Characterization of intracellular membrane structures derived from a massive expansion of endoplasmic reticulum (ER) membrane due to synthetic ER-membrane-resident polyproteins.

The endoplasmic reticulum (ER) is a dynamic organelle that is amenable to major restructuring. Introduction of recombinant ER-membrane-resident proteins that form homo oligomers is a known method of inducing ER proliferation: interaction of the proteins with each other alters the local structure of the ER network, leading to the formation large aggregations of expanded ER, sometimes leading to the formation of organized smooth endoplasmic reticulum (OSER). However, these membrane structures formed by ER proliferation are poorly characterized and this hampers their potential development for plant synthetic biology. Here, we characterize a range of ER-derived membranous compartments in tobacco and show how the nature of the polyproteins introduced into the ER membrane affect the morphology of the final compartment. We show that a cytosol-facing oligomerization domain is an essential component for compartment formation. Using fluorescence recovery after photobleaching, we demonstrate that although the compartment retains a connection to the ER, a diffusional barrier exists to both the ER and the cytosol associated with the compartment. Using quantitative image analysis, we also show that the presence of the compartment does not disrupt the rest of the ER network. Moreover, we demonstrate that it is possible to recruit a heterologous, bacterial enzyme to the compartment, and for the enzyme to accumulate to high levels. Finally, transgenic Arabidopsis constitutively expressing the compartment-forming polyproteins grew and developed normally under standard conditions.

Phylogenetic analysis of sacbrood virus structural polyprotein and non-structural RNA dependent RNA polymerase gene: Differences in Turkish strains.

Sacbrood virus (SBV) is one of the most damaging viruses in honey bee colonies. Genetic differences among sacbrood viruses detected in honey bees in different locales have been reported in previous studies. The aim of this study was to construct phylogenetic trees based on the structural polyprotein and non-structural RNA dependent RNA polymerase gene regions and to make a molecular characterization of the Tur/Bur/Sac01 and Tur/Bur/Sac02 strains identified in Apis mellifera in Turkey. As a result of the study, the tree based on the structural polyprotein region separated into four lineages: Tur/Bur/Sac01 and Tur/Bur/Sac02 were in the same branch as the Turkish sacbrood virus strains identified in previous studies and formed the Turkish clade. Strains isolated from adjacent geographical areas were in the same clade in this tree. The phylogenetic tree based on the non-structural RNA dependent RNA polymerase gene region divides into two main branches, reflecting host affiliation: Apis cerana and A. mellifera. Strains formed clusters based on their geographic distribution and host affiliation. The Tur/Bur/Sac01 and Tur/Bur/Sac02 strains formed a separate cluster among the European strains. Sacbrood viruses from Turkey were genetically different from SBV strains detected in other countries and in A. cerana.

ERVK polyprotein processing and reverse transcriptase expression in human cell line models of neurological disease.

Enhanced expression of the reverse transcriptase (RT) protein encoded by human endogenous retrovirus-K (ERVK) is a promising biomarker for several inflammatory and neurological diseases. However, unlike RT enzymes encoded by exogenous retroviruses, little work has been done to identify ERVK RT isoforms, their expression patterns, and cellular localization. Using Western blot, we showcase the ERVK gag-pro-pol polyprotein processing leading to the production of several ERVK RT isoforms in human neuronal (ReNcell CX) and astrocytic (SVGA) models of neuroinflammatory disease. Since the pro-inflammatory cytokine IFNγ plays a key role in the pathology of several ERVK-associated neurological diseases, we sought to determine if IFNγ can drive ERVK RT expression. IFNγ signalling markedly enhanced ERVK polyprotein and RT expression in both human astrocytes and neurons. RT isoforms were expressed in a cell-type specific pattern and the RT-RNase H form was significantly increased with IFNγ treatment. Fluorescent imaging revealed distinct cytoplasmic, perinuclear and nuclear ERVK RT staining patterns upon IFNγ stimulation of astrocytes and neurons. These findings indicate that ERVK expression is inducible under inflammatory conditions such as IFNγ exposure-and thus, these newly established in vitro models may be useful in exploring ERVK biology in the context of neuroinflammatory disease.

A new, modular mass calibrant for high-mass MALDI-MS.

The application of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of high-mass proteins requires suitable calibration standards at high m/z ratios. Several possible candidates were investigated, and concatenated polyproteins based on recombinantly expressed maltodextrin-binding protein (MBP) are shown here to be well-suited for this purpose. Introduction of two specific recognition sites into the primary sequence of the polyprotein allows for the selective cleavage of MBP3 into MBP and MBP2. Moreover, these MBP2 and MBP3 oligomers can be dimerized specifically, such that generation of MPB4 and MBP6 is possible as well. With the set of calibrants presented here, the m/z range of 40-400 kDa is covered. Since all calibrants consist of the same species and differ only in mass, the ionization efficiency is expected to be similar. However, equimolar mixtures of these proteins did not yield equal signal intensities on a detector specifically designed for detecting high-mass molecules.

Identification of CD8+ T cell epitopes in the West Nile virus polyprotein by reverse-immunology using NetCTL.

BACKGROUND: West Nile virus (WNV) is a growing threat to public health and a greater understanding of the immune response raised against WNV is important for the development of prophylactic and therapeutic strategies. METHODOLOGY/PRINCIPAL FINDINGS: In a reverse-immunology approach, we used bioinformatics methods to predict WNV-specific CD8(+) T cell epitopes and selected a set of peptides that constitutes maximum coverage of 20 fully-sequenced WNV strains. We then tested these putative epitopes for cellular reactivity in a cohort of WNV-infected patients. We identified 26 new CD8(+) T cell epitopes, which we propose are restricted by 11 different HLA class I alleles. Aiming for optimal coverage of human populations, we suggest that 11 of these new WNV epitopes would be sufficient to cover from 48% to 93% of ethnic populations in various areas of the World. CONCLUSIONS/SIGNIFICANCE: The 26 identified CD8(+) T cell epitopes contribute to our knowledge of the immune response against WNV infection and greatly extend the list of known WNV CD8(+) T cell epitopes. A polytope incorporating these and other epitopes could possibly serve as the basis for a WNV vaccine.

Exploitation of a high genomic mutation rate in Solenopsis invicta virus 1 to infer demographic information about its host, Solenopsis invicta.

The RNA-dependent RNA polymerase (RdRp) region of Solenopsis invicta virus 1 (SINV-1) was sequenced from 47 infected colonies of S. invicta, S. richteri, S. geminata, and S. invicta/richteri hybrids collected from across the USA, northern Argentina, and northern Taiwan in an attempt to infer demographic information about the recent S. invicta introduction into Taiwan by phylogenetic analysis. Nucleotide sequences were calculated to exhibit an overall identity of >90% between geographically-separated samples. A total of 171 nucleotide variable sites (representing 22.4% of the region amplified) were mapped across the SINV-1 RdRp alignment and no insertions or deletions were detected. Phylogenetic analysis at the nucleotide level revealed clustering of Argentinean sequences, distinct from the USA sequences. Moreover, the SINV-1 RdRp sequences derived from recently introduced populations of S. invicta from northern Taiwan resided within the multiple USA groupings implicating the USA as the source for the recent introduction of S. invicta into Taiwan. Examination of the amino acid alignment for the RdRp revealed sequence identity >98% with only nine amino acid changes observed. Seven of these changes occurred in less than 4.3% of samples, while 2 (at positions 1266 and 1285) were featured prominently. Changes at positions 1266 and 1285 accounted for 36.2% and 34.0% of the samples, respectively. Two distinct groups were observed based on the amino acid residue at position 1266, Threonine or Serine. In cases where this amino acid was a Threonine, 90% of these sequences possessed a corresponding Valine at position 1285; only 10% of the Threonine(1266)-containing sequences possessed an Isoleucine at the 1285 position. Among the Serine(1266) group, 76% possessed an Isoleucine at position 1285, while only 24% possessed a Valine. Thus, it appears that the Threonine(1266)/Valine(1285) and Serine(1266)/Isoleucine(1285) combinations are predominant phenotypes.

Detection of 6K1 as a mature protein of 6 kDa in plum pox virus-infected Nicotiana benthamiana.

The RNA genome of Plum pox virus (PPV) encodes one large polyprotein that is subsequently cleaved into mature viral proteins. One of the products of proteolytic processing, the 6K1 protein, has not yet been identified in vivo for any member of the genus Potyvirus. In this study, 6K1-specific polyclonal antiserum was raised against PPV 6K1 expressed in Escherichia coli as a translational fusion with the N terminus of avian troponin C and an unusual metal-binding cluster of troponin T-1. For detection of 6K1 in vivo, a pPPV-H6K1-NAT infectious clone was constructed, enabling concentration of histidine-tagged 6K1 by affinity chromatography. Affinity-purified 6K1 was detected in locally infected Nicotiana benthamiana leaves at 4, 7 and 14 days post-inoculation (d.p.i.) and, in addition, in systemically infected leaves at 14 d.p.i., 6K1 was detected exclusively as a protein of 6 kDa and no polyprotein precursors were identified with the raised anti-6K1 antiserum.

Biophysical investigations of engineered polyproteins: implications for force data.

Dynamic force spectroscopy is rapidly becoming a standard biophysical technique. Significant advances in the methods of analysis of force data have resulted in ever more complex systems being studied. The use of cloning systems to produce homologous tandem repeats rather than the use of endogenous multidomain proteins has facilitated these developments. What is poorly addressed are the physical properties of these constructed polyproteins. Are the properties of the individual domains in the construct independent of one another or attenuated by adjacent domains? We present data for a construct of eight fibronectin type III domains from the human form of tenascin that exhibits approximately 1 kcal mol(-1) increase in stability compared to the monomer. This effect is salt and pH dependent, suggesting that the stabilization results from electrostatic interactions, possibly involving charged residues at the interfaces of the domains. Kinetic analysis shows that this stabilization reflects a slower unfolding rate. Clearly, if domain-domain interactions affect the unfolding force, this will have implications for the comparison of absolute forces between types of domains. Mutants of the tenascin 8-mer construct exhibit the same change in stability as that observed for the corresponding mutation in the monomer. And when Phi-values are calculated for the 8-mer construct, the pattern is similar to that observed for the monomer. Therefore, mutational analyses to resolve mechanical unfolding pathways appear valid. Importantly, we show that interactions between the domains may be masked by changes in experimental conditions.

The polyprotein and FAR lipid binding proteins of nematodes: shape and monomer/dimer states in ligand-free and bound forms.

Nematodes produce two classes of small, helix-rich fatty acid- and retinol-binding proteins whose structures and in vivo functions remain to be elucidated. These are the polyprotein allergens (NPA) and the FAR proteins. The solution properties of recombinant forms of these proteins from parasitic [Ascaris suum (As) and Onchocerca volvulus (Ov)] and free-living [Caenorhabditis elegans (Ce)] nematodes have been examined. Analytical ultracentrifugation (AUC) showed that, contrary to previous findings, the rAs-NPA-1A polyprotein unit (approximately 15 kDa) is a monomer, and this stoichiometry is unaltered by ligand (oleic acid) binding. The rOv-FAR-1 and rCe-FAR-5 proteins differ in that the former forms a tight dimer and the latter a monomer, and these oligomeric states are also unaffected by ligand binding or protein concentration. Sedimentation equilibrium experiments showed that the partial specific volume v of the unliganded proteins agree well with the value calculated from amino acid composition extrapolated to experimental temperature, and was unaffected upon ligand binding. Data from small-angle X-ray scattering (SAXS) indicated that both of the monomeric proteins rAs-NPA-1A and rCe-FAR-5 are globular, although slightly elongated and flattened. These data are in good agreement with shapes predicted from sedimentation velocity experiments and hydrodynamic bead modelling. On the basis of functional and secondary structural homology with the ligand-binding domain of the retinoic acid receptor RXRalpha, de novo atomic resolution structures for rAs-NPA-1A and rCe-FAR-5 have been constructed which are consistent with the SAXS and hydrodynamic data.

African swine fever virus polyproteins pp220 and pp62 assemble into the core shell.

African swine fever virus (ASFV), a complex enveloped DNA virus, expresses two polyprotein precursors, pp220 and pp62, which after proteolytic processing give rise to several major components of the virus particle. We have analyzed the structural role of polyprotein pp62, the precursor form of mature products p35 and p15, in virus morphogenesis. Densitometric analysis of one- and two-dimensional gels of purified virions showed that proteins p35 and p15, as well as the pp220-derived products, are present in equimolecular amounts in the virus particle. Immunoelectron microscopy revealed that the pp62-derived products localize at the core shell, a matrix-like domain placed between the DNA-containing nucleoid and the inner envelope, where the pp220-derived products are also localized. Pulse-chase experiments indicated that the processing of both polyprotein precursors is concomitant with virus assembly. Furthermore, using inducible ASFV recombinants, we show that pp62 processing requires the expression of the pp220 core precursor, whereas the processing of both precursors pp220 and pp62 is dependent on expression of the major capsid protein p72. Interestingly, when p72 expression is blocked, unprocessed pp220 and pp62 polyproteins assemble into aberrant zipper-like elements consisting of an elongated membrane-bound protein structure reminiscent of the core shell. Moreover, the two polyproteins, when coexpressed in COS cells, interact with each other to form zipper-like structures. Together, these findings indicate that the mature products derived from both polyproteins, which collectively account for about 30% of the virion protein mass, are the basic components of the core shell and that polyprotein processing represents a maturational process related to ASFV morphogenesis.

Complete nucleotide sequence and infectious cDNA clone of the RNA1 of a Chinese isolate of broad bean wilt virus 2.

The nucleotide sequence of the RNA1 of broad bean wilt virus 2 (BBWV2) isolate B935 has been determined from overlapping cDNA clones. It contains 5956 nucleotides in length excluding the 3' terminal poly(A) tail and contains a single long open reading frame (ORF) of 5613 nucleotides extending from nucleotide 234 to 5846. A repeated motif has been found in the 5' non-coding region. The predicted polyprotein encoded by the long ORF is 1870 amino acid in length with a molecular weight of 210 K. Amino acid sequence comparisons between portions of the BBWV2 RNA1-encoded polyprotein and proteins encoded by several species in Comoviridae revealed the putative functions of BBWV2 RNA1-encoded proteins and the same general genetic organization as that of comoviruses and nepoviruses. Based on the determined sequence, full-length cDNA clone of RNA1 designated as pU1FL was constructed. Together with transcripts from full-length cDNA clone of RNA2 (pU2FL), transcripts from pU1FL infected Chenopodium quinoa successfully.