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Nourishing the Microbiota to Promote Mucosal Immunity

Cell Host Microbe

2020 Meghan Koch; Meera Shenoy

Childhood undernutrition is associated with dysbiosis and dampened vaccine responses. Understanding how nutrients influence the microbiota and immunity is critical for vaccine efficacy. In this issue of Cell Host & Microbe, Di Luccia et al. and Huus et al. reveal that nutrition affects IgA responses to the microbiota and oral vaccines.

Different genetic barriers for resistance to HA stem antibodies in influenza H3 and H1 viruses


2020 Juhye Lee; Jesse Bloom

The discovery and characterization of broadly neutralizing human antibodies (bnAbs) to the highly conserved stem region of influenza hemagglutinin (HA) have contributed to considerations of a universal influenza vaccine. However, the potential for resistance to stem bnAbs also needs to be more thoroughly evaluated. Using deep mutational scanning, with a focus on epitope residues, we found that the genetic barrier to resistance to stem bnAbs is low for the H3 subtype but substantially higher for the H1 subtype owing to structural differences in the HA stem. Several strong resistance mutations in H3 can be observed in naturally circulating strains and do not reduce in vitro viral fitness and in vivo pathogenicity. This study highlights a potential challenge for development of a truly universal influenza vaccine.

High-throughput, microscope-based sorting to dissect cellular heterogeneity

Mol Syst Biol

2020 Raymond Monnat; Douglas Fowler; Emily Hatch; Cole Trapnell

Microscopy is a powerful tool for characterizing complex cellular phenotypes, but linking these phenotypes to genotype or RNA expression at scale remains challenging. Here, we present Visual Cell Sorting, a method that physically separates hundreds of thousands of live cells based on their visual phenotype. Automated imaging and phenotypic analysis directs selective illumination of Dendra2, a photoconvertible fluorescent protein expressed in live cells; these photoactivated cells are then isolated using fluorescence-activated cell sorting. First, we use Visual Cell Sorting to assess hundreds of nuclear localization sequence variants in a pooled format, identifying variants that improve nuclear localization and enabling annotation of nuclear localization sequences in thousands of human proteins. Second, we recover cells that retain normal nuclear morphologies after paclitaxel treatment, and then derive their single-cell transcriptomes to identify pathways associated with paclitaxel resistance in cancers. Unlike alternative methods, Visual Cell Sorting depends on inexpensive reagents and commercially available hardware. As such, it can be readily deployed to uncover the relationships between visual cellular phenotypes and internal states, including genotypes and gene expression programs.

Retrocopying expands functional repertoire of APOBEC3 antiviral proteins in primates


2020 Michael Emerman; Harmit Malik; Richard McLaughlin

Host-virus arms races are inherently asymmetric; viruses evolve much more rapidly than host genomes. Thus, there is high interest in discovering mechanisms by which host genomes keep pace with rapidly evolving viruses. One family of restriction factors, the APOBEC3 (A3) cytidine deaminases, has undergone positive selection and expansion via segmental gene duplication and recombination. Here, we show that new copies of A3 genes have also been created in primates by reverse transcriptase-encoding elements like LINE-1 or endogenous retroviruses via a process termed retrocopying. First, we discovered that all simian primate genomes retain the remnants of an ancient A3 retrocopy: A3I. Furthermore, we found that some New World monkeys encode up to ten additional APOBEC3G (A3G) retrocopies. Some of these A3G retrocopies are transcribed in a variety of tissues and able to restrict retroviruses. Our findings suggest that host genomes co-opt retroelement activity in the germline to create new host restriction factors as another means to keep pace with the rapid evolution of viruses. (163).

Antibody Neutralization of an Influenza Virus that Uses Neuraminidase for Receptor Binding


2020 Jesse Bloom; Lauren Gentles

Influenza virus infection elicits antibodies against the receptor-binding protein hemagglutinin (HA) and the receptor-cleaving protein neuraminidase (NA). Because HA is essential for viral entry, antibodies targeting HA often potently neutralize the virus in single-cycle infection assays. However, antibodies against NA are not potently neutralizing in such assays, since NA is dispensable for single-cycle infection. Here we show that a modified influenza virus that depends on NA for receptor binding is much more sensitive than a virus with receptor-binding HA to neutralization by some anti-NA antibodies. Specifically, a virus with a receptor-binding G147R N1 NA and a binding-deficient HA is completely neutralized in single-cycle infections by an antibody that binds near the NA active site. Infection is also substantially inhibited by antibodies that bind NA epitopes distant from the active site. Finally, we demonstrate that this modified virus can be used to efficiently select mutations in NA that escape antibody binding, a task that can be laborious with typical influenza viruses that are not well neutralized by anti-NA antibodies. Thus, viruses dependent on NA for receptor binding allow for sensitive in vitro detection of antibodies binding near the catalytic site of NA and enable the selection of viral escape mutants.

BAF facilitates interphase nuclear membrane repair through recruitment of nuclear transmembrane proteins

Mol Biol Cell

2020 Emily Hatch; Alexandra Young; Amanda Gunn

Nuclear membrane rupture during interphase occurs in a variety of cell contexts, both healthy and pathological. Membrane ruptures can be rapidly repaired, but these mechanisms are still unclear. Here we show BAF, a nuclear envelope protein that shapes chromatin and recruits membrane proteins in mitosis, also facilitates nuclear membrane repair in interphase, in part through recruitment of the nuclear membrane proteins emerin and LEMD2 to rupture sites. Characterization of GFP-BAF accumulation at nuclear membrane rupture sites confirmed BAF is a fast, accurate, and persistent mark of nucleus rupture whose kinetics are partially dictated by membrane resealing. BAF depletion significantly delayed nuclear membrane repair, with a larger effect on longer ruptures. This phenotype could be rescued by GFP-BAF, but not by a BAF mutant lacking the LEM-protein binding domain. Depletion of the BAF interactors LEMD2 or emerin, and to a lesser extent lamin A/C, increased the duration of nucleus ruptures, consistent with LEM-protein binding being a key function of BAF during membrane repair. Overall our results suggest a model where BAF is critical for timely repair of large ruptures in the nuclear membrane, potentially by facilitating membrane attachment to the rupture site. [Media: see text].

When two are better than one: Modeling the mechanisms of antibody mixtures

PLoS Comput Biol

2020 Jesse Bloom; Tal Einav

It is difficult to predict how antibodies will behave when mixed together, even after each has been independently characterized. Here, we present a statistical mechanical model for the activity of antibody mixtures that accounts for whether pairs of antibodies bind to distinct or overlapping epitopes. This model requires measuring n individual antibodies and their [Formula: see text] pairwise interactions to predict the 2n potential combinations. We apply this model to epidermal growth factor receptor (EGFR) antibodies and find that the activity of antibody mixtures can be predicted without positing synergy at the molecular level. In addition, we demonstrate how the model can be used in reverse, where straightforward experiments measuring the activity of antibody mixtures can be used to infer the molecular interactions between antibodies. Lastly, we generalize this model to analyze engineered multidomain antibodies, where components of different antibodies are tethered together to form novel amalgams, and characterize how well it predicts recently designed influenza antibodies.

New Solutions to Old Problems: Molecular Mechanisms of Meiotic Crossover Control

Trends Genet

2020 Gerald Smith; Mridula Nambiar

During scientific investigations, the explanation of remarkably interesting phenomena must often await development of new methods or accrual of new observations that in retrospect can lead to lucid answers to the initial problem. A case in point is the control of genetic recombination during meiosis, which leads to crossovers between chromosomes critical for production of healthy offspring. Crossovers must be properly placed along meiotic chromosomes for their accurate segregation. Here, we review observations on two aspects of meiotic crossover control - crossover interference and repression of crossovers near centromeres, both observed more than 85 years ago. Only recently have relatively simple molecular mechanisms for these phenomena become clear through advances in both methods and understanding the molecular basis of meiotic recombination.

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing


2020 Michael Emerman; Harmit Malik; Janet Young

The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 2.3 million people, killed over 160,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven clinical efficacy, nor are there vaccines for its prevention, and these efforts are hampered by limited knowledge of the molecular details of SARS-CoV-2 infection. To address this, we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), identifying 332 high-confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (29 FDA-approved drugs, 12 drugs in clinical trials, and 28 preclinical compounds). Screening a subset of these in multiple viral assays identified two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the Sigma1 and Sigma2 receptors. Further studies of these host factor targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.

TRIM34 restricts HIV-1 and SIV capsids in a TRIM5 alpha-dependent manner

PLoS Pathog

2020 Michael Emerman; Molly OhAinle; Abby Felton

The HIV-1 capsid protein makes up the core of the virion and plays a critical role in early steps of HIV replication. Due to its exposure in the cytoplasm after entry, HIV capsid is a target for host cell factors that act directly to block infection such as TRIM5α and MxB. Several host proteins also play a role in facilitating infection, including in the protection of HIV-1 capsid from recognition by host cell restriction factors. Through an unbiased screening approach, called HIV-CRISPR, we show that the CPSF6-binding deficient, N74D HIV-1 capsid mutant is sensitive to restriction mediated by human TRIM34, a close paralog of the well-characterized HIV restriction factor TRIM5α. This restriction occurs at the step of reverse transcription, is independent of interferon stimulation, and limits HIV-1 infection in key target cells of HIV infection including CD4+ T cells and monocyte-derived dendritic cells. TRIM34 can also restrict some SIV capsids. TRIM34 restriction requires TRIM5α as knockout or knockdown of TRIM5α results in a loss of antiviral activity. Through immunofluorescence studies, we show that TRIM34 and TRIM5α colocalize to cytoplasmic bodies and are more frequently observed to be associated with infecting N74D capsids than with WT HIV-1 capsids. Our results identify TRIM34 as an HIV-1 CA-targeting restriction factor and highlight the potential role for heteromultimeric TRIM interactions in contributing to restriction of HIV-1 infection in human cells.