Cucumber Mosaic Virus Infection in Arabidopsis: A Conditional Mutualistic Symbiont?

A cucumber mosaic virus isolate, named Ho [CMV(Ho)], was isolated from a symptomless Arabidopsis halleri field sample containing low virus titers. An analysis of CMV(Ho) RNA molecules indicated that the virus isolate, besides the usual cucumovirus tripartite RNA genome, additionally contained defective RNA3 molecules and a satellite RNA. To study the underlying mechanism of the persistent CMV(Ho) infection in perennial A. halleri, infectious cDNA clones were generated for all its genetic elements. CMV, which consists of synthetic transcripts from the infectious tripartite RNA genomes, and designated CMV(Ho)tr, multiplied in A. halleri and annual Arabidopsis thaliana Col-0 to a similar level as the virulent strain CMV(Y), but did not induce any symptoms in them. The response of Col-0 to a series of reassortant CMVs between CMV(Ho)tr and CMV(Y) suggested that the establishment of an asymptomatic phenotype of CMV(Ho) infection was due to the 2b gene of CMV RNA2, but not due to the presence of the defective RNA3 and satellite RNA. The accumulation of CMV(Ho) 2b protein tagged with the FLAG epitope (2b.Ho-FLAG) in 2b.Ho-FLAG-transformed Col-0 did not induce any symptoms, suggesting a 2b-dependent persistency of CMV(Ho)tr infection in Arabidopsis. The 2b protein interacted with Argonaute 4, which is known to regulate the cytosine methylation levels of host genomic DNA. Whole genomic bisulfite sequencing analysis of CMV(Ho)tr- and mock-inoculated Col-0 revealed that cytosine hypomethylation in the promoter regions of 82 genes, including two genes encoding transcriptional regulators (DOF1.7 and CBP1), was induced in response to CMV(Ho)tr infection. Moreover, the increased levels of hypomethylation in the promoter region of both genes, during CMV(Ho)tr infection, were correlated with the up- or down-regulation of their expression. Taken altogether, the results indicate that during persistent CMV(Ho) infection in Arabidopsis, host gene expression may be epigenetically modulated resulting from a 2b-mediated cytosine hypomethylation of host genomic DNA.

The Weak Small RNA-Binding Activity of the 2b Proteins of Subgroup II Cucumber Mosaic Virus Strains Is Insufficient for RNA Silencing Suppression

The 2b proteins encoded by cucumber mosaic virus (CMV) subgroup I strains suppress RNA silencing primarily by competitively binding small RNAs (sRNAs) in the host cell cytoplasm. Interestingly, 2b proteins encoded by CMV subgroup II strains accumulate predominantly in nuclei. Here we determined that whereas the 2b protein (Fny2b) of subgroup IA strain Fny-CMV is highly effective in suppressing both sense RNA-induced and inverted repeat-induced posttranscriptional gene silencing, the 2b protein (LS2b) of the subgroup II strain LS-CMV was not as effective. Reducing nuclear accumulation of LS2b by mutating a residue in its nuclear localization sequence had no effect on RNA silencing suppressor activity, while attenuated viral symptoms. Electrophoretic mobility shift assays showed that the sRNA binding of LS2b was weaker and more selective than that of Fny2b. The domain determining the differential sRNA-binding ability was delimited to the putative helix α1 region. Moreover, LS2b mutants that completely lost suppressor activity still retained their weak sRNA-binding ability, suggesting that sRNA binding is not sufficient for LS2b to suppress RNA silencing. Considering the subgroup I strain-encoded 2b proteins that require sRNA-binding ability for the suppression of RNA silencing, we suggest that in addition to binding sRNA, the 2b proteins of subgroup II CMV strains would require extra biological activities to achieve RNA silencing inhibition.

Role of Disulfide Bonds in Membrane Partitioning of a Viral Peptide

The importance of disulfide bond in mediating viral peptide entry into host cells is well known. In the present work, we elucidate the role of disulfide (SS) bond in partitioning mechanism of membrane active Hepatitis A Virus-2B (HAV-2B) peptide, which harbours three cysteine residues promoting formation of multiple SS-bonded states. The inclusion of SS-bond not only results in a compact conformation but also induces distorted α-helical hairpin geometry in comparison to SS-free state, resulting in reduced hydrophobic exposure. Owing to this, the partitioning of HAV-2B peptide is completely or partly abolished. In a way, the disulfide bond regulates the partitioning of HAV-2B peptide, such that the membrane remodelling effects of this viral peptide are significantly reduced. The current findings may have potential implications in drug designing, targeting the HAV-2B protein by promoting disulfide bond formation within its membrane active region.

Novel drug for COVID-19: Virafin a therapeutic breakthrough

Background: Pegylated Interferon Alpha-2b is synthesized via the use of PEG (polyethylene glycol). It is formulated via attaching a 12kDa mono methoxy polyethylene glycol moiety to the recombinant human Interferon Alpha-2b protein. Pegylated Interferon Alpha-2b acts via binding to the (JAK/STAT) Janus kinase signal transducer and activator of the transcription interferon receptor. The Pegylated Interferon Alpha-2b /Virafin binds to the (IFNAR1/2) alpha interferon receptor 1 and 2. Pegylated Interferon Alpha-2b better known as Virafin has been approved by the Drugs Controller General of India. This therapeutic agent is currently under a multicentric phase 3 trial with very promising results being reported. A single subcutaneous dose of Virafin has been shown to decrease the need for oxygen therapy in patients. This reduction in the need for oxygen therapy is a vital factor needed to provide relief to the Indian medical system in light of the recent oxygen shortages faced due to India’s worst wave of COVID-19 cases since the onset of the global pandemic. Pegylated Interferon Alpha-2b/ Virafin confers enhanced viral clearance and bolsters the immune response to induce a quicker recovery in patients with mild to moderate symptoms. Conclusion: It is of paramount importance that further research on Virafin is undertaken as it can hinder the progression of COVID-19, reduce pressure on the inundated health systems, and save countless lives.

Elevated Carbon Dioxide Levels Decreases Cucumber Mosaic Virus Accumulation in Correlation with Greater Accumulation of rgs-CaM, an Inhibitor of a Viral Suppressor of RNAi

Plant viruses cause a range of plant diseases symptoms that are often responsible for significant crop production losses and the severity and spread of the symptoms may be affected by climate change. While the increase in anthropogenic activities has caused a critical problem of increased CO2 levels in the atmosphere, these elevated CO2 levels have been reported to reduce virus disease severity in some plant species. In such instances, it is not clear if the plant defense mechanisms are being enhanced or virus-mediated mechanisms to overcome plant resistance are being defeated. Additionally, a few studies have been attempted in this area to determine if reduced disease is the norm or the exception under enhanced CO2 levels. In the present study, the effects of elevated CO2 levels (750 ppm vs. 390 ppm) on RNAi-mediated resistance of Nicotiana tabacum against the cucumber mosaic virus (CMV), and the activity of viral suppressor of RNAi (VSR) 2b protein of CMV were evaluated. On the one hand, our results showed that elevated CO2 decreased the transcription of dicer-like protein 2 (DCL2), DCL4, and argonaut 1 (AGO1) genes with functions related to RNAi-mediated resistance when infected by CMV, which is contradictory with the decreased CMV copy numbers under elevated CO2. On the other hand, we found that elevated CO2 increased the calcium concentration and expression of the calcium-binding protein rgs-CaM in tobacco plants when infected by CMV, which directly weakened the function of 2b protein, the VSR of CMV, and therefore decreased the infection efficiency of the virus and suppressed the severity of CMV in tobacco plants under elevated CO2. This study provides molecular insights into the ecological implications underlying the development of prevention strategies against plant virus infection in the context of climate change.

The cucumber mosaic virus 1a protein regulates interactions between the 2b protein and ARGONAUTE 1 while maintaining the silencing suppressor activity of the 2b protein

The cucumber mosaic virus (CMV) 2b viral suppressor of RNA silencing (VSR) is a potent counter-defense and pathogenicity factor that inhibits antiviral silencing by titration of short double-stranded RNAs. It also disrupts microRNA-mediated regulation of host gene expression by binding ARGONAUTE 1 (AGO1). But in Arabidopsis thaliana complete inhibition of AGO1 is counterproductive to CMV since this triggers another layer of antiviral silencing mediated by AGO2, de-represses strong resistance against aphids (the insect vectors of CMV), and exacerbates symptoms. Using confocal laser scanning microscopy, bimolecular fluorescence complementation, and co-immunoprecipitation assays we found that the CMV 1a protein, a component of the viral replicase complex, regulates the 2b-AGO1 interaction. By binding 2b protein molecules and sequestering them in P-bodies, the 1a protein limits the proportion of 2b protein molecules available to bind AGO1, which ameliorates 2b-induced disease symptoms, and moderates induction of resistance to CMV and to its aphid vector. However, the 1a protein-2b protein interaction does not inhibit the ability of the 2b protein to inhibit silencing of reporter gene expression in agroinfiltration assays. The interaction between the CMV 1a and 2b proteins represents a novel regulatory system in which specific functions of a VSR are selectively modulated by another viral protein. The finding also provides a mechanism that explains how CMV, and possibly other viruses, modulates symptom induction and manipulates host-vector interactions.