Potato (Solanum tuberosum L.) non-specific lipid transfer protein StLTP6 promotes viral infection by inhibiting virus-induced RNA silencing.

MAIN CONCLUSION: For the first time it is reported that members of the nsLTP protein family could promote viral infection by inhibiting virus-induced RNA silencing. Non-specific lipid transfer proteins (nsLTPs) are a class of soluble proteins with low relative molecular weight and widely present in higher plants. The role of nsLTPs in biotic and abiotic stresses has been studied, but no report has shown that nsLTPs play a role in the process of viral infection. We report the function and mechanism of the classical nsLTP protein StLTP6 in viral infection. We found that StLTP6 expression was remarkably upregulated in potato infected with potato virus Y and potato virus S. The infection efficiency and virus content of StLTP6-overexpressed potato and Nicotiana benthamiana were remarkable increased. Further study found that the overexpression of StLTP6 inhibited the expression of multiple genes in the RNA silencing pathway, thereby inhibiting virus-induced RNA silencing. This result indicated that StLTP6 expression was induced during viral infection to inhibit the resistance of virus-induced RNA silencing and promote viral infection. In summary, we reported the role of StLTP6 in viral infection, broadening the biological function range of the nsLTP family and providing valuable information for the study of viral infection mechanism.

Comparative evaluation of resistance to potato virus Y (PVY) in three different RNAi-based transgenic potato plants.

Small interfering RNAs (siRNAs) produced from template double-stranded RNAs (dsRNAs) can activate the immune system in transgenic plants by detecting virus transcripts to degrade. In the present study, an RNA interference (RNAi) gene silencing mechanism was used for the development of transgenic potato plants resistant to potato virus Y (PVY), the most harmful viral disease. Three RNAi gene constructs were designed based on the coat protein (CP) and the untranslated region parts of the PVY genome, being highly conserved among all strains of the PVY viruses. Transgenic potato plants were generated using Agrobacterium containing pCAMRNAiCP, pCAMRNAiUR, and pCAMRNAiCP-UR constructs. The transgene insertions were confirmed by molecular analysis containing polymerase chain reaction (PCR) and southern blotting. The resistance of transgenic plants to PVY virus was determined using bioassay and evaluating the amount of viral RNA in plants by RT-PCR, dot blotting of PVY coating protein, and enzyme-linked immunosorbent assay (ELISA). Bioassay analysis revealed that more than 67% of transgenic potato plants were resistant to PVY compared with the non-transgenic plants, which showed viral disease symptoms. No phenotypic abnormalities were observed in transgenic plants. Out of six lines in southern blot analysis, four lines had one copy of the transgene and two lines had two copies of the target genes. No correlation was detected between the copy number of the genes and the resistance level of the plant to PVY. Transgenic lines obtained from all three constructs indicated more or less similar levels of resistance against viral infection; however, CP-UR lines exhibited relatively high resistance followed by CP and UR expressing lines, respectively. Meanwhile, some lines showed a delay in symptoms 35 days after infection which were classified as susceptible.

Multifaceted Functions of Host Cell Caveolae/Caveolin-1 in Virus Infections.

Virus infection has drawn extensive attention since it causes serious or even deadly diseases, consequently inducing a series of social and public health problems. Caveolin-1 is the most important structural protein of caveolae, a membrane invagination widely known for its role in endocytosis and subsequent cytoplasmic transportation. Caveolae/caveolin-1 is tightly associated with a wide range of biological processes, including cholesterol homeostasis, cell mechano-sensing, tumorigenesis, and signal transduction. Intriguingly, the versatile roles of caveolae/caveolin-1 in virus infections have increasingly been appreciated. Over the past few decades, more and more viruses have been identified to invade host cells via caveolae-mediated endocytosis, although other known pathways have been explored. The subsequent post-entry events, including trafficking, replication, assembly, and egress of a large number of viruses, are caveolae/caveolin-1-dependent. Deprivation of caveolae/caveolin-1 by drug application or gene editing leads to abnormalities in viral uptake, viral protein expression, or virion release, whereas the underlying mechanisms remain elusive and must be explored holistically to provide potential novel antiviral targets and strategies. This review recapitulates our current knowledge on how caveolae/caveolin-1 functions in every step of the viral infection cycle and various relevant signaling pathways, hoping to provide a new perspective for future viral cell biology research.

Functional analysis reveals G/U pairs critical for replication and trafficking of an infectious non-coding viroid RNA.

While G/U pairs are present in many RNAs, the lack of molecular studies to characterize the roles of multiple G/U pairs within a single RNA limits our understanding of their biological significance. From known RNA 3D structures, we observed that the probability a G/U will form a Watson-Crick (WC) base pair depends on sequence context. We analyzed 17 G/U pairs in the 359-nucleotide genome of Potato spindle tuber viroid (PSTVd), a circular non-coding RNA that replicates and spreads systemically in host plants. Most putative G/U base pairs were experimentally supported by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). Deep sequencing PSTVd genomes from plants inoculated with a cloned master sequence revealed naturally occurring variants, and showed that G/U pairs are maintained to the same extent as canonical WC base pairs. Comprehensive mutational analysis demonstrated that nearly all G/U pairs are critical for replication and/or systemic spread. Two selected G/U pairs were found to be required for PSTVd entry into, but not for exit from, the host vascular system. This study identifies critical roles for G/U pairs in the survival of an infectious RNA, and increases understanding of structure-based regulation of replication and trafficking of pathogen and cellular RNAs.

DEAD-box helicases: the Yin and Yang roles in viral infections.

Viruses hijack the host cell machinery and recruit host proteins to aid their replication. Several host proteins also play vital roles in inhibiting viral replication. Emerging class of host proteins central to both of these processes are the DEAD-box helicases: a highly conserved family of ATP-dependent RNA helicases, bearing a common D-E-A-D (Asp-Glu-Ala-Asp) motif. They play key roles in numerous cellular processes, including transcription, splicing, miRNA biogenesis and translation. Though their sequences are highly conserved, these helicases have quite diverse roles in the cell. Interestingly, often these helicases display contradictory actions in terms of the support and/or clearance of invading viruses. Increasing evidence highlights the importance of these enzymes, however, little is known about the structural basis of viral RNA recognition by the members of the DEAD-box family. This review summarizes the current knowledge in the field for selected DEAD-box helicases and highlights their diverse actions upon viral invasion of the host cell. We anticipate that through a better understanding of how these helicases are being utilized by viral RNAs and proteins to aid viral replication, it will be possible to address the urgent need to develop novel therapeutic approaches to combat viral infections.

Virus-host interactomes--antiviral drug discovery.

One of the key questions in virology is how viruses, encoding relatively few genes, gain temporary or constant control over their hosts. To understand pathogenicity of a virus it is important to gain knowledge on the function of the individual viral proteins in the host cell, on their interactions with viral and cellular proteins and on the consequences of these interactions on cellular signaling pathways. A combination of transcriptomics, proteomics, high-throughput technologies and the bioinformatical analysis of the respective data help to elucidate specific cellular antiviral drug target candidates. In addition, viral and human interactome analyses indicate that different viruses target common, central human proteins for entering cellular signaling pathways and machineries which might constitute powerful broad-spectrum antiviral targets.

Nuclear factor κB subunits RelB and cRel negatively regulate Toll-like receptor 3-mediated ß-interferon production via induction of transcriptional repressor protein YY1.

The induction of ß-interferon (IFN-ß) is a key anti-viral response to infection by RNA viruses. Virus-induced expression of IFN-ß requires the co-operative action of the transcription factors IRF-3/7, NF-κB, and ATF-2/c-Jun on the IFN-ß promoter leading to the orderly recruitment of chromatin remodeling complexes. Although viruses strongly activate NF-κB and promote its binding to the IFN-ß promoter, recent studies have indicated that NF-κB is not essential for virus-induced expression of IFN-ß. Herein, we examined the role of NF-κB in regulating IFN-ß expression in response to the viral-sensing Toll-like receptor 3 (TLR3). Intriguingly pharmacological inhibition of the NF-κB pathway augments late phase expression of IFN-ß expression in response to TLR3 stimulation. We show that the negative effect of NF-κB on IFN-ß expression is dependent on the induction of the transcriptional repressor protein YinYang1. We demonstrate that the TLR3 ligand polyriboinosinic:polyribocytidylic acid (poly(I:C)) induces expression and nuclear translocation of YinYang1 where it interacts with the IFN-ß promoter and inhibits the binding of IRF7 to the latter. Evidence is also presented showing that the NF-κB subunits c-Rel and RelB are the likely key drivers of these negative effects on IFN-ß expression. These findings thus highlight for the first time a novel self-regulatory mechanism that is employed by TLR3 to limit the level and duration of IFN-ß expression.

Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays.

Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1×), tryptose phosphate broth (2.95 gl⁻¹), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 µg ml⁻¹ chloramphenicol, 100 µg ml⁻¹ streptomycin and 100 IU ml⁻¹ penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8 days. 5-Bromo-2'-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24h. Susceptibility of the cells to WSSV was confirmed by immunofluorescence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT(50)/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC(50). The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals.

A top-down approach for construction of hybrid polymer-virus gene delivery vectors.

Safe and efficient delivery of therapeutic nucleic acids remains the primary hurdle for human gene therapy. While many researchers have attempted to re-engineer viruses to be suited for gene delivery, others have sought to develop non-viral alternatives. We have developed a complementary approach in which viral and synthetic components are combined to form hybrid nanoparticulate vectors. In particular, we complexed non-infectious retrovirus-like particles lacking a viral envelope protein, from Moloney murine leukemia virus (M-VLP) or human immunodeficiency virus (H-VLP), with poly-L-lysine (PLL) or polyethylenimine (PEI) over a range of polymer/VLP ratios. At appropriate stoichiometry (75-250 microg polymer/10(6) VLP), the polymers replace the function of the viral envelope protein and interact with the target cell membrane, initiate cellular uptake and facilitate escape from endocytic vesicles. The viral particle, once in the cytosol, efficiently completes its normal infection process including integration of viral genes with the host genome as demonstrated by long-term (at least 5 weeks) transgene expression. In addition, hybrid vectors comprising H-VLP were shown to be capable of infecting non-dividing cells.

Characterization, nucleotide sequence and genome organization of leek white stripe virus, a putative new species of the genus Necrovirus.

White stripe is a disease affecting leek in France with which an isometric virus c. 30 nm in diameter is associated. The most evident symptom is the presence of white stripes on the leaves extending to the stem. Attempts to demonstrate transmission through the soil by sowing or transplanting leek in contaminated soil were unsuccessful. The virus was transmitted by sap inoculation to a narrow range of herbaceous hosts, all of which were infected only locally. Virus purification was from infected leek tissues, where it accumulated in large amounts, as demonstrated by ultrastructural observations. RNA was extracted from purified virus preparations and cDNA clones were prepared. The complete nucleotide sequence of the viral RNA was determined: The genome is 3,662 nucleotides long and contains five open reading frames (ORFs). The first (ORF 1) encodes a putative translation product of M(r) 23,803 (p24) and read through of its amber stop codon results in a protein of M(r) 82,625 (p83) (ORF 2). ORF 3 and ORF 4 encode two small polypeptides of M(r) 11,280 (p11) and M(r) 6,261 (p6), respectively. ORF 5 encodes the capsid protein of M(r) 27,460 (p27). The genome organization and sequence alignments with the corresponding products of necroviruses suggest that the virus isolated from leek is a new species in the genus Necrovirus, for which the name of leek white stripe virus (LWSV) is proposed.