Protease Sequences Research Articles

Sexual intermingling of Arab and Jewish MSM in Israel: results of a molecular epidemiology study.

MSM comprise ∼30% of new HIV infections in Israel, a country with mixed Jewish and Arab populations. We molecularly characterized HIV-1 in the Arab and Jewish MSM (AMSM, JMSM) populations to reveal possible interethnical connections. Cross-sectional study. All Israeli-born, HIV-1-infected MSM diagnosed between 2005 and 2016 (n = 1143) were cross-matched with the National Civil Registry to identify religion (Jews/Muslims/Christians). Transmitted drug-resistance mutations (TDRM) and HIV-1 subtypes were determined on the first partial protease and reverse transcriptase sequences from treatment-naive patients and phylogenetic trees were constructed. Among MSM, 6.4% (73/1143) were Arabs and 93.6% (1070/1143) were Jews. Interestingly, a higher proportion of Arabs was identified among non-MSM (19%, 46/247 versus 6.4%, 73/1143, P < 0.01). Subtype analysis of 62 HIV-1 AMSM and 440 randomly selected HIV-1 JMSM sequences revealed 80.6, 8.1, 4.8 and 6.5% of AMSM and 82.3, 9.5, 4.1 and 4.1% of JMSM had B, A, C and non-A/B/C, respectively. Overall, 13.1% (66/502) had TDRM; reverse transcriptase-K103N/S, M184 V, T215S and protease-L90M were the most common. TDRM prevalence was not significantly higher in JMSM compared to AMSM (P = 0.1) and no temporal changes were observed in their frequency. Phylogenetic analysis demonstrated AMSM and JMSM clusters including L90M, K103N/S or T215S TDRM. Intermingling of AMSM and JMSM HIV-1 in clusters of HIV-1 sequences suggest interethnical sexual contacts among these MSM. Interventions aiming to prevent HIV-transmission in MSM should similarly address both populations groups. The high TDRM frequency requires continuation of resistance testing.

HIV Drug Resistance Mutations in Patients with HIV and HIV-TB Coinfection After Failure of First-Line Therapy: A Prevalence Study in a Resource-Limited Setting

Introduction:The present study aimed to report the prevalent HIV-1 drug-resistant mutations in patients with HIV-1 alone and tuberculosis (TB) coinfection alone to improve our understanding of the mutation patterns and aid treatment decisions.Methods:Patients with HIV-1 and HIV-TB on treatment for more than 1 year with suspected failure were recruited. Sequencing of protease and two-thirds of the region of reverse transcriptase gene was done for drug-resistant mutations.Results:In the HIV-TB group (n = 25), 88%, 92%, and 12% had mutations to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs), respectively. In the HIV-alone group (n = 25), 84%, 100%, and 4% had mutations to NRTIs, NNRTIs, and PIs, respectively. M184V, M41L, D67N, G190A, A98G, and K103N were the most common mutations seen.Conclusion:There is a high prevalence of drug-resistant mutations in HIV and HIV-TB coinfected patients.

Virtual screening and molecular docking of Anti-Antileishmanial for selected pharmacophore for visceral Leishmaniasis

Objective: DNA amplification of Cysteine protease of Leishmania donovani and study the interaction of cysteine protease inhibitors, antileishmanial compounds with cysteine protease receptor in various computational programs.&#x0D; Materials and methods: Cysteine protease DNA of Leishmania donovani was amplified by PCR. The sequence of cysteine protease has been modeled and docked with suitable inhibitors by using various servers and computational tools. The model was designed, compared and validated by DOPE and Verify 3D scores. The model and the compound interaction were studied by LibDock and other programs.&#x0D; Results: Cysteine protease DNA of Leishmania donovani was successfully amplified by PCR. The structural modeling was done to achieve effective enzyme inhibition, inhibitors block the binding sites of that protein. Homology modeling of cysteine protease has been done and docked with suitable inhibitors by using various servers and computational tools. The model was designed, compared and validated by DOPE and Verify 3D scores by using DSv3.5. Licochalcone-a alone showed 37 LibDock conformations with 6 different poses, were suitably docked at the site 1 with hydrogen bond formation. The study would help to design the novel drugs in respect of resistant one for the treatment of harmful visceral Leishmaniasis.&#x0D; Conclusion: The molecular interaction of vinyl sulfones, hydrazide derivatives, antileishmanial drugs molecules and carbohydrazide derivatives have exhibited ideal molecular interaction with cathepsin B, a cysteine protease of L. donovani, amino acids such as Cys29, Hisl88 and Asn208 has been found to be active residues. Licochalcone-a and hydrazide derivative may become future antileishmanial compounds, which needs to be tested in in vitro and in vivo.&#x0D; Keywords: Cysteine Protease, Vinyl Sulfone, Hydrazide, Antileishmanial Drugs, Licochalcone, Visceral Leishmaniasis.

Prevalence of resistance-associated substitutions to direct-acting antiviral agents in hemodialysis and renal transplant patients infected with hepatitis C virus.

BackgroundDirect-acting antiviral agents (DAAs) permit the use of interferon (IFN)-free regimens to treat hepatitis C (HCV) in patients with chronic kidney disease (CKD) on hemo-dialysis (HD) or renal transplant (RTx) recipients, with excellent response rates and safety. However, the occurrence of basal or therapy-induced resistance-associated substitutions (RASs) to DAAs can result in treatment failure. The aim of this study was to estimate the prevalence of RASs to NS3A, NS5A and NS5B inhibitors, and particularly the Q80K polymorphism, in CKD patients on HD and RTx recipients infected with HCV.Patients and methodsHD and RTx patients infected with HCV-genotype 1 (GT1) were subjected to sequencing of the NS3, NS5A and NS5B regions.ResultsDirect sequencing of NS3 protease, NS5A and NS5B was performed in 76 patients (HD, n=37; RTx, n=39). The overall prevalence of RASs was 38.2%, but only 5.3% of the patients had mutations in more than one region. Substitutions were detected in NS3A (17.8%), NS5A (21.9%) and NS5B (8.4%). Q80K was detected in 1.5 % of the patients. Highly inhibitory RASs were uncommon (L31M, 2.6%; L159F+C316N, 2.6%). RASs were more prevalent in HCV-GT1a (42.9%) than in HCV-GT1b (32.4%), P=0.35. RASs were detected in 52.4% of treatment-naive patients and 27.8% of peg-IFN/ribavirin-experienced patients (P=0.12). The presence of RASs was associated with time of RTx (P=0.01).ConclusionThe Q80K polymorphism was uncommon in our sample of HD and RTx patients. Despite the high prevalence of naturally occurring RASs, most of the substitutions detected were associated with a low level of resistance to DAAs.

Features in HIV genotypes associated with failure in the computational prediction of patients' response to antiretroviral treatment

HIV acts by attacking the immune system and gradually destroying the TCD4+ defense cells. Without adequate treatment, the carriers develop the most severe form of the infection, AIDS, when the patient can be afflicted by opportunistic diseases that inevitably lead to death. Fortunately, with the advent of the highly active antiretroviral therapy (HAART), the mortality of people with HIV is decreasing. However, mutations can occur in the genotype of the virus, generating drug-resistant phenotypes. Computational methods have been used to predict whether a given strain is drug-resistant, and to which drugs this resistance occurs, thereby increasing the chances of success of the prescribed treatment regimen. However, these methods are not always accurate in their task. In this context, by applying Feature Selection methods and estimating Decision Tree models, we investigated patterns in Protease and Reverse Transcriptase enzyme sequences, as well as in patients’ clinical data, which can lead to correct or incorrect computational prediction. As a result, we identified 21 features that are highly informative, 11 which tend to lead the methods to error, and eight that present both behaviors simultaneously, being able to predict the patient's response to therapy and at the same time may lead the predictor's methods to failure.

HIV-1 proviral DNA as a potential target for studying antiretroviral drug resistance. Preliminary analyses

Background: Selection of mutations associated to resistance (MARS) is one of the factors contributing to virological failure in HIV-1 treatment. Therefore, identification of MARS in circulating virus (plasmatic viral RNA) is critical. For viral loads < 500-1000 copies/ml is very difficult to genotype. Assuming that the HIV-1 reservoirs are a genetic archive of the viral population, analyses of genotypes in proviral DNA would be useful in these patients and those that need switch with undetectable viral loads. Some limitations are the low proportion of circulating infected cells together with the sensitivity of routine tests. Objetives: Comparison of MARS and basal mutations in viral RNA and proviral DNA in HIV-1 patients from our Institution. Evaluate if the proviral DNA genotype could be a predictor of virological failure. Methods & Materials: We studied from 20 HIV-1-patients, plasma (P) and peripheral blood (B) samples, and 10 B samples from patients in failure with low viral load. Nucleic acids were extracted and the pol gene was amplified by nested PCR. Sequencing of protease (PR) and reverse transcriptase (RT) were analyzed in the Stanford database. Results: We observed a good concordance of the mutational profiles in P and B samples in 18 of 20 patients. The 2 remaining ones demonstrated mutations only in B: D30 N (highly resistant to Nelfinavir) and T215NT (low resistant to AZT and d4 T). We also observed mutations in unusual residues and more proportion of mixed bases in proviral DNA. Five of the 10 patients in failure, demonstrated MARS (M41L, K65R, A98G, K101E, K103 N, M184 V/I, G190S) coincidentally with their therapeutic schemas Conclusion: We conclude that P and B are generally concordant for studying HIV-1 genotype in certain patients. Two discordant patients were naïve explaining the lack of mutations in circulation and their presence in archival proviral DNA. The absence of mutations in DNA does not exclude their existence in lymphocytic reservoirs. It appears that genotyping the proviral HIV-1 DNA is useful in patients virologically suppressed in need of switch for treatment simplification or better tolerance/adherence and in those in failure that failed the plasma study. More data is required to evaluate the clinical impact in therapeutic decisions.

Related Endogenous Retrovirus-K Elements Harbor Distinct Protease Active Site Motifs

Background: Endogenous retrovirus-K is a group of related genomic elements descending from retroviral infections in human ancestors. HML2 is the clade of these viruses which contains the most intact provirus copies. These elements can be transcribed and translated in healthy and diseased tissues, and some of them produce active retroviral enzymes, such as protease. Retroviral gene products, including protease, contribute to illness in exogenous retroviral infections. There are ongoing efforts to test anti-retroviral regimens against endogenous retroviruses. Herein, we examine the potential activity and diversity of human endogenous retrovirus-K proteases, and their potential for impact on immunity and human disease.Results: Sequences similar to the endogenous retrovirus-K HML2 protease and reverse transcriptase were identified in the human genome, classified by phylogenetic inference and compared to Repbase reference sequences. The topologies of trees inferred from protease and reverse transcriptase sequences were similar and agreed with the classification using reference sequences. Surprisingly, only 62/480 protease sequences identified by BLAST were classified as HML2; the remainder were classified as other HML groups, with the majority (216) classified as HML3. Variation in functionally significant protease motifs was explored, and two major active site variants were identified – the DTGAD variant is common in all groups, but the DTGVD motif appears limited to HML3, HML5, and HML6. Furthermore, distinct RNA expression patterns of protease variants are seen in disease states, such as amyotrophic lateral sclerosis, breast cancer, and prostate cancer.Conclusion: Transcribed ERVK proteases exhibit a diversity which could impact immunity and inhibitor-based treatments, and these facets should be considered when designing therapeutic regimens.

Abstract 4200: New potential therapeutic applications of WX-UK1, as a specific and potent inhibitor of human trypsin-like proteases

Abstract WX-UK1 (the active metabolite of upamostat) was originally developed as an inhibitor of the serine protease urokinase (uPA) with a Ki ~1 uM. To identify more sensitive targets, we performed a bioinformatic analysis of the ~200 human trypsin-like serine proteases, many of which play crucial roles in homeostasis and disease. Among these we selected a subset for biochemical analysis based on an inspection of modelled 3D structures of WX-UK1:protease complexes and sequence alignment of binding site residues. Samples of the selected proteases were prepared and characterized for their binding to WX-UK1; enzymatically with respect to inhibition constant (Ki) and by surface plasmon resonance with respect to dissociation constant (kd). We now report that WX-UK1 is a potent and specific inhibitor of five human serine proteases (trypsin-3, trypsin-2, trypsin-1 and matriptase-1 and trypsin-6), with Ki's down to the low nanomolar range, 19 nM for trypsin-3. Several of these serine proteases are known to be associated with cancer progression and metastasis. As a compound with an established clinical safety profile, targeted use of upamostat in oncology and extension to non-oncology indications may be assessed. Citation Format: Emil Oldenburg, Christine R. Schar, Eva Lange, Terry F. Plasse, Danielle T. Abramson, Reza Fathi, Eric M. Towler, Mark Levitt, Jan K. Jensen. New potential therapeutic applications of WX-UK1, as a specific and potent inhibitor of human trypsin-like proteases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4200.

Molecular identification and functional characterization of the cathepsin B gene (Ab-cb-1) in the plant parasitic nematode Aphelenchoides besseyi.

The rice white tip nematode, Aphelenchoides besseyi, is widely distributed in rice planting areas worldwide and causes serious economic losses. Cathepsin genes have been demonstrated to have importance in studying the reproduction, development, pathogenicity, and control methods of plant nematodes. In this paper, a novel cathepsin B gene, Ab-cb-1, was found and cloned. The Ab-cb-1 gene was 1347 bp in length and encodes 369 amino acids. The Ab-CB-1 protein contains characteristic occluding loops but no signal peptide. A homology analysis showed that Ab-CB-1 had the highest identity value (64%) to the known amino acid sequence of cathepsin B-like cysteine protease 6 from Toxocara canis. When Ab-cb-1 was expressed in a prokaryotic system, the protein massed approximately 45 kDa and could decompose carrot callus. Ab-cb-1 mRNA was localized in the nematode intestine. The relative expression level of Ab-cb-1 in the A. besseyi Ab-S24 population, which had high reproductivity, was approximately 6.9 times that in the Ab-N10 population, which had low reproductivity, and the difference was significant (p<0.05). The Ab-cb-1 expression level was highest in females; the expression levels in males, juveniles and eggs were 30%, 12.2% and 5% of that in females, respectively, and the differences were significant among all four stages (p<0.05). Nematodes of the Ab-S24 population were treated with Ab-cb-1 dsRNA for 12 h, 24 h, 36 h and 48 h, and their reproduction decreased with increasing time. These results demonstrated that Ab-CB-1 was a digestive enzyme with hydrolytic protease properties and that Ab-cb-1 played an important role in the reproduction of A. besseyi.

Exploiting protein modification systems to boost crop productivity: SUMO proteases in focus.

In recent years, post-translational modification (PTM) of proteins has emerged as a key process that integrates plant growth and response to a changing environment. During the processes of domestication and breeding, plants were selected for various yield and adaptational characteristics. The post-translational modifier small ubiquitin-like modifier (SUMO) protein is known to have a role in the regulation of a number of these characteristics. Using bioinformatics, we mined the genomes of cereal and Brassica crops and their non-crop relatives Arabidopsis thaliana and Brachypodium distachyon for ubiquitin-like protease (ULP) SUMO protease sequences. We discovered that the SUMO system in cereal crops is disproportionately elaborate in comparison with that in B. distachyon. We use these data to propose deSUMOylation as a mechanism for specificity in the SUMO system.

Evaluation of different analysis pipelines for the detection of HIV-1 minority resistant variants.

ObjectiveReliable detection of HIV minority resistant variants (MRVs) requires bioinformatics analysis with specific algorithms to obtain good quality alignments. The aim of this study was to analyze ultra-deep sequencing (UDS) data using different analysis pipelines.MethodsHIV-1 protease, reverse transcriptase (RT) and integrase sequences from antiretroviral-naïve patients were obtained using GS-Junior® (Roche) and MiSeq® (Illumina) platforms. MRVs were defined as variants harbouring resistance-mutation present at a frequency of 1%–20%. Reads were analyzed using different alignment algorithms: Amplicon Variant Analyzer®, Geneious® compared to SmartGene® NGS HIV-1 module.Results101 protease and 51 RT MRVs identified in 139 protease and 124 RT sequences generated with a GS-Junior® platform were analyzed using AVA® and SmartGene® software. The correlation coefficients for the MRVs were R2 = 0.974 for protease and R2 = 0.972 for RT. Discordances (n = 13 in protease and n = 15 in RT) mainly concerned low-level MRVs (i.e., with frequencies of 1%–2%, n = 18/28) and they were located in homopolymeric regions (n = 10/15). Geneious® and SmartGene® software were used to analyze 143 protease, 45 RT and 26 integrase MRVs identified in 172 protease, 69 RT, and 72 integrase sequences generated with a MiSeq® platform. The correlation coefficients for the MRVs were R2 = 0.987 for protease, R2 = 0.995 for RT and R2 = 0.993 for integrase. Discordances (n = 9 in protease, n = 3 in RT, and n = 3 in integrase) mainly concerned low-level MRVs (n = 13/15).ConclusionWe found an excellent correlation between the various UDS analysis pipelines that we tested. However, our results indicate that specific attention should be paid to low-level MRVs, for which the use of two different analysis pipelines and visual inspection of sequences alignments might be beneficial. Thus, our results argue for use of a 2% threshold for MRV detection, rather than the 1% threshold, to minimize misalignments and time-consuming sight reading steps essential to ensure accurate results for MRV frequencies below 2%.

HIV Reverse Transcriptase and Protease Genes Variability Can Be a Biomarker Associated with HIV and Hepatitis B or C Coinfection

Variability of the HIV reverse transcriptase (RT) and protease (PR) genes has been used as indicators of drug resistance and as a mean to evaluate phylogenetic relationships among circulating virus. However, these studies have been carried in HIV mono-infected populations. The goal of this study was to evaluate, for the first time, the HIV PR and RT sequences from HIV/HBV and HIV/HCV co-infected patients. HIV PR and RT genes were amplificated and sequenced to resistance analysis. The bioinformatics analysis was performed to infer about sequences clustering and molecular evolution. The results showed that the most frequent amino acid substitutions in RT were L214F (67.6%), I135T (55.9%), and in PR was V15I (41.2%). The molecular clock analysis showed that the HIV circulating in co-infected patients were separated in two clusters in the years 1999–2000. Some patients included as HIV mono-infected according patients’ medical records and inside the co-infected cluster were, in fact, co-infected by PCR analysis. Analysis of the decision trees showed susceptibility to lamivudine and emtricitabine were important attribute to characterize co-infected patients. In conclusion, the results obtained in this study suggest, for the first time, that HIV RT and PR genes variability could be a genetic biomarker to coinfection.

Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted Human Immunodeficiency Virus Type 1 Drug Resistance in a Large US Clinic Population.

There are few large studies of transmitted drug resistance (TDR) prevalence and the drug resistance mutations (DRMs) responsible for TDR in the United States. Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and protease sequences were obtained from 4253 antiretroviral therapy (ART)-naive individuals in a California clinic population from 2003 to 2016. Phylogenetic analyses were performed to study linkages between TDR strains and selection pressure on TDR-associated DRMs. From 2003 to 2016, there was a significant increase in overall (odds ratio [OR], 1.05 per year [95% confidence interval {CI}, 1.03-1.08]; P < .001) and nonnucleoside RT inhibitor (NNRTI)-associated TDR (OR, 1.11 per year [95% CI, 1.08-1.15]; P < .001). Between 2012 and 2016, TDR rates to any drug class ranged from 15.7% to 19.2%, and class-specific rates ranged from 10.0% to 12.8% for NNRTIs, 4.1% to 8.1% for nucleoside RT inhibitors (NRTIs), and 3.6% to 5.2% for protease inhibitors. The thymidine analogue mutations, M184V/I and the tenofovir-associated DRMs K65R and K70E/Q/G/N/T accounted for 82.9%, 7.3%, and 1.4% of NRTI-associated TDR, respectively. Thirty-seven percent of TDR strains clustered with other TDR strains sharing the same DRMs. Although TDR has increased significantly in this large cohort, many TDR strains are unlikely to influence the activity of currently preferred first-line ART regimens. The high proportion of DRMs associated with infrequently used regimens combined with the clustering of TDR strains suggest that some TDR strains are being transmitted between ART-naive individuals.

Full-length and defective enterovirus G genomes with distinct torovirus protease insertions are highly prevalent on a Chinese pig farm.

Recombination occurs frequently between enteroviruses (EVs) which are classified within the same species of the Picornaviridae family. Here, using viral metagenomics, the genomes of two recombinant EV-Gs (strains EVG 01/NC_CHI/2014 and EVG 02/NC_CHI/2014) found in the feces of pigs from a swine farm in China are described. The two strains are characterized by distinct insertion of a papain-like protease gene from toroviruses classified within the Coronaviridae family. According to recent reports the site of the torovirus protease insertion was located at the 2C/3A junction region in EVG 02/NC_CHI/2014. For the other variant EVG 01/NC_CHI/2014, the inserted protease sequence replaced the entire viral capsid protein region up to the VP1/2A junction. These two EV-G strains were highly prevalent in the same pig farm with all animals shedding the full-length genome (EVG 02/NC_CHI/2014) while 65% also shed the capsid deletion mutant (EVG 01/NC_CHI/2014). A helper-defective virus relationship between the two co-circulating EV-G recombinants is hypothesized.

Emerging resistance mutations in PI-naive patients failing an atazanavir-based regimen (ANRS multicentre observational study).

Atazanavir is a PI widely used as a third agent in combination ART. We aimed to determine the prevalence and the patterns of resistance in PI-naive patients failing on an atazanavir-based regimen. We analysed patients failing on an atazanavir-containing regimen used as a first line of PI therapy. We compared the sequences of reverse transcriptase and protease before the introduction of atazanavir and at failure [two consecutive viral loads (VLs) >50 copies/mL]. Resistance was defined according to the 2014 Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS) algorithm. Among the 113 patients, atazanavir was used in the first regimen in 71 (62.8%) patients and in the first line of a PI-based regimen in 42 (37.2%). Atazanavir was boosted with ritonavir in 95 (84.1%) patients and combined with tenofovir/emtricitabine or lamivudine (n = 81) and abacavir/lamivudine or emtricitabine (n = 22). At failure, median VL was 3.05 log10 copies/mL and the median CD4+ T cell count was 436 cells/mm3. The median time on atazanavir was 21.2 months. At failure, viruses were considered resistant to atazanavir in four patients (3.5%) with the selection of the following major atazanavir-associated mutations: I50L (n = 1), I84V (n = 2) and N88S (n = 1). Other emergent PI mutations were L10V, G16E, K20I/R, L33F, M36I/L, M46I/L, G48V, F53L, I54L, D60E, I62V, A71T/V, V82I/T, L90M and I93L/M. Emergent NRTI substitutions were detected in 21 patients: M41L (n = 2), D67N (n = 3), K70R (n = 1), L74I/V (n = 3), M184V/I (n = 16), L210W (n = 1), T215Y/F (n = 3) and K219Q/E (n = 2). Resistance to atazanavir is rare in patients failing the first line of an atazanavir-based regimen according to the ANRS. Emergent NRTI resistance-associated mutations were reported in 18% of patients.

A cell surface display fluorescent biosensor for measuring MMP14 activity in real-time

Despite numerous recent advances in imaging technologies, one continuing challenge for cell biologists and microscopists is the visualization and measurement of endogenous proteins as they function within living cells. Achieving this goal will provide a tool that investigators can use to associate cellular outcomes with the behavior and activity of many well-studied target proteins. Here, we describe the development of a plasmid-based fluorescent biosensor engineered to measure the location and activity of matrix metalloprotease-14 (MMP14). The biosensor design uses fluorogen-activating protein technology coupled with a MMP14-selective protease sequence to generate a binary, “switch-on” fluorescence reporter capable of measuring MMP14 location, activity, and temporal dynamics. The MMP14-fluorogen activating protein biosensor approach is applicable to both short and long-term imaging modalities and contains an adaptable module that can be used to study many membrane-bound proteases. This MMP14 biosensor promises to serve as a tool for the advancement of a broad range of investigations targeting MMP14 activity during cell migration in health and disease.

Elucidation of a 2.3 Å Resolution Norovirus GII.4 Protease Structure by X‐Ray Crystallography

According to a current report from the Centers for Disease Control (CDC), it is estimated that noroviruses are responsible for 685 million cases of acute gastroenteritis each year with an estimated $60 billion medically and socioeconomically. This overwhelming global burden drives the need for identification and characterization of potential norovirus drug targets to exploit for design and development. Specifically, the GII.4 norovirus genotype accounts for more than 50% of all outbreaks worldwide. Up until now, the only GII.4 model was based on an alignment of the GI.1 and GII.4 protease sequences which resulted in a sequence identity of only 67%. This high genetic diversity among human noroviruses enhances the need for an improved model of the GII.4 protease. We report the crystallization and structure elucidation of a 2.3Å resolution structure of the GII.4 norovirus protease (GII.4 NoV PR) through X‐ray crystallography. Additionally, extensive investigation and characterization of the GII.4 norovirus protease structure and mechanism were carried out through computational studies and enzyme kinetics. With the elucidation of the GII.4 norovirus protease structure, we hope to significantly improve the structure‐based design of both potent therapeutic inhibitors and potential vaccine strategies for future studies.Support or Funding InformationWayne State University School of MedicineThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Next generation sequencing of HIV-1 protease in the PIVOT trial of protease inhibitor monotherapy

BackgroundThe PIVOT trial examined whether patients with suppressed viral load on combination antiretroviral therapy could be safely switched long-term to ritonavir-boosted protease inhibitor (PI) monotherapy. The main trial publication reported that only one of 296 patients allocated to PI monotherapy experienced a loss of drug options due to protease mutations (identified by local Sanger sequencing resistance tests) likely selected by study drug. ObjectivesTo assess if we had missed low frequency mutations, using a more sensitive methodology. Study designWe performed next generation sequencing (NGS) on all available frozen plasma samples with VL >1000 copies/ml from patients who were randomised to PI monotherapy. Assays were performed at Public Health England laboratories using a previously described method.Median coverage depth was 76,000 and the threshold for detection of minority variants was 2%. Drug susceptibility was predicted using the Stanford HIVdb algorithm. Results17 of 26 potential samples, all from different patients, were identified and successfully tested. The median viral load was 6780 copies/ml and the median time since randomisation was 43 weeks. NGS revealed previously unidentified minority variant protease mutations (G73D, I54T, L89V) in three samples, at frequencies ranging between 2% and 10%. None of these mutations predicted intermediate or high level resistance, the trial primary outcome. DiscussionThis report adds to the body of evidence that ritonavir-boosted PI monotherapy, when used as a switch strategy with prompt detection of viral load rebound and early re-introduction of combination therapy, rarely leads to the development of clinically important protease resistance mutations.

Minority resistant variants are also present in HIV-2-infected antiretroviral-naive patients.

To assess the prevalence of minority resistant variants (MRV) and X4-tropic minority variants in ART-naive HIV-2-infected patients. ART-naive HIV-2-infected patients with detectable plasma viral load (>100 copies/mL) included in the ANRS HIV-2 CO5 Cohort were assessed. We performed ultra-deep sequencing (UDS) of protease, RT, integrase and gp105 regions. Only mutations in the HIV-2 ANRS list >1% were considered. HIV-2 tropism was assessed by V3 loop region UDS, and each read was interpreted with determinants of CXCR4-coreceptor use. Among the 47 patients assessed, three displayed plasma viruses with a resistance-associated mutation (RAM) above the 20% detection threshold, all in RT, resulting in a prevalence of transmitted drug resistance for NRTI of 7.9% (95% CI 0.0%-16.5%). No RAM above the 20% detection threshold was found in protease or integrase. At the 1% detection threshold the transmitted drug resistance prevalence was 9.8% (95% CI 0.6%-19.0%), 13.2% (95% CI 3.5%-22.9%) and 4.5% (95% CI 0%-17.5%) for PI, NRTI and integrase inhibitors. The most prevalent MRV was the PI RAM I50V detected in three samples. Tropism analysis showed that 21% of patients (4 of 19) exhibited X4-tropic viruses: two in majority proportion and two in minority proportions (1.5% and 1.9%). In this first study assessing the prevalence of MRV in HIV-2 infection among ART-naive patients, we observed a 2-3-fold higher prevalence of RAM when a 1% detection threshold of mutations was used compared with a 20% threshold. Similarly, the proportion of patients with X4-tropic viruses was twice as high when UDS was used.

A safe an easy method for building consensus HIV sequences from 454 massively parallel sequencing data

ObjectiveTo show how to generate a consensus sequence from the information of massive parallel sequences data obtained from routine HIV anti-retroviral resistance studies, and that may be suitable for molecular epidemiology studies. Material and methodsPaired Sanger (Trugene-Siemens) and next-generation sequencing (NGS) (454 GSJunior-Roche) HIV RT and protease sequences from 62 patients were studied. NGS consensus sequences were generated using Mesquite, using 10%, 15%, and 20% thresholds. Molecular evolutionary genetics analysis (MEGA) was used for phylogenetic studies. ResultsAt a 10% threshold, NGS-Sanger sequences from 17/62 patients were phylogenetically related, with a median bootstrap-value of 88% (IQR 83.5–95.5). Association increased to 36/62 sequences, median bootstrap 94% (IQR 85.5–98), using a 15% threshold. Maximum association was at the 20% threshold, with 61/62 sequences associated, and a median bootstrap value of 99% (IQR 98–100). ConclusionA safe method is presented to generate consensus sequences from HIV-NGS data at 20% threshold, which will prove useful for molecular epidemiological studies.