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.

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.

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.

Manejo de pacientes con síndrome de Budd-Chiari. Revisión de tema / Treatment of patients with Budd-Chiari syndrome. Literature review

El síndrome de Budd-Chiari (SBC), descrito en 1845, se define como la obstrucción del flujo venoso hepático en ausencia de enfermedad cardíaca o pericárdica. En Colombia no se tienen datos epidemiológicos claros de esta patología, la cual alrededor del mundo se considera poco frecuente. Se diagnostica al demostrar la obstrucción del flujo de las venas hepáticas. Tiene diversas manifestaciones clínicas como fiebre, ascitis, dolor abdominal y circulación colateral, entre otras. En ciertos casos es asintomática y en su gran mayoría se acompaña de patologías protrombóticas. El manejo inicial depende de la condición del paciente; sin embargo, se ha propuesto el manejo escalonado, donde se inicia con anticoagulación, se continúa con angioplastia, luego con desvío portosistémico intrahepático transyugular (TIPS), y se termina con trasplante hepático. El pronóstico depende de un diagnóstico precoz y un tratamiento adecuado. En las mejores circunstancias se alcanza una sobrevida a cinco años en el 90% de los casos, mientras que en ausencia de manejo, la tasa de mortalidad a un año alcanza el mismo porcentaje.

Budd-Chiari syndrome (SBC), described in 1845, is defined as the obstruction of hepatic venous flow in the absence of heart or pericardial disease. In Colombia there are no clear epidemiological data of this pathology, that around the world is considered rare. It is diagnosed by demonstrating the obstruction of the flow of the hepatic veins. It has various clinical manifestations such as fever, ascites, abdominal pain and collateral circulation, among others. In certain cases, it is asymptomatic but in the great majority it is accompanied by prothrombotic pathologies. Initial management depends on the patient's condition; however, staggered management has been proposed, beginning with anticoagulation, continuing with angioplasty, then with transjugular intrahepatic portosystemic shunt (TIPS), and finally, with liver transplantation. The prognosis depends on an early diagnosis and proper treatment. In the best circumstances, a five-year survival is achieved in 90% of cases, while in the absence of treatment, the one-year mortality rate reaches the same percentage.

Colangiocarcinoma en pacientes con colangitis esclerosante primaria / Cholangiocarcinoma in patients with primary sclerosing cholangitis

Resumen La colangitis esclerosante primaria (CEP) es una enfermedad inflamatoria poco común que afecta los conductos biliares, produciendo colestasis. Actualmente, el único tratamiento disponible es el manejo sintomático con ácido ursodesoxicólico y el trasplante hepático. Además de ser una etiología de cirrosis a largo plazo, en su historia natural tiene una asociación importante con el colangiocarcinoma (CCA), una neoplasia agresiva que es casi exclusiva de este grupo de pacientes, y hoy en día constituye su principal causa de muerte debido a que es de difícil diagnóstico y cuenta con opciones muy limitadas de tratamiento. En el presente artículo se exponen conceptos actuales sobre esta patología, enfatizando la importancia de realizar una adecuada tamización con pruebas diagnósticas efectivas (CA 19-9 y colangiorresonancia) que ayuden a diferenciar el CCA de procesos benignos y poder detectarlo en estadios tempranos donde la probabilidad de tratamiento curativo es mucho mayor.

Abstract Primary sclerosing cholangitis (PSC) is an uncommon inflammatory disease that affects the bile ducts to produce cholestasis. Currently, the only treatment available is management of symptoms with ursodeoxycholic acid and liver transplantation. In addition to its relation to long-term cirrhosis, its natural history has an important association with cholangiocarcinoma (CCA), an aggressive neoplasia which is almost exclusive to this group of patients. Today CCA is the main cause of death of these patients, primarily due to the difficultly of diagnosis and the very limited treatment options. This article discusses current ideas about this pathology and emphasizes the importance of appropriate screening with effective diagnostic tests (CA 19-9 and magnetic resonance cholangiopancreatography (MRC)) that help differentiate CCA from benign processes and can detect it in early stages when the probability of curative treatment is much greater.

Fusion Stage of HIV-1 Entry Depends on Virus-Induced Cell Surface Exposure of Phosphatidylserine.

HIV-1 entry into host cells starts with interactions between the viral envelope glycoprotein (Env) and cellular CD4 receptors and coreceptors. Previous work has suggested that efficient HIV entry also depends on intracellular signaling, but this remains controversial. Here we report that formation of the pre-fusion Env-CD4-coreceptor complexes triggers non-apoptotic cell surface exposure of the membrane lipid phosphatidylserine (PS). HIV-1-induced PS redistribution depends on Ca2+ signaling triggered by Env-coreceptor interactions and involves the lipid scramblase TMEM16F. Externalized PS strongly promotes Env-mediated membrane fusion and HIV-1 infection. Blocking externalized PS or suppressing TMEM16F inhibited Env-mediated fusion. Exogenously added PS promoted fusion, with fusion dependence on PS being especially strong for cells with low surface density of coreceptors. These findings suggest that cell-surface PS acts as an important cofactor that promotes the fusogenic restructuring of pre-fusion complexes and likely focuses the infection on cells conducive to PS signaling.

pH regulation in early endosomes and interferon-inducible transmembrane proteins control avian retrovirus fusion.

Enveloped viruses infect host cells by fusing their membranes with those of the host cell, a process mediated by viral glycoproteins upon binding to cognate host receptors or entering into acidic intracellular compartments. Whereas the effect of receptor density on viral infection has been well studied, the role of cell type-specific factors/processes, such as pH regulation, has not been characterized in sufficient detail. Here, we examined the effects of cell-extrinsic factors (buffer environment) and cell-intrinsic factors (interferon-inducible transmembrane proteins, IFITMs), on the pH regulation in early endosomes and on the efficiency of acid-dependent fusion of the avian sarcoma and leukosis virus (ASLV), with endosomes. First, we found that a modest elevation of external pH can raise the pH in early endosomes in a cell type-dependent manner and thereby delay the acid-induced fusion of endocytosed ASLV. Second, we observed a cell type-dependent delay between the low pH-dependent and temperature-dependent steps of viral fusion, consistent with the delayed enlargement of the fusion pore. Third, ectopic expression of IFITMs, known to potently block influenza virus fusion with late compartments, was found to only partially inhibit ASLV fusion with early endosomes. Interestingly, IFITM expression promoted virus uptake and the acidification of endosomal compartments, resulting in an accelerated fusion rate when driven by the glycosylphosphatidylinositol-anchored, but not by the transmembrane isoform of the ASLV receptor. Collectively, these results highlight the role of cell-extrinsic and cell-intrinsic factors in regulating the efficiency and kinetics of virus entry and fusion with target cells.

Screening and Functional Profiling of Small-Molecule HIV-1 Entry and Fusion Inhibitors.

HIV-1 entry and fusion with target cells is an important target for antiviral therapy. However, a few currently approved treatments are not effective as monotherapy due to the emergence of drug resistance. This consideration has fueled efforts to develop new bioavailable inhibitors targeting different steps of the HIV-1 entry process. Here, a high-throughput screen was performed of a large library of 100,000 small molecules for HIV-1 entry/fusion inhibitors, using a direct virus-cell fusion assay in a 384 half-well format. Positive hits were validated using a panel of functional assays, including HIV-1 specificity, cytotoxicity, and single-cycle infectivity assays. One compound-4-(2,5-dimethyl-pyrrol-1-yl)-2-hydroxy-benzoic acid (DPHB)-that selectively inhibited HIV-1 fusion was further characterized. Functional experiments revealed that DPHB caused irreversible inactivation of HIV-1 Env on cell-free virions and that this effect was related to binding to the third variable loop (V3) of the gp120 subunit of HIV-1 Env. Moreover, DPHB selectively inhibited HIV-1 strains that use CXCR4 or both CXCR4 and CCR5 co-receptors for entry, but not strains exclusively using CCR5. This selectivity was mapped to the gp120 V3 loop using chimeric Env glycoproteins. However, it was found that pure DPHB was not active against HIV-1 and that its degradation products (most likely polyanions) were responsible for inhibition of viral fusion. These findings highlight the importance of post-screening validation of positive hits and are in line with previous reports of the broad antiviral activity of polyanions.

Click labeling of unnatural sugars metabolically incorporated into viral envelope glycoproteins enables visualization of single particle fusion.

Enveloped viruses infect target cells by fusing their membrane with cellular membrane through a process that is mediated by specialized viral glycoproteins. The inefficient and highly asynchronous nature of viral fusion complicates studies of virus entry on a population level. Single virus imaging in living cells has become an important tool for delineating the entry pathways and for mechanistic studies of viral fusion. We have previously demonstrated that incorporation of fluorescent labels into the viral membrane and trapping fluorescent proteins in the virus interior enables the visualization of single virus fusion in living cells. Here, we implement a new approach to non-invasively label the viral membrane glycoproteins through metabolic incorporation of unnatural sugars followed by click-reaction with organic fluorescent dyes. This approach allows for efficient labeling of diverse viral fusion glycoproteins on the surface of HIV pseudoviruses. Incorporation of a content marker into surface-labeled viral particles enables sensitive detection of single virus fusion with live cells.

Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus.

BACKGROUND: HIV-1 Vpr is recruited into virions during assembly and appears to remain associated with the viral core after the reverse transcription and uncoating steps of entry. This feature has prompted the use of fluorescently labeled Vpr to visualize viral particles and to follow trafficking of post-fusion HIV-1 cores in the cytoplasm. RESULTS: Here, we tracked single pseudovirus entry and fusion and observed that fluorescently tagged Vpr gradually dissociates from post-fusion viral cores over the course of several minutes and accumulates in the nucleus. Kinetics measurements showed that fluorescent Vpr released from the cores very rapidly entered the cell nucleus. More than 10,000 Vpr molecules can be delivered into the cell nucleus within 45 min of infection by HIV-1 particles pseudotyped with the avian sarcoma and leukosis virus envelope glycoprotein. The fraction of Vpr from cell-bound viruses that accumulated in the nucleus was proportional to the extent of virus-cell fusion and was fully blocked by viral fusion inhibitors. Entry of virus-derived Vpr into the nucleus occurred independently of envelope glycoproteins or target cells. Fluorescence correlation spectroscopy revealed two forms of nuclear Vpr-monomers and very large complexes, likely involving host factors. The kinetics of viral Vpr entering the nucleus after fusion was not affected by point mutations in the capsid protein that alter the stability of the viral core. CONCLUSIONS: The independence of Vpr shedding of capsid stability and its relatively rapid dissociation from post-fusion cores suggest that this process may precede capsid uncoating, which appears to occur on a slower time scale. Our results thus demonstrate that a bulk of fluorescently labeled Vpr incorporated into HIV-1 particles is released shortly after fusion. Future studies will address the question whether the quick and efficient nuclear delivery of Vpr derived from incoming viruses can regulate subsequent steps of HIV-1 infection.

Distinct requirements for HIV-cell fusion and HIV-mediated cell-cell fusion.

Whether HIV-1 enters cells by fusing with the plasma membrane or with endosomes is a subject of active debate. The ability of HIV-1 to mediate fusion between adjacent cells, a process referred to as "fusion-from-without" (FFWO), shows that this virus can fuse with the plasma membrane. To compare FFWO occurring at the cell surface with HIV-cell fusion through a conventional entry route, we designed an experimental approach that enabled the measurements of both processes in the same sample. The following key differences were observed. First, a very small fraction of viruses fusing with target cells participated in FFWO. Second, whereas HIV-1 fusion with adherent cells was insensitive to actin inhibitors, post-CD4/coreceptor binding steps during FFWO were abrogated. A partial dependence of HIV-cell fusion on actin remodeling was observed in CD4(+) T cells, but this effect appeared to be due to the actin dependence of virus uptake. Third, deletion of the cytoplasmic tail of HIV-1 gp41 dramatically enhanced the ability of the virus to promote FFWO, while having a modest effect on virus-cell fusion. Distinct efficiencies and actin dependences of FFWO versus HIV-cell fusion are consistent with the notion that, except for a minor fraction of particles that mediate fusion between the plasma membranes of adjacent cells, HIV-1 enters through an endocytic pathway. We surmise, however, that cell-cell contacts enabling HIV-1 fusion with the plasma membrane could be favored at the sites of high density of target cells, such as lymph nodes.

Pinpointing retrovirus entry sites in cells expressing alternatively spliced receptor isoforms by single virus imaging.

BACKGROUND: The majority of viruses enter host cells via endocytosis. Current knowledge of viral entry pathways is largely based upon infectivity measurements following genetic and/or pharmacological interventions that disrupt vesicular trafficking and maturation. Imaging of single virus entry in living cells provides a powerful means to delineate viral trafficking pathways and entry sites under physiological conditions. RESULTS: Here, we visualized single avian retrovirus co-trafficking with markers for early (Rab5) and late (Rab7) endosomes, acidification of endosomal lumen and the resulting viral fusion measured by the viral content release into the cytoplasm. Virus-carrying vesicles either merged with the existing Rab5-positive early endosomes or slowly accumulated Rab5. The Rab5 recruitment to virus-carrying endosomes correlated with acidification of their lumen. Viral fusion occurred either in early (Rab5-positive) or intermediate (Rab5- and Rab7-positive) compartments. Interestingly, different isoforms of the cognate receptor directed virus entry from distinct endosomes. In cells expressing the transmembrane receptor, viruses preferentially entered and fused with slowly maturing early endosomes prior to accumulation of Rab7. By comparison, in cells expressing the GPI-anchored receptor, viruses entered both slowly and quickly maturing endosomes and fused with early (Rab5-positive) and intermediate (Rab5- and Rab7-positive) compartments. CONCLUSIONS: Since the rate of low pH-triggered fusion was independent of the receptor isoform, we concluded that the sites of virus entry are determined by the kinetic competition between endosome maturation and viral fusion. Our findings demonstrate the ability of this retrovirus to enter cells via alternative endocytic pathways and establish infection by releasing its content from distinct endosomal compartments.

Anionic lipids are required for vesicular stomatitis virus G protein-mediated single particle fusion with supported lipid bilayers.

Viral glycoproteins mediate fusion between viral and cellular membranes upon binding to cognate receptors and/or experiencing low pH. Although activation of viral glycoproteins is thought to be necessary and sufficient for fusion, accumulating evidence suggests that additional cellular factors, including lipids, can modulate the fusion process. Understanding the role of lipids in virus entry via endocytosis is impeded by poor accessibility and the highly diverse nature of endosomes. Here we imaged fusion of single retroviral particles pseudotyped with the vesicular stomatitis virus (VSV) G protein with dextran-supported lipid bilayers. Incorporation of diffusible fluorescent labels into the viral membrane and the viral interior enabled detection of the lipid mixing (hemifusion) and content transfer (full fusion) steps of VSV G-mediated fusion at low pH. Although single virus fusion with supported bilayers made of zwitterionic lipids could not be detected, inclusion of anionic lipids, phosphatidylserine, and bis(monoacylglycero)phosphate (BMP), greatly enhanced the efficiency of hemifusion and permitted full fusion. Importantly, lipid mixing always preceded the opening of a fusion pore, demonstrating that VSV G-mediated fusion proceeds through a long-lived hemifusion intermediate. Kinetic analysis of lipid and content transfer showed that the lags between lipid and content mixing defining the lifetime of a hemifusion intermediate were significantly shorter for BMP-containing compared with PS-containing bilayers. The strong fusion-enhancing effect of BMP, a late endosome-resident lipid, is consistent with the model that VSV initiates fusion in early endosomes but releases its core into the cytosol after reaching late endosomal compartments.

Quantitative imaging of endosome acidification and single retrovirus fusion with distinct pools of early endosomes.

Diverse enveloped viruses enter host cells through endocytosis and fuse with endosomal membranes upon encountering acidic pH. Currently, the pH dynamics in virus-carrying endosomes and the relationship between acidification and viral fusion are poorly characterized. Here, we examined the entry of avian retrovirus that requires two sequential stimuli--binding to a cognate receptor and low pH--to undergo fusion. A genetically encoded sensor incorporated into the viral membrane was used to measure the pH in virus-carrying endosomes. Acid-induced virus fusion was visualized as the release of a fluorescent viral content marker into the cytosol. The pH values in early acidic endosomes transporting the virus ranged from 5.6 to 6.5 but were relatively stable over time for a given vesicle. Analysis of viral motility and luminal pH showed that cells expressing the transmembrane isoform of the receptor (TVA950) preferentially sorted the virus into slowly trafficking, less acidic endosomes. In contrast, viruses internalized by cells expressing the GPI-anchored isoform (TVA800) were uniformly distributed between stationary and mobile compartments. We found that the lag times between acidification and fusion were significantly shorter and fusion pores were larger in dynamic endosomes than in more stationary compartments. Despite the same average pH within mobile compartments of cells expressing alternative receptor isoforms, TVA950 supported faster fusion than TVA800 receptor. Collectively, our results suggest that fusion steps downstream of the low-pH trigger are modulated by properties of intracellular compartments harboring the virus.

Synchronized retrovirus fusion in cells expressing alternative receptor isoforms releases the viral core into distinct sub-cellular compartments.

Disparate enveloped viruses initiate infection by fusing with endosomes. However, the highly diverse and dynamic nature of endosomes impairs mechanistic studies of fusion and identification of sub-cellular sites supporting the nucleocapsid release. We took advantage of the extreme stability of avian retrovirus-receptor complexes at neutral pH and of acid-dependence of virus-endosome fusion to isolate the latter step from preceding asynchronous internalization/trafficking steps. Viruses were trapped within endosomes in the presence of NH4Cl. Removal of NH4Cl resulted in a quick and uniform acidification of all subcellular compartments, thereby initiating synchronous viral fusion. Single virus imaging demonstrated that fusion was initiated within seconds after acidification and often culminated in the release of the viral core from an endosome. Comparative studies of cells expressing either the transmembrane or GPI-anchored receptor isoform revealed that the transmembrane receptor delivered the virus to more fusion-permissive compartments. Thus the identity of endosomal compartments, in addition to their acidity, appears to modulate viral fusion. A more striking manifestation of the virus delivery to distinct compartments in the presence of NH4Cl was the viral core release into the cytosol of cells expressing the transmembrane receptor and into endosomes of cells expressing the GPI-anchored isoform. In the latter cells, the newly released cores exhibited restricted mobility and were exposed to a more acidic environment than the cytoplasm. These cores appear to enter into the cytosol after an additional slow temperature-dependent step. We conclude that the NH4Cl block traps the virus within intralumenal vesicles of late endosomes in cells expressing the GPI-anchored receptor. Viruses surrounded by more than one endosomal membrane release their core into the cytoplasm in two steps--fusion with an intralumenal vesicle followed by a yet unknown temperature-dependent step that liberates the core from late endosomes.

Imaging single retrovirus entry through alternative receptor isoforms and intermediates of virus-endosome fusion.

A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA)-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950) internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800). Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes.

Reprocessamento de artigos de uso único: conhecimento de funcionários do CME / Reprodessing of single-use items: knowledge of supply material staff

Preocupadas com a problemática do reprocessamento de artigos de suso único, as autoras objetivaram verificar o grau de conhecimento de 31 elementos da equipe de enfermagem (24 auxiliares, 4 técnicos e 3 enfermeiros) de um centro de material e esterilização sobre os aspectos legais que envolvem o processamento, bem como quais são os artigos de uso único reprocessados, a existência de controle e os critérios para avaliação desses artigos...

The anti-HIV-1 editing enzyme APOBEC3G binds HIV-1 RNA and messenger RNAs that shuttle between polysomes and stress granules.

Deoxycytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) (members of the apolipoprotein B mRNA-editing catalytic polypeptide 3 family) have RNA-binding motifs, invade assembling human immunodeficiency virus (HIV-1), and hypermutate reverse transcripts. Antagonistically, HIV-1 viral infectivity factor degrades these enzymes. A3G is enzymatically inhibited by binding RNA within an unidentified large cytosolic ribonucleoprotein, implying that RNA degradation during reverse transcription may activate intravirion A3G at the necessary moment. We purified a biologically active tandem affinity-tagged A3G from human HEK293T cells. Mass spectrometry and coimmunoprecipitation from HEK293T and T lymphocyte extracts identified many RNA-binding proteins specifically associated with A3G and A3F, including poly(A)-binding proteins (PABPs), YB-1, Ro-La, RNA helicases, ribosomal proteins, and Staufen1. Most strikingly, nearly all A3G-associated proteins were known to bind exclusively or intermittently to translating and/or dormant mRNAs. Accordingly, A3G in HEK293T and T lymphocyte extracts was almost completely in A3G-mRNA-PABP complexes that shifted reversibly between polysomes and dormant pools in response to translational inhibitors. For example arsenite, which inhibits 5'-cap-dependent translational initiation, shifted mRNA-A3G-PABP from polysomes into stress granules in a manner that was blocked and reversed by the elongation inhibitor cycloheximide. Immunofluorescence microscopy showed A3G-mRNA-PABP stress granules only partially overlapping with Staufen1. A3G coimmunoprecipitated HIV-1 RNA and many mRNAs. Ribonuclease released nearly all A3G-associated proteins, including A3G homo-oligomers and A3G-A3F hetero-oligomers, but the viral infectivity factor remained bound. Many proteins and RNAs associated with A3G are excluded from A3G-containing virions, implying that A3G competitively partitions into virions based on affinity for HIV-1 RNA.