Biology subjects

Smith, A. B.

Publications and source records attributed to Smith, A. B..

At least 19 recordsLinked to original sources

bioRxiv2025

Ecological niche modeling reveals habitat differentiation and climatic vulnerability in two imperiled, sympatric southern Appalachian carnivorous plants

PREMISEUnderstanding the habitat requirements of imperiled flora is critical for informing ex situ conservation practices, designing effective reintroduction strategies, and understanding how climate change will impact such species, especially in montane regions with high levels of environmental heterogeneity. In southern Appalachia, USA, the Mountain Sweet Pitcher Plant (Sarracenia rubra ssp. jonesii) and Mountain Purple Pitcher Plant (Sarracenia purpurea var. montana) inhabit overlapping ranges. These taxa rarely co-occur in the same mountain bogs but frequently hybridize at sites where they do co-occur. METHODSWe assessed patterns of climatic niche differentiation in these imperiled taxa to explore whether they naturally co-occur or may have been brought into secondary contact through human translocations. In addition, we constructed ecological niche models to evaluate the comparative availability of suitable habitat for each taxon under present and future climates. RESULTSWe 1) find evidence that the two taxa inhabit distinct niches, and 2) predict that current populations of Sarracenia rubra ssp. jonesii will experience climates markedly different from those it currently inhabits in the future, while 3) suitable habitat for S. purpurea var. montana will remain comparatively stable and may expand in its current range. CONCLUSIONSDespite high spatial overlap, these two related taxa exhibit divergent climatic niches, resulting in highly different management needs and conservation approaches. We raise concerns about the future of mountain bog plant assemblages, and the rare species they include, under climate change.

ecology↗

bioRxiv2025

Enterococcus faecalis modulates phase variation in Clostridioides difficile

To adapt and persist in the gastrointestinal tract, many enteric pathogens, including Clostridioides difficile, employ strategies such as phase variation to generate phenotypically heterogeneous populations. Notably, the role of the gut microbiota and polymicrobial interactions in shaping population heterogeneity of invading pathogens has not been explored. Here, we show that Enterococcus faecalis, an opportunistic pathogen that thrives in the inflamed gut during C. difficile infection, can impact the phase variable CmrRST signal transduction system in C. difficile. The CmrRST system controls multiple phenotypes including colony morphology, cell elongation, and cell chaining in C. difficile. Here we describe how interactions between E. faecalis and C. difficile on solid media lead to a marked shift in C. difficile phenotypes associated with phase variation of CmrRST. Specifically, E. faecalis drives a switch of the C. difficile population to the cmr-ON state leading to chaining and a rough colony morphology. This phenomenon preferentially occurs with E. faecalis among the enterococci, as other enterococcal species do not show a similar effect, suggesting that the composition of the polymicrobial environment in the gut is likely critical to shaping C. difficile population heterogeneity. Our findings shed light on the complex role that microbial ecology and polymicrobial interactions can have in the phenotypic heterogeneity of invading pathogens. IMPORTANCE STATEMENTClostridioides difficile is an enteric pathogen with critical implications for public health. The microbial ecosystem in which C. difficile resides shapes the behavior and fitness of C. difficile; however, the mechanisms underlying these interactions are not well defined. Here, we demonstrate that Enterococcus faecalis, an opportunistic pathogen known to co-colonize the gut with C. difficile, influences phase variation and downstream growth phenotypes in C. difficile. This phenomenon represents a new paradigm by which co-residing bacteria can modulate phase variation dynamics in C. difficile or other enteric pathogens. Understanding factors that influence C. difficile behavior may elucidate new therapeutic strategies, especially in complex polymicrobial infections.

microbiology↗

bioRxiv2025

A Novel Heme-Degrading Enzyme that Regulates Heme and Iron Homeostasis and Promotes Virulence in Enterococcus faecalis

Enterococcus faecalis, a gut commensal, is a leading cause of opportunistic infections. Its virulence is linked to its ability to thrive in hostile environments, which includes host-imposed metal starvation. We recently showed that E. faecalis evades iron starvation using five dedicated transporters that collectively scavenge iron from host tissues and other iron-deprived conditions. Interestingly, heme, the most abundant source of iron in the human body, supported growth of a strain lacking all five iron transporters ({Delta}5Fe). To release iron from heme, many bacterial pathogens utilize heme oxygenase enzymes to degrade the porphyrin that coordinates the iron ion of heme. Although E. faecalis lacks these enzymes, bioinformatics revealed a potential ortholog of the anaerobic heme-degrading enzyme anaerobilin synthase, found in Escherichia coli and a few other Gram-negative bacteria. Here, we demonstrated that deletion of OG1RF_RS05575 in E. faecalis ({Delta}RS05575) or in the {Delta}5Fe background ({Delta}5Fe{Delta}RS05575) led to intracellular heme accumulation and hypersensitivity under anaerobic conditions, suggesting RS05575 encodes an anaerobilin synthase, the first of its kind described in Gram- positive bacteria. Additionally, deletion of RS05575, either alone or in the {Delta}5Fe background, impaired E. faecalis colonization in the mouse gastrointestinal tract and virulence in mouse peritonitis and rabbit infective endocarditis models. These results reveal that RS05575 is responsible for anaerobic degradation of heme and identify this relatively new enzyme class as a novel factor in bacterial pathogenesis. Findings from this study are likely to have broad implications, as homologues of RS05575 are found in other Gram-positive facultative anaerobes. IMPORTANCEHeme is an important nutrient for bacterial pathogens, mainly for its ability to serve as an iron source during infection. While bacteria are known to release iron from heme using enzymes called heme oxygenases, a new family of anaerobic heme-degrading enzymes has been described recently in Gram-negative bacteria. Here, we report the first description of anaerobic heme degradation by a Gram-positive bacterium, the opportunistic pathogen Enterococcus faecalis, and link activity of this enzyme to their ability to colonize and infect the host. We also show that homologues of this enzyme are found in many Gram-positive facultative anaerobes, implying that the ability to degrade heme under anaerobic conditions may be an overlooked fitness and virulence factor of bacterial pathogens.

microbiology↗

bioRxiv2024

Stoichiometry of HIV-1 Envelope Glycoprotein Protomers with Changes That Stabilize or Destabilize the Pretriggered Conformation

During human immunodeficiency virus (HIV-1) entry into host cells, binding to the receptors, CD4 and CCR5/CXCR4, triggers conformational changes in the metastable envelope glycoprotein (Env) trimer ((gp120-gp41)3). CD4 binding induces Env to make transitions from its pretriggered conformation (PTC) to more "open" conformations that are sensitive to inhibition by antibodies, CD4-mimetic compounds (CD4mcs) and exposure to cold. Changes in functional membrane Envs have been identified that either stabilize or destabilize the PTC. Here, we investigate the stoichiometric requirements for the PTC-stabilizing and -destabilizing changes in the Env protomers. To this end, we generated viruses bearing Envs with mixed protomers exhibiting different degrees of PTC stability and determined the sensitivity of the viruses to cold (0{degrees}C) and, in some cases, to a CD4mc. The number of stabilized Env protomers required to achieve stabilization of the PTC was inversely related to the degree of PTC stabilization that resulted from the introduced Env change. For strongly stabilizing Env changes, modification of a single protomer was sufficient to achieve PTC stabilization; given adequate stability, the modified protomer can apparently constrain the conformation of the other two protomers to maintain the PTC. Weakly stabilizing Env changes needed to be present in all three protomers to achieve efficient stabilization of the PTC. In many cases, the PTC was disrupted when destabilizing changes were present in only a single protomer. These complementary results suggest that conformational symmetry among the protomers of the functional Env trimer is conducive to the integrity of the PTC. IMPORTANCEThe human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer consists of three protomers. In response to receptor binding, the flexible Env changes its conformation to mediate virus entry into host cells. The shape-shifting ability of Env also contributes to HIV-1s capacity to evade the host immune system. The pretriggered (State-1) conformation (PTC) of Env is an important target for virus entry inhibitors and host antibodies, but is unstable and therefore incompletely characterized. Changes in Env amino acids that either stabilize or destabilize the PTC have been identified. Here, we define how many Env protomers need to be modified by these changes to achieve stabilization or destabilization of the PTC. These results can guide the placement of changes in the HIV-1 Env protomers to control the movement of the Env trimer from the PTC, allowing better characterization of this elusive conformation and testing of its utility in vaccines.

microbiology↗

bioRxiv2024

The asymmetric opening of HIV-1 Env by a potent CD4 mimetic enables anti-coreceptor binding site antibodies to mediate ADCC

HIV-1 envelope glycoproteins (Env) from primary HIV-1 isolates typically adopt a pretriggered "closed" conformation that resists to CD4-induced (CD4i) non-neutralizing antibodies (nnAbs) mediating antibody-dependent cellular cytotoxicity (ADCC). CD4-mimetic compounds (CD4mcs) "open-up" Env allowing binding of CD4i nnAbs, thereby sensitizing HIV-1-infected cells to ADCC. Two families of CD4i nnAbs, the anti-cluster A and anti-coreceptor binding site (CoRBS) Abs, are required to mediate ADCC in combination with the indane CD4mc BNM-III-170. Recently, new indoline CD4mcs with improved potency and breadth have been described. Here, we show that the lead indoline CD4mc, CJF-III-288, sensitizes HIV-1-infected cells to ADCC mediated by anti-CoRBS Abs alone, contributing to improved ADCC activity. Structural and conformational analyses reveal that CJF-III-288, in combination with anti-CoRBS Abs, potently stabilizes an asymmetric "open" State-3 Env conformation, This Env conformation orients the anti-CoRBS Ab to improve ADCC activity and therapeutic potential.

microbiology↗

bioRxiv2024

The combination of three CD4-induced antibodies targeting highly conserved Env regions with a small CD4-mimetic achieves potent ADCC activity

The majority of naturally-elicited antibodies against the HIV-1 envelope glycoproteins (Env) are non-neutralizing (nnAbs), because they are unable to recognize the Env timer in its native "closed" conformation. Nevertheless, it has been shown that nnAbs have the potential to eliminate HIV-1-infected cells by Antibody-Dependent Cellular Cytotoxicity (ADCC) provided that Env is present on the cell surface in its "open" conformation. This is because most nnAbs recognize epitopes that become accessible only after Env interaction with CD4 and the exposure of epitopes that are normally occluded in the closed trimer. HIV-1 limits this vulnerability by downregulating CD4 from the surface of infected cells, thus preventing a premature encounter of Env with CD4. Small CD4-mimetics (CD4mc) sensitize HIV-1-infected cells to ADCC by opening the Env glycoprotein and exposing CD4-induced (CD4i) epitopes. There are two families of CD4i nnAbs, termed anti-cluster A and anti-CoRBS Abs, which are known to mediate ADCC in the presence of CD4mc. Here, we performed Fab competition experiments and found that anti-gp41 cluster I antibodies comprise a major fraction of the plasma ADCC activity in people living with HIV (PLWH). Moreover, addition of gp41 cluster I antibodies to cluster A and CoRBS antibodies greatly enhanced ADCC mediated cell killing in the presence of a potent indoline CD4mc, CJF-III-288. This cocktail outperformed broadly-neutralizing antibodies and even showed activity against HIV-1 infected monocyte-derived macrophages. Thus, combining CD4i antibodies with different specificities achieves maximal ADCC activity, which may be of utility in HIV cure strategies. IMPORTANCEThe elimination of HIV-1-infected cells remains an important medical goal. While current antiretroviral therapy decreases viral loads below detection levels, it does not eliminate latently infected cells which form the viral reservoir. Here, we developed a cocktail of non-neutralizing antibodies targeting highly conserved Env regions and combined it with a potent indoline CD4mc. This combination exhibited very potent ADCC activity against HIV-1-infected primary CD4+ T cells as well as monocyte-derived macrophages, suggesting its potential utility in decreasing the size of the viral reservoir.

microbiology↗

bioRxiv2024

Pervasive but lagged responses in the composition of small mammal communities to a century of climate change

Climate change has widespread effects on the distribution, abundance, and behavior of species around the world, leading to the reshuffling of ecological communities. However, it remains unclear whether individual species range shifts scale up to result in communities whose rate of change lag, lead, or track the rate of climate change. We capitalized on a century-old data set originally collected by Joseph Grinnell and his students, plus modern resurveys, to measure long-term compositional responses of small mammal communities to climate change in historical and modern eras across three regions in the Sierra Nevada of California (Lassen, Yosemite, and Sequoia and Kings Canyon National Parks). Across this period, mean annual temperature in each region increased and mean annual precipitation decreased. We tested whether small mammal communities have shifted their composition in favor of species more adapted to hot and dry conditions, processes known as thermophilization and mesophilization respectively. We found positive thermophilization rates (communities composed of more warm-adapted species) in all three regions and negative mesophilization rates (communities composed of dry-adapted species) in one of the three regions. Importantly, thermophilization and mesophilization rates lagged behind corresponding rates of climate change on average by 0.90-1.2 {degrees}C and 26.5-111.0 mm, but the magnitude of lags was unrelated to region, amount of climate change, or their interaction. Previous work demonstrated high intraspecific variability in range shifts across the three regions in our study. Our results suggest that the net effects of climate change will be directional at the scale of the ecological community, despite variability in individual species responses to environmental change and the varied mechanisms that govern them. Communities, like many individual species, may already be out of equilibrium with ambient climate.

ecology↗

bioRxiv2023

V3 tip determinants of susceptibility to inhibition by CD4-mimetic compounds in natural clade A human immunodeficiency virus (HIV-1) envelope glycoproteins

CD4-mimetic compounds (CD4mcs) bind the human immunodeficiency virus (HIV-1) gp120 exterior envelope glycoprotein (Env) and compete for binding to CD4, the host receptor. CD4mcs prematurely trigger conformational changes in Env similar to those induced by CD4, leading to transient activation of infectivity followed by irreversible virus inactivation. Natural HIV-1 variants exhibit a wide range of susceptibilities to CD4mc inhibition, only a small fraction of which can be explained by variation in the gp120 Phe-43 cavity/vestibule where CD4mcs bind. Here, we study Envs from the resistant HIV-1BG505 and the more sensitive HIV-1191955_A4 clade A strains. The major determinant of the relative sensitivity of the HIV-1191955_A4 Env to CD4mcs mapped to a single residue change (F317Y) in the tip of the gp120 V3 variable loop. In the Envs of several HIV-1 strains, replacement of the more prevalent Phe 317 with a tyrosine residue increased virus sensitivity to multiple CD4mcs. Tryptophan substitutions at residues 317 and 316 resulted in increases and decreases, respectively, in sensitivity to CD4mcs. Some of the gp120 V3 changes increased virus sensitivity to inactivation by both CD4mc and cold exposure, phenotypes indicative of increased Env triggerability. Infection of CD4-negative cells expressing the CCR5 coreceptor by these Env variants was triggered more efficiently by CD4mcs. For the panel of studied HIV-1 Envs, resistance to the CD4mcs was associated with decreased ability to support virus entry. These studies illustrate how variation in gp120 outside the CD4mc binding site can influence the sensitivity of natural HIV-1 strains to inhibition by these compounds. IMPORTANCECD4-mimetic compounds (CD4mcs) are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. CD4mcs target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor, CD4, and is highly conserved among HIV-1 strains. Nonetheless, naturally occurring HIV-1 strains exhibit a wide range of sensitivities to CD4mcs. Our study identifies changes distant from the binding pocket that can influence the susceptibility of natural HIV-1 strains to the antiviral effects of multiple CD4mcs. We relate the antiviral potency of the CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate entry-related changes in Env conformation prematurely. These findings will guide efforts to improve the potency and breadth of CD4mcs against natural HIV-1 variants.

microbiology↗

bioRxiv2023

Piperidine CD4-mimetic compounds expose vulnerable Env epitopes sensitizing HIV-1-infected cells to ADCC

The ability of HIV-1 accessory proteins Nef and Vpu to decrease CD4 levels contributes to the protection of infected cells from antibody-dependent cellular cytotoxicity (ADCC) by preventing the exposure of Env vulnerable epitopes. Small-molecule CD4 mimetics (CD4mc) based on the indane and piperidine scaffolds such as (+)-BNM-III-170 and (S)-MCG-IV-210 sensitize HIV-1 infected cells to ADCC by exposing CD4-induced (CD4i) epitopes recognized by non-neutralizing antibodies abundantly present in plasma from people living with HIV. Here, we characterize a new family of CD4mc, (S)-MCG-IV-210 derivatives, based on the piperidine scaffold which engage the gp120 within the Phe43 cavity by targeting the highly-conserved Asp368 Env residue. We utilized structure-based approaches and developed a series of piperidine analogs with improved activity to inhibit infection of difficult-to-neutralize tier-2 viruses and sensitize infected cells to ADCC mediated by HIV+ plasma. Moreover, the new analogs formed an H-bond with the -carboxylic acid group of Asp368, opening a new avenue to enlarge the breadth of this family of anti-Env small molecules. Overall, the new structural and biological attributes of these molecules make them good candidates for strategies aimed at the elimination HIV-1-infected cells.

immunology↗

bioRxiv2023

Conformations of the Human Immunodeficiency Virus (HIV-1) Envelope Glycoproteins in Detergents and Styrene-Maleic Acid Lipid Particles (SMALPs)

The mature human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer, which consists of non-covalently associated gp120 exterior and gp41 transmembrane subunits, mediates virus entry into cells. The pretriggered (State-1) Env conformation is the major target for broadly neutralizing antibodies (bNAbs), whereas receptor-induced downstream Env conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. To examine the contribution of membrane anchorage to the maintenance of the metastable pretriggered Env conformation, we compared wild-type and State-1-stabilized Envs solubilized in detergents or in styrene-maleic acid (SMA) copolymers. SMA directly incorporates membrane lipids and resident membrane proteins into lipid nanodiscs (SMALPs). The integrity of the Env trimer in SMALPs was maintained at both 4{degrees}C and room temperature. By contrast, Envs solubilized in Cymal-5, a non-ionic detergent, were unstable at room temperature, although their stability was improved at 4{degrees}C and after incubation with the entry inhibitor BMS-806. Envs solubilized in ionic detergents were relatively unstable at either temperature. Comparison of Envs solubilized in Cymal-5 and SMA at 4{degrees}C revealed subtle differences in bNAb binding to the gp41 membrane-proximal external region (MPER), consistent with these distinct modes of Env solubilization. Otherwise, the antigenicity of the Cymal-5- and SMA- solubilized Envs was remarkably similar, both in the absence and presence of BMS-806. However, both solubilized Envs were recognized differently from the mature membrane Env by specific bNAbs and pNAbs. Thus, detergent-based and detergent-free solubilization at 4{degrees}C alters the pretriggered membrane Env conformation in consistent ways, indicating that loss of Env association with the membrane results in default state(s). IMPORTANCEThe human immunodeficiency virus (HIV-1) envelope glycoproteins (Envs) in the viral membrane mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins rely on purification procedures that allow the proteins to maintain their natural conformation. In this study, we show that a styrene-maleic acid (SMA) copolymer can extract HIV-1 Env from a membrane without the use of detergents. The Env in SMA is more stable at room temperature than Env in detergents. The purified Env in SMA maintains many but not all of the characteristics expected of the natural membrane Env. Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful tools for future studies of HIV-1 Env structure and its interaction with receptors and antibodies.

microbiology↗

bioRxiv2023

Indoline CD4-mimetic Compounds Mediate Potent and Broad HIV-1 Inhibition and Sensitization to Antibody-dependent Cellular Cytotoxicity

Binding to the host cell receptors, CD4 and CCR5/CXCR4, triggers large-scale conformational changes in the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer [(gp120/gp41)3] that promote virus entry into the cell. CD4-mimetic compounds (CD4mcs) comprise small organic molecules that bind in the highly conserved CD4-binding site of gp120 and prematurely induce inactivating Env conformational changes, including shedding of gp120 from the Env trimer. By inducing more "open," antibody-susceptible Env conformations, CD4mcs also sensitize HIV-1 virions to neutralization by antibodies and infected cells to antibody-dependent cellular cytotoxicity (ADCC). Here, we report the design, synthesis and evaluation of novel CD4mcs based on an indoline scaffold. Compared with our current lead indane scaffold CD4mc, BNM-III-170, several indoline CD4mcs exhibit increased potency and breadth against HIV-1 variants from different geographic clades. Viruses that were selected for resistance to the lead indane CD4mc, BNM-III-170, are susceptible to inhibition by the indoline CD4mcs. The indoline CD4mcs also potently sensitize HIV-1-infected cells to ADCC mediated by plasma from HIV-1-infected individuals. Crystal structures indicate that the indoline CD4mcs gain potency compared to the indane CD4mcs through more favorable {pi}-{pi} overlap from the indoline pose and by making favorable contacts with the vestibule of the CD4-binding pocket on gp120. The rational design of indoline CD4mcs thus holds promise for further improvements in antiviral activity, potentially contributing to efforts to treat and prevent HIV-1 infection.

microbiology↗

bioRxiv2022

Small CD4 mimetics sensitize HIV-1-infected macrophages to antibody-dependent cellular cytotoxicity

HIV-1 Envelope (Env) conformation determines the susceptibility of infected CD4+ T cells to Antibody Dependent Cellular Cytotoxicity (ADCC). Upon interaction with CD4, Env adopts more "open" conformations, exposing ADCC epitopes. HIV-1 limits Env-CD4 interaction and protects infected cells against ADCC by downregulating CD4 via Nef, Vpu and Env. Limited data exists however of the role of these proteins in downmodulating CD4 on infected macrophages and how this impacts Env conformation. While Nef, Vpu and Env are all required to efficiently downregulate CD4 on infected CD4+T cells, we show here that any one of these proteins is sufficient to downmodulate most CD4 from the surface of infected macrophages. Consistent with this finding, Nef and Vpu have a lesser impact on Env conformation and ADCC sensitivity in infected macrophages compared to CD4+T cells. However, treatment of infected macrophages with small CD4-mimetics expose vulnerable CD4-induced Env epitopes and sensitize them to ADCC.

microbiology↗

bioRxiv2022

Modeling the rarest of the rare: A comparison between joint species distribution models, ensembles of small models, and single-species models at extremely low sample sizes

Determining the distribution and environmental preferences of rare species threatened by global change has long been a focus of conservation. Typical minimum suggested number of occurrences ranges from [~]5 to 30, but many species are represented by even fewer occurrences. However, several newer methods may be able to accommodate such low samples sizes. These include Bayesian joint species distribution models (JSDMs) which allow rare species to statistically "borrow strength" from more common species with similar niches, and ensembles of small models (ESMs), which reduce the number of parameters by averaging smaller models. Here we explore how niche breadth and niche position relative to other species influence model performance at low sample sizes (N=2, 4, 8, 16, 32, 64) using virtual species within a community of real species. ESMs were better at discrimination tasks for most species, and yielded better-than-random accuracy even for N=2. In contrast, "traditional" single species or JSDMs were better able to estimate the underlying response curves of variables that influenced the niche, but at low sample sizes also were more likely to incorrectly identify unimportant factors as influential. Species with niches that were narrow and peripheral to the available environmental space yielded models with better discrimination capacity than species with broad niches or niches that were similar to those of other species, regardless of whether the modeling algorithm allowed for borrowing of strength. Our study suggests that some rare species may be able to be modeled reliably at very low sample sizes, although the best algorithm depends on number of occurrences and whether the niche or distribution is the focus.

ecology↗

bioRxiv2022

Dynamics of mRNA fate during light stress and recovery: from transcription to stability and translation

Transcript stability is an important determinant of its abundance and, consequently, translation. However, it is unclear the extent to which it is modulated between environmental conditions. We previously hypothesised that recovery-induced transcript destabilisation facilitated a phenomenon of rapid recovery gene down-regulation (RRGD) in Arabidopsis thaliana following stress, based on mathematical calculations to account for ongoing transcription. Here, we test this hypothesis, and investigate processes regulating transcript abundance and fate, by quantifying changes in transcription, stability, and translation before, during, and after light stress. We adapt syringe infiltration to apply a transcriptional inhibitor to soil-grown plants in combination with stress. Compared to measurements in juvenile plants and cell culture, we find reduced stability in a range of transcripts. We also observe transcript destabilisation during light stress, followed by stabilisation upon recovery. Alongside fast transcriptional shut-off in recovery, this destabilisation appears to facilitate RRGD. Translation was dynamic over the course of light stress and recovery, with substantial transcript-specific increases in ribosome-association, independent of changes in total transcript abundance, observed after 30 minutes of light stress. Taken together, we provide evidence for the combinatorial regulation of transcription, stability, and translation that occurs to facilitate responses to light stress and recovery.

plant biology↗

bioRxiv2022

Characterization of human immunodeficiency virus (HIV-1) envelope glycoprotein variants selected for resistance to a CD4-mimetic compound

Binding to host cell receptors, CD4 and CCR5/CXCR4, triggers conformational changes in the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer that promote virus entry. CD4 binding allows the gp120 exterior Env to bind CCR5/CXCR4 and induces a pre-hairpin intermediate conformation in the gp41 transmembrane Env. Small-molecule CD4-mimetic compounds (CD4mcs) bind within the conserved Phe-43 cavity of gp120, near the binding site for CD4. CD4mcs inhibit HIV-1 infection by competing with CD4 and by prematurely activating Env, leading to irreversible inactivation. BNM-III-170 is a CD4mc that inhibits the infection of approximately 70% of HIV-1 strains at micromolar concentrations. We selected and analyzed variants of the primary HIV-1AD8 strain resistant to BNM-III-170. Two changes (S375N and I424T) in gp120 residues that flank the Phe-43 cavity each conferred [~]5-fold resistance to BNM- III-170 with minimal fitness cost. A third change (E64G) in Layer 1 of the gp120 inner domain resulted in [~]100-fold resistance to BNM-III-170, [~]2-3-fold resistance to soluble CD4-Ig, and a moderate decrease in viral fitness. The gp120 changes additively or synergistically contributed to BNM-III-170 resistance. The sensitivity of the Env variants to BNM-III-170 inhibition of virus entry correlated with their sensitivity to BNM-III-170- induced Env activation and shedding of gp120. The S375N and I424T changes, but not the E64G change, conferred resistance to BMS-806, a potent HIV-1 entry inhibitor that blocks Env conformational transitions. These studies identify pathways whereby HIV-1 can develop resistance to CD4mcs and BMS-806 conformational blockers, two classes of entry inhibitors that target the conserved gp120 Phe-43 cavity. IMPORTANCECD4-mimetic compounds (CD4mcs) and BMS-806 are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. Although CD4mcs and BMS-806 inhibit HIV-1 entry by different mechanisms, they both target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor, CD4, and is highly conserved among HIV-1 strains. Our study identifies changes near this pocket that can confer various levels of resistance to the antiviral effects of both a CD4mc and BMS-806. We relate the antiviral potency of a CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate changes in Env conformation and to induce the shedding of the gp120 exterior Env from the spike. These findings will guide efforts to improve the potency and breadth of small-molecule HIV-1 entry inhibitors.

microbiology↗

bioRxiv2022

HIV-1 Vpu restricts Fc-mediated effector functions in vivo

Non-neutralizing antibodies (nnAbs) can eliminate HIV-1-infected cells via antibody-dependent cellular cytotoxicity (ADCC) and were identified as a correlate of protection in the RV144 vaccine trial. Fc-mediated effector functions of nnAbs were recently shown to alter the course of HIV-1 infection in vivo using a vpu-defective virus. Since Vpu is known to downregulate cell surface CD4, which triggers conformational changes in the viral envelope glycoprotein (Env), we ask whether the lack of Vpu expression was linked to the observed nnAbs activity. We found that restoring Vpu expression greatly reduces nnAb recognition of infected cells, rendering them resistant to ADCC responses. Moreover, administration of a nnAb in humanized mice reduces viral loads only in animals infected with a vpu-defective but not with a wildtype virus. Finally, nnAb Fc-effector functions are observed only on cells expressing Env in the "open" conformation. This work highlights the importance of Vpu-mediated evasion of humoral responses.

microbiology↗

bioRxiv2022

VE607 Stabilizes SARS-CoV-2 Spike In the "RBD-up" Conformation and Inhibits Viral Entry

SARS-CoV-2 infection of host cells starts by binding of the Spike glycoprotein (S) to the ACE2 receptor. The S-ACE2 interaction is a potential target for therapies against COVID-19 as demonstrated by the development of immunotherapies blocking this interaction. Here, we present the commercially available VE607, comprised of three stereoisomers, that was originally described as an inhibitor of SARS-CoV-1. We show that VE607 specifically inhibits infection of SARS-CoV-1 and SARS-CoV-2 S-expressing pseudoviral particles as well as authentic SARS-CoV-2. VE607 stabilizes the receptor binding domain (RBD) in its "up" conformation. In silico docking and mutational analysis map the VE607 binding site at the RBD-ACE2 interface. The IC50 values are in the low micromolar range for pseudoparticles derived from SARS-CoV-2 Wuhan/D614G as well as from variants of concern (Alpha, Beta, Gamma, Delta and Omicron), suggesting that VE607 has potential for the development of drugs against SARS-CoV-2 infections.

microbiology↗

bioRxiv2021

Global Increases in Human Immunodeficiency Virus Neutralization Sensitivity Due to Alterations in the Membrane-Proximal External Region of the Envelope Glycoprotein Can be Minimized by Distant State 1-Stabilizing Changes

Binding to the receptor, CD4, drives the pretriggered, "closed" (State-1) conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer ((gp120/gp41)3) into more "open" conformations. HIV-1 Env on the viral membrane is maintained in a State-1 conformation that resists binding and neutralization by commonly elicited antibodies. Premature triggering of Env before the virus engages a target cell typically leads to increased susceptibility to spontaneous inactivation or ligand-induced neutralization. Here, we show that single amino acid substitutions in the gp41 membrane-proximal external region (MPER) of a primary HIV-1 strain result in viral phenotypes indicative of premature triggering of Env to downstream conformations. Specifically, the MPER changes reduced viral infectivity and globally increased virus sensitivity to poorly neutralizing antibodies, soluble CD4, a CD4-mimetic compound, and exposure to cold. By contrast, the MPER mutants exhibited decreased sensitivity to the State-1 preferring inhibitor, BMS-806, and to the PGT151 broadly neutralizing antibody. Depletion of cholesterol from virus particles did not produce the same State 1-destabilizing phenotypes as MPER alterations. Notably, State 1-stabilizing changes in Env distant from the MPER could minimize the phenotypic effects of MPER alteration, but did not affect virus sensitivity to cholesterol depletion. Thus, membrane-proximal gp41 elements contribute to the maintenance of the pretriggered Env conformation. The conformationally disruptive effects of MPER changes can be minimized by distant State 1-stabilizing Env modifications, a strategy that may be useful in preserving the native pretriggered state of Env. IMPORTANCEThe pretriggered shape of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) is a major target for antibodies that can neutralize many strains of the virus. An effective HIV-1 vaccine may need to raise these types of antibodies, but this goal has proven difficult. One reason is that the pretriggered shape of Env is unstable and dependent on interactions near the viral membrane. Here we show that the membrane-proximal external region (MPER) of Env plays an important role in maintaining Env in a pretriggered shape. Alterations in the MPER resulted in global changes in Env conformation that disrupted its pretriggered shape. We also found that these disruptive effects of MPER changes can be minimized by distant Env modifications that stabilize the pretriggered shape. These modifications may be useful for preserving the native shape of Env for structural and vaccine studies.

microbiology↗