Viral genome-capsid core senses host environments to prime RNA release

Viruses are metastable macromolecular assemblies containing a nucleic acid core packaged by capsid proteins that are primed to disassemble in host-specific environments leading to genome release and replication. The mechanism of how viruses sense environmental changes associated with host entry to prime them for disassembly is unknown. We have applied a combination of mass spectrometry, cryo-EM, and simulation-assisted structure refinement to Turnip crinkle virus (TCV), which serves as a model non-enveloped icosahedral virus (Triangulation number = 3, 180 copies/icosahedron). Our results reveal genomic RNA tightly binds a subset of viral coat proteins to form a stable RNA-capsid core which undergoes conformational switching in response to host-specific environmental changes. These changes include: i) Depletion of Ca 2+ which triggers viral particle expansion ii) Increase in osmolytes further disrupt interactions of outer coat proteins from the RNA-capsid core to promote complete viral disassembly. A cryo-EM structure of the expanded particle shows that RNA is asymmetrically extruded from a single 5-fold axis during disassembly. The genomic RNA:capsid protein interactions confer metastability to the TCV capsid and drive release of RNA from the disassembling virion within the plant host cell.

Nucleus

In our application of AuNPs on the leaf surface, we were pushing the Barley Yellow Dwarf Virus (BYDV-PAV) source and Gold nanoparticles (AuNPs) into the plant cell system up on the events of systemic plant defense response. In the infected host cell, the viral coat protein is the first obvious in the cytoplasm. When nanoparticles are applied on leaf surfaces, a large surface area relative to their volume happens. AuNPs solutions are more active and dispersed ooplasm. The correlation between Zeta potential value and Zeta sizer is inverse proportion. Filaments are visible in the nucleopores, the nuclear outline is distorted, and massive clumping of heterochromatin begins as declared. It was mostly found in or around regions of ribosome-associated filaments. Our present study combines TEM and nucleus content in the presence of AuNPS to explore the level of repair mechanism illustrating in TEM micrographs, showing Polyploidy nucleus and segregated chromatin. Multi membranous structure, imaging the AuNPs inside and around the nucleus and Pseudo crystal array is enveloped in an endoplasmic reticulum cisterna (ER).

Two evolutionary distinct effectors from a nematode and virus target RanGAP1 and 2 via the WPP domain to promote disease

The Gpa2 and Rx1 intracellular immune receptors are canonical CC-NB-LRR proteins belonging to the same R gene cluster in potato. Despite sharing high sequence homology, they have evolved to provide defence against unrelated pathogens. Gpa2 detects Gp-RBP-1 effectors secreted by the potato cyst nematode Globodera pallida whereas Rx1 recognizes the viral coat protein (CP) of Potato Virus X (PVX). How Gpa2 and Rx1 perceive their matching effectors remains unknown. Using a combination of in planta Co-Immunoprecipitation and cellular imaging, we show that both Gp-RBP-1 and PVX-CP physically interact with RanGAP2 and RanGAP1 in the cytoplasm of plant cells. Interestingly, this was also demonstrated for the eliciting variants of Gp-RBP-1 and PVX-CP indicating a role for RanGAP1 and RanGAP2 in pathogenicity independent from Gpa2 and Rx1 recognition. Indeed, knocking down both RanGAP homologs reduce cyst nematode and PVX infection. These findings show that RanGAP1/2 act as common host targets of evolutionary distinct effectors from two plant pathogens with different lifestyles. The involvement of RanGAP1/2 to pathogen virulence is a novel role not yet reported for these key host cell components and as such, their possible role in cyst nematode parasitism and viral pathogenicity are discussed. Moreover, from these findings a model emerges for their possible role as co-factor in pathogen recognition by the potato immune receptors Gpa2/Rx1.

Analysis of the Role of Bradysiaimpatiens (Diptera: Sciaridae) as a Vector Transmitting Peanut Stunt Virus on the Model Plant Nicotiana benthamiana

Bradysia species, commonly known as fungus gnats, are ubiquitous in greenhouses, nurseries of horticultural plants, and commercial mushroom houses, causing significant economic losses. Moreover, the insects from the Bradysia genus have a well-documented role in plant pathogenic fungi transmission. Here, a study on the potential of Bradysia impatiens to acquire and transmit the peanut stunt virus (PSV) from plant to plant was undertaken. Four-day-old larvae of B. impatiens were exposed to PSV-P strain by feeding on virus-infected leaves of Nicotiana benthamiana and then transferred to healthy plants in laboratory conditions. Using the reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR (RT-qPCR), and digital droplet PCR (RT-ddPCR), the PSV RNAs in the larva, pupa, and imago of B. impatiens were detected and quantified. The presence of PSV genomic RNA strands as well as viral coat protein in N. benthamiana, on which the viruliferous larvae were feeding, was also confirmed at the molecular level, even though the characteristic symptoms of PSV infection were not observed. The results have shown that larvae of B. impatiens could acquire the virus and transmit it to healthy plants. Moreover, it has been proven that PSV might persist in the insect body transstadially. Although the molecular mechanisms of virion acquisition and retention during insect development need further studies, this is the first report on B. impatiens playing a potential role in plant virus transmission.

GmGSTU13 is related to the development of mosaic symptoms in soybean plants infected with Soybean mosaic virus

The leaves of soybean cv. ZheA8901 show various symptoms (necrosis, mosaic and symptomless) when infected with different strains of Soybean mosaic virus (SMV). Based on a proteomic analysis performed with tandem mass tags (TMT), 736 proteins were differentially expressed from soybean samples that showed asymptomatic, mosaic and necrosis symptoms induced by SMV strains SC3, SC7, and SC15, respectively. Among these, GmGSTU13 and APX (ascorbate peroxidase) were only upregulated in mosaic and symptomless leaves, respectively. The protein level of GmGSTU13 determined by Western blot was consistent with TMT analysis, qRT-PCR analysis showed that GmGSTU13 mRNA levels in mosaic plants was 5.26- and 3.75-fold higher than that in necrotic and symptomless plants, respectively. Additionally, the expression of viral coat protein (CP) gene was increased, and serious mosaic symptoms were observed in GmGSTU13-overexpressing plants inoculated with all three SMV strains. These results showed that GmGSTU13 is associated with the development of SMV-induced mosaic symptoms in soybean and that APX is upregulated in symptomless leaves at both the transcriptional and protein levels. In APX gene-silenced soybean plants, the relative expression of the viral CP gene was 1.50, 7.59 and 1.30 times higher than in positive control plants inoculated with the three SMV strains, suggesting that the upregulation of APX may be associated with lack of symptoms in soybean infected with SMV. This work provides a useful dataset for identifying key proteins responsible for symptom development in soybean infected with different SMV strains.

The p23 of Citrus Tristeza Virus Interacts with Host FKBP-Type Peptidyl-Prolylcis-Trans Isomerase 17-2 and Is Involved in the Intracellular Movement of the Viral Coat Protein

Citrus tristeza virus is a member of the genus Closterovirus in the family Closteroviridae. The p23 of citrus tristeza virus (CTV) is a multifunctional protein and RNA silencing suppressor. In this study, we identified a p23 interacting partner, FK506-binding protein (FKBP) 17-2, from Citrus aurantifolia (CaFKBP17-2), a susceptible host, and Nicotiana benthamiana (NbFKBP17-2), an experimental host for CTV. The interaction of p23 with CaFKBP17-2 and NbFKBP17-2 were individually confirmed by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Subcellular localization tests showed that the viral p23 translocated FKBP17-2 from chloroplasts to the plasmodesmata of epidermal cells of N. benthamiana leaves. The knocked-down expression level of NbFKBP17-2 mRNA resulted in a decreased CTV titer in N. benthamiana plants. Further, BiFC and Y2H assays showed that NbFKBP17-2 also interacted with the coat protein (CP) of CTV, and the complexes of CP/NbFKBP17-2 rapidly moved in the cytoplasm. Moreover, p23 guided the CP/NbFKBP17-2 complexes to move along the cell wall. To the best of our knowledge, this is the first report of viral proteins interacting with FKBP17-2 encoded by plants. Our results provide insights for further revealing the mechanism of the CTV CP protein movement.

Bistability in a model of tumor-immune system interactions with an oncolytic viral therapy

<abstract><p>A mathematical model of tumor-immune system interactions with an oncolytic virus therapy for which the immune system plays a twofold role against cancer cells is derived. The immune cells can kill cancer cells but can also eliminate viruses from the therapy. In addition, immune cells can either be stimulated to proliferate or be impaired to reduce their growth by tumor cells. It is shown that if the tumor killing rate by immune cells is above a critical value, the tumor can be eradicated for all sizes, where the critical killing rate depends on whether the immune system is immunosuppressive or proliferative. For a reduced tumor killing rate with an immunosuppressive immune system, that bistability exists in a large parameter space follows from our numerical bifurcation study. Depending on the tumor size, the tumor can either be eradicated or be reduced to a size less than its carrying capacity. However, reducing the viral killing rate by immune cells always increases the effectiveness of the viral therapy. This reduction may be achieved by manipulating certain genes of viruses via genetic engineering or by chemical modification of viral coat proteins to avoid detection by the immune cells.</p></abstract>

A general solution to broad-spectrum vaccine design for rapidly mutating viruses

Rapidly evolving pathogens pose a challenge to vaccine design, as their mutations render previous vaccine responses obsolete. For influenza, conserved epitopes on the viral coat proteins have been identified, but mysteriously they are missed by the antibodies elicited by most vaccine recipients [1, 2]. In simulated immunizations using 263 million antibody-hemagglutinin (HA) structural docking solutions, non-conserved epitopes were immunodominant when HAs were immunized at standard concentrations. However, by vaccinating with a pool of 30 diverse and dilute HA variants, B-cells that recognize broadly-conserved epitopes across HA receive up to 30-fold higher antigen dose, with concentration being linearly correlated to conservation on a per epitope basis. If individual variants are at concentrations below the minimum threshold of immune activation, then cross-reactive B-cells will be preferentially elicited. In pig immunizations, the approach induced a broad-spectrum antibody response against a panel of 36 strains from 1918-2014, including all pandemic strains from the past century and multiple strains not in the vaccine. In further swine studies with a vaccine containing HAs from 1918-2008, we observe broad-spectrum neutralizing responses against 6 future heterologous strains, including pandemic strains, spanning H1N1 2009-2017 and H3N2 2009-2014. Our results support a greater understanding of why non-conserved epitopes are immunodominant, as well as indicate a general solution to overcome this in broad-spectrum vaccine design.

Artificially Edited Alleles of the Eukaryotic Translation Initiation Factor 4E1 Gene Differentially Reduce Susceptibility to Cucumber Mosaic Virus and Potato Virus Y in Tomato

Eukaryotic translation initiation factors, including eIF4E, are susceptibility factors for viral infection in host plants. Mutation and double-stranded RNA-mediated silencing of tomato eIF4E genes can confer resistance to viruses, particularly members of the Potyvirus genus. Here, we artificially mutated the eIF4E1 gene on chromosome 3 of a commercial cultivar of tomato (Solanum lycopersicum L.) by using CRISPR/Cas9. We obtained three alleles, comprising two deletions of three and nine nucleotides (3DEL and 9DEL) and a single nucleotide insertion (1INS), near regions that encode amino acid residues important for binding to the mRNA 5' cap structure and to eIF4G. Plants homozygous for these alleles were termed 3DEL, 9DEL, and 1INS plants, respectively. In accordance with previous studies, inoculation tests with potato virus Y (PVY; type member of the genus Potyvirus) yielded a significant reduction in susceptibility to the N strain (PVYN), but not to the ordinary strain (PVYO), in 1INS plants. 9DEL among three artificial alleles had a deleterious effect on infection by cucumber mosaic virus (CMV, type member of the genus Cucumovirus). When CMV was mechanically inoculated into tomato plants and viral coat accumulation was measured in the non-inoculated upper leaves, the level of viral coat protein was significantly lower in the 9DEL plants than in the parental cultivar. Tissue blotting of microperforated inoculated leaves of the 9DEL plants revealed significantly fewer infection foci compared with those of the parental cultivar, suggesting that 9DEL negatively affects the initial steps of infection with CMV in a mechanically inoculated leaf. In laboratory tests, viral aphid transmission from an infected susceptible plant to 9DEL plants was reduced compared with the parental control. Although many pathogen resistance genes have been discovered in tomato and its wild relatives, no CMV resistance genes have been used in practice. RNA silencing of eIF4E expression has previously been reported to not affect susceptibility to CMV in tomato. Our findings suggest that artificial gene editing can introduce additional resistance to that achieved with mutagenesis breeding, and that edited eIF4E alleles confer an alternative way to manage CMV in tomato fields.

In vitro immunization approach to generate specific murine monoclonal IgG antibodies

Generating monoclonal antibodies to date is a time intense process requiring immunization of laboratory animals. The transfer of the humoral immune response into in vitro settings shortens this process and circumvents the necessity of animal immunization. However, orchestrating the complex interplay of immune cells in vitro is very challenging. We aimed for a simplified approach focusing on the protagonist of antibody production: the B lymphocyte. We activated purified murine B lymphocytes in vitro with combinations of antigen and stimuli. Within ten days of culture we induced specific IgM and IgG antibody responses against a viral coat protein. Permanently antibody-producing hybridomas were generated. Furthermore we used this method to induce a specific antibody response against Legionella pneumophila. We thus established an effective protocol to generate monoclonal antibodies in vitro. By overcoming the necessity of in vivo immunization it may be the first step towards a universal strategy to generate antibodies from various species.