bioRxiv @biorxivpreprint@qoto.org

Cell-cell communication is mediated by membrane receptors and their cognate ligands, such as the Eph/ephrin system, and dictates physiological processes, including cell proliferation and migration. However, whether and how Eph/ephrin signaling culminates in transcriptional regulation is largely unknown. Epigenetic mechanisms are key for integrating external signals, e.g., from neighboring cells, into the transcriptome. We have previously reported that ephrinA5 stimulation of immortalized cerebellar granule (CB) cells elicits transcriptional changes of lncRNAs and protein-coding genes. LncRNAs represent important adaptors for epigenetic writers through which they regulate gene expression. Here, we investigate the interaction of lncRNA with protein-coding genes by the combined power of in silico modeling of RNA/DNA interactions and respective wet lab approaches, in the context of ephrinA5-dependent regulation of cellular motility. We found that Snhg15, a cancer-related lncRNA, forms a triplex structure with the Ncam1 promoter and interacts with DNMT1. EphrinA5 stimulation leads to reduced Snhg15 expression, diminished Snhg15/DNMT1 interaction and decreased DNMT1 association with the Ncam1 promoter. These findings can explain the attenuated Ncam1 promoter methylation and elevated Ncam1 expression that in turn elicits decreased cell motility of CB cells. Hence, we propose that ephrinA5 influences gene transcription via lncRNA-targeted DNA methylation underlying the regulation of cellular motility. ### Competing Interest Statement The authors have declared no competing interest.

Plasmids are extrachromosomal genetic elements that often encode fitness enhancing features. However, many bacteria carry 'cryptic' plasmids that do not confer clear beneficial functions. We identified one such cryptic plasmid, pBI143, which is ubiquitous across industrialized gut microbiomes, and is 14 times as numerous as crAssphage, currently established as the most abundant genetic element in the human gut. The majority of mutations in pBI143 accumulate in specific positions across thousands of metagenomes, indicating strong purifying selection. pBI143 is monoclonal in most individuals, likely due to the priority effect of the version first acquired, often from one's mother. pBI143 can transfer between Bacteroidales and although it does not appear to impact bacterial host fitness in vivo, can transiently acquire additional genetic content. We identified important practical applications of pBI143, including its use in identifying human fecal contamination and its potential as an inexpensive alternative for detecting human colonic inflammatory states. ### Competing Interest Statement The authors have declared no competing interest.

The intestine is the primary colonisation site for carbapenem-resistant Enterobacteriaceae (CRE) and serves as a reservoir of CRE that cause invasive infections (e.g. bloodstream infections). Antibiotics disrupt colonisation resistance mediated by the gut microbiota, promoting the expansion of CRE within the intestine. We used ex vivo faecal cultures to measure the impact of antibiotics (that promote CRE intestinal colonisation) on the faecal microbiota from healthy human donors. We demonstrated that antibiotics decreased the abundance of gut commensals (including Bifidobacteriaceae and Bacteroidales) in human faecal microbiota, resulting in an enrichment of nutrients and a depletion of microbial metabolites. The nutrient utilisation abilities, nutrient preferences, and metabolite inhibition susceptibilities of carbapenem-resistant Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae strains were measured. Nutrients (which were elevated with antibiotics) acted as carbon and nitrogen sources to support CRE growth, where CRE showed an ordered preference for specific nutrients. These nutrients were also increased in faeces from antibiotic-treated mice but decreased following intestinal colonisation with carbapenem-resistant E. coli. Microbial metabolites (which decreased with antibiotics) were inhibitory towards CRE growth in vitro. Carbapenem-resistant E. coli growth was decreased in faecal samples from mice treated with a mixture of inhibitory metabolites compared with PBS-treated mice. These findings demonstrate that killing gut commensals with antibiotics disrupts colonisation resistance by enriching nutrients that support CRE growth and depleting metabolites that inhibit CRE growth. These results support the development of new microbiome therapeutics to prevent CRE intestinal colonisation, which would also prevent the subsequent development of invasive CRE infections. ### Competing Interest Statement JAKM, AYGY, and OGK have filed a patent application related to this work (patent application number 2217266.2). BHM received consultancy fees from Finch Therapeutics Group, Ferring Pharmaceuticals, and Summit Therapeutics, outside of the submitted work. JRM received consultancy fees from EnteroBiotix Ltd. and Cultech Ltd, outside of the submitted work. All other authors disclose no conflicts.

Dieback or decline caused by Lasiodiplodia spp. is a major disease of mango trees (Mangifera indica L.). The main objectives of this study were to identify Lasiodiplodia species associated with mango decline in Burkina Faso, and to asses the climatic and edaphic factors affecting the geographic distribution of the disease in the country. The genetic diversity of 47 Lasiodiplodia isolates was studied based on sequence data of the translation elongation factor 1-alpha gene (tef1-a) and the rDNA internal transcribed spacer region (ITS). Phylogeny analyses grouped the isolates from Burkina Faso with 6 different Lasiodiplodia species, including L. euphorbicola that accounted for 36 of the 47 isolates. Lasiodiplodia isolates tested on mango seedlings induced the typical dieback symptoms. Disease incidence and severity were generally higher in the drier and warmer regions (eastern) of the country. This study provides the required information to establish control strategies against mango decline in Burkina Faso. ### Competing Interest Statement The authors have declared no competing interest.

Prior studies have identified genetic, infectious, and biological associations with immune competence and disease severity; however, there have been few integrative analyses of these factors and study populations are often limited in demographic diversity. Utilizing samples from 1,705 individuals in 5 countries, we examined putative determinants of immunity, including: single nucleotide polymorphisms, ancestry informative markers, herpesvirus status, age, and sex. In healthy subjects, we found significant differences in cytokine levels, leukocyte phenotypes, and gene expression. Transcriptional responses also varied by cohort, and the most significant determinant was ancestry. In influenza infected subjects, we found two disease severity immunophenotypes, largely driven by age. Additionally, cytokine regression models show each determinant differentially contributes to acute immune variation, with unique and interactive, location-specific herpesvirus effects. These results provide novel insight into the scope of immune heterogeneity across diverse populations, the integrative effects of factors which drive it, and the consequences for illness outcomes. ### Competing Interest Statement P.G.T. has consulted or received honorarium and/or travel support from Illumina, JNJ, Pfizer, and 10X. P.G.T. serves on the Scientific Advisory Board of ImmunoScape and CytoAgents. All remaining authors declare no competing interests.

The vast majority of chemistry and biology occurs in solution, and new label-free analytical techniques that can help resolve solution-phase complexity at the single-molecule level can provide new microscopic perspectives of unprecedented detail. Here, we use the increased light-molecule interactions in high-finesse fiber Fabry-Perot microcavities to detect individual biomolecules as small as 1.2 kDa with signal-to-noise ratios >100, even as the molecules are freely diffusing in solution. Our method delivers 2D intensity and temporal profiles, enabling the distinction of sub-populations in mixed samples. Strikingly, we observe a linear relationship between passage time and molecular radius, unlocking the potential to gather crucial information about diffusion and solution-phase conformation. Furthermore, mixtures of biomolecule isomers of the same molecular weight can also be resolved. Detection is based on a novel molecular velocity filtering and dynamic thermal priming mechanism leveraging both photo-thermal bistability and Pound-Drever-Hall cavity locking. This technology holds broad potential for applications in life and chemical sciences and represents a major advancement in label-free in vitro single-molecule techniques. ### Competing Interest Statement The authors have declared no competing interest.

Complex mechanisms regulate the cellular distribution of cholesterol, a critical component of eukaryote membranes involved in regulation of membrane protein functions directly and through the physiochemical properties of membranes. StarD4, a member of the steroidogenic acute regulator-related lipid-transfer (StART) domain (StARD)-containing protein family, is a highly efficient sterol-specific transfer protein involved in cholesterol homeostasis. Its mechanism of cargo loading and release remains unknown despite recent insights into the key role of phosphatidylinositol phosphates in modulating its interactions with target membranes. We have used large-scale atomistic Molecular dynamics (MD) simulations to study how the dynamics of cholesterol bound to the StarD4 protein can affect interaction with target membranes, and cargo delivery. We identify the two major cholesterol (CHL) binding modes in the hydrophobic pocket of StarD4, one near S136&S147 (the Ser-mode), and another closer to the putative release gate located near W171, R92&Y117 (the Trp-mode). We show that conformational changes of StarD4 associated directly with the transition between these binding modes facilitate the opening of the gate. To understand the dynamics of this connection we apply a machine-learning algorithm for the detection of rare events in MD trajectories (RED), which reveals the structural motifs involved in the opening of a front gate and a back corridor in the StarD4 structure occurring together with the spontaneous transition of CHL from the Ser-mode of binding to the Trp-mode. Further analysis of MD trajectory data with the information-theory based NbIT method reveals the allosteric network connecting the CHL binding site to the functionally important structural components of the gate and corridor. Mutations of residues in the allosteric network are shown to affect the performance of the allosteric connection. These findings outline an allosteric mechanism which prepares the CHL-bound StarD4 to release and deliver the cargo when it is bound to the target membrane. ### Competing Interest Statement The authors have declared no competing interest.

Bacteriophages, which naturally shape bacterial communities, can be co-opted as a biological technology to help eliminate pathogenic bacteria from our bodies and food supply. Phage genome editing is a critical tool to engineer more effective phage technologies. However, editing phage genomes has traditionally been a low efficiency process that requires laborious screening, counter selection, or in vitro construction of modified genomes. These requirements impose limitations on the type and throughput of phage modifications, which in turn limit our knowledge and potential for innovation. Here, we present a scalable approach for engineering phage genomes using recombitrons: modified bacterial retrons that generate recombineering donor DNA paired with single stranded binding and annealing proteins to integrate those donors into phage genomes. This system can efficiently create genome modifications in multiple phages without the need for counterselection. Moreover, the process is continuous, with edits accumulating in the phage genome the longer the phage is cultured with the host, and multiplexable, with different editing hosts contributing distinct mutations along the genome of a phage in a mixed culture. In lambda phage, as an example, recombitrons yield single-base substitutions at up to 99% efficiency and up to 5 distinct mutations installed on a single phage genome, all without counterselection and only a few hours of hands-on time. ### Competing Interest Statement C.B.F., S.B.K., and S.L.S. are named inventors on a patent application related to the technologies described in this work.

Short amphipathic peptides are capable of binding to transcriptional coactivators, often targeting the same binding surfaces as native transcriptional activation domains. However, they do so with modest affinity and generally poor selectivity, limiting their utility as synthetic modulators. Here we show that incorporation of a medium-chain, branched fatty acid to the N-terminus of one such heptameric lipopeptidomimetic (34913-8) increases the affinity for the coactivator Med25 >10-fold (Ki >>100 micromolar to 10 micromolar). Importantly, the selectivity of 34913-8 for Med25 compared to other coactivators is excellent. 34913-8 engages Med25 through interaction with the H2 face of its Activator Interaction Domain and in doing so stabilizes full-length protein in the cellular proteome. Further, genes regulated by Med25-activator PPIs are inhibited in a cell model of triple-negative breast cancer. Thus, 34913-8 is a useful tool for studying Med25 and the Mediator complex biology and the results indicate that lipopeptidomimetics may be a robust source of inhibitors for activator-coactivator complexes. ### Competing Interest Statement The authors have declared no competing interest.

Bacteriophage enzymes synthesize varied and complex DNA hypermodifications. The enzyme encoded by the phage Mu gene mom is necessary for post-replicative carbamoylmethyl addition to the exocyclic amine of deoxyadenosine in DNA during the lytic phase of the viral life-cycle. The molecular details of this modification reaction, including the molecular origins of the modification itself, have long eluded understanding. Here, we demonstrate that Mom co-opts the translational machinery of the host by harvesting activated glycine from charged tRNAGly to hypermodify adenine. Based on this insight, we report the first in vitro reconstitution of the Mu hypermodification from purified components. Using isotope labeling, we demonstrate that the carbamoyl nitrogen of the Mom modification is derived from the N 6 of adenine, indicating an on-base rearrangement of the N 6 aminoacylation product, possibly via a cyclic intermediate. Informed by the X-ray crystal structure of Mom, we have probed the location of the active site, identified a novel insertion, and established substrate specificities of the Mom enzyme. ### Competing Interest Statement R.M.B.S, Y.J.L., C.G., E.A.S., S.R.L., and P.R.W. are employees of New England Biolabs, a manufacturer and vendor of molecular biology reagents. This affiliation does not affect the authors' impartiality, adherence to journal standards and policies, or availability of data.

Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic ″unknown unknowns″, but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded His6-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved ~5-fold more effective than the canonical nitroreductase NfsB. ### Competing Interest Statement The authors have declared no competing interest.

Telomeres protect chromosome ends from unscheduled DNA repair. Of particular concern to telomere integrity is the action of the MRN (MRE11, RAD50, NBS1) complex, which plays a critical role in the recognition and processing of double-stranded DNA breaks (DSBs). MRN orchestrates activation of the ATM kinase in the cellular response to DNA damage, promotes DNA end-tethering thus aiding the nonhomologous end joining (NHEJ) pathway, and initiates DSB resection through the nuclease activity of MRE11. A previously identified amino acid motif (which we refer to as MIN, for MRN inhibitor) is capable of regulating MRN activity via binding to a RAD50 interface. The motif has independently arisen at least twice in yeasts, through convergent evolution of telomeric proteins Rif2 and Taz1, in budding and fission yeast respectively. We now provide a third example of convergent evolution for this mechanism controlling the activity of MRN at telomeres, by demonstrating that the iDDR motif of shelterin protein TRF2 binds to RAD50 at the same site engaged by the MIN motif in the yeast proteins, despite not sharing any sequence conservation with the latter. Modelling for the human CtIP interaction with RAD50 (known to be necessary for activation of MRE11), as well as for the budding and fission yeast counterparts Sae2 and Ctp1, indicates that the interaction is mutually exclusive with binding of the iDDR/MIN motifs, thus pointing to a clear conserved mechanism for inhibition of MRN nuclease activity at telomeres. ### Competing Interest Statement The authors have declared no competing interest.

Dinoflagellates in the Family Symbiodiniaceae (Order Suessiales) represent diverse, predominantly symbiotic lineages that associate with taxa such as corals and jellyfish. Their ancestor is believed to have been free-living, and the establishment of symbiosis (i.e., symbiogenesis) is hypothesised to have occurred multiple times during Symbiodiniaceae evolution. Among Symbiodiniaceae taxa, the genus Effrenium is an early diverging, free-living lineage that is phylogenetically positioned between two robustly supported groups of genera within which symbiotic taxa have emerged. The lack of symbiogenesis in Effrenium suggests that the ancestral features of Symbiodiniaceae may have been retained in this lineage. Here we present de novo assembled genomes and associated transcriptome data from three isolates of Effrenium voratum . We compared the Effrenium genomes (1.2-1.9 Gbp in size) and gene features with those of 16 Symbiodiniaceae taxa and other outgroup dinoflagellates. Surprisingly, we find that genome reduction predates the origin of Symbiodiniaceae and is not primarily explained by their symbiotic lifestyle. We postulate that adaptation to an extreme habitat (e.g., as in Polarella glacialis ) or life in oligotrophic conditions resulted in the Suessiales ancestor having a haploid genome size < 2Gbp, which is retained (or smaller) among all extant algae in this lineage. Our data reveal that the free-living lifestyle distinguishes Effrenium from symbiotic Symbiodiniaceae vis-à-vis longer introns, more-extensive mRNA editing, fewer (~30%) lineage-specific gene families, and lower (~10%) level of pseudogenisation. These results demonstrate how genome reduction and the adaptation to symbiotic versus free-living lifestyles intersect, and have driven the diversification and genome evolution of Symbiodiniaceae. ### Competing Interest Statement The authors have declared no competing interest.

Understanding the DNA methylation patterns in the human genome is a key step to decipher gene regulatory mechanisms and model mutation rate heterogeneity in the human genome. While methylation rates can be measured e.g. with bisulfite sequencing, such measures do not capture historical patterns. Here we present a new method, Methylation Hidden Markov Model (MHMM), to estimate the accumulated germline methylation signature in human population history leveraging two properties: (1) Mutation rates of cytosine to thymine transitions at methylated CG dinucleotides are orders of magnitude higher than that in the rest of the genome. (2) Methylation levels are locally correlated, so the allele frequencies of neighboring CpGs can be used jointly to estimate methylation status. We applied MHMM to allele frequencies from the TOPMed and the gnomAD genetic variation catalogs. Our estimates are consistent with whole genome bisulfite sequencing (WGBS) measured human germ cell methylation levels at 90% of CpG sites, but we also identified ~442,000 historically methylated CpG sites that could not be captured due to sample genetic variation, and inferred methylation status for ~721,000 CpG sites that were missing from WGBS. Hypo-methylated regions identified by combining our results with experimental measures are 1.7 times more likely to recover known active genomic regions than those identified by WGBS alone. Our estimated historical methylation status can be leveraged to enhance bioinformatic analysis of germline methylation such as annotating regulatory and inactivated genomic regions and provide insights in sequence evolution including predicting mutation constraint. ### Competing Interest Statement The authors have declared no competing interest.

The African fig fly, Zaprionus indianus (Gupta), has spread globally from its native range in tropical Africa, becoming an invasive crop pest in select areas such as Brazil. Z. indianus was first reported in the United States in 2005 and has since been documented as far north as Canada. As a tropical species, Z. indianus is expected to have low cold tolerance, likely limiting its ability to persist at northern latitudes. In North America, the geographic regions where Z. indianus can thrive and seasonal fluctuations in its abundance are not well understood. The purpose of this study was to characterize the temporal and spatial variation in Z. indianus abundance to better understand its invasion of the eastern United States. We sampled drosophilid communities over the growing season at two orchards in Virginia from 2020-2022 and several locations along the East Coast during the fall of 2022. Virginia abundance curves showed similar seasonal dynamics across years with individuals first detected around July and becoming absent around December. Massachusetts was the northernmost population and no Z. indianus were detected in Maine. Variation in Z. indianus relative abundance was high between nearby orchards and across different fruits within orchards but was not correlated with latitude. Fitness of wild-caught females decreased later in the season and at higher latitudes. The patterns of Z. indianus abundance shown here demonstrate an apparent susceptibility to cold and highlight a need for systematic sampling to accurately characterize the range and spread of Z. indianus. ### Competing Interest Statement The authors have declared no competing interest.

Microalgae play an essential role in global net primary productivity and global biogeochemical cycling, but despite their phototrophic lifestyle, over half of algal species depend on a supply of the corrinoid vitamin B12 (cobalamin) for growth. This essential organic micronutrient is produced only by a subset of prokaryotic organisms, which implies that for algal species to use this compound, they must first acquire it from external sources. Previous studies have identified protein components involved in vitamin B12 uptake in bacterial species and humans. However, little is known about how it is taken up in algae. Here, we demonstrate the essential role of a protein, CBA1 (for cobalamin acquisition protein 1), in B12 uptake in Phaeodactylum tricornutum, using CRISPR-Cas9 to generate targeted knockouts, and in Chlamydomonas reinhardtii, by insertional mutagenesis. In both cases, CBA1 knockout lines are no longer able to take up exogenous vitamin B12. Complementation of the C. reinhardtii mutants with the wildtype CBA1 gene restores B12 uptake, and regulation of CBA1 expression via a riboswitch element can be used to control the phenotype. When visualised by confocal microscopy, a YFP-fusion with C. reinhardtii CBA1 shows association with membranes. A bioinformatics analysis found that CBA1-like sequences are present in all the major eukaryotic phyla. Its presence is correlated with B12-dependent enzymes in many, although not all, taxa, suggesting CBA1 has a conserved role. Our results thus provide insight into the molecular basis of algal B12 acquisition, a process that likely underpins many interactions in aquatic microbial communities.

A universal feature of living systems is that natural variation in genotype underpins variation in phenotype. Yet, research in model organisms is often constrained to a single genetic background, the reference strain. Further, genomic studies that do evaluate wild strains typically rely on the reference strain genome for read alignment, leading to the possibility of biased inferences based on incomplete or inaccurate mapping; the extent of reference bias can be difficult to quantify. As an intermediary between genome and organismal traits, gene expression is well positioned to describe natural variability across genotypes generally and in the context of environmental responses, which can represent complex adaptive phenotypes. C. elegans sits at the forefront of investigation into small-RNA gene regulatory mechanisms, or RNA interference (RNAi), and wild strains exhibit natural variation in RNAi competency following environmental triggers. Here, we examine how genetic differences among five wild strains affect the C. elegans transcriptome in general and after inducing RNAi responses to two germline target genes. Approximately 34% of genes were differentially expressed across strains; 411 genes were not expressed at all in at least one strain despite robust expression in others, including 49 genes not expressed in reference strain N2. Despite the presence of hyper-diverse hotspots throughout the C. elegans genome, reference mapping bias was of limited concern: over 92% of variably expressed genes were robust to mapping issues. Overall, the transcriptional response to RNAi was strongly strain-specific and highly specific to the target gene, and the laboratory strain N2 was not representative of the other strains. Moreover, the transcriptional response to RNAi was not correlated with RNAi phenotypic penetrance; the two germline RNAi incompetent strains exhibited substantial differential gene expression following RNAi treatment, indicating an RNAi response despite failure to reduce expression of the target gene. We conclude that gene expression, both generally and in response to RNAi, differs across C. elegans strains such that choice of strain may meaningfully influence scientific conclusions. To provide a public, easily accessible resource for querying gene expression variation in this dataset, we introduce an interactive website at https://wildworm.biosci.gatech.edu/rnai/. ### Competing Interest Statement The authors have declared no competing interest.

Viruses are the most abundant biological entities on Earth and play a significant role in the evolution of many organisms and ecosystems. In pathogenic protozoa, the presence of endosymbiotic viruses has been linked to an increased risk of treatment failure and severe clinical outcome. Here, we studied the molecular epidemiology of the zoonotic disease cutaneous leishmaniasis in Peru and Bolivia through a joint evolutionary analysis of Leishmania braziliensis parasites and their endosymbiotic Leishmania RNA virus. We show that parasite populations circulate in isolated pockets of suitable habitat and are associated with single viral lineages that appear in low prevalence. In contrast, groups of hybrid parasites were geographically and ecologically dispersed, and commonly infected from a pool of genetically diverse viruses. Our results suggest that parasite hybridization, likely due to increased human migration and ecological perturbations, increased the frequency of endosymbiotic interactions known to play a key role in disease severity. ### Competing Interest Statement The authors have declared no competing interest.

Genetic monitoring of populations currently attracts interest in the context of the Convention on Biological Diversity but needs long-term planning and investments. Genetic diversity has been largely neglected in biodiversity monitoring, and when addressed is treated separately, detached from other conservation issues, such as habitat alteration due to climate change. Genetic monitoring supports the conservation and management of fisheries, game, and threatened populations. It also can contribute to the assessment of predicted and realized impacts of climate change, and their management. We report the first accounting of genetic monitoring efforts among countries in Europe (their genetic monitoring capacity, GMC) to determine where GMC suggests the combination of national infrastructure, political support and resources for continued and expanded monitoring. Overlaying GMC with areas where species ranges approach current and future climate niche limits (i.e., niche marginality) helps identify whether GMC coincides with anticipated climate change effects on biodiversity. Our analysis suggests that country area extent, financial resources, and conservation policy influence GMC, high values of which inconsistently match joint species patterns of climate niche marginality. Populations at niche margins likely hold genetic diversity that is important to adaptation to changing climate, and our results illuminate the need in Europe for expanded genetic monitoring across the climate gradients occupied by species, a need arguably greatest in southeastern European countries. ### Competing Interest Statement The authors have declared no competing interest.

Human immunodeficiency virus (HIV) and other lentiviruses adapt to new hosts by evolving to evade host-specific innate immune proteins that differ in sequence and often viral recognition between host species. Understanding how these host antiviral proteins, called restriction factors, constrain lentivirus replication and transmission is key to understanding the emergence of pandemic viruses like HIV-1. Human TRIM34, a paralogue of the well-characterized lentiviral restriction factor TRIM5α, was previously identified by our lab via CRISPR-Cas9 screening as a restriction factor of certain HIV and SIV capsids. Here, we show that diverse primate TRIM34 orthologues from non-human primates can restrict a range of Simian Immunodeficiency Virus (SIV) capsids including SIVAGM-SAB, SIVAGM-TAN and SIVMAC capsids, which infect sabaeus monkeys, tantalus monkeys, and rhesus macaques, respectively. All primate TRIM34 orthologues tested, regardless of species of origin, were able to restrict this same subset of viral capsids. However, in all cases, this restriction also required the presence of TRIM5α. We demonstrate that TRIM5α is necessary, but not sufficient, for restriction of these capsids, and that human TRIM5α functionally interacts with TRIM34 from different species. Finally, we find that both the TRIM5α SPRY v1 loop and the TRIM34 SPRY domain are essential for TRIM34-mediated restriction. These data support a model in which TRIM34 is a broadly-conserved primate lentiviral restriction factor that acts in tandem with TRIM5α, such that together, these proteins can restrict capsids that neither can restrict alone. ### Competing Interest Statement The authors have declared no competing interest.