Re-emergence of Peste des Petits Ruminants virus in 2015 in Morocco: Molecular characterization and experimental infection in Alpine goats.

Peste des Petits Ruminants (PPR) is a transboundary viral disease of small ruminants that causes huge economic losses in Africa, The Middle East and Asia. In Morocco, the first PPR outbreak was notified in 2008. Since then no cases were reported for seven years, probably due to three successive vaccination campaigns during 2008-2011 and close surveillance at the border areas. In June 2015, the disease re-emerged in Morocco, raising questions about the origin of the virus. The PPR virus was confirmed by qRT-PCR and virus was isolated from clinical samples on VeroNectin-4 cells. The disease was experimentally reproduced in Alpine goats using both sheep and goat derived outbreak isolates. Molecular characterization of the 2015 Moroccan PPR isolate confirmed the identity of the virus as lineage IV, closely related to the 2012 Algerian (KP793696) and 2012 Tunisian (KM068121) isolates and significantly distinct from the previous PPRV Morocco 2008 strain (HQ131927). Therefore this study confirms a new incursion of PPR virus in Morocco during 2015 and highlights the urgency of implementation of a common control strategy to combat PPR in Maghreb region in North Africa.

Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes.

The Cas9 endonuclease can be targeted to genomic sequences by programming the sequence of an associated single guide RNA (sgRNA). For unknown reasons, the activity of these Cas9-sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting genes in polyploid cell lines or when using poorly performing sgRNAs can require extensive downstream screening to identify homozygous clones. Here we find that non-homologous single-stranded DNA greatly stimulates Cas9-mediated gene disruption in the absence of homology-directed repair. This stimulation increases the frequency of clones with homozygous gene disruptions and rescues otherwise ineffective sgRNAs. The molecular outcome of enhanced gene disruption depends upon cellular context, stimulating deletion of genomic sequence or insertion of non-homologous DNA at the edited locus in a cell line specific manner. Non-homologous DNA appears to divert cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of gene disruption.

VeroNectin-4 is a highly sensitive cell line that can be used for the isolation and titration of Peste des Petits Ruminants virus.

Peste des Petits Ruminants virus (PPRV) is a member of the Morbillivirus subgroup of the family Paramyxoviridae, and is one of the most contagious diseases of small ruminants throughout Africa and the rest of the world. Different cell lines have previously been used to isolate PPRV but with limited success. Thus, to improve the isolation of Morbilliviruses, human, canine, and goat homologues of the lymphocyte receptor signaling lymphocyte activation molecule (SLAM) have been introduced into cells that can support virus replication. However, the amino acid sequence of SLAM varies between species, and often requires adaptation of a particular virus to different versions of the receptor. The protein sequence of Nectin-4 is highly conserved between different mammals, which eliminate the need for receptor adaptation by the virus. Cell lines expressing Nectin-4 have previously been used to propagate measles and canine distemper viruses. In this study, we compared infections in Vero cells expressing canine SLAM (VeroDogSLAM) to those in Vero cells expressing Nectin-4 (VeroNectin-4), following inoculations with wild-type strains of PPRV. Virus isolation using VeroNectin-4 cells was successful with 23% of swabbed samples obtained from live infected animals, and was 89% effective using post-mortem tissues of infected sheep. By contrast, only 4.5% efficiency was observed from swab samples and 67% efficiency was obtained in virus isolation from post-mortem tissues using VeroDogSLAM cells. The average incubation period for virus recovery from post-mortem tissues was 3.4 days using VeroNectin-4 cells, compared with 5.5 days when using VeroDogSLAM cells. The virus titers of PPRV obtained from VeroNectin-4 cells were also higher than those derived from VeroDogSLAM cells. A comparison of the growth kinetics for PPRV in the two cell lines confirmed the superiority of VeroNectin-4 cells for PPR diagnostic purposes and vaccine virus titration.

Future promise of siRNA and other nucleic acid based therapeutics for the treatment of chronic HCV.

RNA interference (RNAi) is gaining favor as a potential therapeutic option for the treatment of Hepatitis C virus infections. RNAi, first discovered in plants, induces sequence specific degradation of messenger RNA following the introduction of short interference RNA (siRNA). RNAi is a natural defense mechanism used by plants to combat viral infections, and the discovery of RNAi activity in mammalian cells has prompted several drug companies to investigate and exploit RNAi based drugs as a potential therapy against HCV infections. A number of research groups have demonstrated that strong RNAi activity can be induced against HCV using synthetic siRNA duplexes as triggers, or by expressing short hairpin RNAs from plasmid or viral vectors. However, much work remains to improve delivery, maintain specificity and limit the development of virus resistance. HCV is capable of evading RNAi activity through the incorporation escape mutations within the siRNA target sequence, highlighting the importance of implementing strategies to limit the development of resistance. Other nucleic acid based therapies such as antisense oligonucleotides, RNA aptamers and ribozymes have also been considered for use as HCV therapeutics, and we will outline the potential opportunities and obstacles to their use as well as RNAi.

X protein of hepatitis B virus modulates cytokine and growth factor related signal transduction pathways during the course of viral infections and hepatocarcinogenesis.

Hepatitis B virus produces chronic infections of the liver leading to cirrhosis and hepatocellular carcinoma. The X protein of hepatitis B virus (HBx) is a multifunctional protein that can interact with p53 but can also influence a variety of signal transduction pathways within the cell. In most instances this small viral protein favors cell survival and probably initiates hepatocarcinogenesis. HBx upregulates the activity of a number of transcription factors including NF-kappa B, AP-1, CREB, and TBP. However, the majority of HBx is localized to the cytoplasm where it interacts with and stimulates protein kinases such as protein kinase C, Janus kinase/STAT, IKK, PI-3-K, stress-activated protein kinase/Jun N-terminal kinase, and protein kinase B/Akt. This small viral protein can localize to the mitochondrion. HBx may act as an adaptor or kinase activator to influence signal transduction pathways. This review will attempt to analyze the involvement of HBx in signal transduction pathways during hepatitis B viral infections and hepatocellular carcinoma development.

CD45 is a JAK phosphatase and negatively regulates cytokine receptor signalling.

The regulation of tyrosine phosphorylation and associated signalling through antigen, growth-factor and cytokine receptors is mediated by the reciprocal activities of protein tyrosine kinases and protein tyrosine phosphatases (PTPases). The transmembrane PTPase CD45 is a key regulator of antigen receptor signalling in T and B cells. Src-family kinases have been identified as primary molecular targets for CD45 (ref. 4). However, CD45 is highly expressed in all haematopoietic lineages at all stages of development, indicating that CD45 could regulate other cell types and might act on additional substrates. Here we show that CD45 suppresses JAK (Janus kinase) kinases and negatively regulates cytokine receptor signalling. Targeted disruption of the cd45 gene leads to enhanced cytokine and interferon-receptor-mediated activation of JAKs and STAT (signal transducer and activators of transcription) proteins. In vitro, CD45 directly dephosphorylates and binds to JAKs. Functionally, CD45 negatively regulates interleukin-3-mediated cellular proliferation, erythropoietin-dependent haematopoieisis and antiviral responses in vitro and in vivo. Our data identify an unexpected and novel function for CD45 as a haematopoietic JAK phosphatase that negatively regulates cytokine receptor signalling.

CDw150(SLAM) is a receptor for a lymphotropic strain of measles virus and may account for the immunosuppressive properties of this virus.

Natural isolates of measles virus readily infect several lymphocyte cell lines. These viruses appear to use a receptor other than CD46, the molecule to which most laboratory strains of virus bind. Methods used to identify and characterize this lymphocyte receptor for measles virus are described in this study. A binding assay with a soluble form of measles virus H protein demonstrated that B-cell lines, activated with Epstein-Barr virus, or T cells, transformed with human T-cell leukemia virus, exhibit this receptor on their cell surfaces. On the other hand, resting lymphocytes, monocytes, or immature leukocytes either failed to express or possessed reduced levels of this receptor. A cDNA library derived from B95-8 marmoset B-cell lines was used to identify this receptor through expression cloning. This molecule was shown to be CDw150, which is also known as the signaling lymphocytic activation molecule (SLAM). When the lymphocyte receptor was expressed in Chinese hamster ovary (CHOP) or human embryonic kidney (293T) cells, these cells became susceptible to lymphotropic as well as laboratory strains of measles virus. Binding assays confirmed that either lymphotropic or laboratory strains of measles virus could adhere to human or marmoset CDw150, but interaction with the mouse homolog was weak. These infections were independent of the presence of CD46 on the host cell surface. Interaction of measles virus with CDw150(SLAM) could explain the immunosuppressive properties of this virus.

X protein of hepatitis B virus inhibits Fas-mediated apoptosis and is associated with up-regulation of the SAPK/JNK pathway.

The X protein from a chronic strain of hepatitis B virus (HBx) was determined to inhibit Fas-mediated apoptosis and promote cell survival. Fas-mediated apoptosis is the major cause of hepatocyte damage during liver disease. Experiments demonstrated that cell death caused by anti-Fas antibodies was blocked by the expression of HBx in human primary hepatocytes and mouse embryo fibroblasts. This effect was also observed in mouse erythroleukemia cells that lacked p53, indicating that protection against Fas-mediated apoptosis was independent of p53. Components of the signal transduction pathways involved in this protection were studied. The SAPK/JNK pathway has previously been suggested to be a survival pathway for some cells undergoing Fas-mediated apoptosis, and kinase assays showed that SAPK activity was highly up-regulated in cells expressing the HBx protein. Normal mouse fibroblasts expressing HBx were protected from death, whereas identical fibroblasts lacking the SEK1 component from the SAPK pathway succumbed to Fas-mediated apoptosis, whether HBx was present or not. Assays showed that caspase 3 and 8 activities and the release of cytochrome c from mitochondria were inhibited, in the presence of HBx, following stimulation with anti-Fas antibodies. Coprecipitation and confocal immunofluorescence microscopy experiments demonstrated that HBx localizes with a cytoplasmic complex containing MEKK1, SEK1, SAPK, and 14-3-3 proteins. Finally, mutational analysis of HBx demonstrated that a potential binding region for 14-3-3 proteins was essential for induction of SAPK/JNK activity and protection from Fas-mediated apoptosis.

Universal GFP reporter for the study of vascular development.

We report the generation and characterization of transgenic mouse and zebrafish expressing green fluorescent protein (GFP) specifically in vascular endothelial cells in a relatively uniform fashion. These reporter lines exhibit fluorescent vessels in developing embryos and throughout adulthood, allowing visualization of the general vascular patterns with single cell resolution. Furthermore, we show the ability to purify endothelial cells from whole embryos and adult organs by a single step fluorescence activated cell sorting. We expect that these transgenic reporters will be useful tools for imaging vascular morphogenesis, global gene expression profile analysis of endothelial cells, and high throughput screening for vascular mutations.

The coxsackie-adenovirus receptor (CAR) is used by reference strains and clinical isolates representing all six serotypes of coxsackievirus group B and by swine vesicular disease virus.

Group B coxsackieviruses are etiologically linked to many human diseases, and cell surface receptors are postulated to play an important role in mediating their pathogenesis. The coxsackievirus adenovirus receptor (CAR) has been shown to function as a receptor for selected strains of coxsackievirus group B (CVB) serotypes 3, 4, and 5 and is postulated to serve as a receptor for all six serotypes. In this study, we demonstrate that CAR can serve as a receptor for laboratory reference strains and clinical isolates of all six CVB serotypes. Infection of CHO cells expressing human CAR results in a 1000-fold increase in CVB progeny virus titer compared to mock transfected cells. CAR was shown to be a functional receptor for swine vesicular disease virus (SVDV), as CHO-CAR cells but not CHO mock transfected controls were susceptible to SVDV infection, produced progeny SVDV, and developed cytopathic effects. Moreover, SVDV infection could be specifically blocked by monoclonal antibody to CAR (RmcB). SVDV infection of HeLa cells was also inhibited by an anti-CD55 MAb, suggesting that this virus, like some CVB, may interact with CD55 (decay accelerating factor) in addition to CAR. Finally, pretreatment of CVB or SVDV with soluble CAR effectively blocks virus infection of HeLa cell monolayers.

Alpha2-adrenergic receptors in human dorsal root ganglia: predominance of alpha2b and alpha2c subtype mRNAs.

BACKGROUND: Nonselective alpha2-adrenergic receptor (alpha2AR) agonists (e.g., clonidine) mediate antinociception in part through alpha2ARs in spinal cord dorsal horn; however, use of these agents for analgesia in humans is limited by unwanted sedation and hypotension. The authors previously demonstrated alpha2a approximately alpha2b > > > alpha2c mRNA in human spinal cord dorsal horn cell bodies. However, because 20% of dorsal horn alpha2ARs derive from cell bodies that reside in the associated dorsal root ganglion (DRG), it is important to evaluate alpha2AR expression in this tissue as well. Therefore, the authors evaluated the hypothesis that alpha2b mRNA, alpha2c mRNA, or both are present in human DRG. METHODS: Molecular approaches were used to determine alpha2AR expression in 28 human DRGs because of low overall receptor mRNA expression and small sample size. After creation of synthetic competitor cDNA and establishment of amplification conditions with parallel efficiencies, competitive reverse transcription polymerase chain reaction was performed using RNA isolated from human DRG. RESULTS: Overall expression of alpha2AR mRNA in DRG is low but reproducible at all spinal levels. alpha2b and alpha2cAR subtype mRNAs predominate (alpha2b approximately alpha2c), accounting for more than 95% of the total alpha2AR mRNA in DRG at all human spinal nerve root levels. CONCLUSIONS: Predominance of alpha2b and alpha2cAR mRNA in human DRG is distinct from alpha2AR mRNA expression in cell bodies originating in human spinal cord dorsal horn, where alpha2a and alpha2b predominate with little or absent alpha2c expression. These findings also highlight species heterogeneity in alpha2AR expression in DRG. If confirmed at a protein level, these findings provide an additional step in unraveling mechanisms involved in complex neural pathways such as those for pain.

Subtype specific regulation of human vascular alpha(1)-adrenergic receptors by vessel bed and age.

BACKGROUND: alpha(1)-adrenergic receptors (alpha(1)ARs) regulate blood pressure, regional vascular resistance, and venous capacitance; the exact subtype (alpha(1a), alpha(1b), alpha(1 d)) mediating these effects is unknown and varies with species studied. In order to understand mechanisms underlying cardiovascular responses to acute stress and chronic catecholamine exposure (as seen with aging), we tested two hypotheses: (1) human alpha(1)AR subtype expression differs with vascular bed, and (2) age influences human vascular alpha(1)AR subtype expression. METHODS AND RESULTS: Five hundred vessels from 384 patients were examined for alpha(1)AR subtype distribution at mRNA and protein levels (RNase protection assays, ligand binding, contraction assays). Overall vessel alpha(1)AR density is 16+/-2.3fmol/mg total protein. alpha(1a)AR predominates in arteries at mRNA (P<0.001) and protein (P<0.05) levels; all 3 subtypes are present in veins. Furthermore, alpha(1)AR mRNA subtype expression varies with vessel bed (alpha(1a) higher in splanchnic versus central arteries, P<0.05); competition analysis (selected vessels) and functional assays demonstrate alpha(1a) and alpha(1b)-mediated mammary artery contraction. Overall alpha(1)AR expression doubles with age (<55 versus > or = 65 years) in mammary artery (no change in saphenous vein), accompanied by increased alpha(1b)>alpha(1a) expression (P< = 0.001). CONCLUSIONS: Human vascular alpha(1)AR subtype distribution differs from animal models, varies with vessel bed, correlates with contraction in mammary artery, and is modulated by aging. These findings provide potential novel targets for therapeutic intervention in many clinical settings.

Use of site-specific mutagenesis and monoclonal antibodies to map regions of CD46 that interact with measles virus H protein.

Researchers at our laboratory have been dissecting the binding domains of the receptor for the Edmonston laboratory strain of measles virus (CD46) through site-specific mutagenesis. We initially substituted most of the hydrophilic amino acids in the two external short consensus regions (SCRI and SCRII) of CD46 with the amino acid alanine [Hsu et al. (1997) J. Virol. 71:6144-6154] and found that the glutamic-arginine residues at positions 58 and 59 were particularly sensitive to change. Here we consider the roles of hydrophobic amino acids in the binding between measles virus H protein and CD46. Hydrophobic amino acids in the SCRI and SCRII domains of CD46 were systematically replaced with serine. The effects of these changes were monitored through the interaction of Sf9 insect cells expressing the H protein and mouse OST-7 cells synthesizing the mutant CD46 molecules. Binding was quantified through a colorimetric assay for beta-galactosidase that was also produced by the insect cells. Our results indicate that E45, Y54, 58E/R59, Y68, F69, Y101, I102, R103, D104, and Y117 seem to be critical residues for the binding of CD46 to measles virus H protein. The hydrophilic amino acid R59 in SCR1 and hydrophobic residues Y101, I102, and Y117 in SCR2 seem to be especially important for interaction between H protein and CD46. In addition, we mapped the antigenic epitopes of five monoclonal antibodies that are known to inhibit the binding between H protein and CD46. Three of these antibodies recognized regions in SCR1, and two reacted with amino acids in SCR2. For the most part, the determinants recognized by the monoclonal antibody corresponded to the amino acids that were most sensitive to change in the binding process. The SCR1 and SCR2 domains of CD46 were modeled from an analogous region in another complement regulatory protein, factor H, whose three-dimensional structure has been previously reported. Amino acids implicated in binding seem to lie on one planar face of the SCR1 and SCR2 domains. These studies serve as a prelude to understanding the structural interactions that occur between CD46 and the measles virus H protein.

The V protein of the paramyxovirus SV5 interacts with damage-specific DNA binding protein.

The simian parainfluenza virus 5 (SV5) V/P gene encodes two proteins: V and the phosphoprotein P. The V and P proteins are amino coterminal for 164 residues, but they have unique carboxyl termini. The unique carboxyl terminus of V contains seven cysteine residues, resembles a zinc finger, and binds two atoms of zinc. In a glutathione-S-transferase (GST)-fusion protein selection of cell lysate assay, the GST-V protein was found to interact with the 127-kDa subunit (DDB1) of the damage-specific DNA binding protein (DDB) [also known as UV-damaged DNA binding protein (UV-DDB), xeroderma pigmentosum group E binding factor (XPE-BF), and the hepatitis B virus X-associated protein 1 (XAP-1)]. A reciprocal GST-DDB1 fusion protein selection assay of SV5-infected cell lysates showed that DDB1 and V interact, and it was found that V and DDB1 could be coimmunoprecipitated from SV5-infected cells or from cells expressing V and DDB1 using the vaccinia virus T7 expression system. The interaction of V and DDB1 involves the carboxyl-terminal domain of V in that either deletion of the V carboxyl-terminal domain or substitution of the cysteine residues (C189, C193, C205, C207, C210, C214, and C217) in the zinc-binding domain with alanine was able to disrupt binding to DDB1. The V proteins of the mumps virus, human parainfluenza virus 2 (hPIV2), and measles virus have also been found to interact with DDB1 in GST-fusion protein selection assays using in vitro transcribed and translated DDB1.

The late expression factors 8 and 9 and possibly the phosphoprotein p78/83 of Autographa californica multicapsid nucleopolyhedrovirus are components of the virus-induced RNA polymerase.

Attempts were made to identify some of the subunits of the baculovirus-induced RNA polymerase following purification of its enzymatic activity by conventional chromatography. Polymerase activity was extracted from lysates of insect cells infected with Autographa californica multicapsid nucleopolyhedrovirus by polyethylenimine precipitation and subsequently purified by phosphocellulose, anion exchange, poly(A) Sepharose affinity, and gel filtration chromatography. The presence of the polymerase was monitored by its alpha-amanitin-resistant activity in in vitro transcription assays. A number of polypeptides associated with the enzymatic activity were identified. Peptide-specific antibodies were generated against a variety of late-expression factors (LEFs) and these antibodies, along with antisera directed against several other baculovirus proteins, were used in an immunoblot analysis of the purified polymerase. The results revealed that both the viral helicase (p143) and the virogenic stroma protein, pp31, copurify with the baculovirus-induced RNA polymerase activity through several chromatographic steps and may be loosely associated with the RNA polymerase. LEF8, LEF9 and p78/83, a nucleocapsid-associated phosphoprotein, were found to associate with the viral-induced polymerase activity. LEF8 and LEF9 contain regions of sequence homology with components of other DNA-directed RNA polymerases, while a portion of p78/83 exhibits some homology to the sigma factor of bacterial RNA polymerase, the RAP30 protein found in the mammalian transcription complex TFIIF, and the RAP94 polypeptide associated with vaccinia virus RNA polymerase. The p78/83 protein has previously been shown by our laboratory to be a capsid protein, but it may also play some role with the RNA polymerase. These results represent a first attempt to identify specific components of the RNA polymerase associated with infections of insect cells by A. californica nucleopolyhedrovirus.

Alpha1-adrenergic receptor subtypes in human detrusor.

PURPOSE: To identify and quantitate alpha1-adrenergic receptor (alpha1AR) subtype expression in human detrusor. MATERIALS AND METHODS: Initial studies to determine alpha1AR expression in human detrusor were performed using saturation binding with [125I]HEAT. Once the presence of alpha1ARs was documented, subtype (alpha1a, alpha1b, alpha1d) expression at the mRNA level (and comparison with rat) was determined with RNase protection assays (human detrusor) and RT-PCR (human detrusor, rat whole bladder). Competition binding analysis with the alpha1dAR-selective ligand BMY7378 was used to measure alpha1AR subtype expression at a protein level in human detrusor. RESULTS: Alpha1AR expression in human detrusor was low but reproducible (6.3 +/- 1.0 fmol./mg. total protein). RNase protection assays performed on total RNA extracted from human detrusor revealed the following alpha1AR subtype expression: alpha1d (66%) > alpha1a (34%), and no alpha1b. RT-PCR confirmed alpha1AR subtype mRNA distribution in human detrusor with alpha1d (approximately 60-70%) > alpha1a (approximately 30-40%), and a lack of alpha1b mRNA. Rat whole bladder expressed different alpha1AR subtype mRNA than human detrusor, with alpha1a approximately alpha1b approximately alpha1d. The presence of alpha1d > alpha1a expression in human detrusor was confirmed at a protein level by competition analysis utilizing BMY7378 which revealed a two-site fit, with Ki and high affinity binding (66%) consistent with the alpha1dAR subtype. CONCLUSIONS: Human detrusor contained two alpha1AR subtypes (alpha1d > alpha1a), a finding that is different from rat, another commonly used animal model. Since non-subtype selective alpha1AR antagonists ameliorate irritative bladder symptoms (in men and women with/without outlet obstruction), and Rec 15/2739 (alpha1a selective antagonist) does not improve symptom scores in BPH, our findings suggest bladder alpha1dARs may provide a potentially novel mechanism underlying these therapeutic benefits.

Cardiovirulent coxsackieviruses and the decay-accelerating factor (CD55) receptor.

Group B coxsackieviruses are etiologically linked with many human diseases including acute myocarditis and associated chronic dilated cardiomyopathy. Well-established CVB3 cardiovirulent strains (CVB3c(s)) with known phenotypic difference have been used to study the pathogenesis of virus-induced heart disease. The receptor-binding characteristics of cardiovirulent CVB3 are not known, but may represent one mechanism accounting for differences in disease virulence. In this study, interactions between CVB3c(s) and the decay-accelerating factor (DAF or CD55) cell surface receptor were examined. Anti-DAF monoclonal antibodies (MAbs) blocked virus binding and infection of susceptible HeLa cells. Virus binding was significantly reduced by treatment of these cells with phosphatidylinositol phospholipase C enzyme, which rendered them DAF-deficient CVB3c(s) exhibited a differential propensity for the DAF receptor, as several cardiovirulent strains interacted more strongly than others. However, virus binding and infection was always most effectively blocked by MAbs directed against the SCR 2 and 3 domains of DAF, suggesting that binding occurs at a similar site(s) on the molecule for all strains. Virus binding and internalization were associated with DAF down-regulation at the cell surface, as monitored by flow cytometry analysis. Cardiovirulent CVB3 did not interact with molecules functionally and/or structurally related to DAF, including CD35, CD46, Factor H, or C4-binding protein. Adenovirus type 2 (Ad2) does not use the DAF receptor. However, competitive binding assays between Ad2 and CVB1-6, CVB3c(s), anti-DAF MAbs, or DAF-reduced cells indicated that DAF is associated with Ad2 receptors on the HeLa cell membrane. In summary, this study indicates that DAF is an attachment receptor for cardiovirulent CVB3 and that DAF interaction may be important in the pathogenesis of CVB-mediated heart disease.

A single amino acid change in the hemagglutinin protein of measles virus determines its ability to bind CD46 and reveals another receptor on marmoset B cells.

This paper provides evidence for a measles virus receptor other than CD46 on transformed marmoset and human B cells. We first showed that most tissues of marmosets are missing the SCR1 domain of CD46, which is essential for the binding of Edmonston measles virus, a laboratory strain that has been propagated in Vero monkey kidney cells. In spite of this deletion, the common marmoset was shown to be susceptible to infections by wild-type isolates of measles virus, although they did not support Edmonston measles virus production. As one would expect from these results, measles virus could not be propagated in owl monkey or marmoset kidney cell lines, but surprisingly, both a wild-type isolate (Montefiore 89) and the Edmonston laboratory strain of measles virus grew efficiently in B95-8 marmoset B cells. In addition, antibodies directed against CD46 had no effect on wild-type infections of marmoset B cells and only partially inhibited the replication of the Edmonston laboratory strain in the same cells. A direct binding assay with insect cells expressing the hemagglutinin (H) proteins of either the Edmonston or Montefiore 89 measles virus strains was used to probe the receptors on these B cells. Insect cells expressing Edmonston H but not the wild-type H bound to rodent cells with CD46 on their surface. On the other hand, both the Montefiore 89 H and Edmonston H proteins adhered to marmoset and human B cells. Most wild-type H proteins have asparagine residues at position 481 and can be converted to a CD46-binding phenotype by replacement of the residue with tyrosine. Similarly, the Edmonston H protein did not bind CD46 when its Tyr481 was converted to asparagine. However, this mutation did not affect the ability of Edmonston H to bind marmoset and human B cells. The preceding results provide evidence, through the use of a direct binding assay, that a second receptor for measles virus is present on primate B cells.

Insect virus proteins (FALPE and p10) self-associate to form filaments in infected cells.

Entomopoxviruses and baculoviruses are pathogens of insects which replicate in the cytoplasm and nuclei of their host cells, respectively. During the late stages of infection, both groups of viruses produce occlusion bodies which serve to protect virions from the external environment. Immunofluorescence and electron microscopy studies have shown that large bundles of filaments are associated with these occlusion bodies. Entomopoxviruses produce cytoplasmic fibrils which appear to be composed of the filament-associated late protein of entomopoxviruses (FALPE). Baculoviruses, on the other hand, yield filaments in the nuclei and cytoplasm of the infected cell which are composed of a protein called p10. Despite significant differences in their sequences, FALPE and p10 have similar hydrophilicity profiles, and each has a proline-rich stretch of amino acids at its carboxyl terminus. Evidence that FALPE and p10 could produce filaments in the absence of other viral proteins is presented. When FALPE was expressed in insect cells from a recombinant baculovirus, filaments similar to those produced by the wild-type Amsacta moorei entomopoxvirus were observed. In addition, when expression plasmids containing FALPE or p10 genes were transfected into Vero monkey kidney cells, filament structures similar to those found in infected insect cells were produced. The manner in which FALPE and p10 subunits interact to form polymers was investigated through deletion and site-specific mutagenesis in conjunction with immunofluorescence microscopy, yeast two-hybrid protein interaction analysis, and chemical cross-linking of adjacent molecules. These studies indicated that the amino termini of FALPE and p10 were essential for subunit interaction. Although deletion of the carboxy termini did not affect this interaction, it did inhibit filament formation. In addition, modification of several potential sites for phosphorylation also abolished filament assembly. We concluded that although the sequences of FALPE and p10 were different, the structural and functional properties of the two polypeptides appeared to be similar.