Hemagglutinin Tag Research Articles

Activation of soluble AC10 induces cytoskeletal reorganisation

The cellular monolayer of the pulmonary endothelium forms a semi-selective barrier intersecting the vascular space, the underlying interstitium, and adjacent airspace. This endothelial barrier maintains tissue homeostasis critically regulating vessel permeability. Endothelial cAMP signals regulate endothelial permeability; however, cAMP signals are highly compartmentalized, such that cAMP signals generated by transmembrane adenylyl cyclase protect the barrier, while cAMP signals generated by soluble AC disrupt the barrier. Indeed, bicarbonate stimulation of soluble AC10 disrupts the endothelial barrier increasing permeability and is required for LPS-induced lung edema. Yet, how AC10 cAMP signals coordinate endothelial barrier regulation is unclear. The endothelial cytoskeleton alters cell shape which in turn regulates endothelial permeability. Thus, we hypothesized that bicarbonate stimulation of AC10 induces cytoskeletal remodeling. PMVECs were engineered to overexpress AC10 fused to a C-terminus hemagglutinin tag (HA). Gene and protein expression were controlled using a two-tier gene expression approach in which the doxycycline inducible promoter couples with a protein destabilizing variant of FKBP12 fused to the AC10-HA construct rendering the protein unstable in the absence of Shield1. In the presence of doxycycline and Shield1, AC10-HA protein began to appear 4-hours after induction and was absent in control cells. In this system, following addition of doxycycline and Shield1 and in the presence of elevated bicarbonate, whole cell cAMP increased over the same time course as protein expression while control cells showed no additional increase above normal bicarbonate stimulation. As cAMP increases above controls, endothelial cells begin to retract inducing inter-endothelial gaps between adjacent cells. This increase in inter-endothelial gaps was consistent with decreased resistance across the endothelial monolayer. To further address cytoskeletal mediated changes during AC10-induced endothelial barrier disruption, we examined actin cytoskeleton dynamics. Significant loss of the cortical actin rim and formation of actin stress fibers progress to complete actin cytoskeletal disruption by 24 hours. Interestingly, over the same course as the restructuring of the actin cytoskeleton, we detected phosphorylation of actin binding proteins, filamin and VASP, at PKA mediated sites. Thus, it appears that AC10 mediated pulmonary endothelial barrier disruption is coupled to actin remodeling.

Protein Levels of Several Arabidopsis Auxin Response Factors Are Regulated by Multiple Factors and ABA Promotes ARF6 Protein Ubiquitination.

The auxin response factor (ARF) transcription factors are a key component in auxin signaling and play diverse functions in plant growth, development, and stress response. ARFs are regulated at the transcript level and posttranslationally by protein modifications. However, relatively little is known regarding the control of ARF protein levels. We expressed five different ARFs with an HA (hemagglutinin) tag and observed that their protein levels under the same promoter varied considerably. Interestingly, their protein levels were affected by several hormonal and environmental conditions, but not by the auxin treatment. ABA (abscisic acid) as well as 4 °C and salt treatments decreased the levels of HA-ARF5, HA-ARF6, and HA-ARF10, but not that of HA-ARF19, while 37 °C treatment increased the levels of the four HA-ARFs, suggesting that the ARF protein levels are regulated by multiple factors. Furthermore, MG132 inhibited the reduction of HA-ARF6 level by ABA and 4 °C treatments, suggesting that these treatments decrease HA-ARF6 level through 26S proteasome-mediated protein degradation. It was also found that ABA treatment drastically increased HA-ARF6 ubiquitination, without strongly affecting the ubiquitination profile of the total proteins. Together, these results reveal another layer of control on ARFs, which could serve to integrate multiple hormonal and environmental signals into the ARF-regulated gene expression.

Characterization of HA-tagged \u03b19 and \u03b110 nAChRs in the mouse cochlea

Neurons of the medial olivary complex inhibit cochlear hair cells through the activation of α9α10-containing nicotinic acetylcholine receptors (nAChRs). Efforts to study the localization of these proteins have been hampered by the absence of reliable antibodies. To overcome this obstacle, CRISPR-Cas9 gene editing was used to generate mice in which a hemagglutinin tag (HA) was attached to the C-terminus of either α9 or α10 proteins. Immunodetection of the HA tag on either subunit in the organ of Corti of adult mice revealed immunopuncta clustered at the synaptic pole of outer hair cells. These puncta were juxtaposed to immunolabeled presynaptic efferent terminals. HA immunopuncta also occurred in inner hair cells of pre-hearing (P7) but not in adult mice. These immunolabeling patterns were similar for both homozygous and heterozygous mice. All HA-tagged genotypes had auditory brainstem responses not significantly different from those of wild type littermates. The activation of efferent neurons in heterozygous mice evoked biphasic postsynaptic currents not significantly different from those of wild type hair cells. However, efferent synaptic responses were significantly smaller and less frequent in the homozygous mice. We show that HA-tagged nAChRs introduced in the mouse by a CRISPR knock-in are regulated and expressed like the native protein, and in the heterozygous condition mediate normal synaptic function. The animals thus generated have clear advantages for localization studies.

MUSCLE FUNCTION & HOMEOSTASIS / MOLECULAR THERAPEUTIC APPROACHES: P.69 Generation and characterization of Pax7-HA knock-in mice

Satellite cells are residential muscle stem cells that express a paired box transcription factor PAX7. Pax7 has been shown to be involved in determination of myogenic cell lineage during development, yet it is also required for the postnatal muscle growth, muscle regeneration and maintenance of the satellite cell pool. Although the functions of Pax7 have been studied thoroughly, its interacting partners in myogenic cells in vivo are largely unknown due to the lack of proper antibodies for immunoprecipitation. To solve this problem, we generated knock-in mice expressing human influenza hemagglutinin (HA) tags at the C-terminus of Pax7 by using the CRISPR/Cas9 system. Homozygous Pax7-HA knock-in mice (Pax7-HA/HA) showed normal viability, growth, and fertility. The HA expression in Pax7-HA knock-in embryos clearly recapitulated the endogenous expression of PAX7 and HA protein were colocalized with Pax7 protein in satellite cells in adult muscles. In addition, muscle regeneration following injury was also normal. Isolated Pax7-HA/HA satellite cells proliferated and differentiated as efficiently as those from wild type mice. By immunoprecipitation with the anti-HA antibody coupled with mass spectrometry, we identified several hundred candidates of Pax7-interacting proteins during muscle development. Taken together, our Pax7-HA knock-in mouse line is a useful tool to uncover unknown Pax7 functions in muscle progenitor cells during development and also in satellite cells in adult skeletal muscles.

Recombinant bluetongue virus with hemagglutinin epitopes in VP2 has potential as a labeled vaccine

Bluetongue (BT) is an arbovirus-borne disease of ruminants caused by bluetongue virus (BTV) that has the potential to have a serious economic impact. Currently available commercial vaccines include attenuated vaccines and inactivated vaccines, both of which have achieved great success in the prevention and control of BTV. However, these vaccines cannot distinguish between infected animals and immunized animals. To control outbreaks of BTV, the development of labeled vaccines is urgently needed. In this study, we used the plasmid-based reverse genetics system (RGS) of BTV to rescue four recombinant viruses in which HA (influenza hemagglutinin) tags were inserted at different sites of VP2. In vitro, the recombinant tagged viruses exhibited morphologies, plaque, and growth kinetics similar to the parental BTV-16, and expressed both VP2 and HA tag. Subsequently, the selected recombinant tagged viruses were prepared as inactivated vaccines to immunize IFNAR(−/−) mice and sheep, and serological detection results of anti-HA antibody provided discriminative detection. In summary, we used plasmid-based RGS to rescue BTV recombinant viruses with HA tags inserted into VP2, and detected several sites on VP2 that can accommodate HA tags. Some of the recombinant tagged viruses have potential to be developed into distinctive inactivated vaccines.

Protein Kinase A‐mediated Regulation of SLC5A8 Surface Expression

SLC5A8 (solute carrier gene family 5A, member 8) encodes the sodium monocarboxylate transporter, SMCT1. SMCT1 mediates cotransport of sodium and monocarboxylates, such as short chain fatty acids, in many tissues, including thyroid, kidney, and colon. Inspection of SMCT1’s primary amino acid sequence identifies not only a carboxyl terminus post‐synaptic density/discs large/zona occuldens 1 (PDZ) binding motif postulated to be important in macromolecular transport protein complexing, but also consensus sites for phosphorylation by multiple classes of kinases. Notably, SMCT1 contains a single protein kinase A (PKA) consensus site: RRMT (amino acids 50–53), with T53 potentially functioning as the site of phosphorylation. The present study considers the hypothesis that PKA regulates SMCT1 surface expression, as reported for the sodium‐glucose cotransporter (SGLT1, the product of the SLC5A1 gene). The following experiments test whether elimination of the phosphorylation site disrupts PKA‐mediated regulation of surface expression. Using site‐directed mutagenesis, we disrupted the consensus motif and concomitantly disabled phosphorylation by replacing T53 with an alanine residue (T53A). To facilitate downstream assays, we generated T53A on the background of a human SMCT1 expression construct containing an amino‐terminus, extracellular hemagglutinin (HA) tag. In transiently expressing COS‐7 cells, surface luminescence measurements confirmed T53A resides in the cell surface at levels similar to wild‐type SMCT1, and immunoblot measurements indicate similar total expression levels. Wild‐type SMCT1‐expressing cells displayed increased luminescence signal (~20%) subsequent to forskolin (10 μM) treatment to stimulate adenylate cyclase and activate PKA, consistent with augmented surface expression. In contrast, cells expressing T53A were indifferent to forskolin treatment. As an independent measure of surface expression, plans are underway to perform cell‐surface immunoprecipitation (CS‐IP) experiments. Taken together, these data suggest that, although constitutive plasma membrane residency does not depend on an intact PKA consensus site, forskolin‐activated, PKA‐dependent phosphorylation can increase SMCT1 levels in the plasma membrane, and thereby augment potential transport capacity.Support or Funding InformationNIH/NIGMS Award 1R15GM101674‐01A1 (to PF)

Induction of Effective Immunity against Trypanosoma cruzi.

Chagas disease, caused by Trypanosoma cruzi, is a major public health issue. Limitations in immune responses to natural T. cruzi infection usually result in parasite persistence with significant complications. A safe, effective, and reliable vaccine would reduce the threat of T. cruzi infections; however, no suitable vaccine is currently available due to a lack of understanding of the requirements for induction of fully protective immunity. We established a T. cruzi strain expressing green fluorescent protein (GFP) under the control of dihydrofolate reductase degradation domain (DDD) with a hemagglutinin (HA) tag, GFP-DDDHA, which was induced by trimethoprim-lactate (TMP-lactate), which results in the death of intracellular parasites. This attenuated strain induces very strong protection against reinfection. Using this GFP-DDDHA strain, we investigated the mechanisms underlying the protective immune response in mice. Immunization with this strain led to a response that included high levels of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α), as well as a rapid expansion of effector and memory T cells in the spleen. More CD8+ T cells differentiate to memory cells following GFP-DDDHA infection than after infection with a wild-type (WT) strain. The GFP-DDDHA strain also provides cross-protection against another T. cruzi isolate. IFN-γ is important in mediating the protection, as IFN-γ knockout (KO) mice failed to acquire protection when infected with the GFP-DDDHA strain. Immune cells demonstrated earlier and stronger protective responses in immunized mice after reinfection with T. cruzi than those in naive mice. Adoptive transfers with several types of immune cells or with serum revealed that several branches of the immune system mediated protection. A combination of serum and natural killer cells provided the most effective protection against infection in these transfer experiments.

Visualization of Endogenous Type I TGF-\u03b2 Receptor Baboon in the Drosophila Brain

The transforming growth factor β (TGF-β) signaling pathway is evolutionarily conserved and widely used in the animal kingdom to regulate diverse developmental processes. Prior studies have shown that Baboon (Babo), a Drosophila type I TGF-β receptor, plays essential roles in brain development and neural circuit formation. However, the expression pattern for Babo in the developing brain has not been previously reported. We generated a knock-in fly with a human influenza hemagglutinin (HA) tag at the C-terminus of Babo and assessed its localization. Babo::HA was primarily expressed in brain structures enriched with neurites, including the mushroom body lobe and neuropils of the optic lobe, where Babo has been shown to instruct neuronal morphogenesis. Since the babo 3' untranslated region contains a predicted microRNA-34 (miR-34) target sequence, we further tested whether Babo::HA expression was affected by modulating the level of miR-34. We found that Babo was upregulated by mir-34 deletion and downregulated by miR-34 overexpression, confirming that it is indeed a miR-34 target gene. Taken together, our results demonstrate that the baboHA fly permits accurate visualization of endogenous Babo expression during brain development and the construction of functional neural circuits.

PP2AC Phospho-Tyr307 Antibodies Are Not Specific for this Modification but Are Sensitive to Other PP2AC Modifications Including Leu309 Methylation

Protein phosphatase 2A (PP2A) is an important regulator of signal transduction pathways and a tumor suppressor. Phosphorylation of the PP2A catalytic subunit (PP2AC) at tyrosine 307 has been claimed to inactivate PP2A and was examined in more than 180 studies using commercial antibodies, but this modification was never identified using mass spectrometry. Here we show that the most cited pTyr307 monoclonal antibodies, E155 and F-8, are not specific for phosphorylated Tyr307 but instead are hampered by PP2AC methylation at leucine 309 or phosphorylation at threonine 304. Other pTyr307 antibodies are sensitive to PP2AC methylation as well, and some cross-react with pTyr residues in general, including phosphorylated hemagglutinin tags. We identify pTyr307 using targeted mass spectrometry after transient overexpression of PP2AC and Src kinase. Yet under such conditions, none of the tested antibodies show exclusive pTyr307 specificity. Thus, data generated using these antibodies need to be revisited, and the mechanism of PP2A inactivation needs to be redefined.

Rapid Single-Step Affinity Purification of HA-Tagged Plant Mitochondria.

Photosynthesis in plant cells would not be possible without the supportive role of mitochondria. However, isolating mitochondria from plant cells for physiological and biochemical analyses is a lengthy and tedious process. Established isolation protocols require multiple centrifugation steps and substantial amounts of starting material. To overcome these limitations, we tagged mitochondria in Arabidopsis (Arabidopsis thaliana) with a triple hemagglutinin tag for rapid purification via a single affinity-purification step. This protocol yields a substantial quantity of highly pure mitochondria from 1 g of Arabidopsis seedlings. The purified mitochondria were suitable for enzyme activity analyses and yielded sufficient amounts of proteins for deep proteomic profiling. We applied this method for the proteomic analysis of the Arabidopsis bou-2 mutant deficient in the mitochondrial Glu transporter À BOUT DE SOUFFLE (BOU) and identified 27 differentially expressed mitochondrial proteins compared with tagged Col-0 controls. Our work sets the stage for the development of advanced mitochondria isolation protocols for distinct cell types.

High yield recombinant expression and purification of oncogenic NSD1, NSD2, and NSD3 with human influenza hemagglutinin tag

The nuclear receptor-binding SET Domain (NSD) family consists of NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1 histone methyltransferases that are crucial for chromatin remodeling. NSDs are implicated in developmental disorders such as Wolf-Hirschhorn and Sotos syndromes as well as various cancers including t(4; 14)(p16; q32) myeloma, an incurable cancer in plasma cells. NSDs have been the target of intensive study to understand their biological functions more fully and inform anti-cancer drug design. Recombinant protein expression and purification of human NSDs using an E. coli expression system are notoriously challenging, but the production of pure, stable, and active NSDs is essential for further studies. To overcome production challenges, we propose a cost-efficient approach optimized to produce a high yield of NSDs using a modified E. coli expression system. We found that tagging the NSDs with a human influenza hemagglutinin (HA) tag greatly improved the quality of the recombinant NSDs, resulting in more than 95% pure, stable, and active NSD-HAs, with an increase in production yield up to 22.4-fold and up to 6.25 mg/L from LB E. coli culture, and without further purification such as ion-exchange or size-exclusion chromatography.

Ultrastructural localization of DREADDs in monkeys.

Designer receptors exclusively activated by designer drugs (DREADDs) are extensively used to modulate neuronal activity in rodents, but their use in primates remains limited. An essential need that remains is the demonstration that DREADDs are efficiently expressed on the plasma membrane of primate neurons. To address this issue, electron microscopy immunogold was used to determine the subcellular localization of the AAV vector-induced DREADDs hM4Di and hM3Dq fused to different tags in various brain areas of rhesus monkeys and mice. When hM4Di was fused to mCherry, the immunogold labelling was mostly confined to the intracellular space, and poorly expressed at the plasma membrane in monkey dendrites. In contrast, the hM4Di-mCherry labelling was mostly localized to the dendritic plasma membrane in mouse neurons, suggesting species differences in the plasma membrane expression of these exogenous proteins. The lack of hM4Di plasma membrane expression may limit the functional effects of systemic administration of DREADD-actuators in monkey neurons. Removing the mCherry and fusing of hM4Di with the haemagglutinin (HA) tag resulted in strong neuronal plasma membrane immunogold labelling in both monkeys and mice neurons. Finally, hM3Dq-mCherry was expressed mostly at the plasma membrane in monkey neurons, indicating that the fusion of mCherry with hM3Dq does not hamper membrane incorporation of this specific DREADD. Our results suggest that the pattern of ultrastructural expression of DREADDs in monkey neurons depends on the DREADD/tag combination. Therefore, a preliminary characterization of plasma membrane expression of specific DREADD/tag combinations is recommended when using chemogenetic approaches in primates.

CteG is a Chlamydia trachomatis effector protein that associates with the Golgi complex of infected host cells

Chlamydia trachomatis is a bacterial pathogen causing ocular and genital infections in humans. C. trachomatis multiplies exclusively inside host cells within a characteristic vacuole, from where it manipulates host cells by injecting them with type III secretion effector proteins. Here, we identified CteG as the first C. trachomatiseffector associated with the Golgi. For this, C. trachomatis strains expressing candidate effectors fused to a double hemagglutinin (2HA) tag were constructed. Then, among these strains, immunofluorescence microscopy revealed that CteG-2HA was delivered into the cytoplasm of infected cells. Between 16–20 h post-infection, CteG-2HA mostly associated with the Golgi; however, CteG-2HA also appeared at the host cell plasma membrane, and at 30 or 40 h post-infection this was its predominant localization. This change in the main localization of CteG-2HA was independent of intact microfilaments or microtubules. Ectopic expression of different regions of CteG (656 amino acid residues) in uninfected cells revealed that its first 100 residues contain a Golgi targeting region. Although a C. trachomatis cteG mutant did not display a defect in intracellular multiplication, CteG induced a vacuolar protein sorting defect when expressed in Saccharomyces cerevisiae. This suggested that CteG might function by subverting host cell vesicular transport.

Determining the association of the TrmB‐like protein OxsR to chromatin binding and oxidative stress in Haloferax volcanii

Archaea are single‐celled microorganisms that are well known for their ability to survive and thrive in harsh environmental conditions; however, the factors that mediate homeostasis during redox and/or oxidative stress are not fully understood this domain of life. OxsR (redox stress responsive regulator, HVO_2970) is a TrmB‐like protein of arCOG002242 that is increased several‐fold in abundance during hypochlorite stress based on quantitative proteomic analysis of the halophilic archaeon Haloferax volcanii. TrmB (transcription regulator of mal operon) family proteins are typically transcription factors that bind sugar/effector molecules and regulate the expression of carbon metabolism and transport genes. OxsR has a winged‐helix DNA binding domain but lacks the sugar/effector binding domain common to members of the TrmB family. Here we find that OxsR has a conserved cysteine residue (C24) at a predicted homodimer interface that may to be intimately associated with redox homeostasis in this archaeon. OxsR is required: i) for tolerance to oxidative stress and ii) for an increase in the level of transcripts associated with DNA repair and oxidative stress response. Current work is focused on determining how OxsR senses redox stress, binds DNA, and regulates transcript levels by use of RNAseq, ChIP‐Seq and ChIP‐PCR methods. To this end, we have prepared an RNA library for RNAseq analysis and integrated a C‐terminal hemagglutinin (HA) tag onto the oxsR gene locus for the ChIP methods. Overall, OxsR is an apparent redox sensor required for elevated transcript levels of DNA repair and oxidative stress response pathways. The distribution of OxsR homologs with conserved cysteine residue(s) in Euryarchaeota and Crenarchaeota phyla suggests this type of redox sensor is widespread in Archaea.Support or Funding InformationFunds for this project were awarded to JM‐F through the Bilateral NSF/BIO‐BBSRC program (NSF 1642283), the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences, Physical Biosciences Program (DOE DE‐FG02‐05ER15650) and the National Institutes of Health (NIH R01 GM57498).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tandem Immunoaffinity Purification Using Anti-FLAG and Anti-HA Antibodies.

The immunoaffinity purification of target proteins followed by the identification and characterization of associated proteins by mass spectrometry is a widely used technique. An immunoaffinity purification bears resemblance to a standard immunoprecipitation; however, the end product for mass spectrometric analysis in the femtomole (10-15) to attomole (10-18) range is required to be of exceptional purity. This high degree of sensitivity in detection renders it of extreme importance to eliminate most if not all of the nonspecific background proteins and can be achieved by performing a tandem affinity purification (TAP). In TAP, the cDNA of the target protein is engineered to contain at least two different epitope tags, and the target protein is extracted under nondenaturing conditions upon expression using an appropriate protein expression platform (CHO cells, HEK 293 cells, or yeast). The expressed protein is initially immunoprecipitated using an antibody against one epitope tag and is eluted in the presence of excess peptide by competition for antibody-binding sites, before being reimmunoprecipitated using an antibody that specifically recognizes the second epitope. These sequential immunoprecipitations significantly reduce the presence of associated nonspecific proteins. Numerous combinations of epitope tags have been applied for tandem affinity purification, and this protocol illustrates the use of tandem hemagglutinin (HA) and FLAG epitope tags. The first immunoprecipitation uses an anti-FLAG antibody followed by the elution in the presence of a competing FLAG peptide before the reimmunoprecipitation of the protein using an anti-HA antibody. Numerous high-quality antiepitope tag antibodies are commercially available from different antibody manufacturers.

Global polysome analysis of normal and injured podocytes.

Podocyte injury is a key event for progressive renal failure. We have previously established a mouse model of inducible podocyte injury (NEP25) that progressively develops glomerulosclerosis after immunotoxin injection. We performed polysome analysis of intact and injured podocytes utilizing the NEP25 and RiboTag transgenic mice, in which a hemagglutinin tag is attached to ribosomal protein L22 selectively in podocytes. Podocyte-specific polysomes were successfully obtained by immunoprecipitation with an antihemagglutinin antibody from glomerular homogenate and analyzed using a microarray. Compared with glomerular cells, 353 genes were highly expressed and enriched in podocytes; these included important podocyte genes and also heretofore uncharacterized genes, such as Dach1 and Foxd2. Podocyte injury by immunotoxin induced many genes to be upregulated, including inflammation-related genes despite no infiltration of inflammatory cells in the glomeruli. MafF and Egr-1, which structurally have the potential to antagonize MafB and WT1, respectively, were rapidly and markedly increased in injured podocytes before MafB and WT1 were decreased. We demonstrated that Maff and Egr1 knockdown increased the MafB targets Nphs2 and Ptpro and the WT1 targets Ptpro, Nxph3, and Sulf1, respectively. This indicates that upregulated MafF and Egr-1 may promote deterioration of podocytes by antagonizing MafB and WT1. Our systematic microarray study of the heretofore undescribed behavior of podocyte genes may open new insights into the understanding of podocyte pathophysiology.

A Mutation in the UL24 Gene Abolishes Expression of the Newly Identified UL24.5 Protein of Herpes Simplex Virus 1 and Leads to an Increase in Pathogenicity in Mice.

Herpes simplex virus 1 (HSV-1) infects the host via epithelia and establishes latency in sensory neurons. The UL24 gene is conserved throughout the Herpesviridae family, and the UL24 protein is important for efficient viral replication and pathogenesis. Multiple transcripts are expressed from the UL24 gene. The presence of a transcription initiation site inside the open reading frame of UL24 and an ATG start codon in the same open reading frame led us to suspect that another protein was expressed from the UL24 locus. To test our hypothesis, we constructed a recombinant virus that expresses a hemagglutinin tag at the C terminus of UL24. Western blot analysis revealed the expression of an 18-kDa protein that is not a degradation product of the full-length UL24, which we refer to as UL24.5. Ectopically expressed UL24.5 did not induce the dispersal of nucleolar proteins, as seen for UL24. In order to characterize the role of UL24.5, we constructed a mutant virus encoding a substitution of the predicted initiation methionine to a valine. This substitution eliminated the expression of the 18-kDa polypeptide. Unlike the UL24-null mutant (UL24X), which exhibits reduced viral yields, the UL24.5-null mutant exhibited the same replication phenotype in cell culture as the parental strain. However, in a murine ocular infection model, we observed an increase in the incidence of neurological disorders with the UL24.5 mutant. Alignment of amino acid sequences for various herpesviruses revealed that the initiation site of UL24.5 is conserved among HSV-1 strains and is present in many herpesviruses.IMPORTANCE We discovered a new HSV-1 protein, UL24.5, which corresponds to the C-terminal portion of UL24. In contrast to the replication defects observed with HSV-1 strains that do not express full-length UL24, the absence of UL24.5 did not affect viral replication in cell culture. Moreover, in mice, the absence of UL24.5 did not affect viral titers in epithelia or trigeminal ganglia during acute infection; however, it was associated with a prolonged persistence of signs of inflammation. Strikingly, the absence of UL24.5 also led to an increase in the incidence of severe neurological impairment compared to results for wild-type control viruses. This increase in pathogenicity is in stark contrast to the reduction in clinical signs associated with the absence of full-length UL24. Bioinformatic analyses suggest that UL24.5 is conserved among all human alphaherpesviruses and in some nonhuman alphaherpesviruses. Thus, we have identified UL24.5 as a new HSV-1 determinant of pathogenesis.

The epitope-mediated MMP activation assay: detection and quantification of the activation of Mmp2 in vivo in the zebrafish embryo.

Matrix remodeling is a consequence of tightly regulated matrix metalloproteinase (MMP) activity. MMPs are synthesized as inactive precursors with auto-inhibitory N-terminal propeptides, the proteolytic removal of which exposes the catalytic zinc ion, rendering the protease active. The regulation of MMP activation has been investigated primarily in tissue culture and biochemical assays that lack important biological context. Here we present the epitope-mediated MMP activation (EMMA) assay and use it to observe the activation of Mmp2 (gelatinase A) by endogenous mechanisms in the intact zebrafish embryo. The hemagglutinin (HA) and GFP-tagged reporter construct becomes activated on the surface of specific cells and this activation is abolished by broad-spectrum inhibition of metalloproteinase activity, consistent with existing models of gelatinase A activation. The mechanism(s) acting on the construct are spatially restricted, metalloproteinase-dependent and replacing the HA tag with mCherry abolishes activation, showing that the mechanism(s) are sensitive to the structure of the N-terminal domain. The construct is activated strongly in maturing myotome boundaries, but also intracellularly within myofibrils, consistent with reports implicating this protease in muscle development and function. In addition to general-purpose tools for the production of "EMMAed" MMPs and other proteins, we have established a transgenic line of zebrafish expressing EMMAedMmp2 under control of an inducible promoter to facilitate further investigation into the regulation of this ubiquitous ECM-remodeling protease in vivo.

Targeting of severe fever with thrombocytopenia syndrome virus structural proteins to the ERGIC (endoplasmic reticulum Golgi intermediate compartment) and Golgi complex.

Severe fever with thrombocytopenia syndrome phlebovirus (SFTSV) is a newly emerged phlebovirus identified in China, Japan, and South Korea. Phlebovirus glycoproteins (GP) play a key role in targeting viral structural components to the budding compartments in the ER-Golgi intermediate compartment (ERGIC) and Golgi complex. However, the role of SFTSV GP in targeting structural proteins to the ERGIC and Golgi complex remains unresolved. In this study, we show that SFTSV GP plays a significant role in targeting RNA-dependent RNA polymerase (L) and nucleocapsid protein (NP) to the budding sites. Confocal microscopy was used to investigate the subcellular localization of SFTSV structural proteins. In SFTSV-infected cells, GP and L localized to the ER, ERGIC and Golgi complex, whereas NP localized to the ERGIC and Golgi complex. In addition, GP colocalized with L and NP in infected cells. In cells singly transfected with GP, L or NP, GP localized to the same subcellular compartments as in infected cells. However, L or NP alone did not localize to the ER, ERGIC, or Golgi complex. Cotransfection experiments showed that GP altered the localization of L to the ERGIC and Golgi complex but not that of NP. Interestingly, plasmid-expressed NP fused with a hemagglutinin tag localized to the ERGIC and Golgi complex when expressed in SFTSV-infected cells and colocalised with GP, suggesting that GP plays a role in the subcellular localization of L and NP in infected cells. Thus, the SFTSV structural components start to assemble at the ERGIC to Golgi complex. GP is required for transporting L and NP to the ERGIC and Golgi complex. In addition, targeting of NP requires interaction with other factors besides GP.

Residues 28 to 39 of the Extracellular Loop 1 of Chicken Na+/H+ Exchanger Type I Mediate Cell Binding and Entry of Subgroup J Avian Leukosis Virus.

Chicken Na+/H+ exchanger type I (chNHE1), a multispan transmembrane protein, is a cellular receptor of the subgroup J avian leukosis virus (ALV-J). To identify the functional determinants of chNHE1 responsible for the ALV-J receptor activity, a series of chimeric receptors was created by exchanging the extracellular loops (ECL) of human NHE1 (huNHE1) and chNHE1 and by ECL replacement with a hemagglutinin (HA) tag. These chimeric receptors then were used in binding and entry assays to map the minimal ALV-J gp85-binding domain of chNHE1. We show that ECL1 of chNHE1 (chECL1) is the critical functional ECL that interacts directly with ALV-J gp85; ECL3 is also involved in ALV-J gp85 binding. Amino acid residues 28 to 39 of the N-terminal membrane-proximal region of chECL1 constitute the minimal domain required for chNHE1 binding of ALV-J gp85. These residues are sufficient to mediate viral entry into ALV-J nonpermissive cells. Point mutation analysis revealed that A30, V33, W38, and E39 of chECL1 are the key residues mediating the binding between chNHE1 and ALV-J gp85. Further, the replacement of residues 28 to 39 of huNHE1 with the corresponding chNHE1 residues converted the nonfunctional ALV-J receptor huNHE1 to a functional one. Importantly, soluble chECL1 and huECL1 harboring chNHE1 residues 28 to 39 both could effectively block ALV-J infection. Collectively, our findings indicate that residues 28 to 39 of chNHE1 constitute a domain that is critical for receptor function and mediate ALV-J entry.IMPORTANCE chNHE1 is a cellular receptor of ALV-J, a retrovirus that causes infections in chickens and serious economic losses in the poultry industry. Until now, the domains determining the chNHE1 receptor function remained unknown. We demonstrate that chECL1 is critical for receptor function, with residues 28 to 39 constituting the minimal functional domain responsible for chNHE1 binding of ALV-J gp85 and efficiently mediating ALV-J cell entry. These residues are located in the membrane-proximal region of the N terminus of chECL1, suggesting that the binding site of ALV-J gp85 on chNHE1 is probably located on the apex of the molecule; the receptor-binding mode might be different from that of retroviruses. We also found that soluble chECL1, as well as huECL1 harboring chNHE1 residues 28 to 39, effectively blocked ALV-J infection. These findings contribute to a better understanding of the ALV-J infection mechanism and also provide new insights into the control strategies for ALV-J infection.