HIV - 1 ودعمت دراسات uncoating في المختبر أيكن من منح المعاهد الوطنية للصحة AI40364 ، وAI50423 AI076121. وقدمت العديد من المواد الأساسية ، بما في ذلك الكواشف المستخدمة في ELISA P24 ، من قبل المعاهد الوطنية للصحة لأبحاث الايدز والبرنامج المرجعي ، شعبة الإيدز ، NIAID ، المعاهد الوطنية للصحة.

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Molecular Biology. 485, 41-53 (2009).</li><li>Kotov, A., Zhou, J., Flicker, P., Aiken, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+Nef+with+the+human+immunodeficiency+virus+type+1+core.">Association of Nef with the human immunodeficiency virus type 1 core.</a> J. Virol. 73, 8824-8830 (1999).</li><li>Kewalramani, V. N., Emerman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vpx+association+with+mature+core+structures+of+HIV-2.">Vpx association with mature core structures of HIV-2.</a> Virology. 218, 159-168 (1996).</li><li>Wehrly, K., Chesebro, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p24+antigen+capture+assay+for+quantification+of+human+immunodeficiency+virus+using+readily+available+inexpensive+reagents.">p24 antigen capture assay for quantification of human immunodeficiency virus using readily available inexpensive reagents.</a> Methods. 12, 288-293 (1997).</li><li>Welker, R., Hohenberg, H., Tessmer, U., Huckhagel, C., Kr&#228;usslich, H. 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الإعلان عن أي تضارب في المصالح.

وصف الأسلوب هنا لتنقية HIV - 1 النوى لدراسة uncoating في المختبر هو مفيد لدراسة هذه المرحلة من دورة الحياة HIV - 1 ، خصوصا بسبب عدم وجود طريقة لتحليل التحقق من صحة HIV - 1 uncoating في الخلايا المستهدفة. على الرغم من أن هذا الأسلوب يستخدم لتنقية تنبيذ فائق التوازن النوى ، والبعض الآخر ق...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td width="190"> اسم الكاشف </td><td width="197"> شركة </td><td width="114"> فهرس العدد </td><td width="161"> تعليق (اختياري) </td></tr><tr><td> DMEM </td><td> Cellgro </td><td> 10-013 - CV </td><td></td></tr><tr><td> برنامج تلفزيوني </td><td> Cellgro </td><td> 21-031 - CV </td><td></td></tr><tr><td> تريتون - X - 100 </td><td> مالينكرودت شركة بيكر </td><td> 9002-93-1 </td><td></td></tr><tr><td> توين 20 </td><td> Acros </td><td> 9005-64-5 </td><td></td></tr><tr><td> 0.25 ٪ التربسين - EDTA </td><td> Cellgro </td><td> 25-053 - CI </td><td></td></tr><tr><td> 2XBBS </td><td></td><td></td><td> التكوين : 50mM BES [ع ه 6،95] ، 280 ملي مول كلوريد الصوديوم و 1.5 ملي نا 2 هبو 4. ضبط درجة الحموضة إلى 6.95في درجة حرارة الغرفة. تصفية وتعقيم في متجر في aliquots -20 درجة مئوية. </td></tr><tr><td> طلاء الأضداد : ريتوكسيماب إلى P24 (183 - H12 - 5C) </td><td> المعاهد الوطنية للصحة والبحوث الإيدز المرجعي برنامج الكاشف </td><td> 3537 </td><td></td></tr><tr><td> الابتدائية الأضداد : فيروس نقص المناعة البشرية الإيج (مصل المريض مفرط المناعة البشرية) </td><td> NIH أبحاث الإيدز والبرنامج المرجعي الكاشف </td><td> 3957 </td><td></td></tr><tr><td> الأجسام المضادة الثانوية : الماعز المضادة للمفتش الإنسان ، مترافق البيروكسيداز </td><td> ثقب </td><td> 31130 </td><td></td></tr><tr><td> HRP الركيزة </td><td> KPL المؤتمر الوطني العراقي </td><td> 50-76-11 </td><td></td></tr><tr><td> Immulon 2HB 96 لوحات جيدة </td><td> الحرارية العلمية </td><td> 3455 </td><td></td></tr><tr><td> وSW32Ti SW32.1 الدوارات تي نابذة فائقة السرعة ومتوافق </td> &lt;TD&gt; بيكمان كولتر </td><td></td><td></td></tr><tr><td> TLA - 55 الدوار ونابذة فائقة السرعة منضدية </td><td> بيكمان كولتر </td><td></td><td></td></tr><tr><td> سرعة عالية أنابيب microfuge </td><td> بيكمان كولتر </td><td> 326823 ، 358123 ، 357448 </td><td></td></tr><tr><td> التدرج التلقائي Densi كثافة تدفق fractionator </td><td> Labconco كورب </td><td> 4517000 </td><td></td></tr><tr><td> التدرج السابقة </td><td> شركة سي بي اس العلمية المحدودة </td><td> GM - 20 </td><td></td></tr></tbody></table>

in vitro uncoating of hiv-1 cores

الجينوم من الفيروسات القهقرية هو المغطى في قفيصة محاطة المغلف الدهون. لlentiviruses ، مثل HIV - 1 ، وتتألف القشرة قفيصة المخروطية من البروتين CA رتبت بوصفها شعرية من مسدس. إغلاق قفيصة pentamers بنسبة 7 في نهاية اسعة و 5 في نهاية الضيقة للمخروط 1 و 2. المغطى في هذه القذيفة قفيصة هو مجمع بروتين نووي ريبوزي الفيروسية ، وأنهم يشكلون معا جوهر. بعد الانصهار في الغشاء الفيروسية مع غشاء الخلية المستهدفة ، يتم الافراج عن HIV - 1 في السيتوبلازم. قفيصة ثم يفكك الافراج CA الحرة في شكل قابل للذوبان 3 في عملية يشار إلى uncoating. ولا يزال الغموض يكتنف مكان وتوقيت الخلايا HIV - 1 uncoating. الأحماض الأمينية واحد التبديلات في كاليفورنيا التي تغير على استقرار قفيصة يضعف أيضا قدرة HIV - 1 إلى 4 تصيب الخلايا. هذا يدل على أن الاستقرار في قفيصة هو أمر حاسم لHIV - 1 العدوى. </p&gt; وقد HIV - 1 uncoating الصعب دراسة نظرا لعدم توافر فحوصات حساسة وموثوقة لهذه العملية. نحن هنا وصف الأسلوب الكمي للدراسة في المختبر باستخدام uncoating النوى معزولة عن جسيمات HIV - 1 المعدية. هذا النهج ينطوي على عزل النوى بالترسيب من خلال virions تتركز طبقة من المنظفات والى الانحدار الخطي السكروز ، في البرد. لتحديد uncoating ، يتم تحضين النوى معزولة عند 37 درجة مئوية لفترات مختلفة وتوقيتها لاحقا من قبل مكعبات تنبيذ فائق. ويتم تحليل مدى uncoating عن طريق قياس نسبة ضئيلة من CA في طاف. وقد استخدمت هذه الطريقة لتحليل آثار الطفرات الفيروسية على HIV - 1 الاستقرار قفيصة 4 ، 5 ، 6. وينبغي أن يكون من المفيد أيضا لدراسة دور العوامل الخلوية في HIV - 1 uncoating.

Watch this Scientific Journal Video about In vitro Uncoating of HIV-1 Cores at JoVE.com

In vitro Uncoating of HIV-1 Cores

Uncoating هو خطوة ضرورية في مرحلة مبكرة من دورة الحياة HIV - 1 ، ويعرف بأنه تفكك قذيفة قفيصة والإفراج عن مجمع بروتين نووي ريبوزي الفيروسي (vRNP). هنا ، علينا أن نظهر تقنيات لعزل نواة سليمة من virions HIV - 1 لقياس وuncoating بهم<em&gt; في المختبر</em&gt;.

Uncoating في المختبر من HIV - 1 النوى

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Research

JoVE Journal

Immunology and Infection

In vitro Uncoating of HIV-1 Cores

15.6K Views.  Vanderbilt University School of Medicine. Uncoating هو خطوة ضرورية في مرحلة مبكرة من دورة الحياة HIV - 1 ، ويعرف بأنه تفكك قذيفة قفيصة والإفراج عن مجمع بروتين نووي ريبوزي الفيروسي (vRNP). هنا ، علينا أن نظهر تقنيات لعزل نواة سليمة من virions HIV - 1 لقياس وuncoating بهم في المختبر.

Video: In vitro Uncoating of HIV-1 Cores

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

الاستدلال الوراثي من HIV - 1 Tropism باستخدام السكان على أساس تسلسل V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

التصوير من HIV-1 مغلف من صنع المشبك الفيروسية، واشارة على طبقات ثنائية الدهون الاصطناعية

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

و<em&gt; في المختبر</em&gt; المشارك عدوى نموذج لدراسة<em&gt; المتصورة المنجلية</em&gt; HIV-1-التفاعلات في الإنسان خلايا الوحيدات المشتقة الابتدائية المناعي

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

أدوات جديدة لتوسيع الخلايا التائية التنظيمية من الأفراد HIV-1-المصابة

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

تقييد أنزيم الاستنساخ القائم على أسلوب لتقييم<em&gt; في المختبر</em&gt; القدرة تكرار HIV-1 النوع الفرعي C-الكمامة MJ4 همي الفيروسات

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

تقييم بتكوين لHIV-1 مغلف مثلوثي بروتينات سكرية باستخدام خلية القاعدة ELISA الفحص

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

عزل Exosomes من البلازما من HIV-1 الأفراد ايجابية

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

تقييم فعالية وسمية الرنا استهداف HIV-1 الإنتاج للاستخدام في الجينات أو العلاج بالعقاقير

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

يبلوع الهجرة والسرعة تقاس في لايف الابتدائية الضامة الإنسان المصابة بفيروس نقص المناعة البشرية-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

قياس ل<em&gt; في المختبر</em&gt; التكامل آخر من HIV-1 Preintegration مجمعات

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...