The barley (Hordeum vulgare) CCR4 protein (HvCCR4) was identified to interact with the BYSMV phosphoprotein (P). The BYSMV P protein recruited HvCCR4 from processing bodies (PBs) into viroplasm-like bodies. Overexpression of HvCCR4 promoted BYSMV replication in plants. Conversely, knockdown of the small brown planthopper CCR4 inhibited viral accumulation in the insect vector. Biochemistry experiments revealed that HvCCR4 was recruited into N–RNA complexes by the BYSMV P protein and triggered turnover of N-bound cellular mRNAs, thereby releasing RNA-free N protein to bind viral genomic RNA for optimal viral replication. Our results demonstrate that the co-opted CCR4-mediated RNA decay facilitates cytorhabdovirus replication in plants and insects.Carbon catabolite repression 4 (CCR4) is a conserved mRNA deadenylase regulating posttranscriptional gene expression. However, regulation of CCR4 in virus infections is less understood. Here, we characterized a pro-viral role of CCR4 in replication of a plant cytorhabdovirus In eukaryotic cells, mRNA levels determined by biosynthesis and turnover tightly regulate protein production in response to cellular environment changes . The mRNIn the CCR4-NOT complex, carbon catabolite repression 4 (CCR4) and CCR4 associated factor (CAF1) are two deadenylases responsible for removing mRNA poly(A) tails . CAF1 in0) by forming an N0–P complex that prevent N0 from binding nonspecifically to cellular RNAs, and attaches L protein polymerase complexes to the N-RNA complex , a member of Cytorhabdovirus genus, infects cereal crops and causes yield losses worldwide. In 2015, BYSMV was first reported in wheat fields of northern China  P protein interacting proteins from infected barley leaves. The IP products were separated in SDS-PAGE gels and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and a barley CCR4 fragment was identified amongst the IP products (Hordeum vulgare (HvCCR4) and Laodelphax striatellus (LsCCR4) were obtained from NCBI protein sequence databases  and P fused with a GST tag (GST-P) were purified from  control . To furtr P6-GFP .C) or N (YN) halves of sYFP, and co-expressed in N. benthamiana leaves. BiFC fluorescence was not observed in the negative control combinations of either YC-P/Rub-YN or YN-P/Rub-YC , a central domain , and a C-terminal domain  , P60-183 (aa 60–183), and P208-295 (aa 208–295) were individually fused to YC for BiFC assays. Coexpression of YC-P and YN-HvCCR4N resulted in production of BiFC fluorescent punctate granules, whereas YN-HvCCR4C did not associate with YC-P  . The HvC208–270) . To deteith YC-P . Both P6 BYSMV P . Moreove BYSMV P . Collect60-183) are responsible for the P–HvCCR4 body formation. Besides, we found that the P protein of northern cereal mosaic virus (NCMV), a close related cytorhabdovirus, interacts with the N-terminal zf-MYND-like domain of HvCCR4 using BiFC assays  that are foci for mRNA turnover in the cytoplasm . The decsmic PBs . Interessmic PBs , but thesmic PBs , implyinus study .N. benthamiana leaves, and the results revealed that HvCCR4-GFP and CFP-P fully overlapped in punctate granules that were adjacent to DCP1-mCherry-labelled PBs , viral suppressor of RNA silencing (VSRs), N-Flag, L-Myc, P, along with HvCCR4-GFP or GFP. At five dpi, total proteins from infiltrated leaves were immunoprecipitated with anti-Flag affinity beads and analyzed by western blotting analysis. Co-IP assays showed that HvCCR4-GFP, but not GFP, coprecipitated with N-Flag, L-Myc, and P , the N, P, L proteins, and VSRs  also did not increase the fluorescence foci, compared with empty vector , and then coexpressed with HvCCR4N-YN or HvCCR4C-YN for BiFC assays. As expected, BiFC analysis shows that HvCAF1 interacts with the HvCCR4 N terminus, but has compromised affinity with the HvCCR4 C terminus  that has been described previously . We firsLsCCR4 dsRNA to evaluate knocked down LsCCR4 functions during BY-RFP insect infections. For these experiments, crude extraction of BY-RFP-infected barley leaves were mixed with synthesized GFP or LsCCR4 dsRNAs , and coinjected with 13.8 nl of the mixtures extractions into healthy SBPH thoraxes. Then, the injected insects were maintained on healthy rice seedlings and RFP fluorescence was monitored at 3-, 7-, and 10- dpi  of 6 × His tagged GFP, N, P, and HvCCR4 were used for Northwestern blotting with digoxigenin-labeled luciferase (Luc) mRNA. As expected, the BYSMV N protein exhibited high RNA-binding affinity for Luc mRNA  was commercially synthesized as a substrate for HvCCR4. The labelled RNA probe was not obviously degraded after 30 min of incubation with the N–P complex, whereas approximately 44% of substrate remaining was not degraded after 30 min of incubation with HvCCR4 alone  tails, and hence are not CCR4 substrates. Thus, CCR4-mediated turnover of the N-bound cellular mRNAs could potentially release RNA-free N0 to specifically bind genome RNA. To test this hypothesis, Agrobacterium harboring plasmids for expression of agMR, N, P, HvCCR4 and HvCCR4m was mixed in different combinations and coinfiltrated into N. benthamiana leaves. At two dpi, Co-IP assays showed that the N-Flag protein could be co-precipitated with P, HvCCR4, and HvCCR4eEEP proteins  families, respectively . HvCAF1 0 molecule to form soluble N0–P complexes that maintains the N0 molecule in an RNA-free state  plants were grown on soil at 25 ± 2°C with a day/night cycle of a 14/10 hr. SBPHs were isolated from Hebei province, China and reared in illumination incubators. BYSMV was initially isolated from wheat fields in Hebei province, China and maintained in barley plants as previously described (N.escribed . SBPH trescribed . BrieflyHvCCR4 (AK374808), HvCAF1 (AK361706), LsCCR4 (RZF46990.1) and BYSMV (KM213865) GenBank accession sequences were used throughout this study. To clone HvCCR4, HvCAF1, and LstCCR4, total RNA was extracted from barley leaves or insects, and used as templates for reverse transcription PCR (RT-PCR) with the specific primers shown in HvCCR4 ORF, the HvCCR4 N terminus (NHvCCR4) and C terminus (CHvCCR4), as well as the HvCCR4 point mutant (N260A/E305A) were cloned into pMDC32−3 × Flag , 20% glycerol, 4% SDS, and 0.2% bromophenol blue], separated in SDS-PAGE gels and transferred to nitrocellulose membranes. Protein accumulation was detected with corresponding anti-BYSMV N (1:3000), P (1:3000), RFP (1:2000), or HA (1:3000) polyclonal antibodies, and goat anti-rabbit IgG horseradish peroxidase conjugate (1:30000) was used as secondary antibodies, followed by incubation with Pierce ECL Plus chemiluminescent substrate before exposure to x-ray films.Total proteins were extracted from agroinfiltrated N.benthamiana leaves were agroinfiltrated with expression vectors of CCR4-Flag with P-GFP, P6-GFP, or GFP. Agroinfiltrated leaves were homogenized in liquid nitrogen and extracted with IP buffer  polyvinylpolypyrrolidone (PVPP) and protease inhibitor cocktail). After centrifugation and filtration, the supernatants were mixed with anti-Flag M2 affinity gel (Sigma) at 4°C for 4 hr with gentle mixing, followed by centrifugation at 800 g for 1 min. The precipitate was washed three times with IP buffer and analyzed by western blotting analysis with anti-Flag or anti-GFP antibodies.Co-IP assays were performed as described previously . BrieflyE. coli strain BL21. GST or GST-P were incubated with HvCCR4-His or LsCCR4-His in 500 µL reaction buffer  with glutathione-agarose beads at 4°C for 2 hr. After centrifugation at 800 g for 1 min, beads were washed five times with reaction buffer, boiled in SDS buffer for western blotting analysis with anti-GST (1:5000) and anti-His (1:5000) antibodies.GST pull down assays were performed as described previously . GST-P, CCR4 in insects, LsCCR4 and GFP control fragments fused with the T7 promoter at their two termini were amplified, and the resulting PCR products served as templates for in vitro synthesis of double-stranded RNA of CCR4 (dsCCR4) and dsGFP using the T7 RiboMAX Express RNAi System kit (Promega). Virus extract of BY-RFP-infected barley leaves  were mixed with synthesized GFP or CCR4 dsRNAs , and 13.8 nl of the mixed extracts were microinjected into second instar nymph thoraxes as described previously  for RT reactions using M-MLV Reverse Transcriptase . The full-length MR RNA abundance was analyzed with BYS-RT-1 as RT primer and the PCR primer corresponding to the trailer fragment. Accumulation of the BYSMV N, RFP, and the EF1A mRNA were analyzed with oligo dT as RT primer and their specific primers. QPCR were carried out using 2 × SsoFast EvaGreen Supermix (Bio-Rad). The primers used in this study are listed in UCUAAAUAAAAAAAAAAAAAAAAAAAA-3') served as a substrate for deadenylation assays. The labeled RNA substrate was incubated with HvCCR4-His or its derivative proteins in reaction buffer  at 37°C for different times as indicated. Then, reaction mixtures were fractioned on denaturing polyacrylamide gels , and the RNA gels were analyzed with a fluorescence imager (Bio-Rad). To quantify the rates of deadenylated RNA, we fist quantified the 5’FAM-RNA band intensities by ImageJ software. The graphs plot the relative accumulation of remaining substrates estimated by electronic autoradiography in the gels. The values of 5’FAM-RNA band intensities at 0 min were set to 100%. Data points represent mean values from three independent repeats. The curve diagrams was drawn with GraphPad Prism.In vitro deadenylase assays were performed as described . BrieflyNorthwestern blotting assays were performed as described previously . BrieflyN.benthamiana leaves. At three dpi, total proteins were extracted from the agroinfiltrated N.benthamiana leaves and incubated with anti-Flag M2 affinity gel (Sigma) in IP buffer  at 4°C for 4 hr. After washing five times, the IP products were used for protein detection by western blotting analysis with corresponding antibodies. In addition, IP product RNA was extracted with TRIzol Reagent for qRT-PCR analysis of minigenomic RNA and the EF1A gene accumulation with specific primers as shown in The BYSMV N-Flag and P proteins, CCR4, and minigenome RNA in different combinations were transiently expressed in agroinfiltrated  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This manuscript teaches us that a plant virus, BYMSV, uses its phosphoprotein (P) to hijack the host deadenylation factor CCR4 to promote viral replication. A particularly striking finding is that the virus uses the same trick to manipulate both its plant host and its insect vector. The reviewers were pleased that the authors were overall very aware of the many pitfalls inherent in the complex experimental setups and data analysis. The work advances our understanding of the molecular arms race in virus-host interactions and will be of great interest to the fields of viral interaction and RNA turnover.Decision letter after peer review:Thank you for submitting your article "A co-opted CCR4 RNA decay factor enhances plant cytorhabdovirus replication" for consideration by eLife. Your article has been reviewed by Detlef Weigel as Senior and Reviewing Editor and three reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: Yi Li (Reviewer #3).The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:RNA quality control and regulated RNA decay are conserved eukaryotic mechanisms that protect host cells from dysfunctional endogenous or invasive RNAs. Therefore, viruses have evolved strategies not only to evade, but also to manipulate these mechanisms in order to successfully complete their replication cycle.In this manuscript, you address the roles of carbon catabolite repression 4 (CCR4) in a plant virus infection both in the host plant and the insect vector. In eukaryotes, mRNA deadenylation is primarily mediated by the CCR4-NOT complex in RNA processing bodies (PBs) known from yeast and human cells, but regulation of CCR4 and the role of its deadenylase activity during viral infections is not well understood. You discovered that BYMSV uses its phosphoprotein (P) to hijack host CCR4 to promote viral replication. You convincingly show that P protein interacts directly with CCR4 and recruits it into viroplasm-like bodies to improve viral RNA replication; P-recruited CCR4 proteins are responsible for turnover of the N-bound cellular mRNAs and thereby releasing RNA-free N protein to bind viral genomic RNA for optimal viral replication.0) to prevent binding to cellular mRNAs and to facilitate specific viral gRNA/agRNA interactions. A particularly nice aspect of the work is that you also tested CCR4 orthologues in the transmitting vector, which led to similar conclusions: CCR4 knockdown inhibits BYSMV infection of insects and significantly reduces virus transmissibility, indicating a potential evolutionarily conserved mechanism that BYSMV has adopted to outcompete and inhibit N protein binding of cellular mRNAs. You are overall very aware of the many pitfalls inherent in the complex experimental setups and data analysis. The manuscript is well written and does an especially effective job presenting complex interactions.You present a potential mechanism for how P protein binds to RNA-free N protein  You used transient expression to test N-bound RNA ratios in Figure 8E and F, with the key proteins all expressed from 35S promoters. To obtain more physiological relevant ratios, please repeat the experiments for Figure 8E and F using BYR-HvCCR4/HVCCR40) by forming the N0-P complex that prevents N0 from binding non-specifically to cellular RNAs, and attaches L protein polymerase complexes to the N-RNA complex. In Figure 1, you show that P interacts with CCR4 through the central and/or the C-terminal domain. Does the CCR4-P interaction interfere with the dimerization of P protein? Also, what is the effect on the function of P protein? Is the functional P form a dimer or a monomer?2) Rhabdovirus P protein has been shown to form dimers through the central dimerization domain and the dimerization is important in the process of chaperoning nascent RNA-free N (N3) The deadenylation assays showed strong and direct evidence that P protein could tether CCR4 and that the N protein triggers degradation of RNA containing poly(A). However, the quality of Figure 8B should be improved; no significant increase of the degradation products can be seen in the figure at 15, 30, and 60 minutes. You should also describe the method and details how to quantify the deadenylated RNA in Figure 8B and C.Arabidopsis CCR4a is localized to both P-bodies and siRNA bodies  and BYSMV infection promotes the generation of virus-derived siRNAs as shown in one of the authors' previous study. Do BYSMV P protein (or viroplasm-like structures) co-localize with siRNA bodies and HvCCR4? Is the accumulation of virus-derived siRNAs affected by HvCCR4 overexpression ? Please consider this possibility and discuss it in depth.4) RQC and RNA decay act as antiviral mechanisms, but they also play a role as endogenous suppressors of PTGS. This entails that HvCCR4 overexpression may promote viral replication through repressing PTGS. HvCCR4 is localized to viroplasm-like structures where viral mRNA is transcribed. In another scenario, it is possible that HvCCR4 degrades aberrant or excessive viral mRNAs to suppress antiviral RNA silencing, thereby promoting viral replication. 5) Figure 2D and E requires quantitative data showing how many foci were detected and how many were completely or adjacently co-localized in the absence/presence of P proteins.Thank you for resubmitting your work entitled "A co-opted CCR4 RNA decay factor enhances plant cytorhabdovirus replication" for further consideration by eLife. Your revised article has been evaluated by Detlef Weigel (Senior Editor) and a Reviewing Editor.The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined by reviewer 4. Could you please address these in the final revision?Reviewer #1:Deadenylation of mRNA is the first and rate-limiting step of mRNA and is primarily mediated by the CCR4-NOT complex in RNA processing bodies (PBs). In this manuscript, Zhang et al. addressed the important roles of carbon catabolite repression 4 (CCR4) in a plant virus infection both in plant and insect vector. The quality of the manuscript is significantly improved after revisions. I recommend acceptance.Reviewer #2:The authors have successfully addressed most of my concerns, which improve the clarity and the significance of the study. Regarding the determination of the N-bound RNA ratio, I still think that in vivo date with stable lines would be much more convincing. However, in the revised manuscript, the additional explanations and experiments supporting the promotion of viral replication by HvCCR4 and the fact that the level of CCR4 does not affect RNA silencing strengthen the conclusion of this study revealing a new aspect of the function of a RNA decay component, HvCCR4, in the interaction between viruses and plants, and I agree with it.Reviewer #4:In this manuscript, Zhang et al. report that CCR4 RNA decay factor is hijacked by BYSMV P protein into viroplasm-like bodies to trigger degradation of cellular mRNAs bound by BYSMV N protein, allowing N protein to bind viral genomic RNA for viral replication. The findings deepen our understanding of the molecular mechanisms in host-virus arm race.In general, the conclusions supported by the data they presented and the data are of good quality. The authors also addressed most of the concerns raised during the last round of review. Listed below are my comments:1) The Title should be revised to reflect that CCR4 RNA decay factor is hijacked by BYSMV P.2) CCR4 was identified to interact with BYSMV P protein through IP-MS, proteins identified in IP-MS should be listed in a table.3) Figure1—figure supplement 6A, labeling for the fifth lane is missing. The bottom western blot image only has 4 lanes, other images have 5 lanes.4) In Figure 6D-F, the authors show that barley plants infected with BYR-HvCCR4 developed more severe symptoms and have a higher level of BYSMV accumulation. Is it because that SBPH infected with BYR-HvCCR4 carries a higher amount of BYSMV?5) For all the western blot results, the loading control should be specified. For some of the IP results, for instance, Figure 1C and D, only the symbol "+" was used. The symbol "-" was missing above the images.The writing should be improved. The authors should check throughout the manuscript for writing problems. Here are some examples.6) Introduction, "Thus, shortening of mRNA poly (A) tail..."The word "thus" should be removed.7) Introduction, "Thus, studying the interplay between.. . understanding the molecular mechanisms of host-pathogen interactions"The sentence should be removed.”, "when N. benthamiana leaves... in contrast to the highly dynamic movement along ER tubules observed in the DMSO control"8) Subsection “BYSMV P interacts with barley HvCCR4 in vitroand in vivoThe sentence should be rephrased.”, the results should be described at the end of the preceding paragraph.9) Subsection “BYSMV P interacts with barley HvCCR4 in vitroand in vivo10) Subsection “HvCAF1 has a negative effect on BYSMV minigenome replication”, "Western blotting analyses revealed that.. .in different expression level of HvCAF1-Flag"The sentence should be rephrased.11) Subsection “HvCAF1 has a negative effect on BYSMV minigenome replication”, "thereby weakening the HvCCR4-P interactions that compromise HvCCR4 proviral functions in BYSMV replication"The sentence should be rephrased.12) Subsection “CCR4 triggers decay of the N-bound cellular mRNA”, "exhibit specificity improvement”. Essential revisions:mEEP-rescued barley plants.1) You used transient expression to test N-bound RNA ratios in Figure 8E and F, with the key proteins all expressed from 35S promoters. To obtain more physiological relevant ratios, please repeat the experiments for Figure 8E and F using BYR-HvCCR4/HVCCR4mEEP, could significantly increase the replication of minireplicon genome. Therefore, different accumulation of agMR would affect the final ratios of N-bound agMR verse cellular RNA in the presence of HvCCR4 or HvCCR4mEEP. To rule out the different accumulation of viral genomic RNA, we did not express the BYSMV L protein in the Figure 8E and F. Therefore, accumulation of the agRNA in vivo is similar in different samples, because all agRNA is transcribed from 35S promoter without virus replication. Then, we carried out the IP assays using N-Flag to show HvCCR4, but not HvCCR4mEEP, could improve the binding specificity of N to agMR.Thanks for suggestions. As shown in Figure 4B, transiently expressed HvCCR4, but not HvCCR4mEEP plasmid as the pBYR-HvCCR4 using the BYSMV vector. We first inoculated BYR-HvCCR4mEEP and BYR-HvCCR4 in two half parts of N. benthamiana leaves to compare their infectivity. In consistence with transiently expressed HvCCR4mEEP and HvCCR4 in minireplicon assays shown in Figure 4B, BYR-HvCCR4mEEP exhibited a significantly reduced infectivity compared with BYR-HvCCR4, which was revealed by the RFP fluorescence and accumulation of RFP as shown in Figure 6—figure supplement 1. Since replication of BYR-HvCCR4mEEP is very weak in N. benthamiana leaves, we cannot rescue the BYR-HvCCR4mEEP infections in planthoppers and barley plants. We will examine the BYR-HvCCR4mEEP infections in barley plants in future studies.We also constructed the pBYR-HvCCR40) by forming the N0-P complex that prevents N0 from binding non-specifically to cellular RNAs, and attaches L protein polymerase complexes to the N-RNA complex. In Figure 1, you show that P interacts with CCR4 through the central and/or the C-terminal domain. Does the CCR4-P interaction interfere with the dimerization of P protein? Also, what is the effect on the function of P protein? Is the functional P form a dimer or a monomer?2) Rhabdovirus P protein has been shown to form dimers through the central dimerization domain and the dimerization is important in the process of chaperoning nascent RNA-free N . Thus, the direct CCR4-P interaction has no effect on the function of P protein. Moreover, the EEP domain is required for the positive effect of on BYSMV replication , indicating that the CCR4 deadenylation activity is essential for the CCR4 function in BYSMV infections.In this Figure 4B, the CCR4The rhabdovirus P protein should form functional dimer structures in virus replication according to the review paper. Ivanov et al. (2011).3) The deadenylation assays showed strong and direct evidence that P protein could tether CCR4 and that the N protein triggers degradation of RNA containing poly(A). However, the quality of Figure 8B should be improved; no significant increase of the degradation products can be seen in the figure at 15, 30, and 60 minutes. You should also describe the method and details how to quantify the deadenylated RNA in Figure 8B and C.Thanks for your suggestions.There is no increase of the degradation products at 15, 30, and 60 minutes, which should be resulted from the long reaction time. We have repeated the deadenylation assays in 10, 20, and 30 minutes. The new results clearly show significant increase of the degradation products. Please see the new Figure 8B and C.To quantify the rates of deadenylated RNA, we fist quantified the 5’FAM-RNA band intensities by ImageJ software. The graphs plot the relative accumulation of remaining substrates estimated by electronic autoradiography in the gels of Figure 8D. The values of 5’FAM-RNA band intensities at 0 minutes were set to 100%. Data points represent mean values from three independent repeats. Error bars indicate standard errors of three independent experiments. The curve diagram was drown with GraphPad Prism. The detail method has been updated, please see subsection “Real-time quantitative RT-PCR (qRT-PCR)”.4) RQC and RNA decay act as antiviral mechanisms, but they also play a role as endogenous suppressors of PTGS. This entails that HvCCR4 overexpression may promote viral replication through repressing PTGS. HvCCR4 is localized to viroplasm-like structures where viral mRNA is transcribed. In another scenario, it is possible that HvCCR4 degrades aberrant or excessive viral mRNAs to suppress antiviral RNA silencing, thereby promoting viral replication. Arabidopsis CCR4a is localized to both P-bodies and siRNA bodies  and BYSMV infection promotes the generation of virus-derived siRNAs as shown in one of the authors' previous study. Do BYSMV P protein (or viroplasm-like structures) co-localize with siRNA bodies and HvCCR4? Is the accumulation of virus-derived siRNAs affected by HvCCR4 overexpression ? Please consider this possibility and discuss it in depth.N. benthamiana leaves, and some but not all P-GFP bodies were adjacent to but not overlap with SGS-RFP bodies that is a siRNA body marker. These results indicate that some transient connection between BYSMV P bodies and siRNA bodies. Please see Figure 8—figure supplement 1A.Thanks for these suggestions. RNA decay and RNA silencing have some relationship and the two pathways have some common complements such as AGO1. BYSMV GFP-P and SGS3-RFP were co-expressed in N. benthamiana leaves. Please see Figure 8—figure supplement 1B and C.To evaluate whether HvCCR4 has suppressor activities, we used the classical co-infiltration assays to show that neither CCR4 RNAi nor CCR4 overexpression has effects on GFP-induced RNA silencing in The possibility of CCR4 effect in RNAi-mediated silencing will examined in our future studies. We also add some discussion, please see the Discussion section.5) Figure 2D and E requires quantitative data showing how many foci were detected and how many were completely or adjacently co-localized in the absence/presence of P proteins.Thanks, and we agree that quantitative data would provide solid conclusions.We have done the quantitative analyses and clearly show that most the CCR4-GFP and DCP1-mCherry bodies  are overlapped in the absence of the BYSMV P protein . When the P protein was co-expressed, 58 among 60 CCR4-GFP bodies were adjacent to DCP1-mCherry bodies. Please see the new Figure 2—figure supplement 2.The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined by reviewer 4. Could you please address these in the final revision?Reviewer #4:In this manuscript, Zhang et al. report that CCR4 RNA decay factor is hijacked by BYSMV P protein into viroplasm-like bodies to trigger degradation of cellular mRNAs bound by BYSMV N protein, allowing N protein to bind viral genomic RNA for viral replication. The findings deepen our understanding of the molecular mechanisms in host-virus arm race.In general, the conclusions supported by the data they presented and the data are of good quality. The authors also addressed most of the concerns raised during the last round of review. Listed below are my comments:1) The Title should be revised to reflect that CCR4 RNA decay factor is hijacked by BYSMV P.Agreed, we have changed the Title as suggested as “CCR4, a RNA decay factor, is hijacked by a plant cytorhabdovirus phosphoprotein to facilitate virus replication”2) CCR4 was identified to interact with BYSMV P protein through IP-MS, proteins identified in IP-MS should be listed in a table.Agreed, we added a new supplementary file to list the P interacting proteins in IP-MS. Please new Supplementary file 1.3) Figure 1—figure supplement 6A, labeling for the fifth lane is missing. The bottom western blot image only has 4 lanes, other images have 5 lanes.Thanks, we changed the bottom image. The fifth lane is empty and cut out mistakenly. Please see the new Figure1—figure supplement 6A.4) In Figure 6D-F, the authors show that barley plants infected with BYR-HvCCR4 developed more severe symptoms and have a higher level of BYSMV accumulation. Is it because that SBPH infected with BYR-HvCCR4 carries a higher amount of BYSMV?Thanks for your suggestions. SBPH infected with BYR-HvCCR4 probably carry a higher virus amount than BYR-GUS. However, the SBPH with viruses were only incubated with healthy barley plants for a two-day inoculation, and then removed. The systemically infected leaves were collected for virus detection. Therefore, we propose that overexpression of CCR4 facilitate virus infections in systemically infected leaves.N. benthamiana leaves . Collectively, these results clearly show that overexpression of CCR4 could facilitate virus infections in N. benthamiana and barley plants, as well as in planthopper vectors.In addition, accumulation of BYR-HvCCR4 is higher than BYR-GUS in infiltrated 5) For all the western blot results, the loading control should be specified. For some of the IP results, for instance, Figure 1C and D, only the symbol "+" was used. The symbol "-" was missing above the images.Changed. Rubisco complex large subunit (RbcL) detected by Stain-Free technology was used as a loading control. Please see updated Figure 1, Figure 4, Figure 6 and Figure 7.The symbol "-" was added in the IP figures. Please see updated Figure 1, Figure 3, Figure 5 and Figure 7.The writing should be improved. The authors should check throughout the manuscript for writing problems. Here are some examples.6) Introduction, "Thus, shortening of mRNA poly (A) tail..."The word "thus" should be removed.7) Introduction, "Thus, studying the interplay between.. . understanding the molecular mechanisms of host-pathogen interactions"The sentence should be removed.8) Subsection “BYSMV P interacts with barley HvCCR4 in vitro and in vivo”, "when N. benthamiana leaves... in contrast to the highly dynamic movement along ER tubules observed in the DMSO control"The sentence should be rephrased.9) Subsection “BYSMV P interacts with barley HvCCR4 in vitro and in vivo”, the results should be described at the end of the preceding paragraph.10) Subsection “HvCAF1 has a negative effect on BYSMV minigenome replication”, "Western blotting analyses revealed that.. .in different expression level of HvCAF1-Flag"The sentence should be rephrased.11) Subsection “HvCAF1 has a negative effect on BYSMV minigenome replication”, "thereby weakening the HvCCR4-P interactions that compromise HvCCR4 proviral functions in BYSMV replication"The sentence should be rephrased.12) Subsection “CCR4 triggers decay of the N-bound cellular mRNA”, "exhibit specificity improvement”.Many thanks. All the writing errors have been changed or rephrased.


Trichomonas vaginalis, is the most common nonviral sexually transmitted infection in humans. The millions of cases each year have sequelae that may include complications during pregnancy and increased risk of HIV infection.Trichomoniasis, caused by the protozoan  Trichomonas vaginalis is the causative agent of trichomoniasis, the most prevalent nonviral sexually transmitted infection worldwide. Repetitive elements, including transposable elements (TEs) and virally derived repeats, comprise more than half of the ∼160-Mb T. vaginalis genome. An intriguing question is how the parasite controls its potentially lethal complement of mobile elements, which can disrupt transcription of protein-coding genes and genome functions. In this study, we generated high-throughput RNA sequencing (RNA-Seq) and small RNA-Seq data sets in triplicate for the T. vaginalis G3 reference strain and characterized the mRNA and small RNA populations and their mapping patterns along all six chromosomes. Mapping the RNA-Seq transcripts to the genome revealed that the majority of genes predicted within repetitive elements are not expressed. Interestingly, we identified a novel species of small RNA that maps bidirectionally along the chromosomes and is correlated with reduced protein-coding gene expression and reduced RNA-Seq coverage in repetitive elements. This novel small RNA family may play a regulatory role in gene and repetitive element expression. Our results identify a possible small RNA pathway mechanism by which the parasite regulates expression of genes and TEs and raise intriguing questions as to the role repeats may play in shaping T. vaginalis genome evolution and the diversity of small RNA pathways in general.IMPORTANCE Trichomoniasis, caused by the protozoan Trichomonas vaginalis, is the most common nonviral sexually transmitted infection in humans. The millions of cases each year have sequelae that may include complications during pregnancy and increased risk of HIV infection. Given its evident success in this niche, it is paradoxical that T. vaginalis harbors in its genome thousands of transposable elements that have the potential to be extremely detrimental to normal genomic function. In many organisms, transposon expression is regulated by the activity of endogenously expressed short (∼21 to 35 nucleotides [nt]) small RNA molecules that effect gene silencing by targeting mRNAs for degradation or by recruiting epigenetic silencing machinery to locations in the genome. Our research has identified small RNA molecules correlated with reduced expression of T. vaginalis genes and transposons. This suggests that a small RNA pathway is a major contributor to gene expression patterns in the parasite and opens up new avenues for investigation into small RNA biogenesis, function, and diversity. Trichomonas vaginalis colonizes the human urogenital tract and has an estimated global incidence of ∼270 million new cases per year, making it the most common nonviral sexually transmitted infection (STI) , they can also cause vaginitis, urethritis, and pelvic inflammatory disease  . While T disease –5. In exdisease –. MetroniGuinea 7–.T. vaginalis screening, surveillance, or control programs for women or men in the United States, resulting in the disease being considered a “neglected” STI , and genes that encode the protein machinery required for their own transposition, such as transposase and integrase genes. T. vaginalis contains class 2 DNA transposons, which rely on cut-and-paste mechanisms to replicate and transpose  that are produced from long double-stranded RNAs (dsRNAs) —short (∼20 to 35 nucleotides [nt]) RNA molecules that effect gene silencing either by targeting mRNAs for degradation or by recruiting epigenetic silencing machinery to specific locations in the genome –31. In a–32–(dsRNAs) , 39. Whi(dsRNAs) , 41. AllRNAs) 41–. In prota brucei , 45, rettolytica , and pretolytica –49.T. vaginalis genome  mapping to endogenous T. vaginalis protein-coding genes . We also identified putative piRNA clusters (regions that generate the sRNAs for sRNA-guided gene silencing by Argonaute proteins) in the T. vaginalis genome, indicating that the 34-nt sRNAs are likely piRNAi guides. Combined, these data suggest that a small RNA pathway is a major contributor to gene expression patterns in this sexually transmitted parasite, and they open up new avenues for investigation into small RNA biogenesis, function, and diversity.The enormous burden of TEs and repetitive sequences, and our previous identification of a putative RNase III enzyme and two putative Argonaute proteins (AGO1 and AGO2) encoded in the s genome , led us ng genes , 51. HerT. vaginalis AGO1 and AGO2 proteins with Argonaute proteins from a range of phylogenetically diverse organisms , Caenorhabditis elegans WAGO, AGO-like, and PIWI-like. AGO orthologs from T. vaginalis form a monophyletic cluster within the PIWI-like clade, which includes AGO proteins from the ciliates Tetrahymena thermophila, Paramecium tetraurelia, and Oxytricha trifallax, which function in the germ line micronucleus to excise TE sequences by RNAi-mediated programmed DNA elimination , account for 62.8% of or 24.6% . We founor 24.6% . Using aor 24.6% . In addior 24.6% . We alsoor 24.6% .T. vaginalis TE groups have at least one element that was covered by RNA-Seq data, while all of the TE groups have at least one member that was covered by sRNA-Seq reads, and in 11 of the 16 families, 100% of elements are covered by sRNA-Seq reads (We next questioned whether members of each of the described TE families was covered by RNA-Seq and sRNA-Seq reads. We found that while the majority (92.8%) of RNA-Seq reads that map to repeats appear to align to “unknown repeats,” the sRNA-Seq reads map to each of the different repeat and TE families in proportions that more closely reflect their presence in the genome, with the Maverick TE family accounting for 61.6% of all repeats in the genome and 48.8% of the small RNA-Seq reads . All buteq reads . Again, eq reads .Many TEs contain open reading frames (ORFs) coding for transposases or integrases. For example, using Northern blot methods, we previously identified active transcription of the single ORF that encodes a transposase in Mariners . We inteTvMar1 element ±1 kb upstream and downstream. Tvmar1 transposase ORF. This revealed that sRNA-Seq reads map unevenly along both strands of the Tvmar1 element, while the RNA-Seq signal comes from small mapping regions that overlap at the 5′ and 3′ ends of the TE coordinates. It appears that the Mariner elements are not covered by RNA-Seq reads and that sRNA-Seq reads notably map where the RNA-Seq reads are not (To account for the possibility that RNA-Seq (and sRNA-Seq) reads were being distributed across the ∼600 annotated Mariners and thus leading to misleadingly low expression level results, we summed both sRNA-Seq and RNA-Seq reads across every  are not .T. vaginalis small RNAs. We plotted the read length of the unique small RNA reads for each replicate and found that they have a modal length of 34 nt  showed the midpoint at 34 nt, exactly matching the size distribution from our sequencing data. A Northern blot using a DNA probe identical to the 1.3-kb consensus sequence of the Tvmar1 TE family (MAR1) showed a band between the 30- and 40-nt size markers congruent with the band observed on our RNA gels and in our sequencing libraries .Tvmar1 element close to a protein-coding gene is associated with a decrease or lack of expression , we asked whether these small RNAs were associated with silencing. To determine this, we categorized genes as “transcribed” or “silent” according to whether they had an RNA-Seq FPKM above or below the described threshold  and repeats as “with coverage” or “without coverage” according to whether they had an RNA-Seq FPKM above or below the described threshold. We found that sRNA-Seq RPKM is higher in protein-coding genes and repeats that are silent/without RNA-Seq coverage , and expressed protein-coding genes have >80% of their sRNA-Seq reads mapping to the sense strand . To inveTvmar1 elements, because the orientation of members of the Tvmar1 family is known, unlike for the other repeat families. This revealed a 50% sense/antisense distribution, with reads mapping to both strands having a strong modal peak at 34 nt  Proportion of sRNA-Seq reads mapping to the sense and antisense strands of Tvmar1 elements. (B) Length distributions of sRNA-Seq reads mapping to sense and antisense strands of Tvmar1 elements. Download FIG S3, PDF file, 0.1 MB.Strandedness and length distributions of sRNA-Seq reads mapping to Copyright © 2021 Warring et al.2021Warring et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.01061-20.4FIG S4FIG S4, PDF file, 0.2 MB.Nucleotide composition of the first 16 nucleotides of small RNAs aligning to different genomic features. The top row of plots show nucleotide compositions for features which do not have a known sense/antisense orientation, and the sRNA-Seq reads are split by chromosome strand mapping. The bottom row of plots show nucleotide compositions for features that have a known sense/antisense orientation, and the sRNA-Seq reads are split by feature strand mapping. Download Copyright © 2021 Warring et al.2021Warring et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the These findings lead us to hypothesize that the antisense- or bidirectionally mapping ∼34-nt small RNAs are correlated with gene and repeat silencing, while the sRNA-Seq reads mapping to the sense strand of expressed genes are most likely a mix of small RNAs and library contaminants produced by mRNA degradation.T. vaginalis genome that fit the description of piRNA clusters and may generate the sRNAs for sRNA-guided gene silencing by PIWI-like Argonaute proteins  of piRNA-like sRNA-Seq reads  reported by ShortStack identified an ∼10-Mb candidate piRNA locus on chromosome IV (14 to 24 Mb). Download Copyright © 2021 Warring et al.2021Warring et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.01061-20.9TABLE S2https://github.com/MikeAxtell/ShortStack). Download Table S2, XLSX file, 4.9 MB.Candidate piRNA loci predicted by ShortStack. Detailed descriptions of each metric are provided in the documentation for ShortStack  to guide TE excision from germ line DNA in the macronucleus  to regulate its repeats. Our small RNA sequencing results revealed a population of bidirectionally mapping small RNAs with a mode length of ∼34 nt, in the range of piRNAs which are 21 to 35 nt  than AGO-like (miRNA and siRNA-guided) Argonaute proteins , the exng genes . In the bidopsis .T. vaginalis Argonaute proteins. The differences in functional domains encoded by the two genes indicate that they likely perform different functions. Experiments to generate antibodies against the T. vaginalis AGO1 and AGO2 are ongoing in our laboratory. If differential localization and piRNA binding propensity of the proteins are identified, this may alter either of the two previously characterized methods of piRNA-guided expression regulation: DNA modification   , 68 and nucleus) , 69, 70.pathways .T. vaginalis parasites grown under standard laboratory conditions. These small RNAs are correlated with reduced expression of protein-coding genes and repeats at the mRNA level. This finding raises the possibility that a small RNA pathway is a major contributor to gene and TE expression patterns in this parasite’s genome, opening up new avenues for further investigation into the nature and function of the T. vaginalis small RNAs and the diversity of small RNA biogenesis, structure, and function on a wider scale. This mechanism presents an opportunity for harnessing such a system to control gene expression in T. vaginalis in a laboratory setting.In summary, we have identified a novel species of small RNA molecule expressed in T. vaginalis reference strain G3, which has been submitted to the eukaryotic pathogen genomics database TrichDB (http://trichdb.org/trichdb/). Additional identification and annotation of the Maverick family, the largest family of TEs in the genome, and other TE families and repeats were undertaken. Briefly, predicted protein sequences of the 18 ORFs encoding putative proteins of ≥50 amino acid residues in Maverick Tv1.1  pipeline , a more general repeat annotation program that uses RECON and RepeatScout, was used at the end of the pipeline for the identification of any remaining unannotated transposon families using default parameters.Consensus TE sequences from Repbase  were used in BLASTn queries to identify complete and nearly complete non-Maverick transposable elements in the G3 assembly. Additional non-Maverick transposable elements were identified using the Extensive pipeline . The pipT. vaginalis strain G3, the genome reference strain commonly used in research, isolated from Kent, United Kingdom, in 1963, was used for all experiments in this study + RNA (8 μl) with 0.5 μl of random primers , 10 mM dithiothreitol (DTT), and 0.25 μl of anti-RNase  in 1× first-strand synthesis buffer , with incubation at 65°C for 3 min to remove RNA secondary structures, and then placed on ice. A total of 0.5 μl of SuperScript III enzyme  and deoxynucleoside triphosphates (dNTPs) to a final concentration of 0.125 mM were added to the mixture, and reverse transcription was carried out using the following incubations: 25°C for 10 min, 42°C for 50 min, and 70°C for 15 min. The resulting cDNA/RNA hybrid was purified from the mix using Agencourt RNAClean XP beads according to the manufacturer’s instructions. Second-strand synthesis was carried out by mixing the purified cDNA/RNA hybrid with 1 μl of dUTP mix , 0.5 μl of RNase H , and 1 μl of DNA polymerase I  in 1× NEBuffer 2 with 2.5 mM DTT. This mixture was incubated at 16°C for 2.5 h. The resulting double-stranded cDNA was purified using Agencourt AMPure XP beads according to the manufacturer’s instructions. The cDNA was end repaired by mixing 5 μl of T4 DNA polymerase , 2 μl of Klenow DNA polymerase , and 5 μl of T4 polynucleotide kinase  in 1× T4 DNA ligase buffer with 10 mM ATP (NEB) with 0.4 mM dNTPs. The mixture was incubated at room temperature for 30 min. The end-repaired cDNA was purified using Agencourt AMPure XP beads according to the manufacturer’s instructions. The purified end-repaired cDNA was then taken through A-tailing, adapter ligation, and PCR enrichment using the Illumina TruSeq stranded mRNA sample preparation kit, with different barcodes for each sample. The libraries were pooled and sequenced on an Illumina HiSeq 2500 with 101 cycles, paired-end reads, and multiplexing.mirVana kit (Ambion) according to the manufacturer’s instructions. Approximately1 μg of total RNA from each replicate was taken through library preparation using the Illumina TruSeq small RNA sample preparation kit, with different barcodes for each sample. The libraries were pooled and sequenced on an Illumina HiSeq 2500 with 50 cycles, single-end reads, and multiplexing.For small RNA-Seq (sRNA-Seq), total RNA was isolated from overnight cultures using the T. vaginalis cultures using TRIzol. Smaller (<200 bp) RNAs were enriched using the mirVana kit, and 15 μg of this small RNA-enriched RNA was separated on a 15% polyacrylamide gel, transferred to a nitrocellulose membrane, and RNA cross-linked to the membrane using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). A radioactive probe was prepared by cloning the Tvmar1 (TVAG_TE_DS113512_1) consensus sequence into a TOPO TA Cloning vector (Invitrogen), amplified from T. vaginalis G3 genomic DNA using the following primers: forward primer sequence 5′-GAAATCTGTCGTTTAGATCTTCG-3′ and reverse primer sequence 5′- ATTAAATATTTGAGCTTGTGCAC-3′, with an amplicon size of 4,121 bp. After propagation of the cloned insert in TOP10 electrocompetent Escherichia coli, the probe was amplified from the vector using primers that bind to the ends of the Tvmar1 element (5′-GCACAGCGCTCTATATGAGACT-3′ and 5′-GCACAAACCTGAATACTGCG-3′), producing a 1,304-bp probe. A random primer DNA labeling system (Invitrogen) was used to label the probe using [γ32-P]ATP. Size markers were custom 30-nt  and 40-nt  oligonucleotides complementary to the probe DNA.Total RNA was extracted from 15 ml of overnight 32-P]ATP and incubation at 37°C for 1 h. Equal volumes of RNA loading buffer were added, and 1 μl of the sample was run on a 15% polyacrylamide gel for 1 to 1.5 h, including 1 μl of Ambion Decade Marker end labeled with [γ32-P]ATP as a ladder. The gel was exposed to a phosphorimaging screen for 10 min and developed on a Typhoon FLA 9000 laser scanner . The length of the ∼34-nt band was determined by extrapolation using the fR plot method.A total of 1 μg of total RNA was dephosphorylated using shrimp alkaline phosphatase , and the reaction was stopped by heat inactivation (65°C for 10 min). Radioactive labeling of RNA was achieved by the addition of polynucleotide kinase in the presence of 0.5 μl of [γFor determination of the 5′ end of the small RNAs, a synthetic 34-nt RNA oligonucleotide  with no 5′ or 3′ modifications was made . Approximately10 μg each of total RNA and synthetic 34-nt oligonucleotide were treated with Terminator 5′-phosphate-dependent exonuclease (Epicentre), which digests RNA that has a 5′-monophosphate end, according to the manufacturer’s instructions. The reaction was stopped by phenol extraction, and RNA was recovered by ethanol precipitation. RNA dephosphorylation, end labeling, and visualization from a polyacrylamide gel were performed as described above.https://www.ncbi.nlm.nih.gov/; accessed 24 March 2020). Amino acid sequences were aligned with ClustalW version 2.0 .Amino acid sequences of Argonaute proteins from a phylogenetically diverse range of organisms were downloaded from the NCBI website  for RNA-Seq data, and RPKM  for sRNA-Seq data. Genes are transcribed and repeats covered for RNA-Seq if they have a logeplicate . This th10.1128/mSphere.01061-20.7FIG S72 F/RPKM distributions across the three replicates. Shaded areas indicate values that were considered above the threshold. Download FIG S7, PDF file, 0.1 MB.Histograms showing logCopyright © 2021 Warring et al.2021Warring et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.01061-20.10TABLE S3Table S3, XLS file, 8.1 MB.Gene and repeat annotations, including a description of whether they are expressed in RNA-Seq and/or covered with sRNA-Seq. Download Copyright © 2021 Warring et al.2021Warring et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Candidate piRNA loci were identified using ShortStack  version T. vaginalis strain G3 in triplicate have been deposited in NCBI's Sequence Read Archive under accession no. SRX1122976, SRX1122977, and SRX1122978 and small RNA-seq data for T. vaginalis strain G3 under BioProject identifier PRJNA647375.Whole-genome RNA-seq data for


H234A and UL36(480)D232A, but not C98 mutants. These in vitro results suggested that the C98 and H234 are essential catalytic residues of UL36-DUB. UL36-DUB exhibited a strict substrate specificity. Inhibition assay revealed that UL36-DUB exhibits resistance to the Roche protease inhibitor cocktail and serine protease inhibitor, but not to the Solarbio protease inhibitor cocktail. (4) Conclusions: UL36-DUB exhibited a strict substrate preference, and the protocol developed in the current study for obtaining active UL36-DUB protein should promote the high-throughput screening of UL36 inhibitors and the study on the function of MDV-encoded UL36.(1) Background: Deubiquitinase (DUB) regulates various important cellular processes via reversing the protein ubiquitination. The N-terminal fragment of a giant tegument protein, UL36, encoded by the Marek’s disease (MD) virus (MDV), encompasses a putative DUB (UL36-DUB) and shares no homology with any known DUBs. The N-terminus 75 kDa fragment of UL36 exists in MD T lymphoma cells at a high level and participates in MDV pathogenicity. (2) Methods: To characterize deubiquitinating activity and substrate specificity of UL36-DUB, the UL36 N-terminal fragments, UL36(323), UL36(480), and mutants were prepared using the Bac-to-Bac system. The deubiquitinating activity and substrate specificity of these recombinant UL36-DUBs were analyzed using various ubiquitin (Ub) or ubiquitin-like (UbL) substrates and activity-based deubiquitinating enzyme probes. (3) Results: The results indicated that wild type UL36-DUBs show a different hydrolysis ability against varied types of ubiquitin chains. These wild type UL36-DUBs presented the highest activity to K11, K48, and K63 linkage Ub chains, weak activity to K6, K29, and K33 Ub chains, and no activity to K27 linkage Ub chain. UL36 has higher cleavage efficiency for K48 and K63 poly-ubiquitin than linear ubiquitin chain (M1-Ub4), but no activity on various ubiquitin-like modifiers. The mutation of C98 and H234 residues eliminated the deubiquitinating activity of UL36-DUB. D232A mutation impacted, but did not eliminated UL36(480) activity. The Ub-Br probe can bind to wild type UL36-DUB and mutants UL36(480) Ubiquitination is one of the most important regulatory machinery of post-translational modification of intracellular proteins . The modIn human, approximately 100 proteases for deconjugating Ub or Ub chains were found and classified into two main families, cysteine proteases and metalloproteases . The cysPathogens have evolved a series of molecular strategies to promote infection, proliferation, and survival during host–pathogen interactions. Many viruses encode viral DUBs to hijack host cell defense mechanisms, such as Herpes simplex virus (HSV), Kaposi’s sarcoma-associated herpesvirus (KSHV), Epstein–Barr virus (EBV), MDV, pseudorabies virus (PRV), Human cytomegalovirus (HCMV), severe acute respiratory syndrome-coronavirus (SARS-CoV), and Middle East respiratory syndrome-related coronavirus (MERS-CoV) which encode USP type DUBs ,11,12. E+ T cells, and the N-terminal 75 kDa fragment of UL36, which contains the viral DUB catalytic domain (UL36-DUB), exists in MDV-induced T lymphoma cells in high level )/([S] + KM). A Lineweaver–Burk plot was used to determine the KM and Vmax, and then the Vmax/enzyme concentration to calculate the catalytic constant kcat. The catalytic efficiency of each type of UL36 was compared using the ratio kcat/KM.The kinetics of the deubiquitinating activity of wild type UL36(323), UL36(480), and the mutants, UL36(480)C98A, UL36(480)C98S, UL36(480)D232A, and UL36(480)H234A, was characterized by testing the hydrolysis efficiency on a variety of Ub substrates. A reaction system containing 98 μM of substrate , 1× reaction buffer in a final volume of 100 μL was initiated by adding 200 nM UL36-DUBs at 37 °C for different incubation times. The hydrolyzed products were separated by SDS-PAGE and stained with Coomassie brilliant blue R-250. Standard Western blotting was performed using primary antibody against ubiquitin , and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody  was used for ECL imaging.The substrate preference of wild type UL36(323), UL36(480), and the mutants, UL36(480)SUMO1-GST, SUMO2-GST, SUMO3-GST ,48, di-Subiquitin gene (GenBank ID M14693.1) and the NEDD8 gene (GenBank ID XP_015130268.1), the sequence of two genes were optimized using software Codon OptimWiz (Genewiz Inc.). The genes of chicken Ub(75) and NEDD8(75) proteins from which the Gly76 of chicken Ub(76) and NEDD8(76) were absent were amplified by PCR with primers, Ub-probe-F and Ub-probe-R, and NEDD8-probe-F and NEDD8-probe-R (E. coli BL21 (DE3) with the induction by 0.1 mM isopropyl-β-d-thiogalactoside (IPTG). The whole purification process was performed according to the manual of IMPACT™-CN System , except that the elution was carried out post the incubating column with cleavage buffer ) for 12 h at 25 °C to generate Strep-Ub(75)-MESNa or Strep-Nedd8(75)-MESNa. Strep-Ub(75)-MESNa or Strep-Nedd8(75)-MESNa was incubated with 400 mM of bromoethylamine at pH 8.0 for 20 min at 25 °C to generate inhibitor probe, Strep-Ub(75)-Br or Strep-Nedd8(75)-Br. Finally, the prepared probe was dialyzed in a storage buffer  glycerol, pH 7.5), aliquoted for use or stored at −80 °C.Some animal or pathogen DUBs have dual deubiquitinating/de-NEDDylating activity ,31,32,33-probe-R , and theFor the direct binding test, 5 pmol of the Strep-Ub(75)-Br or Strep-Nedd8(75)-Br probe was incubated with 4 pmol of UL36(480) for 1 h at 37 °C. The reaction products were separated with SDS-PAGE and identified by Western blotting using standard protocols  with priFor the catalytic activity assay of the probe treated UL36(480), 125 nM UL36(480) or mutants, and 4 μM Strep-Ub(75)-Br or Strep-Nedd8(75)-Br were preincubated in 1× reaction buffer for 1 h at 37 °C, in this case, a final concentration of 100 nM Ub-AMC was added to initiate the deubiquitinating reaction. The deubiquitinating activity was evaluated by measuring the fluorescence intensity of the AMC released from the reaction on a FluoroMax 4 fluorescence spectrophotometer (HORIBA Scientific) with excitation and emission wavelengths of 380 nm and 460 nm, respectively.For the assay of inhibitor interference to the binding between probes and UL36(480), UL36(480) protein was pre-incubated with 1× working concentration (1 mM) of phenylmethanesulfonyl fluoride (PMSF)  and two types of protease inhibitor cocktails, respectively, manufactured by Solarbio Lifesciences  or Roche of Millipore Sigma  for 1 h at 4 °C, and then followed by the catalytic activity assay as described above. Western blotting was carried out to assess the inhibition effect using primary antibodies against ubiquitin, as described above. Two hundred millimolar stock solution of PMSF was freshly dissolved in DMSO, and the stock solutions of both inhibitor cocktails were prepared fresh before use according to the instructions.


SINEUP RNAs, performing co-transfection of expression vectors for these transcripts into human embryonic kidney cells (HEK293T/17), to investigate the network of translational regulation. The results showed that co-localization of target mRNAs and SINEUP RNAs in the cytoplasm was a key phenomenon. We identified PTBP1 and HNRNPK as essential RNA binding proteins. These proteins contributed to SINEUP RNA sub-cellular distribution and to assembly of translational initiation complexes, leading to enhanced target mRNA translation. These findings will promote a better understanding of the mechanisms employed by regulatory RNAs implicated in efficient protein translation.SINEUPs are long non-coding RNAs (lncRNAs) that contain a SINE element, and which up-regulate the translation of target mRNA. They have been studied in a wide range of applications, as both biological and therapeutic tools, although the underpinning molecular mechanism is unclear. Here, we focused on the sub-cellular distribution of target mRNAs and  More than 70% of the mammalian genome is transcribed into RNAs ,2. The mUchl1, a natural antisense (AS) lncRNA, was able to enhance the translation of its sense Uchl1  mRNA in mouse dopaminergic cells (Uchl1 is an inverted SINEB2 (Short Interspersed Nuclear Element B2) that acts as an effector domain (ED) and is essential for up-regulating protein synthesis  to shed light on the mechanism behind the translation enhancement of target mRNAs by synthetic SINEUPs, with a specific focus on RNA subcellular distribution and its contribution to protein translation.We also found that other natural antisense lncRNAs containing SINEs specifically up-regulate translation of partially overlapping sense mRNAs, and termed this functional class of lncRNAs ‘SINEUPs’ . In addiSINEUP RNA interacts with nucleocytoplasmic proteins such as PTBP1 (polypyrimidine tract binding protein-1) and HNRNPK (heterogeneous nuclear ribonucleoprotein K) that are essential for RNA localization and translational initiation assembly.Here, we show that 2.Human Embryonic Kidney (HEK) 293T/17 cells purchased from ATCC were maintained in DMEM, high glucose, GlutaMAX™ Supplement, pyruvate (Gibco) supplemented with 10% fetal bovine serum (Sigma) and 1% penicillin–streptomycin (Wako) at 37°C, 5% COet al. (-) and SINEUP-ΔHNRNPK binding regions (Δ-) were designed with deletion of the binding sites from SINEUP-GFP. The BD (−34/+10) of SINEUP-UCHL1 is designed to replace the BD of SINEUP-GFP.The pEGFP-C2 vector (expression vector for EGFP) was purchased from Clontech. SINEUP-GFP in pcDNA3.1 (−) vector was described previously in Carrieri et al. . SINEUP-et al. . SINEUP-EGFP mRNA translation at various time points after transfection confirmed that up-regulation of translation of EGFP mRNA occurred from 24 to 48 h post-transfection. SINEUP-UCHL1 vector was transfected in HEK293T/17 cells as mentioned above and harvested after 48 h of vector transfection.The pEGFP-C2 and SINEUP vectors were co-transfected into HEK293T/17 cells by using Lipofectamine 2000 (Invitrogen) with OptiMEM (1×) Reduced Serum Medium (Gibco). Testing Cells were plated in twelve-well plates, transfected with plasmid(s), lysed in Cell Lysis buffer , and incubated at 4°C for 1 h. The cell lysates were applied to 10% precast polyacrylamide gels (Bio-Rad) for SDS-PAGE and transferred to nitrocellulose membranes (Amersham). The membranes were incubated for 1 h at room temperature with the primary antibody, anti-GFP rabbit polyclonal antibody , and then for 45 min at room temperature with the secondary antibody, anti-rabbit IgG conjugated with HRP , and EGFP was detected by ECL detection reagent (Amersham). As a control, anti-β actin mouse monoclonal antibody  was used as the primary antibody, and anti-mouse IgG–conjugated HRP  was used as the secondary antibody. To detect endogenous target proteins, anti-hnRNPK mouse monoclonal antibody [3C2]-ChIP Grade , anti-PTBP1 mouse monoclonal antibody , RPL7A rabbit polyclonal antibody , RPS3A rabbit polyclonal antibody , and anti-UCHL1 mouse monoclonal antibody  were used at 1:1000 dilution with overnight incubation at 4°C.Taq II (TAKARA) and the 7900HT Fast Real-Time PCR System (Applied Biosystems). The thermocycling protocol was 95°C for 30 s followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The RNA expression level was normalized to the level of GAPDH mRNA in each fraction. The primer sequence of SINEUP RNAs and GFP mRNAs was described in published paper  following the manufacturer's instructions. For separation of nuclear and cytoplasmic fractions, the PARIS kit (ThermoFisher) was used to obtain nucleic and cytoplasmic fractionated lysate. TURBO DNA-free Kit (Invitrogen) was used for DNase I treatment to remove plasmid DNAs. For RT-qPCR, cDNA was synthesized using the PrimeScript 1st strand cDNA synthesis kit (TAKARA), and PCR was performed with SYBR Premix Ex ed paper . UCHL1 mhttps://www.biosearchtech.com/support/tools/design-software/stellaris-probe-designer), and fluorescently labelled with Quasar 570 (for SINEUP RNAs) or Quasar 670 (for EGFP mRNA and UCHL1 mRNA)  . Stellaretector; , and per_Studio; .2 of 254 nm UV light . The cell pellet (2 × 107 cells) was suspended with 2 ml of Lysis buffer  supplemented with Protease Inhibitor Cocktail Set III (Merck Millipore), mixed by rotation at 4°C for 30 min, and then sonicated for 8 cycles  using a Picoruptor Sonicator (Diagenode). Each tube of lysate was incubated at 37°C for 30 min with MagCapture Tamavidin 2-REV magnetic beads (WAKO) to remove nonspecific binding proteins, which improves specificity of capture of specific RBPs. The lysate was incubated overnight at 37°C in hybridization buffer , 1 mM EDTA) with additional 15% (v/v) formamide (Sigma), Phenylmethanesulfonyl Fluoride (PMSF) , Protease Inhibitor Cocktail Set III (Merck Millipore) and SUPERase• In RNase Inhibitor (Thermo Fisher Scientific) just before use, and 100 pmol of probe. Each lysate was then incubated at 37°C for 30 min with washed beads. After sequential washes, the bead samples were separated into two halves for protein and RNA extraction. Proteins were extracted as reported by Chu et al. 2015 (The protocol for detecting SINEUP RBPs was based on the protocol for the Magna ChIRP RNA Interactome kit (Merck Millipore) with the modification of cross-linking with 300 mJ/cm2 for 24 h. Following this, the vectors were transfected into the cells as described above. Targeted proteins were detected by using the following anti-mouse monoclonal antibodies purchased from Santa Cruz: DnaJC1[D-10] (sc-514244), EF-1 α1 [CBP-KK1] (sc-21758), EF-2 [C-9] (sc-166415), hnRNP K/J [3C2] (sc-32307), hnRNP M [A-12] (sc-515008), hnRNP I [SH54] (sc-56701), hnRNP U [3G6] (sc-32315) and Lamin B1 [8D1] (sc-56144). The above primary antibodies were diluted 1 in 500 and then incubated at 4°C overnight. HRP-conjugated anti-mouse IgG  was used as secondary antibody diluted 1 in 1000 and then incubated at room temperature for 45 min for protein visualization.All siRNAs listed below were purchased from Thermo Fisher Scientific. DNAJC1 Silencer Select Pre-designed siRNA (ID: s34557), EEF1A1 Silencer Select Pre-designed siRNA (ID: s4479), EEF2 Silencer Select Pre-designed siRNA (ID: s4492), HNRNPK Silencer Select siRNA: Standard (ID: s6739 was used in main figures and ID: s6738 and ID: s6737 were used in https://www.abcam.com/epigenetics/rna-immunoprecipitation-rip-protocol) with some modifications. HEK293T/17 cells were plated into 10-cm plates, followed by plasmid transfection described above. On the following day, the cells were irradiated with 300 mJ/cm2 of 254 nm UV light, and nuclei and cytoplasmic fractions were isolated. Nuclear pellets were sheared by sonication with 5 cycles  using a Bioruptor Pico device. To immunoprecipitate RNA with the antibodies for target proteins, each lysate was incubated at 4°C overnight. Protein A/G magnetic beads (Invitrogen) were added to bind the target antibodies, and sequential washing was conducted to remove unbound antibodies. The anti-hnRNP K mouse monoclonal antibody [3C2]-ChIP Grade  and anti-PTBP1 mouse monoclonal antibody  were used to immunoprecipitate HNRNPK and PTBP1, respectively, in each nuclear or cytoplasmic fraction. To purify the RNA, the lysates were incubated with protease K at 55°C overnight, followed by Trizol (Thermo Fisher Scientific) and chloroform (WAKO) extraction. RNA levels were quantified by RT-qPCR.RIP was performed using the Abcam protocol . The transfected cells were maintained in DMEM (1×) + GlutaMAX-1 (Gibco) supplemented with 10% fetal bovine serum (Sigma) without penicillin-streptomycin (Wako) at 37°C, 5% COHEK293T/17 were prepared as described above for RNA FISH. After the cells were permeabilized, the primary antibodies, anti-hnRNP K mouse monoclonal antibody [3C2]-ChIP Grade  and anti-PTBP1 mouse monoclonal antibody , were added and hybridized overnight. Alexa Fluor 647–conjugated goat anti-mouse IgG secondary antibodies  were used to visualize the results. Several organelle marker antibodies were tested as follows; anti-SC35 , anti-nmt55/p54nrb , anti-ILF3 , anti-EF1A , anti-ATP5 , anti-Calnexin  and J2 monoclonal antibody . MALAT1 probes labeled with Atto 633 .6 cells were plated into 10-cm plates, followed by hnRNPK and PTBP1 clone vector transfection after 18 h, and EGFP and SINEUP vector transfection 6 h later. All transfections were performed as described above. The transfected cells were maintained in growth media without antibiotics for 48 h following clone vector transfection, then incubated with 0.1 mg/ml cycloheximide for 5 min at 37°C followed by washing with ice-cold PBS containing 0.1 mg/ml cycloheximide. The harvested cells were centrifuged at 300 × g for 10 min at 4°C. The cell pellets were suspended with 200 μL of ice-cold lysis buffer . The cell lysate was incubated for 10 min on ice followed by centrifugation at 2000 × g at 4°C for 5 min to separate the nuclei. The cytoplasmic fraction was subjected to further centrifugation at 17,000 × g at 4°C for 5 min to remove cell debris. The cytoplasmic lysate was layered onto a 15–45% sucrose gradient and centrifuged in an SW41Ti Beckman rotor at 190 000 × g at 4°C for 3.5 h. The sucrose gradient was separated into 12 fractions calculated by Triax flow cell (Biocomp). Half of each fraction was treated with Proteinase K at 55°C overnight then followed by Trizol and chloroform extractions as described above. The eluent was treated with DNase I (Ambion), and the RNA expression level was quantified by RT-qPCR.Polysome fractionation was performed as reported previously . Brieflyg for 10 min at 4°C. Each pellet was suspended in PBS and subjected to Western blotting analysis.Using the other half of each fraction, proteins were isolated using the Thermo Fisher Scientific acetone precipitation protocol, 4 volumes of cold acetone were added to each sample, and then the samples were incubated at −20°C overnight, and centrifuged at 13 000 × BS3 (bis [sulfosuccinimidyl] suberate) cross-linking was performed based on a published protocol . The celhttps://www.abcam.co.jp/protocols/cross-linking-chromatin-immunoprecipitation-x-chip-protocol), and nuclei and cytoplasmic fractions were isolated using the PARIS kit (ThermoFisher). To immunoprecipitate dsRNA with J2 monoclonal antibody , fractionated lysates were incubated at 4°C overnight. The beads were washed three times with 1× RIPA buffer at 37°C for 5 min, and nucleic fraction beads underwent two further DNase treatments incubated at 37°C for 30 min. RNA was extracted as mentioned in the RIP protocol and quantified by RT-qPCR.Formaldehyde cross-linking was performed based on the Abcam X-ChIP protocol  or anti-PTBP1  antibodies were coupled to 125 μl magnetic beads (Dynabeads M-280 Sheep Anti-mouse IgG). To capture RBP-RNA complexes on beads, the cell lysate was incubated with a magnetic beads-coupled antibody at 4°C overnight. Next, 2% input was saved and RNA in IP samples was dephosphorylated and end treated with Poly Nucleotide Kinase followed by on-beads 3′ RNA linker    (FASTX Toolkit/)  (versionoolkit/)  merge fuSINEUP-GFP deletion mutants lacking PTBP1 or HNRNPK binding regions were synthesized by the commercial preparation service at GENEWIZ . HEK293T/17 cells were prepared as described above (see cell culture and plasmid transfection), and EGFP up-regulation was measured by Western blotting assay (see Measuring protein up-regulation by Western blotting assay).https://www.ncbi.nlm.nih.gov/sra/?term=SRP111756). Raw FASTQ files for MCF7 (paired end) were downloaded and processed to remove rRNA reads, check overall quality and remove any orphan reads from the paired end samples. Resultant files were mapped to the human genome (hg38) using STAR   . The bact-test. Bar graphs were described as mean ± standard deviation (SD) from at least three independent experiments. Statistically significant changes relative to a negative control were represented with *P < 0.05, **P < 0.01. To test normality of all data sets, Kolmogorov–Smirnov test was used.Statistical differences were measured using a paired, two-sided Student's EGFP mRNA  was ∼1.5-fold higher for pCS2+ than for pcDNA3.1  is transported to the cytoplasm upon rapamycin treatment, enhancing Uchl1 mRNA translation  following co-transfection of EGFP and SINEUP expression vectors (SINEUP-GFP or the deletion mutants) into HEK293T/17 cells. We observed that EGFP mRNAs were predominantly localized in the cytoplasm  and SINEUP-ΔAlu RNAs (31.12%) than for SINEUP RNAs with impaired translational up-regulation activity (i.e. SINEUP-SCR and SINEUP-ΔSB2)  as compared to SINEUP vector with EGFP vector co-transfection (SINEUP RNAs into the cytoplasm in the presence of EGFP mRNA.) Figure ; SINEUP-) Figure . This inytoplasm , as was  mutants . In conts Figure , e, h, kDetector . The pere Figure ; this waEUP-ΔSB2 . As suppsfection . ConsistSINEUP RNA binding proteins (RBPs) may play a crucial role in EGFP expression. To identify SINEUP RBPs in the cells, we used a modified version of the Chromatin Isolation by RNA Purification (ChIRP) method  following PTBP1 knockdown was not significantly changed, with reduction of SINEUP RNA only seen at the cytoplasmic level following PTBP1 knockdown. This suggests that PTBP1 does not have an effect on the transcript expression level and EGFP mRNA translation machineries, but instead has an effect on SINEUP RNA subcellular localization, consistent with changes in subcellular localization following PTBP1 overexpression (see We hypothesized that ) method  (Supplem) method  followed) method  while no) method . We then) method  and obse) method . After e) method  to asses) method . The exp) method  or HNRNP) method  significod , and . Knockdood , and , suggestod , and , f–j andod , and , k–o sig. Figure , a-e  assay of RNA–protein interactions with PTBP1 and HNRNPK proteins in the nucleus and cytoplasm. SINEUP-GFP RNAs were pulled down with PTBP1 both in the nucleus  HNRNPK protein was able to bind to EGFP mRNA or the EGFP–SINEUP RNA complex in either the nucleus or the cytoplasm, but did not bind to SINEUP-GFP RNA alone. Taken together, these findings indicate that PTBP1 and HNRNPK play a role in the formation of the RNA-protein complexes and participate in the kinetic distribution of these RNA–protein complexes.In the knockdown of HNRNPK, we observed that the reduction of rocesses , and assrocesses , we inverocesses . As resus Figure A1 and cys Figure A2 and EGs Figure B1 and cys Figure B2. TheseSINEUP RNA complex and functionally enhanced EGFP levels.We next wondered whether EGFP levels would be further enhanced by overexpression of PTBP1 and HNRNPK proteins. To address this question, we transfected cells with either PTBP1 or HNRNPK expression vector. The EGFP level was moderately, but significantly increased by overexpression of PTBP1 Figure A1 and A3SINEUP RNA, we overexpressed PTBP1  and monosomes (37.8%)  Figure , m. Thiss Figure , j, k, ls Figure , l. PTBPs Figure , k. Addie Figure , m. ThisSINEUP-GFP RNA, we performed seCLIP; single-end enhanced crosslinking and immunoprecipitation assay  with different annealing sites , were ineffective to induce SINEUP activity  into HEK293T/17 cells. We observed that Uchl1 mRNAs were mainly localized in the cytoplasm  and SINEUP-SCR Figure . On the l Figure . In ordem Figure , c, g, km Figure , f, j. Cs Figure . When eis Figure , a1, 2 os Figure , b1,2 wad Figure , d. Noteockdowns . These rSINEUP RNA plays a critical role in the enhancement of the translation of its target mRNA by (i) co-localizing with the target mRNA in the cytoplasm, (ii) interacting with SINEUP RBPs to influence the SINEUP RNA’s distribution and (iii) increasing target mRNA shifting to the polysome by participating in translational initiation assembly.In this study, we demonstrated that a synthetic EGFP mRNA and SINEUP-GFP RNA, and also Uchl1 mRNA and SINEUP-UCHL1 RNA in the cytoplasm was required for positive translational regulation. We previously reported that AS-Uchl1 is enriched in the nucleus and exported to the cytoplasm under rapamycin-induced stress, which inhibits the mTOR  pathway, resulting in up-regulation of UCHL1 translation  is a multifunctional RNA binding protein that participates in alternative splicing, mRNA stabilization, and nucleocytoplasmic shuttling by binding to the polypyrimidine-rich tract in pre-mRNAs . PTBP1 i subunit . In this subunit ,38. In o subunit . In this subunit , j–m. Totriplets . AlthougVEGF mRNA and stimulated the ribosome to bind the mRNA resulting in a shift to heavier polysomes  domains, which bind RNAs, and unique nuclear localization signals with bi-directional transport, that enables its export to the nuclear envelope with target mRNAs . HNRNPK olysomes . In contnslation . In our SINEUP-GFP RNA and EGFP mRNA coexisted, i.e. not when the cells were transfected with EGFP construct alone. Similarly, when PTBP1 was overexpressed, SINEUP-GFP RNA distribution was affected only when SINEUP-GFP RNA and EGFP mRNAs co-existed. These findings imply that the SINEUP RBPs, SINEUP-GFP RNA, and EGFP mRNAs together form RNA–protein complexes, and that these multiple components are essential for a functional complex. Several studies report protein–protein direct interactions between PTBP1 and HNRNPK, supporting that they function cooperatively in biological processes . The result showed that SINEUP-GFP RNAs and EGFP mRNAs formed double strands in the cytoplasm , which mainly participate in alternative mRNA splicing, conformation of RNP assembly to compact transcripts in the nucleus, and nucleocytoplasmic shuttling . RNA–RNAtin loci ,47, and tin loci ,49. NONO nucleus . ILF3 .The eCLIP-seq data for HNRNPK and PTBP1 discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus  and are We have uploaded bigWig files for each of the samples to a UCSC genome browser session which can be accessed using the following link.https://genome.ucsc.edu/s/mnzvalentine/HNRNPK_PTBP1_eCLIP_hg38_BAMgkaa814_Supplemental_FilesClick here for additional data file.


Aedes flavivirus (AEFV), Menghai rhabdovirus (MERV), and Shinobi tetra virus (SHTV), we infected adult Aedes albopictus females and males and generated small RNA libraries from ovaries, testes, and the remainder of the body. Overall, both female and male mosquitoes showed unique small RNA profiles to each co-infecting ISV regardless of the sex or tissue tested. While all three ISVs generated virus-derived siRNAs, only MERV generated virus-derived piRNAs. We also studied the expression of PIWI genes in reproductive tissues and carcasses. In contrast to Piwi5-9, Piwi1-4 were abundantly expressed in ovaries and testes, suggesting that Piwi5-9 are involved in exogenous viral piRNA production. Together, our results show that ISV-infected Aedes albopictus produce viral small RNAs in a virus-specific manner and that male mosquitoes mount a similar small RNA-mediated antiviral response to that of females.The mosquito antiviral response has mainly been studied in the context of arthropod-borne virus (arbovirus) infection in female mosquitoes. However, in nature, both female and male mosquitoes are frequently infected with insect-specific viruses (ISVs). ISVs are capable of infecting the reproductive organs of both sexes and are primarily maintained by vertical transmission. Since the RNA interference (RNAi)-mediated antiviral response plays an important antiviral role in mosquitoes, ISVs constitute a relevant model to study sex-dependent antiviral responses. Using a naturally generated viral stock containing three distinct ISVs,  Aedes and Culex are two major genera of mosquitoes that transmit medically important arthropod-borne viruses (arboviruses) such as dengue, West Nile and Zika viruses. Recent metagenomics studies have shed light on the virome of these mosquito vectors, revealing diverse virus communities largely composed of insect-specific viruses (ISVs) that specifically infect invertebrates but not vertebrates [Aedes (Ae.) albopictus C6/36 cell line from the Japanese Collection of Research Bioresources (JCRB), suppressed Zika virus replication in vitro [Mosquitoes are efficient vectors for various human pathogens. tebrates ,4,5,6,7.tebrates ,12,13,14in vitro ,16,17. TThe small interfering RNA (siRNA) pathway is considered to be a major antiviral defense mechanism in insects, including vector mosquitoes . The celDrosophila melanogaster and reviewed in [Drosophila melanogaster, where vpiRNAs are not produced during virus infections [Drosophila; Ae. aegypti has eight PIWI genes (Piwi1-7 and Ago3) and Ae. albopictus was estimated to have ten PIWI genes (Piwi1-9 and Ago3) [Ae. albopictus remains to be determined. Functional studies of PIWI proteins have been primarily conducted in the Ae. aegypti cell line Aag2 using arboviruses. Gene silencing of Piwi5 and Ago3 decreased vpiRNA biogenesis without impacting the replication of the Semliki Forest virus (SFV), Sindbis virus (SINV) or dengue virus [The P-element induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway has been demonstrated to control transposable elements (TEs) in the germ line. The biogenesis of piRNAs is well characterized in iewed in . piRNAs iewed in ,30,31,32fections . The prond Ago3) ,35. Thisue virus ,29,31,36ue virus . More reue virus . Aedes flavivirus (AEFV) in a field-collected Ae. albopictus individual [Flaviviridae family and is a positive-sense single-stranded RNA (ssRNA) virus, MERV is from the Rhabdoviridae and is a negative-sense ssRNA virus, and SHTV is from the Tetraviridae and is a positive-sense ssRNA virus. To compare the small RNA responses generated by these distant ISVs, we deep-sequenced small RNAs from ovaries, testes and carcasses of Ae. albopictus triple-infected with AEFV, MERV and SHTV. All three viruses infected the reproductive tissues and carcasses. Each ISV showed a unique small RNA profile independent of the sex and tissue of the mosquito tested. Similar amounts of viral small RNAs were produced in the testes and carcasses, while the ovaries produced reduced amounts. We also observed differential gene expression for some PIWI-genes between reproductive tissues and the carcasses. Our results showed that Ae. albopictus responds to co-infecting ISVs in a virus-specific manner at the small RNA level and that male adult mosquitoes mount a similar small RNA response to that of females during viral infection. Unlike arboviruses, ISVs are likely to be commensal in mosquito hosts and are considered to be maintained in nature by vertical transmission ,41,42,43dividual . The AEFdividual . ConsequAe. albopictus were maintained at 28 °C in L-15 Leibovitz’s medium  supplemented with 10% fetal bovine serum , 1% nonessential amino acids (Gibco), 2% tryptose phosphate broth , and 1% penicillin-streptomycin . The C6/36 cells used in this study are free of known viruses in the NCBI viruses database, confirmed by BLASTn searches using high throughput DNA and RNA sequencing data from the cells with the following parameters: reward/penalty value of 1/-1, gap opening penalty of 2, gap extension of 1, and the expected value of 0.001.C6/36 cells (ATCC CRL-1660) derived from The mixed virus stock containing AEFV strain Narita-21, MERV isolate Menghai, and SHTV strain Shinobi was kindly provided by Haruhiko Isawa, National Institute of Infectious Diseases, Japan. The virus stock was amplified in naïve C6/36 cells grown in L-15 Leibovitz’s medium and the supernatant was used for the co-infection experiment in vivo. To create a standard curve for AEFV, the non-structural gene 4 (NS4) was amplified with primers containing the T7 promoter sequence followed by reverse transcription with MEGAscript T7 Transcription Kit  to generate AEFV NS4 RNA. The copy number of AEFV RNA was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) based on a serial dilution of the AEFV NS4 RNA with Maxima H minus First Strand cDNA Synthesis kit  and Fast SYBR Green Master Mix . MERV and SHTV RNA copy numbers in the virus stock were calculated using the Qgene template program .Ae. albopictus were established using field-collected mosquitoes from Binh Duong Province, Vietnam (2011) [Laboratory colonies of m (2011)  and Kawam (2011) . All theAe. albopictus were intrathoracically injected with an AEFV/MERV/SHTV mixture containing 1 × 107 genome copies of AEFV, 3.2 × 105 genome copies of MERV, and 1.6 × 105 genome copies of SHTV with a nanoinjector . Following the injection, mosquitoes were incubated at 28 °C, 70% relative humidity and a 12 h light: 12 h dark cycle with access to a 10% sucrose solution at all times.Four to seven days after emergence, adult female and male Ovaries, testes and the rest of the bodies, which we define as carcasses for this study, from the AEFV/MERV/SHTV-injected mosquitoes were manually dissected at day 8 post-infection. Total RNA was extracted from 3 pools of 20 ovaries, testes or carcasses with TRIzol reagent . cDNA synthesis and qPCR were performed as described above for AEFV. To normalize ISV, DCR2, Ago2, and PIWI gene expression, two different housekeeping genes were used, actin and ribosomal protein L18 (RPL18). All PCR primer sequences are listed in Three pools of 20 ovaries, testes or carcasses from the ISV-infected mosquitoes were combined for the generation of small RNA libraries. 19-33 nt long RNAs were cut and extracted from a 15% acrylamide/bis-acrylamide (37.5:1), 7M urea gel and the purified RNAs were subjected to small RNA library preparation using the NEBNext Multiplex Small RNA Library Prep  kit with a 3’ adaptor from Integrated DNA Technologies  and in-house designed indexed primers. Libraries diluted to 4 nM were sequenced with the NextSeq 500 High Output Kit v2 (75 cycles) using a NextSeq 500 . Small RNA libraries have been submitted to the NCBI sequence read archive (SRA) under BioProject PRJNA587399.www.bioinformatics.babraham.ac.uk/projects/fastqc/). Low-quality bases and adaptors were trimmed from each read using the Cutadapt program and only reads showing an acceptable quality  were retained. A second set of graphics was generated by the FastQC software using the trimmed fastq files. Reads were mapped to the genome sequences of AEFV strain Narita-21 (GenBank accession no. AB488408.1), MERV isolate Menghai (GenBank accession no. KX785335.1) and SHTV strain shinobi (GenBank accession no. LC270813.1) using Bowtie [Ae. albopictus Vietnam strain, a BLASTn search against its genomic DNA library, deposited in SRA under BioProject accession number PRJNA587399, was performed. The quality of the fastq files was assessed with FastQC software  adult females and males (4–7 days old post-emergence) were intrathoracically injected with a naturally derived viral stock containing a mixture of three ISVs. The relative genome copies for each virus injected in the mosquitoes were as follows: 1 × 107 genome copies of AEFV, 3.2 × 105 genome copies of MERV, and 1.6 × 105 genome copies of SHTV. At 8 days post-injection (dpi), ovaries, testes and carcasses, which refers to the rest of the body parts without ovaries or testes, were dissected and 3 pools of 20 tissues each were prepared to examine ISV infections. RNA levels of each ISV were quantified by RT-qPCR and normalized based on the levels of actin or RPL18 RNA  co-infected with the three ISVs by intrathoracic injection. Analysis of this library confirmed that, despite the high number of reads corresponding to SHTV-derived siRNAs, 26-32 nt-long small RNAs derived from SHTV were nearly undetectable in carcasses of female mosquitoes , the 21 nt vsiRNAs and 26-30 nt vpiRNA-like small RNAs were mapped to the AEFV genome sequence. The vsiRNAs were detected across the entire viral genome in carcasses with a notably high density of vsiRNAs mapping to the 3’ UTR A. The 26Mapping of the small RNA reads to the genome of SHTV (a positive-sense ssRNA virus) showed that the vast majority were vsiRNAs. SHTV-derived vsiRNAs mapped across the entire genome and lacked any obvious hotspots A. Very fAe. albopictus genome (Bioproject PRJNA587399). No MERV-like DNA sequences were found in the Ae. albopictus-Vietnam genome. These results indicate that the vpiRNAs were derived from MERV RNA.The small RNA profile of MERV (a negative-sense ssRNA virus) showed high levels of both vsiRNAs and putative vpiRNAs . The 21-Ae. aegypti found differential expression of some PIWI genes between ovaries and carcasses [Ae. albopictus in vivo. This prompted us to determine if a link between the production of vpiRNAs and PIWI gene expression levels in co-infected mosquitoes existed. We examined mRNA levels of the main components of the piRNA pathway, together with the siRNA pathway, in ovaries, testes, and carcasses. Based on the Ae. aegypti PIWI genes, a recent bioinformatics study [Ae. albopictus mosquitoes - Piwi1-9 and Ago3. Using the same RNA samples we used to measure ISV RNA levels in different mosquito tissues, we performed RT-qPCR to determine the expression levels of genes predicted to be involved in the production of siRNAs and piRNAs in Ae. albopictus. Due to the sequence similarity of some PIWI genes, we assessed gene expression as a group or as pairs for Piwi1-4, Piwi5/6, and Piwi8/9. Relative gene expression was calculated by normalization based on the levels of actin or RPL18 RNA. Overall, ovaries displayed a significantly higher level of expression for all genes examined, especially PIWI genes, as compared to other samples , Merida virus (Rhabdoviridae), La Crosse virus, or Rift Valley fever virus (both Bunyaviridae). In addition, both primary and secondary vpiRNAs were generated from some alphaviruses (positive-sense single-stranded viruses) such as SINV, SFV, and chikungunya virus in vitro and/or in vivo in Aedes mosquitoes [The production of different types of small RNAs is in agreement with previous studies in r piRNAs ,30,32. Wll lines ,30,51,52squitoes ,27,51,53The vsiRNAs derived from AEFV and MERV clustered in the 3’ UTR and 5’ UTR, respectively. This accumulation was also observed for 26-30 nt small RNAs, although the number of reads was much lower than the number of vsiRNAs. We speculate that these viral small RNAs from hotspots could derive from the secondary structure within the viral genome rather than from replication intermediates. DCR2 could cleave the hairpin structure of viral RNAs and the loop structure adjacent to the cleavage site might result in 26-30 nt long small RNAs as a by-product of vsiRNA production. Interestingly, previous studies suggested that the subgenomic RNA of flaviviruses, which corresponds to the 3’ UTR, is capable of suppressing the RNAi machinery, possibly by acting as an RNA decoy or by inhibiting the loading of vsiRNA into the RISC ,23,24. TCx. quinquefasciatus cell lines infected with Merida virus, also a rhabdovirus, showed a similar vpiRNA profile, suggesting that the regions that generate vpiRNAs are conserved regardless of the mosquito species and depend on the viral replication strategy [Aedes mosquitoes [Ae. aegypti, Piwi5 is likely to play a role in producing primary piRNAs from both TEs and exogenous viruses, however, how Piwi5 selects the transcript to process into primary piRNAs is still unknown [MERV-derived piRNAs were largely produced from the region of the viral genome encoding the structural proteins, corresponding to nucleotide positions 130 to 4162. squitoes ,51,52,53 unknown . Ae. albopictus based on PIWI genes in Ae. aegypti using a tBLASTx search [Ae. albopictus is not as well developed as compared to Ae. aegypti. We quantified the expression levels of the putative Piwi1-9 and Ago3 genes as well as the DCR2 and Ago2 genes by qPCR in ISVs-infected Ae. albopictus samples. A previous study examined PIWI gene expression in midgut and non-midgut tissues of female Ae. albopictus mosquitoes by transcriptome analysis and RT-PCR [Ae. albopictus. Nevertheless, a better genome assembly and annotation for Ae. albopictus is required to conduct studies that will allow the determination of the PIWI genes involved in vpiRNA biogenesis.A recent study identified ten PIWI genes, Piwi1-9 and Ago3, in x search . Howeverd RT-PCR . Our resThe present study demonstrated that, regardless of the mosquito sex and the reproductive nature of the tissue, the production of different types of viral small RNAs from ISVs is virus-dependent. Our results highlight the value of ISVs as models to better understand small RNA pathways in mosquitoes as well as the potential countermeasures of viruses to escape the RNA interference response. Future studies should address the small RNA responses to ISVs in mosquitoes that are naturally infected. This could lead to new insights into mosquito immunity and mosquito–ISV–arbovirus relationships.


Latinos have high risk of Alzheimer’s disease and related dementias (ADRD). Self-rated health (SRH) has been used to predict cognitive decline. Early detection of executive function changes may help identify those at higher risk of cognitive decline. The purpose of this study was to examine the relationship between SRH and executive function in Latinos. Latinos  from the BAILA randomized controlled trial self-rated their health as 1) poor/fair, 2) good, and 3) very good/excellent. Executive function was assessed by the Trail-making B, Verbal Fluency, Stroop C & CW, and the Digit Modality tests and stratified by SRH. One-way analysis of variance showed that the effect of SRH was significant for Trails B, F=4.01, p=.019 and Stroop CW, F=3.07, p=.048. Tukey’s test indicated that participants who rated their health as fair/poor took longer to complete Trails B (M=196.78±83.0 seconds) compared to those who rated their health as good (M=185.25 ± 85.1 seconds) and very good/excellent (M=149.25±95.3 seconds). Stroop CW results demonstrated that those in the fair/poor health category scored lower (M=17.22±6.6) than those in good (M=19.70±8.5 words/minutes) and very good/excellent health categories (M=18.73±8.2 words/minute). In sum, the results suggest SRH is related to executive function such that lower categories of SRH are indicative of poorer executive function. SRH might be used as a proxy for executive function and as a tool that community leaders can use to identify individuals at high risk of ADRD in need of behavioral interventions.