Lost Interactions Research Articles

Recessive resistance against beet chlorosis virus is conferred by the eukaryotic translation initiation factor (iso)4E in Beta vulgaris.

Eukaryotic translation initiation factors (eIFs) are important for mRNA translation but also pivotal for plant-virus interaction. Most of these plant-virus interactions were found between plant eIFs and the viral protein genome-linked (VPg) of potyviruses. In case of lost interaction due to mutation or deletion of eIFs, the viral translation and subsequent replication within its host is negatively affected, resulting in a recessive resistance. Here we report the identification of the Beta vulgaris Bv-eIF(iso)4E as a susceptibility factor towards the VPg-carrying beet chlorosis virus (genus Polerovirus). Using yeast two-hybrid and bimolecular fluorescence complementation assays, the physical interaction between Bv-eIF(iso)4E and the putative BChV-VPg was detected, while the VPg of the closely related beet mild yellowing virus (BMYV) was found to interact with the two isoforms Bv-eIF4E and Bv-eIF(iso)4E. These VPg-eIF interactions within the polerovirus-beet pathosystem were demonstrated to be highly specific, as single mutations within the predicted cap-binding pocket of Bv-eIF(iso)4E resulted in a loss of interaction. To investigate the suitability of eIFs as a resistance resource against beet infecting poleroviruses, B.vulgaris plants were genome edited by CRISPR/Cas9 resulting in knockouts of different eIFs. Asimultaneous knockout of the identified BMYV-interaction partners Bv-eIF4E and Bv-eIF(iso)4E was not achieved, but Bv-eIF(iso)4EKO plants showed a significantly lowered BChV accumulation and decrease in infection rate from 100% to 28.86%, while no influence on BMYV accumulation was observed. Still, these observations support that eIFs are promising candidate genes for polerovirus resistance breeding in sugar beet.

Vinyl Halide‐Modified Unsaturated Cyclitols are Mechanism‐Based Glycosidase Inhibitors

AbstractSuitably configured allyl ethers of unsaturated cyclitols act as substrates of β‐glycosidases, reacting via allylic cation transition states. Incorporation of halogens at the vinylic position of these carbasugars, along with an activated leaving group, generates potent inactivators of β‐glycosidases. Enzymatic turnover of these halogenated cyclitols (F, Cl, Br) displayed a counter‐intuitive trend wherein the most electronegative substituents yielded the most labile pseudo‐glycosidic linkages. Structures of complexes with the Sulfolobus β‐glucosidase revealed similar enzyme‐ligand interactions to those seen in complexes with a 2‐fluorosugar inhibitor, the lone exception being displacement of tyrosine 322 from the active site by the halogen. Mutation of Y322 to Y322F largely abolished glycosidase activity, consistent with lost interactions at O5, but minimally affected (7‐fold) rates of carbasugar hydrolysis, yielding a more selective enzyme for unsaturated cyclitol ether hydrolysis.

Vinyl Halide-Modified Unsaturated Cyclitols are Mechanism-Based Glycosidase Inhibitors.

Suitably configured allyl ethers of unsaturated cyclitols act as substrates of β-glycosidases, reacting via allylic cation transition states. Incorporation of halogens at the vinylic position of these carbasugars, along with an activated leaving group, generates potent inactivators of β-glycosidases. Enzymatic turnover of these halogenated cyclitols (F, Cl, Br) displayed a counter-intuitive trend wherein the most electronegative substituents yielded the most labile pseudo-glycosidic linkages. Structures of complexes with the Sulfolobus β-glucosidase revealed similar enzyme-ligand interactions to those seen in complexes with a 2-fluorosugar inhibitor, the lone exception being displacement of tyrosine 322 from the active site by the halogen. Mutation of Y322 to Y322F largely abolished glycosidase activity, consistent with lost interactions at O5, but minimally affected (7-fold) rates of carbasugar hydrolysis, yielding a more selective enzyme for unsaturated cyclitol ether hydrolysis.

Perturbation of the Normalized Laplacian Matrix for the Prediction of Missing Links in Real Networks

The problem of predicting missing links in real-world networks is an active and challenging research area in both science and engineering. The goal is to model the process of link formation in a complex network based on its observed structure to unveil lost or unseen interactions. In this paper, we use perturbation theory to develop a general link prediction procedure, called Laplacian Perturbation Method (LPM), that relies on relevant structural information encoded in the normalized Laplacian matrix of the network. We implement a general algorithm for our perturbation method valid for different Laplacian-based link prediction schemes that successfully surpass the prediction accuracy of their standard non-perturbed versions in real-world and model networks. The suggested LPM for link prediction also exhibits higher accuracy compared to other extensively used local and global state-of-the-art techniques and, in particular, it outperform the Structural Perturbation Method (SPM), a popular procedure that perturbs the adjacency matrix of a network for inferring missing links, in many real-world and in synthetic networks. Taken together, our results show that perturbation methods can significantly improve Laplacian-based link prediction techniques, and feeds the debate on which representation, Laplacian or adjacency, better represents structural information for link prediction tasks in networks.

The impact of COVID-19 on professional identity.

BackgroundThe COVID‐19 pandemic has had a profound impact on medical care and medical student education as clinical rotations were halted and students' clinical activities were drastically curtailed. Learning experiences in medical school are known to promote identity formation through teamwork, reflection, and values‐based community discussion. This study explored the impact of the COVID‐19 pandemic on medical students' professional identity formation (PIF).MethodsStudents in all cohorts of medical education were invited by email in May 2020 to submit a written reflection about their learning experiences and impact of the pandemic on their PIF. We used iterative individual and team reviews, known as the “immersion/crystallisation” method, to code and analyse the data.FindingsTwenty‐six students (20%) submitted reflections in which they discussed “changing conceptions of the role and image of a physician,” “views about medical education,” and the “role of students in a pandemic.” Students viewed physicians as altruistic, effective communicators, and pledged to be like them in the future. Their perceptions of virtual learning were mixed, along with considerations of lost interactions with patients, and wanting to be more useful as professionals‐in‐training.DiscussionCOVID‐19 has impacted students' views of themselves and reshaped their ideas, both negatively and positively, about the profession they are entering and their role(s) in it.ConclusionExploring PIF and the impact of disruptions has allowed us to address the issues raised regarding clinical learning now and into the future. Reflection enhances PIF and unexpected events, such as COVID‐19, offer opportunities for reflection and development.

Structure of a dimer of the Sulfolobus solfataricus MCM N-terminal domain reveals a potential role in MCM ring opening.

Cells strongly regulate DNA replication to ensure genomic stability and prevent several diseases, including cancers. Eukaryotes and archaea strictly control DNA-replication initiation by the regulated loading of hexameric minichromosome maintenance (MCM) rings to encircle both strands of the DNA double helix followed by regulated activation of the loaded rings such that they then encircle one DNA strand while excluding the other. Both steps involve an open/closed ring transformation, allowing DNA strands to enter or exit. Here, the crystal structure of a dimer of the N-terminal domain of Sulfolobus solfataricus MCM with an intersubunit interface that is more extensive than in closed-ring structures, while including common interactions to enable facile interconversion, is presented. It is shown that the identified interface could stabilize open MCM rings by compensating for lost interactions at an open neighbor interface and that the prior open-ring cryo-EM structure of MCM loading has a similar extended interface adjacent to its open interface.

Synthesis and biological evaluation of benzodiazepines containing a pentafluorosulfanyl group

The widely used pentafluorosulfanyl group (SF 5 ) was deployed as a bioisosteric replacement for a chloro-group in the benzodiazepine diazepam (Valium™). Reaction of 2-amino-5-pentafluorosulfanyl-benzophenone with chloroacetyl chloride followed by hexamethylenetetramine, in the presence of ammonia, led to 7-sulfurpentafluoro-5-phenyl-1 H -benzo[ 1,4 ]diazepin-2( 3H )-one (2c). The latter was able to undergo a Pd-catalysed ortho-arylation, demonstrating that these highly fluorinated benzodiazepines can be further modified to form more complicated scaffolds. The replacement of Cl by the SF 5 group, led to a loss of potency for potentiating GABA A receptor activation, most likely because of a lost ligand interaction with His102 in the GABA A receptor α subunit. Dedicated to Professor Jonathan Williams, an inspirational and humble pioneer, a colleague and mentor in chemistry.

New directions in pollinator research: diversity, conflict and response to global change.

Interactions between pollinators and their plant hosts are central to maintaining global biodiversity and ensuring our food security. In this special issue, we compile reviews that summarize existing knowledge and point out key outstanding research areas to understand and safeguard pollinators, pollinators-host plant interactions and the pollination ecosystem services they provide. The vast diversity of the pollinator-plant interactions that exists on this planet still remains poorly explored, with many being associations involving a specialist pollinator partner, although historically most focus has been given to generalist pollinators, such as the honeybee. Two areas highlighted here are the ecology and evolution of oligolectic bee species, and the often-neglected groups of pollinators that forage solely at night. Advances in automated detection technologies could offer potential and complementary solutions to the current shortfall in knowledge on interactions occurring between less well-documented plant-pollinator associations, by increasing the collection range and capacity of flower visitation data over space and time. Pollinator-host plant interactions can be affected by external biotic factors, with herbivores and pathogens playing particularly important roles. Such interactions can be disrupted by modifying plant volatile and reward chemistry, with possible effects on pollinator attraction and pollination success. Mechanisms which underpin interactions between plants and their pollinators also face many anthropogenic disturbances. Reviews in this issue discuss threats from parasites and climate change to pollinator populations and plant-pollinator networks, and suggest new ways to mitigate these threats. While the protection of existing plant-pollinator networks will be a crucial goal for conservation biology, more research is needed to understand how lost interactions in degraded habitats may be restored with mutual benefits to plants and pollinators.

Targeting the protein-protein interface pocket of Aurora-A-TPX2 complex: rational drug design and validation

Aurora-A is a novel therapeutic target that belongs to the serine/threonine kinase family of proteins. Several cancers are associated with gene amplification or over-expression of Aurora-A. The somatic mutation (S155R) in Aurora-A results in a loss of interaction with its binding partner TPX2. The S155R mutation thus leads to ectopic expression of Aurora-A, resulting in centrosome amplification, chromosomal instability, aneuploidy, and oncogenic transformations. In order to restore the interaction between mutant Aurora-A and TPX2, we predicted a binding pocket in the interface of the Aurora-A-TPX2 complex. We performed molecular docking of potential bioactive molecules of the Himalayan region at the predicted site. Alantolactone and Dactylose-A were selected as potential molecules that could bind to the interface pocket and restore the lost interaction between mutant Aurora-A and TPX2. The molecular docking results were validated by performing explicit long term molecular dynamics simulations (4.0 μs) and MM-PBSA analysis. The molecular dynamics results confirmed that both the selected molecules could act as potent drugs to tackle the abnormal expression of Aurora-A manifested due to the somatic mutation (S155R). Communicated by Ramaswamy H. Sarma

Management measures to control pine wood nematode spread in Europe

Abstract Invasive pests, such as the pine wood nematode (PWN), threaten European forests. In De la Fuente, Saura, and Beck, Journal of Applied Ecology, 2018, 55, 2374, we modelled the natural spread of the PWN by the longhorn beetle (Monochamus galloprovincialis) on the Iberian Peninsula. We used the model to evaluate the effectiveness of several management measures to control the PWN spread: early detection and removal of infected trees, beetle mass trapping and clear‐cut belts. We simulated these particular measures because they have been or are being applied in PWN containment efforts on the Iberian Peninsula. Martínez‐Abraín and Jiménez, Journal of Applied Ecology, 2019, suggested that two other management measures would be appropriate to halt the PWN expansion: installing nest boxes for insectivorous birds in order to increase predation on vector beetles and increasing the populations of large mammalian herbivores to lower forest tree density. The results of De la Fuente, Saura, and Beck, Journal of Applied Ecology, 2018, 55, 2374 showed that a combination of measures is more likely to control PWN spread than any single one in isolation, indicating that halting this, and many other invasive species, requires a comprehensive approach. Here, we review current evidence on the efficacy of the measures proposed by Martínez‐Abraín and Jiménez, Journal of Applied Ecology, 2019, as PWN management tools. This evidence suggests that, by themselves, these measures are unlikely to significantly slow down, let alone halt, the PWN spread on the Iberian Peninsula. Furthermore, we indicate how the model presented in De la Fuente, Saura, and Beck, Journal of Applied Ecology, 2018, 55, 2374 lends itself to assessing this quantitatively in future research. Synthesis and applications. Lost biotic interactions in ecosystems can make them more vulnerable to invasions, and restoring interactions can in turn increase resilience. Ecosystem restoration can thus, in theory, also benefit invasive species management. Current evidence indicates however that managing natural species interactions, such as beetle predation from insectivorous birds or herbivory by large mammals, would need to be applied in combination with other measures if they are to halt or significantly slow down the expansion of the PWN in Europe.

Drug Resistance Mutation L76V Alters Nonpolar Interactions at the Flap-Core Interface of HIV-1 Protease.

Four HIV-1 protease (PR) inhibitors, clinical inhibitors lopinavir and tipranavir, and two investigational compounds 4 and 5, were studied for their effect on the structure and activity of PR with drug-resistant mutation L76V (PRL76V). Compound 5 exhibited the best Ki value of 1.9 nM for PRL76V, whereas the other three inhibitors had Ki values of 4.5–7.6 nM, 2–3 orders of magnitude worse than for wild-type enzymes. Crystal structures showed only minor differences in interactions of inhibitors with PRL76V compared to wild-type complexes. The shorter side chain of Val76 in the mutant lost hydrophobic interactions with Lys45 and Ile47 in the flap, and with Asp30 and Thr74 in the protein core, consistent with decreased stability. Inhibitors forming additional polar interactions with the flaps or dimer interface of PRL76V were unable to compensate for the decrease in internal hydrophobic contacts. These structures provide insights for inhibitor design.

Structure-based methods to predict mutational resistance to diarylpyrimidine non-nucleoside reverse transcriptase inhibitors

Resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) is a leading cause of HIV treatment failure. Often included in antiviral therapy, NNRTIs are chemically diverse compounds that bind an allosteric pocket of enzyme target reverse transcriptase (RT). Several new NNRTIs incorporate flexibility in order to compensate for lost interactions with amino acid conferring mutations in RT. Unfortunately, even successful inhibitors such as diarylpyrimidine (DAPY) inhibitor rilpivirine are affected by mutations in RT that confer resistance. In order to aid drug design efforts, it would be efficient and cost effective to pre-evaluate NNRTI compounds in development using a structure-based computational approach. As proof of concept, we applied a residue scan and molecular dynamics strategy using RT crystal structures to predict mutations that confer resistance to DAPYs rilpivirine, etravirine, and investigational microbicide dapivirine. Our predictive values, changes in affinity and stability, are correlative with fold-resistance data for several RT mutants. Consistent with previous studies, mutation K101P is predicted to confer high-level resistance to DAPYs. These findings were further validated using structural analysis, molecular dynamics, and an enzymatic reverse transcription assay. Our results confirm that changes in affinity and stability for mutant complexes are predictive parameters of resistance as validated by experimental and clinical data. In future work, we believe that this computational approach may be useful to predict resistance mutations for inhibitors in development.

Opportunistic attachment assembles plant–pollinator networks

Species and interactions are being lost at alarming rates and it is imperative to understand how communities assemble if we have to prevent their collapse and restore lost interactions. Using an 8-year dataset comprising nearly 20000 pollinator visitation records, we explore the assembly of plant-pollinator communities at native plant restoration sites in an agricultural landscape. We find that species occupy highly dynamic network positions through time, causing the assembly process to be punctuated by major network reorganisations. The most persistent pollinator species are also the most variable in their network positions, contrary to what preferential attachment - the most widely studied theory of ecological network assembly - predicts. Instead, we suggest assembly occurs via an opportunistic attachment process. Our results contribute to our understanding of how communities assembly and how species interactions change through time while helping to inform efforts to reassemble robust communities.

Gene duplication can impart fragility, not robustness, in the yeast protein interaction network.

The maintenance of duplicated genes is thought to protect cells from genetic perturbations, but the molecular basis of this robustness is largely unknown. By measuring the interaction of yeast proteins with their partners in wild-type cells and in cells lacking a paralog, we found that 22 out of 56 paralog pairs compensate for the lost interactions. An equivalent number of pairs exhibit the opposite behavior and require each other's presence for maintaining their interactions. These dependent paralogs generally interact physically, regulate each other's abundance, and derive from ancestral self-interacting proteins. This reveals that gene duplication may actually increase mutational fragility instead of robustness in a large number of cases.

Differences in aggressive behaviour along the expanding range of an invasive crayfish: an important component of invasion dynamics

Aggressive interactions are essential for resource distribution and population dynamics of many animal species. Aggression can also help invasive species to wrestle the resources from other species and invade new habitat. To examine the effects of intra-species aggression on range expansion, we compared aggression levels of signal crayfish (Pacifastacus leniusculus) at the core and front of the invasion range in recently invaded regions of Croatia. More pronounced aggression was observed in core populations with high population densities, indicating potentially greater importance of highly aggressive behaviour in populations with higher competitive pressure. Despite better overall physical condition, individuals at the invasion front generally displayed lower levels of aggression and regularly lost interactions to individuals from the invasion core. Rather than providing a competitive advantage during range expansion, aggression may be more expressed in established populations, priming the individuals for future expansions while also driving the dispersal outward. The observed difference in aggression along the invasion pathway demonstrates that traits that help individuals to overcome challenges of their environment, such as competition against conspecifics, can drive the invasion dynamics of a successful invader in a new environment in terms of both niche competition and intrinsic expansion dynamics.

Impaired Association of Retinal Degeneration-3 with Guanylate Cyclase-1 and Guanylate Cyclase-activating Protein-1 Leads to Leber Congenital Amaurosis-1

One-fifth of all cases of Leber congenital amaurosis are type 1 (LCA1). LCA1 is a severe form of retinal dystrophy caused by loss-of-function mutations in guanylate cyclase 1 (GC1), a key member of the phototransduction cascade involved in modulating the photocurrents. Although GC1 has been studied for some time, the mechanisms responsible for its regulation and membrane targeting are not fully understood. We reported earlier that retinal degeneration 3 (RD3) protein interacts with GC1 and promotes its targeting to the photoreceptor outer segments (POS). Here, we extend our studies to show a direct association between RD3 and guanylate cyclase activating protein 1 (GCAP1). Furthermore, we demonstrate that this functional interaction is important for GC1 targeting to POS. We also show that most LCA1-causing mutations in GC1 result in lost GC1 interaction with RD3 or GC1 being targeted to the plasma membrane. Our data suggest that GC1, GCAP1, and RD3 form a complex in the endoplasmic reticulum that targets GC1 to POS. Interruption of this assembly is likely the underlying mechanism for a subset of LCA1. This study offers insights for the development of therapeutic strategies to treat this severe form of blindness.

Inhibition of hepatitis C viral RNA-dependent RNA polymerase by α-P-boranophosphate nucleotides: Exploring a potential strategy for mechanism-based HCV drug design

Improved treatments for chronic HCV infections remain a challenge, and new chemical strategies are needed to expand the current paradigm. The HCV RNA polymerase (RdRP) has been a target for antiviral development. For the first time we show that the boranophosphate (BP) modification increases the substrate efficiency of ATP analogs into HCV NS5BΔ55 RdRP-catalyzed RNA. Boranophosphate nucleotides contain a borane (BH3) group substituted for a non-bridging phosphoryl oxygen of a normal phosphate group, resulting in a class of modified isoelectronic DNA and RNA mimics capable of modulating the reading and writing of genetic information. We determine that HCV NS5BΔ55, being a stereospecific enzyme, incorporates the Rp isomer of both ATPαB and the two boranophosphate analogs: 2′-O-methyladenosine 5′-(α-P-borano) triphosphate (2′-OMe ATPαB, 5a) and 3′-deoxyadenosine 5′-(α-P-borano) triphosphate (3′-dATPαB, 5b). The Rp diastereomer of ATPαB (6), having no ribose modifications, was found to be a slightly better substrate than natural ATP, showing a 42% decrease in the apparent Michaelis–Menten constant (Km). The IC50 of both 2′-O-Me and 3′-deoxy ATP was decreased with the boranophosphate modification up to 16-fold. This “borano effect” was further confirmed by determining the steady-state inhibitory constant (Ki), showing a comparable potency shift (21-fold). These experiments also indicate that the boranophosphate analogs 5a and 5b inhibit HCV NS5B through a competitive mode of inhibition. This evidence, together with previous crystal structure data, further supports the idea that HCV NS5B (in a similar manner to HIV-1 RT) discriminates against the 3′-deoxy modification via lost interactions between the 3′-OH on the ribose and the active site residues, or lost intramolecular hydrogen bonding interactions between the 3′-OH and the pyrophosphate leaving group during phosphoryl transfer. To our knowledge, these data represent the first time a phosphate modified NTP has been studied as a substrate for HCV NS5B RdRP.

A Lost Link between a Flightless Parrot and a Parasitic Plant and the Potential Role of Coprolites in Conservation Paleobiology

Late Quaternary extinctions and population fragmentations have severely disrupted animal-plant interactions globally. Detection of disrupted interactions often relies on anachronistic plant characteristics, such as spines in the absence of large herbivores or large fruit without dispersers. However, obvious anachronisms are relatively uncommon, and it can be difficult to prove a direct link between the anachronism and a particular faunal taxon. Analysis of coprolites (fossil feces) provides a novel way of exposing lost interactions between animals (depositors) and consumed organisms. We analyzed ancient DNA to show that a coprolite from the South Island of New Zealand was deposited by the rare and threatened kakapo (Strigops habroptilus), a large, nocturnal, flightless parrot. When we analyzed the pollen and spore content of the coprolite, we found pollen from the cryptic root-parasite Dactylanthus taylorii. The relatively high abundance (8.9% of total pollen and spores) of this zoophilous pollen type in the coprolite supports the hypothesis of a former direct feeding interaction between kakapo and D. taylorii. The ranges of both species have contracted substantially since human settlement, and their present distributions no longer overlap. Currently, the lesser short-tailed bat (Mystacina tuberculata) is the only known native pollinator of D. taylorii, but our finding raises the possibility that birds, and other small fauna, could have once fed on and pollinated the plant. If confirmed, through experimental work and observations, this finding may inform conservation of the plant. For example, it may be possible to translocate D. taylorii to predator-free offshore islands that lack bats but have thriving populations of endemic nectar-feeding birds. The study of coprolites of rare or extinct taxonomic groups provides a unique way forward to expand existing knowledge of lost plant and animal interactions and to identify pollination and dispersal syndromes. This approach of linking paleobiology with neoecology offers significant untapped potential to help inform conservation and restoration plans.

Investigation of Catalytic Loop Structure, Dynamics, and Function Relationship ofYersiniaProtein Tyrosine Phosphatase by Temperature-Jump Relaxation Spectroscopy and X-ray Structural Determination

Yersinia protein tyrosine phosphatase (YopH) is the most efficient enzyme among all PTPases. YopH is hyperactive compared to human PTPases, interfering with mammalian cellular pathways to achieve the pathogenicity of Yersinia. Two properties related to the catalytic loop structure differences have been proposed to affect its dynamics and enzyme efficiency. One is the ability of the loop to form stabilizing interactions to bound ligand after loop closure, which has long been recognized. In addition, the loop flexibility/mobility was suggested in a previous study to be a factor as well, based on the observation that incremental changes in PTPase loop structure by single point mutations to alanine often induce incremental changes in enzyme catalytic efficiency. In this study, the temperature jump relaxation spectroscopy (T-jump) has been used to discern the subtle changes of the loop dynamics due to point loop mutations. As expected, our results suggest a correlation between loop dynamics and the size of the residue on the catalytic loop. The stabilization of the enzyme-ligand complex is often enthalpy driven, achieved by formation of additional favorable hydrogen bonding/ionic interactions after loop closure. Interestingly, our T-jump and X-ray crystallography studies on YopH suggest that the elimination of some ligand-protein interactions by mutation does not necessarily destabilize the ligand-enzyme complex after loop closure, since the increased entropy in the forms of more mobile protein residues may be sufficient to compensate the free energy loss due to lost interactions and may even lead to enhanced efficiency of the enzyme catalysis. How these competing loop properties may affect loop dynamics and enzyme function are discussed.

Vpx is Critical for SIVmne infection of pigtail macaques

BackgroundViral protein X (Vpx) of SIV has been reported to be important for establishing infection in vivo. Vpx has several different activities in vitro, promoting preintegration complex import into the nucleus in quiescent lymphocytes and overcoming a block in reverse transcription in macrophages. Vpx interacts with the DDB1-CUL4-DCAF1 E3 ligase complex, which may or may not be required for the ascribed functions. The goal of the current study was to determine whether these activities of Vpx are important in vivo.ResultsAn infectious, pathogenic clone of SIVmne was used to examine correlations between Vpx functions in vitro and in vivo. Three previously described HIV-2 Vpx mutants that were shown to be important for nuclear import of the preintegration complex in quiescent lymphocytes were constructed in SIVmne: A vpx-deleted virus, a truncation of Vpx at amino acid 102 that deletes the C-terminal proline-rich domain (X(102)), and a mutant with tyrosines 66, 69, and 71 changed to alanine (X(y-a)). All mutant viruses replicated similarly to wild type SIVmne027 in primary pigtail macaque PBMCs, and were only slightly retarded in CEMx174 cells. However, all the vpx mutant viruses were defective for replication in both human and pigtail monocyte-derived macrophages. PCR assays demonstrated that the efficiency of reverse transcription and the levels of viral integration in macrophages were substantially reduced for the vpx mutant viruses. In vitro, the X(y-a) mutant, but not the X(102) mutant lost interaction with DCAF1. The wild type SIVmne027 and the three vpx mutant SIVs were inoculated by the intra-rectal route into pigtail macaques. Peak levels of plasma viremia of the vpx mutant SIVs were variable, but consistently lower than that observed in macaques infected with wild type SIVmne. In situ hybridization for SIV demonstrated that compared to wild type SIVmne infected macaques five of the six animals inoculated with the vpx mutant SIVs had only low levels of SIV-expressing cells in the rectum, most intestinal epithelial tissues, spleen, and mesenteric and peripheral nodes.ConclusionsThis work demonstrates that the activities of Vpx to overcome restrictions in culture in vitro are also likely to be important for establishment of infection in vivo and suggest that both the nuclear localization and DCAF1-interaction functions of Vpx are critical in vivo.