Disruption of Transmembrane Phosphatidylserine Asymmetry by HIV-1 Incorporated SERINC5 Is Not Responsible for Virus Restriction.

Host restriction factor SERINC5 (SER5) incorporates into the HIV-1 membrane and inhibits infectivity by a poorly understood mechanism. Recently, SER5 was found to exhibit scramblase-like activity leading to the externalization of phosphatidylserine (PS) on the viral surface, which has been proposed to be responsible for SER5's antiviral activity. This and other reports that document modulation of HIV-1 infectivity by viral lipid composition prompted us to investigate the role of PS in regulating SER5-mediated HIV-1 restriction. First, we show that the level of SER5 incorporation into virions correlates with an increase in PS levels in the outer leaflet of the viral membrane. We developed an assay to estimate the PS distribution across the viral membrane and found that SER5, but not SER2, which lacks antiviral activity, abrogates PS asymmetry by externalizing this lipid. Second, SER5 incorporation diminished the infectivity of pseudoviruses produced from cells lacking a flippase subunit CDC50a and, therefore, exhibited a higher baseline level of surface-accessible PS. Finally, exogenous manipulation of the viral PS levels utilizing methyl-alpha-cyclodextrin revealed a lack of correlation between external PS and virion infectivity. Taken together, our study implies that the increased PS exposure to SER5-containing virions itself is not directly linked to HIV-1 restriction.

IFITM1 and IFITM3 Proteins Inhibit the Infectivity of Progeny HIV-1 without Disrupting Envelope Glycoprotein Clusters.

Human interferon-induced transmembrane (IFITM) proteins inhibit the fusion of a broad spectrum of enveloped viruses, both when expressed in target cells and when present in infected cells. Upon expression in infected cells, IFITMs incorporate into progeny virions and reduce their infectivity by a poorly understood mechanism. Since only a few envelope glycoproteins (Envs) are present on HIV-1 particles, and Env clustering has been proposed to be essential for optimal infectivity, we asked if IFITM protein incorporation modulates HIV-1 Env clustering. The incorporation of two members of the IFITM family, IFITM1 and IFITM3, into HIV-1 pseudoviruses correlated with a marked reduction of infectivity. Super-resolution imaging of Env distribution on single HIV-1 pseudoviruses did not reveal significant effects of IFITMs on Env clustering. However, IFITM3 reduced the Env processing and incorporation into virions relative to the control and IFITM1-containing viruses. These results show that, in addition to interfering with the Env function, IFITM3 restricts HIV-1 Env cleavage and incorporation into virions. The lack of notable effect of IFITMs on Env clustering supports alternative restriction mechanisms, such as modification of the properties of the viral membrane.

Human Immunodeficiency Virus 1 Preferentially Fuses with pH-Neutral Endocytic Vesicles in Cell Lines and Human Primary CD4+ T-Cells.

Despite extensive efforts, the principal sites of productive HIV-1 entry in different target cells─plasma membrane (PM) vs endosomes─remain controversial. To delineate the site(s) of HIV-1 fusion, we implemented a triple labeling approach that involves tagging pseudoviruses with the fluid-phase viral content marker, iCherry, the viral membrane marker, DiD, and the extraviral pH sensor, ecliptic pHluorin. The viral content marker iCherry is released into the cytoplasm upon virus-cell fusion irrespective of the sites of fusion. In contrast, the extent of dilution of the membrane marker upon fusion with the PM (loss of signal) vs the endosomal membrane (no change in punctate DiD appearance) discriminates between the principal sites of viral fusion. Additionally, ecliptic pHluorin incorporated into the viral membrane reports whether virus fusion occurs in acidic endosomes. Real-time single virus imaging in living HeLa-derived cells, a CD4+ T-cell line, and activated primary human CD4+ T-cells revealed a strong (80-90%) HIV-1 preference for fusion with endosomes. Intriguingly, we observed HIV-1 fusion only with pH-neutral intracellular vesicles and never with acidified endosomes. These endocytic fusion events are likely culminating in productive infection since endocytic inhibitors, such as EIPA, Pitstop2, and Dynasore, as well as a dominant-negative dynamin-2 mutant, inhibited HIV-1 infection in HeLa-derived and primary CD4+ T-cells. Furthermore, the inhibition of endocytosis in HeLa-derived cells promoted hemifusion at the PM but abrogated complete fusion. Collectively, these data reveal that the primary HIV-1 entry pathway in diverse cell types is through fusion with pH-neutral intracellular vesicles.

Cyclosporines Antagonize the Antiviral Activity of IFITMProteins by Redistributing Them toward the Golgi Apparatus.

Interferon-induced transmembrane proteins (IFITMs) block the fusion of diverse enveloped viruses, likely through increasing the cell membrane's rigidity. Previous studies have reported that the antiviral activity of the IFITM family member, IFITM3, is antagonized by cell pretreatment with rapamycin derivatives and cyclosporines A and H (CsA and CsH) that promote the degradation of IFITM3. Here, we show that CsA and CsH potently enhance virus fusion with IFITM1- and IFITM3-expressing cells by inducing their rapid relocalization from the plasma membrane and endosomes, respectively, towards the Golgi. This relocalization is not associated with a significant degradation of IFITMs. Although prolonged exposure to CsA induces IFITM3 degradation in cells expressing low endogenous levels of this protein, its levels remain largely unchanged in interferon-treated cells or cells ectopically expressing IFITM3. Importantly, the CsA-mediated redistribution of IFITMs to the Golgi occurs on a much shorter time scale than degradation and thus likely represents the primary mechanism of enhancement of virus entry. We further show that rapamycin also induces IFITM relocalization toward the Golgi, albeit less efficiently than cyclosporines. Our findings highlight the importance of regulation of IFITM trafficking for its antiviral activity and reveal a novel mechanism of the cyclosporine-mediated modulation of cell susceptibility to enveloped virus infection.

Rolosense: Mechanical detection of SARS-CoV-2 using a DNA-based motor.

Assays detecting viral infections play a significant role in limiting the spread of diseases such as SARS-CoV-2. Here we present Rolosense, a virus sensing platform that transduces the motion of synthetic DNA-based motors transporting 5-micron particles on RNA fuel chips. Motors and chips are modified with virus-binding aptamers that lead to stalling of motion. Therefore, motors perform a "mechanical test" of viral target and stall in the presence of whole virions which represents a unique mechanism of transduction distinct from conventional assays. Rolosense can detect SARS-CoV-2 spiked in artificial saliva and exhaled breath condensate with a sensitivity of 103 copies/mL and discriminates among other respiratory viruses. The assay is modular and amenable to multiplexing, as we demonstrated one-pot detection of influenza A and SARS-CoV-2. As a proof-of-concept, we show readout can be achieved using a smartphone camera in as little as 15 mins without any sample preparation steps. Taken together, mechanical detection using Rolosense can be broadly applied to any viral target and has the potential to enable rapid, low-cost, point-of-care screening of circulating viruses.

Copper(II) Oxide Nanoparticles Embedded within a PEDOT Matrix for Hydrogen Peroxide Electrochemical Sensing.

The aim of this study is the preparation of nanostructured copper(II) oxide-based materials (CuONPs) through a facile additive-free polyol procedure that consists of the hydrolysis of copper(II) acetate in 1,4-butane diol and its application in hydrogen peroxide sensing. The nonenzymatic electrochemical sensor for hydrogen peroxide determination was constructed by drop casting the CuONP sensing material on top of a glassy carbon electrode (GCE) modified by a layer of poly(3,4-ethylenedioxythiophene) conducting polymer (PEDOT). The PEDOT layer was prepared on GCE using the sinusoidal voltage method. The XRD pattern of the CuONPs reveals the formation of the monoclinic tenorite phase, CuO, with average crystallite sizes of 8.7 nm, while the estimated band gap from UV-vis spectroscopy is of 1.2 eV. The SEM, STEM, and BET analyses show the formation of quasi-prismatic microaggregates of nanoparticles, with dimensions ranging from 1 µm up to ca. 200 µm, with a mesoporous structure. The developed electrochemical sensor exhibited a linear response toward H2O2 in the concentration range from 0.04 to 10 mM, with a low detection limit of 8.5 µM of H2O2. Furthermore, the obtained sensor possessed an excellent anti-interference capability in H2O2 determination in the presence of interfering compounds such as KNO3 and KNO2.

SERINC5-Mediated Restriction of HIV-1 Infectivity Correlates with Resistance to Cholesterol Extraction but Not with Lipid Order of Viral Membrane.

Serine incorporator 5 (SER5) is a protein that upon incorporation into virions inhibits HIV-1 infectivity by interfering with the ability of the Env glycoprotein to promote viral fusion. The mechanisms by which SER5 antagonizes HIV-1 fusion are not well understood. A recent study of SER5's structure revealed a lipid-binding pocket, suggesting the ability to sequester lipids. This finding, along with the well-documented modulation of HIV-1 infectivity by viral lipids, especially cholesterol, prompted our examination of SER5's effect on the general lipid order of the HIV-1 membrane. Pseudoviruses bearing the SER5-sensitive HXB2-Env and containing SER5 or SER2, a control protein that lacks antiviral activity, were analyzed using two distinct lipid-order probes. We show that SER5 incorporation does not noticeably affect the lipid order of pseudoviruses. Although viral cholesterol extraction reduces HIV-1 infectivity, SER5+ viruses are less sensitive to cholesterol extraction than the control samples. In contrast, the virus' sensitivity to cholesterol oxidation was not affected by SER5 incorporation. The hydrolytic release of sphingomyelin-sequestered cholesterol had a minimal impact on the apparent resistance to cholesterol extraction. Based on these results, we propose that a subpopulation of more stable Env glycoproteins responsible for the residual infectivity of SER5+ viruses is less sensitive to the cholesterol content of the viral membrane.

SERINC5 Restricts HIV-1 Infectivity by Promoting Conformational Changes and Accelerating Functional Inactivation of Env.

SERINC5 incorporates into HIV-1 particles and inhibits the ability of Env glycoprotein to mediate virus-cell fusion. SERINC5-resistance maps to Env, with primary isolates generally showing greater resistance than laboratory-adapted strains. Here, we examined a relationship between the inhibition of HIV-1 infectivity and the rate of Env inactivation using a panel of SERINC5-resistant and -sensitive HIV-1 Envs. SERINC5 incorporation into pseudoviruses resulted in a faster inactivation of sensitive compared to resistant Env strains. A correlation between fold reduction in infectivity and the rate of inactivation was also observed for multiple Env mutants known to stabilize and destabilize the closed Env structure. Unexpectedly, most mutations disfavoring the closed Env conformation rendered HIV-1 less sensitive to SERINC5. In contrast, functional inactivation of SERINC5-containing viruses was significantly accelerated in the presence of a CD4-mimetic compound, suggesting that CD4 binding sensitizes Env to SERINC5. Using a small molecule inhibitor that selectively targets the closed Env structure, we found that, surprisingly, SERINC5 increases the potency of this compound against a laboratory-adapted Env which prefers a partially open conformation, indicating that SERINC5 may stabilize the closed trimeric Env structure. Our results reveal a complex effect of SERINC5 on Env conformational dynamics that promotes Env inactivation and is likely responsible for the observed restriction phenotype.

Marine and Agro-Industrial By-Products Valorization Intended for Topical Formulations in Wound Healing Applications.

Over the past years, research attention has been focusing more on waste-derived, naturally derived, and renewable materials, in the view of a more sustainable economy. In this work, different topical formulations were obtained from the valorization of marine and agro-industrial by-products and the use of Carbopol 940 as gelling agent. In particular, the combination of extracts obtained from the marine snail, Rapanosa venosa, with Cladophora vagabunda and grape pomace extracts, was investigated for wound healing purposes. Rapana venosa has demonstrated wound healing properties and antioxidant activity. Similarly, grape pomace extracts have been shown to accelerate the healing process. However, their synergic use has not been explored yet. To this aim, four different formulations were produced. Three formulations differed for the presence of a different extract of Rapana venosa: marine collagen, marine gelatin, and collagen hydrolysate, while another formulation used mammalian gelatin as further control. Physico-chemical properties of the extracts as well as of the formulations were analyzed. Furthermore, thermal stability was evaluated by thermogravimetric analysis. Antioxidant capacity and biological behavior, in terms of cytocompatibility, wound healing, and antimicrobial potential, were assessed. The results highlighted for all the formulations (i) a good conservation and thermal stability in time, (ii) a neutralizing activity against free radicals, (iii) and high degree of cytocompatibility and tissue regeneration potential. In particular, collagen, gelatin, and collagen hydrolysate obtained from the Rapana venosa marine snail represent an important, valuable alternative to mammalian products.

A prática de atividade física por acadêmicos de medicina durante a pandemia causada pelo SARS-CoV-2: um estudo transversal / The practice of physical activity of medical students during the pandemic caused by SARS-CoV-2: a cross-sectional study

Introdução: o contexto de pandemia instaurada pelo SARS-CoV-2 acarretou um cenário de isolamento social, dificultando a prática de atividade física regular. Somado a isso, os estudantes de Medicina ainda possuem uma carga horária sobrecarregada. O objetivo do trabalho foi conhecer a qualidade de vida e a prevalência da prática de atividade física e seus efeitos durante o período de pandemia em acadêmicos de Medicina. Métodos: estudo observacional transversal realizado a partir da aplicação do questionário "Prática de atividade física por acadêmicos de Medicina durante a pandemia" em 286 estudantes de ambos os sexos, de todos os períodos de uma faculdade. Resultados: notou-se que os participantes, cursando Medicina com ensino remoto, que tinham mais motivação e tempo livre praticavam mais atividade física, enquanto os participantes que tinham menos motivação e tempo livre diminuíram a prática no período analisado. Discussão: Os estudantes compreendem que a prática de atividade física é benéfica, mesmo ela sendo impedida por cargas horárias extenuantes, até mesmo devido à educação que recebem durante a graduação. Conclusão: constatou-se que acadêmicos de Medicina que possuem aulas de educação remota durante o período estabelecido de isolamento social, consideraram apresentar mais tempo livre para a prática de atividade física. Entretanto, nem todos dedicaram esse tempo para a realização de exercícios [au]

Introduction: the context of the SARS-CoV-2 pandemic led to a scenario of social isolation, hindering the practice of regular physical activity. In addition, medical students still have a very high workload. The objective of this study was to assess the quality of life and the prevalence of physical activity and its effects among medical students during the pandemic period. Methods: cross-sectional observational study conducted through the application of the questionnaire "Engagement in physical activity among medical students during the pandemic" in 286 students of both genders, from all course periods. Results: it was found that the participants on remote medical training who had more motivation and free time exercised more, while the participants who had less motivation and free time decreased their practice of exercises in the analyzed period. Discussion: Students understand that physical activity is beneficial, even though it is hindered by strenuous workloads, due to the education they receive in the course. Conclusion: it was found that medical students who had remote classes during the period of social isolation considered they had more free time for the practice of exercises. However, not all of the dedicated this time to physical activity [au]

The late endosome-resident lipid bis(monoacylglycero)phosphate is a cofactor for Lassa virus fusion.

Arenavirus entry into host cells occurs through a low pH-dependent fusion with late endosomes that is mediated by the viral glycoprotein complex (GPC). The mechanisms of GPC-mediated membrane fusion and of virus targeting to late endosomes are not well understood. To gain insights into arenavirus fusion, we examined cell-cell fusion induced by the Old World Lassa virus (LASV) GPC complex. LASV GPC-mediated cell fusion is more efficient and occurs at higher pH with target cells expressing human LAMP1 compared to cells lacking this cognate receptor. However, human LAMP1 is not absolutely required for cell-cell fusion or LASV entry. We found that GPC-induced fusion progresses through the same lipid intermediates as fusion mediated by other viral glycoproteins-a lipid curvature-sensitive intermediate upstream of hemifusion and a hemifusion intermediate downstream of acid-dependent steps that can be arrested in the cold. Importantly, GPC-mediated fusion and LASV pseudovirus entry are specifically augmented by an anionic lipid, bis(monoacylglycero)phosphate (BMP), which is highly enriched in late endosomes. This lipid also specifically promotes cell fusion mediated by Junin virus GPC, an unrelated New World arenavirus. We show that BMP promotes late steps of LASV fusion downstream of hemifusion-the formation and enlargement of fusion pores. The BMP-dependence of post-hemifusion stages of arenavirus fusion suggests that these viruses evolved to use this lipid as a cofactor to selectively fuse with late endosomes.

Human Defensins Inhibit SARS-CoV-2 Infection by Blocking Viral Entry.

Innate immunity during acute infection plays a critical role in the disease severity of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), and is likely to contribute to COVID-19 disease outcomes. Defensins are highly abundant innate immune factors in neutrophils and epithelial cells, including intestinal Paneth cells, and exhibit antimicrobial and immune-modulatory activities. In this study, we investigated the effects of human α- and ß-defensins and RC101, a θ-defensin analog, on SARS-CoV-2 infection. We found that human neutrophil peptides (HNPs) 1-3, human defensin (HD) 5 and RC101 exhibited potent antiviral activity against pseudotyped viruses expressing SARS-CoV-2 spike proteins. HNP4 and HD6 had weak anti-SARS-CoV-2 activity, whereas human ß-defensins (HBD2, HBD5 and HBD6) had no effect. HNP1, HD5 and RC101 also inhibited infection by replication-competent SARS-CoV-2 viruses and SARS-CoV-2 variants. Pretreatment of cells with HNP1, HD5 or RC101 provided some protection against viral infection. These defensins did not have an effect when provided post-infection, indicating their effect was directed towards viral entry. Indeed, HNP1 inhibited viral fusion but not the binding of the spike receptor-binding domain to hACE2. The anti-SARS-CoV-2 effect of defensins was influenced by the structure of the peptides, as linear unstructured forms of HNP1 and HD5 lost their antiviral function. Pro-HD5, the precursor of HD5, did not block infection by SARS-CoV-2. High virus titers overcame the effect of low levels of HNP1, indicating that defensins act on the virion. HNP1, HD5 and RC101 also blocked viral infection of intestinal and lung epithelial cells. The protective effects of defensins reported here suggest that they may be useful additives to the antivirus arsenal and should be thoroughly studied.

Interferon-Induced Transmembrane Protein 3 Blocks Fusion of Diverse Enveloped Viruses by Altering Mechanical Properties of Cell Membranes.

Interferon-induced transmembrane protein 3 (IFITM3) potently inhibits entry of diverse enveloped viruses by trapping the viral fusion at a hemifusion stage, but the underlying mechanism remains unclear. Here, we show that recombinant IFITM3 reconstituted into lipid vesicles induces negative membrane curvature and that this effect maps to its small amphipathic helix (AH). We demonstrate that AH (i) partitions into lipid-disordered domains where IAV fusion occurs, (ii) induces negative membrane curvature, and (iii) increases lipid order and membrane stiffness. These effects on membrane properties correlate with the fusion-inhibitory activity, as targeting the ectopically expressed AH peptide to the cytoplasmic leaflet of the cell plasma membrane diminishes IAV-cell surface fusion induced by exposure to acidic pH. Our results thus imply that IFITM3 inhibits the transition from hemifusion to full fusion by imposing an unfavorable membrane curvature and increasing the order and stiffness of the cytoplasmic leaflet of endosomal membranes. Our findings reveal a universal mechanism by which cells block entry of diverse enveloped viruses.

Changes in Body Size during Early Growth Are Independently Associated with Arterial Properties in Early Childhood.

Nutritional status in early life stages has been associated with arterial parameters in childhood. However, it is still controversial whether changes in standardized body weight (z-BW), height (z-BH), BW for height (z-BWH) and/or body mass index (z-BMI) in the first three years of life are independently associated with variations in arterial structure, stiffness and hemodynamics in early childhood. In addition, it is unknown if the strength of the associations vary depending on the growth period, nutritional characteristics and/or arterial parameters analyzed. AIMS: First, to compare the strength of association between body size changes (Δz-BW, Δz-BH, Δz-BWH, Δz-BMI) in different time intervals (growth periods: 0-6, 0-12, 0-24, 0-36, 12-24, 12-36, 24-36 months (m)) and variations in arterial structure, stiffness and hemodynamics at age 6 years. Second, to determine whether the associations depend on exposure to cardiovascular risk factors, body size at birth and/or on body size at the time of the evaluation (cofactors). Anthropometric (at birth, 6, 12, 24, 36 m and at age 6 years), hemodynamic (peripheral and central (aortic)) and arterial (elastic (carotid) and muscular (femoral) arteries; both hemi-bodies) parameters were assessed in a child cohort (6 years; n =632). The association between arterial parameters and body size changes (Δz-BW, Δz-BH, Δz-BWH, Δz-BMI) in the different growth periods was compared, before and after adjustment by cofactors. RESULTS: Δz-BW 0-24 m and Δz-BWH 0-24 m allowed us to explain inter-individual variations in structural arterial properties at age 6 years, with independence of cofactors. When the third year of life was included in the analysis (0-36, 12-36, 24-36 m), Δz-BW explained hemodynamic (peripheral and central) variations at age 6 years. Δz-BH and Δz-BMI showed limited associations with arterial properties. CONCLUSION: Δz-BW and Δz-BWH are the anthropometric variables with the greatest association with arterial structure and hemodynamics in early childhood, with independence of cofactors.

Stroke volume and cardiac output non-invasive monitoring based on brachial oscillometry-derived pulse contour analysis: Explanatory variables and reference intervals throughout life (3-88 years).

BACKGROUND: Non-invasive assessment of stroke volume (SV), cardiac output (CO) and cardiac index (CI) has shown to be useful for the evaluation, diagnosis and/or management of different clinical conditions. Through pulse contour analysis (PCA) cuff­based oscillometric devices would enable obtaining ambulatory operator-independent non-invasive hemodynamic monitoring. There are no reference intervals (RIs), when considered as a continuum in childhood, adolescence and adult life, for PCA-derived SV [SV(PCA)], CO [CO(PCA)] and CI [CI(PCA)]. The aim of the study were to analyze the associations of SV(PCA), CO(PCA) and CI(PCA) with demographic, anthropometric, cardiovascular risk factors (CVRFs) and hemodynamic parameters, and to define RIs and percentile curves for SV(PCA), CO(PCA) and CI(PCA), considering the variables that should be considered when expressing them. METHODS: In 1449 healthy subjects (3-88 years) SV(PCA), CO(PCA) and CI(PCA) were non-invasively obtained (Mobil-O-Graph; Germany). ANALYSIS: associations between subject characteristics and SV(PCA), CO(PCA) and CI(PCA) levels (correlations; regression models); RIs and percentiles for SV(PCA), CO(PCA) and CI(PCA) (parametric methods; fractional polynomials). RESULTS: Sex, age, and heart rate would be explanatory variables for SV, CO, and CI levels. SV levels were also examined by body height, while body surface area (BSA) contributing to evaluation of CO and CI. CVRFs exposure did not contribute to independently explain the values of the dependent variables. SV, CO and CI levels were partially explained by the oscillometric-derived signal quality. RIs and percentiles were defined. CONCLUSIONS: Reference intervals and percentile for SV(PCA), CO(PCA) and CI(PCA), were defined for subjects from 3-88 years of age, results are expressed according to sex, age, heart rate, body height and/or BSA.

A Sensitive Electrochemical Sensor Based on Sonogel-Carbon Material Enriched with Gold Nanoparticles for Melatonin Determination.

In this work, the development of an electrochemical sensor for melatonin determination is presented. The sensor was based on Sonogel-Carbon electrode material (SNGCE) and Au nanoparticles (AuNPs). The low-cost and environmentally friendly SNGCE material was prepared by the ultrasound-assisted sonogel method. AuNPs were prepared by a chemical route and narrow size distribution was obtained. The electrochemical characterization of the SNGCE/AuNP sensor was carried out by cyclic voltammetry in the presence of a redox probe. The analytical performance of the SNGCE/AuNP sensor in terms of linear response range, repeatability, selectivity, and limit of detection was investigated. The optimized SNGCE/AuNP sensor displayed a low detection limit of 8.4 nM melatonin in synthetic samples assessed by means of the amperometry technique. The potential use of the proposed sensor in real sample analysis and the anti-matrix capability were assessed by a recovery study of melatonin detection in human peripheral blood serum with good accuracy.

Publisher Correction: HIV-1 replication complexes accumulate in nuclear speckles and integrate into speckle-associated genomic domains.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20152-w.

HIV-1 Uncoating and Nuclear Import Precede the Completion of Reverse Transcription in Cell Lines and in Primary Macrophages.

An assembly of capsid proteins (CA) form the mature viral core enclosing the HIV-1 ribonucleoprotein complex. Discrepant findings have been reported regarding the cellular sites and the extent of core disassembly (uncoating) in infected cells. Here, we combined single-virus imaging and time-of-drug-addition assays to elucidate the kinetic relationship between uncoating, reverse transcription, and nuclear import of HIV-1 complexes in cell lines and monocyte-derived macrophages (MDMs). By using cyclophilin A-DsRed (CDR) as a marker for CA, we show that, in contrast to TZM-bl cells, early cytoplasmic uncoating (loss of CDR) is limited in MDMs and is correlated with the efficiency of reverse transcription. However, we find that reverse transcription is dispensable for HIV-1 nuclear import, which progressed through an uncoating step at the nuclear pore. Comparison of the kinetics of nuclear import and the virus escape from inhibitors targeting distinct steps of infection, as well as direct quantification of viral DNA synthesis, revealed that reverse transcription is completed after nuclear import of HIV-1 complexes. Collectively, these results suggest that reverse transcription is dispensable for the uncoating step at the nuclear pore and that vDNA synthesis is completed in the nucleus of unrelated target cells.

HIV-1 replication complexes accumulate in nuclear speckles and integrate into speckle-associated genomic domains.

The early steps of HIV-1 infection, such as uncoating, reverse transcription, nuclear import, and transport to integration sites are incompletely understood. Here, we imaged nuclear entry and transport of HIV-1 replication complexes in cell lines, primary monocyte-derived macrophages (MDMs) and CD4+ T cells. We show that viral replication complexes traffic to and accumulate within nuclear speckles and that these steps precede the completion of viral DNA synthesis. HIV-1 transport to nuclear speckles is dependent on the interaction of the capsid proteins with host cleavage and polyadenylation specificity factor 6 (CPSF6), which is also required to stabilize the association of the viral replication complexes with nuclear speckles. Importantly, integration site analyses reveal a strong preference for HIV-1 to integrate into speckle-associated genomic domains. Collectively, our results demonstrate that nuclear speckles provide an architectural basis for nuclear homing of HIV-1 replication complexes and subsequent integration into associated genomic loci.

Super-Resolution Fluorescence Imaging Reveals That Serine Incorporator Protein 5 Inhibits Human Immunodeficiency Virus Fusion by Disrupting Envelope Glycoprotein Clusters.

Serine incorporator protein 5 (SERINC5) is the host antiretroviral factor that reduces HIV-1 infectivity by incorporating into virions and inhibiting the envelope glycoprotein (Env) mediated virus fusion with target cells. We and others have shown that SERINC5 incorporation into virions alters the Env structure and sensitizes the virus to broadly neutralizing antibodies targeting cryptic Env epitopes. We have also found that SERINC5 accelerates the loss of Env function over time compared to control viruses. However, the exact mechanism by which SERINC5 inhibits HIV-1 fusion is not understood. Here, we utilized 2D and 3D super-resolution microscopy to examine the effect of SERINC5 on the distribution of Env glycoproteins on single HIV-1 particles. We find that, in agreement with a previous report, Env glycoproteins form clusters on the surface of mature virions. Importantly, incorporation of SERINC5, but not SERINC2, which lacks antiviral activity, disrupted Env clusters without affecting the overall Env content. We also show that SERINC5 and SERINC2 also form clusters on single virions. Unexpectedly, Env and SERINC molecules exhibited poor codistribution on virions, as evidenced by much greater Env-SERINC pairwise distances compared to Env-Env distances. This observation is inconsistent with the previously reported interaction between Env and SERINC5 and suggests an indirect effect of SERINC5 on Env cluster formation. Collectively, our results reveal a multifaceted mechanism of SERINC5-mediated restriction of HIV-1 fusion that, aside from the effects on individual Env trimers, involves disruption of Env clusters, which likely serve as sites of viral fusion with target cells.