Host-pathogen Conflict Research Articles

Inverse correlation between Leishmania-induced TLR1/2 and TGF-β differentially regulates parasite persistence in bone marrow during the chronic phase of infection.

Host-tissue preference is a critical aspect of parasitic infections and is directly correlated with species diversity. Even the same species, Leishmania donovani, infects the host's bone marrow, spleen, and liver differentially. The tissue-specific persistence of Leishmania results from host-pathogen immune conflicts and arguments. The protective pro-host or destructive pro-parasitic role of TLRs during L. donovani infection has been well established, but what entirely missing is the influence of TLRs on tissue-specific parasite persistence. We observed that the parasites induced differential expression of TLR1/2 in the bone marrow but not in the spleen. Interestingly, the rate of Leishmania infection was found to be positively correlated with TLR1/2-mediated upregulation of myelopoietic cytokines, M-CSF, GM-CSF, IL-6, and IL-3, leading to the expansion of Ly6ChiCCR2+ monocytes, however, negatively correlated with the expression of the disease hallmark cytokines, TNF-α, TGF-β, and IL-10, along the course of infection in the bone marrow. Leishmania induced the activation of bone marrow-specific TLR1/2 to promote Ly6ChiCCR2+ monocytes for its safe shelter vis-à-vis infection establishment. Consequently, the established infection initiated the release of TNF-α, TGF-β, and IL-10 in the bone marrow. Post-infection time-kinetic study affirmed that TGF-β had a significant negative influence on the expression of TLR1/2 heterodimer in the bone marrow niche. To the best of our knowledge, this is the first report to show that the inverse correlation of TLR1/2 - TGF-β can be instrumental in tissue-specific parasite persistence during Leishmania infection.

Anti-viral defence by an mRNA ADP-ribosyltransferase that blocks translation

Host–pathogen conflicts are crucibles of molecular innovation1,2. Selection for immunity to pathogens has driven the evolution of sophisticated immunity mechanisms throughout biology, including in bacterial defence against bacteriophages3. Here we characterize the widely distributed anti-phage defence system CmdTAC, which provides robust defence against infection by the T-even family of phages4. Our results support a model in which CmdC detects infection by sensing viral capsid proteins, ultimately leading to the activation of a toxic ADP-ribosyltransferase effector protein, CmdT. We show that newly synthesized capsid protein triggers dissociation of the chaperone CmdC from the CmdTAC complex, leading to destabilization and degradation of the antitoxin CmdA, with consequent liberation of the CmdT ADP-ribosyltransferase. Notably, CmdT does not target a protein, DNA or structured RNA, the known targets of other ADP-ribosyltransferases. Instead, CmdT modifies the N6 position of adenine in GA dinucleotides within single-stranded RNAs, leading to arrest of mRNA translation and inhibition of viral replication. Our work reveals a novel mechanism of anti-viral defence and a previously unknown but broadly distributed class of ADP-ribosyltransferases that target mRNA.

Rapid Evolution of Glycan Recognition Receptors Reveals an Axis of Host-Microbe Arms Races beyond Canonical Protein-Protein Interfaces.

Detection of microbial pathogens is a primary function of many mammalian immune proteins. This is accomplished through the recognition of diverse microbial-produced macromolecules including proteins, nucleic acids, and carbohydrates. Pathogens subvert host defenses by rapidly changing these structures to avoid detection, placing strong selective pressures on host immune proteins that repeatedly adapt to remain effective. Signatures of rapid evolution have been identified in numerous immunity proteins involved in the detection of pathogenic protein substrates, but whether similar signals can be observed in host proteins engaged in interactions with other types of pathogen-derived molecules has received less attention. This focus on protein-protein interfaces has largely obscured the study of fungi as contributors to host-pathogen conflicts, despite their importance as a formidable class of vertebrate pathogens. Here, we provide evidence that mammalian immune receptors involved in the detection of microbial glycans have been subject to recurrent positive selection. We find that rapidly evolving sites in these genes cluster in key functional domains involved in carbohydrate recognition. Further, we identify convergent patterns of substitution and evidence for balancing selection in one particular gene, MelLec, which plays a critical role in controlling invasive fungal disease. Our results also highlight the power of evolutionary analyses to reveal uncharacterized interfaces of host-pathogen conflict by identifying genes, like CLEC12A, with strong signals of positive selection across mammalian lineages. These results suggest that the realm of interfaces shaped by host-microbe conflicts extends beyond the world of host-viral protein-protein interactions and into the world of microbial glycans and fungi.

APEX3 – An Optimized Tool for Rapid and Unbiased Proximity Labeling

Macromolecular interactions regulate all aspects of biology. The identification of interacting partners and complexes is important for understanding cellular processes, host-pathogen conflicts, and organismal development. Multiple methods exist to label and enrich interacting proteins in living cells. Notably, the soybean ascorbate peroxidase, APEX2, rapidly biotinylates adjacent biomolecules in the presence of biotin-phenol and hydrogen peroxide. However, during initial experiments with this system, we found that APEX2 exhibits a cytoplasmic-biased localization and is sensitive to the nuclear export inhibitor leptomycin B (LMB). This led us to identify a putative nuclear export signal (NES) at the carboxy-terminus of APEX2 (NESAPEX2), structurally adjacent to the conserved heme binding site. This putative NES is functional as evidenced by cytoplasmic localization and LMB sensitivity of a mCherry-NESAPEX2 chimeric construct. Single amino acid substitutions of multiple hydrophobic residues within NESAPEX2 eliminate cytoplasm-biased localization of both mCherry-NESAPEX2 as well as full-length APEX2. However, all but one of these NES substitutions also compromises peroxide-dependent labeling. This unique separation-of-function mutant, APEX2-L242A, is termed APEX3. Localization and functionality of APEX3 are confirmed by fusion to the nucleocytoplasmic shuttling transcriptional factor, RELA. APEX3 is therefore an optimized tool for unbiased proximity labeling of cellular proteins and interacting factors..

Lipopolysaccharide -mediated resistance to host antimicrobial peptides and hemocyte-derived reactive-oxygen species are the major Providencia alcalifaciens virulence factors in Drosophila melanogaster.

Bacteria from the genus Providencia are ubiquitous Gram-negative opportunistic pathogens, causing "travelers' diarrhea", urinary tract, and other nosocomial infections in humans. Some Providencia strains have also been isolated as natural pathogens of Drosophila melanogaster. Despite clinical relevance and extensive use in Drosophila immunity research, little is known about Providencia virulence mechanisms and the corresponding insect host defenses. To close this knowledge gap, we investigated the virulence factors of a representative Providencia species-P. alcalifaciens which is highly virulent to fruit flies and amenable to genetic manipulations. We generated a P. alcalifaciens transposon mutant library and performed an unbiased forward genetics screen in vivo for attenuated mutants. Our screen uncovered 23 mutants with reduced virulence. The vast majority of them had disrupted genes linked to lipopolysaccharide (LPS) synthesis or modifications. These LPS mutants were sensitive to cationic antimicrobial peptides (AMPs) in vitro and their virulence was restored in Drosophila mutants lacking most AMPs. Thus, LPS-mediated resistance to host AMPs is one of the virulence strategies of P. alcalifaciens. Another subset of P. alcalifaciens attenuated mutants exhibited increased susceptibility to reactive oxygen species (ROS) in vitro and their virulence was rescued by chemical scavenging of ROS in flies prior to infection. Using genetic analysis, we found that the enzyme Duox specifically in hemocytes is the source of bactericidal ROS targeting P. alcalifaciens. Consistently, the virulence of ROS-sensitive P. alcalifaciens mutants was rescued in flies with Duox knockdown in hemocytes. Therefore, these genes function as virulence factors by helping bacteria to counteract the ROS immune response. Our reciprocal analysis of host-pathogen interactions between D. melanogaster and P. alcalifaciens identified that AMPs and hemocyte-derived ROS are the major defense mechanisms against P. alcalifaciens, while the ability of the pathogen to resist these host immune responses is its major virulence mechanism. Thus, our work revealed a host-pathogen conflict mediated by ROS and AMPs.

Patterns of Coevolutionary Adaptations across Time and Space in Mouse Gammaretroviruses and Three Restrictive Host Factors.

The classical laboratory mouse strains are genetic mosaics of three Mus musculus subspecies that occupy distinct regions of Eurasia. These strains and subspecies carry infectious and endogenous mouse leukemia viruses (MLVs) that can be pathogenic and mutagenic. MLVs evolved in concert with restrictive host factors with some under positive selection, including the XPR1 receptor for xenotropic/polytropic MLVs (X/P-MLVs) and the post-entry restriction factor Fv1. Since positive selection marks host-pathogen genetic conflicts, we examined MLVs for counter-adaptations at sites that interact with XPR1, Fv1, and the CAT1 receptor for ecotropic MLVs (E-MLVs). Results describe different co-adaptive evolutionary paths within the ranges occupied by these virus-infected subspecies. The interface of CAT1, and the otherwise variable E-MLV envelopes, is highly conserved; antiviral protection is afforded by the Fv4 restriction factor. XPR1 and X/P-MLVs variants show coordinate geographic distributions, with receptor critical sites in envelope, under positive selection but with little variation in envelope and XPR1 in mice carrying P-ERVs. The major Fv1 target in the viral capsid is under positive selection, and the distribution of Fv1 alleles is subspecies-correlated. These data document adaptive, spatial and temporal, co-evolutionary trajectories at the critical interfaces of MLVs and the host factors that restrict their replication.

Pathogenic ubiquitination of GSDMB inhibits NK cell bactericidal functions

Gasdermin B (GSDMB) belongs to a large family of pore-forming cytolysins that execute inflammatory cell death programs. While genetic studies have linked GSDMB polymorphisms to human disease, its function in the immunological response to pathogens remains poorly understood. Here, we report a dynamic host-pathogen conflict between GSDMB and the IpaH7.8 effector protein secreted by enteroinvasive Shigella flexneri. We show that IpaH7.8 ubiquitinates and targets GSDMB for 26S proteasome destruction. This virulence strategy protects Shigella from the bacteriocidic activity of natural killer cells by suppressing granzyme-A-mediated activation of GSDMB. In contrast to the canonical function of most gasdermin family members, GSDMB does not inhibit Shigella by lysing host cells. Rather, it exhibits direct microbiocidal activity through recognition of phospholipids found on Gram-negative bacterial membranes. These findings place GSDMB as a central executioner of intracellular bacterial killing and reveal a mechanism employed by pathogens to counteract this host defense system.

Diversification of CD1 Molecules Shapes Lipid Antigen Selectivity.

Molecular studies of host–pathogen evolution have largely focused on the consequences of variation at protein–protein interaction surfaces. The potential for other microbe-associated macromolecules to promote arms race dynamics with host factors remains unclear. The cluster of differentiation 1 (CD1) family of vertebrate cell surface receptors plays a crucial role in adaptive immunity through binding and presentation of lipid antigens to T-cells. Although CD1 proteins present a variety of endogenous and microbial lipids to various T-cell types, they are less diverse within vertebrate populations than the related major histocompatibility complex (MHC) molecules. We discovered that CD1 genes exhibit a high level of divergence between simian primate species, altering predicted lipid-binding properties and T-cell receptor interactions. These findings suggest that lipid–protein conflicts have shaped CD1 genetic variation during primate evolution. Consistent with this hypothesis, multiple primate CD1 family proteins exhibit signatures of repeated positive selection at surfaces impacting antigen presentation, binding pocket morphology, and T-cell receptor accessibility. Using a molecular modeling approach, we observe that interspecies variation as well as single mutations at rapidly-evolving sites in CD1a drastically alter predicted lipid binding and structural features of the T-cell recognition surface. We further show that alterations in both endogenous and microbial lipid-binding affinities influence the ability of CD1a to undergo antigen swapping required for T-cell activation. Together these findings establish lipid–protein interactions as a critical force of host–pathogen conflict and inform potential strategies for lipid-based vaccine development.

Signatures of host-pathogen evolutionary conflict reveal MISTR-A conserved MItochondrial STress Response network.

Host–pathogen conflicts leave genetic signatures in genes that are critical for host defense functions. Using these “molecular scars” as a guide to discover gene functions, we discovered a vertebrate-specific MItochondrial STress Response (MISTR) circuit. MISTR proteins are associated with electron transport chain (ETC) factors and activated by stress signals such as interferon gamma (IFNγ) and hypoxia. Upon stress, ultraconserved microRNAs (miRNAs) down-regulate MISTR1(NDUFA4) followed by replacement with paralogs MItochondrial STress Response AntiViral (MISTRAV) and/or MItochondrial STress Response Hypoxia (MISTRH). While cells lacking MISTR1(NDUFA4) are more sensitive to chemical and viral apoptotic triggers, cells lacking MISTRAV or expressing the squirrelpox virus-encoded vMISTRAV exhibit resistance to the same insults. Rapid evolution signatures across primate genomes for MISTR1(NDUFA4) and MISTRAV indicate recent and ongoing conflicts with pathogens. MISTR homologs are also found in plants, yeasts, a fish virus, and an algal virus indicating ancient origins and suggesting diverse means of altering mitochondrial function under stress. The discovery of MISTR circuitry highlights the use of evolution-guided studies to reveal fundamental biological processes.

Structures of diverse poxin cGAMP nucleases reveal a widespread role for cGAS-STING evasion in host-pathogen conflict.

DNA viruses in the family Poxviridae encode poxin enzymes that degrade the immune second messenger 2'3'-cGAMP to inhibit cGAS-STING immunity in mammalian cells. The closest homologs of poxin exist in the genomes of insect viruses suggesting a key mechanism of cGAS-STING evasion may have evolved outside of mammalian biology. Here we use a biochemical and structural approach to discover a broad family of 369 poxins encoded in diverse viral and animal genomes and define a prominent role for 2'3'-cGAMP cleavage in metazoan host-pathogen conflict. Structures of insect poxins reveal unexpected homology to flavivirus proteases and enable identification of functional self-cleaving poxins in RNA-virus polyproteins. Our data suggest widespread 2'3'-cGAMP signaling in insect antiviral immunity and explain how a family of cGAS-STING evasion enzymes evolved from viral proteases through gain of secondary nuclease activity. Poxin acquisition by poxviruses demonstrates the importance of environmental connections in shaping evolution of mammalian pathogens.

The Hippo Kinase LATS2 Controls Helicobacter pylori-Induced Epithelial-Mesenchymal Transition and Intestinal Metaplasia in Gastric Mucosa.

Background & AimsGastric carcinoma is related mostly to CagA+-Helicobacter pylori infection, which disrupts the gastric mucosa turnover and elicits an epithelial-mesenchymal transition (EMT) and preneoplastic transdifferentiation. The tumor suppressor Hippo pathway controls stem cell homeostasis; its core, constituted by the large tumor suppressor 2 (LATS2) kinase and its substrate Yes-associated protein 1 (YAP1), was investigated in this context.MethodsHippo, EMT, and intestinal metaplasia marker expression were investigated by transcriptomic and immunostaining analyses in human gastric AGS and MKN74 and nongastric immortalized RPE1 and HMLE epithelial cell lines challenged by H pylori, and on gastric tissues of infected patients and mice. LATS2 and YAP1 were silenced using small interfering RNAs. A transcriptional enhanced associated domain (TEAD) reporter assay was used. Cell proliferation and invasion were evaluated.ResultsLATS2 and YAP1 appear co-overexpressed in the infected mucosa, especially in gastritis and intestinal metaplasia. H pylori via CagA stimulates LATS2 and YAP1 in a coordinated biphasic pattern, characterized by an early transient YAP1 nuclear accumulation and stimulated YAP1/TEAD transcription, followed by nuclear LATS2 up-regulation leading to YAP1 phosphorylation and targeting for degradation. LATS2 and YAP1 reciprocally positively regulate each other’s expression. Loss-of-function experiments showed that LATS2 restricts H pylori–induced EMT marker expression, invasion, and intestinal metaplasia, supporting a role of LATS2 in maintaining the epithelial phenotype of gastric cells and constraining H pylori–induced preneoplastic changes.ConclusionsH pylori infection engages a number of signaling cascades that alienate mucosa homeostasis, including the Hippo LATS2/YAP1/TEAD pathway. In the host–pathogen conflict, which generates an inflammatory environment and perturbations of the epithelial turnover and differentiation, Hippo signaling appears as a protective pathway, limiting the loss of gastric epithelial cell identity that precedes gastric carcinoma development.

Post‐translational modifications in effectors and plant proteins involved in host–pathogen conflicts

Molecular interplay between two species is largely driven by protein–protein interactions and protein modifications that set the pace of co‐evolution in these species. During host–pathogen interactions, proteins involved in virulence and defence impart tempospatial dynamic post‐translational modifications (PTMs) to gain advantage for the causative species. Pathogens mainly cause disease in plant hosts by secreting elicitors (peptides and small molecules) or proteins in the inter‐ and intracellular space of host cells. These pathogen proteins have evolved a wide array of sophisticated mechanisms to manipulate host responses, including resistance. Through a set of diverse events ranging from PTMs to post‐translational oligomerization, these proteins are able to enhance virulence and suppress the otherwise elaborate plant immune system. Similarly, PTMs adapted by host proteins often lead to the activation of a robust defence response. Insights into the PTMs of pathogen and host proteins are therefore germane to the understanding of the co‐evolutionary arms race. This review summarizes the characterization of PTMs in pathogen effectors and their target host proteins. Based on this, a metaphorical view of host–pathogen conflicts is proposed, where PTMs act as molecular pivots in a 3D combinatorial game model – a novel abstraction of the arms race, where these molecular pivots restore the balance of competition between the two organisms.

Immune genes are hotspots of shared positive selection across birds and mammals.

Consistent patterns of positive selection in functionally similar genes can suggest a common selective pressure across a group of species. We use alignments of orthologous protein-coding genes from 39 species of birds to estimate parameters related to positive selection for 11,000 genes conserved across birds. We show that functional pathways related to the immune system, recombination, lipid metabolism, and phototransduction are enriched for positively selected genes. By comparing our results with mammalian data, we find a significant enrichment for positively selected genes shared between taxa, and that these shared selected genes are enriched for viral immune pathways. Using pathogen-challenge transcriptome data, we show that genes up-regulated in response to pathogens are also enriched for positively selected genes. Together, our results suggest that pathogens, particularly viruses, consistently target the same genes across divergent clades, and that these genes are hotspots of host-pathogen conflict over deep evolutionary time.

Phosphoserine acidic cluster motifs bind distinct basic regions on the μ subunits of clathrin adaptor protein complexes

Protein trafficking in the endosomal system involves the recognition of specific signals within the cytoplasmic domains (CDs) of transmembrane proteins by clathrin adaptors. One such signal is the phosphoserine acidic cluster (PSAC), the prototype of which is in the endoprotease furin. How PSACs are recognized by clathrin adaptors has been controversial. We reported previously that HIV-1 Vpu, which modulates cellular immunoreceptors, contains a PSAC that binds to the μ subunits of clathrin adaptor protein (AP) complexes. Here, we show that the CD of furin binds the μ subunits of AP-1 and AP-2 in a phosphorylation-dependent manner. Moreover, we identify a potential PSAC in a cytoplasmic loop of the cellular transmembrane Serinc3, an inhibitor of the infectivity of retroviruses. The two serines within the PSAC of Serinc3 are phosphorylated by casein kinase II and mediate interaction with the μ subunits in vitro The sites of these serines vary among mammals in a manner suggesting host-pathogen conflict, yet the Serinc3 PSAC seems dispensable for anti-HIV activity and for counteraction by HIV-1 Nef. The CDs of Vpu and furin and the PSAC-containing loop of Serinc3 each bind the μ subunit of AP-2 (μ2) with similar affinities, but they appear to utilize different basic regions on μ2. The Serinc3 loop requires a region previously reported to bind the acidic plasma membrane lipid phosphatidylinositol 4,5-bisphosphate. These data suggest that the PSACs within different proteins recognize different basic regions on the μ surface, providing the potential to inhibit the activity of viral proteins without necessarily affecting cellular protein trafficking.

Chlorosis caused by two recessively interacting genes reveals a role of RNA helicase in hybrid breakdown in Arabidopsis thaliana.

Hybrids often differ in fitness from their parents. They may be superior, translating into hybrid vigour or heterosis, but they may also be markedly inferior, because of hybrid weakness or incompatibility. The underlying genetic causes for the latter can often be traced back to genes that evolve rapidly because of sexual or host-pathogen conflicts. Hybrid weakness may manifest itself only in later generations, in a phenomenon called hybrid breakdown. We have characterized a case of hybrid breakdown among two Arabidopsis thaliana accessions, Shahdara (Sha, Tajikistan) and Lövvik-5 (Lov-5, Northern Sweden). In addition to chlorosis, a fraction of the F2 plants have defects in leaf and embryo development, and reduced photosynthetic efficiency. Hybrid chlorosis is due to two major-effect loci, of which one, originating from Lov-5, appears to encode an RNA helicase (AtRH18). To examine the role of the chlorosis allele in the Lövvik area, in addition to eight accessions collected in 2009, we collected another 240 accessions from 15 collections sites, including Lövvik, from Northern Sweden in 2015. Genotyping revealed that Lövvik collection site is separated from the rest. Crosses between 109 accessions from this area and Sha revealed 85 cases of hybrid chlorosis, indicating that the chlorosis-causing allele is common in this area. These results suggest that hybrid breakdown alleles not only occur at rapidly evolving loci, but also at genes that code for conserved processes.

The conserved macrodomains of the non-structural proteins of Chikungunya virus and other pathogenic positive strand RNA viruses function as mono-ADP-ribosylhydrolases

Human pathogenic positive single strand RNA ((+)ssRNA) viruses, including Chikungunya virus, pose severe health problems as for many neither efficient vaccines nor therapeutic strategies exist. To interfere with propagation, viral enzymatic activities are considered potential targets. Here we addressed the function of the viral macrodomains, conserved folds of non-structural proteins of many (+)ssRNA viruses. Macrodomains are closely associated with ADP-ribose function and metabolism. ADP-ribosylation is a post-translational modification controlling various cellular processes, including DNA repair, transcription and stress response. We found that the viral macrodomains possess broad hydrolase activity towards mono-ADP-ribosylated substrates of the mono-ADP-ribosyltransferases ARTD7, ARTD8 and ARTD10 (aka PARP15, PARP14 and PARP10, respectively), reverting this post-translational modification both in vitro and in cells. In contrast, the viral macrodomains possess only weak activity towards poly-ADP-ribose chains synthesized by ARTD1 (aka PARP1). Unlike poly-ADP-ribosylglycohydrolase, which hydrolyzes poly-ADP-ribose chains to individual ADP-ribose units but cannot cleave the amino acid side chain - ADP-ribose bond, the different viral macrodomains release poly-ADP-ribose chains with distinct efficiency. Mutational and structural analyses identified key amino acids for hydrolase activity of the Chikungunya viral macrodomain. Moreover, ARTD8 and ARTD10 are induced by innate immune mechanisms, suggesting that the control of mono-ADP-ribosylation is part of a host-pathogen conflict.

The Binding Interface between Human APOBEC3F and HIV-1 Vif Elucidated by Genetic and Computational Approaches

APOBEC3 family DNA cytosine deaminases provide overlapping defenses against pathogen infections. However, most viruses have elaborate evasion mechanisms such as the HIV-1 Vif protein, which subverts cellular CBF-β and a polyubiquitin ligase complex to neutralize these enzymes. Despite advances in APOBEC3 and Vif biology, a full understanding of this direct host-pathogen conflict has been elusive. We combine virus adaptation and computational studies to interrogate the APOBEC3F-Vif interface and build a robust structural model. A recurring compensatory amino acid substitution from adaptation experiments provided an initial docking constraint, and microsecond molecular dynamics simulations optimized interface contacts. Virus infectivity experiments validated a long-lasting electrostatic interaction between APOBEC3F E289 and HIV-1 Vif R15. Taken together with mutagenesis results, we propose a “wobble model” to explain how HIV-1 Vif has evolved to bind different APOBEC3 enzymes and, more generally, explain how pathogens may evolve to escape innate host defenses.

The Binding Interface between Human APOBEC3F and HIV-1 Vif Elucidated by Genetic and Computational Approaches.

APOBEC3 family DNA cytosine deaminases provide overlapping defenses against pathogen infections. However, most viruses have elaborate evasion mechanisms such as the HIV-1 Vif protein, which subverts cellular CBF-β and a polyubiquitin ligase complex to neutralize these enzymes. Despite advances in APOBEC3 and Vif biology, a full understanding of this direct host-pathogen conflict has been elusive. We combine virus adaptation and computational studies to interrogate the APOBEC3F-Vif interface and build a robust structural model. A recurring compensatory amino acid substitution from adaptation experiments provided an initial docking constraint, and microsecond molecular dynamic simulations optimized interface contacts. Virus infectivity experiments validated a long-lasting electrostatic interaction between APOBEC3F E289 and HIV-1 Vif R15. Taken together with mutagenesis results, we propose a wobble model to explain how HIV-1 Vif has evolved to bind different APOBEC3 enzymes and, more generally, how pathogens may evolve to escape innate host defenses.

Rac-Null Leukocytes Are Associated with Increased Inflammation-Mediated Alveolar Bone Loss

Periodontitis is characterized by altered host-biofilm interactions that result in irreversible inflammation-mediated alveolar bone loss. Genetic and epigenetic factors that predispose to ineffective control of biofilm composition and maintenance of tissue homeostasis are not fully understood. We elucidated how leukocytes affect the course of periodontitis in Rac-null mice. Mouse models of acute gingivitis and periodontitis were used to assess the early inflammatory response and patterns of chronicity leading to loss of alveolar bone due to inflammation in Rac-null mice. Leukocyte margination was differentially impaired in these mice during attachment in conditional Rac1-null (granulocyte/monocyte lineage) mice and during rolling and attachment in Rac2-null (all blood cells) mice. Inflammatory responses to subgingival ligatures, assessed by changes in peripheral blood differential leukocyte numbers, were altered in Rac-null compared with wild-type mice. In response to persistent subgingival ligature-mediated challenge, Rac-null mice had increased loss of alveolar bone with patterns of resorption characteristic of aggressive forms of periodontitis. These findings were partially explained by higher osteoclastic coverage of the bone-periodontal ligament interface in Rac-null compared with wild-type mice. In conclusion, this study demonstrates that leukocyte defects, such asdecreased endothelial margination and tissue recruitment, are rate-limiting steps in the periodontal inflammatory process that lead to more aggressive forms of periodontitis.

Evolution of Functional and Sequence Variants of the Mammalian XPR1 Receptor for Mouse Xenotropic Gammaretroviruses and the Human-Derived Retrovirus XMRV

Genetic conflicts between retroviruses and their receptors result in the evolution of novel host entry restrictions and novel virus envelopes, and such variants can influence trans-species transmission. We screened rodents and other mammals for sequence variation in the Xpr1 receptor for the mouse xenotropic or polytropic mouse leukemia viruses (X-MLVs or P-MLVs, respectively) of the gammaretrovirus family and for susceptibility to mouse-derived X/P-MLVs and to XMRV (xenotropic murine leukemia virus-related virus), an X-MLV-like virus isolated from humans with prostate cancer and chronic fatigue syndrome. We identified multiple distinct susceptibility phenotypes; these include the four known Xpr1 variants in Mus and a novel fifth Xpr1 gene found in Mus molossinus and Mus musculus. We describe the geographic and species distribution of the Mus Xpr1 variants but failed to find the X-MLV-restrictive laboratory mouse allele in any wild mouse. We used mutagenesis and phylogenetic analysis to evaluate the functional contributions made by constrained, variable, and deleted residues. Rodent Xpr1 is under positive selection, indicating a history of host-pathogen conflicts; several codons under selection have known roles in virus entry. All non-Mus mammals are susceptible to mouse X-MLVs, but some restrict other members of the X/P-MLV family, and the resistance of hamster and gerbil cells to XMRV indicates that XMRV has unique receptor requirements. We show that the hypervariable fourth extracellular XPR1 loop (ECL4) contains three evolutionarily constrained residues that do not contribute to receptor function, we identify two novel residues important for virus entry (I579 and T583), and we describe a unique pattern of ECL4 variation in the three virus-restrictive Xpr1 variants found in MLV-infected house mice; these mice carry different deletions in ECL4, suggesting either that these sites or loop size affects receptor function.