Coxsackievirus and Adenovirus Receptor Cytoplasmic and Transmembrane Domains Are Not Essential for Coxsackievirus and Adenovirus Infection

Coxsackievirus and adenovirus receptor (CAR) from which the cytoplasmic domain had been deleted and glycosylphosphatidylinositol (GPI)-anchored CAR lacking both transmembrane and cytoplasmic domains were both capable of facilitating adenovirus 5-mediated gene delivery and infection by coxsackievirus B3. These results indicate that the CAR extracellular domain is sufficient to permit virus attachment and entry and that the presence of a GPI anchor does not prevent infection.     

Complex Patterns of Histidine, Hydroxylated Amino Acids and the GxxxG Motif Mediate High-affinity Transmembrane Domain Interactions

Specific interactions of transmembrane helices play a pivotal role in the folding and oligomerization of integral membrane proteins. The helix-helix interfaces frequently depend on specific amino acid patterns. In this study, a heptad repeat pattern was randomized with all naturally occurring amino acids to uncover novel sequence motifs promoting transmembrane domain interactions. Self-interacting transmembrane domains were selected from the resulting combinatorial library by means of the ToxR/POSSYCCAT system. A comparison of the amino acid composition of high-and low-affinity sequences revealed that high-affinity transmembrane domains exhibit position-specific enrichment of histidine. Further, sequences containing His preferentially display Gly, Ser, and/or Thr residues at flanking positions and frequently contain a C-terminal GxxxG motif. Mutational analysis of selected sequences confirmed the importance of these residues in homotypic interaction. Probing heterotypic interaction indicated that His interacts in trans with hydroxylated residues. Reconstruction of minimal interaction motifs within the context of an oligo-Leu sequence confirmed that His is part of a hydrogen bonded cluster that is brought into register by the GxxxG motif. Notably, a similar motif contributes to self-interaction of the BNIP3 transmembrane domain.     

Cytidine Deaminase Motifs within the DYW Domain of Two Pentatricopeptide Repeat-containing Proteins Are Required for Site-specific Chloroplast RNA Editing

In angiosperm organelles, cytidines are converted to uridines by a deamination reaction in the process termed RNA editing. The C targets of editing are recognized by members of the pentatricopeptide repeat (PPR) protein family. Although other members of the editosome have begun to be identified, the enzyme that catalyzes the C-U conversion is still unknown. The DYW motif at the C terminus of many PPR editing factors contains residues conserved with known cytidine deaminase active sites; however, some PPR editing factors lack a DYW motif. Furthermore, in many PPR-DYW editing factors, the truncation of the DYW motif does not affect editing efficiency, so the role of the DYW motif in RNA editing is unclear. Here, a chloroplast PPR-DYW editing factor, quintuple editing factor 1 (QED1), was shown to affect five different plastid editing sites, the greatest number of chloroplast C targets known to be affected by a single PPR protein. Loss of editing at the five sites resulted in stunted growth and accumulation of apparent photodamage. Adding a C-terminal protein tag to QED1 was found to severely inhibit editing function. QED1 and RARE1, another plastid PPR-DYW editing factor, were discovered to require their DYW motifs for efficient editing. To identify specific residues critical for editing, conserved deaminase residues in each PPR protein were mutagenized. The mutant PPR proteins, when expressed in qed1 or rare1 mutant protoplasts, could not complement the editing defect. Therefore, the DYW motif, and specifically, the deaminase residues, of QED1 and RARE1 are required for editing efficiency.     

A Unique Phenylalanine in the Transmembrane Domain Strengthens Homodimerization of the Syndecan-2 Transmembrane Domain and Functionally Regulates Syndecan-2

The syndecans are a type of cell surface adhesion receptor that initiates intracellular signaling events through receptor clustering mediated by their highly conserved transmembrane domains (TMDs). However, the exact function of the syndecan TMD is not yet fully understood. Here, we investigated the specific regulatory role of the syndecan-2 TMD. We found that syndecan-2 mutants in which the TMD had been replaced with that of syndecan-4 were defective in syndecan-2-mediated functions, suggesting that the TMD of syndecan-2 plays one or more specific roles. Interestingly, syndecan-2 has a stronger tendency to form sodium dodecyl sulfate (SDS)-resistant homodimers than syndecan-4. Our structural studies showed that a unique phenylalanine residue (Phe¹⁶⁷) enables an additional molecular interaction between the TMDs of the syndecan-2 homodimer. The presence of Phe¹⁶⁷ was correlated with a higher tendency toward oligomerization, and its replacement with isoleucine significantly reduced the SDS-resistant dimer formation and cellular functions of syndecan-2 (e.g. cell migration). Conversely, replacement of isoleucine with phenylalanine at this position in the syndecan-4 TMD rescued the defects observed in a mutant syndecan-2 harboring the syndecan-4 TMD. Taken together, these data suggest that Phe¹⁶⁷ in the TMD of syndecan-2 endows the protein with specific functions. Our work offers new insights into the signaling mediated by the TMD of syndecan family members.     

Mutational Mapping of UL130 of Human Cytomegalovirus Defines Peptide Motifs within the C-Terminal Third as Essential for Endothelial Cell Infection

The UL130 gene is one of the major determinants of endothelial cell (EC) tropism of human cytomegalovirus (HCMV). In order to define functionally important peptides within this protein, we have performed a charge-cluster-to-alanine (CCTA) mutational scanning of UL130 in the genetic background of a bacterial artificial chromosome-cloned endotheliotropic HCMV strain. A total of 10 charge clusters were defined, and in each of them two or three charged amino acids were replaced with alanines. While the six N-terminal clusters were phenotypically irrelevant, mutation of the four C-terminal clusters each caused a reduction of EC tropism. The importance of this protein domain was further emphasized by the fact that the C-terminal pentapeptide PNLIV was essential for infection of ECs, and the cell tropism could not be rescued by a scrambled version of this sequence. We conclude that the C terminus of the UL130 protein serves an important function for infection of ECs by HCMV. This makes UL130 a promising molecular target for antiviral strategies, e.g., the development of antiviral peptides.Human cytomegalovirus (HCMV) is a widespread betaherpesvirus that causes lifelong persistent infections with occasional reactivations. While HCMV infection is usually clinically unapparent in the immunocompetent host, it can cause severe disseminated infections under conditions of immunosuppression, with manifestations in the lung, retina, and gastrointestinal tract, among others (12). Various cell types support viral replication, including epithelial cells and endothelial cells (ECs), smooth muscle cells, fibroblasts, and cells of hematopoietic origin (13, 14, 18, 19, 25, 26, 37). Among these target cells, endothelial cells are assumed to contribute particularly to hematogenous dissemination of HCMV (24).While recent clinical HCMV isolates are characterized by this broad cell tropism, the target cell range becomes restricted during long-term propagation on fibroblasts (28, 33). The underlying mechanism for this cell culture adaptation is a modulation within the viral genes UL128, UL130, and UL131A (8, 11). These three genes have been shown to be essential for infection of granulocytes, dendritic cells, epithelial cells, and endothelial cells but are dispensable for infection of fibroblasts (1, 9, 11, 34, 35). The encoded proteins pUL128, pUL130, and pUL131A were reported to form a complex with the viral glycoproteins gH and gL that is distinct from the glycoprotein complex gCIII (gH/gL/gO) (35). Whereas poorly endotheliotropic HCMV strains bear just the gH/gL/gO complex in their envelopes, highly endotheliotropic strains bear both gCIII variants: gH/gL/gO and gH/gL/pUL128-131A. Deletion of any of the three genes UL128-131A results in loss of EC tropism (11), most likely because only a complete complex of gH/gL and pUL128, pUL130, and pUL131A can efficiently function in endocytic entry in ECs (21). However, functional sites within the proteins (e.g., mediating binding to the viral complex partners or interaction with a putative cellular receptor) have not yet been identified. One approach to search for candidate protein-protein interaction sites is charge-cluster-to-alanine (CCTA) mutagenesis. This method is based on the assumption that clusters of charged amino acids tend to be exposed in the tertiary structure of a protein and are thus likely to be sites of interaction with other proteins. Replacement of these charged amino acids by uncharged alanines should then target protein-protein interaction sites without destroying the protein backbone (5, 7). Applying this method to HCMV pUL128, we were able to identify a central core region within pUL128 essential for EC infection as well as contributing sites in the N-terminal half and the C terminus of the protein (22). We now aimed to extend the study to the scanning of UL130 by markerless mutagenesis in the context of a highly endotheliotropic HCMV BACmid, TB40-BAC4. The resulting mutant viruses were then characterized with regard to their ability to infect ECs to identify the relevant parts of the protein.With regard to the role of UL130 in EC infection by endocytosis, the C-terminal part of pUL130 was of special interest. A frameshift mutation that changes the last 11 amino acids (aa) of pUL130 is the most prominent difference between the poorly endotheliotropic HCMV strain Towne and the highly endotheliotropic strain HCMV-TB40-BAC4 in this region (8, 11, 27). Rhee and Davis have described a cell-penetrating pentapeptide (CPP) motif (PFVYLI) mediating internalization by endocytosis, which is clathrin and caveolin independent but may involve lipid rafts (17). Not only do the last five amino acids of pUL130 (PNLIV) bear a striking similarity to this motif, but also the entry of HCMV into ECs has been reported to occur by an endocytic pathway (20, 23). Thus, we hypothesized that the pentapeptide motif PNLIV in pUL130 might be involved in mediating endocytic uptake of HCMV in ECs, and if so, deletion of this motif should result in a nonendotheliotropic virus. A number of CPPs that are thought to be taken up by endocytosis have now been described, including VPMLK, PMLKE, VPTLK, KLPVM, and others (32). These CPPs all bear some similarity, but the exact amino acid sequence seems to be irrelevant. We thus hypothesized for UL130 that a scrambled mutant (PNLIV changed to PINVL) should still be able to mediate endocytosis of HCMV in ECs. To test these assumptions we generated a series of mutant viruses where the PNLIV motif was either deleted, scrambled (PNLIV changed to PINVL), or exchanged against a known CPP (PFVYLI [17]) and characterized them with regard to EC infectivity.     

Amino Acids of Conserved Kinase Motifs of Cytomegalovirus Protein UL97 Are Essential for Autophosphorylation

Thirteen point mutations targeting predicted domains conserved in homologous protein kinases were introduced into the UL97 coding region of the human cytomegalovirus. All mutagenized proteins were expressed in cells infected with recombinant vaccinia viruses (rVV). Several mutations drastically reduced ganciclovir (GCV) phosphorylation. Mutations at amino acids G340, A442, L446, and F523 resulted in a complete loss of pUL97 phosphorylation, which was strictly associated with a loss of GCV phosphorylation. Our results confirm that in rVV-infected cells pUL97 phosphorylation is due to autophosphorylation and show that several amino acids conserved within domains of protein kinases are essential for this pUL97 phosphorylation. GCV phosphorylation is dependent on pUL97 phosphorylation.     

The S100A10 Subunit of the Annexin A2 Heterotetramer Facilitates L2-Mediated Human Papillomavirus Infection

Mucosotropic, high-risk human papillomaviruses (HPV) are sexually transmitted viruses that are causally associated with the development of cervical cancer. The most common high-risk genotype, HPV16, is an obligatory intracellular virus that must gain entry into host epithelial cells and deliver its double stranded DNA to the nucleus. HPV capsid proteins play a vital role in these steps. Despite the critical nature of these capsid protein-host cell interactions, the precise cellular components necessary for HPV16 infection of epithelial cells remains unknown. Several neutralizing epitopes have been identified for the HPV16 L2 minor capsid protein that can inhibit infection after initial attachment of the virus to the cell surface, which suggests an L2-specific secondary receptor or cofactor is required for infection, but so far no specific L2-receptor has been identified. Here, we demonstrate that the annexin A2 heterotetramer (A2t) contributes to HPV16 infection and co-immunoprecipitates with HPV16 particles on the surface of epithelial cells in an L2-dependent manner. Inhibiting A2t with an endogenous annexin A2 ligand, secretory leukocyte protease inhibitor (SLPI), or with an annexin A2 antibody significantly reduces HPV16 infection. With electron paramagnetic resonance, we demonstrate that a previously identified neutralizing epitope of L2 (aa 108-120) specifically interacts with the S100A10 subunit of A2t. Additionally, mutation of this L2 region significantly reduces binding to A2t and HPV16 pseudovirus infection. Furthermore, downregulation of A2t with shRNA significantly decreases capsid internalization and infection by HPV16. Taken together, these findings indicate that A2t contributes to HPV16 internalization and infection of epithelial cells and this interaction is dependent on the presence of the L2 minor capsid protein.     

Point Mutations in Dimerization Motifs of the Transmembrane Domain Stabilize Active or Inactive State of the EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L⁵³⁵X₃G⁵³⁹X₂A⁵⁴²X₃V⁵⁴⁶X₂L⁵⁴⁹ rather than through the alternative glycine zipper motif A⁵³⁶X₃G⁵⁴⁰X₃G⁵⁴⁴ (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr⁵⁸⁸ and/or Tyr⁵⁹⁴) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.     

Sequences within the Herpesvirus-Conserved pac1 and pac2 Motifs Are Required for Cleavage and Packaging of the Murine Cytomegalovirus Genome

The DNA sequence motifs pac1 [an A-rich region flanked by poly(C) runs] and pac2 (CGCGGCG near an A-rich region) are conserved near herpesvirus genomic termini and are believed to mediate cleavage of genomes from replicative concatemers. To determine their importance in the cleavage process, we constructed a number of recombinant murine cytomegaloviruses with a second cleavage site inserted at an ectopic location within the viral genome. Cleavage at a wild-type ectopic site occurred as frequently as at the natural cleavage site, whereas mutation of this ectopic site revealed that some of the conserved motifs of pac1 and pac2 were essential for cleavage whereas others were not. Within pac1, the left poly(C) region was very important for cleavage and packaging but the A-rich region was not. Within pac2, the A-rich region and adjacent sequences were essential for cleavage and packaging and the CGCGGCG region contributed to, but was not strictly essential for, efficient cleavage and packaging. A second A-rich region was not important at all. Furthermore, mutations that prevented cleavage also blocked duplication and deletion of the murine cytomegalovirus 30-bp terminal repeat at the ectopic site, suggesting that repeat duplication and deletion are consequences of cleavage. Given that the processes of genome cleavage and packaging appear to be highly conserved among herpesviruses, these findings should be relevant to other members of this family.     

Gln-222 in Transmembrane Domain 4 and Gln-526 in Transmembrane Domain 9 Are Critical for Substrate Recognition in the Yeast High Affinity Glutathione Transporter, Hgt1p

Hgt1p, a member of the oligopeptide transporter family, is a high affinity glutathione transporter from the yeast Saccharomyces cerevisiae. We have explored the role of polar or charged residues in the putative transmembrane domains of Hgt1p to obtain insights into the structural features of Hgt1p that govern its substrate specificity. A total of 22 charged and polar residues in the predicted transmembrane domains and other conserved regions were subjected to alanine mutagenesis. Functional characterization of these 22 mutants identified 11 mutants which exhibited significant loss in functional activity. All 11 mutants except T114A had protein expression levels comparable with wild type, and all except E744A were proficient in trafficking to the cell surface. Kinetic analyses revealed differential contributions toward the functional activity of Hgt1p by these residues and identified Asn-124 in transmembrane domain 1 (TMD1), Gln-222 in TMD4, Gln-526 in TMD9, and Glu-544, Arg-554, and Lys-562 in the intracellular loop region 537–568 containing the highly conserved proline-rich motif to be essential for the transport activity of the protein. Furthermore, mutants Q222A and Q526A exhibited a nearly 4- and 8-fold increase in the K_m for glutathione. Interestingly, although Gln-222 is widely conserved among other functionally characterized oligopeptide transporter family members including those having a different substrate specificity, Gln-526 is present only in Hgt1p and Pgt1, the only two known high affinity glutathione transporters. These results provide the first insights into the substrate recognition residues of a high affinity glutathione transporter and on residues/helices involved in substrate translocation in the structurally uncharacterized oligopeptide transporter family.Hgt1p or ScOpt1p, a polytopic membrane protein, from the yeast Saccharomyces cerevisiae, was the first high affinity glutathione transporter to be identified in any system (1). Hgt1p belongs to a relatively novel family of transporters, the oligopeptide transporter (OPT)³ family, that contains a large number of fungal, plant, and prokaryotic members (2). The functional characterizations of a few of the fungal and plants members have demonstrated their ability to transport oligopeptides, glutathione, and metal-secondary amino acid conjugates by harnessing the proton gradient across the plasma membrane (3–7). Furthermore, these studies have also highlighted the physiological significance of this family in assimilation/mobilization of oligopeptides as nutrients in fungi and plants and in maintenance of metal homeostasis in plants. However, the majority of the members are yet uncharacterized and need to be defined with respect to their substrate specificity and physiological role.A complete lack of information on the structural features of the OPT family further limits our understanding of this large, uncharacterized family. Identification of residues or motifs critical for substrate recognition among the functionally characterized members would enable functional characterization of the new members within the family. This has prompted us to initiate a systematic study on the structure-function characterization of Hgt1p as a representative of the OPT family. Not only is Hgt1p the best characterized member of the OPT family in terms of its substrate specificity, being also able to transport some oligopeptides albeit with low affinity (1, 7, 8), its native host S. cerevisiae is a well established model system and easily amendable for mutagenesis-based structure-function studies. We have recently investigated the role of the 12 native cysteine residues in the structural stability and the transporter activity of the protein where 2 of the cysteines were found to be essential for functionality (9). However, no hints on the important motifs or conserved amino acids of Hgt1p (or any other member of the OPT family) that could be involved in substrate recognition have been obtained so far. In the current study we have focused on the polar and charged residues in the transmembrane domains of Hgt1p to explore their role in substrate recognition.Glutathione, the substrate for Hgt1p, is a hydrophilic substrate. Prior studies on structural characterization of transporters of the other hydrophilic substrates using biochemical and genetic strategies, such as site-directed mutagenesis and random mutagenesis, have established the role of polar and charged residues in the transmembrane domains of transporters in recognition, binding, and translocation of substrates (10–18). The availability of the crystal structures of a few transporter proteins have further enabled direct visualization of such interactions between the key residues in the transmembrane domains and the substrate molecule (19–22). In light of these studies we anticipated that few of the charged or polar residues in the predicted transmembrane domains of Hgt1p would be involved in substrate recognition and translocation across the membrane. Hence, a total of 22 polar or charged amino acids spanning the predicted transmembrane domains of Hgt1p were subjected to alanine scanning and functionally characterized. Detailed biochemical characterizations of these mutants revealed that Asn-124, Gln-222, Gln-526, Glu-544, Arg-554, and Lys-562 are key residues for the transport activity of Hgt1p. As replacement of Gln-222 in TMD4 and Gln-526 in TMD9 with alanine resulted in a significant decrease in the affinity of the transporter for glutathione, it suggested that the two residues might directly participate in glutathione recognition as a substrate. These observations provide the first insights into substrate binding residues in Hgt1p, a member of a novel and important transporter family (OPT family).     

Protein Disorder and Short Conserved Motifs in Disordered Regions Are Enriched near the Cytoplasmic Side of Single-Pass Transmembrane Proteins

Intracellular juxtamembrane regions of transmembrane proteins play pivotal roles in cell signalling, mediated by protein-protein interactions. Disordered protein regions, and short conserved motifs within them, are emerging as key determinants of many such interactions. Here, we investigated whether disorder and conserved motifs are enriched in the juxtamembrane area of human single-pass transmembrane proteins. Conserved motifs were defined as short disordered regions that were much more conserved than the adjacent disordered residues. Human single-pass proteins had higher mean disorder in their cytoplasmic segments than their extracellular parts. Some, but not all, of this effect reflected the shorter length of the cytoplasmic tail. A peak of cytoplasmic disorder was seen at around 30 residues from the membrane. We noted a significant increase in the incidence of conserved motifs within the disordered regions at the same location, even after correcting for the extent of disorder. We conclude that elevated disorder within the cytoplasmic tail of many transmembrane proteins is likely to be associated with enrichment for signalling interactions mediated by conserved short motifs.     

GuaA and GuaB Are Essential for Borrelia burgdorferi Survival in the Tick-Mouse Infection Cycle

Pathogens lacking the enzymatic pathways for de novo purine biosynthesis are required to salvage purines and pyrimidines from the host environment for synthesis of DNA and RNA. Two key enzymes in purine salvage pathways are IMP dehydrogenase (GuaB) and GMP synthase (GuaA), encoded by the guaB and guaA genes, respectively. While these genes are typically found on the chromosome in most bacterial pathogens, the guaAB operon of Borrelia burgdorferi is present on plasmid cp26, which also harbors a number of genes critical for B. burgdorferi viability. Using molecular genetics and an experimental model of the tick-mouse infection cycle, we demonstrate that the enzymatic activities encoded by the guaAB operon are essential for B. burgdorferi mouse infectivity and provide a growth advantage to spirochetes in the tick. These data indicate that the GuaA and GuaB proteins are critical for the survival of B. burgdorferi in the infection cycle and highlight a potential difference in the requirements for purine salvage in the disparate mammalian and tick environments.Purine metabolism is critical for the growth and virulence in mammals of many bacterial pathogens (11, 26, 29, 33, 51). Borrelia burgdorferi, the infectious agent of Lyme borreliosis, lacks the genes encoding the enzymes required for de novo nucleotide synthesis (8, 12) and therefore must rely on salvage of purines and pyrimidines from its hosts for nucleic acid biosynthesis (21, 35). Furthermore, B. burgdorferi lacks the genes encoding key enzymes required for a classic purine salvage pathway, including hpt (hypoxanthine-guanine phosphoribosyltransferase), purA (adenylosuccinate synthase), purB (adenylosuccinate lyase), and the locus encoding a ribonucleotide reductase (4, 8, 12, 35, 66). Despite the absence of a ribonucleotide reductase, an enzyme critical for the generation of deoxynucleotides through enzymatic reduction of ribonucleotides (32), a novel purine salvage pathway that involves salvage of deoxynucleosides from the host and interconversion of purine bases to deoxynucleosides by BB0426, a deoxyribosyl transferase, has recently been demonstrated for B. burgdorferi (23) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Pivotal role of the GuaAB proteins in the purine salvage pathway of B. burgdorferi. A novel pathway for purine salvage has recently been elucidated for B. burgdorferi (23). Extracellular adenine and hypoxanthine are salvaged by B. burgdorferi from mammalian and tick host environments (61). Following transport, adenine can be converted to hypoxanthine by adenine deaminase (BBK17) (21). This pathway proposes two possible fates for hypoxanthine, as follows. (i) Hypoxanthine is converted to IMP by a putative xanthine-guanine phosphoribosyl transferase (BB0103), IMP is converted to XMP by IMPDH (GuaB or BBB17), and XMP is converted to GMP by GMP synthase (GuaA or BBB18), resulting in guanine nucleotides for RNA synthesis. (ii) Direct transport of deoxynucleosides appears to provide a source of deoxyribose for interconversion of hypoxanthine to deoxyinosine by a deoxyribosyl transferase (BB0426) (23). dIMP is generated by a putative deoxynucleotide kinase (BB0239). GuaB converts dIMP to dXMP, and GuaA converts dXMP to dGMP, providing guanine deoxynucleotides for DNA synthesis (23). Salvage of free guanine nucleosides and guanine deoxynucleosides, when they are available in the host environment, may allow B. burgdorferi to circumvent the GuaAB requirement for GMP and dGMP biosynthesis. The dashed arrows indicate dephosphorylation of nucleotide monophosphate or deoxynucleotide monophosphate prior to transport by the spirochete and rephosphorylation of nucleoside and deoxynucleoside to nucleotide triphosphate and deoxynucleotide triphosphate, respectively, for RNA and DNA synthesis. NMP, nucleotide monophosphate; N, nucleoside; dN, deoxynucleoside; dNMP, deoxynucleotide monophosphate; OM, outer membrane; IM, inner membrane.In its infection cycle, B. burgdorferi passages between two disparate environments with potentially distinct purine availabilities, the tick vector and a mammalian host. Hypoxanthine is the most abundant purine in mammalian blood (17), and it is available for salvage by B. burgdorferi during the blood meal of an infected tick and during the spirochete''s transient presence in the mammalian bloodstream. Despite the absence of the hpt gene, we and others have shown that B. burgdorferi is able to transport and incorporate low levels of hypoxanthine (23, 35). During mammalian infection B. burgdorferi resides in various tissues, including the skin, heart, bladder, and joints. Adenine has been shown to be ubiquitous in mammalian tissues (61) and therefore is available for salvage by B. burgdorferi. Guanine is present at low levels in mammalian blood and tissues (17, 61); however, the amount may not be sufficient for survival of the spirochete.The limiting step in guanine nucleotide biosynthesis from adenine and hypoxanthine is the conversion of IMP to XMP, which is catalyzed by IMP dehydrogenase (IMPDH) (65). Guanine nucleotides are essential for DNA and RNA synthesis, signal transduction, and cell cycle control; thus, IMPDH activity is critical for the survival of most organisms (60). The enzymes required for the final two steps of guanine nucleotide biosynthesis, IMPDH and GMP synthase, are encoded by the guaB and guaA genes, respectively. The guaA and guaB genes and the corresponding activities of their protein products are conserved in B. burgdorferi (28, 67). These genes are typically carried on the chromosomes of bacterial species. However, in B. burgdorferi, the guaAB operon resides on a 26-kbp circular plasmid, cp26, and it shares an approximately 185-bp intergenic region with, and is transcribed divergently from, the essential virulence gene ospC (8, 12, 28, 50, 54, 62). The cp26 plasmid has been shown to harbor numerous genes important for B. burgdorferi survival in vivo and in vitro, including ospC (16, 34, 50, 53, 56) and resT (7), as well as BBB26 and BBB27 (20). Because of these critical functions, this plasmid is the only plasmid of the approximately 21 B. burgdorferi plasmids that is present in all natural isolates and has never been shown to be lost during in vitro culture (2, 7, 18, 20, 44, 52).Here we establish that the enzymatic activities of GuaA and GuaB are critical for the survival of B. burgdorferi in the infectious cycle and highlight a potential difference in this spirochete''s requirement for purine salvage in the disparate mammalian and tick environments.     

A Conserved Basic Patch and Central Kink in the Nipah Virus Phosphoprotein Multimerization Domain Are Essential for Polymerase Function

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A Novel Assay for Assessing Juxtamembrane and Transmembrane Domain Interactions Important for Receptor Heterodimerization

Understanding the basis of specificity in receptor homodimerization versus heterodimerization is essential in determining the role receptor plays in signal transduction. Specificity in each of the interfaces formed during signal transduction involves cooperative interactions between receptor extracellular, transmembrane (TM), and cytoplasmic domains. While methods exist for studying receptor heterodimerization in cell membranes, they are limited to either TM domains expressed in an inverted orientation or capture only heterodimerization in a single assay. To address this limitation, we have developed an assay (DN-AraTM) that enables simultaneous measurement of homodimerization and heterodimerization of type I receptor domains in their native orientation, including both soluble and TM domains. Using integrin α_IIb and RAGE (receptor for advanced glycation end products) as model type I receptor systems, we demonstrate both specificity and sensitivity of our approach, which will provide a novel tool to identify specific domain interactions that are important in regulating signal transduction.     

Nod Factor Receptors Form Heteromeric Complexes and Are Essential for Intracellular Infection in Medicago Nodules

Rhizobial Nod factors are the key signaling molecules in the legume-rhizobium nodule symbiosis. In this study, the role of the Nod factor receptors NOD FACTOR PERCEPTION (NFP) and LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) in establishing the symbiotic interface in root nodules was investigated. It was found that inside Medicago truncatula nodules, NFP and LYK3 localize at the cell periphery in a narrow zone of about two cell layers at the nodule apex. This restricted accumulation is narrower than the region of promoter activity/mRNA accumulation and might serve to prevent the induction of defense-like responses and/or to restrict the rhizobium release to precise cell layers. The distal cell layer where the receptors accumulate at the cell periphery is part of the meristem, and the proximal layer is part of the infection zone. In these layers, the receptors can most likely perceive the bacterial Nod factors to regulate the formation of symbiotic interface. Furthermore, our Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicates that NFP and LYK3 form heteromeric complexes at the cell periphery in M. truncatula nodules.     

Human Papillomavirus Infection and Fertility Alteration: A Systematic Review

BackgroundHPV is the most prevalent sexually transmitted infection and its effect in cancer induction is well documented. HPV infections are mostly asymptomatic, but it is unclear whether HPV infections can result in alterations of reproductive health.ObjectiveTo determine the relationship between human papillomavirus infections and reproductive health in both men and women.MethodsA systematic literature review was performed in PubMed and ScienceDirect data bases from January 1994 through August 2014.ResultsHPV infections are shown to be significantly associated to many adverse effects in the reproductive function. These adverse effects were reported in different levels from cells production to pregnancy and may be related to the infecting genotype.ConclusionsIt appears from this study that HPV detection and genotyping could be of great value in infertility diagnosis at least in idiopathic infertility cases. Like for the risk of carcinogenesis, another classification of HPV regarding the risk of fertility alteration may be considered after deep investigations.