克里米亚-刚果出血热病毒(CCHFV),Orthonairovirus属的蜱传播病毒,持续感染蜱细胞。据报道,在非人类灵长类动物中建立了持续感染,但病毒学分析尚未在人类细胞中进行。这里,我们研究了是否以及如何使用Hazara正负病毒(HAZV)持续感染人类细胞,CCHFV的代理模型。我们建立了持续感染HAZV的人细胞系。令人惊讶的是,在培养上清液中未观察到持续感染的HAZV(HAZVpi)的病毒体。HAZVpi的L蛋白中存在5个突变(mut1、mut2、mut3、mut4和mut5)。HAZVpi的L蛋白中的突变有助于在上清液中检测不到病毒体。Lmut4被发现导致低病毒生长速率,尽管它的聚合酶活性很高。Lmut2、Lmut3和Lmut5恢复了低生长速率。Lmut1的聚合酶活性极低,并且携带Lmut1的重组HAZV(rHAZV/Lmut1)不释放到上清液中。然而,rHAZV/Lmut1的基因组保留在感染的细胞中。HAZVpi的L蛋白中发现的所有突变(Lmut1-5)都是HAZVpi的实验性繁殖所必需的,只有Lmut1和Lmut4是不够的。我们证明了病毒聚合酶中的点突变有助于持续HAZV感染的建立。此外,在HAZVpi感染的细胞中发现先天免疫受到抑制,这也可能有助于病毒的持久性。这是nairovirus如何在人细胞中建立持续感染背后的可能机制的首次呈现。重要性我们研究了是否以及如何持续感染人体细胞,使用Hazara正交流感病毒(HAZV),克里米亚-刚果出血热病毒的替代模型。我们建立了持续感染HAZV的人细胞系。在持续感染的HAZV(HAZVpi)的L蛋白中发现了五个突变:mut1,mut2,mut3,mut4和mut5。其中,Lmut1和Lmut4通过低聚合酶活性和低生长速率限制病毒生长,分别,导致抑制病毒过度生长。由Lmut1和Lmut4引起的病毒生长的限制被其他突变所补偿,包括Lmut2、Lmut3和Lmut5。在HAZVpi的L蛋白中发现的每个突变都被认为共同调节病毒生长,这有助于持续感染的建立。先天免疫的抑制也可能有助于病毒的持久性。这是nairovirus如何在人细胞中建立持续感染背后的可能机制的首次呈现。
Crimean-Congo hemorrhagic fever virus (CCHFV), a tick-borne virus of the Orthonairovirus genus, persistently infects tick cells. It has been reported to establish persistent infection in non-human primates, but virological analysis has not yet been performed in human cells. Here, we investigated whether and how nairoviruses persistently infect human cells using Hazara orthonairovirus (HAZV), a surrogate model for CCHFV. We established a human cell line that was persistently infected with HAZV. Surprisingly, virions of persistently infected HAZV (HAZVpi) were not observed in the culture supernatants. There were five mutations (mut1, mut2, mut3, mut4, and mut5) in L protein of HAZVpi. Mutations in L protein of HAZVpi contribute to non-detection of virion in the supernatants. Lmut4 was found to cause low viral growth rate, despite its high
polymerase activity. The low growth rate was restored by Lmut2, Lmut3, and Lmut5. The
polymerase activity of Lmut1 was extremely low, and recombinant HAZV carrying Lmut1 (rHAZV/Lmut1) was not released into the supernatants. However, genomes of rHAZV/Lmut1 were retained in the infected cells. All mutations (Lmut1-5) found in L protein of HAZVpi were required for experimental reproduction of HAZVpi, and only Lmut1 and Lmut4 were insufficient. We demonstrated that point mutations in viral
polymerase contribute to the establishment of persistent HAZV infection. Furthermore, innate immunity was found to be suppressed in HAZVpi-infected cells, which also potentially contributes to viral persistence. This is the first presentation of a possible mechanism behind how nairoviruses establish persistent infection in human cells.
OBJECTIVE: We investigated whether and how nairoviruses persistently infect human cells, using Hazara orthonairovirus (HAZV), a surrogate model for Crimean-Congo hemorrhagic fever virus. We established a human cell line that was persistently infected with HAZV. Five mutations were found in L protein of persistently infected HAZV (HAZVpi): mut1, mut2, mut3, mut4, and mut5. Among them, Lmut1 and Lmut4 restricted viral growth by low
polymerase activity and low growth rate, respectively, leading to inhibition of viral overgrowth. The restriction of viral growth caused by Lmut1 and Lmut4 was compensated by other mutations, including Lmut2, Lmut3, and Lmut5. Each of the mutations found in L protein of HAZVpi was concluded to cooperatively modulate viral growth, which facilitates the establishment of persistent infection. Suppression of innate immunity also potentially contributes to virus persistence. This is the first presentation of a possible mechanism behind how nairoviruses establish persistent infection in human cells.