Subunit-specific mutational analysis of residue N348 in HIV-1 reverse transcriptase

<p>Abstract</p> <p>Background</p> <p>N348I in HIV-1 reverse transcriptase (RT) confers resistance to zidovudine (AZT) and nevirapine. Biochemical studies demonstrated that N348I indirectly increases AZT resistance by decreasing the frequency of secondary ribonuclease H (RNase H) cleavages that reduce the RNA/DNA duplex length of the template/primer (T/P) and diminish the efficiency of AZT-monophosphate (MP) excision. By contrast, there is some discrepancy in the literature in regard to the mechanisms associated with nevirapine resistance: one study suggested that it is due to decreased inhibitor binding while others suggest that it may be related to the decreased RNase H cleavage phenotype. From a structural perspective, N348 in both subunits of RT resides distal to the enzyme's active sites, to the T/P binding tract and to the nevirapine-binding pocket. As such, the structural mechanisms associated with the resistance phenotypes are not known.</p> <p>Results</p> <p>Using a novel modelled structure of RT in complex with an RNA/DNA T/P, we identified a putative interaction between the β14-β15 loop in the p51 subunit of RT and the RNA template. Substitution of the asparagine at codon 348 in the p51 subunit with either isoleucine or leucine abrogated the observed protein-RNA interaction, thus, providing a possible explanation for the decreased RNase H phenotype. By contrast, alanine or glutamine substitutions exerted no effect. To validate this model, we introduced the N348I, N348L, N348A and N348Q mutations into RT and purified enzymes that contained subunit-specific mutations. N348I and N348L significantly decreased the frequency of secondary RNase H cleavages and increased the enzyme's ability to excise AZT-MP. As predicted by the modelling, this phenotype was due to the mutation in the p51 subunit of RT. By contrast, the N348A and N348Q RTs exhibited RNase H cleavage profiles and AZT-MP excision activities similar to the wild-type enzyme. All N348 mutant RTs exhibited decreased nevirapine susceptibility, although the N348I and N348L mutations conferred higher fold resistance values compared to N348A and N348Q. Nevirapine resistance was also largely due to the mutation present in the p51 subunit of RT.</p> <p>Conclusions</p> <p>This study demonstrates that N348I-mediated AZT and nevirapine resistance is due to the mutation in the p51 subunit of RT.</p

Mechanisms by which Nonnucleoside Reverse Transcriptase Inhibitors Block HIV-1 Replication Alone and in Combination with other Reverse Transcriptase Inhibitors

Inhibition of reverse transcriptase (RT) is a vital tactic in the prevention of human immunodeficiency virus 1 (HIV-1). Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are a class of compounds demonstrated to act as allosteric inhibitors of RT DNA polymerization. However, several lines of evidence suggest that polymerization may not be the main mechanism of inhibition of reverse transcription. It has been demonstrated that NNRTIs also have the ability to modulate RT ribonuclease (RNase) H cleavage. Additionally, recent evidence suggests that resistance to chain-terminating nucleoside reverse transcriptase inhibitors (NRTIs) is dependent on a balance between the polymerase and RNase H activities of the enzyme. In light of this, I hypothesize that NNRTIs block reverse transcription by exerting effects on both the DNA polymerase and RNase H active sites of the enzyme, significantly disrupting the equilibrium between these two enzymatic activities. Therefore, the ability for NNRTIs to be combined with other classes of RT inhibitors in antiretroviral therapies will depend on how these compounds respond to the NNRTI-induced shift in the polymerase/RNase H activity equilibrium. This study demonstrates that NNRTIs cause the accelerated appearance of secondary RNase H cleavage products that have decreased RNA/DNA hybrid structures. As a result, these template/primers(T/Ps) are not sufficient substrates for NRTI removal and therefore, excision is less efficient in the presence of NNRTIs. Additionally, fluorescent resonance energy transfer experiments demonstrate that NNRTIs cause a shift in the binding of RT and T/P such that the RNase H domain is moved away from the 5'end of the primer. Finally, subunit-specific analysis shows that resistance to RTI combination therapy facilitated by the N348I mutation is a result of effects from the p51 subunit. I propose that the binding of NNRTIs cause RT to bind to T/P in a polymerase-incompetent mode, resulting in decreased polymerization and shorted RNase H cleavage products. Additionally, N348I can facilitate dual resistance by favoring the polymerase-competent binding mode. This work is of public health significance because it lays the foundation for the development of new reverse transcriptase inhibitors and highlights the importance of resistance in the connection domain of HIV-1 RT

Early detection of Mycobacterium avium subsp. paratuberculosis infection in cattle with multiplex-bead based immunoassays

Johne’s Disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), results in significant economic loss to livestock production. The early detection of MAP infection in animals with extant serological assays has remained challenging due to the low sensitivity of commercially available ELISA tests, a fact that has hampered the development of effective JD control programs. Our recent protein microarray-based studies identified several promising candidate antigens that are immunogenic during different stages of MAP infection. To evaluate these antigens for use in diagnostic assays and reliably identify animals with MAP infection, a multiplex (Luminex®) assay was developed using color-coded flourescent beads coupled to 6 MAP recombinant proteins and applied to screen 180 serum and 90 milk samples from cows at different stages of MAP infection including negative (NL), fecal test positive/ELISA negative (F+E-), and fecal positive/ELISA positive (F+E+). The results show that while serum antibody reactivities to each of the 6 anti-gens were highest in F+E+ group, antibody reactivity to three of the six antigens were identified in the F+E- group, suggesting that these three antigens are expressed and provoke antibody responses during the early infection stages with MAP. Further, antibodies against all six antigens were elevated in milk samples from both the F+E- and F+E+ groups in comparison to the NL group (

Identification of Sero-Diagnostic Antigens for the Early Diagnosis of Johne’s Disease using MAP Protein Microarrays

Considerable effort has been directed toward controlling Johne’s disease (JD), a chronic granulomatous intestinal inflammatory disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) in cattle and other ruminants. However, progress in controlling the spread of MAP infection has been impeded by the lack of reliable diagnostic tests that can identify animals early in the infection process and help break the transmission chain. To identify reliable antigens for early diagnosis of MAP infection, we constructed a MAP protein array with 868 purified recombinant MAP proteins, and screened a total of 180 well-characterized serum samples from cows assigned to 4 groups based on previous serological and fecal test results: negative low exposure (NL, n = 30); negative high exposure (NH, n = 30); fecal- positive, ELISA-negative (F + E−, n = 60); and both fecal- and ELISA-positive (F + E+, n = 60). The analyses identified a total of 49 candidate antigens in the NH, F + E−, and F + E+ with reactivity compared with the NL group (p \u3c 0.01), a majority of which have not been previously identified. While some of the antigens were identified as reactive in only one of the groups, others showed reactivity in multiple groups, including NH (n = 28), F + E− (n = 26), and F + E+ (n = 17) groups. Using combinations of top reactive antigens in each group, the results reveal sensitivities of 60.0%, 73.3%, and 81.7% in the NH, F + E−, and F + E+, respectively at 90% specificity, suggesting that early detection of infection in animals may be possible and enable better opportunities to reduce within herd transmission that may be otherwise missed by traditional serological assays that are biased towards more heavily infected animals. Together, the results suggest that several of the novel candidate antigens identified in this study, particularly those that were reactive in the NH and F + E− groups, have potential utility for the early sero-diagnosis of MAP infection

Transcriptional Innate Immune Response of the Developing Chicken Embryo to Newcastle Disease Virus Infection

Traditional approaches to assess the immune response of chickens to infection are through animal trials, which are expensive, require enhanced biosecurity, compromise welfare, and are frequently influenced by confounding variables. Since the chicken embryo becomes immunocompetent prior to hatch, we here characterized the transcriptional response of selected innate immune genes to Newcastle disease virus (NDV) infection in chicken embryos at days 10, 14, and 18 of embryonic development. The results suggest that the innate immune response 72 h after challenge of 18-day chicken embryo is both consistent and robust. The expression of CCL5, Mx1, and TLR3 in lung tissues of NDV challenged chicken embryos from the outbred Kuroiler and Tanzanian local ecotype lines showed that their expression was several orders of magnitude higher in the Kuroiler than in the local ecotypes. Next, the expression patterns of three additional innate-immunity related genes, IL-8, IRF-1, and STAT1, were examined in the highly congenic Fayoumi (M5.1 and M15.2) and Leghorn (Ghs6 and Ghs13) sublines that differ only at the microchromosome bearing the major histocompatibility locus. The results show that the Ghs13 Leghorn subline had a consistently higher expression of all genes except IL-8 and expression seemed to be subline-dependent rather than breed-dependent, suggesting that the innate immune response of chicken embryos to NDV infection may be genetically controlled by the MHC-locus. Taken together, the results suggest that the chicken embryo may represent a promising model to studying the patterns and sources of variation of the avian innate immune response to infection with NDV and related pathogens

Molecular species identification of bushmeat recovered from the Serengeti ecosystem in Tanzania.

This research article published by PLOS ONE, 2020Bushmeat harvesting and consumption represents a potential risk for the spillover of endemic zoonotic pathogens, yet remains a common practice in many parts of the world. Given that the harvesting and selling of bushmeat is illegal in Tanzania and other parts of Africa, the supply chain is informal and may include hunters, whole-sellers, retailers, and individual resellers who typically sell bushmeat in small pieces. These pieces are often further processed, obscuring species-identifying morphological characteristics, contributing to incomplete or mistaken knowledge of species of origin and potentially confounding assessments of pathogen spillover risk and bushmeat offtake. The current investigation sought to identify the species of origin and assess the concordance between seller-reported and laboratory-confirmed species of origin of bushmeat harvested from in and around the Serengeti National Park in Tanzania. After obtaining necessary permits, the species of origin of a total of 151 bushmeat samples purchased from known intermediaries from 2016 to 2018 were characterized by PCR and sequence analysis of the cytochrome B (CytB) gene. Based on these sequence analyses, 30%, 95% Confidence Interval (CI: 24.4-38.6) of bushmeat samples were misidentified by sellers. Misreporting amongst the top five source species (wildebeest, buffalo, impala, zebra, and giraffe) ranged from 20% (CI: 11.4-33.2) for samples reported as wildebeest to 47% (CI: 22.2-72.7) for samples reported as zebra although there was no systematic bias in reporting. Our findings suggest that while misreporting errors are unlikely to confound wildlife offtake estimates for bushmeat consumption within the Serengeti ecosystem, the role of misreporting bias on the risk of spillover events of endemic zoonotic infections from bushmeat requires further investigation