30K Protein Research Articles

Virtual Discovery of Immune-Stimulating Epitopes in Chikungunya Virus for Vaccine Design

Epitope identification is a key step in vaccine development, and this can be achieved much faster and less expensively with in silico methods, compared to traditional methods for vaccine production. In silico methods applied in this research utilised both bioinformatics and immunoinformatics approaches for chikungunya virus vaccine design, which involved the retrieval of sequences from databases, and identification of conserved regions within the sequences by multiple sequence alignment on the MEGA X software (Pennsylvania State University, State College, USA). The epitopes in the conserved regions were selected, and various immunological predictions and screenings were carried out by employing immunological databases and tools. This process identifies epitopes such as conservation of cytotoxic T lymphocyte, helper T lymphocytes, and B cell epitopes. The primary, secondary, and tertiary structure of the vaccine was also predicted using structure predicting servers, and finally, the vaccine candidate was docked to toll-like receptor 4 to study its binding affinity and configuration. A total of 125 conserved antigenic epitopes were selected from capsid, 6K, and E1 proteins, which were found to be non-allergens and conform to acceptable physicochemical standards, as reported by other authors with similar work. The epitopes were predicted to be capable of inducing cytotoxic T lymphocytes, helper T lymphocytes, and B cell production. Construction of secondary structure was done using the Self-Optimized Prediction Method with Alignment (SOPMA), which predicted 17.96% α-helices, and 4.69% β-turns, among others. Predicting the tertiary structure provided five models, of which Model 1 was selected on the bases of its confidential score of 0.59, estimated TM-score of 0.79±0.09, and root mean square deviation of 8.0±4.4Å. Validity analysis revealed a Ramachandran plot where 97.2% of the vaccine residue was within the favoured region, and the peptide showed a Z-score of -1.52. The predicted peptide effectively docked with toll-like receptor 4 with a binding energy of -1,072.8. From the data obtained, it was revealed that the selected epitopes are highly immunogenic, non-allergenic, conform to native protein, and form a peptide capable of vaccine application. The authors can conclude this is a promising candidate for vaccine design and development.

Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery.

Sweet orange (Citrus sinensis) is one of the most important fruit crops worldwide. Virus infections in this crop can interfere with cellular processes, causing dramatic economic losses. By performing RT-qPCR analyses, we demonstrated that citrus psorosis virus (CPsV)-infected orange plants exhibited higher levels of unprocessed microRNA (miRNA) precursors than healthy plants. This result correlated with the reported reduction of mature miRNAs species. The protein 24K, the CPsV suppressor of RNA silencing (VSR), interacts with miRNA precursors in vivo. Thus, this protein becomes a candidate responsible for the increased accumulation of unprocessed miRNAs. We analyzed 24K RNA-binding and protein-protein interaction domains and described patterns of its subcellular localization. We also showed that 24K colocalizes within nuclear D-bodies with the miRNA biogenesis proteins DICER-LIKE 1 (DCL1), HYPONASTIC LEAVES 1 (HYL1), and SERRATE (SE). According to the results of bimolecular fluorescence complementation and co-immunoprecipitation assays, the 24K protein interacts with HYL1 and SE. Thus, 24K may inhibit miRNA processing in CPsV-infected citrus plants by direct interaction with the miRNA processing complex. This work contributes to the understanding of how a virus can alter the regulatory mechanisms of the host, particularly miRNA biogenesis and function.IMPORTANCESweet oranges can suffer from disease symptoms induced by virus infections, thus resulting in drastic economic losses. In sweet orange plants, CPsV alters the accumulation of some precursors from the regulatory molecules called miRNAs. This alteration leads to a decreased level of mature miRNA species. This misregulation may be due to a direct association of one of the viral proteins (24K) with miRNA precursors. On the other hand, 24K may act with components of the cell miRNA processing machinery through a series of predicted RNA-binding and protein-protein interaction domains.

NtG3BPL1 confers resistance to chilli veinal mottle virus through promoting the degradation of 6K2 in tobacco.

The RNA regulatory network is a complex and dynamic regulation in plant cells involved in mRNA modification, translation, and degradation. Ras-GAP SH3 domain-binding protein (G3BP) is a scaffold protein for the assembly of stress granules (SGs) and is considered an antiviral component in mammals. However, the function of G3BP during virus infection in plants is still largely unknown. In this study, four members of the G3BP-like proteins (NtG3BPLs) were identified in Nicotiana tabacum and the expression levels of NtG3BPL1 were upregulated during chilli veinal mottle virus (ChiVMV) infection. NtG3BPL1 was localized in the nucleus and cytoplasm, forming cytoplasmic granules under transient high-temperature treatment, whereas the abundance of cytoplasmic granules was decreased under ChiVMV infection. Overexpression of NtG3BPL1 inhibited ChiVMV infection and delayed the onset of symptoms, whereas knockout of NtG3BPL1 promoted ChiVMV infection. In addition, NtG3BPL1 directly interacted with ChiVMV 6K2 protein, whereas 6K2 protein had no effect on NtG3BPL1-derived cytoplasmic granules. Further studies revealed that the expression of NtG3BPL1 reduced the chloroplast localization of 6K2-GFP and the NtG3BPL1-6K2 interaction complex was localized in the cytoplasm. Furthermore, NtG3BPL1 promoted the degradation of 6K2 through autophagy pathway, and the accumulation of 6K2 and ChiVMV was affected by autophagy activation or inhibition in plants. Taken together, our results demonstrate that NtG3BPL1 plays a positive role in tobacco resistance against ChiVMV infection, revealing a novel mechanism of plant G3BP in antiviral strategy.

AtHVA22a, a plant-specific homologue of Reep/DP1/Yop1 family proteins is involved in turnip mosaic virus propagation.

The movement of potyviruses, the largest genus of single-stranded, positive-sense RNA viruses responsible for serious diseases in crops, is very complex. As potyviruses developed strategies to hijack the host secretory pathway and plasmodesmata (PD) for their transport, the goal of this study was to identify membrane and/or PD-proteins that interact with the 6K2 protein, a potyviral protein involved in replication and cell-to-cell movement of turnip mosaic virus (TuMV). Using split-ubiquitin membrane yeast two-hybrid assays, we screened an Arabidopsis cDNA library for interactors of TuMV6K2. We isolated AtHVA22a (Hordeum vulgare abscisic acid responsive gene 22), which belongs to a multigenic family of transmembrane proteins, homologous to Receptor expression-enhancing protein (Reep)/Deleted in polyposis (DP1)/Yop1 family proteins in animal and yeast. HVA22/DP1/Yop1 family genes are widely distributed in eukaryotes, but the role of HVA22 proteins in plants is still not well known, although proteomics analysis of PD fractions purified from Arabidopsis suspension cells showed that AtHVA22a is highly enriched in a PD proteome. We confirmed the interaction between TuMV6K2 and AtHVA22a in yeast, as well as in planta by using bimolecular fluorescence complementation and showed that TuMV6K2/AtHVA22a interaction occurs at the level of the viral replication compartment during TuMV infection. Finally, we showed that the propagation of TuMV is increased when AtHVA22a is overexpressed in planta but slowed down upon mutagenesis of AtHVA22a by CRISPR-Cas9. Altogether, our results indicate that AtHVA22a plays an agonistic effect on TuMV propagation and that the C-terminal tail of the protein is important in this process.

Barley yellow dwarf virus-GAV 17K protein disrupts thiamine biosynthesis to facilitate viral infection in plants.

Thiamine functions as a crucial activator modulating plant health and broad-spectrum stress tolerances. However, the role of thiamine in regulating plant virus infection is largely unknown. Here, we report that the multifunctional 17K protein encoded by barley yellow dwarf virus-GAV (BYDV-GAV) interacted with barley pyrimidine synthase (HvTHIC), a key enzyme in thiamine biosynthesis. HvTHIC was found to be localized in chloroplast via an N-terminal 74-amino acid domain. However, the 17K-HvTHIC interaction restricted HvTHIC targeting to chloroplasts and triggered autophagy-mediated HvTHIC degradation. Upon BYDV-GAV infection, the expression of the HvTHIC gene was significantly induced, and this was accompanied by accumulation of thiamine and salicylic acid. Silencing of HvTHIC expression promoted BYDV-GAV accumulation. Transcriptomic analysis of HvTHIC silenced and non-silenced barley plants showed that the differentially expressed genes were mainly involved in plant-pathogen interaction, plant hormone signal induction, phenylpropanoid biosynthesis, starch and sucrose metabolism, photosynthesis-antenna protein, and MAPK signaling pathway. Thiamine treatment enhanced barley resistance to BYDV-GAV. Taken together, our findings reveal a molecular mechanism underlying how BYDV impedes thiamine biosynthesis to uphold viral infection in plants.

A Novel Role for the Adenovirus L4 Region 22K and 33K Proteins in Adeno-Associated Virus Production.

Despite decades of research in adeno-associated virus (AAV) and the role of adenovirus in production, the interplay of AAV and adenovirus is not fully understood. Specific regions of the adenoviral genome containing E1, E2a, E4 open reading frame (ORF), and VA RNA have been demonstrated as necessary for AAV production; however, incorporating these regions into either a producer cell line or subcloning into an Ad helper plasmid may lead to inclusion of neighboring adenoviral sequence or ORFs with unknown function. Because AAV is frequently used in gene therapies, removing excessive adenovirus sequences improves the Ad helper plasmid size and manufacturability, and may lead to safer vectors for patients. Furthermore, deepening our understanding of the helper virus genes required for recombinant AAV (rAAV) production has the potential to increase yields and manufacturability of rAAV for clinical and commercial applications. One region continuously included in various Ad helper plasmid iterations is the adenoviral E2a promoter region that appears to be necessary for E2a expression. Due to the compact nature of viral genomes, the E2a promoter region overlaps with the Hexon Assembly/100K protein and the L4 region. The L4 region, which contains the coding sequences for 22K and 33K proteins, had not been thought to be necessary for AAV production. Through molecular techniques, this study demonstrates that the adenoviral 22K protein is essential for rAAV production in HEK293 cells by triple transfection and that the 33K protein synergistically increases rAAV yield.

The roles of 6K protein on Getah virus replication and pathogenicity.

Alphavirus is a type of arbovirus that can infect both humans and animals. The amino acid sequence of the 6K protein, being one of the structural proteins of the alphavirus, is not conserved. Deletion of this protein will result in varying effects on different alphaviruses. Our study focuses on the function of the Getah virus (GETV) 6K protein in infected cells and mice. We successfully constructed infectious clone plasmids and created resulting viruses (rGETV and rGETV-Δ6K). Our comprehensive microscopic analysis revealed that the 6 K protein mainly stays in the endoplasmic reticulum. In addition, rGETV-Δ6K has lower thermal stability and sensitivity to temperature than GETV. Although the deletion of the 6K protein does not reduce virion production in ST cells, it affects the release of virions from host cells by inhibiting the process of E2 protein transportation to the plasma membrane. Subsequent in vivo testing demonstrated that neonatal mice infected with rGETV-Δ6K had a lower virus content, less significant pathological changes in tissue slices, and milder disease than those infected with the wild-type virus. Our results indicate that the 6K protein effectively reduces the viral titer by influencing the release of viral particles. Furthermore, the 6K protein play a role in the clinical manifestation of GETV disease.

ATG8f Interacts with Chilli Veinal Mottle Virus 6K2 Protein to Limit Virus Infection.

Autophagy, as a conserved protein degradation pathway in plants, has also been reported to be intricately associated with antiviral defense mechanisms. However, the relationship between chilli veinal mottle virus (ChiVMV) and autophagy has not been investigated in the existing research. Here, we reveal that ChiVMV infection caused the accumulation of autophagosomes in infected Nicotiana benthamiana leaves and the upregulation of autophagy-related genes (ATGs). Moreover, the changes in gene expression were correlated with the development of symptoms. Treatment with autophagy inhibitors (3-MA or E-64D) could increase the infection sites and facilitate virus infection, whereas treatment with the autophagy activator (Rapamycin) limited virus infection. Then, ATG8f was identified to interact with ChiVMV 6K2 protein directly in vitro and in vivo. The silencing of ATG8f promoted virus infection, whereas the overexpression of ATG8f inhibited virus infection. Furthermore, the expression of 6K2-GFP in ATG8f- or ATG7-silenced plants was significantly higher than that in control plants. Rapamycin treatment reduced the accumulation of 6K2-GFP in plant cells, whereas treatment with the inhibitor of the ubiquitin pathway (MG132), 3-MA, or E-64D displayed little impact on the accumulation of 6K2-GFP. Thus, our results demonstrated that ATG8f interacts with the ChiVMV 6K2 protein, promoting the degradation of 6K2 through the autophagy pathway.

Sugarcane mosaic virus employs 6K2 protein to impair ScPIP2;4 transport of H2O2 to facilitate virus infection.

Sugarcane mosaic virus (SCMV), one of the main pathogens causing sugarcane mosaic disease, is widespread in sugarcane (Saccharum spp. hybrid) planting areas and causes heavy yield losses. RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) NADPH oxidases and plasma membrane intrinsic proteins (PIPs) have been associated with the response to SCMV infection. However, the underlying mechanism is barely known. In the present study, we demonstrated that SCMV infection upregulates the expression of ScRBOHs and the accumulation of hydrogen peroxide (H2O2), which inhibits SCMV replication. All eight sugarcane PIPs (ScPIPs) interacted with SCMV-encoded protein 6K2, whereby two PIP2s (ScPIP2;1 and ScPIP2;4) were verified as capable of H2O2 transport. Furthermore, we revealed that SCMV-6K2 interacts with ScPIP2;4 via transmembrane domain 5 to interfere with the oligomerization of ScPIP2;4, subsequently impairing ScPIP2;4 transport of H2O2. This study highlights a mechanism adopted by SCMV to employ 6K2 to counteract the host resistance mediated by H2O2 to facilitate virus infection and provides potential molecular targets for engineering sugarcane resistance against SCMV.

Appropriate reduction of importin-α gene expression enhances yellow dwarf disease resistance in common wheat.

Barley yellow dwarf viruses (BYDVs) cause widespread damage to global cereal crops. Here we report a novel strategy for elevating resistance to BYDV infection. The 17K protein, a potent virulence factor conserved in BYDVs, interacted with barley IMP-α1 and -α2 proteins that are nuclear transport receptors. Consistently, a nuclear localization signal was predicted in 17K, which was found essential for 17K to be transported into the nucleus and to interact with IMP-α1 and -α2. Reducing HvIMP-α1 and -α2 expression by gene silencing attenuated BYDV-elicited dwarfism, accompanied by a lowered nuclear accumulation of 17K. Among the eight common wheat CRISPR mutants with two to four TaIMP-α1 and -α2 genes mutated, the triple mutant α1aaBBDD /α2AAbbdd and the tetra-mutant α1aabbdd /α2AAbbDD displayed strong BYDV resistance without negative effects on plant growth under field conditions. The BYDV resistance exhibited by α1aaBBDD /α2AAbbdd and α1aabbdd /α2AAbbDD was correlated with decreased nuclear accumulation of 17K and lowered viral proliferation in infected plants. Our work uncovers the function of host IMP-α proteins in BYDV pathogenesis and generates the germplasm valuable for breeding BYDV-resistant wheat. Appropriate reduction of IMP-α gene expression may be broadly useful for enhancing antiviral resistance in agricultural crops and other economically important organisms.

First identification of canine adenovirus 1 in mink and bioinformatics analysis of its 100 K protein.

Animal trade favors the spreading of emerging canine adenovirus 1 (CAdV-1) in mink. Because the 100K protein is not exposed to the viral surface at any stage, it can be used to differentiate the vaccine from wild virus infection. However, no related research has been conducted. This study aimed to find evidence of CAdV-1 in mink and predict the character of the 100K protein in the current circulating CAdV-1 strain of mink. In this experiment, the identification of CAdV-1, the phylogenetic tree, homology, and bioinformatics analysis of 100K were conducted. The results showed that the CAdV-1 was identified in the mink and that its Fiber was located in a separate branch. It was closely related to strains isolated from Norwegian Arctic fox and Red fox. 100K was located in a separate branch, which had the closest genetic relationship with skunks, porcupines, raccoons, and hedgehogs and a far genetic relationship with the strains in dogs. 100K protein is an unstable and hydrophobic protein. It had evidence of selective pressure and recombination, 1 glycosylation site, 48 phosphorylation sites, 60 dominant B cell epitopes, and 9 peptides of MHC-I and MHC-II. Its subcellular localization was mainly in the endoplasmic reticulum and mitochondria. The binding sites of 100K proteins were DBP proteins and 33K proteins. The stains in the mink were different from fox. The exploration of its genomic characteristics will provide us with a deeper understanding of the prevention of canine adenovirus.

P3 and NIa-Pro of Turnip Mosaic Virus Are Independent Elicitors of Superinfection Exclusion.

Superinfection exclusion (SIE) is an antagonistic interaction between identical or closely related viruses in host cells. Previous studies by us and others led to the hypothesis that SIE was elicited by one or more proteins encoded in the genomes of primary viruses. Here, we tested this hypothesis using Turnip mosaic virus (TuMV), a member of the genus Potyvirus of the family Potyviridae, with significant economic consequences. To this end, individual TuMV-encoded proteins were transiently expressed in the cells of Nicotiana benthamiana leaves, followed by challenging them with a modified TuMV expressing the green fluorescent protein (TuMV-GFP). Three days after TuMV-GFP delivery, these cells were examined for the replication-dependent expression of GFP. Cells expressing TuMV P1, HC-Pro, 6K1, CI, 6K2, NIa-VPg, NIb, or CP proteins permitted an efficient expression of GFP, suggesting that these proteins failed to block the replication of a superinfecting TuMV-GFP. By contrast, N. benthamiana cells expressing TuMV P3 or NIa-Pro did not express visible GFP fluorescence, suggesting that both of them could elicit potent SIE against TuMV-GFP. The SIE elicitor activity of P3 and NIa-Pro was further confirmed by their heterologous expression from a different potyvirus, potato virus A (PVA). Plants systemically infected with PVA variants expressing TuMV P3 or NIa-Pro blocked subsequent infection by TuMV-GFP. A +1-frameshift mutation in P3 and NIa-Pro cistrons facilitated superinfection by TuMV-GFP, suggesting that the P3 and NIa-Pro proteins, but not the RNA, are involved in SIE activity. Additionally, deletion mutagenesis identified P3 amino acids 3 to 200 of 352 and NIa-Pro amino acids 3 to 40 and 181 to 242 of 242 as essential for SIE elicitation. Collectively, our study demonstrates that TuMV encodes two spatially separated proteins that act independently to exert SIE on superinfecting TuMV. These results lay the foundation for further mechanistic interrogations of SIE in this virus.

Ebony plays an important role in egg hatching and 30k protein expression of silkworm (Bombyx mori).

QiufengN is a silkworm strain. During the feeding process of QiufengN, a mutant of black pupal cuticle QiufengNBP was found. Some silkworm pupae of the mutant were unable to easily molt during pupation, and some silkworm eggs produced by developed normally but larvae were unable to break out of the eggshells. These phenomena had not been observed in other black pupa mutants. Genetic analysis showed that the melanization trait of QiufengNBP is controlled by a recessive gene located on the autosome and follows Mendelian inheritance. Results of positional cloning and qRT-PCR showed that the occurrence of black pupae was caused by the mutation of the ebony gene on chromosome 26. 2-DE analysis of the pupal cuticle of QiufengN and QiufengNBP found that the 30K protein, the main storage protein for the growth and development of silkworms and an important energy substance for embryonic development, has changed significantly. In addition, the expression level of Bombyx mori hatching enzyme (BmHEL), which can soften the eggshell during the hatching process of silkworm, was significantly higher in the eggs of black pupae before and after hatching than in normal eggs. The mutation of ebony makes hatching difficult for silkworms, and increases in BmHEL is needed to soften the eggshell. This study showed that ebony may have important effects on the formation of silkworm pigment and egg hatching, and its formation mechanism is complex and deserves further study.

The LIM Domain Protein BmFHL2 Inhibits Egg Production in Female Silkworm, Bombyx mori.

The female Bombyx mori accumulates a large amount of egg proteins, mainly Vg and 30K, during egg formation to provide nutrition for embryo development. The synthesis and transport of Vg have been extensively studied, particularly the regulation of Vg transcription induced by 20E; however, the mechanism of 30K protein synthesis is poorly studied. As a model organism of the order Lepidoptera, B. mori has high reproduction potential. In the present study, we found that the FHL2 homologous gene (BmFhl2) in B. mori is involved in inhibiting female egg formation by influencing the synthesis of 30K protein. Interference of BmFhl2 expression in silkworm females increased 30K protein synthesis, accelerated ovarian development, and significantly increased the number of eggs produced and laid; however, the 20E pathway was inhibited. The transcription levels of Vg and 30Kc19 were significantly downregulated following BmFhl2 overexpression in the silkworm ovarian cell line BmN. The Co-IP assay showed that the potential binding protein of BmFHL2 included three types of 30K proteins (30Kc12, 30Kc19, and 30Kc21). These results indicate that BmFHL2 participates in egg formation by affecting 30K protein in female B. mori.

Vitellogenin receptor transports the 30K protein LP1 without cell-penetrating peptide, into the oocytes of the silkworm, Bombyx mori.

Vitellogenin receptors (VgRs) transport vitellogenin (Vg) into oocytes, thereby promoting egg growth and embryonic development. VgRs recognize and transport multiple ligands in oviparous animals, but their role in insects is rarely reported. In this study, we investigated whether Bombyx mori VgR (BmVgR) binds and transports lipoprotein-1 (BmLP1) and lipoprotein-7 (BmLP7) of the 30kDa lipoproteins (30K proteins), which are essential for egg formation and embryonic development in B. mori. Protein sequence analysis showed BmLP7, similar to reported lipoprotein-3 (BmLP3), contains the cell-penetrating peptides and Cysteine position, while BmLP1 has not. Assays using Spodoptera frugiperda ovary cells (sf9) indicated the direct entry of BmLP7 into the cells, whereas BmLP1 failed to enter. However, co-immunoprecipitation (Co-IP) assays indicated that BmVgR could bind BmLP1. Western blotting and immunofluorescence assays further revealed that over-expressed BmVgR could transport BmLP1 into sf9 cells. Co-IP assays showed that SE11C (comprising LBD1+EGF1+OTC domains of BmVgR) or SE22C (comprising LBD2+EGF2+OTC domains of BmVgR) could bind BmLP1. Over-expressed SE11C or SE22C could also transport BmLP1 into sf9 cells. Western blotting revealed that the ability of SE11C to transport BmLP1 might be stronger than that of SE22C. In the vit mutant with BmVgR gene mutation (vit/vit), SDS-PAGE and western blotting showed the content of BmLP1 in the ovary, like BmVg, was lower than that in the normal silkworm. When transgenic with hsp70 promoter over-expressed BmVgR in the vit mutant, we found that the phenotype of the vit mutant was partly rescued after heat treatment. And contents of BmLP1 and BmVg in vit mutant over-expressed BmVgR were higher than in the vit mutant. We conclude that BmVgR and its two repeat domains could bind and transport BmLP1 into the oocytes of the silkworm, besides BmVg. These results will provide a reference for studying the molecular mechanism of VgR transporting ligands in insects.

Sugarcane mosaic virus orchestrates the lactate fermentation pathway to support its successful infection.

Viruses often establish their own infection by altering host metabolism. How viruses co-opt plant metabolism to support their successful infection remains an open question. Here, we used untargeted metabolomics to reveal that lactate accumulates immediately before and after robust sugarcane mosaic virus (SCMV) infection. Induction of lactate-involved anaerobic glycolysis is beneficial to SCMV infection. The enzyme activity and transcriptional levels of lactate dehydrogenase (LDH) were up-regulated by SCMV infection, and LDH is essential for robust SCMV infection. Moreover, LDH relocates in viral replicase complexes (VRCs) by interacting with SCMV-encoded 6K2 protein, a key protein responsible for inducing VRCs. Additionally, lactate could promote SCMV infection by suppressing plant defense responses. Taken together, we have revealed a viral strategy to manipulate host metabolism to support replication compartment but also depress the defense response during the process of infection.

Requirement of a functional ion channel for Sindbis virus glycoprotein transport, CPV-II formation, and efficient virus budding.

Many viruses encode ion channel proteins that oligomerize to form hydrophilic pores in membranes of virus-infected cells and the viral membrane in some enveloped viruses. Alphavirus 6K, human immunodeficiency virus type 1 Vpu (HIV-Vpu), influenza A virus M2 (IAV-M2), and hepatitis C virus P7 (HCV-P7) are transmembrane ion channel proteins that play essential roles in virus assembly, budding, and entry. While the oligomeric structures and mechanisms of ion channel activity are well-established for M2 and P7, these remain unknown for 6K. Here we investigated the functional role of the ion channel activity of 6K in alphavirus assembly by utilizing a series of Sindbis virus (SINV) ion channel chimeras expressing the ion channel helix from Vpu or M2 or substituting the entire 6K protein with full-length P7, in cis. We demonstrate that the Vpu helix efficiently complements 6K, whereas M2 and P7 are less efficient. Our results indicate that while SINV is primarily insensitive to the M2 ion channel inhibitor amantadine, the Vpu inhibitor 5-N, N-Hexamethylene amiloride (HMA), significantly reduces SINV release, suggesting that the ion channel activity of 6K similar to Vpu, promotes virus budding. Using live-cell imaging of SINV with a miniSOG-tagged 6K and mCherry-tagged E2, we further demonstrate that 6K and E2 colocalize with the Golgi apparatus in the secretory pathway. To contextualize the localization of 6K in the Golgi, we analyzed cells infected with SINV and SINV-ion channel chimeras using transmission electron microscopy. Our results provide evidence for the first time for the functional role of 6K in type II cytopathic vacuoles (CPV-II) formation. We demonstrate that in the absence of 6K, CPV-II, which originates from the Golgi apparatus, is not detected in infected cells, with a concomitant reduction in the glycoprotein transport to the plasma membrane. Substituting a functional ion channel, M2 or Vpu localizing to Golgi, restores CPV-II production, whereas P7, retained in the ER, is inadequate to induce CPV-II formation. Altogether our results indicate that ion channel activity of 6K is required for the formation of CPV-II from the Golgi apparatus, promoting glycoprotein spike transport to the plasma membrane and efficient virus budding.

Barley GRIK1‐SnRK1 kinases subvert a viral virulence protein to upregulate antiviral RNAi and inhibit infection

Viruses often usurp host machineries for their amplification, but it remains unclear if hosts may subvert virus proteins to regulate viral proliferation. Here, we show that the 17K protein, an important virulence factor conserved in barley yellow dwarf viruses (BYDVs) and related poleroviruses, is phosphorylated by host GRIK1‐SnRK1 kinases, with the phosphorylated 17K (P17K) capable of enhancing the abundance of virus‐derived small interfering RNAs (vsiRNAs) and thus antiviral RNAi. Furthermore, P17K interacts with barley small RNA‐degrading nuclease 1 (HvSDN1) and impedes HvSDN1‐catalyzed vsiRNA degradation. Additionally, P17K weakens the HvSDN1‐HvAGO1 interaction, thus hindering HvSDN1 from accessing and degrading HvAGO1‐carried vsiRNAs. Importantly, transgenic expression of 17K phosphomimetics (17K5D), or genome editing of SDN1, generates stable resistance to BYDV through elevating vsiRNA abundance. These data validate a novel mechanism that enhances antiviral RNAi through host subversion of a viral virulence protein to inhibit SDN1‐catalyzed vsiRNA degradation and suggest new ways for engineering BYDV‐resistant crops.

LncR26319/miR-2834/EndophilinA axis regulates oogenesis of the silkworm, Bombyx mori.

Oocyte maturation is critical for insect reproduction. Vitellogenesis, the timely production and uptake of vitellogenin (Vg), is crucial for female fecundity. Vg is synthesized in fat body and absorbed by the oocytes through endocytosis during insect oogenesis. In the silkworm, Bombyx mori, we discovered that a nucleus-enriched long-noncoding RNA (lncRNA) lncR26319 regulates Endophilin A (EndoA) - a member of the endophilin family of endocytic proteins - through competitive binding to miR-2834. The lncR26319-miR-2834-EndoA axis was required for Vg endocytosis in the silkworm; loss of EndoA or overexpression of miR-2834 significantly reduced egg numbers in virgin moths. In addition, accumulation of miR-2834 resulted in pupal and adult deformation and reduced fecundity in females. The expression of Vg, 30-kDa (30K) protein, and egg-specific protein (Esp) decreased after knockdown of EndoA or overexpression of miR-2834, while knockdown of miR-2834 had an opposite effect on the expression of Vg, 30K protein gene, and Esp. These results suggest that the lncR26319-miR-2834-EndoA axis contributes to the endocytic activity in the Vg uptake and leads to the normal progression of oogenesis in the silkworm. Thus, miR-2834 and EndoA are crucial for female reproduction and could be potential targets for new pest management strategies in lepidopterans.

Cellular protein HSC70 promotes fowl adenovirus serotype 4 replication in LMH cells via interacting with viral 100K protein

Fowl adenovirus serotype 4 (FAdV-4), the predominant causative agent of hepatitis-hydropericardium syndrome (HHS), has caused severe economic losses to poultry industry since 2015. Although fiber2 and hexon have been confirmed to be the virulence-related factors, the roles of nonstructural viral proteins in pathogenicity of FAdV-4 remain poorly understood. Here, a tandem mass spectrometry (MS) was used to identify host factors interacted with 100K protein of hypervirulent FAdV-4 isolate (CH/HNJZ/2015), and 2595 cellular proteins associated with many biological processes and pathways were identified according to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Among the proteins, HSC70 was verified to interact with 100K through co-immunoprecipitation assay. Notably, overexpression of HSC70 promoted the replication of FAdV-4 in LMH cells, whereas blocking HSC70 with inhibitor ver-155008 markedly suppressed viral replication. Collectively, these findings suggested that many cellular proteins involved in FAdV-4 infection through interacting with 100K and HSC70 positively regulated virus replication.