Novel functional hepatitis C virus glycoprotein isolates identified using an optimized viral pseudotype entry assay.
Novel functional hepatitis C virus glycoprotein isolates identified using an optimized viral pseudotype entry assay.
复制标题
DOI：
10.1099/jgv.0.000537
复制
发表时间：
2016-09
期刊：
影响因子：
--
通讯作者：
Tarr AW
中科院分区：
文献类型：
--
作者：
Urbanowicz RA;McClure CP;King B;Mason CP;Ball JK;Tarr AW
逆转录病毒假型是用于研究包膜病毒进入途径的高度易处理的模型。该模型已被广泛应用于丙型肝炎病毒（HCV）进入途径的研究，抗病毒抗体的临床前筛选和使用具有HCV E1和E2糖蛋白的HCV假颗粒（HCVpp）评估患者来源病毒的表型。然而，并非所有患者分离的克隆都产生在该模型中具有感染性的颗粒。本研究调查了可能限制患者分离的HCV糖蛋白表型的因素。发现个体患者准种中的遗传相关HCV糖蛋白在该进入模型中表现非常不同。经验优化的包装构建体和糖蛋白编码质粒的比例是成功的HCVpp的成因所需的不同的克隆。逆转录病毒包装结构的选择也影响了HCV假颗粒的功能。一些糖蛋白构建体耐受广泛的测定参数，而其他糖蛋白构建体对改变更敏感。此外，先前被表征为不能介导进入的糖蛋白被发现是功能性的。这些发现得到了验证，使用嵌合细胞培养的HCV轴承这些糖蛋白。使用相同的经验方法，我们证明了感染性埃博拉病毒假病毒（EBOVpv）的产生也对所用质粒的量和比例敏感，并且这些假病毒的最佳生产方案取决于确切的病毒糖蛋白构建体。这些发现表明，对于利用假病毒的研究而言，对每个特定糖蛋白序列进行假型生产的经验优化以实现最佳滴度并促进准确的表型分型至关重要。
Retrovirus pseudotypes are a highly tractable model used to study the entry pathways of enveloped viruses. This model has been extensively applied to the study of the hepatitis C virus (HCV) entry pathway, preclinical screening of antiviral antibodies and for assessing the phenotype of patient-derived viruses using HCV pseudoparticles (HCVpp) possessing the HCV E1 and E2 glycoproteins. However, not all patient-isolated clones produce particles that are infectious in this model. This study investigated factors that might limit phenotyping of patient-isolated HCV glycoproteins. Genetically related HCV glycoproteins from quasispecies in individual patients were discovered to behave very differently in this entry model. Empirical optimization of the ratio of packaging construct and glycoprotein-encoding plasmid was required for successful HCVpp genesis for different clones. The selection of retroviral packaging construct also influenced the function of HCV pseudoparticles. Some glycoprotein constructs tolerated a wide range of assay parameters, while others were much more sensitive to alterations. Furthermore, glycoproteins previously characterized as unable to mediate entry were found to be functional. These findings were validated using chimeric cell-cultured HCV bearing these glycoproteins. Using the same empirical approach we demonstrated that generation of infectious ebolavirus pseudoviruses (EBOVpv) was also sensitive to the amount and ratio of plasmids used, and that protocols for optimal production of these pseudoviruses are dependent on the exact virus glycoprotein construct. These findings demonstrate that it is crucial for studies utilizing pseudoviruses to conduct empirical optimization of pseudotype production for each specific glycoprotein sequence to achieve optimal titres and facilitate accurate phenotyping.
登录
查看更多内容
DOI：
10.1084/jem.20090766
发表时间：
2010-08-30
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Fafi-Kremer S;Fofana I;Soulier E;Carolla P;Meuleman P;Leroux-Roels G;Patel AH;Cosset FL;Pessaux P;Doffoël M;Wolf P;Stoll-Keller F;Baumert TF
通讯作者：
Baumert TF
影响因子：
4.8
作者：
Bartosch, B;Vitelli, A;Cosset, FL
通讯作者：
Cosset, FL
影响因子：
4.5
作者：
Bouard, David;Sandrin, Virginie;Cosset, Francois-Loic
通讯作者：
Cosset, Francois-Loic
影响因子：
3.8
作者：
Brown, RJP;Juttla, VS;Ball, JK
通讯作者：
Ball, JK
影响因子：
13.5
作者：
Carlsen, Thomas H. R.;Pedersen, Jannie;Prentoe, Jannick C.;Giang, Erick;Keck, Zhen-Yong;Mikkelsen, Lotte S.;Law, Mansun;Foung, Steven K. H.;Bukh, Jens
通讯作者：
Bukh, Jens