Diagnosis of Ruminant Viral Diseases with Loop-Mediated Isothermal Amplification.

Infectious diseases in livestock industry are major problems for animal health, food safety, and the economy. Zoonotic diseases from farm animals are significant threat to human population as well. These are notifiable diseases listed by the World Organization for Animal Health (OIE). Rapid diagnostic methods can help keep infectious diseases under control in herds. Loop-mediated isothermal amplification (LAMP) is a simple and rapid nucleic acid amplification method that is studied widely for detection of many infectious diseases in the field. LAMP allows biosensing of target DNA or RNA under isothermal conditions with high specificity in a short period of time. An untrained user can analyze results based on color change or turbidity. Here we review LAMP assays to diagnose OIE notifiable ruminant viral diseases in literature highlighting properties of LAMP method considering what is expected from an efficient, field usable diagnostic test.

Exploiting drug-resistant enzymes as tools to identify thienopyrimidinone inhibitors of human immunodeficiency virus reverse transcriptase-associated ribonuclease H.

The thienopyrimidinone 5,6-dimethyl-2-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4(3H)-one (DNTP) occupies the interface between the p66 ribonuclease H (RNase H) domain and p51 thumb of human immunodeficiency virus reverse transcriptase (HIV RT), thereby inducing a conformational change incompatible with catalysis. Here, we combined biochemical characterization of 39 DNTP derivatives with antiviral testing of selected compounds. In addition to wild-type HIV-1 RT, derivatives were evaluated with rationally designed, p66/p51 heterodimers exhibiting high-level DNTP sensitivity or resistance. This strategy identified 3',4'-dihydroxyphenyl (catechol) substituted thienopyrimidinones with submicromolar in vitro activity against both wild type HIV-1 RT and drug-resistant variants. Thermal shift analysis indicates that, in contrast to active site RNase H inhibitors, these thienopyrimidinones destabilize the enzyme, in some instances reducing the Tm by 5 °C. Importantly, catechol-containing thienopyrimidinones also inhibit HIV-1 replication in cells. Our data strengthen the case for allosteric inhibition of HIV RNase H activity, providing a platform for designing improved antagonists for use in combination antiviral therapy.

Multimode, cooperative mechanism of action of allosteric HIV-1 integrase inhibitors.

The multifunctional HIV-1 enzyme integrase interacts with viral DNA and its key cellular cofactor LEDGF to effectively integrate the reverse transcript into a host cell chromosome. These interactions are crucial for HIV-1 replication and present attractive targets for antiviral therapy. Recently, 2-(quinolin-3-yl) acetic acid derivatives were reported to selectively inhibit the integrase-LEDGF interaction in vitro and impair HIV-1 replication in infected cells. Here, we show that this class of compounds impairs both integrase-LEDGF binding and LEDGF-independent integrase catalytic activities with similar IC(50) values, defining them as bona fide allosteric inhibitors of integrase function. Furthermore, we show that 2-(quinolin-3-yl) acetic acid derivatives block the formation of the stable synaptic complex between integrase and viral DNA by allosterically stabilizing an inactive multimeric form of integrase. In addition, these compounds inhibit LEDGF binding to the stable synaptic complex. This multimode mechanism of action concordantly results in cooperative inhibition of the concerted integration of viral DNA ends in vitro and HIV-1 replication in cell culture. Our findings, coupled with the fact that high cooperativity of antiviral inhibitors correlates with their increased instantaneous inhibitory potential, an important clinical parameter, argue strongly that improved 2-(quinolin-3-yl) acetic acid derivatives could exhibit desirable clinical properties.

Differential ordering of the protein backbone and side chains during protein folding revealed by site-specific recombinant infrared probes.

The time scale for ordering of the polypeptide backbone relative to the side chains is a critical issue in protein folding. The interplay between ordering of the backbone and ordering of the side chains is particularly important for the formation of ß-sheet structures, as the polypeptide chain searches for the native stabilizing cross-strand interactions. We have studied these issues in the N-terminal domain of protein L9 (NTL9), a model protein with mixed α/ß structure. We have developed a general approach for introducing site-specific IR probes for the side chains (azide) and backbone ((13)C═(18)O) using recombinant protein expression. Temperature-jump time-resolved IR spectroscopy combined with site-specific labeling enables independent measurement of the respective backbone and side-chain dynamics with single residue resolution. We have found that side-chain ordering in a key region of the ß-sheet structure occurs on a slower time scale than ordering of the backbone during the folding of NTL9, likely as a result of the transient formation of non-native side-chain interactions.

Modulation of p-cyanophenylalanine fluorescence by amino acid side chains and rational design of fluorescence probes of alpha-helix formation.

p-Cyanophenylalanine is an extremely useful fluorescence probe of protein structure that can be recombinantly and chemically incorporated into proteins. The probe has been used to study protein folding, protein-membrane interactions, protein-peptide interactions, and amyloid formation; however, the factors that control its fluorescence are not fully understood. Hydrogen bonding to the cyano group is known to play a major role in modulating the fluorescence quantum yield, but the role of potential side-chain quenchers has not yet been elucidated. A systematic study of the effects of different side chains on p-cyanophenylalanine fluorescence is reported. Tyr is found to have the largest effect followed by deprotonated His, Met, Cys, protonated His, Asn, Arg, and protonated Lys. Deprotonated amino groups are much more effective fluorescence quenchers than protonated amino groups. Free neutral imidazole and hydroxide ion are also effective quenchers of p-cyanophenylalanine fluorescence with Stern-Volmer constants of 39.8 and 22.1 M(-1), respectively. The quenching of p-cyanophenylalanine fluorescence by specific side chains is exploited in developing specific, high-sensitivity, fluorescence probes of helix formation. The approach is demonstrated with Ala-based peptides that contain a p-cyanophenylalanine-His or a p-cyanophenylalanine-Tyr pair located at positions i and i + 4. The p-cyanophenylalanine-His pair is most useful when the His side chain is deprotonated and is, thus, complementary to the Trp-His pair which is most sensitive when the His side chain is protonated.

Interpretation of p-cyanophenylalanine fluorescence in proteins in terms of solvent exposure and contribution of side-chain quenchers: a combined fluorescence, IR and molecular dynamics study.

The use of noncoded amino acids as spectroscopic probes of protein folding and function is growing rapidly, in large part because of advances in the methodology for their incorporation. Recently p-cyanophenylalanine has been employed as a fluorescence and IR probe, as well as a FRET probe to study protein folding, protein-membrane interactions, protein-protein interactions and amyloid formation. The probe has been shown to be exquisitely sensitive to hydrogen bonding interactions involving the cyano group, and its fluorescence quantum yield increases dramatically when it is hydrogen bonded. However, a detailed understanding of the factors which influence its fluorescence is required to be able to use this popular probe accurately. Here we demonstrate the recombinant incorporation of p-cyanophenylalanine in the N-terminal domain of the ribosomal protein L9. Native state fluorescence is very low, which suggests that the group is sequestered from solvent; however, IR measurements and molecular dynamics simulations show that the cyano group is exposed to solvent and forms hydrogen bonds to water. Analysis of mutant proteins and model peptides demonstrates that the reduced native state fluorescence is caused by the effective quenching of p-cyanophenylalanine fluorescence via FRET to tyrosine side-chains. The implications for the interpretation of p-cyanophenylalanine fluorescence measurements and FRET studies are discussed.