Viral P24 Research Articles

P1 of turnip mosaic virus interacts with NOD19 for vigorous infection.

P1 protein, the most divergent protein of virus members in the genus Potyvirus of the family Potyviridae, is required for robust infection and host adaptation. However, how P1 affects viral proliferation is still largely elusive. In this work, a total number of eight potential P1-interacting Arabidopsis proteins were identified by the yeast-two-hybrid screening using the turnip mosaic virus (TuMV)-encoded P1 protein as the bait. Among which, the stress upregulated NODULIN 19 (NOD19) was selected for further characterization. The bimolecular fluorescent complementation assay confirmed the interaction between TuMV P1 and NOD19. Expression profile, structure, and subcellular localization analyses showed that NOD19 is a membrane-associated protein expressed mainly in plant aerial parts. Viral infectivity assay showed that the infection of turnip mosaic virus and soybean mosaic virus was attenuated in the null mutant of Arabidopsis NOD19 and NOD19-knockdown soybean seedlings, respectively. Together, these data indicate that NOD19 is a P1-interacting host factor required for robust infection.

MRP8/14 Is a Molecular Signature Triggered by Dopamine in HIV Latent Myeloid Targets That Increases HIV Transcription and Distinguishes HIV+ Methamphetamine Users with Detectable CSF Viral Load and Brain Pathology.

There is a significant overlap between HIV infection and substance-use disorders. Dopamine (DA) is the most abundantly upregulated neurotransmitter in methamphetamine abuse, with receptors (DRD1-5) that are expressed by neurons as well as by a large diversity of cell types, including innate immune cells that are the targets of HIV infection, making them responsive to the hyperdopaminergic environment that is characteristic of stimulant drugs. Therefore, the presence of high levels of dopamine may affect the pathogenesis of HIV, particularly in the brain. The stimulation of HIV latently infected U1 promonocytes with DA significantly increased viral p24 levels in the supernatant at 24 h, suggesting effects on activation and replication. Using selective agonists to different DRDs, we found that DRD1 played a major role in activating viral transcription, followed by DRD4, which increased p24 with a slower kinetic rate compared to DRD1. Transcriptome and systems biology analyses led to the identification of a cluster of genes responsive to DA, where S100A8 and S100A9 were most significantly correlated with the early increase in p24 levels following DA stimulation. Conversely, DA increased the expression of these genes' transcripts at the protein level, MRP8 and MRP14, respectively, which form a complex also known as calprotectin. Interestingly, MRP8/14 was able to stimulate HIV transcription in latent U1 cells, and this occurred via binding of the complex to the receptor for an advanced glycosylation end-product (RAGE). Using selective agonists, both DRD1 and DRD4 increased MRP8/14 on the surface, in the cytoplasm, as well as secreted in the supernatants. On the other hand, while DRD1/5 did not affect the expression of RAGE, DRD4 stimulation caused its downregulation, offering a mechanism for the delayed effect via DRD4 on the p24 increase. To cross-validate MRP8/14 as a DA signature with a biomarker value, we tested its expression in HIV+ Meth users' postmortem brain specimens and peripheral cells. MRP8/14+ cells were more frequently identified in mesolimbic areas such as the basal ganglia of HIV+ Meth+ cases compared to HIV+ non-Meth users or to controls. Likewise, MRP8/14+ CD11b+ monocytes were more frequent in HIV+ Meth users, particularly in specimens from participants with a detectable viral load in the CSF. Overall, our results suggest that the MRP8 and MRP14 complex may serve as a signature to distinguish subjects using addictive substances in the context of HIV, and that this may play a role in aggravating HIV pathology by promoting viral replication in people with HIV who use Meth.

Affordable paper-based SWNTs field-effect transistor biosensors for nucleic acid amplification-free and label-free detection of micro RNAs

Micro RNAs (miRNAs) are 19–23 nucleotides-long non-coding RNA which have been identified as important biomarkers for many diseases, including cancers. Here, we report an ultrasensitive, highly specific, label-free, and without nucleic acid amplification (NAA) detection of miRNAs using paper-based single-walled carbon nanotubes (SWNTs) field-effect transistor (FET) biosensors. The strategy involved a two-step protocol starting with direct hybridization of the target miRNA with a specific RNA probe immobilized on SWNTs networks deposited on the paper substrate to generate the first response, followed by the recognition of the resulting RNA/miRNA duplexes with the Carnation Italian ringspot virus p19 protein (p19) in a size-dependent manner, giving the second electrical response that magnified the biosensor sensitivity. As a demonstration, we detected the miRNA-122a, a promising biomarker for the diagnosis of early-stage hepatocellular carcinoma (HCC) in various sample matrices, including phosphate buffers, human serum, and synthetic saliva. We achieved the lowest detection of 0.1 aM and a wide dynamic range of 0.1 aM–1 fM, that demonstrated its potential for rapid, facile, low-cost, and point-of-care detection of miRNAs for early cancer diagnosis.

Rice stripe virus p2 protein interacts with ATG5 and is targeted for degradation by autophagy.

Autophagy can be induced by viral infection and plays antiviral roles in plants, but the underlying mechanism is not well understood. In our previous reports, we have demonstrated that the plant ATG5 plays an essential role in activating autophagy in rice stripe virus (RSV)-infected plants. We also showed that eIF4A, a negative factor of autophagy, interacts with and inhibits ATG5. We here found that RSV p2 protein interacts with ATG5 and can be targeted by autophagy for degradation. Expression of p2 protein induced autophagy and p2 protein was shown to interfere with the interaction between ATG5 and eIF4A, while eIF4A had no effect on the interaction between ATG5 and p2. These results indicate an additional information on the induction of autophagy in RSV-infected plants.

Interactions of Tomato Chlorosis Virus p27 Protein with Tomato Catalase Are Involved in Viral Infection.

Tomato chlorosis virus (ToCV) severely threatens tomato production worldwide. P27 is known to be involved in virion assembly, but its other roles in ToCV infection are unclear. In this study, we found that removal of p27 reduced systemic infection, while ectopic expression of p27 promoted systemic infection of potato virus X in Nicotiana benthamiana. We determined that Solanum lycopersicum catalases (SlCAT) can interact with p27 in vitro and in vivo and that amino acids 73 to 77 of the N-terminus of SlCAT represent the critical region for their interaction. p27 is distributed in the cytoplasm and nucleus, and its coexpression with SlCAT1 or SlCAT2 changes its distribution in the nucleus. Furthermore, we found that silencing of SlCAT1 and SlCAT2 can promote ToCV infection. In conclusion, p27 can promote viral infection by binding directly to inhibit anti-ToCV processes mediated by SlCAT1 or SlCAT2.

Structural insights into the multifunctionality of rabies virus P3 protein

Viruses form extensive interfaces with host proteins to modulate the biology of the infected cell, frequently via multifunctional viral proteins. These proteins are conventionally considered as assemblies of independent functional modules, where the presence or absence of modules determines the overall composite phenotype. However, this model cannot account for functions observed in specific viral proteins. For example, rabies virus (RABV) P3 protein is a truncated form of the pathogenicity factor P protein, but displays a unique phenotype with functions not seen in longer isoforms, indicating that changes beyond the simple complement of functional modules define the functions of P3. Here, we report structural and cellular analyses of P3 derived from the pathogenic RABV strain Nishigahara (Nish) and an attenuated derivative strain (Ni-CE). We identify a network of intraprotomer interactions involving the globular C-terminal domain and intrinsically disordered regions (IDRs) of the N-terminal region that characterize the fully functional Nish P3 to fluctuate between open and closed states, whereas the defective Ni-CE P3 is predominantly open. This conformational difference appears to be due to the single mutation N226H in Ni-CE P3. We find that Nish P3, but not Ni-CE or N226H P3, undergoes liquid-liquid phase separation and this property correlates with the capacity of P3 to interact with different cellular membrane-less organelles, including those associated with immune evasion and pathogenesis. Our analyses propose that discrete functions of a critical multifunctional viral protein depend on the conformational arrangements of distant individual domains and IDRs, in addition to their independent functions.

Tomato chlorosis virus p22 interacts with NbBAG5 to inhibit autophagy and regulate virus infection.

Tomato chlorosis virus (ToCV) is a member of the genus Crinivirus in the family Closteroviridae. It has a wide host range and wide distribution, causing serious harm to the vegetable industry. The autophagy pathway plays an important role in plant resistance to virus infection. Viruses and plant hosts coevolve in defence and antidefence processes around autophagy. In this study, the interaction between ToCV p22 and Nicotiana benthamiana B-cell lymphoma2-associated athanogenes5 Nicotiana benthamiana (NbBAG5) was examined. Through overexpression and down-regulation of NbBAG5, results showed that NbBAG5 could negatively regulate ToCV infection. NbBAG5 was found to be localized in mitochondria and can change the original localization of ToCV p22, which is colocalized in mitochondria. NbBAG5 inhibited the expression of mitophagy-related genes and the number of autophagosomes, thereby regulating viral infection by affecting mitophagy. In summary, this study demonstrated that ToCV p22 affects autophagy by interacting with NbBAG5, established the association between viral infection, BAG proteins family, and the autophagy pathway, and explained the molecular mechanism by which ToCV p22 interacts with NbBAG5 to inhibit autophagy to regulate viral infection.

Modeling the accuracy of a novel PCR and antibody ELISA for African swine fever virus detection using Bayesian latent class analysis.

Diagnostic test evaluation for African swine fever (ASF) in field settings like Vietnam is critical to understanding test application in intended populations for surveillance and control strategies. Bayesian latent class analysis (BLCA) uses the results of multiple imperfect tests applied to an individual of unknown disease status to estimate the diagnostic sensitivity and specificity of each test, forgoing the need for a reference test. Here, we estimated and compared the diagnostic sensitivity and specificity of a novel indirect ELISA (iELISA) for ASF virus p30 antibody (Innoceleris LLC.) and the VetAlert™ ASF virus DNA Test Kit (qPCR, Tetracore Inc.) in field samples from Vietnam by assuming that disease status 1) is known and 2) is unknown using a BLCA model. In this cross-sectional study, 398 paired, individual swine serum/oral fluid (OF) samples were collected from 30 acutely ASF-affected farms, 37 chronically ASF-affected farms, and 20 ASF-unaffected farms in Vietnam. Samples were tested using both diagnostic assays. Diagnostic sensitivity was calculated assuming samples from ASF-affected farms were true positives and diagnostic sensitivity by assuming samples from unaffected farms were true negatives. ROC curves were plotted and AUC calculated for each test/sample combination. For comparison, a conditionally dependent, four test/sample combination, three population BLCA model was fit. When considering all assumed ASF-affected samples, qPCR sensitivity was higher for serum (65.2%, 95% Confidence Interval [CI] 58.1-71.8) and OF (52%, 95%CI 44.8-59.2) compared to the iELISA (serum: 42.9%, 95%CI 35.9-50.1; OF: 33.3%, 95%CI 26.8-40.4). qPCR-serum had the highest AUC (0.895, 95%CI 0.863-0.928). BLCA estimates were nearly identical to those obtained when assuming disease status and were robust to changes in priors. qPCR sensitivity was considerably higher than ELISA in the acutely-affected population, while ELISA sensitivity was higher in the chronically-affected population. Specificity was nearly perfect for all test/sample types. The effect of disease chronicity on sensitivity and specificity could not be well characterized here due to limited data, but future studies should aim to elucidate these trends to understand the best use of virus and antibody detection methods for ASF. Results presented here will help the design of surveillance and control strategies in Vietnam and other countries affected by ASF.

Expression of a recombinant ASFV P30 protein and production of monoclonal antibodies.

African Swine Fever (ASF) is an infectious disease that affects domestic pig and wild boar populations. The ASF Virus (ASFV) has a genome characterized by a very complex DNA (170-193 kb) that encodes for more than 200 different proteins. Among these, the highly immunogenic phosphoprotein p30 plays a fundamental role in the induction of specific antibodies. To date, the lack of a vaccine against the disease requires continuous studies to improve knowledge about the virus and the development of new tests in addition to virological ones. The aim of this work was to produce specific monoclonal antibodies (mAbs) against the p30 protein of ASFV, which could find useful applications in routine diagnostics and the implementation of new diagnostic tools. ASFV p30 encoding gene was amplified and used for the generation of the recombinant baculovirus by transfection of the Sf21 insect cells. The recombinant protein was analyzed by immunofluorescence assay, purified, and used for mice Balb-c immunization. The hybridomas obtained were cultured and screened, using an indirect Enzyme-linked Immunosorbent Assay (iELISA), in order to select clones that secrete the mAbs of interest. The expression of recombinant p30 protein was assessed using direct immunofluorescence. The purified p30 protein fractions were analyzed by Coomassie gels staining confirming the presence of bands with a molecular weight of 30 kDa and used for the immunization of Balb-c mice. Six clones of pure hybridomas secreting the specific mAbs against recombinant p30 were obtained and tested in iELISA. The mAbs were also characterized by Western blot and immunofluorescence assay. The best results were obtained with the anti-p30 mAb 2B8E10 clone which showed high reactivity with both recombinant and viral p30 protein, respectively. In this work, a recombinant p30 protein produced in an insect cell system was purified and used to immunize Balb-c mice. Six anti-p30 mAbs-secreting hybridomas clone cells were obtained. These mAbs displayed high reactivity against the recombinant protein, but only 2B8E10 mAb showed excellent functionality against the p30 protein produced by ASFV. These results open the possibility to develop different diagnostic assays.

Indirect ELISA Using Multi-Antigenic Dominants of p30, p54 and p72 Recombinant Proteins to Detect Antibodies against African Swine Fever Virus in Pigs.

African swine fever (ASF) caused by ASF virus (ASFV) is a fatal disease in pigs and results in great economic losses. Due to the lack of available vaccines and treatments, serological diagnosis of ASF plays a key role in the surveillance program, but due to the lack of knowledge and the complexity of the ASFV genome, the candidate target viral proteins are still being researched. False negativity is still a big obstacle during the diagnostic process. In this study, the high antigenic viral proteins p30, p54 and p72 were screened to find the antigenic dominant domains and the tandem His-p30-54-72 was derived. An indirect enzyme-linked immunosorbent assay (iELISA) coated with His-p30-54-72 was developed with a cut-off value of 0.371. A total of 192 clinical samples were detected by His-p30-54-72-coated indirect ELISA (iELISA) and commercial ASFV antibody kits. The results showed that the positive rate of His-p30-54-72-coated iELISA was increased by 4.7% and 14.6% compared with a single viral protein-based commercial ASFV antibody kits. These results provide a platform for future ASFV clinical diagnosis and vaccine immune effect evaluation.

African Swine Fever Virus Exhibits Distinct Replication Defects in Different Cell Types.

African swine fever virus (ASFV) causes one of the most devastating diseases affecting pigs and wild suids, a worldwide epizootic situation exacerbated in recent years due to the lack of vaccine or effective treatment. ASFV has a restricted cell tropism, and is prone to replicate in porcine monocytes and alveolar macrophages with high efficiency. Here, the replication capabilities of ASFV were examined in swine pulmonary alveolar macrophages (PAMs) and compared with 3D4/21, PK-15, MA-104 and Marc-145 cell lines using PCR, qPCR and Western blot with monoclonal antibodies against the viral p30 and p72 proteins. The results showed that ASFV has a variety of infection characteristics in PAMs and showed four cell lines with distinct defects during virus early transcription-translation, genome replication and late protein synthesis. Furthermore, an antiviral role of the stress granule pathway was revealed against ASFV, and ASFV infection inhibited stress granule formation in PAMs but not 3D4/21. These results will help to deepen our knowledge on ASFV infection and to develop ASFV susceptible cell lines.

A click chemistry amplified nanopore assay for ultrasensitive quantification of HIV-1 p24 antigen in clinical samples

Despite major advances in HIV testing, ultrasensitive detection of early infection remains challenging, especially for the viral capsid protein p24, which is an early virological biomarker of HIV-1 infection. Here, To improve p24 detection in patients missed by immunological tests that dominate the diagnostics market, we show a click chemistry amplified nanopore (CAN) assay for ultrasensitive quantitative detection. This strategy achieves a 20.8 fM (0.5 pg/ml) limit of detection for HIV-1 p24 antigen in human serum, demonstrating 20~100-fold higher analytical sensitivity than nanocluster-based immunoassays and clinically used enzyme-linked immunosorbent assay, respectively. Clinical validation of the CAN assay in a pilot cohort shows p24 quantification at ultra-low concentration range and correlation with CD4 count and viral load. We believe that this strategy can improve the utility of p24 antigen in detecting early infection and monitoring HIV progression and treatment efficacy, and also can be readily modified to detect other infectious diseases.

Evaluation of barcoded magnetic bead-based immunoassays for the simultaneous detection of feline leukemia virus antigen and antibody against feline immunodeficiency virus.

Testing platforms that leverage automation, require minimal sample volume, and enable various tests to be performed simultaneously on a single sample have the potential to improve workflow and efficiency in veterinary diagnostic laboratories. We evaluated a barcoded magnetic bead (BMB) technology using established immunoassays for detection of feline leukemia virus (FeLV) p27 antigen and antibody against feline immunodeficiency virus (FIV). Analytical sensitivity, limit of blank, and limit of detection were used to establish a functional sensitivity of 1.00 ng/mL of inactivated FeLV antigen and 35.7 ng/mL of anti-FIV monoclonal antibody. Common interferents, such as hemoglobin, lipid, and bilirubin, were not found to interfere with the performance of the assay. Intra- and inter-assay CVs were <13% for both assays using manufactured samples. Using a set of 116 feline samples, the diagnostic accuracy of our multiplex assay was 100% compared to reference assays. Performance in a convenience set of 1,000 feline samples submitted to a commercial diagnostic laboratory revealed a proportion of positive results of 1.3% for FeLV and 3.7% for FIV. BMB technology should enable rapid screening of samples for various markers in a single immunoassay well.

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.

Synergistic disinfection of aerosolized bacteria and bacteriophage by far-UVC (222-nm) and negative air ions

Air ionizers and 222-nm krypton-chlorine (KrCl) excilamp have proven to be effective disinfection apparatus for bacteria and viruses with limited health risks. We determined inactivation efficiencies by operating them individually and in combined modules. Gram-positive and gram-negative bacteria, non-enveloped dsDNA virus, and enveloped dsRNA virus were examined in a designed air disinfection system. Our results showed that the bioaerosols were inactivated efficiently by negative ionizers and far-UVC (222-nm), either used individually or in combination. Among which the combined modules of negative ionizers and KrCl excilamp had the best disinfection performance for the bacteria. The aerosolized virus P22 and Phi 6 were more susceptible to 222-nm emitted by KrCl excilamp than negative air ions. Significant greater inactivation of bacterial bioaerosols were identified after treated by combined treatment of negative air ion and far-UVC for 2 minutes (Escherichia coli, 6.25 natural log (ln) reduction; Staphylococcus epidermidis, 3.66 ln reduction), as compared to the mean sum value of inactivation results by respective individual treatment of negative ionizers and KrCl excilamp (Escherichia coli, 4.34 ln; Staphylococcus epidermidis, 1.75 ln), indicating a synergistic inactivation effect. The findings provide important baseline data to support the design and development of safe and high-efficient disinfection systems.

Porcine circovirus-like virus P1 activates pancreatic secretion pathway by interacting with CHRM3 protein

The porcine circovirus-like virus P1, a member of the circovirus family, causes post-weaning multisystemic wasting syndrome (PMWS) in weaned piglets with progressive wasting as the main clinical symptom. The pancreatic secretion pathway induces pancreatic acinar cells to secrete various digestive enzymes and as such is an important signaling pathway for the digestive system and somatic growth. This study examined the effects and mechanism of P1 virus infection on the pancreatic secretion pathway. The experiment was conducted by transfecting double-copy plasmid P1 into PK-15 and 3D4 cells and by infecting cells with the P1 virus. Samples were collected at various times after transfection or infection. The pathway's transcription and translation levels of CHRM3, Gq, PLC-β2, PRKCA, Rab3D, RhoA, Rac1, and amyA proteins were detected by real-time PCR and Western blots; these analyses confirmed that the P1 virus infection could upregulate the expression level of key pancreatic secretion signaling molecules. Then, we confirmed that the VP1 protein of the P1 virus could interact with the pathway initiation protein CHRM3 using Co-IP, pull-downs, and confocal fluorescence microscopy. Finally, we demonstrated that the VP1 protein activates the pancreatic secretory pathway through the CHRM3 protein. In conclusion, this study demonstrated that the P1 virus can interact with the CHRM3 receptor protein to activate the pancreatic secretion pathway and promote the secretion of various digestive enzymes downstream of the pathway, thereby providing a basis for P1 virus pathogenesis.

Development of a chromatographic lateral flow immunoassay for detection of African swine fever virus antigen in blood

African swine fever (ASF) is a highly lethal disease of domestic and wild swine caused by African swine fever virus (ASFV). The disease currently circulates in Africa, Europe, Asia and on the island of Hispaniola. The ongoing epizootics in Europe and Asia have produced millions of animal deaths and severe economic losses. No effective vaccine is available for ASF, making rapid and accurate detection of ASFV essential for disease mitigation strategies. Currently available diagnostics for ASFV possess significant limitations related to assay performance, deployability, and/or turn-around time; therefore there is an unmet need for pen-side diagnostic tests with sufficient sensitivity and specificity. A chromatographic lateral flow immunoassay (LFIA) was developed for the detection of ASFV antigen in EDTA-treated whole blood using monoclonal antibodies targeting the viral p30 protein. The assay requires only water to perform and provides results in 25 min, making it well-suited for field use. The LFIA was capable of detecting genotype I and genotype II strains of ASFV in EDTA blood from experimentally infected pigs at varying time-points after infection, though it was unable to detect a genotype X ASFV strain. Diagnostic sensitivity correlated with clinical disease severity, body temperature, and viral DNA levels, and was over 90% in animals showing moderate to severe ASF-related symptoms after challenge with virulent genotype II virus. The LFIA also showed a robust diagnostic specificity of over 98%, which is essential to field testing for a high consequence to foregin animal disease. The LFIA targeting the viral p30 protein can reliably detect ASFV in whole blood from animals showing moderate to severe clinical signs of infection with virulent genotype I and II isolates, making it a promising candidate for use as a field-deployable antigen detection assay. Additional evaluation using field samples and different virus strains is required to further assess the utility of this rapid diagnostic test.

AlkB RNA demethylase homologues and N6 -methyladenosine are involved in Potyvirus infection.

Proteins of the alkylation B (AlkB) superfamily show RNA demethylase activity removing methyl adducts from N 6‐methyladenosine (m6A). m6A is a reversible epigenetic mark of RNA that regulates human virus replication but has unclear roles in plant virus infection. We focused on Potyvirus—the largest genus of plant RNA viruses—and report here the identification of AlkB domains within P1 of endive necrotic mosaic virus (ENMV) and an additional virus of a putative novel species within Potyvirus. We show that Nicotiana benthamiana m6A levels are reduced by infection of plum pox virus (PPV) and potato virus Y (PVY). The two potyviruses lack AlkB and the results suggest a general involvement of RNA methylation in potyvirus infection and evolution. Methylated RNA immunoprecipitation sequencing of virus‐infected samples showed that m6A peaks are enriched in plant transcript 3′ untranslated regions and in discrete internal and 3′ terminal regions of PPV and PVY genomes. Down‐regulation of N. benthamiana AlkB homologues of the plant‐specific ALKBH9 clade caused a significant decrease in PPV and PVY accumulation. In summary, our study provides evolutionary and experimental evidence that supports the m6A implication and the proviral roles of AlkB homologues in Potyvirus infection.

Insights Into the Effect of Rice Stripe Virus P2 on Rice Defense by Comparative Proteomic Analysis.

Rice stripe virus (RSV) has a serious effect on rice production. Our previous research had shown that RSV P2 plays important roles in RSV infection, so in order to further understand the effect of P2 on rice, we used Tandem Mass Tag (TMT) quantitative proteomics experimental system to analyze the changes of protein in transgenic rice expressing P2 for the first time. The results of proteomics showed that a total of 4,767 proteins were identified, including 198 up-regulated proteins and 120 down-regulated proteins. Functional classification results showed that differentially expressed proteins (DEPs) were mainly localized in chloroplasts and mainly involved in the metabolic pathways. Functional enrichment results showed that DEPs are mainly involved in RNA processing and splicing. We also verified the expression of several DEPs at the mRNA level and the interaction of a transcription factor (B7EPB8) with RSV P2. This research is the first time to use proteomics technology to explore the mechanism of RSV infection in rice with the RSV P2 as breakthrough point. Our findings provide valuable information for the study of RSV P2 and RSV infection mechanism.

Strawberry Vein Banding Virus Movement Protein P1 Interacts With Light-Harvesting Complex II Type 1 Like of Fragaria vesca to Promote Viral Infection.

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 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.