Uncovering the processes of microbial community assembly in the near-surface sediments of a climate-sensitive glacier-fed lake.

Glacier-fed lakes are characterized by cold temperatures, high altitudes, and nutrient-poor conditions. Despite these challenging conditions, near-surface sediments of glacier-fed lakes harbor rich microbial communities that are critical for ecosystem functioning and serve as a bridge between aquatic ecology and the deep subsurface biosphere. However, there is limited knowledge regarding the microbial communities and their assembly processes in these sediments, which are highly vulnerable to climate change. To fill this knowledge gap, this study systematically analyzed environmental variables, microbial communities, diversity, co-occurrence relationships, and community assembly processes in the near-surface sediments of a glacier-fed lake in the Tibetan Plateau. The results revealed distinct vertical gradients in microbial diversity and subcommunities, highlighting the significant influence of selection processes and adaptive abilities on microbial communities. Specifically, specialists played a crucial role within the overall microbial communities. Microbial assembly was primarily driven by homogeneous selection, but its influence declined with increasing depth. In contrast, homogenizing dispersal showed an opposite pattern, and the bottom layer exhibited heterogeneous selection and undominated processes. These patterns of microbial assembly were primarily driven by environmental gradients, with significant contributions from processes associated to ammonium and organic matter deposition, as well as chemical precipitation in response to a warming climate. This study enhances our understanding of the microbial communities and assembly processes in the near-surface sediments of glacier-fed lakes and sheds light on geo-microbiological processes in climate-sensitive lacustrine sediments.

Phosphine production in anaerobic wastewater treatment under tetracycline antibiotic pressure.

The influence of tetracycline (TC) antibiotics on phosphine (PH3) production in the anaerobic wastewater treatment was studied. A lab-scale anaerobic baffled reactor with three compartments was employed to simulate this process. The reactor was operated in a TC-absence wastewater and 250µg/L TC-presence wastewater for three months after a start-up period, respectively. The responses of pH, oxidation-reduction potential (ORP), chemical oxygen demand (COD), total phosphorus (TP), enzymes activity (dehydrogenase and acid phosphatase), and microbial community were investigated to reveal the effect of TC on PH3 production. Results suggested that the dehydrogenase (DH) activity, acid phosphatase (ACP) activity and COD have positive relationship with PH3 production, while pH, ORP level and the TP in liquid phase have negative relationship with PH3 production. With prolonged TC exposure, decrease in pH and increase in DH activity are beneficial to PH3 production, while decrease in COD and ACP activity are not the limiting factors for PH3 production.

In vitro characterization of MK-1439, a novel HIV-1 nonnucleoside reverse transcriptase inhibitor.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are a mainstay of therapy for treating human immunodeficiency type 1 virus (HIV-1)-infected patients. MK-1439 is a novel NNRTI with a 50% inhibitory concentration (IC50) of 12, 9.7, and 9.7 nM against the wild type (WT) and K103N and Y181C reverse transcriptase (RT) mutants, respectively, in a biochemical assay. Selectivity and cytotoxicity studies confirmed that MK-1439 is a highly specific NNRTI with minimum off-target activities. In the presence of 50% normal human serum (NHS), MK-1439 showed excellent potency in suppressing the replication of WT virus, with a 95% effective concentration (EC95) of 20 nM, as well as K103N, Y181C, and K103N/Y181C mutant viruses with EC95 of 43, 27, and 55 nM, respectively. MK-1439 exhibited similar antiviral activities against 10 different HIV-1 subtype viruses (a total of 93 viruses). In addition, the susceptibility of a broader array of clinical NNRTI-associated mutant viruses (a total of 96 viruses) to MK-1439 and other benchmark NNRTIs was investigated. The results showed that the mutant profile of MK-1439 was superior overall to that of efavirenz (EFV) and comparable to that of etravirine (ETR) and rilpivirine (RPV). Furthermore, E138K, Y181C, and K101E mutant viruses that are associated with ETR and RPV were susceptible to MK-1439 with a fold change (FC) of <3. A two-drug in vitro combination study indicated that MK-1439 acts nonantagonistically in the antiviral activity with each of 18 FDA-licensed drugs for HIV infection. Taken together, these in vitro data suggest that MK-1439 possesses the desired properties for further development as a new antiviral agent.

Disentangling external loadings, hydrodynamics and biogeochemical controls on the fate of nitrate in a coastal embayment.

Nitrogen, as an essential nutrient, largely contributes to the coastal eutrophication. However, the accurate depiction and evaluation of how external loadings, hydrodynamics, and biogeochemical reactions mediate the occurrence, transport, and transformation of nitrate (NO3-) within coastal embayment still pose ongoing challenges to date. In this study, we took advantage of dual isotopes of NO3- to track external NO3- loadings, radium and dual isotopes of H2O to characterize the influences of hydrodynamic on NO3- transport, δ18O-NO3- and δ18O-H2O along with microbial analysis to explore major NO3- biogeochemical reactions in Tolo Harbour, Hong Kong. The multiple isotopic evidence showed that NO3- in surface harbour water was predominantly contributed by precipitation in wet season and its impact was strengthened by stratification. In dry season, NO3- in the surface harbour water became largely influenced by benthic input and biogeochemical reactions due to intensified vertical mixing. Based on NO3- mass balance model, biogeochemical reaction, especially nitrification, was found to be the major process to secure the closure of NO3- budget and increase NO3- inventory from wet to dry season. Hydrodynamics redistributed the external NO3- loadings and mediated nitrogen biogeochemical reactions, both of which further synergistically regulated the fate of NO3- in the embayment.

A Novel Rotavirus Reverse Genetics Platform Supports Flexible Insertion of Exogenous Genes and Enables Rapid Development of a High-Throughput Neutralization Assay.

Despite the success of rotavirus vaccines, rotaviruses remain one of the leading causes of diarrheal diseases, resulting in significant childhood morbidity and mortality, especially in low- and middle-income countries. The reverse genetics system enables the manipulation of the rotavirus genome and opens the possibility of using rotavirus as an expression vector for heterologous proteins, such as vaccine antigens and therapeutic payloads. Here, we demonstrate that three positions in rotavirus genome-the C terminus of NSP1, NSP3 and NSP5-can tolerate the insertion of reporter genes. By using rotavirus expressing GFP, we develop a high-throughput neutralization assay and reveal the pre-existing immunity against rotavirus in humans and other animal species. Our work shows the plasticity of the rotavirus genome and establishes a high-throughput assay for interrogating humoral immune responses, benefiting the design of next-generation rotavirus vaccines and the development of rotavirus-based expression platforms.

Potent targeted activator of cell kill molecules eliminate cells expressing HIV-1.

Antiretroviral therapy inhibits HIV-1 replication but is not curative due to establishment of a persistent reservoir after virus integration into the host genome. Reservoir reduction is therefore an important HIV-1 cure strategy. Some HIV-1 nonnucleoside reverse transcriptase inhibitors induce HIV-1 selective cytotoxicity in vitro but require concentrations far exceeding approved dosages. Focusing on this secondary activity, we found bifunctional compounds with HIV-1-infected cell kill potency at clinically achievable concentrations. These targeted activator of cell kill (TACK) molecules bind the reverse transcriptase-p66 domain of monomeric Gag-Pol and act as allosteric modulators to accelerate dimerization, resulting in HIV-1+ cell death through premature intracellular viral protease activation. TACK molecules retain potent antiviral activity and selectively eliminate infected CD4+ T cells isolated from people living with HIV-1, supporting an immune-independent clearance strategy.

Antiviral activity and in vitro mutation development pathways of MK-6186, a novel nonnucleoside reverse transcriptase inhibitor.

MK-6186 is a novel nonnucleoside reverse transcriptase inhibitor (NNRTI) which displays subnanomolar potency against wild-type (WT) virus and the two most prevalent NNRTI-resistant RT mutants (K103N and Y181C) in biochemical assays. In addition, it showed excellent antiviral potency against K103N and Y181C mutant viruses, with fold changes (FCs) of less than 2 and 5, respectively. When a panel of 12 common NNRTI-associated mutant viruses was tested with MK-6186, only 2 relatively rare mutants (Y188L and V106I/Y188L) were highly resistant, with FCs of >100, and the remaining viruses showed FCs of <10. Furthermore, a panel of 96 clinical virus isolates with NNRTI resistance mutations was evaluated for susceptibility to NNRTIs. The majority (70%) of viruses tested displayed resistance to efavirenz (EFV), with FCs of >10, whereas only 29% of the mutant viruses displayed greater than 10-fold resistance to MK-6186. To determine whether MK-6186 selects for novel resistance mutations, in vitro resistance selections were conducted with one isolate each from subtypes A, B, and C under low-multiplicity-of-infection (MOI) conditions. The results showed a unique mutation development pattern in which L234I was the first mutation to emerge in the majority of the experiments. In resistance selection under high-MOI conditions with subtype B virus, V106A was the dominant mutation detected in the breakthrough viruses. More importantly, mutant viruses selected by MK-6186 showed FCs of <10 against EFV or etravirine (ETR), and the mutant viruses containing mutations selected by EFV or ETR were sensitive to MK-6186 (FCs of <10).

TM7SF1 (GPR137B): a novel lysosome integral membrane protein.

In the previous proteomic study of human placenta, transmembrane 7 superfamily member 1 (TM7SF1) was found enriched in lysosome compartments. TM7SF1 encodes a 399-amino acid protein with a calculated molecular mass of 45 kDa. Bioinformatic analysis of its amino acid sequence showed that it is a multipass transmembrane protein containing a potential dileucine-based lysosomal targeting signal and four putative N-glycosylation sites. By percoll-gradient centrifugation and further subfraction ways, the lysosomal solute and membrane compartments were isolated respectively. Immunoblotting analysis indicated that TM7SF1 was co-fractioned with lysosome associated membrane protein 2 (LAMP2), which was only detected in lysosomal membrane compartments whereas not detected in the solute compartments. Using specific anti-TM7SF1 antibody and double-immunofluorescence with lysosome membrane protein LAMP1 and Lyso-Tracker Red, the colocalisations of endogenous TM7SF1 with lysosome and late endosome markers were demonstrated. All of this indicated that TM7SF1 is an integral lysosome membrane protein. Rat ortholog of TM7SF1 was found to be strongly expressed in heart, liver, kidney and brain while not or low detected in other tissues. In summary, TM7SF1 was a lysosomal integral membrane protein that shows tissue-specific expression. As a G-protein-coupled receptor in lysosome membrane, TM7SF1 was predicted function as signal transduction across lysosome membrane.

Microbial community assembly and co-occurrence relationship in sediments of the river-dominated estuary and the adjacent shelf in the wet season.

In the estuarine ecosystem, microbial community plays a vital role in controlling biogeochemical processes. However, there is currently limited comprehensive study on the deterministic and stochastic processes that drive the microbial community assembly in the estuaries and adjacent shelves. In this study, we systematically investigated the co-occurrence relationship and microbial community assembly in the sediments along a large river-dominated estuary to shelf in the northern South China Sea during the wet season. The sampling sites were divided into estuary, transection, and shelf sections based on their salinity values. The microbial co-occurrence networks, hierarchical partitioning-based canonical analysis, null model, neutral community model, and the Mantel test were used to investigate the community assembly. Results suggested that microbial community in the estuary section exhibited more interactions and a higher positive interaction ratio than those in the transition and shelf sections. Stochastic processes dominated community assembly in the study, with homogenizing dispersal contributing the most. The estuary exhibited a higher degree of heterogeneous selection than the transition and shelf sections, whereas homogeneous selection showed an opposite trend. Only the estuary section showed dispersal limitation and undominated processes. The river inflow and the resulting environmental heterogeneity were believed to be the key regulators of the community assembly in the studied area. Our study improved the understanding of how microbial community assembly in estuaries and adjacent shelves.

A Phenotypic Screen Identifies Potent DPP9 Inhibitors Capable of Killing HIV-1 Infected Cells.

Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 ("inducer of cell death-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.

Distinct mutation pathways of non-subtype B HIV-1 during in vitro resistance selection with nonnucleoside reverse transcriptase inhibitors.

Studies were conducted to investigate mutation pathways among subtypes A, B, and C of human immunodeficiency virus type 1 (HIV-1) during resistance selection with nonnucleoside reverse transcriptase inhibitors (NNRTIs) in cell culture under low-multiplicity of infection (MOI) conditions. The results showed that distinct pathways were selected by different virus subtypes under increasing selective pressure of NNRTIs. F227C and Y181C were the major mutations selected by MK-4965 in subtype A and C viruses during resistance selection. With efavirenz (EFV), F227C and V106M were the major mutations responsible for viral breakthrough in subtype A viruses, whereas a single pathway (G190A/V106M) accounted for mutation development in subtype C viruses. Y181C was the dominant mutation in the resistance selection with etravirine (ETV) in subtype A, and E138K/H221Y were the mutations detected in the breakthrough viruses from subtype C viruses with ETV. In subtype B viruses, on the other hand, known NNRTI-associated mutations (e.g., Y181C, P236L, L100I, V179D, and K103N) were selected by the NNRTIs. The susceptibility of the subtype A and B mutant viruses to NNRTIs was determined in order to gain insight into the potential mechanisms of mutation development. Collectively, these results suggest that minor differences may exist in conformation of the residues within the NNRTI binding pocket (NNRTIBP) of reverse transcriptase (RT) among the three subtypes of viruses. Thus, the interactions between NNRTIs and the residues in the NNRTIBPs of different subtypes may not be identical, leading to distinct mutation pathways during resistance selection in cell culture.

Purification of untagged HIV-1 reverse transcriptase by affinity chromatography.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) plays an essential role in the life cycle of the virus. Therefore, RT has been a primary target in the development of antiviral agents against HIV-1. Given the prevalence of resistant viruses, evaluation of the resistance profile of potential drug candidates is a key step in drug development. A simplified RT purification protocol would facilitate this process, as it provides an efficient method by which to purify RT variants for compound evaluation. Traditional purification protocols require the use of several columns to purify untagged RT. The entire procedure usually requires at least one week to complete. Herein, we report two novel methods that enable us to purify highly active RT in either one or two steps. First, a one-step purification protocol was developed by employing an affinity column that was prepared by conjugating an RNase H specific inhibitor (RNHI) with NHS-activated resin. Cell lysate containing RT was loaded onto the column followed by washing in the presence of 2mM Mn(2+). The RT retained in the column was eluted after soaking overnight in 10mM EDTA to retrieve the Mn(2+). In the other method, a vector was constructed that encodes RT fused to cleavable intein and AviTag (a biotin tag) sequences at the C-terminus. Cell lysate containing biotinylated RT was passed through a DE-52 column and then loaded onto an avidin column. Untagged RT was released from the column by reductive cleavage of the intein by DTT. These two methods significantly shorten the time required to purify untagged WT and mutant RTs.

Heavy metal contamination in surface sediments: A comprehensive, large-scale evaluation for the Bohai Sea, China.

Heavy metal contamination in the Bohai Sea (China) has been the focus of many studies, but most of them only focused on local pollution levels and thus lacked high spatial resolution for the whole sea. In this study, heavy metals (i.e., As, Cr, Cu, Cd, Pb, Zn, and Fe) in surface sediments were analyzed to assess the spatio-temporal pollution conditions of the Bohai Sea, an important coastal environment consisting of Bohai Bay, Laizhou Bay, and Liaodong Bay. The results indicated that the heavy metal concentration in the sediments was in the range of 6.43-32.18 mg/kg for As, 14.90-58.07 mg/kg for Cr, 3.90-27.19 mg/kg for Cu, 0.04-0.27 mg/kg for Cd, 11.09-30.95 mg/kg for Pb, 18.76-65.58 mg/kg for Zn, and 0.78%-2.55% for Fe. The distribution of heavy metals revealed that the concentrations were relatively low in Laizhou Bay, very high in the northwest coastal region of the Bohai Sea, and decreased from near-shore to off-shore areas. Moreover, both the enrichment factor and geo-accumulation index demonstrated that there was no contamination to be found for Cr, Cu, Zn in the region and a slight to moderate pollution of As, Cd, and Pb. Cd and As presented considerable potential ecological risk as a result of their high toxicity. The potential ecological risk index (RI) suggested that a third of the areas (northwest coastal area of the Bohai Sea) has moderate ecological risk. The risk area was generally decreased as offshore distance increased, which suggested that the contamination and risk of heavy metals are influenced by anthropogenic activities.

Substituted tetrahydroquinolines as potent allosteric inhibitors of reverse transcriptase and its key mutants.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are key elements of multidrug regimens, called HAART (Highly Active Antiretroviral Therapy), that are used to treat HIV-1 infections. Elucidation of the structure-activity relationships of the thiocarbamate moiety of the previous published lead compound 2 provided a series of novel tetrahydroquinoline derivatives as potent inhibitors of HIV-1 RT with nanomolar intrinsic activity on the WT and key mutant enzymes and potent antiviral activity in infected cells. The SAR optimization, mutation profiles, preparation of compounds, and pharmacokinetic profile of compounds are described.

Tidal induced dynamics and geochemical reactions of trace metals (Fe, Mn, and Sr) in the salinity transition zone of an intertidal aquifer.

Biogeochemical reactions in an intertidal aquifer influences the submarine groundwater discharge (SGD) associated trace metal flux to the ocean. Tidal fluctuation greatly affects the physical mixing, and biogeochemical transformation of trace metals in the intertidal aquifer. This study presents the dynamics of trace metals (Fe, Mn, and Sr) and the production of Fe2+ in the salinity transition zone is discovered. The variations of Fe2+ are led by the shifts of both physical mixing and biogeochemical reaction during tidal fluctuation. The transformation from amorphous Fe(OH)3 to FeS is the main reason for the enrichment of Fe2+ in the zone with a salinity of 0.5-10. Mn behaves much less active than Fe in the intertidal aquifer due to the very limited Mn in the solid phase and the major driving force of Mn2+ variation is the physical mixing rather than geochemical reaction. Sr2+ behaves conservatively and shows a synchronous with salinity in the salinity transition zone. This study found that Fe2+ precipitates in a form not limited to Fe (hydro)oxides and the FeS minerals is the most possible form of precipitation in reduced aquifers. In that case, only a small part of Fe2+ discharges to the sea associated with SGD, but Mn2+ has a comparatively conservative property during the transport in the intertidal aquifer and majority of the Mn2+ originated from fresh groundwater will discharge with SGD in this study. The biogeochemical transformation pathways of Fe and Mn observed in this study provides insights into the cycles of Fe and Mn in an intertidal aquifer, which is of significance to accurately estimate the SGD derived Fe and Mn fluxes to the ocean.

Nutrients and heavy metals mediate the distribution of microbial community in the marine sediments of the Bohai Sea, China.

The Bohai Sea, one of the largest marginal seas in China, is extensively influenced by human and industrial activities. The pollutant loads from anthropogenic activities have induced severe ecological problems. The study investigates the physicochemical characteristics of seawater and sediments in Bohai Bay and Laizhou Bay of the Bohai Sea. The diversity and composition of microbial community in sediments are analyzed by 16S rRNA gene amplicon sequencing. The sequencing results present 16 phyla and 31 classes from the samples. Proteobacteria constituted a dominant phylum, of which the classes of Gamma-, Delta-, and Epsilon-are predominant sub-divisions. Nitrogen, phosphorus, and sulfur cycling related microbes present high abundance in both bays. The metabolism of organic matters is the main factor that influences the distribution of microbial communities in Bohai Bay, while the inflow of Yellow River is the dominant factor that influences the distribution of microbial communities in Laizhou Bay. Sulfur oxidizing process is expected to be positively influenced by heavy metals, while ammonia (NH4+) oxidizing process is prone to be negatively affected by heavy metals in both bays. Microbial communities in the offshore sediments of Laizhou Bay and the majority microbial communities in Bohai Bay sediments are subject to similar predominant controlling factors. This phenomenon is likely ascribed to ocean circulation. The results of this study can provide constructive guidelines on ecosystem management of marginal seas in Bohai and elsewhere.

Novel indole-3-sulfonamides as potent HIV non-nucleoside reverse transcriptase inhibitors (NNRTIs).

This Letter describes the design, synthesis, and biological evaluation of novel 3-indole sulfonamides as potent non-nucleoside reverse transcriptase inhibitors (NNRTIs) with balanced profiles against common HIV RT mutants K103N and Y181C.

Endoplasmic reticulum protein 29 is involved in endoplasmic reticulum stress in islet beta cells.

Endoplasmic reticulum stress (ERS) is correlated with insulin resistance and islet­cell function. In the present study, the sub­cellular localization and role of ER protein 29 (Erp29) were investigated in an in vitro ERS model of islet beta cells. The INS­1 islet cell line was treated with various concentrations of tunicamycin to establish the ERS model. Immunofluorescence microscopy demonstrated that Erp29 and anti­ER marker protein calnexin were co­localized in NIH3T3 cells, suggesting that Erp29 is localized to the ER. The ERS model induced by tunicamycin showed significantly increased expression of binding immunoglobulin protein (BIP)/glucose­regulated protein 78 (Grp78), which is a marker for ERS, and the expression of Erp29 was also increased at the mRNA and protein levels. Of note, ERS was blocked following small interfering RNA­mediated silencing of Erp29 expression, as indicated by reduced BIP/Grp78 expression. As an ER protein, Erp29 may have an important role in ERS in islet beta cells.

Biogas generation in anaerobic wastewater treatment under tetracycline antibiotic pressure.

The effect of tetracycline (TC) antibiotic on biogas generation in anaerobic wastewater treatment was studied. A lab-scale Anaerobic Baffled Reactor (ABR) with three compartments was used. The reactor was operated with synthetic wastewater in the absence of TC and in the presence of 250 µg/L TC for 90 days, respectively. The removal rate of TC, volatile fatty acids (VFAs), biogas compositions (hydrogen (H2), methane (CH4), carbon dioxide (CO2)), and total biogas production in each compartment were monitored in the two operational conditions. Results showed that the removal rate of TC was 14.97-67.97% in the reactor. The presence of TC had a large negative effect on CH4 and CO2 generation, but appeared to have a positive effect on H2 production and VFAs accumulation. This response indicated that the methanogenesis process was sensitive to TC presence, but the acidogenesis process was insensitive. This suggested that the presence of TC had less influence on the degradation of organic matter but had a strong influence on biogas generation. Additionally, the decrease of CH4 and CO2 generation and the increase of H2 and VFAs accumulation suggest a promising strategy to help alleviate global warming and improve resource recovery in an environmentally friendly approach.

Mechanistic Study of Common Non-Nucleoside Reverse Transcriptase Inhibitor-Resistant Mutations with K103N and Y181C Substitutions.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are a mainstay of therapy for human immunodeficiency type 1 virus (HIV-1) infections. However, their effectiveness can be hampered by the emergence of resistant mutations. To aid in designing effective NNRTIs against the resistant mutants, it is important to understand the resistance mechanism of the mutations. Here, we investigate the mechanism of the two most prevalent NNRTI-associated mutations with K103N or Y181C substitution. Virus and reverse transcriptase (RT) with K103N/Y188F, K103A, or K103E substitutions and with Y181F, Y188F, or Y181F/Y188F substitutions were employed to study the resistance mechanism of the K103N and Y181C mutants, respectively. Results showed that the virus and RT with K103N/Y188F substitutions displayed similar resistance levels to the virus and RT with K103N substitution versus NNRTIs. Virus and RT containing Y181F, Y188F, or Y181F/Y188F substitution exhibited either enhanced or similar susceptibility to NNRTIs compared with the wild type (WT) virus. These results suggest that the hydrogen bond between N103 and Y188 may not play an important role in the resistance of the K103N variant to NNRTIs. Furthermore, the results from the studies with the Y181 or Y188 variant provide the direct evidence that aromatic π-π stacking plays a crucial role in the binding of NNRTIs to RT.