Kaposi's sarcoma-associated herpesvirus (KSHV) causes several malignancies in people with HIV including Kaposi's sarcoma and primary effusion lymphoma (PEL). We have previously shown that PEL cell lines require myeloid cell leukemia-1 (MCL1) to inhibit apoptosis. MCL1 is an oncogene that is amplified in cancers and causes resistance to chemotherapy regimens. MCL1 is thus an attractive target for drug development. The emerging clinical relevance and therapeutic potential of MCL1 motivated us to study the roles of this oncogene in PEL in depth. Using a systems biology approach, we uncovered an unexpected genetic interaction between MCL1 and MARCHF5 indicating that they function in the same pathway. MARCHF5 is an E3 ubiquitin ligase most known for regulating mitochondrial homeostasis and antiviral signaling, but not apoptosis. We thus investigated how MCL1 and MARCHF5 cooperate to promote PEL cell survival. CRISPR knockout (KO) of MARCHF5 in PEL cell lines resulted in a significant increase in apoptosis despite the presence of MCL1. The anti-apoptotic function of MARCHF5 was dependent on its E3 ligase and dimerization activities. Loss of MARCHF5 or inhibition of the 26S proteasome furthermore stabilized the MCL1 antagonist NOXA without affecting levels of MCL1. Interestingly, NOXA KO provides a fitness advantage to PEL cells suggesting that NOXA is the pro-apoptotic signal that necessitates the anti-apoptotic activities of MCL1 and MARCHF5. Finally, endogenous reciprocal co-immunoprecipitation experiments show that MARCHF5 and NOXA are found in the same protein complex. Our findings thus provide the mechanistic link that underlies the genetic interaction between MCL1 and MARCHF5. We propose that MARCHF5 induces the degradation of the MCL1 antagonist NOXA thus reinforcing the pro-survival role of MCL1 in these tumor cells. This newly appreciated interaction of the MCL1 and MARCHF5 oncogenes may be useful to improve the design of combination therapies for KSHV malignancies.

Introduction Despite the remarkable success of combination treatments, multiple myeloma (MM) still poses significant challenges, with a high percentage of patients experiencing relapse over time. Additional metabolic alterations are found in relapsed and refractory MM; however, their impacts have not been fully elucidated. Expression of thyroid hormone receptor interactor 13 (TRIP13) has been identified by Gene Expression Profile (GEP)-70 to correlate with poor prognosis in MM. TRIP13 is an AAA ATPase that regulates the spindle assembly checkpoint in mitosis. Based on GEP data from 34 paired baseline and relapse samples, TRIP13 continuously increased from baseline to first and to second relapse. Overexpression of TRIP13 induces bortezomib resistance. In this study, we aimed to investigate how TRIP13 might regulate MM cell metabolism and to use this information to develop a novel combination strategy to overcome MM resistance. Methods Knockout (KO) of TRIP13 in OPM2 and MM1.S cells was generated by dual gRNAs CRISPR-Cas9 editing deleting a fragment between exon 4 and exon 5. Cell growth curves and response to bortezomib were recorded in the control and TRIP13-KO cells. Metabolomics was performed to reveal cellular metabolic alterations. Seahorse analysis was used to test the mitochondrial stress and glycolysis stress. To investigate whether TRIP13 KO promotes ferroptosis, an intracellular iron-dependent cell death characterized by the accumulation of lipid peroxides, we applied C11-BODIY staining after treatment with Erastin, a ferroptosis-inducer. Expression levels of cyclooxygenase 2 (encoded by PTGS2, a hallmark of ferroptosis), cystine transporter (xCT), and intracellular glutathione peroxidase 4 (GPX4) were analyzed. In addition, the protein levels of iron metabolic molecules, including iron importer transferrin receptor protein 1 (TFRC), iron exporter ferroportin 1 (FPN1), ferritin heavy chain 1 (FTH1), and nuclear receptor coactivator 4 (NCOA4) were assessed. To study whether TRIP13 KO delays MM progression in an immune-competent MM mouse model, Vκ12653 murine MM cells were genetically modified by CRISPR-mediated TRIP13-KO and then injected into C57BL/6 mice. Mouse survival was monitored. Results The cell-doubling rate of TRIP13-KO cells was decreased by 50% compared to control cells. After 24 hours of treatment with 5 nM bortezomib, cell viability of TRIP13-KO cells decreased by 31% compared with control cells. The metabolomic studies revealed that TRIP13-KO OPM2 and MM1.S cells both had significantly decreased intracellular L-serine (FC = 0.57) but increased pyruvate (FC = 1.79), citric acid (FC = 1.76), and isocitric acid (FC = 1.64), indicating TRIP13 may regulate serine metabolism, mitochondrial function, and cellular redox balance. Seahorse analysis indicated that TRIP13-KO MM cells showed decreased glycolysis (decreased by 30%, p < 0.001) and glycolytic capacity (decreased by 33%, p < 0.001) while oxidative phosphorylation showed little change. Moreover, lipid peroxidation and PTGS2 were increased in TRIP13-KO cells after Erastin treatment, indicating that these cells were more vulnerable to ferroptosis. Mechanistically, inhibition of TRIP13 decreased GPX4, FPN1, FTH1, and increased NCOA4, while overexpression of TRIP13 showed the opposite changes, suggesting that targeting TRIP13 sensitizes MM cells to ferroptosis by impairing cellular redox balance and increasing the labile-iron pool. Lastly, TRIP13-KO significantly prolonged mouse survival in the Vκ12653 mouse model (median survival: TRIP13-KO 93 days vs. Control 60.5 days, p < 0.001). Conclusion Our study revealed that TRIP13 plays a critical role in regulating MM cell metabolism. By suppressing TRIP13, we observed increased sensitivity of MM cells to bortezomib, leading to enhanced ferroptosis-mediated cell death and prolonged mouse survival. These findings strongly suggest that targeting TRIP13 could be a promising approach in MM therapy. Moving forward, our future investigations will concentrate on unraveling the precise molecular mechanisms underlying TRIP13-mediated alterations in cell metabolism. Additionally, we plan to explore the potential synergistic effects of TRIP13 inhibition in combination with metabolic targeted treatments, such as venetoclax, to further enhance therapeutic outcomes in MM.

Kaposi’s sarcoma-associated herpesvirus (KSHV) causes several malignancies in people living with HIV, including primary effusion lymphoma (PEL). PEL cell lines exhibit oncogene addictions to both viral and cellular genes. Using CRISPR screens, we previously identified cellular oncogene addictions in PEL cell lines, including MCL1. MCL1 is a member of the BCL2 family, which functions to prevent intrinsic apoptosis and has been implicated in several cancers. Despite the overlapping functions of the BCL2 family members, PEL cells are only dependent on MCL1 suggesting that MCL1 may have non-redundant functions. To investigate why PEL cells exhibit selective addiction to MCL1, we inactivated the intrinsic apoptosis pathway by engineering BAX/BAK1 double knockout cells. In this context, PEL cells become resistant to MCL1 knockdown or MCL1 inactivation by the MCL1 inhibitor S63845, indicating that the main function of MCL1 in PEL cells is to prevent BAX/BAK1-mediated apoptosis. The selective requirement to MCL1 is due to MCL1 being expressed in excess over the BCL2 family. Ectopic expression of several BCL2 family proteins, as well as the KSHV BCL2 homolog, significantly decreased basal caspase 3/7 activity and buffered against staurosporine-induced apoptosis. Finally, over-expressed BCL2 family members can functionally substitute for MCL1, when it is inhibited by S63845. Together our data indicate that the expression levels of the BCL2 family likely explain why PEL tumor cells are highly addicted to MCL1. Importantly, our results suggest that caution should be taken when considering MCL1i as a monotherapy regimen for PEL, because resistance can easily develop. IMPORTANCE Primary effusion lymphoma (PEL) is caused by Kaposi’s sarcoma-associated herpesvirus. We previously showed that PEL cell lines require the anti-apoptotic protein MCL1 for survival, but not the other BCL2 family proteins. This selective dependence to MCL1 is unexpected as the BCL2 family functions similarly in preventing intrinsic apoptosis. Recently, new roles for MCL1 not shared with the BCL2 family have emerged. Here, we show that non-canonical functions of MCL1 are unlikely essential. Instead, MCL1 mainly functions to prevent apoptosis. The specific requirement to MCL1 is due to MCL1 being expressed in excess over the BCL2 family. Consistent with this model, shifting these expression ratios changes the requirement away from MCL1 and towards the dominant BCL2 family gene. Together, our results indicate that although MCL1 is an attractive chemotherapeutic target to treat PEL, careful consideration must be taken as resistance to MCL1-specific inhibitors easily develops through BCL2 family overexpression.

Next Generation Sequencing (NGS) is the gold standard for the detection of new variants of SARS-CoV-2 including those which have immune escape properties, high infectivity, and variable severity. This test is helpful in genomic surveillance, for planning appropriate and timely public health interventions. But labs with NGS facilities are not available in small or medium research settings due to the high cost of setting up such a facility. Transportation of samples from many places to few centers for NGS testing also produces delays due to transportation and sample overload leading in turn to delays in patient management and community interventions. This becomes more important for patients traveling from hotspot regions or those suspected of harboring a new variant. Another major issue is the high cost of NGS-based tests. Thus, it may not be a good option for an economically viable surveillance program requiring immediate result generation and patient follow-up. The current study used a cost-effective facility which can be set up in a common research lab and which is replicable in similar centers with expertise in Sanger nucleotide sequencing. More samples can be processed at a time and can generate the results in a maximum of 2 days (1 day for a 24 h working lab). We analyzed the nucleotide sequence of the Receptor Binding Domain (RBD) region of SARS-CoV-2 by the Sanger sequencing using in-house developed methods. The SARS-CoV-2 variant surveillance was done during the period of March 2021 to May 2022 in the Northern region of Kerala, a state in India with a population of 36.4 million, for implementing appropriate timely interventions. Our findings broadly agree with those from elsewhere in India and other countries during the period.

We report here a Nipah virus (NiV) outbreak in Kozhikode district of Kerala state, India, which had caused fatal encephalitis in a 12-year-old boy and the outbreak response, which led to the successful containment of the disease and the related investigations. Quantitative real-time reverse transcription (RT)-PCR, ELISA-based antibody detection, and whole genome sequencing (WGS) were performed to confirm the NiV infection. Contacts of the index case were traced and isolated based on risk categorization. Bats from the areas near the epicenter of the outbreak were sampled for throat swabs, rectal swabs, and blood samples for NiV screening by real-time RT-PCR and anti-NiV bat immunoglobulin G (IgG) ELISA. A plaque reduction neutralization test was performed for the detection of neutralizing antibodies. Nipah viral RNA could be detected from blood, bronchial wash, endotracheal (ET) secretion, and cerebrospinal fluid (CSF) and anti-NiV immunoglobulin M (IgM) antibodies from the serum sample of the index case. Rapid establishment of an onsite NiV diagnostic facility and contact tracing helped in quick containment of the outbreak. NiV sequences retrieved from the clinical specimen of the index case formed a sub-cluster with the earlier reported Nipah I genotype sequences from India with more than 95% similarity. Anti-NiV IgG positivity could be detected in 21% of Pteropus medius (P. medius) and 37.73% of Rousettus leschenaultia (R. leschenaultia). Neutralizing antibodies against NiV could be detected in P. medius. Stringent surveillance and awareness campaigns need to be implemented in the area to reduce human-bat interactions and minimize spillover events, which can lead to sporadic outbreaks of NiV.

Primary effusion lymphoma (PEL) is caused by Kaposi’s sarcoma-associated herpesvirus. We showed previously that PEL cell lines require the antiapoptotic protein MCL1 for survival but not the other BCL2 family proteins.

The SARS-CoV-2 Variant of Concern, Delta (B.1.617.2) was first reported in December 2020 in India and has spread colossally throughout the globe. Owing to factors like increased transmissibility, immune escape, and virulence, the delta variant has been considered as a potential public health threat apart from other variants of concern like alpha, beta and gamma. Kerala was one of the first states in India to enroll in the systematic genomic surveillance. In the present report, vaccine breakthrough infections were followed up in 147 patients including 55 healthcare workers who had been vaccinated with ChAdOx1 nCoV- 19/BBV152 across eleven districts from the state of Kerala. The timeline of samples analysed were from April 2021 till June 2021. Severity of the infections reported in the enrolled patients found to be mildly symptomatic, majorly with only 0.7% (n=1) of the cohort to be asymptomatic. Genomic analysis of the samples revealed the Delta variant (B.1.617.2) to constitute about 81.6% (n=120) in the studied cohort. This was followed by the Kappa variant B.1.617.1 (8.35%, n=9), AY.1 (0.6%, n= 1), AY.12 (0.6%, n= 1), AY.4 (1.2%, n= 2), AY.9 (1.2%, n= 2) and Eta variant, B.1.525 (0.6%, n= 1). 11 samples were not assigned any lineage. Evidence from this study suggests the preponderance of the Delta variant in the samples analysed.

We describe genomic analysis of SARS-CoV-2 isolates from breakthrough infections in six healthcare workers following vaccination with AZD1222/Covishield. Four patients were infected by the variant of concern B.1.1.7 while other isolates possessed E484K and S477N mutations in spike protein associated with immune escape.

Coronavirus disease 2019 (COVID-19) rapidly spread from a city in China to almost every country in the world, affecting millions of individuals. The rapid increase in the COVID-19 cases in the state of Kerala in India has necessitated the understanding of SARS-CoV-2 genetic epidemiology. We sequenced 200 samples from patients in Kerala using COVIDSeq protocol amplicon-based sequencing. The analysis identified 166 high-quality single-nucleotide variants encompassing four novel variants and 89 new variants in the Indian isolated SARS-CoV-2. Phylogenetic and haplotype analysis revealed that the virus was dominated by three distinct introductions followed by local spread suggesting recent outbreaks and that it belongs to the A2a clade. Further analysis of the functional variants revealed that two variants in the S gene associated with increased infectivity and five variants mapped in primer binding sites affect the efficacy of RT-PCR. To the best of our knowledge, this is the first and most comprehensive report of SARS-CoV-2 genetic epidemiology from Kerala.

The VPg protein of human Norovirus (hNoV) is a multi-functional protein essential for virus replication. The un-cleaved viral precursor protein, ProPol (NS5-6) was 100-fold more efficient in catalyzing VPg nucleotidylylation than the mature polymerase (Pol, NS6), suggesting a specific intracellular role for ProPol. Sequential and single-point alanine substitutions revealed that several positively charged amino acids in the N-terminal region of VPg regulate its nucleotidylylation by ProPol. We provide evidence that VPg directly binds NTPs, inhibition of binding inhibits nucleotidylylation, and NTP binding appears to involve the first 13 amino acids of the protein. Substitution of multiple positively charged amino acids within the first 12 amino acids of the N-terminal region inhibits nucleotidylylation without affecting binding. Substitution of only Lys20 abolishes nucleotidylylation, but not NTP binding. These studies indicate that positively charged amino acids in the first 20 amino acids of hNoV VPg regulate its nucleotidylylation though several potential mechanisms.

Specific sequence changes in codons 70 and 91 of the hepatitis C virus genotype 1b (HCV GT1b) core gene have been associated with increased risk of hepatocellular carcinoma (HCC). Essentially all previous studies were conducted in Asian populations with a wide range of liver disease, and none were conducted specifically in GT1a-infected individuals. We conducted a pilot study in a multiethnic population in the USA with HCV-related cirrhosis to determine if this association extended to GT1a-infected individuals and to determine if other sequence changes in the HCV core gene were associated with HCC risk. HCV core gene sequences from sera of 90 GT1 HCV carriers with cirrhosis (42 with HCC) were analysed using standard RT-PCR-based procedures. Nucleotide sequence data were compared with reference sequences available from GenBank. The frequency of sequence changes in codon 91 was not statistically different between HCC (7/19) and non-HCC (11/22) GT1b carriers. In GT1a carriers, sequence changes in codon 91 were observed less often than in GT1b carriers but were not observed in non-HCC subjects (4/23 vs 0/26, P = 0.03, Fisher's exact test). Sequence changes in codon 70 were not distributed differently between HCC and non-HCC GT1a and 1b carriers. Most importantly, for GT1a carriers, a panel of specific nucleotide changes in other codons was collectively present in all subjects with HCC, but not in any of the non-HCC patients. The utility of this test panel for early detection of HCC in GT1a-infected individuals needs to be assessed in larger populations, including longitudinal studies.

ABSTRACT Noroviruses (NoV) are members of the family Caliciviridae. The human NoV open reading frame 1 (ORF1) encodes a 200-kDa polyprotein which is cleaved by the viral 20-kDa 3C-like protease (Pro, NS6) into 6 nonstructural proteins that are necessary for viral replication. The NoV ORF1 polyprotein is processed in a specific order, with “early” sites (NS1/2-3 and NS3-4) being cleaved rapidly and three “late” sites (NS4-5, NS5-6, and NS6-7) processed subsequently and less efficiently. Previously, we demonstrated that the NoV polyprotein processing order is directly correlated with the efficiency of the enzyme, which is regulated by the primary amino acid sequences surrounding ORF1 cleavage sites. Using fluorescence resonance energy transfer (FRET) peptides representing the NS2-3 and NS6-7 ORF1 cleavage sites, we now demonstrate that the amino acids spanning positions P4 to P2′ (P4-P2′) surrounding each site comprise the core sequence controlling NoV protease enzyme efficiency. Furthermore, the NoV polyprotein self-processing order can be altered by interchanging this core sequence between NS2-3 and any of the three late sites in in vitro transcription-translation assays. We also demonstrate that the nature of the side chain at the P3 position for the NS1/2-3 (Nterm/NTPase) site confers significant influence on enzyme catalysis (k cat and k cat /Km ), a feature overlooked in previous structural studies. Molecular modeling provides possible explanations for the P3 interactions with NoV protease. IMPORTANCE Noroviruses (NoV) are the prevailing cause of nonbacterial acute gastroenteritis worldwide and pose a significant financial burden on health care systems. Proteolytic processing of the viral nonstructural polyprotein is required for norovirus replication. Previously, the core sequence of amino acids surrounding the scissile bonds responsible for governing the relative processing order had not been determined. Using both FRET-based peptides and full-length NoV polyprotein, we have successfully demonstrated that the core sequences spanning positions P4-P2′ surrounding the NS2-3, NS4-5, NS5-6, and NS6-7 cleavage sites contain all of the structural information necessary to control processing order. We also provide insight into a previously overlooked role for the NS2-3 P3 residue in enzyme efficiency. This article builds upon our previous studies on NoV protease enzymatic activities and polyprotein processing order. Our work provides significant additional insight into understanding viral polyprotein processing and has important implications for improving the design of inhibitors targeting the NoV protease.

The human norovirus (NoV) polyprotein is cleaved into mature non-structural proteins by both mature NoV protease (Pro, NS6) and its un-cleaved precursor (ProPol, NS6-7). Processing order is well-established with ‘early’ and ‘late’ cleavages, but the governing enzymatic mechanisms are unknown. Enzyme kinetics of a GII Pro and ProPol were analyzed using synthetic peptides representing the five natural polyprotein cleavage sites. The relative efficiency of cleavage of the individual peptides was consistent with established polyprotein processing order, and primarily correlated with enzyme turnover (kcat). Enzymatic efficiencies (kcat/Km) of ProPol at all five sites were equivalent to, or greater than, that of Pro. Binding affinities (Km) for the two least efficiently cleaved sites (p20/VPg, VPg/Pro) were 2–4-fold higher than the other sites. This work further defines the role of ProPol in NoV polyprotein cleavage, and demonstrates that human norovirus polyprotein processing order is primarily an inherent property of enzymatic activity.

A highly active, fluorescence-based, in vitro assay for human Norovirus protease from genogroup I and II viruses was optimized utilizing as little as 0.25μM enzyme, pH 7.6, and substrate:enzyme of 50-100. Activity in Tris-HCl or sodium phosphate buffers was 2-fold less than HEPES, and 2-fold lower for buffer concentrations over 10mM. Protease activity at pH 7.6 was 73% (GI) or 63% (GII) of activity at the optimal pH 9.0. Sodium inhibited activity 2-3 fold, while potassium, calcium, magnesium, and manganese inhibited 5-10 fold. Differences in efficiency due to pH, buffer, and cations were due to changes in kcat and not Km. Norovirus protease bound short RNAs representing the 3' or 5' ends of the virus, inhibiting protease activity (IC50 3-5μM) in a non-competitive manner. Previous reports indicated participation of the protease in the Norovirus replicase complex. The current studies provide initial support for a defined role for the viral protease in Norovirus replication.

Nitazoxanide (NTZ) exhibits potent antiviral activity against hepatitis C virus (HCV) in cell culture. Previously, HCV replicon-containing cell lines resistant to NTZ were selected, but transfer the HCV NTZ-resistance phenotype was not observed following transfection of whole cell RNA. To further explore the nature of the resistance of HCV to NTZ, full length HCV replicon sequences were obtained from two NTZ-resistant (NTZ-11, TIZ-9), and the parental (RP7) cell lines. Numerous nucleotide changes were observed in individual HCV genomes relative to the RP7 HCV consensus sequence, but no common mutations in the HCV non-structural genes or 3′-UTR were detected. A cluster of single nucleotide mutations was found within a 5-base portion of the 5′-UTR in 20/21 HCV replicon sequences from both resistant cell lines. Three mutations (5′-UTR G17A, G18A, C20U) were individually inserted into CON1 (‘wild-type’) HCV replicons, showed reduced replication (5 to 50-fold), but none conferred resistance to NTZ. RP7, NTZ-11, and TIZ-9 were cured of HCV genomes by serial passage under interferon. Transfection of cured NTZ-11 and TIZ-9 with either whole cell RNAs from RP7, NTZ-11, or TIZ-9, ‘wild-type’ or the 5′-UTR mutation-containing replicon RNAs exhibited an NTZ-resistance phenotype. TIZ (the active metabolite of NTZ) was found to be inactive against the activity of HCV polymerase, protease, and helicase in enzymatic assays. These data confirm previous speculations that HCV resistance to NTZ is not due to mutations in the virus, and demonstrate that HCV resistance and most likely the antiviral activity of TIZ are due to interactions with cellular target(s).

POSTERSmiR-122 expression and viral load in vivo.In addition, decreased pretreatment level of miR-122 is associated with no response during IFN therapy.In the present study, the miR-122 and miR-221 expression was analysed in chronic HCV infection with and without steatosis compared to non-infected, normal liver tissues.Methods: The microRNA expression was determined in 67 biopsy (12 HCV [genotype 1/b] without steatosis, 36 HCV with steatosis, 19 steatosis) and 6 normal formalin-fixed paraffin-embedded liver samples.TaqMan MicroRNA Assays (ID: 002245 and 000524, Life Technologies) were employed for the analysis of miR-122 and miR-221 expression from isolated total RNA (modified RNeasy FFPE kit, Qiagen).Results: The expression of both microRNAs were decreased in the three sample groups when compared to normal liver tissue.In average, the least miR-122 expression was observed in HCV without steatosis followed by HCV with steatosis and steatosis, while miR-221 expression was equally decreased in HCV without steatosis and HCV with steatosis but moderately decreased in steatosis. Conclusions:The results indicate that steatosis causes a less pronounced, however not significant, decrease in miR-122 expression even when associated with HCV infection, while no increase -observed during hepatocarcinogenesis -but decrease of miR-221 expression is present in chronic hepatitis C.

Recent advances in molecular biology have led to the development of novel small molecules that target specific viral proteins of the hepatitis C virus (HCV) life cycle. These drugs, collectively termed directly acting antivirals (DAA) against HCV, include a range of non-structural (NS) 3/NS4A protease, NS5B polymerase, and NS5A inhibitors at various stages of clinical development. The rapid replication rate of HCV, along with the low fidelity of its polymerase, gives rise to generations of mutations throughout the viral genome resulting in remarkable sequence variation in the HCV population, known as a quasispecies. The efficacy of DAAs is limited by the presence of those mutations that give rise to amino-acid substitutions within the targeted protein, and that affect the viral sensitivity to these compounds. Thus, due to the high genetic variability of HCV, variants with reduced susceptibility to DAA can occur naturally even before treatment begins, but usually at low levels. Not surprisingly then, these changes are selected in patients either breaking through or not responding to potent DAA treatment. In vitro or in vivo, six major position mutations in the NS3 HCV protease (36, 54, 155, 156, 168, and 170) have now been reported associated with different levels of resistance. The amino acid composition at several of the drug resistance sites can vary between the HCV genotypes/subtypes, resulting in different consensus amino acids leading to a reduction in replicative fitness as well as reduced DAA sensitivity. Different amino acid diversity profiles for HCV genotypes/subtypes suggest differences in the position/type of immune escape and drug resistance mutations. Also, different pathways of resistance profiles based on the chemical scaffold (linear or macrocyclic) of the protease inhibitors have been described. This review first describes how resistance to a protease inhibitor can develop and then provides an overview of the mechanism of how particular mutations confer varying levels of resistance to protease inhibitor, which have been identified and characterized using both genotypic and phenotypic tools. Future potential therapeutic strategies to assist patients who do develop resistance to protease inhibitors are also outlined. The challenge developing new HCV protease inhibitors should take into consideration not only the antiviral potency of the drugs, the occurrence and importance of side effects, the frequency of oral administration, but also the resistance profiles of these agents.

The 1-oxo-1, 2, 3, 4-tetrahydroisoquinoline and 1-Oxo-1, 2-dihydroisoquinoline scaffolds were utilized in the design and solution phase synthesis of focused libraries of compounds for screening against West Nile Virus (WNV) protease. Exploratory studies have led to the identification of a WNV protease inhibitor (a 1-oxo-1, 2-dihydroisoquinoline-based derivative, 12j) which could potentially serve as a launching pad for a hit-to-lead optimization campaign. The identified hit was devoid of any inhibitory activity toward a panel of mammalian serine proteases.