Whole-soil-profile warming does not change microbial carbon use efficiency in surface and deep soils.

The paucity of investigations of carbon (C) dynamics through the soil profile with warming makes it challenging to evaluate the terrestrial C feedback to climate change. Soil microbes are important engines driving terrestrial biogeochemical cycles; their carbon use efficiency (CUE), defined as the proportion of metabolized organic C allocated to microbial biomass, is a key regulator controlling the fate of soil C. It has been theorized that microbial CUE should decline with warming; however, empirical evidence for this response is scarce, and data from deeper soils are particularly scarce. Here, based on soil samples from a whole-soil-profile warming experiment (0 to 1 m, +4 °C) and 18O tracing approach, we examined the vertical variation of microbial CUE and its response to ~3.3-y experimental warming in an alpine grassland on the Qinghai-Tibetan Plateau. Microbial CUE decreased with soil depth, a trend that was primarily controlled by soil C availability. However, warming had limited effects on microbial CUE regardless of soil depth. Similarly, warming had no significant effect on soil C availability, as characterized by extractable organic C, enzyme-based lignocellulose index, and lignin phenol-based ratios of vanillyls, syringyls, and cinnamyls. Collectively, our work suggests that short-term warming does not alter microbial CUE in either surface or deep soils, and emphasizes the regulatory role of soil C availability on microbial CUE.

Center extraction method for reflected metallic surface fringes based on line structured light.

Using line structured light to measure metal surface topography, the extraction error of the stripe center is significant due to the influence of the optical characteristics of the metal surface and the scattering noise. This paper proposes a sub-pixel stripe center extraction method based on adaptive threshold segmentation and a gradient weighting strategy to address this issue. First, we analyze the characteristics of the stripe image of the measured metal's surface morphology. Relying on the morphological features of the image, the image is segmented to remove the effect of background noise and to obtain the region of interest in the image. Then, we use the gray-gravity method to get the rough center coordinates of the stripes. We extend the stripes in the width direction using the rough center coordinates as a reference to determine the center of the stripes for extraction after segmentation. Next, we adaptively determine the boundary threshold utilizing the region's grayscale. Finally, we use the gradient weighting strategy to extract the sub-pixel stripe center. The experimental results show that the proposed method effectively eliminates the interference of metal surface scattering on 3D reconstruction. The average height error of the measured standard block is 0.025 mm, and the repeatability of the measurement accuracy is 0.026 mm.

Molecular 14 C evidence for contrasting turnover and temperature sensitivity of soil organic matter components.

Climate projection requires an accurate understanding for soil organic carbon (SOC) decomposition and its response to warming. An emergent view considers that environmental constraints rather than chemical structure alone control SOC turnover and its temperature sensitivity (i.e., Q10 ), but direct long-term evidence is lacking. Here, using compound-specific radiocarbon analysis of soil profiles along a 3300-km grassland transect, we provide direct evidence for the rapid turnover of lignin-derived phenols compared with slower-cycling molecular components of SOC (i.e., long-chain lipids and black carbon). Furthermore, in contrast to the slow-cycling components whose turnover is strongly modulated by mineral association and exhibits low Q10 , lignin turnover is mainly regulated by temperature and has a high Q10 . Such contrasts resemble those between fast-cycling (i.e., light) and mineral-associated slow-cycling fractions from globally distributed soils. Collectively, our results suggest that warming may greatly accelerate the decomposition of lignin, especially in soils with relatively weak mineral associations.

Simulated climate warming decreases fruit number but increases seed mass.

Climate warming is changing plant sexual reproduction, having consequences for species distribution and community dynamics. However, the magnitude and direction of plant reproductive efforts (e.g., number of flowers) and success (e.g., number and mass of fruits or seeds) in response to warming have not been well-characterized. Here, we generated a global dataset of simulated warming experiments, consisting of 477 pairwise comparisons for 164 terrestrial species. We found evidence that warming overall decreased fruit number and increased seed mass, but little evidence that warming influenced flower number, fruit mass, or seed number. The warming effects on seed mass were regulated by the pollination type, and insect-pollinated plants exhibited a stronger response to warming than wind-pollinated plants. We found strong evidence that warming increased the mass of seeds for the nondominant species but no evidence of this for the dominant species. There was no evidence that phylogenetic relatedness explained the effects of warming on plant reproductive effort and success. In addition, the effects of warming on flowering onset negatively related to the responses in terms of the number of fruits and seeds to warming, revealing a cascading effect of plant reproductive development. These findings provide the first quantification of the response of terrestrial plant sexual reproduction to warming and suggest that plants may increase their fitness by producing heavier seeds under a warming climate.

Asymmetric response of aboveground and belowground temporal stability to nitrogen and phosphorus addition in a Tibetan alpine grassland.

Anthropogenic eutrophication is known to impair the stability of aboveground net primary productivity (ANPP), but its effects on the stability of belowground (BNPP) and total (TNPP) net primary productivity remain poorly understood. Based on a nitrogen and phosphorus addition experiment in a Tibetan alpine grassland, we show that nitrogen addition had little impact on the temporal stability of ANPP, BNPP, and TNPP, whereas phosphorus addition reduced the temporal stability of BNPP and TNPP, but not ANPP. Significant interactive effects of nitrogen and phosphorus addition were observed on the stability of ANPP because of the opposite phosphorus effects under ambient and enriched nitrogen conditions. We found that the stability of TNPP was primarily driven by that of BNPP rather than that of ANPP. The responses of BNPP stability cannot be predicted by those of ANPP stability, as the variations in responses of ANPP and BNPP to enriched nutrient, with ANPP increased while BNPP remained unaffected, resulted in asymmetric responses in their stability. The dynamics of grasses, the most abundant plant functional group, instead of community species diversity, largely contributed to the ANPP stability. Under the enriched nutrient condition, the synchronization of grasses reduced the grass stability, while the latter had a significant but weak negative impact on the BNPP stability. These findings challenge the prevalent view that species diversity regulates the responses of ecosystem stability to nutrient enrichment. Our findings also suggest that the ecological consequences of nutrient enrichment on ecosystem stability cannot be accurately predicted from the responses of aboveground components and highlight the need for a better understanding of the belowground ecosystem dynamics.

Carbon-biodiversity relationships in a highly diverse subtropical forest.

Carbon-focused climate mitigation strategies are becoming increasingly important in forests. However, with ongoing biodiversity declines we require better knowledge of how much such strategies account for biodiversity. We particularly lack information across multiple trophic levels and on established forests, where the interplay between carbon stocks, stand age, and tree diversity might influence carbon-biodiversity relationships. Using a large dataset (>4600 heterotrophic species of 23 taxonomic groups) from secondary, subtropical forests, we tested how multitrophic diversity and diversity within trophic groups relate to aboveground, belowground, and total carbon stocks at different levels of tree species richness and stand age. Our study revealed that aboveground carbon, the key component of climate-based management, was largely unrelated to multitrophic diversity. By contrast, total carbon stocks-that is, including belowground carbon-emerged as a significant predictor of multitrophic diversity. Relationships were nonlinear and strongest for lower trophic levels, but nonsignificant for higher trophic level diversity. Tree species richness and stand age moderated these relationships, suggesting long-term regeneration of forests may be particularly effective in reconciling carbon and biodiversity targets. Our findings highlight that biodiversity benefits of climate-oriented management need to be evaluated carefully, and only maximizing aboveground carbon may fail to account for biodiversity conservation requirements.

Microbially enhanced methane uptake under warming enlarges ecosystem carbon sink in a Tibetan alpine grassland.

The alpine grasslands of the Tibetan Plateau store 23.2 Pg soil organic carbon, which becomes susceptible to microbial degradation with climate warming. However, accurate prediction of how the soil carbon stock changes under future climate warming is hampered by our limited understanding of belowground complex microbial communities. Here, we show that 4 years of warming strongly stimulated methane (CH4 ) uptake by 93.8% and aerobic respiration (CO2 ) by 11.3% in the soils of alpine grassland ecosystem. Due to no significant effects of warming on net ecosystem CO2 exchange (NEE), the warming-stimulated CH4 uptake enlarged the carbon sink capacity of whole ecosystem. Furthermore, precipitation alternation did not alter such warming effects, despite the significant effects of precipitation on NEE and soil CH4 fluxes were observed. Metagenomic sequencing revealed that warming led to significant shifts in the overall microbial community structure and the abundances of functional genes, which contrasted to no detectable changes after 2 years of warming. Carbohydrate utilization genes were significantly increased by warming, corresponding with significant increases in soil aerobic respiration. Increased methanotrophic genes and decreased methanogenic genes were observed under warming, which significantly (R2  = .59, p < .001) correlated with warming-enhanced CH4 uptakes. Furthermore, 212 metagenome-assembled genomes were recovered, including many populations involved in the degradation of various organic matter and a highly abundant methylotrophic population of the Methyloceanibacter genus. Collectively, our results provide compelling evidence that specific microbial functional traits for CH4 and CO2 cycling processes respond to climate warming with differential effects on soil greenhouse gas emissions. Alpine grasslands may play huge roles in mitigating climate warming through such microbially enhanced CH4 uptake.

The mechanism and pharmacodynamics of 2-((1H-indol-3-yl)thio/sulfinyl)-N-pheny acetamide derivative as a novel inhibitor against human respiratory syncytial virus.

Human respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection worldwide. Until now, there are no licenced vaccines or effective antiviral drugs against RSV infections. In our previous work, we found 2-((1H-indol-3-yl)thio/sulfinyl)-N-pheny acetamide derivatives (4-49 C and 1-HB-63) being a novel inhibitor against RSV in vitro. Here, we explored the underlying mechanism of 2-((1H-indol-3-yl)thio/sulfinyl)-N-pheny acetamide derivatives to inhibit RSV replication in vitro and disclosed that 4-49 C worked as the inhibitor of membrane fusion and 1-HB-63 functioned at the stage of RSV genome replication/transcription. Yet, both of them could not inhibit RSV infection of BALB/c mice by using RSV-Luc, in vivo imaging and RT-qPCR analyses, for which it may be due to the fast metabolism in vivo. Our work suggests that further structural modification and optimisation of 2-((1H-indol-3-yl) thio/sulfinyl)-N-pheny acetamide derivative are needed to obtain drug candidates with effective anti-RSV activities in vivo.

Clusterin inhibits Aß42 aggregation through a "strawberry model" as detected by FRET-FCS.

Extracellular plaque deposits of ß-amyloid peptide (Aß) are one of the main pathological features of Alzheimer's disease (AD). The aggregation of Aß42 species, especially Aß42 oligomers, is still an active research field in AD pathogenesis. Secretory clusterin protein (sCLU), an extracellular chaperone, plays an important role in AD pathogenesis. Although sCLU interacts directly with Aß42 in vitro and in vivo, the mechanism is not clear. In this paper, His-tagged sCLU (sCLU-His) was cloned, expressed and purified, and we applied florescence resonance energy transfer-fluorescence correlation spectroscopy (FRET-FCS) to investigate the direct interaction of sCLU-His and Aß42 at the single-molecule fluorescence level in vitro. Here, we chose four different fluorescently labeled Aß42 oligomers to form two different groups of aggregation models, easy or difficult to aggregate. The results showed that sCLU-His could form complexes with both aggregation models, and sCLU-His inhibited the aggregation of Aß42/RB  ~ Aß42/Atto647 (easy to aggregate model). The complexes were produced as the Aß42/Label adhered to the sCLU-His, which is similar to a "strawberry model," as strawberry seeds are dotted on the outer surface of strawberries. This work provided additional insight into the interaction mechanism of sCLU and Aß42 .

Inactive and inefficient: Warming and drought effect on microbial carbon processing in alpine grassland at depth.

Subsoils contain >50% of soil organic carbon (SOC) globally yet remain under-investigated in terms of their response to climate changes. Recent evidence suggests that warmer, drier conditions in alpine grasslands induce divergent responses in SOC decomposition and carbon accrual in top- versus subsoils. However, longer term effects on microbial activity (i.e., catabolic respiration vs. anabolic growth) and belowground carbon cycling are not well understood. Here we utilized a field manipulation experiment on the Qinghai-Tibetan Plateau and conducted a 110-day soil incubation with and without 13 C-labeled grass litter to assess microbes' role as both SOC "decomposers" and "contributors" in the top- (0-10 cm) versus subsoils (30-40 cm) after 5 years of warming and drought treatments. Microbial mineralization of both SOC and added litter was examined in tandem with potential extracellular enzyme activities, while microbial biomass synthesis and necromass accumulation were analyzed using phospholipid fatty acids and amino sugars coupled with 13 C analysis, respectively. We found that warming and, to a lesser extent, drought decreased the ratio of inorganic nitrogen (N) to water-extractable organic carbon in the subsoil, intensifying N limitation at depth. Both SOC and litter mineralization were reduced in the subsoil, which may also be related to N limitation, as evidenced by lower hydrolase activity (especially leucine aminopeptidase) and reduced microbial efficiency (lower biomass synthesis and necromass accumulation relative to respiration). However, none of these effects were observed in the topsoil, suggesting that soil microbes became inactive and inefficient in subsoil but not topsoil environments. Given increasing belowground productivity in this alpine grassland under warming, both elevated root deposits and diminished microbial activity may contribute to new carbon accrual in the subsoil. However, the sustainability of plant growth and persistence of subsoil SOC pools deserve further investigation in the long term, given the aggravated N limitation at depth.

Shifting plant species composition in response to climate change stabilizes grassland primary production.

The structure and function of alpine grassland ecosystems, including their extensive soil carbon stocks, are largely shaped by temperature. The Tibetan Plateau in particular has experienced significant warming over the past 50 y, and this warming trend is projected to intensify in the future. Such climate change will likely alter plant species composition and net primary production (NPP). Here we combined 32 y of observations and monitoring with a manipulative experiment of temperature and precipitation to explore the effects of changing climate on plant community structure and ecosystem function. First, long-term climate warming from 1983 to 2014, which occurred without systematic changes in precipitation, led to higher grass abundance and lower sedge abundance, but did not affect aboveground NPP. Second, an experimental warming experiment conducted over 4 y had no effects on any aspect of NPP, whereas drought manipulation (reducing precipitation by 50%), shifted NPP allocation belowground without affecting total NPP. Third, both experimental warming and drought treatments, supported by a meta-analysis at nine sites across the plateau, increased grass abundance at the expense of biomass of sedges and forbs. This shift in functional group composition led to deeper root systems, which may have enabled plant communities to acquire more water and thus stabilize ecosystem primary production even with a changing climate. Overall, our study demonstrates that shifting plant species composition in response to climate change may have stabilized primary production in this high-elevation ecosystem, but it also caused a shift from aboveground to belowground productivity.

Plant diversity enhances productivity and soil carbon storage.

Despite evidence from experimental grasslands that plant diversity increases biomass production and soil organic carbon (SOC) storage, it remains unclear whether this is true in natural ecosystems, especially under climatic variations and human disturbances. Based on field observations from 6,098 forest, shrubland, and grassland sites across China and predictions from an integrative model combining multiple theories, we systematically examined the direct effects of climate, soils, and human impacts on SOC storage versus the indirect effects mediated by species richness (SR), aboveground net primary productivity (ANPP), and belowground biomass (BB). We found that favorable climates (high temperature and precipitation) had a consistent negative effect on SOC storage in forests and shrublands, but not in grasslands. Climate favorability, particularly high precipitation, was associated with both higher SR and higher BB, which had consistent positive effects on SOC storage, thus offsetting the direct negative effect of favorable climate on SOC. The indirect effects of climate on SOC storage depended on the relationships of SR with ANPP and BB, which were consistently positive in all biome types. In addition, human disturbance and soil pH had both direct and indirect effects on SOC storage, with the indirect effects mediated by changes in SR, ANPP, and BB. High soil pH had a consistently negative effect on SOC storage. Our findings have important implications for improving global carbon cycling models and ecosystem management: Maintaining high levels of diversity can enhance soil carbon sequestration and help sustain the benefits of plant diversity and productivity.

Alpine grassland plants grow earlier and faster but biomass remains unchanged over 35 years of climate change.

Satellite data indicate significant advancement in alpine spring phenology over decades of climate warming, but corresponding field evidence is scarce. It is also unknown whether this advancement results from an earlier shift of phenological events, or enhancement of plant growth under unchanged phenological pattern. By analyzing a 35-year dataset of seasonal biomass dynamics of a Tibetan alpine grassland, we show that climate change promoted both earlier phenology and faster growth, without changing annual biomass production. Biomass production increased in spring due to a warming-induced earlier onset of plant growth, but decreased in autumn due mainly to increased water stress. Plants grew faster but the fast-growing period shortened during the mid-growing season. These findings provide the first in situ evidence of long-term changes in growth patterns in alpine grassland plant communities, and suggest that earlier phenology and faster growth will jointly contribute to plant growth in a warming climate.

Phosphorus alleviation of nitrogen-suppressed methane sink in global grasslands.

Grassland ecosystems account for more than 10% of the global CH4 sink in soils. A 4-year field experiment found that addition of P alone did not affect CH4 uptake and experimental addition of N alone significantly suppressed CH4 uptake, whereas concurrent N and P additions suppressed CH4 uptake to a lesser degree. A meta-analysis including 382 data points in global grasslands corroborated these findings. Global extrapolation with an empirical modelling approach estimated that contemporary N addition suppresses CH4 sink in global grassland by 11.4% and concurrent N and P deposition alleviates this suppression to 5.8%. The P alleviation of N-suppressed CH4 sink is primarily attributed to substrate competition, defined as the competition between ammonium and CH4 for the methane mono-oxygenase enzyme. The N and P impacts on CH4 uptake indicate that projected increases in N and P depositions might substantially affect CH4 uptake and alter the global CH4 cycle.

COSORE: A community database for continuous soil respiration and other soil-atmosphere greenhouse gas flux data.

Globally, soils store two to three times as much carbon as currently resides in the atmosphere, and it is critical to understand how soil greenhouse gas (GHG) emissions and uptake will respond to ongoing climate change. In particular, the soil-to-atmosphere CO2 flux, commonly though imprecisely termed soil respiration (RS ), is one of the largest carbon fluxes in the Earth system. An increasing number of high-frequency RS measurements (typically, from an automated system with hourly sampling) have been made over the last two decades; an increasing number of methane measurements are being made with such systems as well. Such high frequency data are an invaluable resource for understanding GHG fluxes, but lack a central database or repository. Here we describe the lightweight, open-source COSORE (COntinuous SOil REspiration) database and software, that focuses on automated, continuous and long-term GHG flux datasets, and is intended to serve as a community resource for earth sciences, climate change syntheses and model evaluation. Contributed datasets are mapped to a single, consistent standard, with metadata on contributors, geographic location, measurement conditions and ancillary data. The design emphasizes the importance of reproducibility, scientific transparency and open access to data. While being oriented towards continuously measured RS , the database design accommodates other soil-atmosphere measurements (e.g. ecosystem respiration, chamber-measured net ecosystem exchange, methane fluxes) as well as experimental treatments (heterotrophic only, etc.). We give brief examples of the types of analyses possible using this new community resource and describe its accompanying R software package.

Exceptional potency and structural basis of a T1249-derived lipopeptide fusion inhibitor against HIV-1, HIV-2, and simian immunodeficiency virus.

Enfuvirtide (T20) is the only viral fusion inhibitor approved for clinical use, but it has relatively weak anti-HIV activity and easily induces drug resistance. In succession to T20, T1249 has been designed as a 39-mer peptide composed of amino acid sequences derived from HIV-1, HIV-2, and simian immunodeficiency virus (SIV); however, its development has been suspended due to formulation difficulties. We recently developed a T20-based lipopeptide (LP-40) showing greatly improved pharmaceutical properties. Here, we generated a T1249-based lipopeptide, termed LP-46, by replacing its C-terminal tryptophan-rich sequence with fatty acid. As compared with T20, T1249, and LP-40, the truncated LP-46 (31-mer) had dramatically increased activities in inhibiting a large panel of HIV-1 subtypes, with IC50 values approaching low picomolar concentrations. Also, LP-46 was an exceptionally potent inhibitor against HIV-2, SIV, and T20-resistant variants, and it displayed obvious synergistic effects with LP-40. Furthermore, we showed that LP-46 had increased helical stability and binding affinity with the target site. The crystal structure of LP-46 in complex with a target surrogate revealed its critical binding motifs underlying the mechanism of action. Interestingly, it was found that the introduced pocket-binding domain in LP-46 did not interact with the gp41 pocket as expected; instead, it adopted a mode similar to that of LP-40. Therefore, our studies have provided an exceptionally potent and broad fusion inhibitor for developing new anti-HIV drugs, which can also serve as a tool to exploit the mechanisms of viral fusion and inhibition.

Warming affects foliar fungal diseases more than precipitation in a Tibetan alpine meadow.

The effects of global change on semi-natural and agro-ecosystem functioning have been studied extensively. However, less well understood is how global change will influence fungal diseases, especially in a natural ecosystem. We use data from a 6-yr factorial experiment with warming (simulated using infrared heaters) and altered precipitation treatments in a natural Tibetan alpine meadow ecosystem, from which we tested global change effects on foliar fungal diseases at the population and community levels, and evaluated the importance of direct effects of the treatments and community-mediated (indirect) effects (through changes in plant community composition and competence) of global change on community pathogen load. At the population level, we found warming significantly increased fungal diseases for nine plant species. At the community level, we found that warming significantly increased pathogen load of entire host communities, whereas no significant effect of altered precipitation on community pathogen load was detected. We concluded that warming influences fungal disease prevalence more than precipitation does in a Tibetan alpine meadow. Moreover, our study provides new experimental evidence that increases in disease burden on some plant species and for entire host communities is primarily the direct effects of warming, rather than community-mediated (indirect) effects.

Climate warming alters subsoil but not topsoil carbon dynamics in alpine grassland.

Subsoil contains more than half of soil organic carbon (SOC) globally and is conventionally assumed to be relatively unresponsive to warming compared to the topsoil. Here, we show substantial changes in carbon allocation and dynamics of the subsoil but not topsoil in the Qinghai-Tibetan alpine grasslands over 5 years of warming. Specifically, warming enhanced the accumulation of newly synthesized (14 C-enriched) carbon in the subsoil slow-cycling pool (silt-clay fraction) but promoted the decomposition of plant-derived lignin in the fast-cycling pool (macroaggregates). These changes mirrored an accumulation of lipids and sugars at the expense of lignin in the warmed bulk subsoil, likely associated with shortened soil freezing period and a deepening root system. As warming is accompanied by deepening roots in a wide range of ecosystems, root-driven accrual of slow-cycling pool may represent an important and overlooked mechanism for a potential long-term carbon sink at depth. Moreover, given the contrasting sensitivity of SOC dynamics at varied depths, warming studies focusing only on surface soils may vastly misrepresent shifts in ecosystem carbon storage under climate change.

Tree species richness increases ecosystem carbon storage in subtropical forests.

Forest ecosystems are an integral component of the global carbon cycle as they take up and release large amounts of C over short time periods (C flux) or accumulate it over longer time periods (C stock). However, there remains uncertainty about whether and in which direction C fluxes and in particular C stocks may differ between forests of high versus low species richness. Based on a comprehensive dataset derived from field-based measurements, we tested the effect of species richness (3-20 tree species) and stand age (22-116 years) on six compartments of above- and below-ground C stocks and four components of C fluxes in subtropical forests in southeast China. Across forest stands, total C stock was 149 ± 12 Mg ha-1 with richness explaining 28.5% and age explaining 29.4% of variation in this measure. Species-rich stands had higher C stocks and fluxes than stands with low richness; and, in addition, old stands had higher C stocks than young ones. Overall, for each additional tree species, the total C stock increased by 6.4%. Our results provide comprehensive evidence for diversity-mediated above- and below-ground C sequestration in species-rich subtropical forests in southeast China. Therefore, afforestation policies in this region and elsewhere should consider a change from the current focus on monocultures to multi-species plantations to increase C fixation and thus slow increasing atmospheric CO2 concentrations and global warming.

Enfuvirtide (T20)-Based Lipopeptide Is a Potent HIV-1 Cell Fusion Inhibitor: Implications for Viral Entry and Inhibition.

The peptide drug enfuvirtide (T20) is the only viral fusion inhibitor used in combination therapy for HIV-1 infection, but it has relatively low antiviral activity and easily induces drug resistance. Emerging studies demonstrate that lipopeptide-based fusion inhibitors, such as LP-11 and LP-19, which mainly target the gp41 pocket site, have greatly improved antiviral potency and in vivo stability. In this study, we focused on developing a T20-based lipopeptide inhibitor that lacks pocket-binding sequence and targets a different site. First, the C-terminal tryptophan-rich motif (TRM) of T20 was verified to be essential for its target binding and inhibition; then, a novel lipopeptide, termed LP-40, was created by replacing the TRM with a fatty acid group. LP-40 showed markedly enhanced binding affinity for the target site and dramatically increased inhibitory activity on HIV-1 membrane fusion, entry, and infection. Unlike LP-11 and LP-19, which required a flexible linker between the peptide sequence and the lipid moiety, addition of a linker to LP-40 sharply reduced its potency, implying different binding modes with the extended N-terminal helices of gp41. Also, interestingly, LP-40 showed more potent activity than LP-11 in inhibiting HIV-1 Env-mediated cell-cell fusion while it was less active than LP-11 in inhibiting pseudovirus entry, and the two inhibitors displayed synergistic antiviral effects. The crystal structure of LP-40 in complex with a target peptide revealed their key binding residues and motifs. Combined, our studies have not only provided a potent HIV-1 fusion inhibitor, but also revealed new insights into the mechanisms of viral inhibition.IMPORTANCE T20 is the only membrane fusion inhibitor available for treatment of viral infection; however, T20 requires high doses and has a low genetic barrier for resistance, and its inhibitory mechanism and structural basis remain unclear. Here, we report the design of LP-40, a T20-based lipopeptide inhibitor that has greatly improved anti-HIV activity and is a more potent inhibitor of cell-cell fusion than of cell-free virus infection. The binding modes of two classes of membrane-anchoring lipopeptides (LP-40 and LP-11) verify the current fusion model in which an extended prehairpin structure bridges the viral and cellular membranes, and their complementary effects suggest a vital strategy for combination therapy of HIV-1 infection. Moreover, our understanding of the mechanism of action of T20 and its derivatives benefits from the crystal structure of LP-40.