The Cucumber leaf spot virus p25 auxiliary replicase protein binds and modifies the endoplasmic reticulum via N-terminal transmembrane domains

AbstractCucumber leaf spot virus (CLSV) is a member of the Aureusvirus genus, family Tombusviridae. The auxiliary replicase of Tombusvirids has been found to localize to endoplasmic reticulum (ER), peroxisomes or mitochondria; however, localization of the auxiliary replicase of aureusviruses has not been determined. We have found that the auxiliary replicase of CLSV (p25) fused to GFP colocalizes with ER and that three predicted transmembrane domains (TMDs) at the N-terminus of p25 are sufficient for targeting, although the second and third TMDs play the most prominent roles. Confocal analysis of CLSV infected 16C plants shows that the ER becomes modified including the formation of punctae at connections between ER tubules and in association with the nucleus. Ultrastructural analysis shows that the cytoplasm contains numerous vesicles which are also found between the perinuclear ER and nuclear membrane. It is proposed that these vesicles correspond to modified ER used as sites for CLSV replication

A 38-Amino-Acid Sequence Encompassing the Arm Domain of the Cucumber Necrosis Virus Coat Protein Functions as a Chloroplast Transit Peptide in Infected Plants

Experiments to determine the subcellular location of the coat protein (CP) of the tombusvirus Cucumber necrosis virus (CNV) have been conducted. By confocal microscopy, it was found that an agroinfiltrated CNV CP-green fluorescent protein (GFP) fusion targets chloroplasts in Nicotiana benthamiana leaves and that a 38-amino-acid (aa) region that includes the complete CP arm region plus the first 4 amino acids of the shell domain are sufficient for targeting. Western blot analyses of purified and fractionated chloroplasts showed that the 38-aa region directs import to the chloroplast stroma, suggesting that the CNV arm can function as a chloroplast transit peptide (TP) in plants. Several features of the 38-aa region are similar to features typical of chloroplast TPs, including (i) the presence of an alanine-rich uncharged region near the N terminus, followed by a short region rich in basic amino acids; (ii) a conserved chloroplast TP phosphorylation motif; (iii) the requirement that the CNV 38-aa sequence be present at the amino terminus of the imported protein; and (iv) specific proteolytic cleavage upon import into the chloroplast stroma. In addition, a region just downstream of the 38-aa sequence contains a 14-3-3 binding motif, suggesting that chloroplast targeting requires 14-3-3 binding, as has been suggested for cellular proteins that are targeted to chloroplasts. Chloroplasts of CNV-infected plants were found to contain CNV CP, but only the shell and protruding domain regions were present, indicating that CNV CP enters chloroplasts during infection and that proteolytic cleavage occurs as predicted from agroinfiltration studies. We also found that particles of a CNV CP mutant deficient in externalization of the arm region have a reduced ability to establish infection. The potential biological significance of these findings is discussed

Induction of Particle Polymorphism by Cucumber Necrosis Virus Coat Protein Mutants In Vivo▿

The Cucumber necrosis virus (CNV) particle is a T=3 icosahedron consisting of 180 identical coat protein (CP) subunits. Plants infected with wild-type CNV accumulate a high number of T=3 particles, but other particle forms have not been observed. Particle polymorphism in several T=3 icosahedral viruses has been observed in vitro following the removal of an extended N-terminal region of the CP subunit. In the case of CNV, we have recently described the structure of T=1 particles that accumulate in planta during infection by a CNV mutant (R1+2) in which a large portion of the N-terminal RNA binding domain (R-domain) has been deleted. In this report we further describe properties of this mutant and other CP mutants that produce polymorphic particles. The T=1 particles produced by R1+2 mutants were found to encapsidate a 1.9-kb RNA species as well as smaller RNA species that are similar to previously described CNV defective interfering RNAs. Other R-domain mutants were found to encapsidate a range of specifically sized less-than-full-length CNV RNAs. Mutation of a conserved proline residue in the arm domain near its junction with the shell domain also influenced T=1 particle formation. The proportion of polymorphic particles increased when the mutation was incorporated into R-domain deletion mutants. Our results suggest that both the R-domain and the arm play important roles in the formation of T=3 particles. In addition, the encapsidation of specific CNV RNA species by individual mutants indicates that the R-domain plays a role in the nature of CNV RNA encapsidated in particles

Recognizing and managing sepsis: what needs to be done?

Sepsis is associated with significant morbidity and mortality if not promptly recognized and treated. Since the development of early goal-directed therapy, mortality rates have decreased, but sepsis remains a major cause of death in patients arriving at the emergency department or staying in hospital. In this forum article, we asked clinicians and researchers with expertise in sepsis care to discuss the importance of rapid detection and treatment of the condition, as well as special considerations in different patient groups

Stress and immune responses in abalone: Limitations in current knowledge and investigative methods based on other models

Increasing mariculture of abalone focuses attention on their immune and stress responses. For abalone, as well as many invertebrates, the function and relationship of these systems and how in vitro tests relate to them are not fully understood. This review focuses on research into the immune system and stress response conducted on abalone and on aspects that can be monitored in vitro. To fill the considerable knowledge gaps, we discuss work on other invertebrate taxa, concentrating on those closest to abalone, and making explicit the phylogenetic relations involved. The stress response appears to be very similar to that in vertebrates, but interpreting most immune responses remains problematic. Phylogeny must be considered: immune function tests derived from research into vertebrates or distantly related invertebrates should not be used in abalone until they have been validated in abalone by studies of susceptibility to pathogens. We suggest phagocytic activity of haemocytes and their efficiency in clearing bacteria are reliable parameters to measure, because they have been directly related to immune competency and are consistently depressed by stress. Carefully designed assays of antimicrobial activity may also be useful. Important aims of future research will be to investigate the relationship between growth, stress and robust immunity, and to develop tests that can be run on production animals, which accurately depict immune status