Structurally divergent and recurrently mutated regions of primate genomes.

We sequenced and assembled using multiple long-read sequencing technologies the genomes of chimpanzee, bonobo, gorilla, orangutan, gibbon, macaque, owl monkey, and marmoset. We identified 1,338,997 lineage-specific fixed structural variants (SVs) disrupting 1,561 protein-coding genes and 136,932 regulatory elements, including the most complete set of human-specific fixed differences. We estimate that 819.47 Mbp or ∼27% of the genome has been affected by SVs across primate evolution. We identify 1,607 structurally divergent regions wherein recurrent structural variation contributes to creating SV hotspots where genes are recurrently lost (e.g., CARD, C4, and OLAH gene families) and additional lineage-specific genes are generated (e.g., CKAP2, VPS36, ACBD7, and NEK5 paralogs), becoming targets of rapid chromosomal diversification and positive selection (e.g., RGPD gene family). High-fidelity long-read sequencing has made these dynamic regions of the genome accessible for sequence-level analyses within and between primate species.

Monkeypox emergency: Urgent questions and perspectives.

Amidst the COVID-19 pandemic, we now face another public health emergency in the form of monkeypox virus. As of August 1, the Centers for Disease Control and Prevention report over 23,000 cases in 80 countries. An inclusive and global collaborative effort to understand the biology, evolution, and spread of the virus as well as commitment to vaccine equity will be critical toward containing this outbreak. We share the voices of leading experts in this space on what they see as the most pressing questions and directions for the community.

Primate hemorrhagic fever-causing arteriviruses are poised for spillover to humans.

Simian arteriviruses are endemic in some African primates and can cause fatal hemorrhagic fevers when they cross into primate hosts of new species. We find that CD163 acts as an intracellular receptor for simian hemorrhagic fever virus (SHFV; a simian arterivirus), a rare mode of virus entry that is shared with other hemorrhagic fever-causing viruses (e.g., Ebola and Lassa viruses). Further, SHFV enters and replicates in human monocytes, indicating full functionality of all of the human cellular proteins required for viral replication. Thus, simian arteriviruses in nature may not require major adaptations to the human host. Given that at least three distinct simian arteriviruses have caused fatal infections in captive macaques after host-switching, and that humans are immunologically naive to this family of viruses, development of serology tests for human surveillance should be a priority.

Macrophages derived from human induced pluripotent stem cells (iPSCs) serve as a high-fidelity cellular model for investigating HIV-1, dengue, and influenza viruses.

Macrophages are important target cells for diverse viruses and thus represent a valuable system for studying virus biology. Isolation of primary human macrophages is done by culture of dissociated tissues or from differentiated blood monocytes, but these methods are both time consuming and result in low numbers of recovered macrophages. Here, we explore whether macrophages derived from human induced pluripotent stem cells (iPSCs)-which proliferate indefinitely and potentially provide unlimited starting material-could serve as a faithful model system for studying virus biology. Human iPSC-derived monocytes were differentiated into macrophages and then infected with HIV-1, dengue virus, or influenza virus as model human viruses. We show that iPSC-derived macrophages support the replication of these viruses with kinetics and phenotypes similar to human blood monocyte-derived macrophages. These iPSC-derived macrophages were virtually indistinguishable from human blood monocyte-derived macrophages based on surface marker expression (flow cytometry), transcriptomics (RNA sequencing), and chromatin accessibility profiling. iPSC lines were additionally generated from non-human primate (chimpanzee) fibroblasts. When challenged with dengue virus, human and chimpanzee iPSC-derived macrophages show differential susceptibility to infection, thus providing a valuable resource for studying the species-tropism of viruses. We also show that blood- and iPSC-derived macrophages both restrict influenza virus at a late stage of the virus lifecycle. Collectively, our results substantiate iPSC-derived macrophages as an alternative to blood monocyte-derived macrophages for the study of virus biology. IMPORTANCE: Macrophages have complex relationships with viruses: while macrophages aid in the removal of pathogenic viruses from the body, macrophages are also manipulated by some viruses to serve as vessels for viral replication, dissemination, and long-term persistence. Here, we show that iPSC-derived macrophages are an excellent model that can be exploited in virology.

RNase L activation in the cytoplasm induces aberrant processing of mRNAs in the nucleus.

The antiviral endoribonuclease, RNase L, is activated by the mammalian innate immune response to destroy host and viral RNA to ultimately reduce viral gene expression. Herein, we show that RNase L and RNase L-mediated mRNA decay are primarily localized to the cytoplasm. Consequently, RNA-binding proteins (RBPs) translocate from the cytoplasm to the nucleus upon RNase L activation due to the presence of intact nuclear RNA. The re-localization of RBPs to the nucleus coincides with global alterations to RNA processing in the nucleus. While affecting many host mRNAs, these alterations are pronounced in mRNAs encoding type I and type III interferons and correlate with their retention in the nucleus and reduction in interferon protein production. Similar RNA processing defects also occur during infection with either dengue virus or SARS-CoV-2 when RNase L is activated. These findings reveal that the distribution of RBPs between the nucleus and cytosol is dictated by the availability of RNA in each compartment. Thus, viral infections that trigger RNase L-mediated cytoplasmic RNA in the cytoplasm also alter RNA processing in the nucleus, resulting in an ingenious multi-step immune block to protein biogenesis.

Just 2% of SARS-CoV-2-positive individuals carry 90% of the virus circulating in communities.

We analyze data from the fall 2020 pandemic response efforts at the University of Colorado Boulder, where more than 72,500 saliva samples were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using qRT-PCR. All samples were collected from individuals who reported no symptoms associated with COVID-19 on the day of collection. From these, 1,405 positive cases were identified. The distribution of viral loads within these asymptomatic individuals was indistinguishable from what has been previously observed in symptomatic individuals. Regardless of symptomatic status, ∼50% of individuals who test positive for SARS-CoV-2 seem to be in noninfectious phases of the disease, based on having low viral loads in a range from which live virus has rarely been isolated. We find that, at any given time, just 2% of individuals carry 90% of the virions circulating within communities, serving as viral "supercarriers" and possibly also superspreaders.

High rates of observed face mask use at Colorado universities align with students' opinions about masking and support the safety and viability of in-person higher education during the COVID-19 pandemic.

BACKGROUND: Over the course of the COVID-19 pandemic, colleges and universities have focused on creating policies, such as mask mandates, to minimize COVID-19 transmission both on their campuses and in the surrounding community. Adherence to and opinions about these policies remain largely unknown. METHODS: The Centers for Disease Control and Prevention (CDC) developed a cross-sectional study, the Mask Adherence and Surveillance at Colleges and Universities Project (MASCUP!), to objectively and inconspicuously measure rates of mask use at institutes of higher education via direct observation. From February 15 through April 11, 2021 the University of Colorado Boulder (CU, n = 2,808 observations) and Colorado State University Fort Collins (CSU, n = 3,225 observations) participated in MASCUP! along with 52 other institutes of higher education (n = 100,353 observations) spanning 21 states and the District of Columbia. Mask use was mandatory at both Colorado universities and student surveys were administered to assess student beliefs and attitudes. RESULTS: We found that 91.7%, 93.4%, and 90.8% of persons observed at indoor locations on campus wore a mask correctly at University of Colorado, Colorado State University, and across the 52 other schools, respectively. Student responses to questions about masking were in line with these observed rates of mask use where 92.9% of respondents at CU and 89.8% at CSU believe that wearing masks can protect the health of others. Both Colorado universities saw their largest surges in COVID-19 cases in the fall of 2020, with markedly lower case counts during the mask observation window in the spring of 2021. CONCLUSION: High levels of mask use at Colorado's two largest campuses aligned with rates observed at other institutes across the country. These high rates of use, coupled with positive student attitudes about mask use, demonstrate that masks were widely accepted and may have contributed to reduced COVID-19 case counts. This study supports an emerging body of literature substantiating masks as an effective, low-cost measure to reduce disease transmission and establishes masking (with proper education and promotion) as a viable tactic to reduce respiratory disease transmission on college campuses.

How host genetics dictates successful viral zoonosis.

Viruses of wild and domestic animals can infect humans in a process called zoonosis, and these events can give rise to explosive epidemics such as those caused by the HIV and Ebola viruses. While humans are constantly exposed to animal viruses, those that can successfully infect and transmit between humans are exceedingly rare. The key event in zoonosis is when an animal virus begins to replicate (one virion making many) in the first human subject. Only at this point will the animal virus first experience the selective environment of the human body, rendering possible viral adaptation and refinement for humans. In addition, appreciable viral titers in this first human may enable infection of a second, thus initiating selection for viral variants with increased capacity for spread. We assert that host genetics plays a critical role in defining which animal viruses in nature will achieve this key event of replication in a first human host. This is because animal viruses that pose the greatest risk to humans will have few (or no) genetic barriers to replicating themselves in human cells, thus requiring minimal mutations to make this jump. Only experimental virology provides a path to identifying animal viruses with the potential to replicate themselves in humans because this information will not be evident from viral sequencing data alone.

Selective use of primate CD4 receptors by HIV-1.

Individuals chronically infected with HIV-1 harbor complex viral populations within their bloodstreams. Recently, it has come to light that when these people infect others, the new infection is typically established by only one or a small number of virions from within this complex viral swarm. An important goal is to characterize the biological properties of HIV-1 virions that seed and exist early in new human infections because these are potentially the only viruses against which a prophylactic HIV-1 vaccine would need to elicit protection. This includes understanding how the Envelope (Env) protein of these virions interacts with the T-cell receptor CD4, which supports attachment and entry of HIV-1 into target cells. We examined early HIV-1 isolates for their ability to infect cells via the CD4 receptor of 15 different primate species. Primates were the original source of HIV-1 and now serve as valuable animal models for studying HIV-1. We find that most primary isolates of HIV-1 from the blood, including early isolates, are highly selective and enter cells through some primate CD4 receptor orthologs but not others. This phenotype is remarkably consistent, regardless of route of transmission, viral subtype, or time of isolation post infection. We show that the weak CD4 binding affinity of blood-derived HIV-1 isolates is what makes them sensitive to the small sequence differences in CD4 from one primate species to the next. To substantiate this, we engineered an early HIV-1 Env to have high, medium, or low binding affinity to CD4, and we show that it loses the ability to enter cells via the CD4 receptor of many primate species as the binding affinity gets weaker. Based on the phenotype of selective use of primate CD4, we find that weak CD4 binding appears to be a nearly universal property of HIV-1 circulating in the bloodstream. Therefore, weak binding to CD4 must be a selected and important property in the biology of HIV-1 in the body. We identify six primate species that encode CD4 receptors that fully support the entry of early HIV-1 isolates despite their low binding affinity for CD4. These findings will help inform long-standing efforts to model HIV-1 transmission and early disease in primates.

A metaanalysis of bat phylogenetics and positive selection based on genomes and transcriptomes from 18 species.

Historically, the evolution of bats has been analyzed using a small number of genetic loci for many species or many genetic loci for a few species. Here we present a phylogeny of 18 bat species, each of which is represented in 1,107 orthologous gene alignments used to build the tree. We generated a transcriptome sequence of Hypsignathus monstrosus, the African hammer-headed bat, and additional transcriptome sequence for Rousettus aegyptiacus, the Egyptian fruit bat. We then combined these data with existing genomic and transcriptomic data from 16 other bat species. In the analysis of such datasets, there is no clear consensus on the most reliable computational methods for the curation of quality multiple sequence alignments since these public datasets represent multiple investigators and methods, including different source materials (chromosomal DNA or expressed RNA). Here we lay out a systematic analysis of parameters and produce an advanced pipeline for curating orthologous gene alignments from combined transcriptomic and genomic data, including a software package: the Mismatching Isoform eXon Remover (MIXR). Using this method, we created alignments of 11,677 bat genes, 1,107 of which contain orthologs from all 18 species. Using the orthologous gene alignments created, we assessed bat phylogeny and also performed a holistic analysis of positive selection acting in bat genomes. We found that 181 genes have been subject to positive natural selection. This list is dominated by genes involved in immune responses and genes involved in the production of collagens.

A glycan shield on chimpanzee CD4 protects against infection by primate lentiviruses (HIV/SIV).

Pandemic HIV-1 (group M) emerged following the cross-species transmission of a simian immunodeficiency virus from chimpanzees (SIVcpz) to humans. Primate lentiviruses (HIV/SIV) require the T cell receptor CD4 to enter into target cells. By surveying the sequence and function of CD4 in 50 chimpanzee individuals, we find that all chimpanzee CD4 alleles encode a fixed, chimpanzee-specific substitution (34T) that creates a glycosylation site on the virus binding surface of the CD4 receptor. Additionally, a single nucleotide polymorphism (SNP) has arisen in chimpanzee CD4 (68T) that creates a second glycosylation site on the same virus-binding interface. This substitution is not yet fixed, but instead alleles containing this SNP are still circulating within chimpanzee populations. Thus, all allelic versions of chimpanzee CD4 are singly glycosylated at the virus binding surface, and some allelic versions are doubly glycosylated. Doubly glycosylated forms of chimpanzee CD4 reduce HIV-1 and SIVcpz infection by as much as two orders of magnitude. Full restoration of virus infection in cells bearing chimpanzee CD4 requires reversion of both threonines at sites 34 and 68, destroying both of the glycosylation sites, suggesting that the effects of the glycans are additive. Differentially glycosylated CD4 receptors were biochemically purified and used in neutralization assays and microscale thermophoresis to show that the glycans on chimpanzee CD4 reduce binding affinity with the lentiviral surface glycoprotein, Env. These glycans create a shield that protects CD4 from being engaged by viruses, demonstrating a powerful form of host resistance against deadly primate lentiviruses.

Control of yeast retrotransposons mediated through nucleoporin evolution.

Yeasts serve as hosts to several types of genetic parasites. Few studies have addressed the evolutionary trajectory of yeast genes that control the stable co-existence of these parasites with their host cell. In Saccharomyces yeasts, the retrovirus-like Ty retrotransposons must access the nucleus. We show that several genes encoding components of the yeast nuclear pore complex have experienced natural selection for substitutions that change the encoded protein sequence. By replacing these S. cerevisiae genes with orthologs from other Saccharomyces species, we discovered that natural sequence changes have affected the mobility of Ty retrotransposons. Specifically, changing the genetic sequence of NUP84 or NUP82 to match that of other Saccharomyces species alters the mobility of S. cerevisiae Ty1 and Ty3. Importantly, all tested housekeeping functions of NUP84 and NUP82 remained equivalent across species. Signatures of natural selection, resulting in altered interactions with viruses and parasitic genetic elements, are common in host defense proteins. Yet, few instances have been documented in essential housekeeping proteins. The nuclear pore complex is the gatekeeper of the nucleus. This study shows how the evolution of this large, ubiquitous eukaryotic complex can alter the replication of a molecular parasite, but concurrently maintain essential host functionalities regarding nucleocytoplasmic trafficking.

Correction: Species-specific vulnerability of RanBP2 shaped the evolution of SIV as it transmitted in African apes.

[This corrects the article DOI: 10.1371/journal.ppat.1006906.].

Species-specific vulnerability of RanBP2 shaped the evolution of SIV as it transmitted in African apes.

HIV-1 arose as the result of spillover of simian immunodeficiency viruses (SIVs) from great apes in Africa, namely from chimpanzees and gorillas. Chimpanzees and gorillas were, themselves, infected with SIV after virus spillover from African monkeys. During spillover events, SIV is thought to require adaptation to the new host species. The host barriers that drive viral adaptation have predominantly been attributed to restriction factors, rather than cofactors (host proteins exploited to promote viral replication). Here, we consider the role of one cofactor, RanBP2, in providing a barrier that drove viral genome evolution during SIV spillover events. RanBP2 (also known as Nup358) is a component of the nuclear pore complex known to facilitate nuclear entry of HIV-1. Our data suggest that transmission of SIV from monkeys to chimpanzees, and then from chimpanzees to gorillas, both coincided with changes in the viral capsid that allowed interaction with RanBP2 of the new host species. However, human RanBP2 subsequently provided no barrier to the zoonotic transmission of SIV from chimpanzees or gorillas, indicating that chimpanzee- and gorilla-adapted SIVs are pre-adapted to humans in this regard. Our observations are in agreement with RanBP2 driving virus evolution during cross-species transmissions of SIV, particularly in the transmissions to and between great ape species.

XRN1 Is a Species-Specific Virus Restriction Factor in Yeasts.

In eukaryotes, the degradation of cellular mRNAs is accomplished by Xrn1 and the cytoplasmic exosome. Because viral RNAs often lack canonical caps or poly-A tails, they can also be vulnerable to degradation by these host exonucleases. Yeast lack sophisticated mechanisms of innate and adaptive immunity, but do use RNA degradation as an antiviral defense mechanism. One model is that the RNA of yeast viruses is subject to degradation simply as a side effect of the intrinsic exonuclease activity of proteins involved in RNA metabolism. Contrary to this model, we find a highly refined, species-specific relationship between Xrn1p and the "L-A" totiviruses of different Saccharomyces yeast species. We show that the gene XRN1 has evolved rapidly under positive natural selection in Saccharomyces yeast, resulting in high levels of Xrn1p protein sequence divergence from one yeast species to the next. We also show that these sequence differences translate to differential interactions with the L-A virus, where Xrn1p from S. cerevisiae is most efficient at controlling the L-A virus that chronically infects S. cerevisiae, and Xrn1p from S. kudriavzevii is most efficient at controlling the L-A-like virus that we have discovered within S. kudriavzevii. All Xrn1p orthologs are equivalent in their interaction with another virus-like parasite, the Ty1 retrotransposon. Thus, the activity of Xrn1p against totiviruses is not simply an incidental consequence of the enzymatic activity of Xrn1p, but rather Xrn1p co-evolves with totiviruses to maintain its potent antiviral activity and limit viral propagation in Saccharomyces yeasts. Consistent with this, we demonstrated that Xrn1p physically interacts with the Gag protein encoded by the L-A virus, suggesting a host-virus interaction that is more complicated than just Xrn1p-mediated nucleolytic digestion of viral RNAs.

Non-human Primate Schlafen11 Inhibits Production of Both Host and Viral Proteins.

Schlafen11 (encoded by the SLFN11 gene) has been shown to inhibit the accumulation of HIV-1 proteins. We show that the SLFN11 gene is under positive selection in simian primates and is species-specific in its activity against HIV-1. The activity of human Schlafen11 is relatively weak compared to that of some other primate versions of this protein, with the versions encoded by chimpanzee, orangutan, gibbon, and marmoset being particularly potent inhibitors of HIV-1 protein production. Interestingly, we find that Schlafen11 is functional in the absence of infection and reduces protein production from certain non-viral (GFP) and even host (Vinculin and GAPDH) transcripts. This suggests that Schlafen11 may just generally block protein production from non-codon optimized transcripts. Because Schlafen11 is an interferon-stimulated gene with a broad ability to inhibit protein production from many host and viral transcripts, its role may be to create a general antiviral state in the cell. Interestingly, the strong inhibitors such as marmoset Schlafen11 consistently block protein production better than weak primate Schlafen11 proteins, regardless of the virus or host target being analyzed. Further, we show that the residues to which species-specific differences in Schlafen11 potency map are distinct from residues that have been targeted by positive selection. We speculate that the positive selection of SLFN11 could have been driven by a number of different factors, including interaction with one or more viral antagonists that have yet to be identified.

The effect of species representation on the detection of positive selection in primate gene data sets.

Over evolutionary time, both host- and virus-encoded genes have been continually selected to modify their interactions with one another. This has resulted in the rapid evolution of the specific codons that govern the physical interactions between host and virus proteins. Virologists have discovered that these evolutionary signatures, acquired in nature, can provide a shortcut in the functional dissection of host-virus interactions in the laboratory. However, the use of evolution studies in this way is complicated by the fact that many nonhuman primate species are endangered, and biomaterials are often difficult to acquire. Here, we assess how the species representation in primate gene data sets affects the detection of positive natural selection. Our results demonstrate how targeted primate sequencing projects could greatly enhance research in immunology, virology, and beyond.

Identification of Owl Monkey CD4 Receptors Broadly Compatible with Early-Stage HIV-1 Isolates.

UNLABELLED: Most HIV-1 variants isolated from early-stage human infections do not use nonhuman primate versions of the CD4 receptor for cellular entry, or they do so poorly. We and others have previously shown that CD4 has experienced strong natural selection over the course of primate speciation, but it is unclear whether this selection has influenced the functional characteristics of CD4 as an HIV-1 receptor. Surprisingly, we find that selection on CD4 has been most intense in the New World monkeys, animals that have never been found to harbor lentiviruses related to HIV-1. Based on this, we sampled CD4 genetic diversity within populations of individuals from seven different species, including five species of New World monkeys. We found that some, but not all, CD4 alleles found in Spix's owl monkeys (Aotus vociferans) encode functional receptors for early-stage human HIV-1 isolates representing all of the major group M clades (A, B, C, and D). However, only some isolates of HIV-1 subtype C can use the CD4 receptor encoded by permissive Spix's owl monkey alleles. We characterized the prevalence of functional CD4 alleles in a colony of captive Spix's owl monkeys and found that 88% of surveyed individuals are homozygous for permissive CD4 alleles, which encode an asparagine at position 39 of the receptor. We found that the CD4 receptors encoded by two other species of owl monkeys (Aotus azarae and Aotus nancymaae) also serve as functional entry receptors for early-stage isolates of HIV-1. IMPORTANCE: Nonhuman primates, particularly macaques, are used for preclinical evaluation of HIV-1 vaccine candidates. However, a significant limitation of the macaque model is the fact that most circulating HIV-1 variants cannot use the macaque CD4 receptor to enter cells and have to be adapted to these species. This is particularly true for viral variants from early stages of infection, which represent the most relevant vaccine targets. In this study, we found that some individuals from captive owl monkey populations harbor CD4 alleles that are compatible with a broad collection of HIV-1 isolates, including those isolated from early in infection in highly affected populations and representing diverse subtypes.

Computational and Functional Analysis of the Virus-Receptor Interface Reveals Host Range Trade-Offs in New World Arenaviruses.

UNLABELLED: Animal viruses frequently cause zoonotic disease in humans. As these viruses are highly diverse, evaluating the threat that they pose remains a major challenge, and efficient approaches are needed to rapidly predict virus-host compatibility. Here, we develop a combined computational and experimental approach to assess the compatibility of New World arenaviruses, endemic in rodents, with the host TfR1 entry receptors of different potential new host species. Using signatures of positive selection, we identify a small motif on rodent TfR1 that conveys species specificity to the entry of viruses into cells. However, we show that mutations in this region affect the entry of each arenavirus differently. For example, a human single nucleotide polymorphism (SNP) in this region, L212V, makes human TfR1 a weaker receptor for one arenavirus, Machupo virus, but a stronger receptor for two other arenaviruses, Junin and Sabia viruses. Collectively, these findings set the stage for potential evolutionary trade-offs, where natural selection for resistance to one virus may make humans or rodents susceptible to other arenavirus species. Given the complexity of this host-virus interplay, we propose a computational method to predict these interactions, based on homology modeling and computational docking of the virus-receptor protein-protein interaction. We demonstrate the utility of this model for Machupo virus, for which a suitable cocrystal structural template exists. Our model effectively predicts whether the TfR1 receptors of different species will be functional receptors for Machupo virus entry. Approaches such at this could provide a first step toward computationally predicting the "host jumping" potential of a virus into a new host species. IMPORTANCE: We demonstrate how evolutionary trade-offs may exist in the dynamic evolutionary interplay between viruses and their hosts, where natural selection for resistance to one virus could make humans or rodents susceptible to other virus species. We present an algorithm that predicts which species have cell surface receptors that make them susceptible to Machupo virus, based on computational docking of protein structures. Few molecular models exist for predicting the risk of spillover of a particular animal virus into humans or new animal populations. Our results suggest that a combination of evolutionary analysis, structural modeling, and experimental verification may provide an efficient approach for screening and assessing the potential spillover risks of viruses circulating in animal populations.

Widespread sequence variations in VAMP1 across vertebrates suggest a potential selective pressure from botulinum neurotoxins.

Botulinum neurotoxins (BoNT/A-G), the most potent toxins known, act by cleaving three SNARE proteins required for synaptic vesicle exocytosis. Previous studies on BoNTs have generally utilized the major SNARE homologues expressed in brain (VAMP2, syntaxin 1, and SNAP-25). However, BoNTs target peripheral motor neurons and cause death by paralyzing respiratory muscles such as the diaphragm. Here we report that VAMP1, but not VAMP2, is the SNARE homologue predominantly expressed in adult rodent diaphragm motor nerve terminals and in differentiated human motor neurons. In contrast to the highly conserved VAMP2, BoNT-resistant variations in VAMP1 are widespread across vertebrates. In particular, we identified a polymorphism at position 48 of VAMP1 in rats, which renders VAMP1 either resistant (I48) or sensitive (M48) to BoNT/D. Taking advantage of this finding, we showed that rat diaphragms with I48 in VAMP1 are insensitive to BoNT/D compared to rat diaphragms with M48 in VAMP1. This unique intra-species comparison establishes VAMP1 as a physiological toxin target in diaphragm motor nerve terminals, and demonstrates that the resistance of VAMP1 to BoNTs can underlie the insensitivity of a species to members of BoNTs. Consistently, human VAMP1 contains I48, which may explain why humans are insensitive to BoNT/D. Finally, we report that residue 48 of VAMP1 varies frequently between M and I across seventeen closely related primate species, suggesting a potential selective pressure from members of BoNTs for resistance in vertebrates.