Host Cell Gene Expression During HIV-1 Latency in Chronically Infected Cell Lines. Homo sapiens

Cells: ACH-2, A3.01, J1.1, and U1 cells were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. U-937 cells were obtained from American Type Culture Collection (Manassas, VA). ACH-2, J1.1 and U1 are chronically infected cell lines harboring HIV-1 LAV strain, while A3.01, Jurkat, and U-937 are the corresponding parental uninfected cell lines. Cells were grown in RPMI-1640 (Invitrogen, San Diego, CA) with 10% fetal bovine serum (FBS, Invitrogen), 5% penicillin-streptomycin (Invitrogen), and 2mM glutamine (Invitrogen). Cells were maintained at a concentration of 1x10e6 cells/ml in T-175 flasks. Cell concentrations and cell viability were monitored throughout the experiment. Cells were harvested by centrifugation at 1000 rpm for 10 minutes. Harvested cells were washed thrice with ice-cold PBS to remove media; cell pellets were snap frozen using ethanol-dry ice mixture and stored at -80°C for subsequent RNA extraction. In order to compare expression profiles of chronically infected cell lines, ACH-2, U1, and J1.1 and their uninfected parental cell lines, A3.01, U-937 and Jurkat cells respectively, were grown under identical conditions but in presence of AZT (250 nM) in growth media and cells were harvested as described above. In these studies, no inducing agent was used in either chronically infected or uninfected parental cell lines so as to study changes in cellular gene expression cells latently infected with HIV and uninfected cells. Total RNA Extraction: Total RNA was extracted using RNEasy Midiprep Kits per manufacturer's protocol (Qiagen, Valencia, CA). RNA concentrations and purity were measured by spectrophotometry, RNA quality (absence of RNA degradation) was assessed by gel electrophoresis. RNA concentration was adjusted to the levels required for subsequent microarray experiment protocols by concentration in a SpeedVac (Savant Instruments, Holbrook, CA). RNA samples (6-7 µg/µL) were stored in 100 µL TE buffer at -80°C. Microarray Studies: Total RNA obtained from chronically infected and corresponding uninfected parental cells were used for microarray experiments. For each cell line, RNA from the chronically infected cells (ACH-2, J1.1 or U1)and RNA from the corresponding induced, uninfected cells (A3.01 , Jurkat or U937) were compared on the same array. Microarrays were obtained from the National Cancer Institute Microarray Facility, Advanced Technology Center (Gaithersburg, MD). The microarrays (Hs. UniGem2) contained 10,368 cDNA spots on each glass slide. The cDNAs were selected for spotting on the slides based on their known or probable involvement in oncogenesis, signal transduction, apoptosis, immune function, inflammatory pathways, cellular transport, transcription, protein translation and other important cellular functions. A number of expressed sequence tags (ESTs) from unknown genes homologous to known genes and cDNAs encoding housekeeping genes were also included in these gene sets. For each array, 50 µg of total RNA from chronically infected cells and 70 µg of total RNA from corresponding uninfected cells was labeled with Cy-3-dUTP and Cy-5-dUTP respectively. Higher amounts of RNA were used for Cy-5 labeling to minimize the disparities in dye incorporation. Following reverse transcription, the labeled cDNAs were then combined and purified using MicroCon YM-30 (Millipore, Bedford, MA) spin column filters, to remove any unincorporated nucleotides. 8-10 µg each of Cot-1 DNA, (Boehringer Mannheim, Indianapolis, IN), yeast tRNA (Sigma) and polyA (Amersham Biosciences) were added to the reaction mixture and heated at 100°C for 1 minute. Hybridization of the labeled cDNA to the microarray was carried out at 65°C overnight, followed by washes with 1X SSC, 0.2X SSC and 0.05X SSC respectively. The slides were dried by centrifugation at 1000 rpm for 3 minutes and then scanned as described below. Microarray Scanning and Data Analysis: The slides were scanned using an Axon GenePix 4000 scanner (Axon Instruments, Union City, CA). The photomultiplier tube values (PMT) were adjusted to obtain equivalent intensities at both wavelengths used, 635 nm and 532 nm for the Cy5 and Cy3 channels respectively. Image analysis was performed using GenePix analysis software (Axon Instruments) and data analysis was performed using the microArray Database (mAdb) system hosted by the Center for Information Technology and Center for Cancer Research at NIH (http://nciarray.nci.nih.gov/). Keywords = HIV Keywords = Latency Keywords: other

细胞系与细胞培养：ACH-2、A3.01、J1.1及U1细胞均购自美国国立卫生研究院（National Institutes of Health, NIH）艾滋病研究与参考试剂项目（NIH AIDS Research and Reference Reagent Program），隶属于国家过敏和传染病研究所（National Institute of Allergy and Infectious Diseases, NIAID）旗下艾滋病分部。U-937细胞购自美国典型培养物保藏中心（American Type Culture Collection, ATCC，马纳萨斯，弗吉尼亚州）。其中ACH-2、J1.1和U1为携带HIV-1 LAV毒株的慢性感染细胞系，A3.01、Jurkat及U-937则为对应的未感染亲本细胞系。
细胞均培养于RPMI-1640培养基（Invitrogen公司，圣地亚哥，加利福尼亚州），培养基添加10%胎牛血清（fetal bovine serum, FBS，Invitrogen）、5%青霉素-链霉素（Invitrogen）及2mM谷氨酰胺（Invitrogen）。细胞在T-175培养瓶中维持于1×10^6 个细胞/毫升的浓度。实验全程监测细胞浓度与细胞活力。通过1000 rpm离心10分钟收集细胞，收集的细胞用预冷磷酸盐缓冲液（phosphate-buffered saline, PBS）洗涤三次以去除培养基，随后使用乙醇-干冰混合物将细胞沉淀速冻，并保存于-80℃以待后续RNA提取。
为比较慢性感染细胞系与其未感染亲本细胞系的表达谱，ACH-2、U1和J1.1分别与其对应未感染亲本细胞系A3.01、U-937及Jurkat在相同条件下培养，但培养基中添加了250 nM的齐多夫定（azidothymidine, AZT），随后按照前述方法收集细胞。本研究未在慢性感染或未感染亲本细胞系中使用诱导剂，以探究HIV潜伏感染细胞与未感染细胞的细胞基因表达变化。
总RNA提取：总RNA提取采用RNEasy Midiprep试剂盒，严格按照制造商说明书操作（Qiagen公司，瓦伦西亚，加利福尼亚州）。通过分光光度法测定RNA浓度与纯度，通过凝胶电泳评估RNA质量（确认无RNA降解）。使用SpeedVac真空浓缩仪（Savant Instruments公司，霍尔布鲁克，加利福尼亚州）将RNA浓度调整至后续微阵列实验所需水平。RNA样品（浓度6-7 µg/µL）溶于100 µL TE缓冲液中，保存于-80℃。
微阵列实验：从慢性感染细胞及对应未感染亲本细胞中提取的总RNA用于微阵列实验。对于每一组细胞系，将慢性感染细胞（ACH-2、J1.1或U1）的RNA与对应未感染亲本细胞（A3.01、Jurkat或U937）的RNA在同一微阵列芯片上进行杂交比较。微阵列芯片购自美国国家癌症研究所微阵列设施中心、先进技术中心（Gaithersburg，马里兰州）。本次使用的Hs. UniGem2微阵列芯片每张玻片包含10368个cDNA斑点，这些cDNA基于其已知或可能参与肿瘤发生、信号转导、细胞凋亡、免疫功能、炎症通路、细胞转运、转录、蛋白质翻译及其他重要细胞功能而被挑选用于芯片点样。本基因集还包含了与已知基因同源的未知基因表达序列标签（expressed sequence tags, ESTs）以及编码持家基因的cDNA。
对于每张芯片，分别使用Cy-3-dUTP和Cy-5-dUTP标记50 µg慢性感染细胞的总RNA，以及70 µg对应未感染细胞的总RNA。为减少染料掺入差异，Cy-5标记使用了更高量的RNA。逆转录后，将标记的cDNA混合，使用MicroCon YM-30离心柱过滤器（Millipore公司，贝德福德，马萨诸塞州）纯化以去除未掺入的核苷酸。向反应体系中加入各8-10 µg的Cot-1 DNA（Boehringer Mannheim公司，印第安纳波利斯，印第安纳州）、酵母tRNA（Sigma公司）及polyA（Amersham Biosciences公司），并于100℃加热1分钟。将标记的cDNA与微阵列芯片进行杂交，杂交条件为65℃过夜，随后依次使用1X 柠檬酸钠缓冲液（saline-sodium citrate, SSC）、0.2X SSC及0.05X SSC进行洗涤。玻片经1000 rpm离心3分钟干燥后，按照下述方法进行扫描。
微阵列扫描与数据分析：使用Axon GenePix 4000扫描仪（Axon Instruments公司，尤宁城，加利福尼亚州）扫描芯片。调整光电倍增管（photomultiplier tube, PMT）参数，使Cy5通道（635 nm）与Cy3通道（532 nm）的信号强度相当。使用GenePix分析软件（Axon Instruments）进行图像分析，使用由美国国立卫生研究院信息技术中心与癌症研究中心托管的微阵列数据库（microArray Database, mAdb）系统进行数据分析（http://nciarray.nci.nih.gov/）。
关键词：HIV；潜伏；其他