Fragaria vesca (FvLHC II-1L) affects viral infection remains unclear. In this study, we demonstrated that the movement protein P1 of strawberry vein banding virus (SVBV P1) interacted with FvLHC II-1L in vivo and in vitro by bimolecular fluorescence complementation and pull-down assays. SVBV P1 was co-localized with FvLHC II-1L at the edge of epidermal cells of Nicotiana benthamiana leaves, and FvLHC II-1L protein expression was upregulated in SVBV-infected F. vesca. We also found that FvLHC II-1L effectively promoted SVBV P1 to compensate for the intercellular movement of movement-deficient potato virus X (PVXΔP25) and the systemic movement of movement-deficient cucumber mosaic virus (CMVΔMP). Transient overexpression of FvLHC II-1L and inoculation of an infectious clone of SVBV showed that the course of SVBV infection in F. vesca was accelerated. Collectively, the results showed that SVBV P1 protein can interact with FvLHC II-1L protein, which in turn promotes F. vesca infection by SVBV.Chlorophyll a/b-binding protein of light-harvesting complex II type 1 like (LHC II-1L) is an essential component of photosynthesis, which mainly maintains the stability of the electron transport chain. However, how the LHC II-1L protein of  The strawberry vein banding virus (SVBV) is a latent virus that severely harms strawberry plants in major strawberry growing areas worldwide . In ChinCaulimovirus of the Caulimoviridae family. It contains seven open reading frames (ORFs), each of which encode a protein ; furthermore, overexpression of AtPATL3 makes AMV MP unable to target PD, thereby affecting the intercellular movement of the virus  to neighboring cells with the help of MPs . MPs canhe virus . Similarnfection . Triple nfection . Moreovehe virus . Thus, hAtLhcb1 in Arabidopsis thaliana hindered the formation of LHC II trimers, affected photosynthesis, and caused plant dwarfing (AtTHF1 decreased the rate of LHC II degradation and delayed leaf yellowing (Fragaria vesca (FvLHC II-1L), based on the results of yeast two-hybrid (Y2H) assay  is a large protein complex located on the thylakoid membrane of chloroplasts. It comprises the PSII core complex and light-harvesting complex II (LHC II), which drive water oxidation and are key components of photosynthesis . LHC II dwarfing ; loss-ofellowing . Our preH) assay , but thein vivo and in vitro. Quantitative real-time polymerase chain reaction (RT-qPCR) showed that FvLHC II-1L expression was significantly upregulated following SVBV infection. The subcellular localization assay revealed that SVBV P1 and FvLHC II-1L co-localized at the edge of the plant cell and that FvLHC II-1L was able to promote SVBV P1 aggregation. Furthermore, we showed that FvLHC II-1L promoted SVBV P1 to compensate for the movement of movement-deficient potato virus X (PVXΔP25) and cucumber mosaic virus (CMVΔMP). Overexpression of FvLHC II-1L enhanced SVBV infection in F. vesca.In this study, we used bimolecular fluorescence complementation (BiFC) and pull-down assays to demonstrate that P1 interacts with FvLHC II-1L All plants used in this study were grown in a growth chamber, set at 25°C, under 60% relative humidity and a 16 h light and 8 h dark photoperiod.N. benthamiana plants were used for Agrobacterium tumefaciens infiltration, as described previously  were mixed before co-infiltration.Six- to eight-week-old eviously . Equal vA. tumefaciens inoculum containing pTRV1 and pTRV2 or its derivatives (OD600 = 1.0) and placed in a vacuum at 101 kPa atmospheric pressure for 30 s; this procedure was repeated. The treated plants were washed with distilled water and cultured in pots containing nutrient solution, and after 24 h, they were transplanted into pots containing nutrient soil.Strawberry plants were infiltrated as described previously . The rooA. tumefaciens (GV3101) and co-infiltrated into N. benthamiana leaves. The agro-infiltrated leaves were observed using confocal microscopy  at approximately 3 days post inoculation (dpi).The coding sequences of SVBV P1 (GenBank No: X97304.1) and FvLHC II-1L (GenBank No: XM_004303830.2) were cloned into pCV-cYFP and pCV-nYFP, respectively , to geneBamHI/SalI restriction sites within the pCAM1307-3*Flag based expression vector to produce pCAM1307-FvLHC II-1L-3*Flag (FvLHC II-1L-Flag) or pCAM1307-CFP-3*Flag (CFP-Flag). To detect the SVBV P1–FvLHC II-1L interaction, a pull-down assay was conducted using protein extracts of FvLHC II-1L-Flag from infiltrated N. benthamiana leaves at 3 dpi. The full-length cDNAs of SVBV P1 were amplified and inserted into the pHMTc. The plasmid pHMTc was kindly supplied by Prof. Xiaorong Tao . The construct pHMTc-SVBV P1 was transformed into Escherichia coli BL21 (DE3) cells. Total protein extracts were incubated with the maltose-binding protein (MBP)-SVBV P1 fusion proteins. The MBP pull-down assay was performed as previously described (The full-length FvLHC II-1L or cyan fluorescent protein (CFP) respectively was inserted between the escribed .A. tumefaciens . Excitation wavelength was 488 nm for GFP, 514 nm for yellow fluorescent protein (YFP), 561 nm for RFP, and 640 nm for chloroplast.For the subcellular localization study, SVBV P1 and FvLHC II-1L were inserted into a modified pCAM2300 containing green fluorescent protein (GFP) or red fluorescent protein (RFP), respectively, and then transformed into efaciens . The cul™ RT reagent kit . RT-qPCR was performed as described previously . cDNA was synthesized using PrimeScripteviously .™ Coomassie Blue Super Fast Staining Solution; Beyotime, Shanghai, China), soaked for 1 h, then decolorized with pure water. Western blotting was performed as previously described  mouse monoclonal antibody, MBP mouse monoclonal antibody , FvLHC II-1L rabbit polyclonal antibody, SVBV P1 rabbit polyclonal antibody, and then incubated using goat anti-rabbit IgG (H + L), HRP Conjugate and goat anti-mouse IgG (H + L), HRP Conjugate  with dilutions at 1:5,000 was used in immunoblot analysis. The detection signal was visualized using the EasySee Western Blot Kit .Total protein was extracted from agro-infiltrated leaves using RIPA lysis buffer II . After electrophoresis, the gel was placed in Coomassie Brilliant Blue stain  and construct P25, expressing the PVX P25 protein, were kindly supplied by Prof. Fei Yan . The segment of FvLHC II-1L fused Myc-tag in the 5-termius was inserted between the BamHI/SalI restriction sites within the pBin438 based expression vector to produce pBin438-Myc-FvLHC II-1L. The full-length SVBV P1 was inserted between the BamHI/SalI restriction sites within the pBin438 based expression vector to produce pBin438-SVBV P1, and the assays were performed as previously described . A. tumefaciens containing pCB301-CMV RNA3 or pCB301-CMV RNA3ΔMP-SVBV P1 was mixed with A. tumefaciens containing pCB301-CMV RNA1 and RNA2 in a 1:1:1 ratio.The CMV complementation assay was performed as previously described . The fulA. tumefaciens and co-transfected into F. vesca using a vacuum-infiltration method, as previously described  according to manufacturer instructions.Strawberry vein banding virus-infected strawberry leaves were harvested at 35 dpi. Total DNA was extracted from the strawberry leaves using the CTAB method. Southern blotting was performed as previously described . SVBV DNXbaI/HindIII restriction sites within the pCAM1307-Myc based expression vector to produce pCAM1307-Myc-FvLHC II-1L. The plasmid was transformed into A. tumefaciens strain LBA4404. The transgenic N. benthamiana plants of T0 generation were obtained by leaf disc transformation method. The homozygous FvLHCII-1L transgenic N. benthamiana was obtained after screening. RT-qPCR and Western blot analyzes were used to detect the expression of FvLHC II-11L. The primers used for vector construction are listed in The full-length FvLHC II-1L was inserted between the F. vesca cDNA library using the Y2H assay to identify the host proteins involved. Consequently, FvLHC II-1L was noted to interact with SVBV P1 . As expected, no fluorescence could be observed in N. benthamiana leaf epidermal cells co-infiltrated with cYFP-SVBV P1 and nYFP or nYFP-FvLHC II-1L and cYFP  into N. benthamiana leaves. At 3 dpi, strong GFP fluorescence was observed in infiltrated and adjacent cells of N. benthamiana leaves co-expressing pBin438-FvLHC II-1L and pBin438-SVBV P1, which was stronger than that observed in N. benthamiana leaves infiltrated with SVBV P1 alone  were generated. The mRNA and protein levels of FvLHC II-1L in transgenic plants were quantified via RT-qPCR and western blotting, respectively , more mild leaf crimping was observed in systemic leaves of N. benthamiana seedlings infiltrated with CMVΔMP-SVBV P1, while the systemic leaves of CMVΔMP-erGFP-infected N. benthamiana seedlings showed no symptoms , the Anhui Provincial Key Research and Development Plan (no. 202004a06020013), the Zhejiang Basic Public Welfare Research Project of China (no. LGN19C140002), and the Postdoctoral Science Foundation of Anhui Province (no. 2019B360).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The emergence and spread of antiviral-resistant influenza viruses are of great concern. To minimize the public health risk, it is important to monitor antiviral susceptibilities of influenza viruses. Analyses of the antiviral susceptibilities of influenza A and B viruses have been conducted globally; however, those of influenza C and D viruses are limited. Here, we determined the susceptibilities of influenza C viruses representing all six lineages  and influenza D viruses representing four lineages  to RNA polymerase inhibitors  by using a focus reduction assay. All viruses tested were susceptible to both drugs. We then performed a genetic analysis to check for amino acid substitutions associated with baloxavir and favipiravir resistance and found that none of the viruses tested possessed these substitutions. Use of the focus reduction assay with the genotypic assay has proven valuable for monitoring the antiviral susceptibilities of influenza C and D viruses as well as influenza A and B viruses. Antiviral susceptibility monitoring of all influenza virus types should continue in order to assess the public health risks posed by these viruses. Influenza viruses are diverse, rapidly changing, enveloped RNA viruses with a widespread global presence. Four types exist: influenza A virus, influenza B virus, influenza C virus, and influenza D virus. Three classes of antivirals have been approved for influenza treatment or prophylaxis in Japan: an M2 inhibitor (amantadine), neuraminidase (NA) inhibitors , and RNA polymerase inhibitors . Laninamivir and favipiravir are approved only in Japan. Because favipiravir increases the risk of teratogenicity and embryotoxicity, it received conditional marketing approval with strict regulations for its production and clinical use . The M2 Antiviral-resistant influenza A viruses have emerged and spread globally. In 2003–2004, the prevalence of M2 inhibitor-resistant A(H3N2) viruses carrying an S31N substitution in the M2 protein increased markedly in China; these viruses spread globally during 2005–2006 ,8. The eInfluenza C and D viruses contain the surface glycoprotein hemagglutinin-esterase-fusion (HEF), which combines the functions of the hemagglutinin (HA) and NA proteins of influenza A and B viruses. Based on the HEF gene sequences, influenza C and D viruses are classified into six   and fiveInfluenza C viruses were propagated in MDCK cells  in Dulbecco’s Modified Eagle Medium (DMEM) containing 0.2% bovine serum albumin (BSA) supplemented with 5 μg/mL N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK)-trypsin at 34 °C. Influenza D viruses were propagated in ST cells (ATCC CRL-1746) in DMEM containing 0.2% BSA supplemented with 0.5 μg/mL TPCK-trypsin at 34 °C. The phylogenetic analyses of the influenza C and D viruses used in this study were previously reported ,24. D/swInfluenza A(H1N1)pdm09, A(H3N2), and B/Yamagata lineage viruses served as reference wild-type and baloxavir-resistant viruses. Influenza A and B viruses were propagated in MDCK cells containing 0.2% BSA supplemented with 1 μg/mL TPCK-trypsin at 34 °C. Influenza A(H1N1)pdm09 and A(H3N2) viruses possessing the PA I38T substitution were reported in our previous studies ,26. RG-BThe active form of baloxavir marboxil  and favipiravir were purchased from Funakoshi Co., Ltd.  and MedChemExpress , respectively. Both compounds were dissolved in dimethyl sulfoxide.50) values, which were calculated by using GraphPad Prism .Baloxavir and favipiravir susceptibilities were determined by using the focus reduction assay as previously described  in MDCK https://gisaid.org): C/Ann Arbor/1/50: EPI_ISL_66438; C/Yamagata/15/2004: EPI_ISL_65156; C/Yamagata/32/2014: EPI_ISL_182756; C/Yamagata/4/92: EPI_ISL_230280; C/Yamagata/13/2014: EPI_ISL_182751; and C/Yamagata/3/2000: EPI_ISL_66420. Accession numbers in GenBank: D/swine/Oklahoma/1334/2011: JQ922305 to JQ922311; D/bovine/Nebraska/9-5/2012: KM392468 to KM392474; D/bovine/Yamagata/10710/2016: LC318665 to LC318671; and D/bovine/Yamagata/1/2019: LC494105 to LC494111. Amino acid substitutions associated with resistance to baloxavir and favipiravir were confirmed by using Analyze Sequence Variation (SNP) tool in the Influenza Research Database through the web site at https://www.fludb.org [The whole genome sequences of the influenza C and D viruses used in this study have been reported elsewhere ,24,28. Iludb.org .p values of <0.05.Statistical analyses were performed using GraphPad Prism and included the unpaired t-test, Mann–Whitney test, and ROUT test. Differences between groups were considered significant for We determined the susceptibilities of all six lineages  of influenza C viruses and four lineages  of influenza D viruses to baloxavir and favipiravir by using the focus reduction assay in MDCK cells and ST cells .50 fold-change thresholds, compared to the median for wild-type viruses of the same type, subtype, and lineage, are used by the WHO-AVWG [50 values in MDCK cells and 110-, 91-, and 19-fold higher IC50 values in ST cells, respectively, than their corresponding wild-type viruses for baloxavir. However, these I38T mutant viruses had comparable IC50 values for favipiravir to their corresponding wild-type viruses in MDCK cells (0.5- to 1.1-fold increase) and ST cells (0.7- to 2.0-fold increase). The wild-type B virus showed higher IC50 values for baloxavir than wild-type A(H1N1)pdm09 and A(H3N2) viruses in both cell types, consistent with previous reports using MDCK cells [Criteria to define RNA polymerase inhibitor susceptibility have not yet been established; therefore, provisional criteria based on ICWHO-AVWG . The proCK cells ,30.50 values for baloxavir among the distinct lineages of influenza C or D viruses. The median IC50 values for baloxavir were 10.89 nM in MDCK cells and 10.32 nM in ST cells for influenza C viruses and 48.10 nM in MDCK cells and 31.08 nM in ST cells for influenza D viruses, respectively. The fold-changes in IC50 values compared with the median value for tested viruses were 0.6- to 2.3-fold in MDCK cells and 0.8- to 1.7-fold in ST cells for influenza C viruses and 0.9- to 1.1-fold in MDCK cells and 0.4- to 1.2-fold in ST cells for influenza D viruses, respectively. These results suggest that influenza C viruses are more susceptible to baloxavir than influenza D viruses, consistent with a previous study [50 values for favipiravir. The median IC50 values for favipiravir were 20.91 μM in MDCK cells and 20.76 μM in ST cells for influenza C viruses and 17.37 μM in MDCK cells and 27.16 μM in ST cells for influenza D viruses, respectively. The fold-changes in IC50 values compared with the median value for tested viruses were 0.6- to 1.4-fold in MDCK cells and 0.8- to 2.8-fold in ST cells for influenza C viruses and 0.7- to 2.1-fold in MDCK cells and 0.4- to 1.7-fold in ST cells for influenza D viruses, respectively. The IC50 values of the reference B viruses for favipiravir were similar to those of influenza C viruses, as previously reported [Based on the internal gene sequences, six lineages  of influenza C viruses and four lineages  of influenza D viruses were classified into two groups, respectively; C/Yamagata and C/Mississippi for influenza C viruses  and D/OKus study . All Infreported , suggest50 values to baloxavir [The I38T substitution in the PA protein that is associated with baloxavir resistance emerged after baloxavir treatment in the Phase II and III clinical trials of baloxavir ,32,33. Naloxavir .Influenza C and D viruses are genetically more closely related to each other than to influenza A or B viruses, suggesting a common ancestor for C and D viruses . InfluenInfluenza D viruses, as well as influenza C viruses, use 9-O-acetylated sialic acids  as the host cell receptor  and repl50 values comparable to or lower than the reference wild-type A and B viruses for baloxavir. These results suggest that baloxavir could be a potential drug for the treatment of influenza C and D virus infections. Clinical data on the efficacy of this drug in patients infected with influenza C and D viruses are needed in the future.At present, there are no antiviral drugs approved for influenza C or D virus infections, although antivirals are widely used for the treatment or prophylaxis of influenza A and B virus infections. Since the RNA-dependent RNA polymerase is highly conserved among influenza A, B, C, and D viruses, RNA polymerase inhibitors, such as baloxavir and favipiravir, may be active against influenza C and D viruses. Baloxavir was approved for the treatment of influenza A and B virus infections in Japan and the USA in 2018 and has since been approved in several other countries; favipiravir was approved for influenza pandemic preparedness in Japan in 2014 and stockpiled for use against novel influenza virus infections for which first-line antivirals are ineffective ,16,30. I50 values among influenza A, B, C, and D viruses.Baloxavir acid, an active form of baloxavir marboxil, forms van der Waals interactions with A20, Y24, K34, A37, and I38 in influenza A virus or with T20, F24, M34, N37, and I38 in influenza B virus in the PA endonuclease domain . In vitrThe WHO-AVWG has been conducting global analysis of antiviral susceptibilities of circulating influenza A and B viruses since 2012–2013 period . To dete


EP84R gene, we show that absence of pEP84R results in the formation of non-infectious core-less icosahedral particles displaying a significant DNA-packaging defect. Concomitantly, aberrant core shell-like structures formed by co-assembly of viral polyproteins pp220 and pp62 are mistargeted to non-ER membranes, as also occurs when these are co-expressed in the absence of other viral proteins. Interestingly, co-expression of both polyproteins with pEP84R led to the formation of ER-targeted core shell-like assemblies and co-immunoprecipitation assays showed that pEP84R binds to the N-terminal region of pp220. Altogether, these results indicate that pEP84R plays a crucial role in core assembly by targeting the core shell polyproteins to the inner viral envelope, which enables subsequent genome packaging and nucleoid formation. These findings unveil a key regulatory mechanism for ASFV morphogenesis and identify a relevant novel target for the development of therapeutic tools against this re-emerging threat.African swine fever virus (ASFV) causes a devastating hemorrhagic disease with worldwide circulation and no widely available therapeutic prevention. The infectious particle has a multilayered architecture that is articulated upon an endoplasmic reticulum (ER)-derived inner envelope. This membrane acts as docking platform for the assembly of the outer icosahedral capsid and the underlying core shell, a bridging layer required for the formation of the central genome-containing nucleoid. While the details of outer capsid assembly are relatively well understood, those of core formation remain unclear. Here we report the functional characterization of pEP84R, a transmembrane polypeptide embedded in the inner envelope that surrounds the viral core. Using an ASFV recombinant inducibly expressing the  African swine fever virus is a complex multilayered DNA virus that produces a highly lethal disease that currently affects domestic and wild pigs in Africa, Europe, Asia, Oceania and more recently the Americas. In spite of extensive research, widely available vaccines or any other antiviral strategy are still lacking and relevant questions on ASFV replication remain to be addressed. The present study characterizes the viral membrane protein pEP84R as a key component for the virion assembly. Using genetical, biochemical and microscopic approaches, we provide evidence that pEP84R acts as a molecular link that bridges the internal genome-containing core to the external viral layers. These findings significantly expand our understanding of the mechanisms underlying the formation of the infectious virus particle, which may be relevant for vaccine and antiviral research. African swine fever virus (ASFV) causes a devastating hemorrhagic disease in both domestic pigs and Eurasian wild boars, whose control is currently based on culling and strict containment measures due to the absence of widely available vaccine or other therapeutic tools available –5. The dAsfarviridae, within the recently established Phylum Nucleocytoviricota [ASFV belongs to the family viricota , a monopviricota . PreviouThe infectious ASFV particle, with an icosahedral morphology and a diameter of about 250 nm between opposite vertices, possesses a multilayered architecture that comprises a central genome-containing nucleoid that is successively enclosed by a thick protein core shell, an inner lipoprotein membrane, an outer protein capsid, and an external lipoprotein membrane –12. The ASFV primarily infects monocytes and macrophages in the porcine host, where it deploys a tightly regulated transcriptional programme including several different gene categories , 15. As Recent elucidation of the ASFV architecture by cryo-electron microscopy (cryo-EM) has provided a detailed map of the outer capsid structure, which consists of a hexagonal lattice (triangulation number T = 277) composed of 2,760 trimeric capsomers of the major capsid protein (MCP) p72, and of 60 copies of a penton complex, which is formed by the minor proteins p49 and pH240R, at the vertices , 11, 12.Less is known about the molecular organization of the viral core and its interaction with the inner envelope. The internal nucleoid contains the viral genome along with the major nucleoproteins pK78R and pA104R and, possibly, a set of viral enzymes and transcription factors involved in viral transcription, mRNA modification and DNA repair . The nucHere we show that the previously uncharacterized structural protein pEP84R is an inner envelope transmembrane protein which guides the assembly of the core shell by interacting with the polyprotein precursor pp220. This process, in turn, is critical for the formation of the genome-containing nucleoid and hence for the generation of infectious ASFV particles.ASFV pEP84R is a small virion protein  with twoThe expression of pEP84R in ASFV-infected cells was analyzed by western blot with an anti-pE84R antibody raised against an N-terminal fragment . As showTo assess the putative interaction of pEP84R with lipid membranes, cytosolic and membrane/particulate fractions of ASFV-infected cells were analyzed by western blotting. As shown in To study the subcellular localization of pEP84R in the absence of other ASFV proteins, a Flag-tagged version was transiently expressed in Cos cells and analyzed by immunofluorescence. As shown in (upper panel), specific pEP84R labeling was detected in ER-derived precursor viral membranes as well as immature and mature icosahedral virus particles. Interestingly, immunogold labeling within the virus particles was mostly detected in close proximity to the interface between the viral core shell and the inner envelope (lower panels) in both immature (left) and mature (middle) particles at the assembly sites as well as in mature budding particles at the PM (right).To analyze the subcellular distribution of pEP84R within the virus factories, immunoelectron microscopy (immuno-EM) was performed on cryosections of ASFV-infected Vero cells. As shown in Altogether, the predicted topology in combination with the biochemical and immunocytochemical evidence, indicate that pEP84R localizes in the inner envelope that encloses the viral core.EP84R gene is under the control of an inducible, IPTG-dependent promoter  and budding areas (right panel) showed, as expected, a significant increase in the proportion of mature particles (from ~30 to ~70%) at the exit sites in control infections with wild type and vEP84Ri virus in the presence of IPTG. The proportion of apparently defective particles remained below 10% for both controls and examined areas. In contrast, more than 90% of the vEP84Ri particles produced in the absence of IPTG were defective in terms of core shell and nucleoid organization in both areas.Next, we investigated the effect of blocking pEP84R expression on virus morphogenesis by EM analysis of cells infected with vEP84Ri under permissive or non-permissive conditions. In the presence of inducer, the appearance of the cytoplasmic virus factories  was simiFinally, we performed EM analysis to confirm the ultrastructural phenotype of recombinant vEP84Ri in porcine macrophages, the primary target cells for ASFV infection. As illustrated in To ascertain how the lack of pEP84R affects the core assembly at the molecular level, immunogold labeling with a set of anti-ASFV antibodies was performed on ultrathin sections of vEP84Ri-infected Vero cells in the presence or absence of IPTG. As illustrated in - particles were analyzed by Western blot using antibodies against representative markers of the different virus layers. As a control, equivalent amounts of wild type and vEP84Ri viral particles produced under permissive conditions were also analyzed. As shown in - particles contained essentially no protein pEP84R as well as substantially reduced levels of major core shell and nucleoid proteins, while the amounts of the outer envelope, outer capsid and inner envelope markers were not significantly affected.In a complementary approach, highly purified extracellular defective vEP84Ri- particles was analyzed. In a first approach, we performed immunogold labeling with an anti-DNA antibody to quantify the percentage of control (+ IPTG) and defective (-IPTG) particles labeled at the budding areas ((EM), the proportion of DNA+ labeled particles was about 3 times lower under restrictive than under permissive conditions. It is worth mentioning that a significant proportion of the nucleoid-containing particles were not labeled (+ IPTG), possibly due to the limited accessibility of the anti-DNA antibody to the highly packaged viral genome.Next, the presence of the viral genome in defective vEP84Ring areas . As show labeled , + IPTG,+ and vEP84Ri- particles were subjected to DNA extraction and ASFV-specific qPCR. As shown in (qPCR), the DNA content of vEP84Ri- particles was about 4 times lower than that detected in control vEP84Ri+ virions, confirming that recombinant vEP84Ri displays a severe DNA packaging defect under restrictive conditions.In a complementary approach, equivalent amounts of purified vEP84RiIn conclusion, immunocytochemical and biochemical approaches indicate that pEP84R repression blocks the recruitment and/or the assembly of the core shell and nucleoid components, including the viral genome, into the virus particles.The core-less phenotype of defective vEP84Ri virus resembles that reported for ASFV recombinants that lack either pp220 or pp62 polyproteins , 30. TheNext, we analyzed the distribution of pp220 precursor in vEP84Ri-infected cells by immunofluorescence. As shown in It is worth mentioning that zipper structures have been also detected at the periphery of the assembly sites of wild type virus infections  Fig 4G)Fig 4G), To evaluate the hypothesis that pEP84R itself may guide the localization of the core shell precursors we performed immunofluorescence  and immuIn keeping with the fluorescence pattern described before  Figs, imCo-transfection experiments showed that the distribution pattern of pp62 was not significantly modified when co-expressed with pEP84R . In contSince polyproteins pp220 and pp62 interact with each other to form zippers linked to the PM and endosomal-lysosomal compartments  Fig 6H , we testSince membrane-anchoring of pp220 occurs through its N-terminal myristoyl group, we speculated that transmembrane pEP84R interacts with the N-terminal region of the polyprotein. To test this, we expressed an N-terminal fragment of pp220 comprising the p5 and p34 regions, fused to a V5-His tag (pp220NT-V5) . As showTo confirm the interaction of pEP84R with the N-terminal region of pp220, clarified lysates of cells transfected with both pEP84R-Flag and pp220NT-V5 constructs were immunoprecipitated with anti-V5 magnetic beads. Western blot analysis demonstrated that pEP84R-Flag co-immunoprecipitated with pp220NT-V5 . No pEP8In conclusion, the co-localization and co-immunoprecipitation of pEP84R and pp220NT in the absence of other viral proteins indicate a direct interaction between both polypeptides. This interaction provides a molecular mechanism to explain the crucial role of pEP84R in the membrane targeting of core assembly.The unique architecture of the infectious ASFV particle, with two icosahedral protein shells and two lipoprotein membranes enclosing the genome-containing nucleoid , 11, 12,As shown above, the absence of pEP84R during ASFV infection leads to the formation of defective icosahedral particles lacking a proper core shell as well as the internal DNA-containing nucleoid. This core-less phenotype  is remarAs already mentioned, ASFV uses polyprotein expression, an uncommon gene expression strategy in DNA viruses , to syntOur present results indicate that pEP84R provides a guiding cue for the correct membrane targeting of the core shell polyproteins. Besides the loss-of-function approaches, transfection experiments showed that pEP84R is able to recruit pp220 to perinuclear ER and Golgi membranes, where pEP84R preferentially targets. Moreover, pEP84R, pp220 and pp62 colocalize to form ER-bound core shell-like structures in transfected cells, as also observed in normal infections. Finally, the colocalization and coimmunoprecipitation of pEP84R with an N-terminal fragment of pp220 provides a mechanistic explanation to these findings.in vitro to phosphatidylserine-enriched liposomes through a positively charged aa patch [On the basis of the previous evidence and the present results, we propose a model for ASFV core assembly  in whichaa patch , which min vitro [The reported defective phenotypes of inducible recombinants for pp220 and p62 along with that of pEP84R indicate that the assembly of the core shell is required for nucleoid formation , 30. It in vitro , 37. It in vitro , which sbona fide nucleocapsid enwrapped by a lipid membrane . In this context, ASFV core formation seems conceptually analogous in some aspects to that of retroviruses, in which the Gag polyprotein precursor acts as a primary driver for both the nucleocapsid assembly and its anchoring to the viral envelope [At present, it is unknown what structural changes are triggered by polyprotein processing in the core structure although the recently identified internal icosahedral capsid that outlines the core shell of the mature virions , 12 seemenvelope –40. Simienvelope , 40. Gagenvelope , 12. CleAt present, no sequence similarity has been found between pEP84R and other viral or cellular proteins. However, the closest relatives of ASFV, the amoeba-infecting faustovirus , pacmanvCurrently, ASFV causes a worldwide animal health emergency that is aggravated by the absence of widely available vaccines or effective anti-viral strategies. In this context, the identification of a key orchestrator for core formation may be exploited for the development of antiviral compounds blocking its interaction with the core shell or for the generation of a safe new class of virus-like particle-based vaccines for the prevention of ASF.2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% fetal bovine serum (FBS), which was reduced to 2% during viral infection. Porcine alveolar macrophages (from a laboratory stock stored in liquid nitrogen) were cultured in DMEM supplemented with 10% heat-inactivated FBS. The Vero cell-adapted ASFV strain BA71V has been described before [Vero and Cos-1 cells, derived from African green monkey kidney epithelia, were obtained from the American Type Culture Collection and grown at 37°C and 5% COd before . Unless The affinity-purified rabbit antibody against the viral protein pEP84R was obtained from Davids Biotechnologie by immunization with the pEP84R-derived peptide ATEPEVGLPLLALQHSKS.The following antibodies against ASFV polypeptides have been previously described , 47–50: The sources of the mouse mAbs for host cell markers and tag epitopes were as follows: anti-β-Actin , anti-CD63 , anti-PDI , anti-ERGIC-53 ; anti-GM130 , anti-DNA , anti-Flag , anti-V5 . The sheep anti-TGN46 (AHP500G) was from Bio-Rad and the rabbit anti-pan-AKT antibody (ab880) from Abcam.EP84R-containing PCR product amplified with oligonucleotides AH406 (5’- CTTTCAGGGTACCGAAGAAGGATCCGCCACCATGCCTTATTCAAGAGATATC) and AH407 (5’- CAGAATTCCTCCTTGGATCCTTAGAATTATCTTTATAAATAAGAAAAAAACC) using purified viral DNA as a template was recombined using InFusion reagents (Clontech) into Bam HI-digested plasmid pIREScOFP (Stratagene). To avoid interference of OFP expression, the fragment containing the IRES sequence and the OFP encoding sequence was deleted from the resulting plasmid using Q5 site directed mutagenesis (NEB) with oligonucleotides AH435 (5’-GAGCATGCATCTAGAGGG) and AH436 (5’- TATGCAGTCGTCGAGGAA) following the manufacturer´s indications.The plasmids pGEM-CP2475L and pGEM-CP530R, containing the pp220- and pp62-encoding genes, respectively, under control of the T7 polymerase promoter, have been described before , 27. PlaCP2475L encoding the N-terminal residues 1 to 368, corresponding to mature proteins p5 and p34, was PCR amplified from plasmid pGEM-CP2475L using Q5 polymerase (NEB) and oligonucleotides AH408 (5’-CACTAGTCCAGTGTGGTGGAATTCGCCACCATGGGTAACCGTGGATC) and AH438 (5’- GCTGGATATCTGCAGAATTCTCACCCCCCTTTTTGG) and assembled with Bam HI-digested plasmid pEF-V5His B (Invitrogen). The final plasmids were sequenced to confirm the absence of unwanted mutations.Plasmid pCP2475L(NT)-V5His to express the pp220NT-V5 fragment was obtained by HiFi Assembly (New England Biolabs). The fragment from U104L early/late viral promoter, a beta-glucuronidase gene under the control of the strong late viral p72.4 promoter and finally, the inducible p72 derived promoter p72I*. The inducible cassette was PCR amplified using high fidelity Q5 polymerase (New England Biolabs) and oligonucleotides AH339 (5’-CGGTACCCGGGGATCGCGGCCGCTCGACGGATTTTAATTAGATTTGTG) and AH340 (5’-CGACTCTAGAGGATCTTAATTAATTGTTATCCGCTCACAATTTATATAATG) and plasmid pE199Li [EP1242L was abrogated by introducing a conservative single nucleotide mutation for cloning purposes. The flanking regions are separated by a short nucleotide stretch containing unique Pac I and Not I restriction sites which allow subcloning of the mentioned inducible cassette placing the initiator codon of EP84R immediately downstream of the inducible promoter, yielding the final plasmid used for recombination, pEP84Ri.In a first step, an inducible cassette was subcloned into Not I/Pac I digested plasmid pUC19 using InFusion (Stratagene) recombination to allow streamlined subcloning. This cassette bears the following relevant elements: E. coli lacI repressor coding gene under the control of the  pE199Li  as a temEP84R gene is under the control of the E coli lac operator/repressor system is shown in A scheme of the ASFV recombinant, vEP84Ri, in which the expression of the Vero cell monolayers, seeded in six-well plates, were infected with recombinant vEP84Ri, or parental BA71V as a control. After 1 h, the inoculum was removed and the cells were overlaid with DMEM containing 0.6% Noble agar and 2% FBS in the presence or absence of 1 mM IPTG. The medium was removed 5–7 days later after paraformaldehyde (PFA) fixation and the monolayers stained with 1% crystal violet.Vero cell monolayers, seeded in 24-well plates, were infected with 5 pfu/cell of recombinant vEP84Ri or parental BA71V. After a 1-h adsorption, the cells were incubated in medium supplemented with 2% FBS in the presence or absence of 1 mM IPTG. Finally, the infected cells were harvested together with their culture supernatants at different times post-infection, sonicated and titrated by plaque assay in the presence of 1 mM IPTG.https://www.uniprot.org/uniprot/Q07383) are considered. The possible protein topology was predicted using TMHMM 2.0 at https://services.healthtech.dtu.dk/service.php?TMHMM-2.0.Putative transmembrane regions in BA71V as annotated on the Uniprot website . For Triton X-114 treatment, the membrane fraction was resuspended in 2% Triton X-114, 150 mM NaCl, and 10 mM Tris-HCl pH 7.5, incubated for 10 min at 4°C and then transferred to 30°C for 20 min. After phase separation by centrifugation, the lower detergent-rich phase and the upper aqueous phase were dissociated as described above. Equivalent amounts of the resulting fractions were analyzed by Western immunoblotting.Mock- and ASFV-infected cells were fractionated at 20 hpi into nuclear, cytoplasmic and membrane fractions, according to the instructions of the Minute Plasma Membrane Protein Isolation and Cell Fractionation Kit (Invent Biotechnologies). For alkaline carbonate extraction, membrane fractions were treated with 0.1 M NaExtracellular vEP84Ri particles were purified from clarified infection supernatants by Percoll gradient sedimentation , with sl+ and vEP84Ri- particles, adjusted by their MCP p72 content, were analyzed by immunoblotting with specific antibodies to representative markers of the different virus layers. As a reference, Percoll-purified wild type virus (BA71V strain) was also analyzed.For the analysis of the vEP84Ri protein composition, equivalent amounts of Percoll-purified vEP84Ri+ and vEP84Ri- particles were mixed with 100 ng of purified genomic DNA from ectromelia virus (ECTV), which was used as spike-in control for normalization, and subjected to DNA extraction and purification with the GeneJET Whole Blood Genomic DNA Purification Mini Kit (Thermo). Purified DNAs (2 ng/well in triplicate) were analyzed by qPCR with specific primers for B646L gene from ASFV strain BA71V (forward: 5´-CCAAACAGCAGGTAAACAAGA and reverse: 5´-GAATGGATACCGAGGGAATAG) and ECTV A10L gene (forward: 5´-CGCAATGCCATACAACATCT and reverse: 5´-TCCAATGCTTGGCTGACTAA) in a CFX384 Real Time System C1000 Thermal Cycler (Bio-Rad) using SSoFast EvaGreen Supermix (Biorad) and the following PCR cycling profile: 3 min x 95°C + (5 sec x 95°C and 5 sec x 60°C) x 40 cycles. After PCR amplification, melting curve analysis was performed for each reaction to verify PCR specificity. Relative quantification (2–ΔΔCq) of ASFV DNA was performed with GenEx software  using spike-in control and MCP p72 content of the virus samples for normalization. Data are expressed as a percentage (± SD) of the normalized DNA content detected in control (vEP84Ri +IPTG) particles.For the analysis of the DNA content, triplicate samples containing 2 μg of purified vEP84RiCells and purified virus samples were dissociated in Laemmli buffer, heated at 90°C for 5 min and electrophoresed on 12% SDS-polyacrylamide gels. Proteins were transferred to PVDF membranes  and incubated overnight at 4°C with the primary antibodies and for 1 h at RT with anti-rabbit, anti-rat or anti-mouse secondary antibodies conjugated to horseradish peroxidase  or TidyBlot (Biorad). Blots were developed with ECL prime reagent  on conventional film autoradiography.and, when necessary, scanned and quantified with ImageStudioLite (Li-cor).For the transient expression of pEP84R-Flag, pp220, pp62 and pp220NT-V5 proteins, Cos cells were grown to 80% confluency in DMEM at 5% FBS onto 35-mm dishes or 7-mm coverslips in 48-well plates. Cells were infected with 5 pfu/cell of a recombinant vaccinia virus (VACV), encoding the bacteriophage T7 RNA polymerase, in DMEM at 2% FBS. After 1h of adsorption, the cells were washed with DMEM and transfected for 12–20 h with the corresponding DNA plasmids in which the target genes are under control of the T7 promoter. Transfections were performed with lipofectamine 3000 transfection reagent (Thermo) in Opti-Mem (Life Technologies) according to the manufacturer’s indications. To prevent VACV late expression and morphogenesis, the DNA replication inhibitor ara-C (50 μg/ml) was added to the media throughout the infection/transfection experiment. At the end of the transfection period, the transfected cells were fixed for immunofluorescence or EM analysis or dissociated for immunoprecipitation or immunoblotting, as indicated.Transfected Cos cells, plated at 80% confluency in 35 mm plates, were lysed in 1ml of 0.1% Triton X-100 in PBS containing a protease inhibitor cocktail . Cell extracts were vortexed and clarified by centrifugation at 16,000 x g for 10 min at 4°C. For immunoprecipitation assays, typically 200 μl of each supernatant were incubated O/N at 4°C under rotation with 5 μg of purified anti-V5 mAb , anti-Flag M2 mAb  or a non-relevant mAb . Then, 40 μl of protein G-conjugated magnetic beads  were added and incubated for 5h at 4°C under rotation. Beads were washed five times with 0.1% Triton in PBS and bound proteins were eluted in 30 μl of 2X Laemmli buffer and boiled at 95°C for 3 min. Input extracts and immunoprecipitated samples were analyzed in 12% polyacrylamide SDS-PAGE followed by western blot with anti-V5 and anti-Flag mAbs.4Cl for 5 min and blocked with 10% FBS for 5 min. The permeabilized infected cells were incubated for 45 min at RT with primary antibodies and 30 min at RT with Alexa-labeled secondary antibodies (Thermo Fisher). Cell nuclei were labeled with Hoechst 33258 (5 μg/ml). Coverslips were mounted with ProLong Glass Antifade Mountant (ThermoFisher) on microscope slides. Images were recorded with a Leica DMI6000B automated inverted microscope equipped with a Hamamatsu Orca R2 digital camera or a Zeiss LSM880 confocal laser scanning microscope.ASFV-infected Vero cells or transfected Cos cells, seeded on glass coverslips, were infected with parental ASFV virus or recombinant vEP84Ri for the indicated times, washed with PBS and fixed with methanol at -20°C for 5 min or with 4% PFA in PBS for 15 min at RT. Aldehyde-fixed cells were permeabilized with 0.1% saponin or 0.1% TX-100 in PBS for 5 min at RT, quenched with 50 mM NH4 and 0.8% K3Fe(CN)6 in water at 4°C for 1 h. Samples were dehydrated with ethanol and embedded in Epoxy, TAAB 812 Resin (TAAB Laboratories) according to standard procedures. After polymerization, ultrathin sections of about 90 nm were obtained and stained with uranyl acetate and lead citrate according to standard procedures. Samples were examined in a Jeol JEM-1010 electron or a Jeol 1400Flash microscope operating at 80 and 100 kV, respectively. Images were recorded with a TemCam-F416  or a OneView  CMOS digital camera.ASFV-infected Vero cells and macrophages as well as transfected Cos cells were fixed with 4% PFA and 2% glutaraldehyde (GA) in 0.1 M phosphate buffer  for 120 min at RT. Post-fixation was carried out with 1% OsOFor virus quantification, a number of 240–260 (virus factories) or 100–120 (budding areas) closed icosahedral virus profiles per condition were counted at a magnification of 10,000–20,000X from 5–10 different areas. Virus particles containing a well-organized core shell and a centered nucleoid of normal size were classified as mature viruses. Particles displaying a well-organized core shell but lacking a centered normal nucleoid were considered as immature. Virus particles lacking a well-organized core shell and a centered normal-sized nucleoid were classified as defective. Data were expressed as the mean percentage (+/- SD) of each virus category per condition.in situ fixed for 2 h at RT with 4% PFA or 2% PFA and 0.2% GA in 0.1 M PB. Then, cells were embedded in 10% (w/v) gelatin, cryoprotected overnight in cold sucrose 2.3 M and rapidly frozen in liquid nitrogen. Ultrathin (90-nm) cryosections were obtained with a Leica EM FCS6 cryo-ultramicrotome at -120°C and retrieved with a 1:1 mixture of sucrose 2.3 M and 2.0% methylcellulose. Immunogold labeling was performed essentially as described [For immunoelectron microscopy, infected or transfected cells were escribed  with sli- particles were expressed as a percentage (+/- SD) of those detected in control (vEP84Ri+) particles. For quantification of DNA labeling, the percentage of virus particles with at least 1 gold particle was estimated in 6–7 different budding areas. Data were expressed as the mean percentage (+/- SD) of DNA+ particles relative to control (vEP84Ri+) particles. Double and triple labelling procedures were set up for each combination and performed following protocols from the Electron Microscopy facility (EMF) at CBMSO. Details are available upon request.For immuno-EM quantification with anti-ASFV antibodies, the mean labeling density (gold particles/virus) was obtained from 5–10 different budding areas (collectively containing from 60 to 130 closed virus profiles) for each antibody and condition (vEP84Ri +/- IPTG). For comparison, the mean labeling densities of vEP84RiUnless otherwise indicated, the data are representative of at least three independent experiments, and values are given as the mean of triplicates ± standard deviation (SD).S1 Data(XLSX)Click here for additional data file.S1 Fighttps://www.ncbi.nlm.nih.gov/) and the corresponding accession numbers for each are: BA71V (NP_042748.1); Ken06.Bus (YP_009702953.1); Georgia_2007 (YP_009927178.1); China_2018(AYW34026.1); NHV (YP_009702621.1); OURT 88/3 (YP_009703662.1); Malawi LIL 20 (P0CAL5.1); Pr4 (P0CAL6.1); Namibia_Wart80 (P0CAL7.1); ASFVK49 (QZK26757.1); Liv13/33 (QID21215.1); Zaire 1977 (QII88576.1); RSA_2_2008 (QGM12834.2); KEN-50 (P0CAL4.1); Uvira (QRY19081.1); Ken05/Tk1 (YP_009702788.1); Ken.rie1 (CAD7112270.1). The sequences were aligned using Clustal Omega software at the EMBL-EBI . Identical aa residues in all aligned sequences are designated with an asterisk at the bottom. Putative transmembrane regions in BA71V as annotated on the Uniprot website (https://www.uniprot.org/uniprot/Q07383) are indicated. The possible topology as predicted by TMHMM 2.0 at  is shown above the sequence, with “in” indicating the cytoplasmic side of the membrane.The aa sequences of pEP84R orthologues from different ASFV strains were retrieved from Genbank at the NCBI ((TIF)Click here for additional data file.S2 FigEP84R gene fused to a C-terminal 3XFlag-tag epitope sequence (pEP84R-Flag). At 20 h, cells were fixed and immunolabelled with rabbit anti-pEP84R , or mAb anti-Flag (D), and antibodies against ER , ERGIC , cis-Golgi  and TGN  markers. Note the partial colocalization of pEP84R-Flag with PDI at the nuclear envelope and peripheral ER, and with ERGIC, Golgi complex and TGN markers at perinuclear areas. Bars, 10 μm.Cos cells were transfected with a plasmid containing (TIF)Click here for additional data file.S3 FigEP84R gene expression, a copy of E. coli lac repressor gene (lacI) and a reporter gene (gusA) used for selection and purification of the recombinant virus. ASFV genes in the proximity of the recombination area as well as the left and right flanking regions are indicated.The recombinant virus was obtained by homologous recombination of parental ASFV genome (wt) with an inducible cassette containing a late, IPTG-dependent strong promoter (p72I*) for (TIF)Click here for additional data file.S4 Fig(A-D) or in the absence (E-H) of IPTG. Panels B and F show higher magnification images of the viral factory areas delimited in A and E, respectively. Note that while under permissive conditions (+IPTG), the viral factories (VF) contain significant amounts of immature particles with a well-organized core shell (arrowheads in B) and mature virions, under non-permissive conditions (-IPTG), they contain essentially core-less particles (F). Note also that defective vEP84Ri- particles exit by budding from the PM (G-H), as occurs with ´full`vEP84Ri+ particles (C-D). The different virus layers (nucleoid (nu), core shell (cs), inner envelope (ie), capsid (ca) and outer envelope (oe)) of budding vEP84Ri+ (inset D) and vEP84Ri- (inset H) particles are indicated. Nucleus (N), plasma membrane (PM). Bars, 1 μm  and 200 nm (B-D and F-H).Porcine alveolar macrophages were infected with vEP84Ri for 18h in the presence (TIF)Click here for additional data file.S5 Fig(A) Extracts of Vero cells mock-infected (Mock) or infected with parental BA71V (wt) or recombinant vEP84Ri viruses in the presence (+I) or absence (-I) of IPTG were analyzed by immunoblotting with antibodies against the mature products derived from polyprotein pp220 (upper) and pp62 (middle) and protease pS273R (bottom). As a control, purified ASFV particles (V) were also analyzed. Note that polyprotein processing is strongly impaired under restrictive conditions. The positions of polyprotein pp220 and pp62, the intermediate processing products pp90 and pp46, the mature products p150, p37, p34, p35 and p15, and the viral protease pS273R are indicated. Molecular masses are indicated on the left. (B) Immunofluorescence labeling of pp220 (green) and MCP p72 (red) in vEP84Ri-infected cells in the presence (+) or absence (-) of IPTG. Nuclear and viral DNA (blue) was stained with Hoechst 33258. The arrows indicate virus factories (VF) whereas the arrowheads indicate cytoplasmic pp220 accumulations outside the virus assembly sites under non-permissive conditions. Bars, 5 μm.(TIF)Click here for additional data file.S6 Fig(A) Transfected Cos cells expressing proteins pEP84R-Flag, pp62 and pp220, individually or in combination, were analyzed by immunoblotting with antibodies to the target proteins. (B) Membrane-association of pEP84R-Flag and pp62. Transfected Cos cells expressing proteins pEP84R-Flag or pp62 were fractionated into cytosolic (C) and membrane/particulate (M) fractions. Also, the membrane fractions were subjected to TX-114 phase separation to obtain aqueous (A) and detergent-rich (D) phases. Equivalent fractions were analyzed by immunoblotting. Note that while pEP84R-Flag behaves as an integral membrane protein, pp62 behaves as a peripheral membrane protein. Molecular masses (in kDa) and pp220, pp62 and pEP84R-Flag bands are indicated.(TIF)Click here for additional data file.S7 Fig, pp62 (C) and pp220 (B and F); mouse mAbs to pp62 , pp220 (E) and Flag tag (A and F) or rat antibody to pp220 (G and H) as indicated. Note that pp220 and pEP84R colocalize (E and F) to perinuclear areas and to a lesser extent to the cell surface (arrowheads). Note also the significant colocalization of pEP84R, pp220 and pp62 (G and H) at perinuclear areas (arrowheads). Bars, 10 μm.Transfected Cos cells expressing proteins pEP84R-Flag, pp62 and pp220 individually or in combination, were fixed and immunolabeled with rabbit antibodies to pEP84R (TIF)Click here for additional data file.S8 Fig(A) or cryosectioning and immunogold labeling (B) for pEP84R , pp220  and pp62 . Note the formation of zipper (z) structures associated to ER cisternae (A), which are immunolabeled for the three viral proteins (B). Bars, 200 nm.Transfected Cos cells co-expressing pEP84R-Flag, pp220 and pp62, were processed for conventional epoxy resin embedding (TIF)Click here for additional data file.S9 FigCos cells transfected with pEP84R-Flag and pp220NT-V5 constructs individually or in combination, were immunolabeled with anti-pEP84R (green) and anti-V5 (red) antibodies. Note the vesicular-like pattern of pp220NT-V5 (middle left) indicated by arrowheads in a detail (bottom left) and colocalization (arrows in right panels) of pEP84R-Flag and pp220NT-V5 in co-transfected cells at perinuclear areas. Bars, 10 μm.(TIF)Click here for additional data file.


The results revealed that the LNPs with a size between 90 and 120 nm showed higher gene silencing activity than those with other sizes. The 3D-iLiNP device is expected to improve DDS performance by precisely controlling the size of LNPs.Sub 100 nm-sized lipid nanoparticles (LNPs) have been widely used in drug delivery systems (DDSs). The size of the LNPs is an important parameter for the DDS performance, such as biodistribution and gene silencing using siRNAs. However, the LNPs prepared by the conventional preparation method show a wide size distribution. To improve the LNP size distribution, we developed a microfluidic device, named the iLiNP™ device, in a previous study. This device could produce LNPs in the size range of 20 to 150 nm, but the size distribution of the large-sized LNPs needs to be further improved. From the viewpoint of the LNP formation process, a homogeneous and slow rate dilution of ethanol plays an important role in improving the large-size LNP size distribution. In this study, we developed a three-dimensional, symmetrically assembled microfluidic device named the 3D-iLiNP device with the aim of precise size control of large-sized LNPs. We designed the 3D-iLiNP device using a computational fluid dynamics simulation and demonstrated that the 3D-iLiNP device can improve the LNP size distribution. The gene silencing activity of four kinds of siRNA-loaded LNPs was investigated  We developed a three-dimensional and symmetrically assembled microfluidic device named the 3D-iLiNP device. The 3D-iLiNP device allowed the precise size control of sub-100 nm sized lipid nanoparticles by the homogeneous and slow ethanol dilution. On the other hand, 100 nm-sized LNPs can deliver drugs or RNA into the hepatocytes of liver tissues passing through the fenestrae (with an approximate diameter of 100–150 nm).8 Recently, the relationship between the LNP size and DDS performance has been investigated widely -based drug delivery systems (DDSs) are among the most advanced nanomedicine systems. The size of LNPs plays an important role in both the LNP biodistribution and LNP performance, such as antitumor effect and gene silencing activity. Table S1. In prev10–13 In the microfluidic-based LNP production method, the LNP size was controlled by the ethanol dilution rate. Therefore, the flow conditions, such as the total flow rate and flow rate ratio (FRR) of aqueous phase to lipid phase, and the microchannel geometry are key parameters for controlling the LNP size.13–16 To control the LNP size, the LNP formation behavior has been investigated using several types of microfluidic devices , symmetrically assembled microchannel, named the 3D-iLiNP device, based on the 2D-iLiNP device. We designed the 3D baffle structures using computational fluid dynamics (CFD) simulations. We demonstrated the homogeneous and slow rate ethanol dilution by the generation of 3D secondary flows at the low flow rate condition. Finally, we investigated the effect of the LNP size distribution on the gene silencing activity 14 With the 3D-iLiNP device, we predicted a secondary flow in the x–z direction in addition to that in the y–z direction at the 3D-baffle structures; this secondary flow would allow the acceleration of the ethanol dilution of the lipid solution. −1 and 3, respectively. We focused on the generation of the secondary flow in the 3D-baffle structures. As expected, the secondary circulation flow was generated at the baffle structures of the 3D-iLiNP devices, whereas no secondary flow was observed in the 2D-iLiNP device. 14,15 This fluid dynamics is significant to control the LNP size at the low flow rate condition. Therefore, we carried out the evaluation of the LNP size controllability of the 3D-iLiNP devices.sn-glycero-3-phosphocholine (POPC) LNPs produced using the 2D and 3D-iLiNP devices, the basic-type, 80–20 μm, and 20–80 μm, at a total flow rate and FRR of 50 μL min−1 and 3, respectively. The basic-type 3D-iLiNP device showed the narrowest LNP size distribution among the four devices, the 2D and three types of 3D-iLiNP devices. We determined the 3D-iLiNP device design with the highest LNP size controllability. −1 and FRRs of 3 and 9. In a previous study, it was reported that the increase in the total flow rate and FRR induces the production of small-sized LNPs.14 From −1 and FRR of 3. 16 At all total flow values, the CV value of the LNPs produced with the 3D-iLiNP device was at least 7% smaller than that of the LNPs produced with the 2D-iLiNP device.15 From the viewpoint of fluid dynamics and mass transportation, it is difficult to achieve a homogeneous distribution and slow dilution rate because the homogeneous and slow rate dilution is a competitive phenomenon to diffusion. The concentration gradient induced by diffusion is the major reason for the formation of heterogeneous-sized LNPs because ethanol dilution is a trigger for LNP formation. Therefore, a microfluidic device suitable for low flow rate conditions is indispensable for producing homogeneous- and large-sized LNPs. On the other hand, a homogeneous size distribution and rapid dilution can be achieved with several types of microfluidic devices, such as the chaotic micromixer and 2D-iLiNP devices. The chaotic micromixer device allows homogeneous mixing under low flow rates;18 however, it could not produce the large-sized LNPs , a commercially available lipid, were used as the main components of the lipid systems. in vivo experimental design. At total flow rates of 50 or 500 μL min−1, the 3D-iLiNP device produced 99 and 59 nm LNPs  expression in mice at a dose of 0.1 mg siRNA per kg. The 99 nm LNPs showed 75% gene silencing activity, while the 59 nm LNPs suppressed 50% of the FVII expression. 3 From the results of the gene silencing activity experiments, intrahepatic distribution, and size of fenestrae (approximately 100–150 nm), the LNPs with a size range of 90–120 nm exhibited the highest RNA delivery performance in vivo using this lipid system. Therefore, the 3D-iLiNP device will be a key technology for the production of size-controlled LNPs for RNA delivery applications.Next, we carried out an ur sizes . The 99 ur sizes . This rein vivo experiment, 100 nm LNPs showed a higher gene silencing activity than the small-sized siRNA LNPs. We believe that the 3D-iLiNP device and size-controlled LNPs could improve not only the RNA delivery and gene silencing activity, but also the transfection or genome-editing performance of LNP-DDSs.We developed a 3D, symmetrically assembled microfluidic device named 3D-iLiNP with the aim of improving the size distribution of large-sized LNPs produced at low flow rate conditions. The 3D-iLiNP device allowed the production of LNPs with a narrow size distribution owing to the 3D secondary flow. We demonstrated that the 3D-iLiNP device could produce POPC LNPs in the size range of 20 to 100 nm depending on the flow conditions with CV values smaller than 10%. We employed the 3D-iLiNP device for the production of siRNA-loaded LNPs to investigate the effect of the LNP size distribution on the gene silencing activity. The 3D-iLiNP device allowed the production of siRNA-loaded LNPs with a size distribution more homogenous than that of the LNPs produced with the 2D-iLiNP device. We clearly distinguished the main LNP size ranges produced by the 3D-iLiNP device and found that the 90–120 nm LNPs suppressed the luciferase expression of HeLa cells. For the sn-glycero-3-phosphocholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K) were purchased from the NOF Corporation . Cholesterol was purchased from Sigma-Aldrich . A pH-sensitive cationic lipid, CL4H6, was synthesized in the laboratory as previously described.19 Ethanol, sodium chloride, citric acid, 2-morpholinoethanesulfonic acid (MES), monohydrate, and phosphate-buffered saline (PBS) were purchased from FUJIFILM Wako Pure Chemical Corporation . The siFVII and Cy-5-labeled siGL4 were purchased from Hokkaido System Science Co. Ltd. . Table S41-Palmitoyl-2-oleoyl-14 The profiles of the ethanol concentration were numerically simulated using 3D models under both the laminar-flow model (no slip) and transport of diluted species (ethanol) condition. The flow was modeled as an incompressible flow using the Navier–Stokes equation. The total flow rate and FRR (water/ethanol) were set to 50 μL min−1 and 3, respectively. For the calculation of the ethanol dilution efficiency, we counted a number of mesh with 20% ethanol concentration at each cross-section and each residence time.20 Then, the ethanol dilution efficiency was calculated from the following equation.COMSOL Multiphysics 5.2  was used for a CFD study. The experimental conditions were the same as those reported in a previous study.21 For the alignment of the 3D-iLiNP device, we employed amino silane coupling with minor modifications  (PDMS) replicas for the 2D- and 3D-iLiNP devices using the standard photolithography method.s Fig. S1.22POPC was used as a model LNP to investigate the size controllability of the 3D-iLiNP device. POPC LNPs were prepared using a 13.4 mM POPC/ethanol solution and saline. The collected LNP suspensions were dialyzed against saline overnight.−1. The siRNA-loaded CL4H6 LNPs were prepared by mixing a 10 mM lipid/ethanol solution composed of CL4H6/DSPC/cholesterol/DMG-PEG 2K (50/10/38.5/1.5 mol%)23 with a 10 mM citrate buffered solution containing siRNAs at a concentration of 132 μg mL−1. The lipid solution and siRNA in the buffer solution were separately introduced into the iLiNP devices at a total flow rates of 50 or 500 μL min−1 and FRR of 3. The collected LNP suspensions were dialyzed against 20 mM MES buffer solution (pH 6.0) for 2 h followed by an overnight dialysis against PBS. The siRNA encapsulation efficiency was measured using a Ribogreen assay.3 For in vivo experiments, the CL4H6-LNP suspensions were concentrated using Amicon Ultra filters .To validate the applicability of the 3D-iLiNP device for the production of siRNA-loaded LNPs, we investigated DODAP- and CL4H6-based LNP systems. The lipid amine-to-oligonucleotide phosphate (N/P) ratio was fixed to 4. The siRNA-loaded DODAP LNPs were prepared by mixing an 8 mM lipid/ethanol solution composed of DODAP/DOPE/cholesterol/DMG-PEG 2K (30/25/40/5 mol%) with a 10 mM citrate buffered solution (pH3.0) containing siRNAs at a concentration of 47.5 μg mLThe size and polydispersity index (PDI) of the LNPs were measured by dynamic light scattering (DLS) measurements obtained with a Zetasizer Nano ZEN3600 .19 Cells were seeded at a concentration of 3 × 103 and 6 × 103 cells per well in a 96 well-microplate for 24 h prior to the LNP treatment for the cell viability assay and gene expression assay. Then, the cells were treated with LNPs and incubated for 24 h. After the incubation, the cell viability was measured using a Cell Counting Kit-8  according to the manufacture's protocol. For gene expression assay, firefly and renilla luciferase activities were measured using a Dual-Glo assay and a GloMax®Explorer System (Promega), according to the manufacture's protocol.HeLa cells stably expressing Firefly and Renilla luciferase (HeLa-dluc) were cultured in the same conditions as described previously.14 with minor modifications. For the observation of the intrahepatic siRNA distributions, we prepared DiI- and (Cy5)-labeled siGL4 encapsulated CL4H6 LNPs by mixing a 10 mM lipid/ethanol solution containing 0.5 mol% DiI with a 10 mM citrate buffered solution containing Cy5-labeled siGL4. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Hokkaido University and approved by the Hokkaido University Animal Ethics Committee.The gene knockdown activity of coagulation factor VII (FVII) and the confocal microscope images of the mice's hepatocytes were analyzed following previously described protocolsThe 3D-iLiNP device used for the investigation of the mass-produced LNPs was made from cycloolefin polymer plates fabricated by a micromachining process .t-test was used to compare the average values of two groups.The results were analyzed with Microsoft® Excel and an unpaired Student's Conceptualization: M. M. and M. T.; data curation: N. K. and M. M.; formal analysis: N. K., M. M., Y. S, A. I., H. T., H. H., and M. T.; methodology: M. M. and M. T.; investigation: N. K., M. M., K. S., and Y. S.; writing-original draft: N. K. and M. M.; writing-review and editing: M. M. and M. T.; funding acquisition: N. K., M. M., H. H., and M. T.; resources: N. K., M. M., and Y. S.; supervision: M. M. and M. T.The authors declare no competing financial interest.RA-011-D0RA08826A-s001


However, not all HLA-B*57:01+ HIV+ patients are affected by AHS, implying the involvement of further patient-specific factors in the development of AHS. The establishment of a reliable assay to classify HLA-B*57:01 carriers as ABC sensitive or ABC tolerant allowed to investigate the T cell receptor (TCR) Vβ chain repertoire of effector cells and revealed Vβ6 and Vβ24 as potential public TCRs in ABC sensitive HLA-B*57:01 carriers. Furthermore, distinct effects of ABC on the cellular proteome of ABC sensitive and tolerant volunteers were observed and suggest enhanced activation and maturation of dentritic cells (DC) in ABC sensitive volunteers. Analysis of ABC-naïve cellular proteomes identified the T cell immune regulator 1 (TCIRG1) as a potential prognostic biomarker for ABC susceptibility and the involvement of significantly upregulated proteins, particularly in peptide processing, antigen presentation, interferon (IFN), and cytokine regulation.Type B adverse drug reactions (ADRs) represent a significant threat as their occurrence arises unpredictable and despite proper application of the drug. The severe immune reaction Abacavir Hypersensitivity Syndrome (AHS) that arises in HIV Adverse drug reactions (ADR) are harmful but unintended reactions triggered by the application of a drug . ADRs ar+ T cells. With their respective T cell receptor (TCR), CD8+ T cells recognize the trimeric complex of HLA class I heavy chain, light chain, and endogenously processed peptide and discriminate between self- and pathogen-derived or foreign peptides [HLA molecules are highly polymorphic cell surface glycoproteins representing a fundamental part of the immune system. They are involved in immune responses through presentation of self- or pathogen-derived peptides to T lymphocytes. Based on the origin of the presented peptide, the HLA system provides information of the individual health condition to prevent and combat infections. The peptide binding region (PBR) of HLA is the region with the highest variability and a basis for the diverse presented immunopeptidome ,16. Almopeptides ,18. Drugs are usually much smaller than peptides, which are bound to HLA molecules; hence, they are not able to induce immune responses. However, to understand how small drug molecules may activate the immune system in an HLA-dependent manner, three models have been postulated. According to the hapten/prohapten model, the drug or its reactive metabolite covalently binds to a host protein or peptide and generates an immunogenic, haptenated, de novo peptide that is presented on HLA molecules. Peptide-independent, non-covalent binding of the drug to either the TCR or HLA protein that directly activates T cell responses is referred to as the pharmacological interaction with the immune receptors (p-i) model. The third model describes an altered peptide repertoire caused by altered biochemical features of the PBR through drug binding , resulting in the presentation of neo-antigens on HLA molecules ,3,8. + T cell engagement is like searching for a needle in a haystack. The HLA-B*57:01-mediated ADR post Abacavir medication represents a strong paradigm for the analysis of drug-induced intracellular conditions. The nucleoside analogue reverse transcriptase inhibitor Abacavir (ABC) used for the treatment of HIV-1 infections causes severe hypersensitivity reactions in certain patients. ABC hypersensitivity syndrome (AHS), which is strongly correlated to the carriage of HLA-B*57:01, was the first discovered HLA-mediated DHR [+ HIV-1+ patients [Unintended activation of immune effector cells post drug administration in an HLA-allele-dependent manner is impossible to preclude post approval of a drug. The variability of the HLA system makes the implication of HLA genotypes in clinical trials prior to drug approval unfeasible. Hence, dissolving the task of personalized drug safety in the context of HLA/drug and CD8ated DHR ,20,21. Spatients .+ T cells [+ T cells elicit polyclonal T cell responses against the neo-antigens [The biophysical mechanism of AHS has already been elucidated. The specific binding of ABC within the peptide binding region of HLA-B*57:01 molecules alters the biochemical features of the F pocket. As a result, self-peptides that have not been involved in thymic selection bind to HLA-B*57:01 and are presented to autologous CD8 T cells ,24,25. Dantigens , resulti+ HIV-1+ patients [Typing for HLA-B*57:01 prevented the occurrence of immunologically confirmed AHS, with a negative predictive value (NPV) of 100%. However, the positive predictive value (PPV) of 49.7% implies ABC sensitive or ABC tolerant characteristics of HLA-B*57:01patients ,28, sugg+ T lymphocytes, resulting in greater maturation of dendritic cells (DCs) and enhanced antigen-presenting cell (APC) co-stimulation, favored AHS in the respective mice [+ HIV-1+ patients defined a soluble cluster of differentiation 14 (sCD14) and endoplasmic reticulum aminopeptidase 1 (ERAP1) allotypes as new potential genetic predictors for ABC tolerance. ABC sensitivity significantly correlates with a functional polymorphism within the CD14 gene, resulting in an increased amount of sCD14. Moreover, HLA-B*57:01+ HIV-1+ patients displayed distinct functional haplotypes of ERAP1 whereby ABC tolerant patients exhibit hypoactive trimming ERAP1 allotypes that conceivably influence the immunopeptidome of the respective patient through differential antigen processing [Initial evidence was provided by examining HLA-B*57:01-linked ABC sensitivity and tolerance in vivo in transgenic (tg) mice. While immunocompetent tg mice tolerated ABC treatment, depletion of CD4ive mice ,30. Furtocessing ,32.The HLA/drug specific TCR is an essential component for HLA-mediated DHRs and is also a potential risk factor . Due to HLA-B*57:01 genotype. For this purpose, we established a reliable assay for the classification of ABC sensitive and ABC tolerant HLA-B*57:01+ healthy volunteers. Based on this classification, we investigated the Vβ TCR repertoire of the respective volunteers to identify a potential public ABC-specific Vβ TCR among ABC sensitive volunteers. Furthermore, we performed full proteome analysis using mass spectrometry to compare the effect of ABC treatment on the proteome of ABC sensitive and ABC tolerant volunteers in order to describe further prognostic risk factors that might contribute to the development of AHS. Knowledge about the involvement of genetic and cellular risk factors in HLA-mediated DHRs will help to improve drug safety and facilitate personalized treatment of patients.The present study aims to provide a step towards a comprehensive understanding of differential immunological AHS through immune profiling of ABC susceptible patients with the LCL721.221  and peripheral blood mononuclear cells (PBMCs) from healthy volunteers were maintained in RPMI 1640 , supplemented with 10% heat-inactivated fetal calf serum , 2 mM L-glutamine , 100 U/mL penicillin, and 100 µg/mL streptomycin (c. c. pro) at 37 °C and 5% CO2.The human B-lymphoblastoid cell line + donor via PCR, as previously described [Constructs encoding for full length HLA-B*57:01  were generated from cDNA of an HLA-B*57:01escribed . The cDNHEK293T cells were transfected with the HLA-B*57:01 encoding transfer vector, the packaging and envelope vectors psPAX, and pmD2G. A produced virus was used to stably transduce LCL721.221 cells, as previously described [LCL721.221 cells was confirmed by flow cytometry using the mab anti-human HLA-I w6/32 .escribed ,37. ExprLCL721.221/mHLA-B*57:01 were incubated with or without 50 µg/mL ABC for 24 h. Afterwards, cells were washed with phosphate-buffered saline . ABC-preincubated target cells were labelled with 0.2 µM CFSE (5(6)-CFDA, SE, Life TechnologiesTM, Carlsbad, CA, USA) and untreated target cells with 2 µM CFSE for 15 min at 37 °C in 5% CO2. Cells were pelleted at 1500 rpm for 5 min and resuspended in RPMI 1640 medium, supplemented with 10% human serum type AB . The two target cell populations were seeded 1:1 into a 48-well microtiter plate. + donors (TM (c. c. pro) density separation was performed in order to isolatePBMCs. CD8+ cells were isolated by negative selection using the human CD8+ T Cell Isolation Kit  and added to the target cells at a 10:1 effector-target ratio. The plate was incubated at 37 °C and 5% CO2. After 4 h, cell viability of the ABC-treated and untreated target cells was detected by 7-AAD (Life TechnologiesTM) staining via flow cytometry. ABC-specific lysis was calculated as follows:Peripheral blood of HLA-B*57:01+ donors  was obta+ healthy blood donors, as described previously. CD8+ cells were isolated directly after LymphoSepTM density separation to obtain naïve CD8+ cells. Additionally, PBMCs were incubated in RPMI 1640, supplemented with 10% human serum type AB for 48 h in the absence (to obtain unspecific CD8+ cells) or presence of 50 µg/mL ABC (to obtain ABC-specific CD8+ cells) prior to isolation of CD8+ cells. In order to isolate total RNA, 5 × 106 CD8+ cells were lysed in RLT buffer  with β-mercaptoethanol  and RNA isolation was performed according to QIAquick RNeasy Mini Kit (Qiagen N. V.) manufacturer’s manual. A total of 1.5 µg RNA was applied for cDNA synthesis according to High-Capacity cDNA Reverse Transcription Kit  manufacturer’s manual. A quantitative RT-PCR (qRT-PCR) was performed, as described previously [PBMCs were isolated from the peripheral blood of four classified ABC sensitive and ABC tolerant HLA-B*57:01eviously . For eac5 LCL721.221 cells were incubated with or without 50 µg/mL ABC for 0 h, 24 h, 48 h, and 72 h. Cells were lysed in 250 µL RIPA buffer, as described by Ho et al. [For ABC tracking in cell lysates, 5 × 10o et al. . Sample o et al. .7 PBMCs of three classified ABC sensitive and three ABC tolerant HLA-B*57:01+ healthy blood donors were treated with 100 µg/mL ABC for 48 h. After 24 h, drug treatment was repeated. Cells were harvested and lysed in RIPA buffer, as previously described [For proteome analysis, 1 × 10escribed . Proteinescribed . + healthy volunteers into ABC sensitive or ABC tolerant, an allogenic in vitro CTA was performed. LCL721.221/mHLA-B*57:01 cells were incubated in the absence or presence of ABC and used as target cells. To verify ABC uptake in LCL721.221 cells and to determine the incubation time sufficient for absorption of ABC, ABC concentration in LCL721.221 cell lysates was detected after various incubation periods  were compared in ABC sensitive and ABC tolerant HLA-B*57:01 carriers.To identify phenotypical differences in CD8+ cells decreased, whereas in ABC tolerant HLA-B*57:01 carriers, Vβ5 usage increased. In contrast, ABC-specific CD8+ cells displayed enhanced TCR Vβ6 and Vβ24 expression in ABC sensitive HLA-B*57:01 carriers and decreased usage in ABC tolerant HLA-B*57:01 carriers . MaxQuant software [+ volunteer exhibited a distinct proteome with up- and downregulation of specific proteins, protein abundance was normalized on the median proteome intensity of each volunteer. A principal component analysis (PCA) was performed to visualize significant differences between no drug treatment and ABC treatment, as well as between ABC sensitive and ABC tolerant HLA-B*57:01 carriers. The impact of ABC-treatment compared to no treatment was clearly distinguishable in HLA-B*57:01+ ABC sensitive and ABC tolerant volunteers  and proteins that were at least altered by factor log2 ± 1.0 in ABC sensitive compared to ABC tolerant HLA-B*57:01+ volunteers were regarded as regulated. In total, 67 quantified proteins were significantly upregulated, and 22 quantified proteins were significantly downregulated in PBMCs of ABC sensitive carrier compared to ABC tolerant HLA-B*57:01 carriers  was utilized to identify upstream regulators that explain the observed proteome changes. Thereby, the tyrosine-protein kinase ZAP70 and The TCR ζ-chain (CD247) were identified as upstream regulators that trigger the upregulation of several proteins in ABC sensitive compared to ABC tolerant HLA-B*57:01+ healthy volunteers is clearly distinguishable  network via the STRING Database that illustrates clusters of significantly upregulated proteins in ABC sensitive compared to ABC tolerant HLA-B*57:01gulation .Type B ADRs display a major economic burden for the public health care system as their occurrence arises despite proper application of the drug and is unpredictable. Thus, ADRs lead to life-threatening consequences for patients, as well as enormous cost for the pharmaceutical industry due to hospital admissions and inpatient treatment . Discove+ cells to provoke ABC-reactive CD8+ T cells with the potential to cause AHS in the respective mice [A Tg mouse model for HLA-B*57:01-linked ABC drug tolerance and reactivity by Cardone et al. in 2018 revealed the necessity of enhanced DC maturation and APC co-stimulation through the depletion of CD4ive mice . In addiive mice ,32, suggive mice  and descive mice , whereasive mice . LCL721.221/mHLA-B*57:01 cells as target cells that ensured constant HLA-B*57:01 expression on the cell surface and allowed the classification of HLA-B*57:01 healthy volunteers as ABC sensitive or ABC tolerant. Chessman et al. demonstrated in 2008 that ABC also induces the activation and proliferation of CD8+ T cells from ABC-naïve healthy HLA-B*57:01+ blood donors in vitro [+ HLA-B*57:01+ patient who previously developed AHS as a positive control and, although HLA-B*57:03 is a closely related natural allotype of HLA-B*57:01, an HIV+ HLA-B*57:03+ patient as a negative control. Application of this assay enabled the identification of four volunteers exhibiting ABC sensitivity and four volunteers exhibiting ABC tolerance. In the present study, we established a standardized protocol for patient screenings by an allogenic in vitro CTA, utilizing in vitro . For val+ volunteers for their TCR Vβ-chain repertoire via RT-qPCR with 24 TCR Vβ-specific primer pairs. For ABC-specific T cells, it has been observed that polyclonal TCR usage is consistent with the altered repertoire model [+ effector cells that might clarify why some, but not all HLA-B*57:01, carriers develop ABC-induced hypersensitivity reactions. TCR bias has been observed in many antigen-specific responses and in autoimmunity and alloreactivity. We proposed differential type 1 TCR bias in ABC sensitive and tolerant HLA-B*57:01 carriers. This bias is characterized by the selection of single TCR Vβ-chains with little or no conservation in CDR3 regions and might originate from thymic selection, immune response initiation, or persistent infection [+ patients [Based on this classification, we investigated and compared ABC sensitive and ABC tolerant HLA-B*57:01re model . Howevernfection . Differepatients . + volunteers revealed the strongest upregulation of the ribosomal protein RPLP1 and the serine/threonine-protein phosphatase 2A that catalyzes the removal of phosphate groups from serine and threonine residues. However, we found no correlation between upregulation of these proteins and ABC-mediated hypersensitivity. Strikingly, upregulation of HLA-DQB1 in ABC sensitive HLA-B*57:01 carriers suggested potential involvement of HLA class II in abacavir sensitivity, as initially reported for HLA-DR7 and HLA-DQ3 [LC-MS-based proteome analysis of untreated and ABC-treated PBMCs from ABC sensitive and tolerant HLA-B*57:01 carriers demonstrated significant differences. We also performed two separate PCAs to show that the proteomic content of ABC-naïve and ABC-treated PBMCs of ABC sensitive and tolerant volunteers are clearly distinguishable . ABC upt HLA-DQ3 . We perf+ T cells [One of these proteins, CD68, is a marker predominantly expressed in monocytes and especially abundant on macrophages . CD68 is T cells ,52. In a T cells . In DCs, T cells , with LY+ T cells. Furthermore, an essential role in the development and maintenance of effector and memory T cells is described for the non-canonical NF-κB pathway [The atypical E3 ubiquitin-protein ligase ZFP91 ubiquitinates NIK, resulting in the induction of the non-canonical NF-κB pathway. This pathway regulates DC development and maturation and is particularly necessary for DC cross-priming of CD8 pathway . In addiIPA. Two upstream regulators, ZAP70 and CD247, are involved in T cell responses [Furthermore, we identified upstream regulators of identified significantly upregulated proteins in ABC sensitive compared to ABC tolerant HLA-B*57:01 carriers via esponses ,57. CD24esponses . ZAP70 pesponses ,57. + T cells in ABC sensitive HLA-B*57:01 carriers. We assume that TCIRG1 might be a prognostic biomarker for ABC-mediated AHS, which requires further investigation. We also investigated the significantly downregulated proteins in untreated PBMCs of ABC sensitive compared to ABC tolerant HLA-B*57:01 carriers, but no clear protein pattern could be detected (The proteomic content of untreated PBMCs of ABC sensitive compared to ABC tolerant HLA-B*57:01 carriers exposed significant differences, suggesting the presence of a potential predictive biomarker among the differentially regulated proteins. Analysis of the ABC-naïve proteomes revealed significant upregulation of the key basement membrane protein NID1 that exhibits a role in the organization of skin, muscle, and nervous system basal laminae and is involved in signal transfer through integrins . PLBD1 hdetected .+ HIV+ patients [We created a PPI network via STRING Database that demonstrated the involvement of significantly upregulated proteins in PBMCs of untreated ABC sensitive compared to ABC tolerant HLA-B*57:01 carriers, especially in peptide processing, antigen presenting, and IFN and cytokine regulation. More efficient peptide processing and antigen presentation correspond to the identification of rather hyperactive ERAP1 allotypes in ABC sensitive HLA-B*57:01patients ,32. + T cell cross priming, and enhanced antigen processing contribute to ABC sensitivity. These data will open the door to comprehend the molecular basis of ADRs prior to approval of a medical product and should be implemented in the data of clinical trials.In conclusion, the data presented in this study emphasize the complex basis of ABC-mediated AHS with an interplay of several immunological processes involved. Despite the occurrence of the HLA genotype HLA-B*57:01, several patient-specific factors, particularly the activation of DCs, increased antigen presentation, more efficient CD8


In this publication we report the creation of a new unc-11 allele that is endogenously-tagged with GFP just upstream of the stop codon. Moreover, we demonstrate that the UNC-11::GFP fusion protein functions like wild type with an expression pattern similar to UNC-11 antibody staining described previously. PICALM facilitates recycling of Vesicle Associated Membrane Proteins (VAMPs) from the plasma membrane  and efficient clathrin-coated endocytosis . unc-11 function in C. elegans is likely conservedasSNB-2 (VAMP2) and the glutamate receptor, GLR-1, both accumulate at the plasma membrane in unc-11(e47) null mutants . Mutant phenotypes outside the worm nervous system have not been described.UNC-11is anortholog of mammalian PICALM (phosphatidylinositol binding clathrin assembly protein) and its neuron-specific paralog, AP180  with the CRISPR knock-in strain, unc-11(vg103) and the null mutant, unc-11(e47). We found no obvious differences between unc-11(+) and unc-11(vg103). Average thrash duration during a 45 second interval was 0.251 seconds  for unc-11(+) and 0.248 seconds  for unc-11(vg103). By contrast, the average thrash duration for unc-11(e47) was significantly higher at 0.566 seconds with a striking increase in variability .To ask if the addition of GFP at the C-terminus of UNC-11 compromises s system . We compet al. 1999) with some noted exceptions. Similar to UNC-11 antibody staining, UNC-11::GFP expression appeared pan-neuronal and was also observed within coelomocytes .Plasmid construction was done in the context of a course entitled Advanced Molecular Techniques at California State University, East Bay. This strain will be made available at the CGC. Given the inclusion of an AID tag and GFP, this strain will be useful for cell-specific protein degradation using auxin-inducible (AID) or GFP nanobody::ZIF-1 degradation methods  and videos were captured at 30 frames per second using an iPhone 7. To open the images in FIJI, the .MOV files were converted to an uncompressed AVI format using ff.WORKS software. The duration of each thrash was measured in FIJI by logging each frame number at which the worm completed a single body bend in one direction. Thrash duration (in seconds) was then calculated as the number of frames per body bend divided by 30 frames per second. Approximately, 1450 frames (~45 seconds) of video were reviewed for each animal. Body bends that did not fall below 150 degrees were not counted as a true body bend. When a body bend angle was suspected to be greater than 150 degrees, the angle ruler in Fiji (Image J) was used. Brief intervals where thrashing occurred along the Z-axis or the worm left the field of view were not included. The data was graphed using Plots of Differences available at https://huygens.science.uva.nl/PlotsOfDifferences/ .Confocal Microscopy:unc-11(vg103) animals were anesthetized with sodium azide (10 mM) and imaged using a Leica SP8 confocal. To visually distinguish true GFP signal from intestinal autofluorescence for Figures 1A and 1D, Z-stacks were captured using a sequential scanning protocol: Seq 1: The 488 laser was set at 2.4%. PMT1 (green) collected wavelengths from 494 to 544 nm (capturing GFP and autofluorescence) while PMT2 (blue) collected wavelengths from 650 to 798 nm (autofluorescence only). Seq 2: The 552 laser was set at 1.0 % and PMT1 (blue) collected wavelengths from 598 to 709 nm (autofluorescence only). Adjustments were made to brightness and contrast of the blue channels to bring out the autofluorescence then the green and blue channels were combined and relevant focal planes were subject to maximum projection. The other images did not emit detectable autofluorescence and so the blue channel images were not included in the Z projection.CTATAATCCAAAgtttaagagctatgctggaa and caagacatctcgcaatagga. The unc-11 target sequence is in all caps and the position of the unc-11 STOP codon is in bold. The GFP::SEC repair template (pDQ1023) was created by Gibson Assembly of pJW1583  digested with AvrII and SpeI and two unc-11 homology arms (HA) amplified by PCR (Q5 DNA polymerase – NEB). Primers used to amplify the 5’HA and 3’HA homology arms from wild type genomic DNA include: ACGACGGCCAGTCGCCGGCATGCGATGGGTTAGTTTATCG and CCTGAGGCTCCCGATGCTCCTAATCCAAATGGATCGGCTG (5’HA); AGGATGACGATGACAAGAGATAGTCTAGTCGTCTATTTCTC and TATGACCATTTATCGATTTCGCAATAGAGATAAAGTTGCA (3’HA). unc-11(vg103[unc-11::gfp]) (DQ3188) was created as described by Dickinson et al. 2015. Successful knock-in was verified by PCR amplification and sequencing of the junction fragments and by GFP expression.The sgRNA/Cas9 plasmid (pDQ1022) was created by Q5 SDM (NEB) using pJW1219 as template (a plasmid containing sgRNA(F+E) and Peft-3::Cas9; Addgene # 61250) and the following primer pair: CGACTAGA