Negative-Strand RNA Virus L Proteins: One Machine, Many Activities.

Structures of L proteins from La Crosse orthobunyavirus and vesicular stomatitis virus reveal insights into RNA synthesis and distinctive mRNA capping mechanisms of segmented and non-segmented negative-sense single-strand RNA viruses.

Structures illustrate step-by-step mitochondrial transcription initiation.

Transcription initiation is a key regulatory step in gene expression during which RNA polymerase (RNAP) initiates RNA synthesis de novo, and the synthesized RNA at a specific length triggers the transition to the elongation phase. Mitochondria recruit a single-subunit RNAP and one or two auxiliary factors to initiate transcription. Previous studies have revealed the molecular architectures of yeast1 and human2 mitochondrial RNAP initiation complexes (ICs). Here we provide a comprehensive, stepwise mechanism of transcription initiation by solving high-resolution cryogenic electron microscopy (cryo-EM) structures of yeast mitochondrial RNAP and the transcription factor Mtf1 catalysing two- to eight-nucleotide RNA synthesis at single-nucleotide addition steps. The growing RNA-DNA is accommodated in the polymerase cleft by template scrunching and non-template reorganization, creating stressed intermediates. During early initiation, non-template strand scrunching and unscrunching destabilize the short two- and three-nucleotide RNAs, triggering abortive synthesis. Subsequently, the non-template reorganizes into a base-stacked staircase-like structure supporting processive five- to eight-nucleotide RNA synthesis. The expanded non-template staircase and highly scrunched template in IC8 destabilize the promoter interactions with Mtf1 to facilitate initiation bubble collapse and promoter escape for the transition from initiation to the elongation complex (EC). The series of transcription initiation steps, each guided by the interplay of multiple structural components, reveal a finely tuned mechanism for potential regulatory control.

Cryo-EM Structures Reveal Transcription Initiation Steps by Yeast Mitochondrial RNA Polymerase.

Mitochondrial RNA polymerase (mtRNAP) is crucial in cellular energy production, yet understanding of mitochondrial DNA transcription initiation lags that of bacterial and nuclear DNA transcription. We report structures of two transcription initiation intermediate states of yeast mtRNAP that explain promoter melting, template alignment, DNA scrunching, abortive synthesis, and transition into elongation. In the partially melted initiation complex (PmIC), transcription factor MTF1 makes base-specific interactions with flipped non-template (NT) nucleotides "AAGT" at -4 to -1 positions of the DNA promoter. In the initiation complex (IC), the template in the expanded 7-mer bubble positions the RNA and NTP analog UTPαS, while NT scrunches into an NT loop. The scrunched NT loop is stabilized by the centrally positioned MTF1 C-tail. The IC and PmIC states coexist in solution, revealing a dynamic equilibrium between two functional states. Frequent scrunching/unscruching transitions and the imminent steric clashes of the inflating NT loop and growing RNA:DNA with the C-tail explain abortive synthesis and transition into elongation.

Structural Basis of Transcription Inhibition by Fidaxomicin (Lipiarmycin A3).

Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial RNA polymerase (RNAP). Here we report a cryo-EM structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5-Å resolution. The structure shows that Lpm binds at the base of the RNAP "clamp." The structure exhibits an open conformation of the RNAP clamp, suggesting that Lpm traps an open-clamp state. Single-molecule fluorescence resonance energy transfer experiments confirm that Lpm traps an open-clamp state and define effects of Lpm on clamp dynamics. We suggest that Lpm inhibits transcription by trapping an open-clamp state, preventing simultaneous interaction with promoter -10 and -35 elements. The results account for the absence of cross-resistance between Lpm and other RNAP inhibitors, account for structure-activity relationships of Lpm derivatives, and enable structure-based design of improved Lpm derivatives.

Structural Basis of Mycobacterium tuberculosis Transcription and Transcription Inhibition.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb RNAP, alone and in complex with Rif, at 3.8-4.4 Å resolution. The results identify an Mtb-specific structural module of Mtb RNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. We also report non-Rif-related compounds-Nα-aroyl-N-aryl-phenylalaninamides (AAPs)-that potently and selectively inhibit Mtb RNAP and Mtb growth, and we report crystal structures of Mtb RNAP in complex with AAPs. AAPs bind to a different site on Mtb RNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-administered with Rif.

Cryo-EM structures of wild-type and E138K/M184I mutant HIV-1 RT/DNA complexed with inhibitors doravirine and rilpivirine.

Structures trapping a variety of functional and conformational states of HIV-1 reverse transcriptase (RT) have been determined by X-ray crystallography. These structures have played important roles in explaining the mechanisms of catalysis, inhibition, and drug resistance and in driving drug design. However, structures of several desired complexes of RT could not be obtained even after many crystallization or crystal soaking experiments. The ternary complexes of doravirine and rilpivirine with RT/DNA are such examples. Structural study of HIV-1 RT by single-particle cryo-electron microscopy (cryo-EM) has been challenging due to the enzyme's relatively smaller size and higher flexibility. We optimized a protocol for rapid structure determination of RT complexes by cryo-EM and determined six structures of wild-type and E138K/M184I mutant RT/DNA in complexes with the nonnucleoside inhibitors rilpivirine, doravirine, and nevirapine. RT/DNA/rilpivirine and RT/DNA/doravirine complexes have structural differences between them and differ from the typical conformation of nonnucleoside RT inhibitor (NNRTI)-bound RT/double-stranded DNA (dsDNA), RT/RNA-DNA, and RT/dsRNA complexes; the primer grip in RT/DNA/doravirine and the YMDD motif in RT/DNA/rilpivirine have large shifts. The DNA primer 3'-end in the doravirine-bound structure is positioned at the active site, but the complex is in a nonproductive state. In the mutant RT/DNA/rilpivirine structure, I184 is stacked with the DNA such that their relative positioning can influence rilpivirine in the pocket. Simultaneously, E138K mutation opens the NNRTI-binding pocket entrance, potentially contributing to a faster rate of rilpivirine dissociation by E138K/M184I mutant RT, as reported by an earlier kinetic study. These structural differences have implications for understanding molecular mechanisms of drug resistance and for drug design.

Combination of ganciclovir and trifluridine prevents drug-resistance emergence in HSV-1.

Ocular herpes simplex virus 1 (HSV-1) infections can lead to visual impairment. Long-term acyclovir (ACV) prophylaxis reduces the frequency of recurrences but is associated with drug resistance. Novel therapies are needed to treat drug-resistant HSV-1 infections. Here, we describe the effects of trifluridine (TFT) in combination with ACV or ganciclovir (GCV) on HSV-1 replication and drug-resistance emergence. Wild-type HSV-1 was grown under increasing doses of one antiviral (ACV, GCV, or TFT) or combinations thereof (ACV + TFT or GCV + TFT). Virus cultures were analyzed by Sanger sequencing and deep sequencing of the UL23 [thymidine kinase (TK)] and UL30 [DNA polymerase (DP)] genes. The phenotypes of novel mutations were determined by cytopathic effect reduction assays. TFT showed overall additive anti-HSV-1 activity with ACV and GCV. Five passages under ACV, GCV, or TFT drug pressure gave rise to resistance mutations, primarily in the TK. ACV + TFT and GCV + TFT combinatory pressure induced mutations in the TK and DP. The DP mutations were mainly located in terminal regions, outside segments that typically carry resistance mutations. TK mutations (R163H, A167T, and M231I) conferring resistance to all three nucleoside analogs (ACV, TFT, and GCV) emerged under ACV, TFT, ACV + TFT pressure and under GCV + TFT pressure initiated from suboptimal drug concentrations. However, higher doses of GCV and TFT prevented drug resistance in the resistance selection experiments. In summary, we identified novel mutations conferring resistance to nucleoside analogs, including TFT, and proposed that GCV + TFT combination therapy may be an effective strategy to prevent the development of drug resistance.

The RNA polymerase "switch region" is a target for inhibitors.

The alpha-pyrone antibiotic myxopyronin (Myx) inhibits bacterial RNA polymerase (RNAP). Here, through a combination of genetic, biochemical, and structural approaches, we show that Myx interacts with the RNAP "switch region"--the hinge that mediates opening and closing of the RNAP active center cleft--to prevent interaction of RNAP with promoter DNA. We define the contacts between Myx and RNAP and the effects of Myx on RNAP conformation and propose that Myx functions by interfering with opening of the RNAP active-center cleft during transcription initiation. We further show that the structurally related alpha-pyrone antibiotic corallopyronin (Cor) and the structurally unrelated macrocyclic-lactone antibiotic ripostatin (Rip) function analogously to Myx. The RNAP switch region is distant from targets of previously characterized RNAP inhibitors, and, correspondingly, Myx, Cor, and Rip do not exhibit crossresistance with previously characterized RNAP inhibitors. The RNAP switch region is an attractive target for identification of new broad-spectrum antibacterial therapeutic agents.

Lung cancer histopathological image classification using wavelets and AlexNet.

Among malignant tumors, lung cancer has the highest morbidity and fatality rates worldwide. Screening for lung cancer has been investigated for decades in order to reduce mortality rates of lung cancer patients, and treatment options have improved dramatically in recent years. Pathologists utilize various techniques to determine the stage, type, and subtype of lung cancers, but one of the most common is a visual assessment of histopathology slides. The most common subtypes of lung cancer are adenocarcinoma and squamous cell carcinoma, lung benign, and distinguishing between them requires visual inspection by a skilled pathologist. The purpose of this article was to develop a hybrid network for the categorization of lung histopathology images, and it did so by combining AlexNet, wavelet, and support vector machines. In this study, we feed the integrated discrete wavelet transform (DWT) coefficients and AlexNet deep features into linear support vector machines (SVMs) for lung nodule sample classification. The LC25000 Lung and colon histopathology image dataset, which contains 5,000 digital histopathology images in three categories of benign (normal cells), adenocarcinoma, and squamous carcinoma cells (both are cancerous cells) is used in this study to train and test SVM classifiers. The study results of using a 10-fold cross-validation method achieve an accuracy of 99.3% and an area under the curve (AUC) of 0.99 in classifying these digital histopathology images of lung nodule samples.

Structure of HIV-1 RT/dsRNA initiation complex prior to nucleotide incorporation.

The initiation phase of HIV reverse transcription has features that are distinct from its elongation phase. The first structure of a reverse transcription initiation complex (RTIC) that trapped the complex after incorporation of one ddCMP nucleotide was published recently [Larsen KP, et al. (2018) Nature 557:118-122]. Here we report a crystal structure of a catalytically active HIV-1 RT/dsRNA complex that mimics the state of the RTIC before the first nucleotide incorporation. The structure reveals that the dsRNA-bound conformation of RT is closer to that of RT bound to a nonnucleoside RT inhibitor (NNRTI) and dsDNA; a hyperextended thumb conformation helps to accommodate the relatively wide dsRNA duplex. The RNA primer 3' end is positioned 5 Å away from the polymerase site; however, unlike in an NNRTI-bound state in which structural elements of RT restrict the movement of the primer, the primer terminus of dsRNA is not blocked from reaching the active site of RT. The observed structural changes and energetic cost of bringing the primer 3' end to the priming site are hypothesized to explain the slower nucleotide incorporation rate of the RTIC. An unusual crystal lattice interaction of dsRNA with its symmetry mate is reminiscent of the RNA architecture within the extended vRNA-tRNALys3 in the RTIC. This RT/dsRNA complex captures the key structural characteristics and components of the RTIC, including the RT conformational changes and interactions with the dsRNA primer-binding site region, and these features have implications for better understanding of RT initiation.

Structure, mechanism, and regulation of mitochondrial DNA transcription initiation.

Mitochondria are specialized compartments that produce requisite ATP to fuel cellular functions and serve as centers of metabolite processing, cellular signaling, and apoptosis. To accomplish these roles, mitochondria rely on the genetic information in their small genome (mitochondrial DNA) and the nucleus. A growing appreciation for mitochondria's role in a myriad of human diseases, including inherited genetic disorders, degenerative diseases, inflammation, and cancer, has fueled the study of biochemical mechanisms that control mitochondrial function. The mitochondrial transcriptional machinery is different from nuclear machinery. The in vitro re-constituted transcriptional complexes of Saccharomyces cerevisiae (yeast) and humans, aided with high-resolution structures and biochemical characterizations, have provided a deeper understanding of the mechanism and regulation of mitochondrial DNA transcription. In this review, we will discuss recent advances in the structure and mechanism of mitochondrial transcription initiation. We will follow up with recent discoveries and formative findings regarding the regulatory events that control mitochondrial DNA transcription, focusing on those involved in cross-talk between the mitochondria and nucleus.

Developing and Evaluating Inhibitors against the RNase H Active Site of HIV-1 Reverse Transcriptase.

We tested three compounds for their ability to inhibit the RNase H (RH) and polymerase activities of HIV-1 reverse transcriptase (RT). A high-resolution crystal structure (2.2 Å) of one of the compounds showed that it chelates the two magnesium ions at the RH active site; this prevents the RH active site from interacting with, and cleaving, the RNA strand of an RNA-DNA heteroduplex. The compounds were tested using a variety of substrates: all three compounds inhibited the polymerase-independent RH activity of HIV-1 RT. Time-of-addition experiments showed that the compounds were more potent if they were bound to RT before the nucleic acid substrate was added. The compounds significantly inhibited the site-specific cleavage required to generate the polypurine tract (PPT) RNA primer that initiates the second strand of viral DNA synthesis. The compounds also reduced the polymerase activity of RT; this ability was a result of the compounds binding to the RH active site. These compounds appear to be relatively specific; they do not inhibit either Escherichia coli RNase HI or human RNase H2. The compounds inhibit the replication of an HIV-1-based vector in a one-round assay, and their potencies were only modestly decreased by mutations that confer resistance to integrase strand transfer inhibitors (INSTIs), nucleoside analogs, or nonnucleoside RT inhibitors (NNRTIs), suggesting that their ability to block HIV replication is related to their ability to block RH cleavage. These compounds appear to be useful leads that can be used to develop more potent and specific compounds.IMPORTANCE Despite advances in HIV-1 treatment, drug resistance is still a problem. Of the four enzymatic activities found in HIV-1 proteins (protease, RT polymerase, RT RNase H, and integrase), only RNase H has no approved therapeutics directed against it. This new target could be used to design and develop new classes of inhibitors that would suppress the replication of the drug-resistant variants that have been selected by the current therapeutics.

Evaluation of Alzheimer's disease progression based on clinical dementia rating scale with missing responses and covariates.

In clinical trials, patient's disease severity is usually assessed on a Likert-type scale. Patients, however, may miss one or more follow-up visits (non-monotone missing). The statistical analysis of non-Gaussian longitudinal data with non-monotone missingness is difficult to handle, particularly when both response and time-dependent covariates are subject to such missingness. Even when the number of patients with intermittent missing data is small, ignoring those patients from analysis seems to be unsatisfactory. The focus of the current investigation is to study the progression of Alzheimer's disease by incorporating a non-ignorable missing data mechanism for both response and covariates in a longitudinal setup. Combining the cumulative logit longitudinal model for Alzheimer's disease progression with the bivariate binary model for the missing pattern, we develop a joint likelihood. The parameters are then estimated using the Monte Carlo Newton Raphson Expectation Maximization (MCNREM) method. This approach is quite easy to handle and the convergence of the estimates is attained in a reasonable amount of time. The study reveals that apolipo-protein plays a significant role in assessing a patient's disease severity. A detailed simulation has also been carried out for justifying the performance of our approach.

Alpha-carboxy nucleoside phosphonates as universal nucleoside triphosphate mimics.

Polymerases have a structurally highly conserved negatively charged amino acid motif that is strictly required for Mg(2+) cation-dependent catalytic incorporation of (d)NTP nucleotides into nucleic acids. Based on these characteristics, a nucleoside monophosphonate scaffold, α-carboxy nucleoside phosphonate (α-CNP), was designed that is recognized by a variety of polymerases. Kinetic, biochemical, and crystallographic studies with HIV-1 reverse transcriptase revealed that α-CNPs mimic the dNTP binding through a carboxylate oxygen, two phosphonate oxygens, and base-pairing with the template. In particular, the carboxyl oxygen of the α-CNP acts as the potential equivalent of the α-phosphate oxygen of dNTPs and two oxygens of the phosphonate group of the α-CNP chelate Mg(2+), mimicking the chelation by the ß- and γ-phosphate oxygens of dNTPs. α-CNPs (i) do not require metabolic activation (phosphorylation), (ii) bind directly to the substrate-binding site, (iii) chelate one of the two active site Mg(2+) ions, and (iv) reversibly inhibit the polymerase catalytic activity without being incorporated into nucleic acids. In addition, α-CNPs were also found to selectively interact with regulatory (i.e., allosteric) Mg(2+)-dNTP-binding sites of nucleos(t)ide-metabolizing enzymes susceptible to metabolic regulation. α-CNPs represent an entirely novel and broad technological platform for the development of specific substrate active- or regulatory-site inhibitors with therapeutic potential.

Marginalized zero-inflated Poisson models with missing covariates.

Unlike zero-inflated Poisson regression, marginalized zero-inflated Poisson (MZIP) models for counts with excess zeros provide estimates with direct interpretations for the overall effects of covariates on the marginal mean. In the presence of missing covariates, MZIP and many other count data models are ordinarily fitted using complete case analysis methods due to lack of appropriate statistical methods and software. This article presents an estimation method for MZIP models with missing covariates. The method, which is applicable to other missing data problems, is illustrated and compared with complete case analysis by using simulations and dental data on the caries preventive effects of a school-based fluoride mouthrinse program.

Structural Insights into HIV Reverse Transcriptase Mutations Q151M and Q151M Complex That Confer Multinucleoside Drug Resistance.

HIV-1 reverse transcriptase (RT) is targeted by multiple drugs. RT mutations that confer resistance to nucleoside RT inhibitors (NRTIs) emerge during clinical use. Q151M and four associated mutations, A62V, V75I, F77L, and F116Y, were detected in patients failing therapies with dideoxynucleosides (didanosine [ddI], zalcitabine [ddC]) and/or zidovudine (AZT). The cluster of the five mutations is referred to as the Q151M complex (Q151Mc), and an RT or virus containing Q151Mc exhibits resistance to multiple NRTIs. To understand the structural basis for Q151M and Q151Mc resistance, we systematically determined the crystal structures of the wild-type RT/double-stranded DNA (dsDNA)/dATP (complex I), wild-type RT/dsDNA/ddATP (complex II), Q151M RT/dsDNA/dATP (complex III), Q151Mc RT/dsDNA/dATP (complex IV), and Q151Mc RT/dsDNA/ddATP (complex V) ternary complexes. The structures revealed that the deoxyribose rings of dATP and ddATP have 3'-endo and 3'-exo conformations, respectively. The single mutation Q151M introduces conformational perturbation at the deoxynucleoside triphosphate (dNTP)-binding pocket, and the mutated pocket may exist in multiple conformations. The compensatory set of mutations in Q151Mc, particularly F116Y, restricts the side chain flexibility of M151 and helps restore the DNA polymerization efficiency of the enzyme. The altered dNTP-binding pocket in Q151Mc RT has the Q151-R72 hydrogen bond removed and has a switched conformation for the key conserved residue R72 compared to that in wild-type RT. On the basis of a modeled structure of hepatitis B virus (HBV) polymerase, the residues R72, Y116, M151, and M184 in Q151Mc HIV-1 RT are conserved in wild-type HBV polymerase as residues R41, Y89, M171, and M204, respectively; functionally, both Q151Mc HIV-1 and wild-type HBV are resistant to dideoxynucleoside analogs.

Clustered data analysis under miscategorized ordinal outcomes and missing covariates.

The primary objective in this article is to look into the analysis of clustered ordinal model where complete information on one or more covariates cease to occur. In addition, we also focus on the analysis of miscategorized data that occur in many situations as outcomes are often classified into a category that does not truly reflect its actual state. A general model structure is assumed to accommodate the information that is obtained via surrogate variables. The theoretical motivation actually developed while encountering an orthodontic data to investigate the effects of age, sex and food habit on the extent of plaque deposit. The model we propose is quite flexible and is capable of tackling those additional noises like miscategorization and missingness, which occur in the data most frequently. A new two-step approach has been proposed to estimate the parameters of model framed. A rigorous simulation study has also been carried out to justify the validity of the model taken up for analysis. Copyright © 2015 John Wiley & Sons, Ltd.

Marginalized zero-inflated negative binomial regression with application to dental caries.

The zero-inflated negative binomial regression model (ZINB) is often employed in diverse fields such as dentistry, health care utilization, highway safety, and medicine to examine relationships between exposures of interest and overdispersed count outcomes exhibiting many zeros. The regression coefficients of ZINB have latent class interpretations for a susceptible subpopulation at risk for the disease/condition under study with counts generated from a negative binomial distribution and for a non-susceptible subpopulation that provides only zero counts. The ZINB parameters, however, are not well-suited for estimating overall exposure effects, specifically, in quantifying the effect of an explanatory variable in the overall mixture population. In this paper, a marginalized zero-inflated negative binomial regression (MZINB) model for independent responses is proposed to model the population marginal mean count directly, providing straightforward inference for overall exposure effects based on maximum likelihood estimation. Through simulation studies, the finite sample performance of MZINB is compared with marginalized zero-inflated Poisson, Poisson, and negative binomial regression. The MZINB model is applied in the evaluation of a school-based fluoride mouthrinse program on dental caries in 677 children.

Exploring the role of the α-carboxyphosphonate moiety in the HIV-RT activity of α-carboxy nucleoside phosphonates.

As α-carboxy nucleoside phosphonates (α-CNPs) have demonstrated a novel mode of action of HIV-1 reverse transcriptase inhibition, structurally related derivatives were synthesized, namely the malonate 2, the unsaturated and saturated bisphosphonates 3 and 4, respectively and the amide 5. These compounds were evaluated for inhibition of HIV-1 reverse transcriptase in cell-free assays. The importance of the α-carboxy phosphonoacetic acid moiety for achieving reverse transcriptase inhibition, without the need for prior phosphorylation, was confirmed. The malonate derivative 2 was less active by two orders of magnitude than the original α-CNPs, while displaying the same pattern of kinetic behavior; interestingly the activity resides in the "L"-enantiomer of 2, as seen with the earlier series of α-CNPs. A crystal structure with an RT/DNA complex at 2.95 Å resolution revealed the binding of the "L"-enantiomer of 2, at the polymerase active site with a weaker metal ion chelation environment compared to 1a (T-α-CNP) which may explain the lower inhibitory activity of 2.