Ultradeep single-molecule real-time sequencing of HIV envelope reveals complete compartmentalization of highly macrophage-tropic R5 proviral variants in brain and CXCR4-using variants in immune and peripheral tissues.
Ultradeep single-molecule real-time sequencing of HIV envelope reveals complete compartmentalization of highly macrophage-tropic R5 proviral variants in brain and CXCR4-using variants in immune and peripheral tissues.
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DOI：
10.1007/s13365-018-0633-5
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发表时间：
2018-08
影响因子：
3.2
通讯作者：
Lamers SL
中科院分区：
文献类型：
--
作者：
Brese RL;Gonzalez-Perez MP;Koch M;O'Connell O;Luzuriaga K;Somasundaran M;Clapham PR;Dollar JJ;Nolan DJ;Rose R;Lamers SL
尽管联合抗逆转录病毒治疗（cART），艾滋病毒+患者仍然发展神经系统疾病，这可能是由于持续的艾滋病毒感染和脑组织的选择性进化。单分子实时（SMRT）测序技术为研究大脑和淋巴组织中HIV分离株之间的关系提供了更好的机会，因为它能够在一次运行中生成数千个长序列读数。在这里，我们使用SMRT测序从来自HIV+/cART+受试者的7个尸检组织中生成约50，000个高质量全长HIV包膜序列（> 2200 bp），包括3个脑和4个非脑位点。桑格测序用于与SMRT数据进行比较，并克隆功能性假病毒用于体外嗜性测定。系统发育分析表明，脑源性艾滋病毒与脑外艾滋病毒区分开来，并且来自三种脑组织的变体独立分组。来自所有外周组织的变体在树上混合，但独立于脑分支。由于大量的序列，聚类分析在三个相似性阈值（99%，99.5%和99.9%）进行。在所有阈值下，所有脑序列聚类排除任何非脑序列;然而额叶序列独立于枕叶和顶叶聚类。翻译的序列揭示了脑和非脑序列在推定的N-连接糖基化位点（N-位点）的位置，V1长度，V3电荷和V4 N-位点的数量之间的潜在功能差异。预测所有脑序列使用CCR 5共受体，而预测大多数非脑序列使用CXCR 4共受体。通过体外感染试验证实了趋向性结果。这项研究是第一个使用SMRT测序方法来研究组织中的HIV区室化，并支持其他关于cART期间大脑和非大脑序列之间有限贩运的报告。由于序列长度较长，我们可以观察到整个包膜基因的沿着变化，这可能是由大脑中的不同选择压力引起的，可能有助于神经疾病。
Despite combined anti-retroviral therapy (cART), HIV+ patients still develop neurological disorders, which may be due to persistent HIV infection and selective evolution in brain tissues. Single-molecule real-time (SMRT) sequencing technology offers an improved opportunity to study the relationship among HIV isolates in the brain and lymphoid tissues because it is capable of generating thousands of long sequence reads in a single run. Here, we used SMRT sequencing to generate ~50,000 high-quality full-length HIV envelope sequences (>2200bp) from 7 autopsy tissues from an HIV+/cART+ subject, including three brain and four non-brain sites. Sanger sequencing was used for comparison with SMRT data and to clone functional pseudoviruses for in vitro tropism assays. Phylogenetic analysis demonstrated that brain-derived HIV was compartmentalized from HIV outside the brain and that the variants from each of the three brain tissues grouped independently. Variants from all peripheral tissues were intermixed on the tree, but independent of the brain clades. Due to the large number of sequences, a clustering analysis at three similarity thresholds (99%, 99.5% and 99.9%) was also performed. All brain sequences clustered exclusive of any non-brain sequences at all thresholds; however frontal lobe sequences clustered independently of occipital and parietal lobes. Translated sequences revealed potentially functional differences between brain and non-brain sequences in the location of putative N-linked glycosylation sites (N-sites), V1 length, V3 charge and the number of V4 N-sites. All brain sequences were predicted to use the CCR5 co-receptor, while most non-brain sequences were predicted to use CXCR4 co-receptor. Tropism results were confirmed by in vitro infection assays. The study is the first to use a SMRT sequencing approach to study HIV compartmentalization in tissues and supports other reports of limited trafficking between brain and non-brain sequences during cART. Due to the long sequence length, we could observe changes along the entire envelope gene, likely caused by differential selective pressure in the brain, that may contribute to neurological disease.
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影响因子：
3.9
作者：
Clifford DB
通讯作者：
Clifford DB
影响因子：
6.7
作者：
Beauparlant D;Rusert P;Magnus C;Kadelka C;Weber J;Uhr T;Zagordi O;Oberle C;Duenas-Decamp MJ;Clapham PR;Metzner KJ;Günthard HF;Trkola A
通讯作者：
Trkola A
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作者：
Duenas-Decamp, Maria Jose;Peters, Paul J.;Clapham, Paul R.
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Clapham, Paul R.
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6.4
作者：
BELL, JE;BUSUTTIL, A;PEUTHERER, JF
通讯作者：
PEUTHERER, JF
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2.7
作者：
Gatanaga, H;Oka, S;Iwamoto, A
通讯作者：
Iwamoto, A