Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America.
Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, United States of America.
PLoS Pathog. 2019 Apr 4;15(4):e1007610. doi: 10.1371/journal.ppat.1007610. eCollection 2019 Apr.
The presence of bottlenecks in the transmission cycle of many RNA viruses leads to a severe reduction of number of virus particles and this occurs multiple times throughout the viral transmission cycle. Viral replication is then necessary for regeneration of a diverse mutant swarm. It is now understood that any perturbation of the mutation frequency either by increasing or decreasing the accumulation of mutations in an RNA virus results in attenuation of the virus. To determine if altering the rate at which a virus accumulates mutations decreases the probability of a successful virus infection due to issues traversing host bottlenecks, a series of mutations in the RNA-dependent RNA polymerase of Venezuelan equine encephalitis virus (VEEV), strain 68U201, were tested for mutation rate changes. All RdRp mutants were attenuated in both the mosquito and vertebrate hosts, while showing no attenuation during in vitro infections. The rescued viruses containing these mutations showed some evidence of change in fidelity, but the phenotype was not sustained following passaging. However, these mutants did exhibit changes in the frequency of specific types of mutations. Using a model of mutation production, these changes were shown to decrease the number of stop codons generated during virus replication. This suggests that the observed mutant attenuation in vivo may be due to an increase in the number of unfit genomes, which may be normally selected against by the accumulation of stop codons. Lastly, the ability of these attenuated viruses to transition through a bottleneck in vivo was measured using marked virus clones. The attenuated viruses showed an overall reduction in the number of marked clones for both the mosquito and vertebrate hosts, as well as a reduced ability to overcome the known bottlenecks in the mosquito. This study demonstrates that any perturbation of the optimal mutation frequency whether through changes in fidelity or by alterations in the mutation frequency of specific nucleotides, has significant deleterious effects on the virus, especially in the presence of host bottlenecks.
许多 RNA 病毒的传播周期中存在瓶颈，这导致病毒颗粒数量严重减少，而且这种情况在整个病毒传播周期中会多次发生。病毒复制对于再生多样化的突变体群是必要的。现在人们已经了解到，任何对突变频率的干扰，无论是通过增加还是减少 RNA 病毒中突变的积累，都会导致病毒衰减。为了确定改变病毒积累突变的速度是否会由于穿越宿主瓶颈而降低病毒成功感染的概率，研究人员对委内瑞拉马脑炎病毒（VEEV）株 68U201 的 RNA 依赖性 RNA 聚合酶（RdRp）进行了一系列突变，以测试其突变率的变化。所有 RdRp 突变体在蚊子和脊椎动物宿主中均表现出衰减，而在体外感染中则没有衰减。含有这些突变的拯救病毒在体外感染中没有表现出衰减，但在传代后没有表现出持续的表型。然而，这些突变体确实表现出了某些保真度变化的证据。使用突变产生模型，这些变化表明在病毒复制过程中产生的终止密码子数量减少。这表明，在体内观察到的突变体衰减可能是由于不适应基因组的数量增加，而这些基因组通常会被终止密码子的积累所选择。最后，使用标记病毒克隆测量了这些衰减病毒在体内通过瓶颈的能力。衰减病毒在蚊子和脊椎动物宿主中标记克隆的数量总体减少，以及在蚊子中已知瓶颈处的克服能力降低。这项研究表明，任何对最佳突变频率的干扰，无论是通过保真度的变化还是通过特定核苷酸突变频率的改变，都会对病毒产生显著的有害影响，尤其是在存在宿主瓶颈的情况下。
