Mah-Lee Ng

West Nile virus capsid protein interaction with importin and HDM2 protein is regulated by protein kinase C-mediated phosphorylation

West Nile virus (WNV) capsid (C) protein was shown to enter the nucleus via importin-mediated pathway and induce apoptosis although the precise regulatory mechanisms for such events have remained elusive. In this study, it was shown that WNV C protein was phosphorylated by protein kinase C (PKC). PKC-mediated phosphorylation influenced nuclear trafficking of C protein by modulating the efficiency of C protein-importin-alpha binding. Combination of bio-informatics, site-directed mutagenesis, co-immunoprecipitation, immuno-fluorescence and mammalian two-hybrid analyses showed that phosphorylation at amino acid residues residing near (Ser83) or within (Ser99 and Thr100) the bipartite nuclear localization motif of WNV C protein was essential for efficient interaction between C protein and importin-alpha. In addition, phosphorylation of WNV C protein by PKC was shown to enhance its binding to HDM2 and could subsequently induce p53-dependent apoptosis. Collectively, this study highlighted that phosphorylation is an important post-translational modification required to execute the functions of C protein.

Specific interaction of capsid protein and importin-α/β influences West Nile virus production.

West Nile virus (WNV) capsid (C) protein has been shown to enter the nucleus of infected cells. However, the mechanism by which C protein enters the nucleus is unknown. In this study, we have unveiled for the first time that nuclear transport of WNV and Dengue virus C protein is mediated by their direct association with importin-alpha. This interplay is mediated by the consensus sequences of bipartite nuclear localization signal located between amino acid residues 85-101 together with amino acid residues 42 and 43 of C protein. Elucidation of biological significance of importin-alpha/C protein interaction demonstrated that the binding efficiency of this association influenced the nuclear entry of C protein and virus production. Collectively, this study illustrated the molecular mechanism by which the C protein of arthropod-borne flavivirus enters the nucleus and showed the importance of importin-alpha/C protein interaction in the context of flavivirus life-cycle.

Human Sec3 protein is a novel transcriptional and translational repressor of flavivirus

The Flaviviridae family consists of several medically important pathogens such as West Nile virus (WNV) and Dengue virus (DENV). Flavivirus capsid (C) protein is a key structural component of virus particles. However, the role of C protein in the pathogenesis of arthropod‐borne flaviviruses is poorly understood. To examine whether flavivirus C protein can associate with cellular proteins, and contribute to viral pathogenesis, WNV/DENV C protein was screened against a human brain/liver cDNA yeast two‐hybrid library. This study identified human Sec3 exocyst protein (hSec3p) as a novel interacting partner of WNV and DENV C protein. Mutagenesis studies showed that the SH2 domain‐binding motif of hSec3p binds to the first 15 amino acids of C protein. We report for the first time that hSec3p can modulate virus production by affecting viral RNA transcription and translation through the sequestration of elongation factor 1α (EF1α). This molecular discovery shed light on the protective role of hSec3p during flavivirus infection. This study also highlighted the antagonistic mechanism adopted by flavivirus C protein that can negatively regulate the formation of hSec3p–EF1α complex by sequestering hSec3p to establish successful infection.

Infection of polarized epithelial cells with flavivirus West Nile: polarized entry and egress of virus occur through the apical surface

Both polarized epithelial Vero (C1008) and non-polarized Vero (control) cells were grown on permeable cell culture inserts and infected either apically or basolaterally with West Nile (WN) or Kunjin (KUN) virus. KUN virus (closely related to WN virus) was used as a comparison. Using indirect immunofluorescence and plaque assays of productive virus titres, entry of WN and KUN viruses was confined to the apical surface of polarized epithelial cells. For the first time, these results provided evidence on the distribution of flavivirus-specific receptor(s) in polarized epithelial cells; that is to say that receptor expression was shown to be predominant at the apical surface. In addition, the release of these viruses from polarized Vero C1008 epithelial cells was also examined. Egress of WN virus strain Sarafend (S) was observed to occur predominantly at the apical surface of Vero C1008 cells. In contrast, the release of KUN virus was bi-directional from polarized Vero C1008 cells. Furthermore, disruption of the cellular microtubule network was shown to inhibit the apical release of WN (S) virus but had no effect on the release of KUN virus. Hence, the difference in the release of these closely related viruses suggested the involvement of a microtubule-dependent, polarized sorting mechanism for WN virus proteins but not for KUN virus proteins in polarized epithelial cells.

Tyrosine 78 of premembrane protein is essential for assembly of West Nile virus.

Flavivirus premembrane (prM) protein plays an important role in conformational folding of the envelope (E) protein and protects it against premature fusion in acidic vesicles of the Golgi network. Currently, molecular determinants on the prM protein ectodomain which mediate critical steps during the flavivirus assembly process are poorly characterized. In this study, bioinformatics analysis and alanine scanning mutagenesis showed that the amino acid triplet valine 76, tyrosine 78 and glycine 79 is absolutely conserved among flavivirus prM ectodomains. Triple mutations engineered at these residues in prM ectodomain of West Nile virus (WNV) completely abrogated virus infectivity. Site-directed mutagenesis of prM protein revealed that tyrosine 78 of the amino acid triplet was required for virus infectivity and secretion. The mutation did not affect folding, post-translational modifications and trafficking of the prM and E proteins. Ultrastructural studies using transmission electron microscopy confirmed that virus particle formation was blocked by tyrosine 78 mutation. Specificity of assembly defect conferred by tyrosine 78 mutation was demonstrated by positive and negative trans complementation studies. Collectively, these results defined tyrosine 78 as a novel critical determinant present on prM protein ectodomain that is required for flavivirus assembly. Molecular dissection of prM protein function provides the crucial knowledge much needed in the elucidation of flavivirus particle formation.

Scanning electron microscopy of the surface coat of Blastocystis hominis

Abstract Scanning electron microscopy of Blastocystis hominis showed that its outer coat has a fibrillar structure and individual fibrils may extend up to 5 μm from the periphery of the parasite. The surface coat remains intact during cell division. Bacteria are often seen adhering to it, but for the first time a trophozoite of Chilomastix mesnili was also seen in this position. It is postulated that breakdown of attached organisms may provide nutrients for Blastocystis.

Dephosphorylation of West Nile virus capsid protein enhances the processes of nucleocapsid assembly.

West Nile virus (WNV) capsid (C) protein is one of the three viral structural proteins and it encapsidates the viral RNA to form the nucleocapsid. It is known to be a multifunctional protein involved in assembly and apoptosis. WNV C protein was previously found to be phosphorylated in infected cells and bioinformatic analysis revealed 5 putative phosphorylation sites at serine 26, 36, 83, 99 and threonine 100. Phosphorylation was abolished through mutagenesis of these putative phosphorylation sites to investigate how phosphorylation could affect the processes of nucleocapsid assembly like RNA binding, oligomerization and cellular localization. It was found that phosphorylation attenuated its RNA binding activity. Although oligomerization was not inhibited by mutagenesis of the putative phosphorylation sites, the rate of dimerization and oligomerization was affected. Hypophosphorylation of C protein reduced its nuclear localization efficiency and hence enhanced cytoplasmic localization. This study also revealed that although WNV C is phosphorylated in infected cells, the relative level of phosphorylation is reduced over the course of an infection to promote RNA binding and nucleocapsid formation in the cytoplasm. This is the first report to describe how dynamic phosphorylation of WNV C protein modulates the processes involved in nucleocapsid assembly.

West Nile virus and dengue virus capsid protein negates the antiviral activity of human Sec3 protein through the proteasome pathway

Flavivirus capsid (C) protein is a key structural component of virus particles. The non‐structural role of C protein in the pathogenesis of arthropod‐borne flaviviruses is not clearly deciphered. This study showed that West Nile virus (WNV) and dengue virus (DENV) utilized C protein to reduce human Sec3p (hSec3p) levels at post‐transcriptional level through activation of chymotrypsin‐like proteolytic function of 20S proteasome. Mutagenesis studies confirmed amino acids 14, 109–114 of WNV C protein and 13, 102–107 of DENV C protein played an important role in activating the proteolytic function of 20S proteasome. Amino acid residues at 14 (WNV) and 13 (DENV) of C protein were important for C protein‐hSec3p binding and physical interaction between C protein and hSec3p was essential to execute hSec3p degradation. Degradation motif required to degrade hSec3p resided between amino acid residues 109–114 of WNV C protein and 102–107 of DENV C protein. Proteasomes, hSec3p binding motif and degradation motif on C protein must be intact for efficient flavivirus production. Clinical isolates of DENV showed more pronounced effect in manipulating the proteasomes and reducing hSec3p levels. This study portrayed the non‐structural function of C protein that helped the flavivirus to nullify the antiviral activity of hSec3p by accelerating its degradation and facilitating efficient binding of elongation factor 1α with flaviviral RNA genome.

Observations on the surface coat of Blastocystis hominis

The surface coat of Blastocystishominis was studied in the electron microscope. In some cells the surface coat was seen in two layers; the external layer was more electron-dense and fragmented than the internal layer. It appears that the surface coat is being continuously formed by the parasite and shed in the environment. The fibrillar material of the surface coat attaches to the bacteria, in some cases, completely surrounding them, possibly causing cytoplasmic damage to the bacterial cell as indicated by loss of electron density.

Ultrastructure of the nucleus of the Iodamoeba bütschlii cyst

Abstract The ultrastructure of the Iodamoeba bütschlii cyst from human feces was studied. The glycogen mass appears as a compact dense body in the cytoplasm without any surrounding membrane. The cytoplasm has no mitochondrion. The nucleus shows a distinct nucleolus filled with electron-dense particles. On one side of the nucleolus are electron-dense cytoplasmic masses measuring 200–400 nm. The nuclear membrane is two-layered and shows pores.