Yonghuan Ma, Hong Lu, Wei Wang, Jiaming Zhu, Wan Zhao, and Feng Cui

PNAS July 27, 2021 118 (30) e2103393118

Significance

The importin α family is well known as a cargo transporter from the cytoplasm to the nucleus, with diverse functions in eukaryotes. However, some members of the importin α family also localize to the cell membrane. Here, we report that a cell membrane–associated importin α takes part in viral transmission by vector insects. Importin α2 of the small brown planthopper binds the rice stripe virus on the proteoglycans of the cell membrane and efficiently controls viral entry into insect salivary glands and subsequent transmission to rice. These findings not only demonstrate the specific function of importin α in the cell membrane utilized by viruses, but also provide a promising target gene for preventing outbreaks of rice stripe disease.

Abstract

The importin α family belongs to the conserved nuclear transport pathway in eukaryotes. However, the biological functions of importin α in the plasma membrane are still elusive. Here, we report that importin α, as a plasma membrane–associated protein, is exploited by the rice stripe virus (RSV) to enter vector insect cells, especially salivary gland cells. When the expression of three importin α genes was simultaneously knocked down, few virions entered the salivary glands of the small brown planthopper, Laodelphax striatellus. Through hemocoel inoculation of virions, only importin α2 was found to efficiently regulate viral entry into insect salivary-gland cells. Importin α2 bound the nucleocapsid protein of RSV with a relatively high affinity through its importin β–binding (IBB) domain, with a dissociation constant K_D of 9.1 μM. Furthermore, importin α2 and its IBB domain showed a distinct distribution in the plasma membrane through binding to heparin in heparan sulfate proteoglycan. When the expression of importin α2 was knocked down in viruliferous planthoppers or in nonviruliferous planthoppers before they acquired virions, the viral transmission efficiency of the vector insects in terms of the viral amount and disease incidence in rice was dramatically decreased. These findings not only reveal the specific function of the importin α family in the plasma membrane utilized by viruses, but also provide a promising target gene in vector insects for manipulation to efficiently control outbreaks of rice stripe disease.

See: https://www.pnas.org/content/118/30/e2103393118

Figure 1:  Effect of the importin α family on RSV transmission by L. striatellus. (A) Relative transcript levels of three importin α genes in viruliferous small brown planthoppers at 8 d after the injection of a mixture of dsRNAs for the three importin α genes (ds3IMP) as measured by RT-qPCR. The transcript levels of these genes were normalized to that of EF2. The control group was injected with dsRNA for GFP (dsGFP). (B) Relative RNA level of RSV NP in viruliferous planthoppers at 8 d after the injection of ds3IMP and in the rice fed to these insects for 1 d as measured by RT-qPCR. The transcript level of planthopper EF2 or rice tubulin was quantified to normalize the cDNA templates. (C) Immunohistochemistry showing the variation in RSV amounts in salivary glands of viruliferous planthoppers at 8 d after injecting ds3IMP compared to that of injecting dsGFP. RSV NP was observed using Alexa Fluor 488-conjugated anti-NP monoclonal antibody (green). F-actin was stained with phalloidin (blue). (Scale bars, 40 μm.) (D) Relative transcript levels of three importin α genes in the nonviruliferous planthoppers after injection with ds3IMP and feeding on RSV-infected rice plants for 8 d measured by RT-qPCR. (E) Relative RNA level of RSV NP in nonviruliferous planthoppers after injection with ds3IMP and feeding on RSV-infected rice plants for 8 d and in the rice fed to these insects for 2 d measured by RT-qPCR. (F) Immunohistochemistry showing the variation in RSV amounts in salivary glands of nonviruliferous planthoppers after injection with ds3IMP and feeding on RSV-infected rice plants for 8 d using Alexa Fluor 488-conjugated anti-NP monoclonal antibody (green). Insects injected with dsGFP were used as controls. F-actin was stained with phalloidin (blue). (Scale bars, 40 μm.) *P < 0.05, **P < 0.01, ***P < 0.001.