Retrovirus Biology

a.在前列腺肿瘤中寻找XMRV最近几篇论文报道了在人类前列腺肿瘤中检测到一种新的MLV，异嗜性小鼠白血病病毒相关病毒（XMRV）（例如，Urisman et al., PLoS Pathog. 2: 25, 2006; schlabberg et al., PNAS 106:16351-16356, 2009）。在与博士合作。来自约翰霍普金斯大学医学院的Karen Sfanos和Angelo De Marzo，我们使用双工实时PCR和免疫组织化学（IHC）相结合的方法对近800例前列腺肿瘤进行了XMRV检测。我们的研究使用已知感染XMRV的22Rv1细胞（Knouf et al.， J. Virol. 83: 7353-7356, 2009）作为阳性对照，而未感染的人类细胞，如293T或HeLa细胞作为阴性对照。每个PCR孔同时分析病毒序列和单拷贝人类基因CCR5，以检验样品DNA的质量和数量。将22Rv1 DNA稀释到HeLa DNA中，我们可以在1.5个22Rv1细胞中检测到XMRV序列，即使存在超过10,000倍的HeLa细胞。我们的免疫组化实验使用的是兔抗血清，分别针对hplc纯化的Moloney MLV p30CA和hplc纯化的Moloney MLV gp70SU。对照不仅包括22Rv1，还包括转染了XMRV分子克隆或模拟转染的293T细胞。在所有情况下，22Rv1细胞和xmrv转染的细胞都被两种抗血清染色，而阴性细胞则没有。这些控制良好的试验都没有在任何前列腺肿瘤样本中显示XMRV感染的任何迹象。我们得出结论（Aloia et al., Cancer Res. 70:10028-10033, 2010），其他实验室的阳性报告可能是假阳性。例如，PCR阳性结果可能是由微量小鼠DNA污染引起的，如果抗血清与宿主细胞以及病毒抗原发生反应，则免疫组化阳性结果可能出现。随后的研究表明，XMRV最初是在22Rv1肿瘤系的异种移植过程中形成的，并且几乎可以肯定人类不会被这种病毒感染（Paprotka等人，Science 333:97-101, 2011； Sfanos等人，Nat. Rev. urrol . 9:111-118, 2012）。一种新的MLV感染检测方法的开发和iGLuc感染检测策略的进一步应用作为我们对XMRV继续研究的一部分，我们采用了一种逆转录法（Curcio和Garfinkel， PNAS 88:936- 940,1991； Heidmann等人，PNAS 85:2219- 2223,1988）来检测具有复制能力的γ -逆转录病毒。在该试验中（称为“iGLuc”），将基于mlv的载体放入293细胞中。载体携带反向的高斯荧光素酶基因，被正向的内含子打断。只有当这个载体被一个有能力的辅助病毒拯救并通过整个逆转录病毒生命周期时，才会表达高斯荧光素酶。这项工作是与博士合作完成的。Gisela Heidecker， Vineet KewalRamani和已故的David Derse （HIV耐药项目，国家癌症研究所；见Mazurov等人，PLoS Pathog. 6:e1000788, 2010）。虽然其他报告者也可用于此分析，但高斯荧光素酶具有独特的优势：它非常明亮，使分析非常敏感，并且它是分泌的，因此可以直接在培养液上进行分析。它很容易以96井的形式进行。与博士一起。Sfanos和De Marzo，我们使用PCR和IHC方法筛选了70个广泛使用的细胞系，以检测mlv的存在（Sfanos等人，PLoS One 6:e20874, 2011）。发现有3个细胞系产生了具有复制能力的mlv。这些病毒具有相当详细的特征：发现其中两种源自内源性MLV Bxv-1，而第三种与另一种异向性MLV有关。iGLuc实验证实了它们的复制能力。在某些情况下，在用于建立细胞系的异种移植传代过程中，mlv感染了人类细胞；在其他实验室，感染可能是发生在其他实验室的交叉污染。我们相信“iGLuc”策略在检测逆转录病毒复制方面具有广泛的应用前景。我们已经与Kenneth Cornetta博士（印第安纳大学）合作，证明了iGLuc测定在检测复制能力γ -逆转录病毒基因治疗载体制备方面的效用（Aloia等人，基因治疗，出版于2012年），我们打算对基于hiv -1的基因治疗载体做同样的工作。与Heidecker博士合作，我们正在开发用于检测小鼠乳腺肿瘤病毒的试剂；一种快速、灵敏的感染试验将极大地促进该病毒的体外生物学研究。几年前，我们和NCI-Frederick的同事描述了一种小分子的HIV-1进入抑制剂，命名为“Stibavirin”或NSC13778 （Yang et al.， J. Virol. 79: 6112 -6133, 2005）。这种化合物已经被fda批准用于其他用途。为了进一步了解其作用机制，匹兹堡大学的John Mellors博士现在选择了部分耐药的HIV-1突变体。我们已经证明，他在gp120的V3环内和附近观察到的变化是造成这种抗性的原因。几种数据表明，Stibavirin通过与CD4结合来阻止进入，阻止gp120结合。这种类型的抑制似乎可能是抗病毒治疗的有用补充。耐药性的机制正在通过分子模型与博士合作进行分析。Rick Gussio和Tam Nguyen（国家癌症研究所发展治疗项目）。[对应2011年10月HIV耐药项目实地考察报告中的Rein项目4]

a. Search for XMRV in prostate tumorsSeveral recent papers reported the detection of a new MLV, xenotropic murine leukemia virus-related virus (XMRV), in human prostate tumors (e.g., Urisman et al., PLoS Pathog. 2:e25, 2006; Schlaberg et al., PNAS 106:16351-16356, 2009). In collaboration with Drs. Karen Sfanos and Angelo De Marzo (Johns Hopkins School of Medicine), we surveyed nearly 800 prostate tumors for XMRV, using a combination of duplex real-time PCR and immunohistochemistry (IHC). Our studies used 22Rv1 cells, which are known to be infected with XMRV (Knouf et al., J. Virol. 83: 7353-7356, 2009), as positive controls, and uninfected human cells, such as 293T or HeLa cells, as negatives. Every PCR well was simultaneously analyzed for viral sequences and for CCR5, a single-copy human gene, as a test of the quality and quantity of sample DNA. Dilution of 22Rv1 DNA into HeLa DNA showed that we could detect the XMRV sequences in 1.5 22Rv1 cells, even in the presence of a 10,000-fold excess of HeLa cells. Our IHC assays used a rabbit antiserum raised against HPLC-purified Moloney MLV p30CA and another against HPLC-purified Moloney MLV gp70SU. Controls included not only 22Rv1, but also 293T cells transfected with a molecular clone of XMRV or mock-transfected. In all cases, the 22Rv1 cells and the XMRV-transfected cells stained with both antisera whereas the negative cells did not.Neither of these well-controlled assays gave any indication of XMRV infection in any of the prostate tumor samples. We concluded (Aloia et al., Cancer Res. 70:10028-10033, 2010) that the positive reports from other laboratories likely represented false positives. For example, positive PCR results can result from contamination with minuscule amounts of mouse DNA, and positive IHC results can arise if the antisera react with host-cell, as well as viral, antigens. Subsequent developments have revealed that XMRV was originally formed during xenotransplantation of the 22Rv1 tumor line, and that humans are almost certainly not infected by this virus (Paprotka et al., Science 333:97-101, 2011; Sfanos et al., Nat. Rev. Urol. 9:111-118, 2012).b. Development of a new MLV infectivity assay and further applications of the iGLuc infectivity assay strategyAs part of our continuing studies on XMRV, we adapted a retrotransposition assay (Curcio and Garfinkel, PNAS 88:936-940, 1991; Heidmann et al., PNAS 85:2219-2223, 1988) for detection of replication-competent gammaretroviruses. In this assay (called "iGLuc"), an MLV-based vector is placed into 293 cells. The vector carries a Gaussia luciferase gene in reverse orientation, interrupted by an intron in forward orientation. Only when this vector has been rescued by a competent helper virus and has progressed through the entire retroviral life cycle is the Gaussia luciferase expressed. This work was performed in collaboration with Drs. Gisela Heidecker, Vineet KewalRamani, and the late David Derse (HIV Drug Resistance Program, National Cancer Institute; see Mazurov et al., PLoS Pathog. 6:e1000788, 2010).Although other reporters can be used in this assay, the Gaussia luciferase offers unique advantages: it is extremely bright, rendering the assay very sensitive, and it is secreted, so that the assay can be performed directly on culture fluids. It is easily performed in a 96-well format. Together with Drs. Sfanos and De Marzo, we screened 70 widely used cell lines for the presence of MLVs, using both PCR and IHC assays (Sfanos et al., PLoS One 6:e20874, 2011). Three lines were found to be producing replication-competent MLVs. The viruses were characterized in considerable detail: two were found to be derived from the endogenous MLV Bxv-1, whereas the third was related to another xenotropic MLV. Their replication-competence was confirmed by the iGLuc assay. In some cases, the MLVs infected the human cells during the xenotransplantation passages used to establish the cell lines; in others, the infections probably represent cross-contamination that occurred in other laboratories.We believe that the "iGLuc" strategy for detection of retroviral replication can have wide application. We have collaborated with Dr. Kenneth Cornetta (Indiana University) to demonstrate the utility of the iGLuc assay for testing gene-therapy vector preparations for replication-competent gammaretroviruses (Aloia et al., Gene Therapy, in press, 2012), and we intend to do the same for HIV-1-based gene-therapy vectors. In collaboration with Dr. Heidecker, we are developing the reagents for assaying mouse mammary tumor virus; a rapid, sensitive infectivity assay should greatly facilitate future in vitro studies on the biology of this virus.c. Characterization of an HIV-1 entry inhibitorSeveral years ago, we and colleagues at the NCI-Frederick described a small-molecule entry inhibitor of HIV-1 entry, named "Stibavirin" or NSC13778 (Yang et al., J. Virol. 79:6122-6133, 2005). This compound is already FDA-approved for other uses. To gain further insight into its mechanism of action, Dr. John Mellors (University of Pittsburgh) has now selected HIV-1 mutants partially resistant to the drug. We have shown that the changes he observed in and near the V3 loop of gp120 are responsible for this resistance. Several kinds of data show that Stibavirin blocks entry by binding to CD4, preventing gp120 from binding. It seems possible that this type of inhibition could be a useful addition to antiviral therapy. The mechanism of the resistance is being analyzed by molecular modeling in collaboration with Drs. Rick Gussio and Tam Nguyen (Developmental Therapeutics Program, National Cancer Institute).[Corresponds to Rein Project 4 in the October 2011 site visit report of the HIV Drug Resistance Program]