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Sialidase releases sialic acid residues from the ends of sugar chains. The sialidases are involved in many physiological processes including cell differentiation and proliferation and immune function as well as pathophysiological conditions such as various human cancers and infections. Therefore visualization of sialidase activities with high sensitivity could provide valuable insights into these isozyme's activity. We developed novel fluorescent sialidase substrates, 2-benzothiazol-2-yl-phenol derivatives-based N-acetylneuraminic acid (Neu5Ac) (BTP-Neu5Ac) substrates, for highly sensitive and specific visualization of sialidase activity in living mammalian tissues and virus-infected cells. We found that BTP-Neu5Ac can visualize sialidase activities sensitively and selectively in rat tissues including brain slices. BTP-Neu5Ac can also clearly detect cancer cells implanted orthotopically in mouse colons and human colon cancers. In this review, I describe imaging of sialidase activity with BTP-Neu5Ac in animal tissues, detection of colon cancer, memory formation, detection of virus-infected cells, and application to drug-resistant influenza virus detection and separation.

Objective: To establish a T7 promoter based reverse genetics system competent for the rescue of bovine parainfluenza virus type 3 (BPIV3)., Methods: We constructed three helper plasmids of px8δT-PT1-bPIV3-NP, px8δT-PT1-bPIV3-P and px8δT-PT1-bPIV3-L and one minigenome plasmid of pSC11-bPIV3-EGFP containing open reading frame (ORF) of the enhanced green fluorescent protein (EGFP) and cis-acting elements including BPIV3 leader region, gene start (GS), gene end (GE) and trailer region. All these plasmids are under the control of T7 promoter and identified by restriction endonuclease analysis. We rescued the pSC11-bPIV3-EGFP by two different methods. Then, we observed the fluorescence expression over time with fluorescence microscopy., Results: We successfully constructed a reverse genetic system based 4 plasmids under the control of T7 promoter and finished the rescue operation to the minigenome of BPIV3., Conclusion: This system can be further applied to investigate the function of BPIV3 genome by deletion and mutation of its genes.

Interferon (IFN) induces an antiviral state in cells that results in alterations of the patterns and levels of parainfluenza virus type 5 (PIV5) transcripts and proteins. This study reports that IFN-stimulated gene 56/IFN-induced protein with tetratricopeptide repeats 1 (ISG56/IFIT1) is primarily responsible for these effects of IFN. It was shown that treating cells with IFN after infection resulted in an increase in virus transcription but an overall decrease in virus protein synthesis. As there was no obvious decrease in the overall levels of cellular protein synthesis in infected cells treated with IFN, these results suggested that ISG56/IFIT1 selectively inhibits the translation of viral mRNAs. This conclusion was supported by in vitro translation studies. Previous work has shown that ISG56/IFIT1 can restrict the replication of viruses lacking a 2'-O-methyltransferase activity, an enzyme that methylates the 2'-hydroxyl group of ribose sugars in the 5'-cap structures of mRNA. However, the data in the current study strongly suggested that PIV5 mRNAs are methylated at the 2'-hydroxyl group and thus that ISG56/IFIT1 selectively inhibits the translation of PIV5 mRNA by some as yet unrecognized mechanism. It was also shown that ISG56/IFIT1 is primarily responsible for the IFN-induced inhibition of PIV5.

Objective: To determine the frequency and effects of nosocomial respiratory viral infections (RVIs) in premature neonates, including those who may be asymptomatic., Study Design: We performed a year-long surveillance for RVIs in infants <33 weeks gestational age admitted to 2 Syracuse neonatal intensive care units. Infants were enrolled within 3 days of neonatal intensive care unit admission and were sampled for RVIs until discharge using a multiplex polymerase chain reaction assay capable of detecting 17 different respiratory viruses or subtypes., Results: Twenty-six of 50 prematurely born infants (52%) tested positive for a respiratory virus at least once during their birth hospitalization. Testing positive for a respiratory virus was significantly associated with longer length of stay (70 days vs 35 days, P = .002) and prolonged ventilatory support (51 vs 13 days, P = .002). Infants who tested positive for a respiratory virus during their birth hospitalization had more than twice the rate of developing bronchopulmonary dysplasia (P < .05)., Conclusion: Nosocomial RVIs were frequent in our study population, despite the absence of clinical indicators of illness. Length of hospital stay was significantly longer and a diagnosis of bronchopulmonary dysplasia was more common in infants who had respiratory viruses detected., (Copyright © 2012 Mosby, Inc. All rights reserved.)

Macrophages regulate immune responses during many viral infections, and can be a major determinant of pathogenesis, virus replication and immune response to infection. Here, we have addressed the question of the outcome of infection of primary human macrophages with parainfluenza virus 5 (PIV5) and a PIV5 mutant (P/V-CPI-) that is unable to counteract interferon (IFN) responses. In cultures of naïve monocyte-derived macrophages (MDMs), WT PIV5 established a highly productive infection, whereas the P/V-CPI- mutant was restricted for replication in MDMs by IFN-beta. Restricted replication in vitro was relieved in MDM that had been activated by prior exposure to heat killed Gram positive bacteria, including Listeria monocytogenes, Streptococcus pyogenes, and Bacillus anthracis. Enhanced replication of the P/V mutant in MDM previously activated by bacterial components correlated with a reduced ability to produce IFN-beta in response to virus infection, whereas IFN signaling was intact. Activated MDM were found to upregulate the synthesis of IRAK-M, which has been previously shown to negatively regulate factors involved in TLR signaling and IFN-beta production. We discuss these results in terms of the implications for mixed bacteria-virus infections and for the use of live RNA virus vectors that have been engineered to be attenuated for IFN sensitivity., (Copyright © 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.)

The paramyxoviruses are a family of negative-sense RNA viruses that includes many important human and animal pathogens. Paramyxovirus RNA synthesis requires the viral phosphoprotein (P) and the large (L) protein. Phosphorylation of P is thought to regulate viral gene expression, though direct proof remains elusive. Recently, we reported that phosphorylation of a specific residue (Ser157) of the P protein of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, correlates with decreased viral gene expression and cytokine expression in infected cells. Here, we show that: Polo-like kinase 1 (PLK1), a serine/theronine kinase that plays a critical role in regulating the cell cycle, interacts with PIV5 P through the S157 residue; PLK1 inhibition increases viral gene expression; PLK1 over-expression inhibits viral gene expression; and PLK1 directly phosphorylates P in vitro, indicating that PLK1 down-regulates viral gene expression by phosphorylating P. Furthermore, we have determined the PLK1 phosphorylation site on P and found that mutant recombinant PIV5 whose P proteins cannot either bind to or be phosphorylated by PLK1 have similar phenotypes. Increased viral gene expression in PIV5 with mutations in the PLK1 binding/phosphorylation sites correlates with increased induction of cell death and cytokine expression, suggesting that PIV5 limits its viral gene expression to avoid these host effects. It is possible that targeting PLK1 will enhance host innate immune responses, leading to a novel strategy of clearing paramyxovirus infections quickly.

Parainfluenza virus 5 (PIV5) is a prototypical paramyxovirus. The V/P gene of PIV5 encodes two mRNA species through a process of pseudotemplated insertion of two G residues at a specific site during transcription, resulting in two viral proteins, V and P, whose N termini of 164 amino acid residues are identical. Previously it was reported that mutating six amino acid residues within this identical region results in a recombinant PIV5 (rPIV5-CPI-) that exhibits elevated viral protein expression and induces production of cytokines, such as beta interferon and interleukin 6. Because the six mutations correspond to the shared region of the V protein and the P protein, it is not clear whether the phenotypes associated with rPIV5-CPI- are due to mutations in the P protein and/or mutations in the V protein. To address this question, we used a minigenome system and recombinant viruses to study the effects of mutations on the functions of the P and V proteins. We found that the P protein with six amino acid residue changes (Pcpi-) was more efficient than wild-type P in facilitating replication of viral RNA, while the V protein with six amino acid residue changes (Vcpi-) still inhibits minigenome replication as does the wild-type V protein. These results indicate that elevated viral gene expression in rPIV5-CPI- virus-infected cells can be attributed to a P protein with an increased ability to facilitate viral RNA synthesis. Furthermore, we found that a single amino acid residue change at position 157 of the P protein from Ser (the residue in the wild-type P protein) to Phe (the residue in Pcpi-) is sufficient for elevated viral gene expression. Using mass spectrometry and (33)P labeling, we found that residue S157 of the P protein is phosphorylated. Based on these results, we propose that phosphorylation of the P protein at residue 157 plays an important role in regulating viral RNA replication.

The paramyxovirus hemagglutinin-neuraminidase (HN) functions in virus attachment to cells, cleavage of sialic acid from oligosaccharides, and stimulating membrane fusion during virus entry into cells. The structural basis for these diverse functions remains to be fully understood. We report the crystal structures of the parainfluenza virus 5 (SV5) HN and its complexes with sialic acid, the inhibitor DANA, and the receptor sialyllactose. SV5 HN shares common structural features with HN of Newcastle disease virus (NDV) and human parainfluenza 3 (HPIV3), but unlike the previously determined HN structures, the SV5 HN forms a tetramer in solution, which is thought to be the physiological oligomer. The sialyllactose complex reveals intact receptor within the active site, but no major conformational changes in the protein. The SV5 HN structures do not support previously proposed models for HN action in membrane fusion and suggest alternative mechanisms by which HN may promote virus entry into cells.

Previous work has documented that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation, whilst the V protein of human parainfluenza virus type 2 (hPIV2) targets STAT2. Here, it was shown that the processes of ubiquitination and degradation could be reconstructed in vitro by using programmed rabbit reticulocyte lysates. Using this system, the addition of bacterially expressed and purified SV5 V protein to programmed lysates was demonstrated to result in the polyubiquitination and degradation of in vitro-translated STAT1, but only if human STAT2 was also present. Surprisingly, in the same assay, purified hPIV2 V protein induced the polyubiquitination of both STAT1 and STAT2. In the light of these in vitro results, the specificity of degradation of STAT1 and STAT2 by SV5 and hPIV2 in tissue-culture cells was re-examined. As previously reported, STAT1 could not be detected in human cells that expressed SV5 V protein constitutively, whilst STAT2 could not be detected in human cells that expressed hPIV2 V protein, although the levels of STAT1 may also have been reduced in some human cells infected with hPIV2. In contrast, STAT1 could not be detected, whereas STAT2 remained present, in a variety of animal cells, including canine (MDCK) cells, that expressed the V protein of either SV5 or hPIV2. Thus, the V protein of SV5 appears to be highly specific for STAT1 degradation, but the V protein of hPIV2 is more promiscuous.

Heptad repeat regions (HR1 and HR2) are highly conserved peptides located in F(1) of paramyxovirus envelope proteins. They are important in the process of virus fusion and form six-helix bundle structure (trimer of HR1 and HR2 heterodimer) post-fusion, similar to those found in the fusion proteins of other enveloped viruses, such as retrovirus HIV. Both HR1 and HR2 show potent inhibition for virus fusion in some members of paramyxovirus. However, in other members, only HR2 gives strong inhibition whereas HR1 does not. Human respiratory syncytial virus (hRSV) is a member of paramyxovirus and its crystal structure of HR1 and HR2 six-helix bundle was solved lately. Although hRSV HR2 inhibition was reported, nevertheless the effect of HR1 on virus fusion is not known. In this study, hRSV HR1 and HR2 were expressed as fusion protein separately in Escherichia coli system and their complex assembly and virus fusion inhibition effect have been analysed. It shows that both HR1 and HR2 (in the fusion form with 50-amino-acid fusion partner) of hRSV F protein give strong inhibition on virus fusion (IC(50) values are 1.68 and 2.93 microM, respectively) and they form stable six-helix bundle in vitro with both in the fusion protein form.

The cis-acting signals found at the 3' ends of the genomic and antigenomic RNAs are a major factor determining the level of paramyxovirus RNA replication from each promoter. Using a minigenome system that reconstitutes SV5 RNA synthesis from cDNA-derived components, we show here that the genomic promoter (GP) for the paramyxovirus SV5 directs RNA replication approximately 14-fold lower than that seen from the antigenomic promoter (AGP). The goal of this study was to identify cis-acting signals responsible for differential levels of RNA replication from the SV5 GP and AGP. We have previously shown that the SV5 AGP contains three sequence-dependent elements (CRI, CRII, and Region III) that are separated by sequence-independent spacer regions. Minigenomes containing chimeric promoters were constructed to test the hypothesis that transfer of discrete cis-acting AGP elements to the GP could confer higher replication properties to the GP. Minigenomes containing a substitution of the AGP CRI, CRII, or Region III elements alone in place of the corresponding GP sequences did not show enhanced levels of RNA replication. However, transfer of both the AGP 3' terminal CRI and Region III elements into the corresponding sites of the GP led to a minigenome which replicated to approximately 40% of the levels seen with the AGP. This enhanced RNA replication from the GP was further increased up to AGP levels by also including the intervening AGP segment (bases 20-50) located between CRI and Region III. Importantly, transfer of nonviral sequences in place of GP bases 20-50 also increased RNA replication to levels approaching that of the AGP, but only in the context of the AGP CRI and Region III substitutions. These data indicate that differential levels of RNA replication from the SV5 GP and AGP are due to a combination of positive-acting signals in the AGP (CRI and Region III) and a negative-acting signal in the GP (bases 20-50). Possible functions for the SV5 promoter elements in determining RNA replication levels are proposed.

Caveolin-1 (Cav-1) is a major protein constituent of caveolae, a type of plasma membrane raft. We observed that coexpression of human Cav-1 with human immunodeficiency virus type 1 (HIV-1) blocked virion production from cells that are ordinarily highly permissive. Further investigation showed that this effect is specific, occurs at low ratios of Cav-1 to HIV-1 DNA, depends on expression of Cav-1 protein, and involves severely impaired expression of HIV-1 proteins. Cav-1 also blocked HIV-2 expression. In contrast, Cav-1 did not inhibit protein expression by a paramyxovirus and did not induce apoptosis or affect cellular morphology, cell viability, or cell cycle progression. Although only small amounts of HIV-1 virions were released from Cav-1-transfected cells, these were fully infectious. Deletion mutagenesis showed that the C-terminal 78 residues were as active as the full-length (178-amino-acid) protein in producing the block. In contrast, the 100 most N-terminal amino acids of Cav-1, which include the previously identified oligomerization and scaffolding domains, were shown to be dispensable. Study of single-amino-acid-exchange mutants of Cav-1 established that palmitoylation was not required. Additional deletion mutants then identified the hydrophobic, membrane-associated domain (residues 101 to 135) as the main determinant. Cellular distribution of wild-type and mutant proteins correlated with ability to block HIV-1 expression. Finally, Cav-2 also blocked HIV-1 expression. These data show that coexpression of caveolins can markedly inhibit expression of HIV proviral DNA and establish that the inhibition is mediated by the hydrophobic, membrane-associated domain.

A lectin-carbohydrate recognition event without enzymatic function is proposed as molecular basis for an important innate immune response to enveloped viruses. It involves the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus (NDV) and sialic acid expressing cellular receptors on human natural interferon (IFN) alpha producing cells. This conclusion is based on two types of experimental evidence: (a) strong UV irradiation of NDV, which destroyed the cell binding and hemadsorption (HAd) but not the neuraminidase (NA) activity of HN, also destroyed its IFN-alpha inducing activity; (b) DNA transfectants expressing HN mutant molecules with greatly reduced NA but not HAd activity induced IFN-alpha while transfectants expressing HN mutant molecules with greatly reduced NA and HAd activity were incapabable of inducing IFN-alpha in human peripheral blood mononuclear cells. The results clarify molecular mechanisms involved in pattern recognition during innate immune responses.

Chimeric bovine respiratory syncytial viruses (BRSV) expressing glycoproteins of bovine parainfluenza virus type 3 (BPIV-3) instead of BRSV glycoproteins were generated from cDNA. In the BRSV antigenome cDNA, the open reading frames of the major BRSV glycoproteins, attachment protein G and fusion protein F, were replaced individually or together by those of the BPIV-3 hemagglutinin-neuraminidase (HN) and/or fusion (F) glycoproteins. Recombinant virus could not be recovered from cDNA when the BRSV F open reading frame was replaced by the BPIV-3 F open reading frame. However, cDNA recovery of the chimeric virus rBRSV-HNF, with both glycoproteins replaced simultaneously, and of the chimeric virus rBRSV-HN, with the BRSV G protein replaced by BPIV-3 HN, was successful. The replication rates of both chimeras were similar to that of standard rBRSV. Moreover, rBRSV-HNF was neutralized by antibodies specific for BPIV-3, but not by antibodies specific to BRSV, demonstrating that the BRSV glycoproteins can be functionally replaced by BPIV-3 glycoproteins. In contrast, rBRSV-HN was neutralized by BRSV-specific antisera, but not by BPIV-3 specific sera, showing that infection of rBRSV-HN is mediated by BRSV F. Hemadsorption of cells infected with rBRSV-HNF and rBRSV-HN proved that BPIV-3 HN protein expressed by rBRSV is functional. Colocalization of the BPIV-3 glycoproteins with BRSV M protein was demonstrated by confocal laser scan microscopy. Moreover, protein analysis revealed that the BPIV-3 glycoproteins were present in chimeric virions. Taken together, these data indicate that the heterologous glycoproteins were not only expressed but were incorporated into the envelope of recombinant BRSV. Thus, the envelope glycoproteins derived from a member of the Respirovirus genus can together functionally replace their homologs in a Pneumovirus background.

The Sendai virus C proteins, C', C, Y1, and Y2, are a nested set of four independently initiated carboxy-coterminal proteins encoded on the P mRNA from an alternate reading frame. Together the C proteins have been shown to inhibit viral transcription and replication in vivo and in vitro and C' binds the Sendai virus L protein, the presumed catalytic subunit of the viral RNA polymerase. To identify amino acids within the C' protein that are important for binding L, site-directed mutagenesis of the gstC' gene was used to change conserved charged amino acids to alanine, generating nine mutants. Additionally, a tenth natural mutant, gstF170S, was also constructed. Six of the gstC' mutants, primarily in the C-terminal half of C', exhibited a defect in the ability to bind L protein. The mutants were assayed for their effect on in vitro transcription and replication from the antigenomic promoter, and the data suggest in all but one case a direct correlation between the ability of C to bind L and to inhibit these steps in RNA synthesis. Further studies with two nonfusion C mutants showed that this correlation was specifically due to the C' portion, and not the gst portion, of the fusion proteins. To study their individual functions, each of the four C proteins was fused downstream of glutathione S-transferase. The gstC', gstC, gstY1, and gstY1 fusion proteins were all able to bind L protein and to inhibit viral mRNA and (+)-leader RNA synthesis, and antigenome replication in vitro. In addition, the nonfusion C, Y1, and Y2 proteins all inhibited transcription. The inhibition of (+)-leader RNA and mRNA synthesis by wt C proteins (nonfusion) showed nearly identical dose-response curves, suggesting that inhibition occurs early in RNA synthesis., (Copyright 2001 Academic Press.)

Peptides derived from heptad repeat regions adjacent to the fusion peptide and transmembrane domains of many viral fusion proteins form stable helical bundles and inhibit fusion specifically. Paramyxovirus SV5 fusion (F) protein-mediated fusion and its inhibition by the peptides N-1 and C-1 were analyzed. The temperature dependence of fusion by F suggests that thermal energy, destabilizing proline residues and receptor binding by the hemagglutinin-neuraminidase (HN) protein collectively contribute to F activation from a metastable native state. F-mediated fusion was reversibly arrested by low temperature or membrane-incorporated lipids, and the resulting F intermediates were characterized. N-1 inhibited an earlier F intermediate than C-1. Co-expression of HN with F lowered the temperature required to attain the N-1-inhibited intermediate, consistent with HN binding to its receptor stimulating a conformational change in F. C-1 bound and inhibited an intermediate of F that could be detected until a point directly preceding membrane merger. The data are consistent with C-1 binding a pre-hairpin intermediate of F and with helical bundle formation being coupled directly to membrane fusion.

The Sendai virus (SeV) C gene codes for a nested set of four C proteins that carry out several functions, including the modulation of viral RNA synthesis and countering of the cellular antiviral response. Using mutant C genes (and in particular a C gene with a deletion of six amino acids present only in the larger pair of C proteins) and recombinant SeV carrying these mutant C genes, we find that the nested set of C proteins carry out a nested set of functions. All of the C proteins interdict interferon (IFN) signaling to IFN-stimulated genes (ISGs) and prevent pY701-Stat1 formation. However, only the larger C proteins can induce STAT1 instability, prevent IFN from inducing an antiviral state, or prevent programmed cell death. Remarkably, interdiction of IFN signaling to ISGs and the absence of pY701-Stat1 formation did not prevent IFN-alpha from inducing an anti-Vesicular stomatitis virus (VSV) state. It is possible that IFN-alpha signaling to induce an anti-VSV state can occur independently of the well-established Jak/Stat/ISGF3 pathway and that it is this parallel pathway that is targeted by the longer C proteins.

The paramyxovirus P protein is an essential component of the viral RNA polymerase composed of P and L proteins. In this study, we characterized the physical and functional interactions between P and L proteins using human parainfluenza virus type 1 (hPIV1) and its counterpart Sendai virus (SV). The hPIV1 P and SV L proteins or the SV P and hPIV1 L proteins formed complexes detected by anti-P antibodies. Functional analysis using the minigenome SV RNA containing CAT gene indicated that the hPIV1 P--SV L complex, but not the SV P--hPIV1 L complex, was biologically active. Mutant SV P or hPIV1 P cDNAs, which do not express C proteins, showed the same phenotype with wild-type P cDNAs, indicating that C proteins are not responsible for the dysfunction of SV P--hPIV1 L polymerase complex. Using the chimeric hPIV1/SV P cDNAs, we identified two regions (residues 387--423 and 511--568) on P protein, which are required for the functional interaction with hPIV1 L. These regions overlap with a previously identified domain for oligomer formation and binding to nucleocapsids. Our results indicate that in addition to a P--L binding domain, hPIV1 L requires a specific region on P protein to be biologically functional as a polymerase., (Copyright 2001 Academic Press.)

The mechanism of Sendai virus membrane fusion to cultured cell membranes was studied. Viral lipids were labeled with the lipophilic dye, 4-(4-(dihexadecylamino)styryl-N-methylquinolinium iodine) (DiQ), and viral proteins were labeled using fluorescein isothiocyanate (FITC). The redistribution of these probes from the virus to cultured cells was followed using the technique of image correlation spectroscopy. This technique assayed the intensity change and the redistribution of these probes as fusion progressed from a more to less aggregated state. The lipid probe DiQ dispersed into the membrane of the target membrane at both 22 and 37 degrees C, while the FITC-labeled proteins dispersed only at 37 degrees C. Simultaneous labeling of virus with both of these probes showed that at 37 degrees C their redistribution proceeded at different rates. These data were consistent with the formation of a hemifusion intermediate during the fusion process.

Extracellular cleavage of virus envelope fusion glycoproteins by host cellular proteases is a prerequisite for the infectivity of mammalian and nonpathogenic avian influenza viruses, and Sendai virus. Here we report a protease present in the airway that, like tryptase Clara, can process influenza A virus haemagglutinin and Sendai virus envelope fusion glycoprotein. This protease was extracted from the membrane fraction of rat lungs, purified and then identified as a mini-plasmin. Mini-plasmin was distributed predominantly in the epithelial cells of the upward divisions of bronchioles and potentiated the replication of broad-spectrum influenza A viruses and Sendai virus, even that of the plasmin-insensitive influenza A virus strain. In comparison with plasmin, its increased hydrophobicity, leading to its higher local concentrations on membranes, and decreased molecular mass may enable mini-plasmin to gain ready access to the cleavage sites of various haemagglutinins and fusion glycoproteins after expression of these viral proteins on the cell surface. These findings suggest that mini-plasmin in the airway may play a pivotal role in the spread of viruses and their pathogenicity.

Natural killer (NK) cells destroy virus-infected and tumour cells, apparently without the need for previous antigen stimulation. In part, target cells are recognized by their diminished expression of major histocompatibility complex (MHC) class I molecules, which normally interact with inhibitory receptors on the NK cell surface. NK cells also express triggering receptors that are specific for non-MHC ligands; but the nature of the ligands recognized on target cells is undefined. NKp46 is thought to be the main activating receptor for human NK cells. Here we show that a soluble NKp46-immunoglobulin fusion protein binds to both the haemagglutinin of influenza virus and the haemagglutinin-neuraminidase of parainfluenza virus. In a substantial subset of NK cells, recognition by NKp46 is required to lyse cells expressing the corresponding viral glycoproteins. The binding requires the sialylation of NKp46 oligosaccharides, which is consistent with the known sialic binding capacity of the viral glycoproteins. These findings indicate how NKp46-expressing NK cells may recognize target cells infected by influenza or parainfluenza without the decreased expression of target-cell MHC class I protein.

The Sendai virus L and P proteins comprise the viral RNA-dependent RNA polymerase. The L subunit is thought to be responsible for all the catalytic activities necessary for viral RNA synthesis. Sequence alignment of the L proteins of negative-stranded RNA viruses revealed six regions of good conservation, domains I-VI, which are thought to correspond to functional domains of the protein. Domain V, amino acids 1129-1378, has no recognizable motifs, and to date its function is unknown. Site-directed mutagenesis was used to construct mutations across domain V. The mutant L proteins were all stably expressed and were tested for activity in several aspects of RNA synthesis. One set of mutants could synthesize more le+ RNA than mRNA, while two mutants showed the opposite phenotype, synthesizing more mRNA than le+ RNA. The majority of the mutants could synthesize mRNA, but not genome RNA in vitro, thus uncoupling transcription and replication. Several mutants could replicate in vivo, but not in vitro, at nearly wildtype L levels, suggesting the importance of the intact host cell for replication in some instances. One L mutant, SS24, was virtually inactive in all viral RNA synthesis. SS24 L was able to form a polymerase complex that recognized the nucleocapsid template, and thus these amino acids are essential for the initiation of RNA synthesis., (Copyright 2000 Academic Press.)

Sendai virus matrix protein (M protein) is critically important for virus assembly and budding and is presumed to interact with viral glycoproteins on the outer side and viral nucleocapsid on the inner side. However, since M protein alone binds to lipid membranes, it has been difficult to demonstrate the specific interaction of M protein with HN or F protein, the Sendai viral glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and flotation in sucrose gradients, we report that the membrane-bound M protein expressed alone or coexpressed with heterologous glycoprotein (influenza virus HA) was totally TX-100 soluble but the membrane-bound M protein coexpressed with HN or F protein either individually or together was predominantly detergent-resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of F protein facilitated binding of M protein to detergent-resistant membranes. Analysis of the membrane association of M protein in the early and late phases of the Sendai virus infectious cycle revealed that the interaction of M protein with mature glycoproteins that associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of the membrane-bound M protein. Immunofluorescence analysis by confocal microscopy also demonstrated that in Sendai virus-infected cells, a fraction of M protein colocalized with F and HN proteins and that some M protein also became associated with the F and HN proteins while they were in transit to the plasma membrane via the exocytic pathway. These studies indicate that F and HN interact with M protein in the absence of any other viral proteins and that F associates with M protein via its cytoplasmic tail and transmembrane domain., (Copyright 2000 Academic Press.)

Receptor aggregation is believed to be an important step in the attachment of membrane enveloped virus' to target cell membranes. A likely receptor for Sendai virus is the ganglioside GD1a. In this work we have studied the membrane diffusion of the fluorescent ganglioside NBD-GD1a on the surface of CV-1 cells with standard photobleaching techniques. Using confocal laser scanning microscopy (CLSM) and Image Correlation Spectroscopy (ICS) NBD-GD1a is shown to exist in at least two populations: dispersed and aggregated. By quantifying the distribution of NBD-GD1a pre- and post-incubation with Sendai virus it is shown that the virus induces a dose-dependent clustering of NBD-GD1a. Image cross-correlation spectroscopy (ICCS) is used to further quantitatively characterize this clustering by demonstrating that it occurs due to binding of virus to the dispersed population of NBD-GD1a.

The P/C mRNA of Sendai virus (SeV) encodes a nested set of accessory proteins, C', C, Y1, and Y2, referred to collectively as C proteins, using the +1 frame relative to the open reading frame of phospho (P) protein and initiation codons at different positions. The C proteins appear to be basically nonstructural proteins as they are found abundantly in infected cells but greatly underrepresented in the virions. We previously created a 4C(-) SeV, which expresses none of the four C proteins, and concluded that the C proteins are categorically nonessential gene products but greatly contribute to viral full replication and infectivity (A. Kurotani et al., Genes Cells 3:111-124, 1998). Here, we further characterized the 4C(-) virus multiplication in cultured cells. The viral protein and mRNA synthesis was enhanced with the mutant virus relative to the parental wild-type (WT) SeV. However, the viral yields were greatly reduced. In addition, the 4C(-) virions appeared to be highly anomalous in size, shape, and sedimentation profile in a sucrose gradient and exhibited the ratios of infectivity to hemagglutination units significantly lower than those of the WT. In the WT infected cells, C proteins appeared to colocalize almost perfectly with the matrix (M) proteins, pretty well with an external envelope glycoprotein (hemagglutinin-neuraminidase [HN]), and very poorly with the internal P protein. In the absence of C proteins, there was a significant delay of the incorporation of M protein and both of the envelope proteins, HN and fusion (F) proteins, into progeny virions. These results strongly suggest that the accessory and basically nonstructural C proteins are critically required in the SeV assembly process. This role of C proteins was further found to be independent of their recently discovered function to counteract the antiviral action of interferon-alpha/beta. SeV C proteins thus appear to be quite versatile.

Sexual dimorphism of the rodent brain is manifested by the epigenetic action of gonadal steroids. Our previous research identified the granulin (grn) precursor gene as a sex steroid-inducible gene, which was shown to be expressed more abundantly in male than female neonates at the mediobasal hypothalamic area. Grn is a 6-kDa polypeptide promoting or inhibiting the growth of epithelial cells and hematocytes in vitro. In this study, effects on male sexual behavior of male were pursued under conditions in which grn gene expression was suppressed during the critical period. To this end, an antisense oligodeoxynucleotide (ODN) of the grn precursor gene was designed, incorporated into inactivated Sendai virus (HVJ)-liposome complexes, and infused into the third ventricle of 2-day-old male rats. Two different control treatments were used: the first consisted of a control sequence ODN that had little homology to known mRNAs; the second of vehicle (HVJ-liposome) alone. After maturation, animals treated with antisense ODN of grn displayed significantly lower scores than control males on various parameters assessing sexual behavior; i.e., mount, intromission, and ejaculation. The antisense ODN, however, did not affect body growth or serum concentrations of testosterone and luteinizing hormone. Further, there was no significant difference in the volume of the sexual dimorphic nucleus of the preoptic area between antisense ODN-treated and control animals. It was shown that inadequate expression of the grn gene in the brain of male neonatal rats during the critical period suppressed the induction of some type of male sexual behavior, suggesting the grn was involved in the process of masculinization of the rat brain.

Sendai virus fuses efficiently with small and large unilamellar vesicles of the lipid 1,2-di-n-hexadecyloxypropyl-4- (beta-nitrostyryl) phosphate (DHPBNS) at pH 7.4 and 37 degrees C, as shown by lipid mixing assays and electron microscopy. However, fusion is strongly inhibited by oligomerization of the head groups of DHPBNS in the bilayer vesicles. The enthalpy associated with fusion of Sendai virus with DHPBNS vesicles was measured by isothermal titration microcalorimetry, comparing titrations of Sendai virus into (i) solutions of DHPBNS vesicles (which fuse with the virus) and (ii) oligomerized DHPBNS vesicles (which do not fuse with the virus), respectively. The observed heat effect of fusion of Sendai virus with DHPBNS vesicles is strongly dependent on the buffer medium, reflecting a partial charge neutralization of the Sendai F and HN proteins upon insertion into the negatively-charged vesicle membrane. No buffer effect was observed for the titration of Sendai virus into oligomerized DHPBNS vesicles, indicating that inhibition of fusion is a result of inhibition of insertion of the fusion protein into the target membrane. Fusion of Sendai virus with DHPBNS vesicles is endothermic and entropy-driven. The positive enthalpy term is dominated by heat effects resulting from merging of the protein-rich viral envelope with the lipid vesicle bilayers rather than by the fusion of the viral with the vesicle bilayers per se., (Copyright 2000 Academic Press.)

The matrix (M) protein of vesicular stomatitis virus (VSV) was reported to form vesicles on the cell surface and subsequently to be released into the cultured medium when expressed from cDNA by virus vectors. To further investigate VSV M activity, we generated a recombinant Sendai virus (SeV) expressing the VSV M protein (SeV-M(VSV)). When cells were infected with SeV-M(VSV), VSV M was found abundantly in the culture medium. Electron microscopy demonstrated the budding of two-membraned vesicles (>/= 0.8 microm in diameter) from the infected cells. The outer membrane of the vesicle was derived from the plasma membrane and the inner one possibly derived from the membrane of an intracellular vesicle. Immuno-gold labeling showed that VSV M was exclusively located in a double-layered region. The released membranes were divided into three parts: the VSV M vesicles with SeV F and HN glycoproteins, SeV particles, and vesicles associated with the cytosolic components. The last abundantly contained phosphorylated SeV matrix (M) protein, which is not released in a usual SeV infection. Furthermore the VSV M protein expressed without using a virus vector was efficiently released into the culture medium. These results suggest that the VSV M protein has a budding activity per se and that SeV proteins are passively involved in the release of VSV M., (Copyright 1999 Academic Press.)

Sendai virus (SeV) renders cells unresponsive to interferon (IFN)-alpha. To identify viral factors involved in this process, we examined whether recombinant SeVs, which could not express V protein, subsets of C proteins (C, C', Y1 and Y2) or any of four C proteins, retained the capability of impeding IFN-alpha-mediated responses. Among these viruses, only the 4C knockout virus completely lost the ability to suppress the induction of IFN-alpha-stimulated gene products and the subsequent establishment of an anti-viral state. These findings reveal crucial roles of the SeV C proteins in blocking IFN-alpha-mediated responses.

Wild-type Sendai virus is exclusively pneumotropic in mice. Protease activation mutants, ts-f1 and F1-R, were isolated from persistently infected tissue culture cells. Additional mutants were isolated from wild-type Sendai virus with phenotypes similar to the pantropic mutant, F1-R. The genome of the mutants was sequenced and mutations were revealed in several proteins encoded by the genes. Three of the six mutations in the fusion (F) proteins were considered prime candidates for the determinant of pantropism. Characterization of the mutants led to the finding that the exchange (Ser to Pro) residue 115 next to the cleavage site of the F protein was the primary determinant that resulted in the enhanced cleavability of the F protein. Another important finding was bipolar budding of F1-R in polarized epithelial cells and mouse bronchial epithelium. This has been attributed to two mutations in the matrix (M) protein, at residues 128 (Asp to Gly) and 210 (Ile to Thr). Thus the determinants of pantropism of F1-R are protease activation of the F protein and bipolar budding attributed to the mutated M protein and enhanced disruption of microtubules.

We have studied the relationship between the Sendai virus (SeV) C proteins (a nested set of four proteins initiated at different start codons) and the interferon (IFN)-mediated antiviral response in IFN-competent cells in culture. SeV strains containing wild-type or various mutant C proteins were examined for their ability (i) to induce an antiviral state (i.e., to prevent the growth of vesicular stomatitis virus [VSV] following a period of SeV infection), (ii) to induce the elevation of Stat1 protein levels, and (iii) to prevent IFN added concomitant with the SeV infection from inducing an antiviral state. We find that expression of the wild-type C gene and, specifically, the AUG114-initiated C protein prevents the establishment of an antiviral state: i.e., cells infected with wild-type SeV exhibited little or no increase in Stat1 levels and were permissive for VSV replication, even in the presence of exogenous IFN. In contrast, in cells infected with SeV lacking the AUG114-initiated C protein or containing a single amino acid substitution in the C protein, the level of Stat1 increased and VSV replication was inhibited. The prevention of the cellular IFN-mediated antiviral response appears to be a key determinant of SeV pathogenicity.

The Sendai virus P protein is an essential component of the viral RNA polymerase (P-L complex) required for RNA synthesis. To identify amino acids important for P-L binding, site-directed mutagenesis of the P gene changed 17 charged amino acids, singly or in groups, and two serines to alanine within the L binding domain from amino acids 408 to 479. Each of the 10 mutants was wild type for P-L and P-P protein interactions and for binding of the P-L complex to the nucleocapsid template, yet six showed a significant inhibition of in vitro mRNA and leader RNA synthesis. To determine if binding was instead hydrophobic in nature, five conserved hydrophobic amino acids in this region were also mutated. Each of these P mutants also retained the ability to bind to L, to itself, and to the template, but two gave a severe decrease in mRNA and leader RNA synthesis. Since all of the mutants still bound L, the data suggest that L binding occurs on a surface of P with a complex tertiary structure. Wild-type biological activity could be restored for defective polymerase complexes containing two P mutants by the addition of wild-type P protein alone, while the activity of two others could not be rescued. Gradient sedimentation analyses showed that rescue was not due to exchange of the wild-type and mutant P proteins within the P-L complex. Mutants which gave a defective RNA synthesis phenotype and could not be rescued by P establish an as-yet-unknown role for P within the polymerase complex, while the mutants which could be rescued define regions required for a P protein function independent of polymerase function.

Alanine substitution mutations in the Sendai virus nucleocapsid (NP) protein have defined highly conserved hydrophobic and charged residues from amino acids (aa) 362 to 371 that are essential for function of the protein in RNA replication. Mutant NP362, which had the change F362A, was incapable of supporting in vitro RNA replication. NP362 expressed alone formed extended oligomers which exhibited an abnormal morphology and density suggesting that these particles were not associated with any RNA. Mutant NP364, which had changes L362A and G365A, was also inactive in RNA replication; however, this was because the protein was unstable and did not form NP-NP complexes. Mutant NP370 mutant, which had changes K370A and D371A, was inactive in in vitro replication, although it could form the required NP0-P and NP-NP protein complexes. The self-assembled nucleocapsid-like particles formed by NP370 alone had a morphology like that of wild-type NP and banded in CsCl as ribonucleoprotein particles, suggesting that they contained cellular RNA. These data suggest that the replication defect of NP370 may be in the ability to specifically encapsidate Sendai virus genome RNA. Mutant NP373, where nonconserved charged residues at aa 373 and 375 were substituted with alanine, gave a wild-type phenotype. Thus these amino acids are not required for either protein-protein interactions or in vitro Sendai virus RNA replication.

RNA-dependent RNA polymerases of single-stranded, negative-sense RNA viruses comprise a phosphoprotein (P) and a large protein. The constitutive phosphorylation of the P protein in these viruses is highly conserved, yet the functional significance of phosphorylation is enigmatic. To approach this problem, phosphorylation sites were determined in two closely related paramyxovirus P proteins. Sendai virus (SV) is a prototypic paramyxovirus. Previously, using a phosphopeptide mapping technique, the primary constitutive phosphorylation site of SV P protein was mapped to Ser-249. Phosphorylation at Ser-249 is dependent on the presence of Pro-250. Human parainfluenza virus type 1 (HPIV-1) P protein has 66% similarity to SV P protein and its predicted secondary structure is highly similar to that of SV P protein. However, there is no obvious conserved phosphorylation site in HPIV-1 P protein. Using the phosphopeptide mapping strategy, the constitutive phosphorylation sites of HPIV-1 P protein were mapped. The HPIV-1 P protein is primarily phosphorylated at Ser-120. Phosphorylation at Ser-120 is dependent on the presence of Pro-121. It also has a minor phosphorylation site at Ser-184. The sequence at Ser-184 does not match any consensus phosphorylation target site for the known kinases. Significantly, the P proteins from both viruses are constitutively and primarily phosphorylated at one serine and the phosphorylation of that serine is dependent on the presence of a proline on its carboxyl side.

The interferon regulatory factor 3 (IRF-3) gene encodes a 55-kDa protein which is expressed constitutively in all tissues. In unstimulated cells, IRF-3 is present in an inactive cytoplasmic form; following Sendai virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues located in the carboxy terminus. Virus-induced phosphorylation of IRF-3 leads to cytoplasmic to nuclear translocation of phosphorylated IRF-3, association with the transcriptional coactivator CBP/p300, and stimulation of DNA binding and transcriptional activities of virus-inducible genes. Using yeast and mammalian one-hybrid analysis, we now demonstrate that an extended, atypical transactivation domain is located in the C terminus of IRF-3 between amino acids (aa) 134 and 394. We also show that the C-terminal domain of IRF-3 located between aa 380 and 427 participates in the autoinhibition of IRF-3 activity via an intramolecular association with the N-terminal region between aa 98 and 240. After Sendai virus infection, an intermolecular association between IRF-3 proteins is detected, demonstrating a virus-dependent formation of IRF-3 homodimers; this interaction is also observed in the absence of virus infection with a constitutively activated form of IRF-3. Substitution of the C-terminal Ser-Thr phosphorylation sites with the phosphomimetic Asp in the region ISNSHPLSLTSDQ between amino acids 395 and 407 [IRF-3(5D)], but not the adjacent S385 and S386 residues, generates a constitutively activated DNA binding form of IRF-3. In contrast, substitution of S385 and S386 with either Ala or Asp inhibits both DNA binding and transactivation activities of the IRF-3(5D) protein. These studies thus define the transactivation domain of IRF-3, two domains that participate in the autoinhibition of IRF-3 activity, and the regulatory phosphorylation sites controlling IRF-3 dimer formation, DNA binding activity, and association with the CBP/p300 coactivator.

Paramyxoviruses are responsible for significant human mortality and disease worldwide, but the molecular mechanisms underlying their entry into host cells remain poorly understood. We have solved the crystal structure of a fragment of the simian parainfluenza virus 5 fusion protein (SV5 F), revealing a 96 A long coiled coil surrounded by three antiparallel helices. This structure places the fusion and transmembrane anchor of SV5 F in close proximity with a large intervening domain at the opposite end of the coiled coil. Six amino acids, potentially part of the fusion peptide, form a segment of the central coiled coil, suggesting that this structure extends into the membrane. Deletion mutants of SV5 F indicate that putative flexible tethers between the coiled coil and the viral membrane are dispensable for fusion. The lack of flexible tethers may couple a final conformational change in the F protein directly to the fusion of two bilayers.

Sendai virus is able to fuse with liposomes even without virus receptors. To determine the roles of envelope protein, hemagglutinin-neuraminidase (HN) and fusion (F) protein, in Sendai virus-liposome fusion, we treated the virus with proteases and examined its fusion with liposomes and the conditions of HN and F protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting analysis showed that the virus treated with 150 units/ml of trypsin, which inactivated selectively hemolysis activity, maintained intact HN, F and partially digested F (32 kDa) protein, while virus treated with 15,000 units/ml of trypsin, which inactivated both hemolysis and neuraminidase activity, had only a 15-kDa digested HN protein and completely digested F protein. The former fused with liposomes, but the latter did not. In the virus treated with chymotrypsin, which lost both hemolysis and neuraminidase activity, F protein was intact, while HN protein was degraded to 15 kDa; in this case the virus fused with liposomes. As the virus with 15-kDa HN protein fused with liposomes and that with 20-kDa protein did not, HN protein does not appear to play any role in virus-liposome fusion. The virus that fused with liposomes had intact F protein. We conclude that Sendai virus-liposome fusion is strongly dependent on the presence of intact F protein, but not HN protein.

Virus infection of cell cultures induces the synthesis of various cytokines which can either inhibit or stimulate virus replication. The Sendai virus induces large quantities of biologically active interferon (IFN-alphan3) in human peripheral blood leukocytes (hPBL) in vitro, as well as many other cytokines. The supernatants of Sendai virus-infected hPBL contained biologically active IFN-alphan3, significant amounts of immunogenic IFN-gamma, monokines (IL-1alpha, IL-beta, TNF-alpha), lymphokines (IL-6, TNF-beta), growth factor (PDGF-AB) and small concentrations of IL-2 and GM-CSF. The analysis of the influence of the Sendai virus inactivation by lowering pH 2.0 on the cytokine concentrations showed that IL-1alpha, TNF-alpha, TNF-beta and IFN-gamma are susceptible to acid conditions, while IFN-alphan3, IL-1beta, IL-6 and IL-2 concentrations remained unchanged., (Copyright 1999 Academic Press.)

The fusion (F) protein of the paramyxovirus SV5 contains two heptad repeat regions, HRA adjacent to the fusion peptide and HRB proximal to the transmembrane domain. Peptides, N-1 and C-1, respectively, corresponding to these heptad repeat regions form a thermostable, alpha-helical trimer of heterodimers (S. B. Joshi, R. E. Dutch, and R. A. Lamb (1998). Virology 248, 20-34). Further characterization of the N-1/C-1 complex indicated that the C-1 peptides, which are predicted to residue on the outside of the complex, are resistant to digestion by several proteases when present in the complex. Only proteinase K digested most of the C-1 peptide, though the small remaining protease protected fragment of C-1 confers extreme thermostability on the proteinase-K-resistant N-1 trimeric coiled-coil. Carboxypeptidase Y digestion of the N-1/C-1 complex indicates that the C-1 peptides associate in an antiparallel orientation relative to the N-1 peptides. Electron microscopy of the N-1/C-1 complex showed a rod-shaped complex with an average length of 9.7 nm, consistent with all of N-1 existing as an alpha helix. Mutations at heptad repeat a and d residues of N-1, positions that are predicted to point inward to the center of the N-1 trimeric coiled-coil, were found to have varying effects as analyzed by circular dichroism measurements. The mutation I137M did not affect the helical structure of the isolated N-1 peptide but did affect the thermostability of the N-1/C-1 complex. Mutations L140M and L161M perturbed the helical structure formed by N-1 in isolation but did not affect formation of a thermostable N-1/C-1 complex. Finally, a peptide, SV5 F 255-293, corresponding to a proposed leucine zipper region, was analyzed for effects on N-1, C-1, or the N-1/C-1 complex. Circular dichroism analysis demonstrated that while the presence of peptide 255-293 increased the helical signal from either N-1 or the N-1/C-1 complex, no change in thermostability was observed, indicating that this region is not a component of the final, most stable core of the F protein., (Copyright 1999 Academic Press.)

Peptides derived from conserved heptad-repeat regions of several viruses have been shown recently to inhibit virus-cell fusion. To find out their possible role in the fusion process, two biologically active heptad-repeat segments of the fusion protein (F) of Sendai virus, SV-150 (residues 150-186), and SV-473 (residues 473-495) were synthesized, fluorescently labeled and spectroscopically characterized for their structure and organization in solution and within the membrane. SV-150 was found to be 50-fold less active than SV-473 in inhibiting Sendai virus-cell fusion. Circular dichroism (CD) spectroscopy revealed that in aqueous solution, the peptides are self-associated and adopt low alpha-helical structure. However, when the two peptides are mixed together, their alpha-helical content significantly increases. Fluorescence studies, CD, and polarized attenuated total reflection infrared (ATR-FTIR) spectroscopy showed that both peptides, alone or as a complex, bind strongly to negatively charged and zwitterionic phospholipid membranes, dissociate therein into alpha-helical monomers, but do not perturb the lipid packing of the membrane. The ability of the peptides to interact with each other in solution may be correlated with antiviral activity, whereas their ability to interact with the membrane, together with their location near the fusion peptide and the transmembrane domain, suggests a revision to the currently accepted model for viral-induced membrane fusion. In the revised model, in the sequence of events associated with viral entry, the two heptad-repeat sequences may assist in bringing the viral and cellular membranes closer, thus facilitating membrane fusion., (Copyright 1999 Academic Press.)

In the assembly of paramyxoviruses, interactions between viral proteins are presumed to be specific. The focus of this study is to elucidate the protein-protein interactions during the final stage of viral assembly that result in the incorporation of the viral envelope proteins into virions. To this end, we examined the specificity of HN incorporation into progeny virions by transiently transfecting HN cDNA genes into Sendai virus (SV)-infected cells. SV HN expressed from cDNA was efficiently incorporated into progeny Sendai virions, whereas Newcastle disease virus (NDV) HN was not. This observation supports the theory of a selective mechanism for HN incorporation. To identify the region on HN responsible for the selective incorporation, we constructed chimeric SV and NDV HN cDNAs and evaluated the incorporation of expressed proteins into progeny virions. Chimera HN that contained the SV cytoplasmic domain fused to the transmembrane and external domains of the NDV HN was incorporated to SV particles, indicating that amino acids in the cytoplasmic domain are responsible for the observed specificity. Additional experiments using the chimeric HNs showed that 14 N-terminal amino acids are sufficient for the specificity. Further analysis identified five consecutive amino acids (residues 10 to 14) that were required for the specific incorporation of HN into SV. These residues are conserved among all strains of SV as well as those of its counterpart, human parainfluenza virus type 1. These results suggest that this region near the N terminus of HN interacts with another viral protein(s) to lead to the specific incorporation of HN into progeny virions.

The membrane fusion reaction promoted by the paramyxovirus simian virus 5 (SV5) and human parainfluenza virus type 3 (HPIV-3) fusion (F) proteins and hemagglutinin-neuraminidase (HN) proteins was characterized when the surface densities of F and HN were varied. Using a quantitative content mixing assay, it was found that the extent of SV5 F-mediated fusion was dependent on the surface density of the SV5 F protein but independent of the density of SV5 HN protein, indicating that HN serves only a binding function in the reaction. However, the extent of HPIV-3 F protein promoted fusion reaction was found to be dependent on surface density of HPIV-3 HN protein, suggesting that the HPIV-3 HN protein is a direct participant in the fusion reaction. Analysis of the kinetics of lipid mixing demonstrated that both initial rates and final extents of fusion increased with rising SV5 F protein surface densities, suggesting that multiple fusion pores can be active during SV5 F protein-promoted membrane fusion. Initial rates and extent of lipid mixing were also found to increase with increasing influenza virus hemagglutinin protein surface density, suggesting parallels between the mechanism of fusion promoted by these two viral fusion proteins.

We have shown previously that the length of cytoplasmic tails influences the selection of lipid substrates for palmitoylation of influenza viral hemagglutinin esterase fusion (HEF) and hemagglutinin (HA) glycoproteins [Veit et al. (1996) Biochem. J. 318, 163-172; Reverey et al. (1996) J. Biol. Chem. 271, 23607-23610]. Using a series of new chimeric mutant proteins derived from acylated influenza virus HA (subtype H7) and from nonacylated Sendai virus fusion protein (F, strain Z), we report here that palmitoylation levels depend on the type of transmembrane or cytoplasmic domain, or both, present in the expression products and that cysteine residues placed close to the cytoplasmic membrane border are not sufficient for acylation. By inserting stretches of the HA transmembrane domain into a nonacylated mutant of Sendai F (FCys), we induce palmitoylation after expression in CV.1 cells, and the level of fatty acid transfer increases with the length of the HA-derived insert. A five-amino-acid shift of the HA transmembrane domain severely augments fatty acid transfer. Our data suggest that the influenza virus HA contains complex conformational signals for palmitoylation that are mainly located within the transmembrane domain but also involve the C-tail region, whereas the extracellular (luminal) domain has only marginal influence on palmitoylation., (Copyright 1998 Academic Press.)

The Sendai virus P/C mRNA expresses eight primary translation products by using a combination of ribosomal choice and cotranscriptional mRNA editing. The longest open reading frame (ORF) of the mRNA starts at AUG104 (the second initiation site) and encodes the 568-amino-acid P protein, an essential subunit of the viral polymerase. The first (ACG81), third (ATG114), fourth (ATG183), and fifth (ATG201) initiation sites are used to express a C-terminal nested set of polypeptides (collectively named the C proteins) in the +1 ORF relative to P, namely, C', C, Y1, and Y2, respectively. Leaky scanning accounts for translational initiation at the first three start sites (a non-ATG followed by ATGs in progressively stronger contexts). Consistent with this, changing ACG81/C' to ATG (GCCATG81G) abrogates expression from the downstream ATG104/P and ATG114/C initiation codons. However, expression of the Y1 and Y2 proteins remains normal in this background. We now have evidence that initiation from ATG183/Y1 and ATG201/Y2 takes place via a ribosomal shunt or discontinuous scanning. Scanning complexes appear to assemble at the 5' cap and then scan ca. 50 nucleotides (nt) of the 5' untranslated region before being translocated to an acceptor site at or close to the Y initiation codons. No specific donor site sequences are required, and translation of the Y proteins continues even when their start codons are changed to ACG. Curiously, ATG codons (in good contexts) in the P ORF, placed either 16 nt upstream of Y1, 29 nt downstream of Y2, or between the Y1 and Y2 codons, are not expressed even in the ACGY1/ACGY2 background. This indicates that ATG183/Y1 and ATG201/Y2 are privileged start sites within the acceptor site. Our observations suggest that the shunt delivers the scanning complex directly to the Y start codons.

The paramyxovirus fusion (F) protein mediates membrane fusion. The biologically active F protein consists of a membrane distal subunit, F2, and a membrane-anchored subunit, F1. We have identified a highly stable structure composed of peptides derived from the F1 heptad repeat A, which abuts the hydrophobic fusion peptide (peptide N-1), and the F1 heptad repeat B, located 270 residues downstream and adjacent to the transmembrane domain (peptides C-1 and C-2). In isolation, peptide N-1 is 47% alpha-helical and peptide C-1 and C-2 are unfolded. When mixed together, peptides N1 + C1 form a thermostable (Tm >90 degreesC), 82% alpha-helical, discrete trimer of heterodimers (mass 31,300 Mr) that is resistant to denaturation by 2% SDS at 40 degreesC. We suggest that this alpha-helical trimeric complex represents the core most stable form of the F protein that either is fusion competent or forms after fusion has occurred. Peptide C-1 is a potent inhibitor of both the lipid mixing and the aqueous content mixing fusion activity of the SV5 F protein. In contrast, peptides N-1 and N-2 inhibit cytoplasmic content mixing but not lipid mixing, leading to a stable hemifusion state. Thus, these peptides define functionally different steps in the fusion process. The parallels among both the fusion processes and the protein structures of paramyxovirus F proteins, HIV gp41, and influenza virus hemagglutinin are discussed, as the analogies are indicative of a conserved paradigm for fusion promotion among fusion proteins from widely disparate viruses., (Copyright 1998 Academic Press.)

An analysis of the R18 fusion assay was made during the fusion of the Sendai virus with erythrocyte ghosts. The increase in R18 fluorescence, reflecting the interaction process, was evaluated in terms of the different processes that in principle may contribute to this increase, that is, monomeric probe transfer, hemifusion, and complete fusion. To this end, the kinetics of the R18-labeled lipid mixing were compared to those obtained with an assay in which the fusion-monitoring probe, eosin-maleimide, was attached to the viral surface proteins. The experiments relied on the use of native and fusion-inactive viruses and studies involving viral and target membranes that were modified by the incorporation of the lysophospholipid. The total dequenching signal detected in the R18 assay consists of components from probe transferred without fusion and from fusion itself. At 37 degrees C, the initial rate of dequenching (within two minutes) was predominately from the probe diluted by fusion with little contribution from transfer. The dequenching signal due to the probe transfer without fusion occurred at temperatures as low as 10 degrees C and increased linearly with time. Complete fusion started at about 20-25 degrees C and increased sharply at 30 degrees C. The extent of hemifusion was deduced from the total R18 dequenching data and those of the eosin-maleimide labeled protein dilution method for the limiting cases; the analysis indicates that hemifusion started at about 15 degrees C and increased over the range 20-25 degrees C. The initial rate of dequenching of the R18 assay measured within 2 min gives an accurate measure of membrane fusion above 30 degrees C.

Wild-type Sendai virus expresses three proteins containing the N-terminal half of the P protein open reading frame due to mRNA editing; a full-length P protein (ca. 70% of the total), a V protein with the N-terminal half fused to a Cys-rich Zn(2+)-binding domain (ca. 25% of the total), and a W protein representing the N-terminal half alone (ca. 5% of the total). To examine the role of these proteins in the virus life cycle, we have prepared recombinant viruses in which the normal V mRNA expresses a W protein (V-stop; 70% P, 30% W), one which cannot edit its P gene mRNA (delta 6A; 100% P), and one which overedits its mRNA like parainfluenza virus type 3 (swap/8;20-40% P, 30% V, 30% W). All these viruses were readily recovered and grew to similar titers in eggs, and except for the P gene products, cell lines individually infected with these viruses accumulated similar amounts of viral macromolecules. The relative competitive advantage of each virus was determined by multiple cycle coinfections of eggs and found to be rSeV-Vstop = rSeV-wt >> rSeV-delta 6A > rSeV-swap/8. On the other hand, rSeV-swap/8 underwent multiple cycles of replication in C57BI/6 mouse lungs and was highly virulent for these animals, whereas rSeV-delta 6A was avirulent in mice and this infection was quickly cleared. Remarkably, rSeV-Vstop appeared to be more virulent for inbred C57BI/6 mice than rSeV-wt, but was partially attenuated in infections of outbred ICR mice. Thus, the expression of either the V or the W proteins is sufficient for multiple cycles of infection and pathogenesis in C57BI/6 mice, whereas W can only partially substitute for V for pathogenesis in ICR mice.

We have identified a virus-activated factor (VAF) that binds to a regulatory element shared by different virus-inducible genes. We provide evidence that VAF contains two members of the interferon regulatory factor (IRF) family of transcriptional activator proteins (IRF-3 and IRF-7), as well as the transcriptional coactivator proteins p300 and CBP. Remarkably, VAF, as well as recombinant IRF-3 and IRF-7 proteins, binds very weakly to the interferon-beta (IFN-beta) gene promoter in vitro. However, in virus-infected cells, both proteins are recruited to the endogenous IFN-beta promoter as part of a protein complex that includes ATF-2/c-Jun and NF-kappa B. These observations provide a unique example of the coordinate activation of multiple transcriptional activator proteins and their highly cooperative assembly into a transcriptional enhancer complex in vivo.

Purpose: To evaluate the feasibility of introducing exogenous genes and phosphorothioate oligonucleotides into the anterior chamber tissues of rats and monkeys using the authors' fusogenic liposomes., Methods: Hemagglutinating virus of Japan liposomes containing LacZ DNA-high-mobility group 1 complexes or fluorescein isothiocyanate (FITC)-labeled phosphorothioate oligonucleotides were prepared and injected into the anterior chambers of rats (3 microliters) and rhesus monkeys (30 microliters). The expression of LacZ DNA was visualized histochemically by beta-Galactosidase assay and was followed for as long as 60 days in rats and 30 days in monkeys. FITC-labeled phosphorothioate oligonucleotides were observed by fluorescence microscopy for as long as 14 days in rats and 7 days in monkeys., Results: Injection of LacZ DNA-high-mobility group 1 complexes encapsulated in hemagglutinating virus of Japan liposomes resulted in blue staining in the trabecular meshwork and iris-ciliary body of rats and selectively in the trabecular meshwork of monkeys at the concentrations used. This LacZ expression lasted for as long as 14 days after injection in both animals. Phosphorothioate oligonucleotides (3 microM) also were introduced into the rat trabecular meshwork and iris-ciliary body and into the primate trabecular meshwork when encapsulated in hemagglutinating virus of Japan liposomes, although the injection of naked FITC-labeled phosphorothioate oligonucleotides at the same concentration resulted in little fluorescence in any anterior chamber tissue., Conclusions: This study shows that the use of hemagglutinating virus of Japan liposomes can transfer LacZ DNA and phosphorothioate oligonucleotides to adult rat and primate trabecular meshwork. This system may enable progress in glaucoma research and in the development of nonviral somatic gene therapy of the trabecular meshwork to treat glaucoma.

The Sendai virus fuses with host cell membranes in a pH-independent manner through an unknown mechanism. Here we report that mild trypsin pre-treatments of Sendai virions, for example 15 min at 4 degrees C, give Sendai virions the ability to fuse at a rate up to 10-fold higher than control. By using human erythrocytes as host cell membranes, viral fusion was assessed by hemolysis as well as fluorescence dequenching of octadecyl rhodamine B chloride. The mild protease treatment strikingly shortens the lag time taken by the virus to start the fusion process. Similar data were obtained on reconstituted Sendai virus envelope. Among proteases, tested as fusion enhancer, trypsin is more effective than either endoproteinase Lys-C, chymotrypsin, or endoproteinase Arg-C. After removal of trypsin from treated virions the fusion rate enhancement remains for hours at room temperature. The lack of protease specificity, together with the impossibility to detect any new N-terminal products, suggests that only a small percentage of viral envelope components are cleaved, still a large enough number to set the envelope in a ready-to-fuse state.