DP2 OD004631 ve R01 AI084119 dahil olmak üzere, Bill ve Melinda Gates Vakfı (38.631) ve NIH hibe tarafından desteklenmiştir.

1. Metodoloji <ol><li> Protokol siliko kat isteyen V3 taç sırasını seçmek için ilk adım. R2 gerginlik için, bu parça için sıra KSIPMGPGRAFYT. </li><li> Bilgisayarın sanal uzayda bu sıra ilgili peptid 3D atomik yapı inşa edilmelidir. Bunun için ICM komut: buildpep &quot;KSIPMGPGRAFYT&quot; ya da Dosya: Yeni: Peptid aşağı çekme menüsü altında seçilebilir </li><li> Sonra arama adımları sayısı (arama süresi), simülasyon sıcaklığı, önyargı makul alanları, enerji açısından, farklı enerji hesaplama yöntemleri seçimi doğru arama değişken koltuk da dahil olmak üzere arama stratejisi parametreleri de dahil olmak üzere, prosedürün bazı parametreleri ayarlanır ve arama gibi bir film kaydetmek isteyip olarak kaydedilir ve kaç ara puanlama konformasyonlar nasıl belirten parametreler kayıt altına alınmalıdır. Tüm bu parametreler önceki yayınlar (www.molsoft.com bakınız) tarafından optimize edilmiştir. </li><li> Katlama sonra komut ile başlatılır: montecarlo ya da Dosya: Yeni: Peptid menü Moleküler Mekanik aşağı çekme altında seçilebilir: Küçült: Küresel Birinci durumda, 3. adımdan itibaren parametreler komut satırından tek tek ayarlanması gerekir. İkinci durumda komut çalıştırılmadan önce, en sık seçilen parametreler için onay kutularını bir bölmede sağlanmaktadır. Kolaylık sağlamak için, daha önce yayınlanan bu deneme ve deneyler için kullanılan aynı katlanır ICM komut dosyası aşağıda gösterilmiştir: <img src="/files/ftp_upload/2118/2118output.jpg" alt="Çıktı" /> Bu komut dosyası, bir metin dosyasına kaydedilir ve hızlı bir şekilde komutunu kullanarak bilgisayara işletim sistemi komut satırı (genellikle LINUX) çalıştırmak olabilir: icm _foldingscript </li></ol> 2. Başarı sırları <ol><li> V3 döngü uzunluğu neredeyse bilinen her türlü gerginlik sürekli bir 35 amino asitler, amino asit pozisyonları sistin gümrüklü kaynaklanan disülfit 1 ile başlayan ve 35 sistin gümrüklü ilgili disülfür ile sonlandıran 1 den 35 kadar numaralandırılmış. Bir döngünün parçası olduğundan, V3 taç sınırlarını dikkatle seçilmelidir. Özgür bir peptid sanki çok büyük bir parçası seçilirse, serbestçe esnek bir bölüm olarak davranmaya olası değildir, katlama simülasyon yapısını doğru değerlendirmek olmaz. Çok küçük bir parçası seçilirse, bilgilendirici üçüncül yapısı simülasyon form olmayabilir. Önceki çalışma pozisyonu 10 pozisyonu 22 parça antikor bağlı kristalografik konformasyonlar ile ilişkili olduğunu gösterdi, bu nedenle bu katlama seçilmelidir V3 herhangi bir döngünün parçası olduğunu. </li><li> Grafik kullanıcı arayüzü kullanarak ve açılan menüler kullanıcı dostu olmasına rağmen, özellikle ICM ve V3 döngü taç katlama kullanarak, genel olarak peptid katlama önceden başarılı çalışmalar, sadece buildpep doğrultusunda sırasını değiştirerek ve çalışan, yukarıda kullanılan komut dosyası Yukarıdaki betik komut satırından tavsiye edilir. </li></ol> 3. Temsilcisi Sonuçlar R2 katlama sonuçları herhangi bir V3 döngü için sonuçlar temsilcisi vardır. Sonuçlarını değerlendirmek için proje dosyası (Yukarıdaki betik &quot;newProject1.icb&quot; adlı olacaktır), seçilmiş &quot;Moleküler Mekanik, Stack,&quot; açılmış ve olmalıdır. Yığını konformasyonlarının bir tablo belirecektir. Yığını konformasyonlar Arsa / Histogram simgesini tıklayarak grafiksel olarak görüntülenebilir. &quot;Moleküler Mekanik, Stack, Play&quot; görsel katlanarak ortaya çıkan konformasyonel tercihleri ​​takdir yığının bir film yapacak. R2 dizisi için konformasyon V3 döngüler 2 için beklenen beta-saç tokası gibi pozisyonlarda parçası olarak, özellikle 12 ila 14 net bir β iplikli tercih yığın boyunca görülebilir ve çok az sayıda alfa sarmal konformasyonlar görülür. Ayrıca, yaklaşık 3 adet bir enerji açığı, düşük enerji uygunluğu ve ikinci en düşük enerji konformasyon arasında görülür. Enerjik bir noktadan itibaren, bu demektir ki en düşük enerji konformasyon yapısı sadece titriyor yüzde birden daha az zaman: katlama sonuçlar bu nedenle R2 V3 taç katı bir yerine, esnek, yapı olduğunu düşündürmektedir. Konformasyonlarının topluluk ek önemli yapısal özellikleri, ancak sistematik olarak takdir etmek zor olabilir. <table border="1"><tbody><tr><td></td><td> JRFL </td><td> SF162_V3JRFL </td><td> SF162_V3R2 </td></tr><tr><td> 447 52d GMT 50 </td><td> 15 </td><td> 0,00061 </td><td> 0,00078 </td></tr></tbody></table> Tablo 1: JRFL ve R2 V3 Bağıl nötralizasyon maskeli ve maskesiz ayarları döngüler. Veriler daha önce Cardozo T., ve ark. ARHR (200 rapor edildi8) ve Pinter, A., ve ark. al J. Virol (2004). SF162_V3JRFL içeren JRFL: Kısaca, nötralize aktivite env-arızalı lusiferaz ifade JRFL Env veya yukarıda açıklanan SF162 V3 türevleri ile pseudotyped pNL4 3.Luc.RE-plazmid ile üretilen psVs kullanarak bir tek döngüsü enfektivite assay ile belirlendi SF162 V3 döngü sırası yerine V3 döngü sırası. SF162_V3R2 SF162 V3 döngü sırası yerine R2 V3 döngü sırası içerir. Polybrene (10 mg = ml) varlığında 96-kuyucuğu kaplama ° C, ve sonra CD4 + CCR5 + U87 hedef hücreleri psVs 37 az 1.5 saat süreyle 447-52d mAb seri dilüsyonları ile inkübe edildi. 24 saat sonra, hücreler inkübasyon ek bir 24-48 saat sonra% 10 FBS ve 10 mg = mL polybrene içeren RPMI medyum refed. Lusiferaz aktivitesi 447 52d% 50 nötralizasyon (GMT50) Promega, Inc geometrik ortalama titreleri tahlil reaktifler kullanarak mikrotiter plate luminometre (HARTA, Inc.) 48-72 saat postinfection tespit edildi nötralizasyon eğrileri interpolasyon tarafından belirlenir ve en az üç bağımsız testleri ortalamasıdır.

<ol>
	<li>Almond, D., Kimura, T., Kong, X., Zolla-Pazner, S. &. a. m. p. ;. a. m. p., Cardozo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+characterization+of+the+V3+loop+crown.">Dynamic characterization of the V3 loop crown.</a> Antiviral Therapy. 12, 13-31 (2007).</li><li>Almond, D., Kimura, T., Kong, X., Swetnam, J., Zolla-Pazner, S., Cardozo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+conservation+predominates+over+sequence+variability+in+the+crown+of+HIV+type+1's+V3+loop.">Structural conservation predominates over sequence variability in the crown of HIV type 1's V3 loop.</a> AIDS Res Hum Retroviruses. 26, (2010).</li><li>Abagyan, R., Totrov, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biased+probability+Monte+Carlo+conformational+searches+and+electrostatic+calculations+for+peptides+and+proteins.">Biased probability Monte Carlo conformational searches and electrostatic calculations for peptides and proteins.</a> J Mol Biol. 235, 983-1002 (1994).</li><li>Quinnan, G. V., Zhang, P. F., Fu, D. W., Dong, M., Alter, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+characterization+of+HIV+type+1+envelope+protein+associated+with+a+broadly+reactive+neutralizing+antibody+response.">Expression and characterization of HIV type 1 envelope protein associated with a broadly reactive neutralizing antibody response.</a> AIDS Res Hum Retroviruses. 15, 561-5670 (1999).</li><li>Zhang, P. F., Bouma, P., Park, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variable+region+3+(V3)+mutation+determines+a+global+neutralization+phenotype+and+CD4-independent+infectivity+of+a+human+immunodeficiency+virus+type+1+envelope+associated+with+a+broadly+cross-reactive,+primary+virus-neutralizing+antibody+response.">A variable region 3 (V3) mutation determines a global neutralization phenotype and CD4-independent infectivity of a human immunodeficiency virus type 1 envelope associated with a broadly cross-reactive, primary virus-neutralizing antibody response.</a> J Virol. 76, 644-655 (2002).</li></ol>

Ab initio katlama üçüncül yapısal özellikleri, bireysel amino asitler sırayla okuyan belirgin olmayabilir olan etkilerini ortaya koymaktadır. R2 V3 döngü taç önceden tanınmayan birçok yapısal özellikleri, katlama simülasyonu ile ortaya çıkar. Bu gözlenen yapısal tercihleri, yani R2 zorlanma, nötralizasyon ve hyperinfectivity 5 duyarlılık gözlenen fonksiyonel özellikleri ile ilişkili olabilir . İlk olarak, pozisyonları 12-14 R2 V3 taç N-termin...

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assessment of immunologically relevant dynamic tertiary structural features of the hiv-1 v3 loop crown r2 sequence by ab initio folding

HIV-1 antijenik çeşitliliğinin uzun aşı tasarımı için bir engel olmuştur ve bu değişkenliği, özellikle virüs yüzey zarf glikoprotein V3 döngü içinde telaffuz edilir. Biz daha önce V3 döngü taç rağmen, dinamik ve sıra değişken, HIV-1 virüs immünolojik ilgili bir β-saç tokası üçüncül yapısı nüfus boyunca kısıtlı olduğunu önerdi. Önemlisi, farklı V3 döngü taç dizileri binlerce kristalografisi veya NMR ile zor veya imkansız virüslerin çeşitlilik trendleri 3D yapısal karakterizasyonu, HIV-1 virüsleri vardır. Bizim daha önceki başarılı çalışmaları V3 taç 1, 2, katlama, ICM-Pro moleküler modelleme yazılım paketi (Molsoft LLC, La Jolla, CA) erişilebilir ab initio algoritması 3 kullanılan ve önerilen V3 döngü taç , özellikle 10 ila 22 pozisyonları, esneklik ve serbestçe çözüm katlanır kısıtsız bir peptid sanki büyük ölçüde davranmaya, yan gövde uzunluğu yeterince yararlanıyor. + 60,000 herhangi bir bireysel suşu V3 döngü sadece bu bölümü, hızlı ab initio katlama gibi dolaşan HIV-1 suşlarının bilgilendirici olabilir. Burada, R2 suşu V3 döngü eşsiz özellikleri yapısal olarak içgörü kazanmak için katlanmış. R2, hasta serumu ve monoklonal antikorlar, 4, 5 bu zorlanma nötralizasyon için bu hassasiyeti sorumlu olduğu düşünülen nadir bir V3 döngü sırası taşımaktadır. Zorlanma, CD4-bağımsız enfeksiyon aracılık ve nötralize edici antikorlar ortaya çıkarmak için geniş görünür. Biz katlama sonuçlarının değerlendirilmesi katlama gözlenen yapılar R2 gözlenen immünolojik faaliyetleri ile ilişkilendirmek için ne kadar bilgilendirici olabilir göstermektedir.

Watch this Scientific Journal Video about Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding at JoVE.com

Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

Taç bölgesi yüzeyinde HIV-1 zarf glikoprotein (gp120) farklı V3 döngü dizilerinin yapısal pozisyonları siliko katlama birçok durumda karakterize edilebilir bir state-of-the-art kullanarak döngü 10 ila 22<em&gt; Ab initio</em&gt; Katlanır algoritması. Burada, HIV-1 R2 gerilme, şaşırtıcı fonksiyonel özellikleri ile benzersiz bir nötralizasyon hassas gerginlik V3 döngü bu bölgenin katlama ve değerlendirme göstermektedir.

HIV-1 V3 döngü Taç R2 Sıra immünolojik İlgili Dinamik Tersiyer Yapısal Özellikler Değerlendirilmesi Ab initio Katlanır

The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' ...

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Immunology and Infection

Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

9.5K Görüntüleme.  School of Medicine, New York University. Taç bölgesi yüzeyinde HIV-1 zarf glikoprotein (gp120) farklı V3 döngü dizilerinin yapısal pozisyonları siliko katlama birçok durumda karakterize edilebilir bir state-of-the-art kullanarak döngü 10 ila 22 Ab initio Katlanır algoritması. Burada, HIV-1 R2 gerilme, şaşırtıcı fonksiyonel özellikleri ile benzersiz bir nötralizasyon hassas gerginlik V3 döngü bu bölgenin katlama ve değerlendirme göstermektedir.

Video: Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

Yeşil Floresan Protein ifade Rev-bağımlı lentiviral Vector kullanarak HIV-1 Olumlu Hücreler Özgül İşareti

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

İmmünoloji ve Enfeksiyon

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.

HIV-1 tropizm genotipik Çıkarım kullanarak Nüfus tabanlı Sıralama 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 ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Standart Klinik Tahliller Algılama Sınırı Aşağıda Viral Yükler HIV enfekte kişilerde kuvvetlendirme ve HIV-1 RNA miktarının

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

Cryo-Elektron Mikroskopi tarafından HIV-1 kapsid Meclisleri Yapısı ve iteratif Helisel Gerçek uzay İmar

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. 
The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 20111.
For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3)2. This region carries enough information to perform a reliable prediction. 
The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL&lt;1000 copies/&mu;l roughly increased in the last years (Fig. 3).
The main steps for tropism testing (Fig. 4) demonstrated in this video: 
1. Collection of a blood sample 
 2. Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells 
 3. Amplification of the env region 
 4. Amplification of the V3 region 
 5. Sequence reaction of the V3 amplicon 
 6. Purification of the sequencing samples 
 7. Sequencing the purified samples 
 8. Sequence editing 
 9. Sequencing data interpretation and tropism prediction

Prediction of HIV-1 Coreceptor Usage Tropism by Sequence Analysis using a Genotypic Approach

The prediction of the coreceptor usage of HIV-1 is required for the administration of a new class of antiretroviral drugs, i.e. coreceptor antagonists. It can be performed by sequence analysis of the env gene and subsequent interpretation through an internet based interpretation system (geno2pheno[coreceptor]).

Bir Genotipik Yöntemi&#39;ne Dizi Analizi ile HIV-1 coreceptor Kullanımı (tropizm) Tahmin

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by ...

In vitro Uncoating of HIV-1 Cores

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 composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

HIV-1 Çekirdek in vitro Uncoating

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 ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 Enfekte CD4 Doğuştan Algılama Değerlendirilmesi<sup&gt; +</sup&gt; Bir kullanılması plazmositoid dendritik hücreler tarafından T Hücreleri<em&gt; Ex vivo</em&gt; Co-kültür sistemi.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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.

Bir Hücre temelli bir ELISA testi kullanarak, HIV-1 zarf trimerik Glikoproteinler konformasyonel Değerlendirilmesi

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

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

Nucleocapsid Tavlama Dolayımlı Elektroforez (ADI) Testi HIV-1 Nükieokapsit inhibitörlerinin Hızlı tanımlanması sağlar

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

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.

HIV-1 pozitif Bireylerin Plazma eksozomlann izolasyonu

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. ...