1 and SIV infection often lead to myelodysplasia, anemia, and accumulation of inflammatory monocytes (CD14+ CD16+), with the latter likely involved in neuroAIDS. We found that some transgenic (Tg) mouse models of AIDS also develop accumulation of mature and immature cells of the granulocytic lineage, decreased erythroid precursors, and expansion of MDSC (equivalent to human CD14+ CD16+ cells). We identified Nef as being responsible for these phenotypes, and its expression in mouse DC appears sufficient for their development through a bystander mechanism. Nef expression in myeloid progenitors may also favor myeloid cell expansion, likely in a cell-autonomous way. Hck/Lyn is required for the Nef-mediated accumulation of myeloid cells. Finally, we identified G-CSF under the control of IL-17 as one bystander mediator of MDSC expansion. Our findings provide a framework to determine whether the Nef>Hck/Lyn>IL-17>G-CSF pathway is involved in human AIDS and whether it represents a valid therapeutic target.

A split-voltage amplifier architecture is proposed which improves the power efficiency compared to a conventional implementation. The approach is verified with a prototype fabricated in 0.35 µm CMOS technology using lateral bipolar input transistors. It achieves a measured DC gain of 105 V/V, a differential AC gain of 40.3 dB with a bandwidth of 55 kHz, a CMRR of approximately 75 dB, and a PSRR of 55 dB. The input-referred noise is 7 nV/√Hz and 923 nVrms integrated from 100 Hz to 10 kHz, resulting in a Noise Efficiency Factor (NEF) of 2.84 and a Power Efficiency Factor (PEF) of 18.3. The split-voltage configuration improves power efficiency by nearly 25% compared to a full voltage supply and maintains a small area design. Action potentials of the medial and lateral giant fiber of an earthworm are recorded as an example application.

People living with human immunodeficiency virus (HIV-1) are at increased risk of developing cancer, such as Kaposi sarcoma (KS), non-Hodgkin lymphoma (NHL), cervical cancer, and other cancers associated with chronic viral infections. Traditionally, this is linked to HIV-1-induced immune suppression with depletion of CD4+ T-helper cells, exhaustion of lymphopoiesis and lymphocyte dysfunction. However, the long-term successful implementation of antiretroviral therapy (ART) with an early start did not preclude the oncological complications, implying that HIV-1 and its antigens are directly involved in carcinogenesis and may exert their effects on the background of restored immune system even when present at extremely low levels. Experimental data indicate that HIV-1 virions and single viral antigens can enter a wide variety of cells, including epithelial. This review is focused on the effects of five viral proteins: envelope protein gp120, accessory protein negative factor Nef, matrix protein p17, transactivator of transcription Tat and reverse transcriptase RT. Gp120, Nef, p17, Tat, and RT cause oxidative stress, can be released from HIV-1-infected cells and are oncogenic. All five are in a position to affect "innocent" bystander cells, specifically, to cause the propagation of (pre)existing malignant and malignant transformation of normal epithelial cells, giving grounds to the direct carcinogenic effects of HIV-1.

HIV-associated neurocognitive disorders (HAND) is a term describing a complex set of cognitive impairments accompanying HIV infection. Successful antiretroviral therapy (ART) reduces the most severe forms of HAND, but milder forms affect over 50% of people living with HIV (PLWH). Pathogenesis of HAND in the ART era remains unknown. A variety of pathogenic factors, such as persistent HIV replication in the brain reservoir, HIV proteins released from infected brain cells, HIV-induced neuroinflammation, and some components of ART, have been implicated in driving HAND pathogenesis in ART-treated individuals. Here, we propose another factor--impairment of cholesterol homeostasis and lipid rafts by HIV-1 protein Nef--as a possible contributor to HAND pathogenesis. These effects of Nef on cholesterol may also underlie the effects of other pathogenic factors that constitute the multifactorial nature of HAND pathogenesis. The proposed Nef- and cholesterol-focused mechanism may provide a long-sought unified explanation of HAND pathogenesis that takes into account all contributing factors. Evidence for the impairment by Nef of cellular cholesterol balance, potential effects of this impairment on brain cells, and opportunities to therapeutically target this element of HAND pathogenesis are discussed. [ABSTRACT FROM AUTHOR]

Let X be a complex smooth projective variety such that the exterior power of the tangent bundle ⋀r TX is nef for some 1 ≤ r < dim X. We prove that, up to a finite étale cover, X is a Fano fiber space over an Abelian variety. This gives a generalization of the structure theorem of varieties with nef tangent bundle by Demailly, Peternell and Schneider [5] and that of varieties with nef ⋀² TX by the author [20]. Our result also gives an answer to a question raised by Li, Ou and Yang [15] for varieties with strictly nef ⋀r TX when r < dim X. [ABSTRACT FROM AUTHOR]

• Apparent survival rates of male and female L. reducta differ greatly, male apparent survival is higher. • The populations of L. reducta in Germany consist of less than 130 adults, efficient habitat management is urgently needed. • The dispersal of L. reducta appears limited, only 9 % of the individuals disperse over more than 1 km. • Our study illustrates that mixture distributions provide great opportunities to fit butterfly dispersal data. Species living in spatially structured populations require a network of interconnected habitat patches. Due to changes in forest management, this network of habitat patches has been lost for insect species inhabiting open spots within forests. We studied two of the last populations of the Southern White Admiral (Limenitis reducta) in Germany. The aim of our study was to provide information for the conservation of this species. We conducted a capture-mark-recapture study over three consecutive years and we estimated population sizes and demographic parameters using Jolly-Seber and Cormack-Jolly-Seber models. Furthermore, we used different dispersal kernels to study the dispersal of L. reducta. We found that apparent survival rates differed greatly between the sexes. The mean residence times were eight to nine days for males and only two to four days for females. Apparent survival rates of both sexes decreased with increasing wing deterioration. Total population sizes of L. reducta varied between sites and years and ranged between 61 and 123. Daily abundances were generally low, especially that of females. The mean dispersal distance of individual recaptured butterflies increased in years with higher population densities, suggesting density-dependent dispersal. The dispersal data in our study was clearly bimodal, probably reflecting 'routine movement' at short distances and 'displacement movement' between habitat patches at longer distances. Consequently, the processes generating the dispersal data were better represented by the lognormal mixture model than by the negative exponential and the inverse power function. The mixed kernel predicted that about 9 % of the population disperses over > 1 km but that long-distance dispersal is rare. Our study highlights the urgent need for conservation measures to protect L. reducta in Germany and, based on our data, we recommend to create new habitat patches at distances of 1 to 1.5 km from existing habitats. [ABSTRACT FROM AUTHOR]

The polymorphisms in host genes such as CCR5, CCR2, stromal derived factor (SDF), and MBL (mannose-binding lectin) as well as the viral nef gene have been shown to influence human immunodeficiency virus (HIV) infection, followed by the development of HIV-associated neurocognitive disorder (HAND). In this preliminary study with a limited number of samples, we have tried to associate the genetic polymorphism from the host and viral genetic factors with the neurocognitive status along with immuno-virological parameters. The total RNA was isolated from 10 unlinked plasma samples containing 5 samples from each group with and without HAND based on the International HIV Dementia Scale (IHDS) score <9.5 and >9.5, respectively. The CCR5, CCR2, SDF, MBL, and HIV nef genes were amplified and digested with restriction enzymes, except for the nef gene amplicon. Restrictions fragment length polymorphism (RFLP) was used to determine whether allelic variations were present in the digested host gene products, while sequencing was done for HIV nef amplicons without digestion. CCR5 delta 32 heterozygous variants were present in two samples from the HAND group. Three samples with HAND showed SDF-1 3′ heterozygous allelic variant, while the MBL-2 gene presented with a homozygous mutant allele (D/D) in codon 52, heterozygous mutant allele (A/B) in codon 54, and codon 57 (A/C) for all samples except IHDS-2 irrespective of dementia status. Furthermore, amino acid alignment of Nef sequences confirmed the heterogeneity, while prediction of the human leukocyte antigen binding epitopes further explored its effect on functional motifs with variable binding efficiency such as epitopes GAFDLSFFL (aa 83) and LTFGWCFKL (aa 138) binding with HLA molecules at 60% and 80%, respectively. Thus, host genetics evidently influence predisposition to HIV infection and HAND. The genetic variability in the nef gene from both groups resulted in altering the functionality of specific domains and showing its impact on the progression of the disease, which needs to be explored. [ABSTRACT FROM AUTHOR]

Purpose of Review: This review aims to elucidate the multifaceted role of the tumor suppressor protein p53 in the context of HIV infection. We explore how p53, a pivotal regulator of cellular processes, interacts with various facets of the HIV life cycle. Understanding these interactions could provide valuable insights into potential therapeutic interventions and the broader implications of p53 in viral infections. Recent Findings: Recent research has unveiled a complex interplay between p53 and HIV. Several reports have highlighted the involvement of p53 in restricting the replication of HIV within both immune and nonimmune cells. Various mechanisms have been suggested to unveil how p53 enforces this restriction on HIV replication. However, HIV has developed strategies to manipulate p53, benefiting its replication and evading host defenses. Summary: In summary, p53 plays a multifaceted role in HIV infection, impacting viral replication and disease progression. Recent findings underscore the importance of understanding the intricate interactions between p53 and HIV for the development of innovative therapeutic approaches. Manipulating p53 pathways may offer potential avenues to suppress viral replication and ameliorate immune dysfunction, ultimately contributing to the management of HIV/AIDS. Further research is warranted to fully exploit the therapeutic potential of p53 in the context of HIV infection. [ABSTRACT FROM AUTHOR]

Nef is an HIV-encoded accessory protein that enhances pathogenicity by down-regulating major histocompatibility class I (MHC-I) expression to evade killing by cytotoxic T lymphocytes (CTLs). A potent Nef inhibitor that restores MHC-I is needed to promote immune-mediated clearance of HIV-infected cells. We discovered that the plecomacrolide family of natural products restored MHC-I to the surface of Nef-expressing primary cells with variable potency. Concanamycin A (CMA) counteracted Nef at subnanomolar concentrations that did not interfere with lysosomal acidification or degradation and were nontoxic in primary cell cultures. CMA specifically reversed Nef-mediated down-regulation of MHC-I, but not CD4, and cells treated with CMA showed reduced formation of the Nef:MHC-I:AP-1 complex required for MHC-I down-regulation. CMA restored expression of diverse allotypes of MHC-I in Nef-expressing cells and inhibited Nef alleles from divergent clades of HIV and simian immunodeficiency virus, including from primary patient isolates. Lastly, we found that restoration of MHC-I in HIV-infected cells was accompanied by enhanced CTL-mediated clearance of infected cells comparable to genetic deletion of Nef. Thus, we propose CMA as a lead compound for therapeutic inhibition of Nef to enhance immune-mediated clearance of HIV-infected cells.

The cellular protein SERINC5 inhibits the infectivity of diverse retroviruses, and its activity is counteracted by the glycosylated Gag (glycoGag) protein of murine leukemia virus (MLV), the S2 protein of equine infectious anemia virus (EIAV), and the Nef protein of human immunodeficiency virus type 1 (HIV-1). Determining the regions within SERINC5 that provide restrictive activity or Nef sensitivity should inform mechanistic models of the SERINC5/HIV-1 relationship. Here, we report that deletion of the conserved sequence EDTEE, which is located within a cytoplasmic loop of SERINC5 and which is reminiscent of an acidic-cluster membrane trafficking signal, increases the sensitivity of SERINC5 to antagonism by Nef, while it has no effect on the intrinsic activity of the protein as an inhibitor of infectivity. These effects correlated with enhanced removal of the ΔEDTEE mutant relative to that of wild-type SERINC5 from the cell surface and with enhanced exclusion of the mutant protein from virions by Nef. Mutational analysis indicated that the acidic residues, but not the threonine, within the EDTEE motif are important for the relative resistance to Nef. Deletion of the EDTEE sequence did not increase the sensitivity of SERINC5 to antagonism by the glycoGag protein of MLV, suggesting that its virologic role is Nef specific. These results are consistent with the reported mapping of the cytoplasmic loop that contains the EDTEE sequence as a general determinant of Nef responsiveness, but they further indicate that sequences inhibitory to as well as supportive of Nef activity reside in this region. We speculate that the EDTEE motif might have evolved to mediate resistance against retroviruses that use Nef-like proteins to antagonize SERINC5.IMPORTANCE Cellular membrane proteins in the SERINC family, especially SERINC5, inhibit the infectivity of retroviral virions. This inhibition is counteracted by retroviral proteins, specifically, HIV-1 Nef, MLV glycoGag, and EIAV S2. One consequence of such a host-pathogen "arms race" is a compensatory change in the host antiviral protein as it evolves to escape the effects of viral antagonists. This is often reflected in a genetic signature, positive selection, which is conspicuously missing in SERINC5 Here we show that despite this lack of genetic evidence, a sequence in SERINC5 nonetheless provides relative resistance to antagonism by HIV-1 Nef.

HIV-1-induced cytopathicity of thymocytes is a major cause of reduced peripheral T cells and rapid disease progression observed in HIV-1-infected infants. Understanding the virulence factors responsible for thymocyte depletion has paramount importance in addressing the pathogenesis of disease progression in children. In this study, thymocyte depletion was analyzed following infection with two primary CXCR4-tropic HIV-1 pediatric isolates (PI), PI-2 and PI-2.1, which were serially derived from an in utero-infected infant. Although highly similar to each other, PI-2 showed markedly decreased thymocyte depletion in vitro compared with PI-2.1. Further analysis showed a novel deletion in the Nef protein (NefΔK7S) of PI-2, which was absent in PI-2.1. This deletion inhibited Nef-mediated major histocompatibility complex class I (MHC-I) downregulation in infected thymocytes in vitro and in vivo; in contrast, the mutated Nef continued to downregulate CD4 surface expression in vitro. These results suggest that HIV-1 Nef contributes to thymic damage in infants through selective functions.

Apolipoprotein A-I binding protein (AIBP) is a protein involved in regulation of lipid rafts and cholesterol efflux. AIBP has been suggested to function as a protective factor under several sets of pathological conditions associated with increased abundance of lipid rafts, such as atherosclerosis and acute lung injury. Here, we show that exogenously added AIBP reduced the abundance of lipid rafts and inhibited HIV replication in vitro as well as in HIV-infected humanized mice, whereas knockdown of endogenous AIBP increased HIV replication. Endogenous AIBP was much more abundant in activated T cells than in monocyte-derived macrophages (MDMs), and exogenous AIBP was much less effective in T cells than in MDMs. AIBP inhibited virus-cell fusion, specifically targeting cells with lipid rafts mobilized by cell activation or Nef-containing exosomes. MDM-HIV fusion was sensitive to AIBP only in the presence of Nef provided by the virus or exosomes. Peripheral blood mononuclear cells from donors with the HLA-B*35 genotype, associated with rapid progression of HIV disease, bound less AIBP than cells from donors with other HLA genotypes and were not protected by AIBP from rapid HIV-1 replication. These results provide the first evidence for the role of Nef exosomes in regulating HIV-cell fusion by modifying lipid rafts and suggest that AIBP is an innate factor that restricts HIV replication by targeting lipid rafts.IMPORTANCE Apolipoprotein A-I binding protein (AIBP) is a recently identified innate anti-inflammatory factor. Here, we show that AIBP inhibited HIV replication by targeting lipid rafts and reducing virus-cell fusion. Importantly, AIBP selectively reduced levels of rafts on cells stimulated by an inflammatory stimulus or treated with extracellular vesicles containing HIV-1 protein Nef without affecting rafts on nonactivated cells. Accordingly, fusion of monocyte-derived macrophages with HIV was sensitive to AIBP only in the presence of Nef. Silencing of endogenous AIBP significantly upregulated HIV-1 replication. Interestingly, HIV-1 replication in cells from donors with the HLA-B*35 genotype, associated with rapid progression of HIV disease, was not inhibited by AIBP. These results suggest that AIBP is an innate anti-HIV factor that targets virus-cell fusion.

The accessory protein Nef of human immunodeficiency virus (HIV) is a primary determinant of viral pathogenesis. Nef is abundantly expressed during infection and reroutes a variety of cell surface proteins to disrupt host immunity and promote the viral replication cycle. Nef counteracts host defenses by sequestering and/or degrading its targets via the endocytic and secretory pathways. Nef does this by physically engaging a number of host trafficking proteins. Substantial progress has been achieved in identifying the targets of Nef, and a structural and mechanistic understanding of Nef's ability to command the protein trafficking machinery has recently started to coalesce. Comparative analysis of HIV and simian immunodeficiency virus (SIV) Nef proteins in the context of recent structural advances sheds further light on both viral evolution and the mechanisms whereby trafficking is hijacked. This review describes how advances in cell and structural biology are uncovering in growing detail how Nef subverts the host immune system, facilitates virus release, and enhances viral infectivity.

The plasma membrane is a site of conflict between host defenses and many viruses. One aspect of this conflict is the host's attempt to eliminate infected cells using innate and adaptive cell-mediated immune mechanisms that recognize features of the plasma membrane characteristic of viral infection. Another is the expression of plasma membrane-associated proteins, so-called restriction factors, which inhibit enveloped virions directly. HIV-1 encodes two countermeasures to these host defenses: The membrane-associated accessory proteins Vpu and Nef. In addition to inhibiting cell-mediated immune-surveillance, Vpu and Nef counteract membrane-associated restriction factors. These include BST-2, which traps newly formed virions at the plasma membrane unless counteracted by Vpu, and SERINC5, which decreases the infectivity of virions unless counteracted by Nef. Here we review key features of these two antiviral proteins, and we review Vpu and Nef, which deplete them from the plasma membrane by co-opting specific cellular proteins and pathways of membrane trafficking and protein-degradation. We also discuss other plasma membrane proteins modulated by HIV-1, particularly CD4, which, if not opposed in infected cells by Vpu and Nef, inhibits viral infectivity and increases the sensitivity of the viral envelope glycoprotein to host immunity.

Tetherin is a host defense factor that physically prevents virion release from the plasma membrane. The Nef accessory protein of simian immunodeficiency virus (SIV) engages the clathrin adaptor AP-2 to downregulate tetherin via its DIWK motif. As human tetherin lacks DIWK, antagonism of tetherin by Nef is a barrier to simian-human transmission of non-human primate lentiviruses. To determine the molecular basis for tetherin counteraction, we reconstituted the AP-2 complex with a simian tetherin and SIV Nef and determined its structure by cryoelectron microscopy (cryo-EM). Nef refolds the first α-helix of the β2 subunit of AP-2 to a β hairpin, creating a binding site for the DIWK sequence. The tetherin binding site in Nef is distinct from those of most other Nef substrates, including MHC class I, CD3, and CD4 but overlaps with the site for the restriction factor SERINC5. This structure explains the dependence of SIVs on tetherin DIWK and consequent barrier to human transmission.

Macroautophagy/autophagy is an evolutionarily conserved degradation system with fundamental biological functions. The activation of the class III phosphatidylinositol 3-kinase (PtdIns3K) complexes and the subsequent production of phosphatidylinositol 3-phosphate (PtdIns3P) are pivotal to autophagy. Using a combination of structural biology, biochemistry, and biophysics, we revealed how the non-catalytic subunit BECN1 serves as a membrane-binding switch in the regulation of PtdIns3K complexes and autophagy.

ABSTRACT HIV-1 evades antibody-dependent cellular cytotoxicity (ADCC) responses not only by controlling Env conformation and quantity at the cell surface but also by altering NK cell activation via the downmodulation of several ligands of activating and co-activating NK cell receptors. The signaling lymphocyte activation molecule (SLAM) family of receptors, which includes NTB-A and 2B4, act as co-activating receptors to sustain NK cell activation and cytotoxic responses. These receptors cooperate with CD16 (FcγRIII) and other activating receptors to trigger NK cell effector functions. In that context, Vpu-mediated downregulation of NTB-A on HIV-1-infected CD4 T cells was shown to prevent NK cell degranulation via an homophilic interaction, thus contributing to ADCC evasion. However, less is known on the capacity of HIV-1 to evade 2B4-mediated NK cell activation and ADCC. Here, we show that HIV-1 downregulates the ligand of 2B4, CD48, from the surface of infected cells in a Vpu-dependent manner. This activity is conserved among Vpu proteins from the HIV-1/SIVcpz lineage and depends on conserved residues located in its transmembrane domain and dual phosphoserine motif. We show that NTB-A and 2B4 stimulate CD16-mediated NK cell degranulation and contribute to ADCC responses directed to HIV-1-infected cells to the same extent. Our results suggest that HIV-1 has evolved to downmodulate the ligands of both SLAM receptors to evade ADCC. IMPORTANCE Antibody-dependent cellular cytotoxicity (ADCC) can contribute to the elimination of HIV-1-infected cells and HIV-1 reservoirs. An in-depth understanding of the mechanisms used by HIV-1 to evade ADCC might help develop novel approaches to reduce the viral reservoirs. Members of the signaling lymphocyte activation molecule (SLAM) family of receptors, such as NTB-A and 2B4, play a key role in stimulating NK cell effector functions, including ADCC. Here, we show that Vpu downmodulates CD48, the ligand of 2B4, and this contributes to protect HIV-1-infected cells from ADCC. Our results highlight the importance of the virus to prevent the triggering of the SLAM receptors to evade ADCC.

Animal models are essential for basic and clinical research on virus diseases. Humanized mice (mice reconstituted with human hematopoietic cells) have been effectively used for various virus studies as small animal models. Studies on human-tropic HIV-1 have also been performed using different humanized mouse models. Various humanized mice have been generated using distinct mouse strains and engraftment methods. These different techniques affect the reconstitution of human hematopoietic cells in individual mice, and in turn the HIV-1 replication in vivo. In this report, we describe the details of the generation method of humanized mice, i.e., severely immunodeficient mice (NSG mice) transplanted with human CD133-positive cells via intra-bone marrow injection (IBMI). It has been shown that the CD133-positive cells are highly capable to generate CD34-positive cells in vivo and IBMI is an excellent methodology for lymphoid and myeloid cell repopulation. In humanized mice transplanted with CD133-positive cells into the bone marrow, human lymphocytes were increased 3 months after the transplantation and a steady increase in CD4-positive cells was observed until 6-8 months after the transplantation. In order to test the utility of our system, CXCR4-tropic and CCR5-tropic HIV-1 clones were intraperitoneally inoculated into the resultant humanized mice 6-8 months after the transplantation. Upon inoculation at the same dose of viruses, the plasma viral load in CCR5-tropic HIV-1-inoculated mice peaked earlier than that in CXCR4-tropic HIV-1-inoculated mice (2-3 weeks vs 5-10 weeks postinoculation). While a rapid decrease in CD4-positive cells was observed at the peak or prior to the peak of viremia for CXCR4-tropic HIV-1-inoculated mice, CD4-positive cells were gradually decreased in CCR5-tropic HIV-1-inoculated mice. Upon inoculation at the same dose of viruses, a Nef-deleted R5-tropic HIV-1 exhibited retarded growth kinetics in the inoculated mice compared to the parental virus (around 8 weeks vs 2-3 weeks post-inoculation), which appears to reflect the decrease in replication potential in primary cells. Taken all together, in addition to the humanized mice reported so far, our humanized mice generated by transplanting CD133-positive cells with the IBMI method would be an appropriate prototype model for understanding HIV-1 biology in vivo. [ABSTRACT FROM AUTHOR]

Serine incorporator 5 (SERINC5 or SER5) is a multipass transmembrane protein with ill-defined cellular activities. SER5 was recently described as a human immunodeficiency virus 1 (HIV-1) restriction factor capable of inhibiting HIV-1 that does not express its accessory protein Nef (Δ Nef). SER5 incorporated into the viral membrane impairs the entry of HIV-1 by disrupting the fusion between the viral and the plasma membrane after envelope receptor interaction induced the first steps of the fusion process. The mechanisms of how SER5 prevents membrane fusion are not fully understood and viral envelope proteins were identified that escape the SER5-mediated restriction. Primate lentiviruses, such as HIV-1 and simian immunodeficiency viruses (SIVs), use their accessory protein Nef to downregulate SER5 from the plasma membrane by inducing an endocytic pathway. In addition to being directly antiviral, recent data suggest that SER5 is an important adapter protein in innate signaling pathways leading to the induction of inflammatory cytokines. This review discusses the current knowledge about HIV-1 restriction by SER5. [ABSTRACT FROM AUTHOR]

HIV-associated neurocognitive disorders (HAND) persist in the era of effective combined antiretroviral therapy (cART). A large body of literature suggests that mitochondrial dysfunction is a prospective etiology of HAND in the cART era. While viral load is often suppressed and the immune system remains intact in HIV+ patients on cART, evidence suggests that the central nervous system (CNS) acts as a reservoir for virus and low-level expression of viral proteins, which interact with mitochondria. In particular, the HIV proteins glycoprotein 120, transactivator of transcription, viral protein R, and negative factor have each been linked to mitochondrial dysfunction in the brain. Moreover, cART drugs have also been shown to have detrimental effects on mitochondrial function. Here, we review the evidence generated from human studies, animal models, and in vitro models that support a role for HIV proteins and/or cART drugs in altered production of adenosine triphosphate, mitochondrial dynamics, mitophagy, calcium signaling and apoptosis, oxidative stress, mitochondrial biogenesis, and immunometabolism in the CNS. When insightful, evidence of HIV or cART-induced mitochondrial dysfunction in the peripheral nervous system or other cell types is discussed. Lastly, therapeutic approaches to targeting mitochondrial dysfunction have been summarized with the aim of guiding new investigations and providing hope that mitochondrial-based drugs may provide relief for those suffering with HAND.

HIV-associated neurocognitive disorders (HAND) persist in the era of effective combined antiretroviral therapy (cART). A large body of literature suggests that mitochondrial dysfunction is a prospective etiology of HAND in the cART era. While viral load is often suppressed and the immune system remains intact in HIV+ patients on cART, evidence suggests that the central nervous system (CNS) acts as a reservoir for virus and low-level expression of viral proteins, which interact with mitochondria. In particular, the HIV proteins glycoprotein 120, transactivator of transcription, viral protein R, and negative factor have each been linked to mitochondrial dysfunction in the brain. Moreover, cART drugs have also been shown to have detrimental effects on mitochondrial function. Here, we review the evidence generated from human studies, animal models, and in vitro models that support a role for HIV proteins and/or cART drugs in altered production of adenosine triphosphate, mitochondrial dynamics, mitophagy, calcium signaling and apoptosis, oxidative stress, mitochondrial biogenesis, and immunometabolism in the CNS. When insightful, evidence of HIV or cART-induced mitochondrial dysfunction in the peripheral nervous system or other cell types is discussed. Lastly, therapeutic approaches to targeting mitochondrial dysfunction have been summarized with the aim of guiding new investigations and providing hope that mitochondrial-based drugs may provide relief for those suffering with HAND.

BackgroundHIV-associated neurocognitive disorder (HAND) is a neurodegenerative disease associated with persistent neuroinflammation and subsequent neuron damage. Pro-inflammatory factors and neurotoxins from activated astrocytes by HIV-1 itself and its encoded proteins, including the negative factor (Nef), are involved in the pathogenesis of HAND. This study was designed to find potential lncRNAs that regulate astrocyte functions and inflammation process.MethodsWe performed microarray analysis of lncRNAs from primary mouse astrocytes treated with Nef protein. Top ten lncRNAs were validated through real-time PCR analysis. Gene ontology (GO) and KEGG pathway analysis were applied to explore the potential functions of lncRNAs. RIP and ChIP assays were performed to demonstrate the mechanism of lncRNA regulating gene expression.ResultsThere were 638 co-upregulated lncRNAs and 372 co-downregulated lncRNAs in primary astrocytes treated with Nef protein for both 6 h and 12 h. GO and KEGG pathway analysis showed that the biological functions of top differential-expressed mRNAs were associated with inflammatory cytokines and chemokine. Knockdown of lncRNA AK006025, not AK138360, inhibited significantly CXCL9, CXCL10 (IP-10), and CXCL11 expression in astrocytes treated with Nef protein. Mechanism study showed that AK006025 associated with CBP/P300 was enriched in the promoter of CXCL9, CXCL10, and CXCL11 genes.ConclusionsOur findings uncovered the expression profiles of lncRNAs and mRNAs in vitro, which might help to understand the pathways that regulate astrocyte activation during the process of HAND.

SERINC5 is an inhibitor of retroviral infectivity that is counteracted by viral proteins, including HIV-1 Nef. Inhibition of infectivity by SERINC5 is associated with its incorporation into virions. Nef counteracts this inhibition, presumably by removing SERINC5 from sites of virion assembly at the plasma membrane. While evaluating the virion incorporation of SERINC5, we observed that a relatively high molecular weight form was preferentially present in virions. We used various glycosidases to establish that virion-associated SERINC5 is modified by N-linked, complex glycans, whereas the majority of SERINC5 in cells is of relatively low molecular weight and is modified by high-mannose glycans. Sequence alignment of SERINC family proteins led us to identify a conserved N-glycosylation site, N294, in SERINC5. We mutated this site to evaluate its effect on glycosylation, the restrictive activity of SERINC5, and the sensitivity of SERINC5 to antagonism by Nef. Our results demonstrate that N294 is the major site of N-glycosylation in SERINC5. Although N-glycosylation was required neither for restrictive activity nor for sensitivity to Nef per se, we observed a decrease in the steady-state expression of glycosylation-deficient SERINC5 (the N294A mutant) compared to the wild-type protein. Expression of this mutant was partly restored by treatment of cells with MG132 (a proteasome inhibitor) but not with bafilomycin A1 (a lysosomal inhibitor). We conclude that although not required for restrictive activity or Nef sensitivity, N-linked glycosylation is important for maintaining the steady-state expression of SERINC5 and that nonglycosylated SERINC5 is likely subjected to a quality control mechanism that induces its proteasomal degradation.IMPORTANCE SERINC5 is a member of a family of multipass transmembrane proteins that inhibit the infectivity of retroviruses, including HIV-1. These proteins are incorporated into virions and inhibit infection of target cells unless counteracted by viral antagonists such as HIV-1 Nef. The only other biological function with which these proteins have been associated is the formation of serine-containing membrane lipids. Here we show that SERINC5 is a glycosylated protein and that N-glycosylation is important for its steady-state expression. In the absence of N-glycosylation, SERINC5 is prone to proteasomal degradation. Nonetheless, N-glycosylation per se is required neither for the ability of SERINC5 to inhibit HIV-1 infectivity nor for its sensitivity to antagonism by Nef.

Objectives: HIV infection still remains a leading cause of morbidity and mortality worldwide. The inability of highly-active antiretroviral therapy in HIV-1 eradication led to development of therapeutic vaccines. Exploiting effective immunogenic constructs and potent delivery systems are important to generate effective therapeutic vaccines, and overcome their poor membrane permeability. Among HIV-1 proteins, the Nef and Vpr proteins can be considered as antigen candidates in vaccine design. Methods: In this study, the immunogenicity of Nef-Vpr antigen candidate in different regimens along with antimicrobial peptide LL-37 (as a DNA carrier) and Montanide 720 (as an adjuvant) was studied in mice. Moreover, the secretion of cytokines was assessed in virion-exposed mice lymphocytes in vitro. Results: Our data indicated that groups immunized with the homologous protein + Montanide regimen (group 1), and also the heterologous DNA + LL-37 prime/protein + Montanide boost regimen (group 2) could significantly generate strong immune responses as compared to groups immunized with the DNA constructs (groups 3 & 4). Moreover, immunization of mice with the homologous DNA + LL-37 regimen in low dose of DNA (5 µg) could induce higher immune responses than the homologous naked DNA regimen in high dose of DNA (50 µg) indicating the role of LL-37 as a cell penetrating peptide. Additionally, the heterologous DNA + LL-37 prime/protein + Montanide boost regimen (group 2) induced significantly IFN-gamma secretion from virion-exposed lymphocytes in vitro. Conclusion: Generally, the use of LL-37 for DNA delivery, Montanide 720 as an adjuvant, and heterologous DNA prime/protein boost strategy could significantly increase IgG2a, IFN-gamma, and Granzyme B, and maintain cytokine secretion after exposure to virions. Indeed, the heterologous DNA + LL-37 prime/protein + Montanide boost regimen can be considered as a potent strategy for development of therapeutic HIV vaccines. [ABSTRACT FROM AUTHOR]

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Nef hijacks the clathrin adaptor complex 2 (AP-2) to downregulate the viral receptor CD4 and the antiviral multipass transmembrane proteins SERINC3 and SERINC5, which inhibit the infectivity of progeny virions when incorporated. In Jurkat Tag T lymphoid cells lacking SERINC3 and SERINC5, Nef is no longer required for full progeny virus infectivity and for efficient viral replication. However, in MOLT-3 T lymphoid cells, HIV-1 replication remains highly dependent on Nef even in the absence of SERINC3 and SERINC5. Using a knockout (KO) approach, we now show that the Nef-mediated enhancement of HIV-1 replication in MOLT-3 cells does not depend on the Nef-interacting kinases LCK and PAK2. Furthermore, Nef substantially enhanced HIV-1 replication even in triple-KO MOLT-3 cells that simultaneously lacked the three Nef/AP-2 targets, SERINC3, SERINC5, and CD4, and were reconstituted with a Nef-resistant CD4 to permit HIV-1 entry. Nevertheless, the ability of Nef mutants to promote HIV-1 replication in the triple-KO cells correlated strictly with the ability to bind AP-2. In addition, knockdown and reconstitution experiments confirmed the involvement of AP-2. These observations raise the possibility that MOLT-3 cells express a novel antiviral factor that is downregulated by Nef in an AP-2-dependent manner. IMPORTANCE The HIV-1 Nef protein hijacks a component of the cellular endocytic machinery called AP-2 to downregulate the viral receptor CD4 and the antiviral cellular membrane proteins SERINC3 and SERINC5. In the absence of Nef, SERINC3 and SERINC5 are taken up into viral particles, which reduces their infectivity. Surprisingly, in a T cell line called MOLT-3, Nef remains crucial for HIV-1 spreading in the absence of SERINC3 and SERINC5. We now show that this effect of Nef also does not depend on the cellular signaling molecules and Nef interaction partners LCK and PAK2. Nef was required for efficient HIV-1 spreading even in triple-knockout cells that completely lacked Nef/AP-2-sensitive CD4, in addition to the Nef/AP-2 targets SERINC3 and SERINC5. Nevertheless, our results indicate that the enhancement of HIV-1 spreading by Nef in the triple-knockout cells remained AP-2 dependent, which suggests the presence of an unknown antiviral factor that is sensitive to Nef/AP-2-mediated downregulation.

Long interspersed element type 1 (LINE-1) is the only known type of retroelement that can replicate autonomously, and its retrotransposition activity can trigger interferon (IFN) production. IFN production suppresses the infectivity of exogenous viruses, such as human immunodeficiency virus (HIV). As a counteraction, HIV has been reported to use multiple proteins and mechanisms to suppress LINE-1 replication. However, the mechanisms of HIV-mediated LINE-1 regulation are not fully understood. In this study, we discovered that Nef protein, which is expressed by HIV and is important for HIV pathogenesis, inhibits LINE-1 retrotransposition. Two distinct mechanisms have been uncovered for Nef-induced LINE-1 suppression. Without direct interaction with LINE-1 DNA, Nef potently inhibits the promoter activity of the LINE-1 59-untranslated region (59-UTR) and reduces the expression levels of LINE-1 RNA and proteins. Alternatively, although Nef does not bind to the LINE-1 open reading frame 1 protein (ORF1p) or LINE-1 RNA, it significantly compromises the ORF1p-LINE-1 RNA interaction, which is essential for LINE-1 retrotransposition. Both mechanisms can be suppressed by the G2A mutation, which abolishes myristoylation of Nef, suggesting that membrane attachment is essential for Nef to suppress LINE1. Consequently, through LINE-1 inhibition, Nef downregulates IFN production in host cells. Therefore, our data revealed that Nef is a potent LINE-1 suppressor and an effective innate immune regulator, which not only provides new information on the intricate interaction between HIV, LINE-1, and IFN signaling systems but also strengthens the importance of Nef in HIV infection and highlights the potential of designing novel Nef-targeting anti-HIV drugs. IMPORTANCE Human immunodeficiency viruses are pathogens of AIDS that were first discovered almost 40 years ago and continue to threaten human lives to date. While currently used anti-HIV drugs are sufficient to suppress viral loads in HIV-infected patients, both drug-resistant HIV strains and adverse side effects triggered by the long-term use of these drugs highlight the need to develop novel anti-HIV drugs targeting different viral proteins and/or different steps in viral replication. To achieve this, more information is required regarding HIV pathogenesis and especially its impact on cellular activities in host cells. In this study, we discovered that the Nef protein expressed by HIV potently inhibits LINE-1 retrotransposition. During our attempt to determine the mechanism of Nef-mediated LINE-1 suppression, two additional functions of Nef were uncovered. Nef effectively repressed the promoter activity of LINE-1 59-UTR and destabilized the interaction between ORF1p and LINE-1 RNA. Consequently, Nef not only compromises LINE-1 replication but also reduces LINE-1-triggered IFN production. The reduction in IFN production, in theory, promotes HIV infectivity. Together with its previously known functions, these findings indicate that Nef is a potential target for the development of novel antiHIV drugs. Notably, the G2 residue, which has been reported to be essential for most Nef functions, was found to be critical in the regulation of innate immune activation by Nef, suggesting that compromising myristoylation or membrane attachment of Nef may be a good strategy for the inhibition of HIV infection. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus-1 (HIV-1) infection leads to the development of acquired immunodeficiency syndrome (AIDS). To establish a productive infection, HIV-1 hijacks the cellular machinery and modulates various physiological processes to propagate itself. The pathways altered by HIV-1 include cell cycle, autophagy, apoptosis, cell stress pathways, immune response, antiviral response, etc. Zipper interacting protein kinase (ZIPK) is a member of the death-associated protein kinase (DAPK) family of proteins, known to be one of the key regulators of cell death and cell survival pathways. ZIPK is also involved in regulating many cellular processes that are altered during HIV-1 infection; thus, we have explored the functional role of ZIPK in HIV-1 infection. Our results show that ZIPK protein expression is downregulated during HIV-1 infection in Nef dependent manner. Overexpression of ZIPK leads to downregulation in LTR-driven gene expression and virus production, whereas ZIPK knockdown induces viral gene expression and replication. HIV-1 promoter activity is reportedly enhanced by Nef-mediated activation of some transcription factors like NFκB and STAT3. ZIPK is reported to inhibit the STAT3 activity by phosphorylating it at ser-727. Our results show that STAT3 (ser-727) phosphorylation is decreased upon overexpression of Nef with simultaneous downregulation of ZIPK expression. We finally show that HIV-1 Nef interacts with ZIPK and induces its proteasomal degradation. Overall, our data suggests that Nef is involved in downregulation of ZIPK thereby increasing the virus production through rescue of STAT3 activity. • ZIPK negatively regulates HIV-1 gene expression and replication. • ZIPK suppresses the activity of STAT3 by phosphorylating it at ser-727. • HIV-1 Nef interacts with ZIPK and induces its proteasomal degradation. • Nef increases viral replication by restricting the antiviral activity of ZIPK. [ABSTRACT FROM AUTHOR]

ObjectiveTo address the role of translationally active HIV reservoir in chronic inflammation and non-AIDS related disorders, we first need a simple and accurate assay to evaluate viral protein expression in virally suppressed subjects.DesignWe optimized an HIV Nef enzyme-linked immunosorbent assay (ELISA) and used it to quantify plasma Nef levels as an indicator of the leaky HIV reservoir in an HIV-infected cohort.MethodsThis study accessed 134 plasma samples from a well-characterized cohort study of HIV-infected and uninfected adults in San Francisco (the SCOPE cohort). We optimized an ELISA for detection of plasma Nef in HIV-negative subjects and HIV-infected non-controllers, and evaluated its utility to quantify plasma Nef levels in a cross-sectional study of ART-suppressed and elite controller HIV-infected subjects.ResultsHere, we describe the performance of an optimized HIV Nef ELISA. When we applied this assay to the study cohort we found that plasma Nef levels were correlated with plasma HIV RNA levels in untreated disease. However, we were able to detect Nef in plasma of approximately half of subjects on ART or with elite control, despite the lack of detectable plasma HIV RNA levels using standard assays. Plasma Nef levels were not consistently associated with CD4+ T-cell count, CD8+ T-cell count, self-reported nadir CD4+ T-cell count or the CD4+/CD8+ T-cell ratio in HIV-infected subjects.ConclusionSince plasma HIV RNA levels are undetectable in virally suppressed subjects, it is reasonable to assume that viral protein expression in leaky reservoir, and not plasma virions, is the source of Nef accumulating in plasma. To examine this further, improvements of the assay sensitivity, by lowering the background through improvements in the quality of Nef antibodies, and detailed characterization of the HIV reservoirs are needed.

Patient-derived HIV-1 subtype B Nef clones downregulate HLA-A more efficiently than HLA-B. However, it remains unknown whether this property is common to Nef proteins across primate lentiviruses and how antiviral immune responses may be affected. We examined 263 Nef clones from diverse primate lentiviruses including different pandemic HIV-1 group M subtypes for their ability to downregulate major histocompatibility complex class A (MHC-A) and MHC-B from the cell surface. Though lentiviral Nef proteins differed markedly in their absolute MHC-A and MHC-B downregulation abilities, all lentiviral Nef lineages downregulated MHC-A, on average, 11 to 32% more efficiently than MHC-B. Nef genotype/phenotype analyses in a cohort of HIV-1 subtype C-infected patients (n = 168), together with site-directed mutagenesis, revealed Nef position 9 as a subtype-specific determinant of differential HLA-A versus HLA-B downregulation activity. Nef clones harboring nonconsensus variants at codon 9 downregulated HLA-B (though not HLA-A) significantly better than those harboring the consensus sequence at this site, resulting in reduced recognition of infected target cells by HIV-1-specific CD8+ effector cells in vitro Among persons expressing protective HLA class I alleles, carriage of Nef codon 9 variants was also associated with reduced ex vivo HIV-specific T cell responses. Our results demonstrate that Nef's inferior ability to downregulate MHC-B compared to that of MHC-A is conserved across primate lentiviruses and suggest that this property influences antiviral cellular immune responses.IMPORTANCE Primate lentiviruses encode the Nef protein that plays an essential role in establishing persistent infection in their respective host species. Nef interacts with the cytoplasmic region of MHC-A and MHC-B molecules and downregulates them from the infected cell surface to escape recognition by host cellular immunity. Using a panel of Nef alleles isolated from diverse primate lentiviruses including pandemic HIV-1 group M subtypes, we demonstrate that Nef proteins across all lentiviral lineages downregulate MHC-A approximately 20% more effectively than MHC-B. We further identify a naturally polymorphic site at Nef position 9 that contributes to the MHC-B downregulation function in HIV-1 subtype C and show that carriage of Nef variants with enhanced MHC-B downregulation ability is associated with reduced breadth and magnitude of MHC-B-restricted cellular immune responses in HIV-infected individuals. Our study underscores an evolutionarily conserved interaction between lentiviruses and primate immune systems that may contribute to pathogenesis.

Abstract Background Autophagy plays an important role as a cellular defense mechanism against intracellular pathogens, like viruses. Specifically, autophagy orchestrates the recruitment of specialized cargo, including viral components needed for replication, for lysosomal degradation. In addition to this primary role, the cleavage of viral structures facilitates their association with pattern recognition receptors and MHC-I/II complexes, which assists in the modulation of innate and adaptive immune responses against these pathogens. Importantly, whereas autophagy restricts the replicative capacity of human immunodeficiency virus type 1 (HIV-1), this virus has evolved the gene nef to circumvent this process through the inhibition of early and late stages of the autophagy cascade. Despite recent advances, many details of the mutual antagonism between HIV-1 and autophagy still remain unknown. Here, we uncover the genetic determinants that drive the autophagy-mediated restriction of HIV-1 as well as the counteraction imposed by Nef. Additionally, we also examine the implications of autophagy antagonism in HIV-1 infectivity. Results We found that sustained activation of autophagy potently inhibits HIV-1 replication through the degradation of HIV-1 Gag, and that this effect is more prominent for nef-deficient viruses. Gag re-localizes to autophagosomes where it interacts with the autophagosome markers LC3 and SQSTM1. Importantly, autophagy-mediated recognition and recruitment of Gag requires the myristoylation and ubiquitination of this virus protein, two post-translational modifications that are essential for Gag’s central role in virion assembly and budding. We also identified residues T48 and A49 in HIV-1 NL4-3 Nef as responsible for impairing the early stages of autophagy. Finally, a survey of pandemic HIV-1 transmitted/founder viruses revealed that these isolates are highly resistant to autophagy restriction. Conclusions This study provides evidence that autophagy antagonism is important for virus replication and suggests that the ability of Nef to counteract autophagy may have played an important role in mucosal transmission. Hence, disabling Nef in combination with the pharmacological manipulation of autophagy represents a promising strategy to prevent HIV spread.

While antiretroviral drugs have transformed the lives of HIV-infected individuals, chronic treatment is required to prevent rebound from viral reservoir cells. People living with HIV also are at higher risk for cardiovascular and neurocognitive complications, as well as cancer. Finding a cure for HIV-1 infection is therefore an essential goal of current AIDS research. This review is focused on the discovery of pharmacological inhibitors of the HIV-1 Nef accessory protein. Nef is well known to enhance HIV-1 infectivity and replication, and to promote immune escape of HIV-infected cells by preventing cell surface MHC-I display of HIV-1 antigens. Recent progress shows that Nef inhibitors not only suppress HIV-1 replication, but also restore sufficient MHC-I to the surface of infected cells to trigger a cytotoxic T lymphocyte response. Combining Nef inhibitors with latency reversal agents and therapeutic vaccines may provide a path to clearance of viral reservoirs. [ABSTRACT FROM AUTHOR]

Findings that certain infections induce immunity not only against the causing agent, but also against an unrelated pathogen have intrigued investigators for many years. Recently, underlying mechanisms of this phenomenon have started to come to light. It was found that the key cells responsible for heterologous protection are innate immune cells such as natural killer cells (NKs), dendritic cells, and monocytes/macrophages. These cells are ‘primed’ by initial infection, allowing them to provide enhanced response to subsequent infection by the same or unrelated agent. This phenomenon of innate immune memory was termed ‘trained immunity’. The proposed mechanism for trained immunity involves activation by the first stimulus of metabolic pathways that lead to epigenetic changes, which maintain the cell in a “trained” state, allowing enhanced responses to a subsequent stimulus. Innate immune memory can lead either to enhanced responses or to suppression of subsequent responses (‘tolerance’), depending on the strength and length of the initial stimulation of the immune cells. In the context of HIV infection, innate memory induced by infection is not well understood. In this Hypothesis and Theory article, we discuss evidence for HIV-induced trained immunity in human monocytes, its possible mechanisms, and implications for HIV-associated co-morbidities. [ABSTRACT FROM AUTHOR]