Membrane association of importin α facilitates viral entry into salivary gland cells of vector insects

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.

The importin α family belongs to the conserved importin α/β nuclear transport pathway in eukaryotes (1–3). It is well known that the importin α family plays an indispensable role in transporting proteins from the cytoplasm to the nucleus, with diverse functions in gene regulation, cell physiology, and differentiation (1, 4, 5). In addition to nucleocytoplasmic transport, some members of the importin α family localize to the plasma membrane with palmitoylation modification or through binding to heparin in heparan sulfate proteoglycan (HSPG) (6–8). Increased importin α levels in the plasma membrane are concomitant with decreased importin α levels in the cytoplasm, which affect the nuclear import of cargos and regulates intracellular scaling (7). However, the function of the importin α family in the plasma membrane is still elusive.Many plant viruses are transmitted by vector insects in a persistent, circulative mode, which is characterized by systemic invasion of diverse tissues prior to entering salivary glands and release in saliva (9–13). The salivary glands are the last barriers for viral transmission to overcome (14–18). Unfortunately, the molecular mechanisms underlying viral entry into salivary-gland cells are not well known. The rice stripe virus (RSV) is a typical persistent, circulative plant virus and is capable of proliferating in the midgut epithelial cells and of being efficiently transmitted by the vector insect small brown planthopper (Laodelphax striatellus) (19). This virus causes one of the most destructive rice stripe diseases, showing an incidence of up to 80% and causing yield losses of 30 to 40% in the rice fields of Asian countries (20). RSV is a nonenveloped negative-strand RNA virus of the Tenuivirus genus (21, 22). The genome of RSV contains four single-stranded RNA segments, each of which is encapsidated by a viral nucleocapsid protein (NP). In addition to the NP, RSV encodes an RNA-dependent RNA polymerase and five nonstructural proteins (NS2, NSvc2, NS3, SP, and NSvc4) (23–25).In our recent work, we found that three importin α proteins, importin α1 (GenBank registration number {"type":"entrez-nucleotide","attrs":{"text":"MT036051","term_id":"2078901399","term_text":"MT036051"}}MT036051), α2 ({"type":"entrez-nucleotide","attrs":{"text":"MT036050","term_id":"2078901365","term_text":"MT036050"}}MT036050), and α3 ({"type":"entrez-nucleotide","attrs":{"text":"MT036052","term_id":"2078901435","term_text":"MT036052"}}MT036052), of the small brown planthopper participate in the nuclear entry of RSV through direct interactions with RSV NPs, triggering an antiviral caspase-dependent apoptotic reaction (26). Knockdown of the expression of all the three importin α genes retarded the nuclear entry of RSV and led to an increase in viral load in planthoppers (26). However, we did not determine the influence on viral transmission. In the present study, we surprisingly found that one of the importin α proteins, importin α2, is associated with the plasma membrane and efficiently facilitates viral entry into insect salivary glands and subsequent viral transmission.