The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles

Copyright @ 2008 American Society for Microbiology.The production of virus-like particles (VLPs) constitutes a relevant and safe model to study molecular determinants of virion egress. The minimal requirement for the assembly of VLPs for the coronavirus responsible for severe acute respiratory syndrome in humans (SARS-CoV) is still controversial. Recent studies have shown that SARS-CoV VLP formation depends on either M and E proteins or M and N proteins. Here we show that both E and N proteins must be coexpressed with M protein for the efficient production and release of VLPs by transfected Vero E6 cells. This suggests that the mechanism of SARS-CoV assembly differs from that of other studied coronaviruses, which only require M and E proteins for VLP formation. When coexpressed, the native envelope trimeric S glycoprotein is incorporated onto VLPs. Interestingly, when a fluorescent protein tag is added to the C-terminal end of N or S protein, but not M protein, the chimeric viral proteins can be assembled within VLPs and allow visualization of VLP production and trafficking in living cells by state-of-the-art imaging technologies. Fluorescent VLPs will be used further to investigate the role of cellular machineries during SARS-CoV egress.The University of Hong Kong and the French Ministry of Health

A single mutation in the envelope protein modulates flavivirus antigenicity, stability, and pathogenesis

The structural flexibility or 'breathing' of the envelope (E) protein of flaviviruses allows virions to sample an ensemble of conformations at equilibrium. The molecular basis and functional consequences of virus conformational dynamics are poorly understood. Here, we identified a single mutation at residue 198 (T198F) of the West Nile virus (WNV) E protein domain I-II hinge that regulates virus breathing. The T198F mutation resulted in a ~70-fold increase in sensitivity to neutralization by a monoclonal antibody targeting a cryptic epitope in the fusion loop. Increased exposure of this otherwise poorly accessible fusion loop epitope was accompanied by reduced virus stability in solution at physiological temperatures. Introduction of a mutation at the analogous residue of dengue virus (DENV), but not Zika virus (ZIKV), E protein also increased accessibility of the cryptic fusion loop epitope and decreased virus stability in solution, suggesting that this residue modulates the structural ensembles sampled by distinct flaviviruses at equilibrium in a context dependent manner. Although the T198F mutation did not substantially impair WNV growth kinetics in vitro, studies in mice revealed attenuation of WNV T198F infection. Overall, our study provides insight into the molecular basis and the in vitro and in vivo consequences of flavivirus breathing

Efficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prM and E proteins.

© 2009 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background: Flavivirus infected cells produce infectious virions and subviral particles, both of which are formed by the assembly of prM and E envelope proteins and are believed to undergo the same maturation process. Dengue recombinant subviral particles have been produced in cell cultures with either modified or chimeric proteins but not using the native forms of prM and E. Methodology/Principal Findings: We have used a codon optimization strategy to obtain an efficient expression of native viral proteins and production of recombinant subviral particles (RSPs) for all four dengue virus (DV) serotypes. A stable HeLa cell line expressing DV1 prME was established (HeLa-prME) and RSPs were analyzed by immunofluorescence and transmission electron microscopy. We found that E protein is mainly present in the endoplasmic reticulum (ER) where assembly of RSPs could be observed. Biochemical characterization of DV1 RSPs secretion revealed both prM protein cleavage and homodimerization of E proteins before their release into the supernatant, indicating that RSPs undergo a similar maturation process as dengue virus. Pulse chase experiment showed that 8 hours are required for the secretion of DV1 RSPs. We have used HeLa-prME to develop a semi-quantitative assay and screened a human siRNA library targeting genes involved in membrane trafficking. Knockdown of 23 genes resulted in a significant reduction in DV RSP secretion, whereas for 22 others we observed an increase of RSP levels in cell supernatant. Conclusions/Significance: Our data describe the efficient production of RSPs containing native prM and E envelope proteins for all dengue serotypes. Dengue RSPs and corresponding producing cell lines are safe and novel tools that can be used in the study of viral egress as well as in the development of vaccine and drugs against dengue virus.This work was supported by the 6th European Framework programme DENFRAME and by the Research Fund for the Control of Infectious Diseases of Hong Kong (RFCID#08070952)

Japanese encephalitis virus counteracts BST2 restriction via its envelope protein E.

It has been well documented that BST2 restricts the release of enveloped viruses by cross-linking newly produced virions to the cell membrane. However, it is less clear whether and how BST2 inhibits the release of enveloped viruses which bud via the secretory pathway. Here, we demonstrated that BST2 restricts the release of Japanese encephalitis virus (JEV) whose budding occurs at the ER-Golgi intermediate compartment, and in turn, JEV infection downregulates BST2 expression. We further found that the JEV envelope protein E, but not other viral components, significantly downregulates BST2 with the viral protein M playing an auxiliary role in the process. Envelope protein E-mediated BST2 downregulation appears to undergo lysosomal degradation pathway. Additional study revealed that the transmembrane domain and the coiled-coil domain (CC) of BST2 are the target domains of viral protein E and that the N- and C-terminal membrane anchors and the CC domain of BST2 are essential for blocking JEV release. Our results together indicate that the release of enveloped viruses whose budding take place in an intracellular compartment can be restricted by BST2

Global Functional Atlas of \u3cem\u3eEscherichia coli\u3c/em\u3e Encompassing Previously Uncharacterized Proteins

One-third of the 4,225 protein-coding genes of Escherichia coli K-12 remain functionally unannotated (orphans). Many map to distant clades such as Archaea, suggesting involvement in basic prokaryotic traits, whereas others appear restricted to E. coli, including pathogenic strains. To elucidate the orphans’ biological roles, we performed an extensive proteomic survey using affinity-tagged E. coli strains and generated comprehensive genomic context inferences to derive a high-confidence compendium for virtually the entire proteome consisting of 5,993 putative physical interactions and 74,776 putative functional associations, most of which are novel. Clustering of the respective probabilistic networks revealed putative orphan membership in discrete multiprotein complexes and functional modules together with annotated gene products, whereas a machine-learning strategy based on network integration implicated the orphans in specific biological processes. We provide additional experimental evidence supporting orphan participation in protein synthesis, amino acid metabolism, biofilm formation, motility, and assembly of the bacterial cell envelope. This resource provides a “systems-wide” functional blueprint of a model microbe, with insights into the biological and evolutionary significance of previously uncharacterized proteins

A gorilla adenovirus-based vaccine against Zika virus induces durable immunity and confers protection in pregnancy

The teratogenic potential of Zika virus (ZIKV) has made the development of an effective vaccine a global health priority. Here, we generate two gorilla adenovirus-based ZIKV vaccines that encode for pre-membrane (prM) and envelope (E) proteins (GAd-Zvp) or prM and the ectodomain of E protein (GAd-Eecto). Both vaccines induce humoral and cell-mediated immune responses and prevent lethality after ZIKV challenge in mice. Protection is antibody dependent, CD

Tocopherol and plastoquinone synthesis in spinach chloroplasts subfractions

Subfractions isolated from intact purified spinach chloroplasts are able to prenylate the aromatic moiety of -tocopherol and plastoquinone-9 precursors. The biosynthesis of -tocopherol and plastoquinone-9 is a compartmentalized process. The chloroplast envelope membranes are the only site of the enzymatic prenylation in -tocopherol synthesis whereas the thylakoid membrane is also involved in the prenylation and methylation sequence of plastoquinone-9 biosynthesis. A very active kinase which forms phytyl-PP is localized in the stroma. Phytol but not geranylgeraniol is the polyprenol precursor of the side chain of -tocopherol in spinach chloroplasts

Expression of the envelope antigen F1 of Yersinia pestis is mediated by the product of caf1M gene having homology with the chaperone protein PapD of Escherichia coli.

The effective synthesis of the envelope antigen F1 of Y. pestis in E. coli HB101 is mediated by the expression of the cuf1M gene. This gene was sequenced, and the protein encoded was found to have a significant homology with the chaperone protein PapD of uropathogenic E. coli. The data presented allow one to suppose Caf1M and PapD proteins perform similar functions in the biogenesis of the Y. pestis capsule and E. coli P-pili, respectively. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/905

Ca2+ - Induced Structural Change of Multi-Domain Collagen Binding Segments of Collagenases ColG and ColH from Hathewaya histolytica

Hathewaya histolytica, previously renamed as Clostridium histolyticum, secretes at least two collagenases, ColH and ColG, that allow for degradation of extracellular matrices of animal tissue. Hathewaya histolytica virulence factors are proposed to undergo domain rearrangement upon secretion from the bacteria to the host. In order to accomplish this, collagenases seek the least ordered regions in collagen to efficiently disassemble the fibril. Two types of domains, Polycystic Kidney Disease-like (PKD) and Collagen Binding Domain (CBD), direct the collagenases ability to disassemble the fibril. Calcium dependent structural change have been reported to increase in thermal stability and in tighter collagen binding for CBD. Different surface properties and indications of varied dynamics suggest unique roles for the PKD-like domains in ColG and in ColH. These domains are useful in anchoring fused growth factors to lesions. Most recent results show that use of multi-domain targeting segments result in more efficacious preclinical outcomes in various animal models. Single domain targeting segments have been biophysically characterized; however, multi-domain targeting segments have not been biophysically characterized. In this study, Small Angle X-Ray Scattering (SAXS) methods were used to monitor calcium dependent structural change of multi-domain segments, i.e. PKD1-PKD2-CBD and PKD-CBD1-CBD2 (Ruth and Sakon 2017). All SAXS data were collected at 10°C through the Advanced Light Source at Berkley National Lab (Otwinowski and Minor 1997) (Classen, Hura et al. 2013). The average of 33 beam exposures were collected and processed through primusqt (Konarev, Petoukhov et al. 2006). Genes for type II secretion system are recently reported in the genus. The bacteria apparently evolved to take advantage of calcium concentration differential from inside the bacteria and the extracellular matrix to efficiently secrete virulence factor