Ubiquitylated Rice Stripe Virus NS3 Mediated By Ubiquitin Ligase E3-RING Of Laodelphax Striatellus Translocates Into The Nucleus To Promote Cytosolic Virus Replication

Rice stripe tenuivirus(RSV)is transmitted by the small brown planthopper(Laodelphax striatellus,SBPH)and causes severe rice yield losses in Asian countries.RSV is a type member of single-stranded RNA virus of the genus Tenuivirus.RSV has been reported to be a cytoplasm-replicating virus in the host insect L.striatellus.However,the NS3,NP,and SP proteins of RSV were shown to localize to nuclei in the host plant Nicotiana benthamiana,suggesting that during replication of RSV in plants,its encoded proteins may enter the nucleus.A recent study revealed that NP can enter cell nuclei of vector insects,which regulate the antiviral apoptotic response to control viral replication levels in vectors.Whether other RSV proteins enter the cell nucleus of insect vectors remains unknown.Our prior research indicated that UPS proteins were highly expressed in the salivary glands of RSV-infected SBPH as compared to nonviruliferous insects.However,it is not clear whether any novel mechanism of ubiquitination is involved in RSV replication.This paper uses RSV and its insect vector L.striatellus as the research object,which clarifies the mechanism that RSV NS3 hijacked the ubiquitin-proteasome system of the planthopper vector for nuclear translocation and regulated miRNA to promote replication.The main results are as follows.1 RSV infection significantly increased the expression of UPS related genes in SBPHThe full length of ubiquitin(GenBank accession no.MW76799),RING E3 Ub ligase(LsE3-RING)(GenBank accession no.MW751455),and a HECT E3 Ub ligase(LsE3HECT)(GenBank accession no.MW767162)were obtained using RT-PCR and rapid amplification of cDNA ends(RACE).Differential expression levels of these three genes in the viruliferous and nonviruliferous SBPHs were assayed by RT-qPCR and western blot.The results showed that the transcript and protein levels of LsUBI and LsE3-RING were upregulated in viruliferous SBPHs compared to nonviruliferous SBPHs.However,RSV infection did not impact the mRNA and protein levels of LsE3-HECT.In addition,western blot with anti-ubiquitin antibody showed that the level of ubiquitylated prote was higher in viruliferous SBPHs relative to nonviruliferous SBPHs.Further studies showed that the transcription level of LsUb was significantly elevated in the midguts and salivary glands of the viruliferous SBPHs,upregulated by 176%and 84%,respectively,compared with that of the nonviruliferous SBPHs,but the difference was not significant in the ovaries.LsE3-RING transcription was upregulated in all tissues of the viruliferous SBPHs and was most significantly upregulated in midguts(upregulated by 330%).followed by salivary glands(upregulated by 123%)and ovaries(upregulated by 112%).In contrast.RSV infection did not impact the transcription of LsE3-HECT in different tissues.Further use of RNAi to silence LsE3-RING and examine its effect on RSV accumulation in the SBPHs and the results showed that protein ubiquitylation and RSV titer occurred at lower levels in the whole body and all tested tissues(midguts,salivary glands,and ovaries)of dsLsE3-RING-treated SBPHs than in dsGFP controls and the most significant downregulation of ubiquitination levels and RSV titers was found in the midgut.Silencing of LsE3-RING reduced RSV particle accumulation in the ovaries,midgut.and salivary glands of SBPHs as observed by Laser scanning confocal microscopy(LSCM).These data suggested that LsE3-RING-mediated ubiquitylation played a critical role in RSV accumulation.2 LsE3-RING conjugates K63-linked ubiquitination chains to RSV NS3The direct interaction between LsE3-RING and RSV NS3 was screened and verified by yeast two-hybrid and GST pull-down techniques,followed by the construction of different truncations to further reveal that the interaction sites of LsE3-RING and RSV NS3 were located at amino acids 65-67 and 74-76 of the NS3 protein.In vitro ubiquitylation assays showed that LsE3-RING promoted ubiquitylation of RSV NS3 and conjugated K63-linked ubiquityl chains to RSV NS3.By comparing the changes in RSV titer and RSV NP protein level in the viruliferous SBPHs treated with dsLsE3RING+MG132(26S proteasome inhibitor),MG132+DMSO and dsGFP+DMSO.the results showed that compared with the control(dsGFP+DMSO dual treatment)and MG132+dsGFP dual treatment groups,MG132+dsLsE3-RING dual treatment resulted in significantly lower RSV titers and RSV NP protein level in the viruliferous SBPHs.These results suggest that the effect of LsE3-RING-mediated RSV NS3 ubiquitination on RSV accumulation is independent on the 26S proteasome.3 LsE3-RING-mediated RSV NS3 ubiquitylation mediates NS3 translocation into the nucleusThe immunofluorescence microscopy results showed that the co-location of RSV NS3 with LsE3-RING,as well as with LsUBI,was frequently observed near the nuclei of midgut cells in viruliferous SBPHs and midgut cells of nonviruliferous SBPHs 5 d after access to diseased plants.To investigate whether LsE3-RING-mediated ubiquitylation of RSV NS3 function in the transport of NS3 from the cytoplasm to nuclear,the viruliferous SBPHs were injected with dsGFP or dsLsE3-RING,and Sf9 cells were cotransfected with the respective recombinant bacmids-LsE3-RING or eGFP for protein expression.In dsGFP-treated controls,RSV NS3 co-localized with LsUBI around or in cell nuclei;however,in the dsLsE3-RING-treated group,NS3 was primarily present in the cytoplasm.Western blot of extracted cell nuclear proteins also revealed a decrease accumulation in RSV NS3 protein in the nucleus.In Sf9 cells stably expressing LsE3-RING,a strong NS3 fluorescent signal appeared in nuclei 24 h after RSV exposure;such a signal was not observed in Sf9 cells stably expressing enhanced green fluorescent protein(eGFP).These results were confirmed by western blot of extracted cell nuclear proteins.The RT-qPCR assay also revealed that the increased transcript levels of LsE3-RING were accompanied by a significant increase in RSV titers in Sf9 cells overexpressing LsE3-RING compared to the control,in addition,western blot analysis revealed that NS3 protein content in nuclei was higher than in the controls.To determine the temporal pattern of RSV NS3 entry into the nucleus,Sf9 cells were collected at 30 min intervals 7 h after RSV infection.RSV NS3 was undetectable in the nuclei of Sf9 cells 7-11.5 h after RSV exposure.Beginning at 12.5 h,NS3 was increasingly detected in the nuclei.At 17 and 21 h after RSV exposure,NS3 fluorescent signals were detected in 30 and 85%of nuclei,respectively.This signal remained relatively stable until at least 21.5-24 h after RSV exposure.The subcellular localization of RSV NS3 in SBPHs was investigated by collecting four different instars,and western blot of different cell fractions and immunofluorescence revealed no significant differences in the RSV NS3 protein content in the nuclei of the midgut cells of the four groups of samples.In addition,four different spatial patterns were observed in Sf9 cells and the midguts of viruliferous SBPHs:(1)NS3 and LsUBI dispersed in the cytosol;(2)LsUBI and NS3 co-localized in the perinuclear regions;(3)nuclear co-localization of LsUBI and NS3 resulted in a dot-like matrix;and(4)high levels of NS3 accumulation occurred in the nuclei.Taken together,these results demonstrate d that LsE3-RING-mediated RSV NS3 ubiquitylation mediated NS3 nuclear translocation and thereby promoted the replication of RSV.4 RSV NS3 regulates microRNA(miRNA)processing in the nucleus to promote RSV replicationWe sequenced small RNAs in viruliferous and nonviruliferous SBPHs to identify differentially regulated miRNAs that may be associated with RSV infection.Six differentially upregulated miRNAs and 10 down-regulated miRNAs were identified inviruliferous versus nonviruliferous SBPHs(p<0.05).The differential expression levels of these 16 differentially expressed miRNAs were verified by RT-qPCR in viruliferous and nonviruliferous SBPHs and the results showed that four miRNAs(lst-miR-92,lstmiR-36.lst-miR-8-5p,and lst-miR-275a-5p)were up-regulated and one miRNA(lst-miR79)was down-regulated in response to RSV infection.Since RSV NS3 is localized in the nucleus during viral replication and the nucleus is the processing site of miRNA primary transcription products(pri-miRNA),the differential expression of the primary transcription products of these five miRNAs was further determined in viruliferous and nonviruliferous SBPHs to determine whether RSV infection increases accumulation of these miRNAs through promoting pri-miRNA processing.We first measured the primary transcript levels of lst-miR-92,lst-miR-36,lst-miR-8-5p,lst-miR-276a-5p,and lst-miR79 in viruliferous and nonviruliferous SBPHs by RT-qPCR,the results showed that RSV infection down-regulated three pri-miRNAs(lst-miR-92,lst-miR-36,and lst-miR-276a5p),but did not impact pri-miR-8-5p and pri-miR-79 levels.In addition,RNA immunoprecipitation(RIP)PCR assays demonstrated that pri-miR-92 and pri-miR-36 can be recognized by RSV NS3.Furthermore,co-immunoprecipitation(CoIP)assays and Immunofluorescence co-localization analysis showed that RSV NS3 interacts with Drosha(a component of the microprocessor complex)in viruliferous SBPHs.Colectively,these results indicated that RSV NS3 functions in pri-miRNA processing in nuclei,where it uses the microprocessor complex to induce the accumulation of corresponding mature miRNAs.We investigated whether LsE3-RING influenced the expression of lst-miR-92,1stmiR-36,lst-miR-8-5p,lst-miR-275a-5p,or lst-miR-79.Interestingly,lst-miR-92 was significantly downregulated by dsLsE3-RING treatment compared to the dsGFP-treated control group.To investigate the function of lst-miR-92 on RSV replication,we injected viruliferous SBPHs with lst-miR-92 mimic and lst-miR-92 inhibitor,and then the changes of RSV replication-related gene-RNA-dependent RNA polymerase(RdRp)were detected at different times.RT-qPCR analysis showed that lst-miR-92 mimic upregulated the transcript level of RSV RdRp in SBPHs at 2 d and 4 d after lst-miR-92 mimic treatment by 206%and 195%,respectively,while there was no significant difference after 6 days of treatment.However,there was no significant difference in lst-miR-92 mimic treated group and mimic-NC treated group.lst-miR-92 inhibitor reduced the transcript level of respectively.Again,it returned to the control level after 6 days of treatment.We then examined whether lst-miR-92 affected RSV titers by measuring RSV NP mRNA levels at various times after injecting lst-miR-92 mimic(mimic-NC was used as control)or lstmiR-92 inhibitor(inhibitor-NC was used as control).RSV NP transcript concentration increased at 2 d,4 d,and 6 d after lst-miR-92 mimic treatment by 207%,83,and 59%,respectively.RSV NP transcript concentration declined at 2 d,4 d,and 6 d after lst-miR92 inhibitor treatment by 76%,61%,and 42%,respectively.The results of western blot assay showed that the RSV NP protein level in SBPHs after 2.4 and 6 days of lst-miR-92 mimic treatment were higher than those in the control group,while the viral NP protein levels in SBPHs after 2,4 and 6 days of lst-miR-92 inhibitor treatment were lower than those in the control group.The effect of lst-miR-92 on viral aggregation in the midgut of the viruliferous SBPHs was further observed by immunofluorescence confocal,and the results again showed that lst-miR-92 increased the RSV titer in SBPHs.Using miRanda,PITA,and RNAhybrid to predict the target genes of lst-miR-92,a total of 81 lst-miR-92 candidate target genes were identified.lst-miR-92 is likely to be involved in RSV replication through these target genes.The above studies suggest that lst-miR-92,which is regulated by RSV NS3 entry nucleus,plays an important role in RSV replication.In this study,infection with RSV promoted the protein ubiquitination mediated by a RING-type E3 ligase(LsE3-RING)in SBPH.LsE3-RING catalyzed K63-linked ubiquitination of RSV NS3 which in turn mediated NS3 cytoplasmic-nuclear trafficking.In nuclei,NS3 regulated pri-miR-92 processing through manipulation of the microprocessor complex,which induced miR-92 accumulation.Finally,miR-92 regulated the target gene to promoted viral replication.Our study will also provide new ideas for the prevention and/or management of virus disease by regulation of ubiquitin and miRNAs.