Retrovirus Biology

a.在前列腺肿瘤中搜索XMRV最近的几篇论文报道了在人前列腺肿瘤中检测到一种新的MLV，异嗜性鼠白血病病毒相关病毒（XMRV）（例如，乌里斯曼等人，PLoS Pathog. 2：e25，2006; Schlaberg等人，PNAS 106：16351-16356，2009）。我们与Karen Sfanos和Angelo De Marzo博士（约翰霍普金斯医学院）合作，使用双重实时PCR和免疫组织化学（IHC）相结合的方法，调查了近800例前列腺肿瘤的XMRV。我们的研究使用了已知被XMRV感染的22 Rv 1细胞（Knouf等人，J. Virol. 83：7353-7356，2009）作为阳性对照，未感染的人细胞如293 T或HeLa细胞作为阴性。同时分析每个PCR孔的病毒序列和CCR 5（一种单拷贝人类基因），以检测样品DNA的质量和数量。将22 Rv 1 DNA稀释到HeLa DNA中表明，即使在存在10，000倍过量的HeLa细胞的情况下，我们也可以在1.5个22 Rv 1细胞中检测到XMRV序列。我们的IHC试验使用了针对HPLC纯化的Moloney MLV p30 CA的兔抗血清和针对HPLC纯化的Moloney MLV gp 70 SU的兔抗血清。对照不仅包括22 Rv 1，而且包括用XMRV的分子克隆转染或模拟转染的293 T细胞。在所有情况下，22 Rv 1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。我们得出结论（Aloia等人，Cancer Res. 70：10028-10033，2010），来自其他实验室的阳性报告可能代表假阳性。例如，阳性PCR结果可由极少量的小鼠DNA污染产生，并且如果抗血清与宿主细胞以及病毒抗原反应，则可产生阳性IHC结果。随后的发展表明，XMRV最初是在22 Rv 1肿瘤系的异种移植过程中形成的，并且人类几乎肯定不会感染这种病毒（Paprotka等人，Science 333：97-101，2011; Sfanos等人，Nat. Rev. Urol. 9：111-118，2012）。新的MLV感染性测定的开发和iGLuc感染性测定策略的进一步应用作为我们对XMRV的持续研究的一部分，我们采用了反转录转座测定（Curcio和Garfinkel，PNAS 88：936-940，1991; Heidmann等人，PNAS 85：2219-2223，1988）用于检测有复制能力的γ逆转录病毒。在该测定法（称为“iGLuc”）中，将基于MLV的载体置于293细胞中。该载体携带反向的Gaussia荧光素酶基因，其被正向的内含子中断。只有当该载体被有能力的辅助病毒拯救并在整个逆转录病毒生命周期中进展时，Gaussia荧光素酶才能表达。这项工作是与Gisela Heidecker博士、Vineet KewalRamani博士和已故的大卫德尔塞博士合作进行的（HIV耐药性计划，国家癌症研究所;参见Mazurov等人，PLoS Pathog.尽管其他报告基因可用于该测定，但Gaussia荧光素酶提供了独特的优点：它非常明亮，使得测定非常灵敏，并且它是分泌的，使得该测定可直接在培养液上进行。它很容易在96孔格式中进行。我们与Sfanos和De Marzo博士一起，使用PCR和IHC测定筛选了70种广泛使用的细胞系中MLV的存在（Sfanos等人，PLoS One 6：e20874，2011）。发现三个品系产生有复制能力的MLV。对病毒进行了相当详细的表征：发现两种病毒来源于内源性MLV Bxv-1，而第三种病毒与另一种异嗜性MLV相关。通过iGLuc试验证实了它们的复制能力。在某些情况下，MLV在用于建立细胞系的异种移植传代过程中感染了人类细胞;在其他情况下，感染可能代表发生在其他实验室的交叉污染。我们相信，用于检测逆转录病毒复制的“iGLuc”策略可以具有广泛的应用。我们与Kenneth Cornetta博士（印第安纳州大学）合作，证明了iGLuc测定法用于检测基因治疗载体制剂对可复制γ逆转录病毒的效用（Aloia et al.，Gene Therapy，in press，2012），我们打算对基于HIV-1的基因治疗载体做同样的事情。在与Heidecker博士的合作中，我们正在开发用于检测小鼠乳腺肿瘤病毒的试剂;快速，灵敏的感染性检测将极大地促进未来对该病毒生物学的体外研究。几年前，我们和NCI-Frederick的同事描述了一种HIV-1进入的小分子进入抑制剂，命名为“Stibavirin”或NSC 13778（Yang et al.，J. Virol. 79：6122-6133，2005）。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，John Mellors博士（匹兹堡大学）现已选择了对该药物部分耐药的HIV-1突变体。我们已经证明，他在gp 120的V3环内和附近观察到的变化是造成这种阻力的原因。几种数据表明，Stibavirin通过与CD 4结合阻止gp 120结合来阻止进入。这种类型的抑制似乎可能是抗病毒治疗的有用补充。目前正在与Rick Gussio和Tam Nguyen博士（国家癌症研究所发展治疗计划）合作，通过分子建模分析耐药机制。[对应于2011年10月艾滋病毒耐药性项目现场访问报告中的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]