Álvarez Ángel L, García-Manso Alberto, Dalton Kevin P, Martín-Alonso José M, Nicieza Inés, Podadera Ana, Acosta-Zaldívar Maikel, de Llano Daniel, Parra Francisco
Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Biotecnología de Asturias (IUBA), Universidad de Oviedo, Oviedo, Spain.
Front Microbiol. 2020 Nov 13;11:596245. doi: 10.3389/fmicb.2020.596245. eCollection 2020.
Most caliciviruses are refractory to replication in cell culture and only a few members of the family propagate . (RaV) is unique due to its ability to grow to high titers in several animal and human cell lines. This outstanding feature makes RaV an ideal candidate for reverse genetics studies, an invaluable tool to understand the molecular basis of virus replication, the biological functions of viral genes and their roles in pathogenesis. The recovery of viruses from a cDNA clone is a prerequisite for reverse genetics studies. In this work, we constructed a RaV infectious cDNA clone using a plasmid expression vector, under the control of bacteriophage T7 RNA-polymerase promoter. The transfection of permissive cells with this plasmid DNA in the presence of T7 RNA-polymerase, provided by a helper recombinant poxvirus, led to synthesis of RNA transcripts that emulated the viral genome. The RaV progeny virus produced the typical virus-induced cytopathic effect after several passages of cell culture supernatants. Similarly, infectious RaV was recovered when the transcription step was performed , prior to transfection, provided that a 5'-cap structure was added to the 5' end of synthetic genome-length RNAs. In this work, we report an efficient and consistent RaV rescue system based on a cDNA transcription vector, as a tool to investigate calicivirus biology through reverse genetics.
大多数杯状病毒在细胞培养中难以复制，该病毒科中只有少数成员能够增殖。兔出血症病毒（RaV）因其能够在多种动物和人类细胞系中生长至高滴度而独具特色。这一突出特性使RaV成为反向遗传学研究的理想候选对象，反向遗传学研究是了解病毒复制分子基础、病毒基因生物学功能及其在发病机制中作用的宝贵工具。从cDNA克隆中拯救出病毒是反向遗传学研究的前提条件。在本研究中，我们使用质粒表达载体，在噬菌体T7 RNA聚合酶启动子的控制下构建了一个RaV感染性cDNA克隆。在辅助重组痘苗病毒提供的T7 RNA聚合酶存在的情况下，用该质粒DNA转染允许细胞，导致合成模拟病毒基因组的RNA转录本。细胞培养上清液经过几次传代后，RaV子代病毒产生了典型的病毒诱导细胞病变效应。同样，当在转染前进行转录步骤时，如果在合成的基因组长度RNA的5'端添加5'-帽结构，也能拯救出感染性RaV。在本研究中，我们报告了一种基于cDNA转录载体的高效且稳定的RaV拯救系统，作为通过反向遗传学研究杯状病毒生物学的工具。
