Viktorova Ekaterina G, Nchoutmboube Jules, Ford-Siltz Lauren A, Belov George A
Department of Veterinary Medicine, University of Maryland, College Park, Maryland, USA.
Department of Veterinary Medicine, University of Maryland, College Park, Maryland, USA Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, USA
J Virol. 2015 Apr;89(8):4372-86. doi: 10.1128/JVI.00055-15. Epub 2015 Feb 4.
It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses.
Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can easily generate mutants resistant to practically any compounds targeting viral proteins. An alternative approach is to target stable cellular factors recruited for the virus-specific functions. In the present study, we analyzed the factors permitting and restricting the establishment of the resistance of poliovirus, a small (+)RNA virus, to brefeldin A (BFA), a drug targeting a cellular component of the viral replication complex. We found that the emergence and replication potential of resistant mutants is cell type dependent and that BFA resistance reduces virus fitness. Our data provide a rational approach to the development of antiviral therapeutics targeting host factors.
据推测，靶向参与病毒复制的稳定细胞因子而非病毒特异性蛋白可能会提高耐药突变体产生的门槛，这对于适应性很强的小（+）RNA病毒尤为重要。然而，与这一假设相反，越来越多的证据表明，至少在某些系统中，这些病毒很容易产生对细胞蛋白抑制剂耐药的突变体。我们在此研究了脊髓灰质炎病毒对布雷菲德菌素A（BFA）产生耐药性的情况，BFA是一种细胞蛋白GBF1的抑制剂，GBF1是细胞小GTP酶Arf1的鸟嘌呤核苷酸交换因子。我们发现，虽然在HeLa细胞中很容易筛选出耐药病毒，但在Vero细胞中却不会出现，尽管在没有药物的情况下两种细胞培养物都能支持强劲的病毒复制。我们的数据表明，病毒复制对BFA的耐受性远高于细胞分泌途径的功能，这表明GBF1在病毒复制中的作用与其激活Arf的功能无关。我们证明，GBF1募集到复制复合物的水平限制了HeLa和Vero细胞中BFA耐药表型的建立和表达。此外，脊髓灰质炎病毒突变体的BFA耐药表型在不同的人源细胞类型中也具有细胞类型依赖性，并且在没有药物的情况下会以RNA复制效率降低的形式导致适应性损失。因此，开发靶向宿主的抗病毒药物的合理方法可能会克服（+）RNA病毒的超强适应性。
与病毒性疾病的数量相比，可用疫苗的数量微不足道。对于一些病毒，经过多次尝试疫苗开发仍未成功，而对于许多其他病毒，接种疫苗并非可行选择。临床实践和公共卫生紧急情况需要抗病毒药物。然而，病毒具有高度适应性，几乎可以轻易产生对任何靶向病毒蛋白的化合物耐药的突变体。另一种方法是靶向为病毒特异性功能募集的稳定细胞因子。在本研究中，我们分析了允许和限制小（+）RNA病毒脊髓灰质炎病毒对布雷菲德菌素A（BFA）产生耐药性的因素，BFA是一种靶向病毒复制复合物细胞成分的药物。我们发现耐药突变体的出现和复制潜力具有细胞类型依赖性，并且BFA耐药性会降低病毒适应性。我们的数据为开发靶向宿主因子的抗病毒疗法提供了合理方法。
