Entropy and Fractal Dimension Study of the TDP-43 Protein Low Complexity Domain Sequence in ALS Disease Severity and SARS-CoV-2 Gene Sequences in Virulence Variability

The low complexity domain (LCD) sequence has been defined in terms of entropy using a 12 amino acid sliding window along a protein sequence in the study of disease-related genes. The amyotrophic lateral sclerosis (ALS)-related TDP-43 protein sequence with intra-LCD structural information based on cryo-EM data was published recently. An application of entropy and Higuchi fractal dimension calculations was described using the Znf521 and HAR1 sequences. A computational analysis of the intra-LCD sequence entropy and Higuchi fractal dimension values at the amino acid level and at the ATCG nucleotide level were conducted without the sliding window requirement. The computational results were consistent in predicting the intermediate entropy/fractal dimension value produced when two subsequences at two different entropy/fractal dimension values were combined. The computational method without the application of a sliding-window was extended to an analysis of the recently reported virulent genes—Orf6, Nsp6, and Orf7a—in SARS-CoV-2. The relationship between the virulence functionality and entropy values was found to have correlation coefficients between 0.84 and 0.99, using a 5% uncertainty on the cell viability data. The analysis found that the most virulent Orf6 gene sequence had the lowest nucleotide entropy and the highest protein fractal dimension, in line with extreme value theory. The Orf6 codon usage bias in relation to vaccine design was discussed.

Dynamic localization of two tobamovirus ORF6 proteins involves distinct organellar compartments

ORF6 is a small gene that overlaps the movement and coat protein genes of subgroup 1a tobamoviruses. The ORF6 protein of tomato mosaic virus (ToMV) strain L (L-ORF6), interacts in vitro with eukaryotic elongation factor 1α, and mutation of the ORF6 gene of tobacco mosaic virus (TMV) strain U1 (U1-ORF6) reduces the pathogenicity in vivo of TMV, whereas expression of this gene from two other viruses, tobacco rattle virus (TRV) and potato virus X (PVX), increases their pathogenicity. In this work, the in vivo properties of the L-ORF6 and U1-ORF6 proteins were compared to identify sequences that direct the proteins to different subcellular locations and also influence virus pathogenicity. Site-specific mutations in the ORF6 protein were made, hybrid ORF6 proteins were created in which the N-terminal and C-terminal parts were derived from the two proteins, and different subregions of the protein were examined, using expression either from a recombinant TRV vector or as a yellow fluorescent protein fusion from a binary plasmid in Agrobacterium tumefaciens. L-ORF6 caused mild necrotic symptoms in Nicotiana benthamiana when expressed from TRV, whereas U1-ORF6 caused severe symptoms including death of the plant apex. The difference in symptoms was associated with the C-terminal region of L-ORF6, which directed the protein to the endoplasmic reticulum (ER), whereas U1-ORF6 was directed initially to the nucleolus and later to the mitochondria. Positively charged residues at the N terminus allowed nucleolar entry of both U1-ORF6 and L-ORF6, but hydrophobic residues at the C terminus of L-ORF6 directed this protein to the ER.

An Adenovirus Type 5 (Ad5) Amplicon-Based Packaging Cell Line for Production of High-Capacity Helper-Independent ΔE1-E2-E3-E4 Ad5 Vectors

ABSTRACT Production of multiply deleted adenoviral (Ad) vectors with increased cloning capacity and reduced immunogenicity to adenovirus gene products requires the concomitant generation of efficient packaging cell lines. High expression levels of the complementing genes must be achieved in a coordinated fashion with viral replication. This is a particularly difficult task in light of the significant cytotoxicity displayed by adenoviral proteins. To this end, we developed a novel adenovirus-based amplicon with an Epstein-Barr virus origin of replication, Ad type 5 (Ad5) inverted terminal repeats, all Ad5 early region 2 (E2) genes, and the early region 4 (E4) open reading frame 6 (ORF6) under the control of a tetracycline-dependent promoter. The amplicon (pE2) was stably maintained in multiple copies in the nuclei of 293 cells stably expressing the Epstein-Barr virus nuclear antigen 1 (EBNA1) and allowed replication as a linear DNA upon induction of E2 and ORF6 gene expression. A stable cell line (2E2) was generated by introducing pE2 into 293EBNATet cells expressing the tetracycline-dependent transcriptional silencer and the reverse Tet transactivator (rtTA2). Upon induction with doxicycline, 2E2 cells produced higher levels of polymerase, precursor terminal protein (pTP), and DNA binding protein than noninduced 2E2 cells infected with first-generation Ad5 vector and supported efficient amplification of a multiply deleted Ad5 vector lacking E1, E2, E3, and E4 genes (Ad5ΔE1-4). The high cloning capacity of Ad5ΔE1-4 (up to 12.6 kb) was exploited to construct a vector encoding the entire hepatitis C virus (HCV) polyprotein. Infection of HeLa cells by the resulting vector showed high levels of correctly processed HCV proteins.

X-ray crystal structure of ornithine acetyltransferase from the clavulanic acid biosynthesis gene cluster

The orf6 gene from the clavulanic acid biosynthesis gene cluster encodes an OAT (ornithine acetyltransferase). Similar to other OATs the enzyme has been shown to catalyse the reversible transfer of an acetyl group from N-acetylornithine to glutamate. OATs are Ntn (N-terminal nucleophile) enzymes, but are distinct from the better-characterized Ntn hydrolase enzymes as they catalyse acetyl transfer rather than a hydrolysis reaction. In the present study, we describe the X-ray crystal structure of the OAT, corresponding to the orf6 gene product, to 2.8 Å (1 Å=0.1 nm) resolution. The larger domain of the structure consists of an αββα sandwich as in the structures of Ntn hydrolase enzymes. However, differences in the connectivity reveal that OATs belong to a structural family different from that of other structurally characterized Ntn enzymes, with one exception: unexpectedly, the αββα sandwich of ORF6 (where ORF stands for open reading frame) displays the same fold as an DmpA (L-aminopeptidase D-ala-esterase/amidase from Ochrobactrum anthropi), and so the OATs and DmpA form a new structural subfamily of Ntn enzymes. The structure reveals an α2β2-heterotetrameric oligomerization state in which the intermolecular interface partly defines the active site. Models of the enzyme–substrate complexes suggest a probable oxyanion stabilization mechanism as well as providing insight into how the enzyme binds its two differently charged substrates.

The adenovirus E4 ORF6 and E1b 55 kDa proteins cooperate in a p53-independent manner to enhance transduction by recombinant adeno-associated virus vectors

The observation that exposure of target cells to genotoxic stress or adenovirus infection enhances recombinant adeno-associated virus (rAAV) transduction is an important lead towards defining the rAAV transduction mechanism, and has significant implications for the exploitation of rAAV in gene therapy applications. The adenovirus-mediated enhancement of rAAV transduction has been mapped to the E4 ORF6 gene, and expression of E4 ORF6 alone has been considered necessary and sufficient to mediate this effect. Since p53 subserves an important function in the cellular response to genotoxic stress, and interacts with the E4 ORF6 gene product during adenovirus infection, we hypothesized that p53 function might be essential to the rAAV enhancement resulting from these cellular insults. In the current study, using the p53-null cell lines H1299 and Saos-2, we find that p53 is not essential to either genotoxic stress or adenovirus-mediated enhancement of rAAV transduction. We further demonstrate using HeLa, H1299 and Saos-2 cells that E4 ORF6 expression alone is not sufficient to enhance rAAV transduction and that coexpression of the adenovirus E1b 55 kDa protein is necessary. Together, these observations indicate that the mechanism by which adenovirus infection enhances rAAV transduction involves cooperative and interdependent functions of the E4 ORF6 and E1b 55 kDa proteins that are p53-independent.

Identification and Expression of a Mycoplasma gallisepticum Surface Antigen Recognized by a Monoclonal Antibody Capable of Inhibiting Both Growth and Metabolism

ABSTRACT In order to identify antigenic proteins of Mycoplasma gallisepticum, monoclonal antibodies (MAbs) against virulentM. gallisepticum R strain were produced in mice. MAb 35A6 was selected for its abilities to inhibit both growth and metabolism ofM. gallisepticum in vitro. The MAb recognized a membrane protein with an apparent molecular mass of 120 kDa. The corresponding gene, designated the mgc3 gene, was cloned from an M. gallisepticum genomic DNA expression library and sequenced. The mgc3 gene is a homologue of the ORF6 gene encoding 130-kDa protein in the P1 operon of M. pneumoniae and is localized downstream of the mgc1 gene, a homologue of the P1 gene. To assess the characteristics of MGC3 protein, all 10 TGA codons in the mgc3 gene, which encode a tryptophan in the Mycoplasma species, were replaced with TGG codons, and recombinant fowlpox viruses (FPV) harboring the altered mgc3 gene were constructed. One of the recombinant FPVs was improved to express MGC3 protein on the cell surface in which the signal peptide of MGC3 protein was replaced with one from Marek's disease virus gB. These results should provide the impetus to develop a vaccine based on MGC3 protein which can induce antibodies with both growth inhibition and metabolic-inhibition activities using a recombinant FPV.