A Survey of the Combinatorial Paraphernalia in Protein Synthesis

Statement of the Problem: The combinatorial paraphernalia in protein synthesis to be surveyed are multifarious, embracing, phenomena, processes, activities and materials, all characterized by plurality and dissimilarity. The materials usable are phenomenal and must be a set of discrete plural and dissimilar objects, e.g. the RNA four bases of Adenine, Uracil, Guanine, Cytosine (A,U,G,C) for the activity of permutation for building genetic code. Sequences for protein type sequence composition, proliferation and diversification as inherent in protein synthesis. Methodology and Theoretical Orientation: We are in for combinatorics which is the scientific study of the phenomenon of input/output productivity exhibited by a duality of numeral entities as in permutation of specified set (n) of dissimilar discrete plural. Things and selection (r) of them. The Dalina apparatus of Input/Output Multiplicative Replication system equipped with Square Kinematics View Mixing Technique sourced from inchoate Numeration Science literature being developed by this author is in use for the computation of 4 from 4 permutations of RNA four bases, A,U,G,C constituting the 24 quadruplet genetic code as the workforce in protein synthesis. Findings: The combinatorial paraphernalia in protein synthesis identified and surveyed comprise 14 characteristics, 3 materials and 11 processes/operatives. Conclusion and Significance: The relevance of the several identified and surveyed combinatorial paraphernalia in protein synthesis has been demonstrated by the test of agreeability with the working of the Dalina apparatus of Input/ Output Multiplicative Replication Combinatorial System using the Square Kinematics View Mixing technique for the computation of permutations of RNA four bases A,U,G,C making up the 24 quadruplet genetic code as the workforce in protein synthesis for the substance of all plants and animals throughout CREATION.

Comparison of Zaire and Bundibugyo ebolavirus polymerase complexes and susceptibility to antivirals through a newly developed Bundibugyo minigenome system

Members of the genus Ebolavirus cause lethal disease in humans with Zaire ebolavirus (EBOV) being the most pathogenic (up to 90% morality) and Bundibugyo ebolavirus (BDBV) the least pathogenic (∼37% mortality). Historically, there has been a lack of research on BDBV and there is no means to study BDBV outside of a high-containment laboratory. Here, we describe a minigenome replication system to study BDBV transcription and compare the efficacy of small molecule inhibtors between EBOV and BDBV. Using this system, we examined the ability of the polymerase complex proteins from EBOV and BDBV to interact and form a functional unit as well as the impact of the genomic untranslated ends, known to contain important signals for transcription (3’-untranslated region) and replication (5’-untranslated region). Varying levels of compatibility were observed between proteins of the polymerase complex from each ebolavirus resulting in differences in genome transcription efficiency. Most pronounced was the effect of the nucleoprotein and the 3’-untranslated region. These data suggest that there are intrinsic specificities in the polymerase complex and untranslated signaling regions that could offer insight regarding observed pathogenic differences. Further adding to the differences in the polymerase complexes, post-transfection/infection treatment with the compound remdesivir (GS-5734) showed a greater inhibitory effect against BDBV compared to EBOV. The delayed growth kinetics of BDBV and the greater susceptibility to polymerase inhibitors indicate that disruption of the polymerase complex may be a viable target for therapeutics. Importance Ebolavirus disease is a viral infection and is fatal in 25-90% of cases, depending on the viral species and the amount of supportive care available. Two species have caused outbreaks in the Democratic Republic of the Congo, Zaire ebolavirus (EBOV) and Bundibugyo ebolavirus (BDBV). Pathogenesis and clinical outcome differ between these two species but there is still limited information regarding the viral mechanism for these differences. Previous studies suggest that BDBV replicates slower than EBOV but it is unknown if this is due to differences in the polymerase complex and its role in transcription and replication. This study details the construction of a minigenome replication system which can be used in a biosafety level (BSL) 2 laboartory. This system will be important for studying the polymerase complex of BDBV and comparing it with other filoviruses and can be used as a tool for screening inhibitors of viral growth.

Construction of a self-directed replication system for label-free and real-time sensing of repair glycosylases with zero background

We construct a self-directed replication system for label-free and real-time sensing of repair glycosylases with zero background.

Rescue of tomato spotted wilt virus entirely from complementary DNA clones

Negative-stranded/ambisense RNA viruses (NSVs) include not only dangerous pathogens of medical importance but also serious plant pathogens of agronomic importance. Tomato spotted wilt virus (TSWV) is one of the most important plant NSVs, infecting more than 1,000 plant species, and poses major threats to global food security. The segmented negative-stranded/ambisense RNA genomes of TSWV, however, have been a major obstacle to molecular genetic manipulation. In this study, we report the complete recovery of infectious TSWV entirely from complementary DNA (cDNA) clones. First, a replication- and transcription-competent minigenome replication system was established based on 35S-driven constructs of the S(−)-genomic (g) or S(+)-antigenomic (ag) RNA template, flanked by the 5′ hammerhead and 3′ ribozyme sequence of hepatitis delta virus, a nucleocapsid (N) protein gene and codon-optimized viral RNA-dependent RNA polymerase (RdRp) gene. Next, a movement-competent minigenome replication system was developed based on M(−)-gRNA, which was able to complement cell-to-cell and systemic movement of reconstituted ribonucleoprotein complexes (RNPs) of S RNA replicon. Finally, infectious TSWV and derivatives carrying eGFP reporters were rescued in planta via simultaneous expression of full-length cDNA constructs coding for S(+)-agRNA, M(−)-gRNA, and L(+)-agRNA in which the glycoprotein gene sequence of M(−)-gRNA was optimized. Viral rescue occurred with the addition of various RNAi suppressors including P19, HcPro, and γb, but TSWV NSs interfered with the rescue of genomic RNA. This reverse genetics system for TSWV now allows detailed molecular genetic analysis of all aspects of viral infection cycle and pathogenicity.