Laboratory of Virology, Wageningen University & Research, PB, Wageningen, The Netherlands.
Laboratory of Entomology, Wageningen University & Research, PB, Wageningen, The Netherlands.
PLoS Negl Trop Dis. 2018 Nov 9;12(11):e0006958. doi: 10.1371/journal.pntd.0006958. eCollection 2018 Nov.
Chikungunya virus (CHIKV) is a re-emerging arthropod-borne (arbo)virus that causes chikungunya fever in humans and is predominantly transmitted by Aedes aegypti mosquitoes. The CHIKV replication machinery consists of four non-structural proteins (nsP1-4) that additionally require the presence of a number of host proteins for replication of the viral RNA. NsP3 is essential for CHIKV replication and has a conserved macro, central and C-terminal hypervariable domain (HVD). The HVD is intrinsically disordered and interacts with various host proteins via conserved short peptide motifs: A proline-rich (P-rich) motif that has affinity for SH3-domain containing proteins and duplicate FGDF motifs with affinity for G3BP and its mosquito homologue Rasputin. The importance of these motifs for infection of mammalian cells has previously been implicated. However, their role during CHIKV infection of mosquito cells and transmission by mosquitoes remains unclear.
METHODOLOGY / PRINCIPAL FINDINGS: Here, we show that in-frame deletion of the P-rich motif is lethal for CHIKV replication in both mosquito and mammalian cells. However, while mutagenesis of the P-rich motif negatively affects replication both in mammalian and mosquito cells, it did not compromise the infection and transmission of CHIKV by Ae. aegypti mosquitoes. Mutagenesis of both FGDF motifs together completely inactivated CHIKV replication in both mammalian and mosquito cells. Importantly, mutation of a single FGDF motif attenuated CHIKV replication in mammalian cells, while replication in mosquito cells was similar to wild type. Surprisingly, CHIKV mutants containing only a single FGDF motif were efficiently transmitted by Ae. aegypti.
CONCLUSIONS / SIGNIFICANCE: The P-rich motif in CHIKV nsP3 is dispensable for transmission by mosquitoes. A single FGDF motif is sufficient for infection and dissemination in mosquitoes, but duplicate FGDF motifs are required for the efficient infection from the mosquito saliva to a vertebrate host. These results contribute to understanding the dynamics of the alphavirus transmission cycle and may help the development of arboviral intervention strategies.
基孔肯雅病毒（CHIKV）是一种重新出现的节肢动物传播（arbovirus）病毒，可引起人类基孔肯雅热，主要由埃及伊蚊传播。CHIKV 复制机制由四个非结构蛋白（nsP1-4）组成，此外还需要一些宿主蛋白的存在才能复制病毒 RNA。nsP3 对 CHIKV 复制至关重要，具有保守的宏、中央和 C 末端超可变结构域（HVD）。HVD 是内在无序的，通过保守的短肽基序与各种宿主蛋白相互作用：富含脯氨酸（P-rich）的基序与 SH3 结构域包含蛋白有亲和力，并且重复的 FGDF 基序与 G3BP 及其蚊子同源物 Rasputin 有亲和力。以前已经暗示了这些基序对感染哺乳动物细胞的重要性。然而，它们在蚊细胞感染 CHIKV 和蚊子传播中的作用仍不清楚。
方法/主要发现：在这里，我们表明，在框内缺失 P-rich 基序会导致 CHIKV 在蚊细胞和哺乳动物细胞中的复制均致死。然而，虽然 P-rich 基序的诱变会负面影响在哺乳动物和蚊细胞中的复制，但它不会影响 Ae. aegypti 蚊子对 CHIKV 的感染和传播。两个 FGDF 基序的同时突变完全使 CHIKV 在哺乳动物和蚊细胞中的复制失活。重要的是，单个 FGDF 基序的突变会减弱 CHIKV 在哺乳动物细胞中的复制，而在蚊细胞中的复制与野生型相似。令人惊讶的是，含有单个 FGDF 基序的 CHIKV 突变体可有效地由 Ae. aegypti 传播。
结论/意义：CHIKV nsP3 中的 P-rich 基序对于蚊子传播是可有可无的。单个 FGDF 基序足以感染和在蚊子中传播，但重复的 FGDF 基序对于从蚊子唾液到脊椎动物宿主的有效感染是必需的。这些结果有助于了解黄病毒传播周期的动态，可能有助于开发虫媒病毒干预策略。
