A natural substitution of a conserved amino acid in eIF4E confers resistance against multiple potyviruses.

Eukaryotic translation initiation factor 4E (eIF4E), which plays a pivotal role in initiating translation in eukaryotic organisms, is often hijacked by the viral genome-linked protein to facilitate the infection of potyviruses. In this study, we found that the naturally occurring amino acid substitution D71G in eIF4E is widely present in potyvirus-resistant watermelon accessions and disrupts the interaction between watermelon eIF4E and viral genome-linked protein of papaya ringspot virus-watermelon strain, zucchini yellow mosaic virus or watermelon mosaic virus. Multiple sequence alignment and protein modelling showed that the amino acid residue D71 located in the cap-binding pocket of eIF4E is strictly conserved in many plant species. The mutation D71G in watermelon eIF4E conferred resistance against papaya ringspot virus-watermelon strain and zucchini yellow mosaic virus, and the equivalent mutation D55G in tobacco eIF4E conferred resistance to potato virus Y. Therefore, our finding provides a potential precise target for breeding plants resistant to multiple potyviruses.

Potato virus Y viral protein 6K1 inhibits the interaction between defense proteins during virus infection.

14-3-3 proteins play vital roles in plant defense against various pathogen invasions. To date, how 14-3-3 affects virus infections in plants remains largely unclear. In this study, we found that Nicotiana benthamiana 14-3-3h interacts with TRANSLATIONALLY CONTROLLED TUMOR PROTEIN (TCTP), a susceptibility factor of potato virus Y (PVY). Silencing of Nb14-3-3h facilitates PVY accumulation, whereas overexpression of Nb14-3-3h inhibits PVY replication. The antiviral activities of 3 Nb14-3-3h dimerization defective mutants are significantly decreased, indicating that dimerization of Nb14-3-3h is indispensable for restricting PVY infection. Our results also showed that the mutant Nb14-3-3hE16A, which is capable of dimerizing but not interacting with NbTCTP, has reduced anti-PVY activity; the mutant NbTCTPI65A, which is unable to interact with Nb14-3-3h, facilitates PVY replication compared with the wild-type NbTCTP, indicating that dimeric Nb14-3-3h restricts PVY infection by interacting with NbTCTP and preventing its proviral function. As a counter-defense, PVY 6K1 interferes with the interaction between Nb14-3-3h and NbTCTP by competitively binding to Nb14-3-3h and rescues NbTCTP to promote PVY infection. Our results provide insights into the arms race between plants and potyviruses.

Corrigendum: Development and Evaluation of Stable Sugarcane Mosaic Virus Mild Mutants for Cross-Protection Against Infection by Severe Strain.

[This corrects the article DOI: 10.3389/fpls.2021.788963.].

A maize triacylglycerol lipase inhibits sugarcane mosaic virus infection.

Triacylglycerol lipase (TGL) plays critical roles in providing energy for seed germination and plant development. However, the role of TGL in regulating plant virus infection is largely unknown. In this study, we adopted affinity purification coupled with mass spectrometry and identified that a maize (Zea mays) pathogenesis-related lipase protein Z. mays TGL (ZmTGL) interacted with helper component-proteinase (HC-Pro) of sugarcane mosaic virus (SCMV). Yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation assays confirmed that ZmTGL directly interacted with SCMV HC-Pro in vitro and in vivo. The 101-460 residues of SCMV HC-Pro were important for its interaction with ZmTGL. ZmTGL and SCMV HC-Pro co-localized at the mitochondria. Silencing of ZmTGL facilitated SCMV infection, and over-expression of ZmTGL reduced the RNA silencing suppression activity, most likely through reducing HC-Pro accumulation. Our results provided evidence that the lipase hydrolase activity of ZmTGL was associated with reducing HC-Pro accumulation, activation of salicylic acid (SA)-mediated defense response, and inhibition of SCMV infection. We show that ZmTGL inhibits SCMV infection by reducing HC-Pro accumulation and activating the SA pathway.

A Predicted Stem Loop in Coat Protein-Coding Sequence of Tobacco Vein Banding Mosaic Virus Is Required for Efficient Replication.

Potyviral coat protein (CP) is involved in the replication and movement of potyviruses. However, little information is available on the roles of CP-coding sequence in potyviral infection. Here, we introduced synonymous substitutions to the codon C574G575C576 coding conserved residue arginine at position 192 (R192) of tobacco vein banding mosaic virus (TVBMV) CP. Substitution of the codon C574G575C576 to A574G575A576 or A574G575G576, but not C574G575A576, C574G575T576, or C574G575G576, reduced the replication, cell-to-cell movement, and accumulation of TVBMV in Nicotiana benthamiana plants, suggesting that C574 was critical for replication of TVBMV. Nucleotides 531 to 576 of the TVBMV CP-coding sequence were predicted to form a stem-loop structure, in which four consecutive C-G base pairs (C576-G531, C532-G575, C574-G533, and C534-G573) were located at the stem. Synonymous substitutions of R178-codon C532G533C534 to A532G533A534 and A532G533G534, but not C532G533A534, C532G533T534, or C532G533G534, reduced the replication levels, cell-to-cell, and systemic movement of TVBMV, suggesting that C532 was critical for TVBMV replication. Synonymous substitutions disrupting base pairs C576-G531 and C534-G573 did not affect viral accumulation. After three serial-passage inoculations, the accumulation of spontaneous mutant viruses was restored, and codons A532G533A534, A532G533G534, A574G575A576, or A574G575G576 of mutants were each separately changed to C532G533A534, C532G533G534, C574G575A576, or C574G575G576. Synonymous mutation of R178 and R192 also reduced viral accumulation in N. tabacum plants. Therefore, we concluded that the two consecutive C532-G575 and C574-G533 base pairs played critical roles in TVBMV replication via maintaining the stability of the stem-loop structures formed by nucleotides 531 to 576 of the CP-coding sequence.

The chloroplast ribosomal protein large subunit 1 interacts with viral polymerase and promotes virus infection.

Chloroplasts play an indispensable role in the arms race between plant viruses and hosts. Chloroplast proteins are often recruited by plant viruses to support viral replication and movement. However, the mechanism by which chloroplast proteins regulate potyvirus infection remains largely unknown. In this study, we observed that Nicotiana benthamiana ribosomal protein large subunit 1 (NbRPL1), a chloroplast ribosomal protein, localized to the chloroplasts via its N-terminal 61 amino acids (transit peptide), and interacted with tobacco vein banding mosaic virus (TVBMV) nuclear inclusion protein b (NIb), an RNA-dependent RNA polymerase. Upon TVBMV infection, NbRPL1 was recruited into the 6K2-induced viral replication complexes in chloroplasts. Silencing of NbRPL1 expression reduced TVBMV replication. NbRPL1 competed with NbBeclin1 to bind NIb, and reduced the NbBeclin1-mediated degradation of NIb. Therefore, our results suggest that NbRPL1 interacts with NIb in the chloroplasts, reduces NbBeclin1-mediated NIb degradation, and enhances TVBMV infection.

Identification of genetic determinants of tomato brown rugose fruit virus that enable infection of plants harbouring the Tm-22 resistance gene.

Tomato cultivars containing the Tm-22 resistance gene have been widely known to resist tobacco mosaic virus (TMV) and tomato mosaic virus. Tomato brown rugose fruit virus (ToBRFV), a new emerging tobamovirus, can infect tomato plants carrying the Tm-22 gene. However, the virulence determinant of ToBRFV that overcomes the resistance conferred by the Tm-22 gene remains unclear. In this study, we substituted the movement protein (MP) encoding sequences between ToBRFV and TMV infectious clones and conducted infectivity assays. The results showed that MP was the virulence determinant for ToBRFV to infect Tm-22 transgenic Nicotiana benthamiana plants and Tm-22 -carrying tomato plants. A TMV MP chimera with amino acid residues 60-186 of ToBRFV MP failed to induce hypersensitive cell death in the leaves of Tm-22 transgenic N. benthamiana plants. Chimeric TMV containing residues 60-186 of ToBRFV MP could, but chimeric ToBRFV containing 61-187 residues of TMV MP failed to infect Tm-22 transgenic N. benthamiana plants, indicating that 60-186 residues of MP were important for ToBRFV to overcome Tm-22 gene-mediated resistance. Further analysis showed that six amino acid residues, H67 , N125 , K129 , A134 , I147 , and I168 of ToBRFV MP, were critical in overcoming Tm-22 -mediated resistance in transgenic N. benthamiana plants and tomato plants. These results increase our understanding of the mechanism by which ToBRFV overcomes Tm-22 -mediated resistance.

Residues R192 and K225 in RNA-Binding Pocket of Tobacco Vein Banding Mosaic Virus CP Control Virus Cell-to-Cell Movement and Replication.

Potyviruses move to neighboring cells in the form of virus particles or a coat protein (CP)-containing ribonucleoprotein complex. However, the precise roles of RNA-binding residues in potyviral CP in viral cell-to-cell movement remain to be elucidated. In this study, we predicted the three-dimensional model of tobacco vein banding mosaic virus (TVBMV)-encoded CP and found nine residues presumably located in the CP RNA-binding pocket. Substitutions of the two basic residues at positions 192 and 225 (R192 and K225) with either alanine, cysteine, or glutamic acid abolished TVBMV cell-to-cell and systemic movement in Nicotiana benthamiana plants. These substitutions also reduced the replication of the mutant viruses. Results from the electrophoretic mobility shift assay showed that the RNA-binding activity of mutant CPs derived from R192 or K225 substitutions was significantly lower than that of wild-type CP. Analysis of purified virus particles showed that mutant viruses with R192 or K225 substitutions formed RNA-free virus-like particles. Mutations of R192 and K225 did not change the CP plasmodesmata localization. The wild-type TVBMV CP could rescue the deficient cell-to-cell movement of mutant viruses. Moreover, deletion of any of the other seven residues also abolished TVBMV cell-to-cell movement and reduced the CP RNA-binding activity. The corresponding nine residues in watermelon mosaic virus CP were also found to play essential roles in virus cell-to-cell movement. In conclusion, residues R192 and K225 in the CP RNA-binding pocket are critical for viral RNA binding and affect both virus replication and cell-to-cell movement.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Development and Evaluation of Stable Sugarcane Mosaic Virus Mild Mutants for Cross-Protection Against Infection by Severe Strain.

Sugarcane mosaic virus (SCMV; genus Potyvirus) induces maize dwarf mosaic disease that has caused serious yield losses of maize in China. Cross-protection is one of the efficient strategies to fight against severe virus strains. Although many mild strains have been identified, the spontaneous mutation is one of the challenging problems affecting their application in cross-protection. In this study, we found that the substitution of cysteine (C) at positions 57 or 60 in the zinc finger-like motif of HC-Pro with alanine (A; C57A or C60A) significantly reduced its RNA silencing suppression activity and SCMV virulence. To reduce the risk of mild strains mutating to virulent ones by reverse or complementary mutations, we obtained attenuated SCMV mutants with double-mutations in the zinc finger-like and FRNK motifs of HC-Pro and evaluated their potential application in cross-protection. The results showed that the maize plants infected with FKNK/C60A double-mutant showed symptomless until 95 days post-inoculation and FKNK/C60A cross-protected plants displayed high resistance to severe SCMV strain. This study provides theoretical and material bases for the control of SCMV through cross-protection.

The conserved aromatic residue W122 is a determinant of potyviral coat protein stability, replication, and cell-to-cell movement in plants.

Coat proteins (CPs) play critical roles in potyvirus cell-to-cell movement. However, the underlying mechanism controlling them remains unclear. Here, we show that substitutions of alanine, glutamic acid, or lysine for the conserved residue tryptophan at position 122 (W122 ) in tobacco vein banding mosaic virus (TVBMV) CP abolished virus cell-to-cell movement in Nicotiana benthamiana plants. In agroinfiltrated N. benthamiana leaf patches, both the CP and RNA accumulation levels of three W122 mutant viruses were significantly reduced compared with those of wild-type TVBMV, and CP accumulated to a low level similar to that of a replication-deficient mutant. The results of polyprotein transient expression experiments indicated that CP instability was responsible for the significantly low CP accumulation levels of the three W122 mutant viruses. The substitution of W122 did not affect CP plasmodesmata localization or virus particle formation; however, the substitution significantly reduced the number of virus particles. The wild-type TVBMV CP could complement the reduced replication and abolished cell-to-cell movement of the mutant viruses. When the codon for W122 was mutated to that for a different aromatic residue, phenylalanine or tyrosine, the resultant mutant viruses moved systemically and accumulated up to 80% of the wild-type TVBMV level. Similar results were obtained for the corresponding amino acids of W122 in the watermelon mosaic virus and potato virus Y CPs. Therefore, we conclude that the aromatic ring in W122 in the core domain of the potyviral CP is critical for cell-to-cell movement through the effects on CP stability and viral replication.

Anxiolytic potency of iridoid fraction extracted from Valeriana jatamansi Jones and its mechanism: a preliminary study.

Valeriana jatamansi Jones (V. jatamansi) has been widely used for treating anxiety and its mechanism involves many aspects including GABA level. This study aimed to evaluate the anxiolytic potency of an iridoid fraction extracted from the radix and rhizomes of V. jatamansi. The iridoid fraction was extracted by using D101 resin; its major components were analysed preliminarily by thin layer chromatography, ultraviolet spectrophotometry and high-performance liquid chromatography; and its anxiolytic effects at 6 mg/kg (low-dose), 9 mg/kg (medium-dose) and 12 mg/kg (high-dose) were evaluated using the elevated plus maze test, the light-dark box test, the Vogel's drinking conflict test, and the open field drink test. Its action mechanism was investigated using the ELISA. This study provided evidence on the anxiolytic potency of the iridoid fraction from V. jatamansi and revealed its action mechanism of regulating the GABA level.

Tobacco vein banding mosaic virus 6K2 Protein Hijacks NbPsbO1 for Virus Replication.

Chloroplast-bound vesicles are key components in viral replication complexes (VRCs) of potyviruses. The potyviral VRCs are induced by the second 6 kDa protein (6K2) and contain at least viral RNA and nuclear inclusion protein b. To date, no chloroplast protein has been identified to interact with 6K2 and involve in potyvirus replication. In this paper, we showed that the Photosystem II oxygen evolution complex protein of Nicotiana benthamiana (NbPsbO1) was a chloroplast protein interacting with 6K2 of Tobacco vein banding mosaic virus (TVBMV; genus Potyvirus) and present in the VRCs. The first 6 kDa protein (6K1) was recruited to VRCs by 6K2 but had no interaction with NbPSbO1. Knockdown of NbPsbO1 gene expression in N. benthamiana plants through virus-induced gene silencing significantly decreased the accumulation levels of TVBMV and another potyvirus Potato virus Y, but not Potato virus X of genus Potexvirus. Amino acid substitutions in 6K2 that disrupted its interaction with NbPsbO1 also affected the replication of TVBMV. NbPsbP1 and NbPsbQ1, two other components of the Photosystem II oxygen evolution complex had no interaction with 6K2 and no effect on TVBMV replication. To conclude, 6K2 recruits 6K1 to VRCs and hijacks chloroplast protein NbPsbO1 to regulate potyvirus replication.

Transcriptomic changes in Nicotiana benthamiana plants inoculated with the wild-type or an attenuated mutant of Tobacco vein banding mosaic virus.

Tobacco vein banding mosaic virus (TVBMV) is a potyvirus which mainly infects solanaceous crops. The helper component proteinase (HCpro) of a potyvirus is an RNA silencing suppressor protein and determines the severity of disease symptoms caused by different potyviruses, including TVBMV. It has been shown that substitution mutations introduced into the HCpro open reading frame (ORF) in a TVBMV infectious clone result in changes of Asp189 to Lys or Ile250 -Gln251 to Asp-Glu (Asp, aspartic acid; Gln, glutamine; Glu, glutamic acid; Ile, isoleucine). These amino acid changes eliminate the RNA silencing suppression activity of the mutant HCpro (HCm) and attenuate the disease symptoms caused by the mutant TVBMV (T-HCm) in Nicotiana benthamiana plants. Here, we used RNA-sequencing technology to compare gene expression in plants inoculated with the wild-type TVBMV (T-WT) with that in plants inoculated with T-HCm at 1, 2 and 10 days post-agroinfiltration (dpai). At 1 and 2 dpai, N. benthamiana genes related to the translation machinery were up-regulated, whereas genes related to lipid biosynthesis and metabolism or to responses to extracellular/external stimuli were down-regulated in leaves inoculated with T-WT or T-HCm. At 10 dpai, T-WT infection repressed photosynthesis-related genes. T-WT and T-HCm infections differentially perturbed the genes involved in the RNA silencing pathway. The salicylic acid and ethylene signalling pathways were induced, but the jasmonic acid signalling pathway was repressed after T-WT infection. Infections of T-WT and T-HCm also differentially regulated the genes involved in auxin signalling transduction, which is known to associate with the stunting phenotypes caused by TVBMV. These results illustrate the dynamic nature of TVBMV infection in N. benthamiana at the transcriptomic level.

[Source Apportionment and Health Risk Assessment of VOCs During the Haze Period in the Winter in Beijing].

A method for determining volatile organic compounds (VOCs) by cryogenic dynamic adsorption in solid adsorbent tubes, subsequent thermal desorption with cryofocusing in a cold trap and analysis by gas chromatography and mass spectrometry was adapted for continuous ambient air monitoring. VOCs pollution characteristics and health risk assessment (HRA)were researched in detail. Moreover, the sources apportionment was reliably analyzed by positive matrix factorization (PMF) model. The results showed that the average concentration of VOCs was 332.34 µg·m-3 per day, the concentrations of aromatic hydrocarbon and halo hydrocarbon were remarkably high compared to the other VOCs. Particularly, the PMF analysis results revealed that solvent/paint use emission, biomass or coal combustion and motor vehicle exhaust emissions were the main pollutants emission sources. Additionally, the cancer risk index of all carcinogenic substances was higher than the suggested value of USEPA(1×10-6), which could cause potential harm to human health.

Influence of post-treatment with 75% (v/v) ethanol vapor on the properties of SF/P(LLA-CL) nanofibrous scaffolds.

In order to improve the water-resistant ability of silk fibroin (SF) and SF/P(LLA-CL) blended nanofibrous scaffolds for tissue engineering applications, 75% (v/v) ethanol vapor was used to post-treat electrospun nanofibers. SEM indicated that the treated SF and SF/P(LLA-CL) nanofibrous scaffolds maintained a nanofibrous structure and possessed good water-resistant ability. Characterization of (13)C CP-MAS NMR clarified that 75% (v/v) ethanol vapor could induce SF conformation from random coil or α-helix to ß-sheet. Although the water contact showed that treated SF/P(LLA-CL) blended nanofibrous scaffolds were hydrophobic, the water uptake demonstrated that their hydrophilicity was greatly superior to those of pure P(LLA-CL) nanofibrous scaffolds. Furthermore, the treated SF/P(LLA-CL) nanofibrous scaffolds, both in dry state and wet state, could retain good mechanical properties. Therefore, 75% (v/v) ethanol vapor treatment might be an ideal method to treat SF and SF/P(LLA-CL) nanofibrous scaffolds for biomedical applications.

[Study on serum differentially expressed proteins in discordance of congenital esotropic phenotypes in monozygotic twins].

OBJECTIVE: To investigate the serum differentially expressed proteins in discordance of congenital esotropia phenotypes in monozygotic twins and non-strabismus ones. METHODS: SELDI-TOF-MS technology was used to detect the changes of protein expression in a couple of twins with phenotype discordance esotropia (twins A is orthotropia and twins B is esotropia). In addition, two non-twins esotropia and 2 orthotropia children with the same age were chosen. The obtained serum differential expression proteome was validated in 12 non-twins esotropia and 18 orthotropia children and initial predication by proteinum database. RESULTS: The result of SELDI-TOF-MS in 6 serums showed that four differential expression protein peaks, the molecular weight of these different proteins were 4146, 4801, 7786 and 5859, and the former 3 proteins were down-regulated and last one was up-regulated. Their features were fairly coincident with glucagons precursor, pituitary adenylate cyclase activating peptide (PACAP), cAMP dependent protein kinase inhibitor α and anti-metastasis gene (antigen). CONCLUSIONS: The expression of different proteins among the esotropic and orthotopic children was existed. The expression of different proteins was main down-regulated in the strabismus patients. Glucagons precursor, protein kinase A inhibitor α and PACAP may be related with occurrence of congenital esotropia.

[Effect of HSV-1 infection on NGF and its receptor expression in human glioma cells].

Nerve growth factor (NGF) is mainly secreted by the neuroglia cells, which can exert biological effect through its receptors on the specific target cell surface. NGF is closely related to neurocyte growth, differentiation and apoptosis. As a neurotropic virus, HSV-1 an easily lead to neurocyte, neuroglia cells death or apoptosis. In this study, the U251 human glioma cells were chosen as target cells to study the change of NGF and its receptors in the apoptosis process of HSV-1 infection. Our results showed that U251 cells were permissive to HSV-1 replication. In the apoptosis process of HSV-1 infected U251 cells, the expression of both NGF and P75NTR increased and then decreased, while the expression of TrkA decreased gradually. These result indicated that HSV-1 was able to induce the abnormal expression of NGF and its receptors in U251 cells.

Effect of human cytomegalovirus infection on nerve growth factor expression in human glioma U251 cells.

OBJECTIVE: To explore the change of endogenic nerve growth factor (NGF) expression in human glioma cells infected with human cytomegalovirus (HCMV). METHODS: U251 cells were cultured in RPMI 1640 culture medium and infected with HCMV AD169 strain in vitro to establish a cell model of viral infection. Morphologic changes of U251 cells were observed under inverted microscope before and after infection with HCMV. Expression of NGF gene and protein of cells was detected by RT-PCR and Western blotting before and after infection with HCMV. RESULTS: The cytopathic effects of HCMV-infected cells appeared on day 5 after infection. However, differential NGF expression was evident on day 7. NGF expression was decreased significantly in U251 cells on day 7 after infection in comparison with control group (P < 0.05). CONCLUSION: HCMV can down-regulate endogenous NGF levels in human glioma cell line U251.

[The expression and anti-apoptotic function of HCMV IE2 protein controlled by Tet-On system].

During the infection of host cells, IE2 protein is one of the first and most abundantly expressed products of HCMV genome, which plays an important role in the controlling of cell cycle and apoptosis. But the correlation between expression level and anti-apoptotic activity of IE2 protein is still not clear. In this study, we successfully established a HCMV IE2 protein expression cell line that was controlled by Tet-On system. The effect of IE2 protein on cell apoptosis and the expression of p53 was detected under different condition of induction. Our results showed that the IE2 protein could inhibit cell apoptosis induced by TNF-alpha. Additionally, the anti-apoptotic activity of IE2 protein seemed to be relevant to its expression level. However, we failed to detect any difference of p53 expression between the IE2 protein expression and non-expression cells. These data indicated that the IE2 protein might inhibit cell apoptosis through regulating different signal pathways.