A I131V Substitution in the Fusion Glycoprotein of Human Parainfluenza Virus Type 1 Enhances Syncytium Formation and Virus Growth.

Human parainfluenza virus type 1 (hPIV1) has two spike glycoproteins, the hemagglutinin-neuraminidase (HN) glycoprotein as a receptor-binding protein and the fusion (F) glycoprotein as a membrane-fusion protein. The F glycoprotein mediates both membrane fusion between the virus and cell and membrane fusion between cells, called syncytium formation. Wild-type C35 strain (WT) of hPIV1 shows little syncytium formation of infected cells during virus growth. In the present study, we isolated a variant virus (Vr) from the WT that showed enhanced syncytium formation of infected cells by using our previously established hPIV1 plaque formation assay. Vr formed a larger focus and showed increased virus growth compared with WT. Sequence analysis of the spike glycoprotein genes showed that the Vr had a single amino acid substitution of Ile to Val at position 131 in the fusion peptide region of the F glycoprotein without any substitutions of the HN glycoprotein. The Vr F glycoprotein showed enhanced syncytium formation in F and HN glycoprotein-expressing cells. Additionally, expression of the Vr F glycoprotein increased the focus area of the WT-infected cells. The single amino acid substitution at position 131 in the F glycoprotein of hPIV1 gives hPIV1 abilities to enhance syncytium formation and increase cell-to-cell spread. The present study supports the possibility that hPIV1 acquires increased virus growth in vitro from promotion of direct cell-to-cell transmission by syncytium formation.

Functional analysis of amino acids at stalk/head interface of human parainfluenza virus type 3 hemagglutinin-neuraminidase protein in the membrane fusion process.

Human parainfluenza virus type 3 (hPIV3) is an important respiratory pathogen that causes the majority of viral pneumonia of infants and young children. hPIV3 can infect host cells through the synergistic action of hemagglutinin-neuraminidase (HN) protein and the homotypic fusion (F) protein on the viral surface. HN protein plays a variety of roles during the virus invasion process, such as promoting viral particles to bind to receptors, cleaving sialic acid, and activating the F protein. Crystal structure research shows that HN tetramer adopted a "heads-down" conformation, at least two heads dimmer on flank of the four-helix bundle stalk, which forms a symmetrical interaction interface. The stalk region determines interactions and activation of F protein in specificity, and the heads in down position statically shield these residues. In order to make further research on the function of these amino acids at the hPIV3 HN stalk/head interface, fifteen mutations (8 sites from stalk and 7 sites from head) were engineered into this interface by site-directed mutagenesis in this study. Alanine substitution in this region of hPIV3 HN had various effects on cell fusion promotion, receptor binding, and neuraminidase activity. Besides, L151A also affected surface protein expression efficiency. Moreover, I112A, D120A, and R122A mutations of the stalk region that were masked by global head in down position had influence on the interaction between F and HN proteins.

'a'-Position-mutated and G4-mutated hemagglutinin-neuraminidase proteins of Newcastle disease virus impair fusion and hemagglutinin-neuraminidase-fusion interaction by different mechanisms.

OBJECTIVES: To determine the effects of heptad repeat regions (HRs) and N-linked carbohydrate sites of the Newcastle disease virus hemagglutinin-neuraminidase (HN) protein on fusion of HN and fusion (F) proteins and HN-F interaction. METHODS: We mutated six 'a' residues in the HRs and four asparagines in N-linked carbohydrate sites to alanine in the HN protein. A vaccinia-T7 RNA polymerase expression system was used to express HN cDNAs in BHK-21 cells to determine the HN functions. Deglycosylation was treated with PGNase F digestion. The formation of HN-F protein complexes was determined by the coimmunoprecipitation assay. RESULTS: Each HR-mutated protein interfered with fusion and the HN-F interaction. The G4-mutated protein not only impaired fusion and HN-F interaction but also decreased activities in cell fusion promotion, hemadsorption and neuraminidase. CONCLUSIONS: It is assumed that two different mechanisms for mutations in these two regions are responsible for the decreased fusion promotion activity and the reduced ability of interaction with F protein. Mutations in the HRs impair fusion and HN-F interaction by altering the transmission of a signal from the globular domain to the F-specific region in the stalk, but the G4 mutation modulates fusion and HN-F interaction by the misfolding of some important structures.

Paramyxovirus entry.

The family Paramyxoviridae consists of a group of large, enveloped, negative-sense, single-stranded RNA viruses and contains many important human and animal pathogens. Molecular and biochemical characterization over the past decade has revealed an extraordinary breadth of biological diversity among this family of viruses. Like all enveloped viruses, paramyxoviruses must fuse their membrane with that of a receptive host cell as a prerequisite for viral entry and infection. Unlike most other enveloped viruses, the vast majority of paramyxoviruses contain two distinct membrane-anchored glycoproteins to mediate the attachment, membrane fusion and particle entry stages of host cell infection. The attachment glycoprotein is required for virion attachment and the fusion glycoprotein is directly involved in facilitating the merger of the viral and host cell membranes. Here we detail important functional, biochemical and structural features of the attachment and fusion glycoproteins from a variety of family members. Specifically, the three different classes of attachment glycoproteins are discussed, including receptor binding preference, their overall structure and fusion promotion activities. Recently solved atomic structures of certain attachment glycoproteins are summarized, and how they relate to both receptor binding and fusion mechanisms are described. For the fusion glycoprotein, specific structural domains and their proposed role in mediating membrane merger are illustrated, highlighting the important features of protease cleavage and associated tropism and virulence. The crystal structure solutions of both an uncleaved and a cleavage-activated metastable F are also described with emphasis on how small conformational changes can provide the necessary energy to mediate membrane fusion. Finally, the different proposed fusion models are reviewed, featuring recent experimental findings that speculate how the attachment and fusion glycoproteins work in concert to mediate virus entry.

A role for caveolin 1 in assembly and budding of the paramyxovirus parainfluenza virus 5.

Caveolin 1 (Cav-1) is an integral membrane protein that forms the coat structure of plasma membrane caveolae and regulates caveola-dependent functions. Caveolae are enriched in cholesterol and sphingolipids and are related to lipid rafts. Many studies implicate rafts as sites of assembly and budding of enveloped virus. We show that Cav-1 colocalizes with the paramyxovirus parainfluenza virus 5 (PIV-5) nucleocapsid (NP), matrix (M), and hemagglutinin-neuraminidase (HN) proteins. Moreover, electron microscopy shows that Cav-1 is clustered at sites of viral budding. HN, M, and F(1)/F(2) are associated with detergent-resistant membranes, and these proteins float on sucrose gradients with Cav-1-rich fractions. A complex containing Cav-1 with M, NP, and HN from virus-infected cells and a complex containing Cav-1 and M from M-transfected cells were found on coimmunoprecipitation. A role of Cav-1 in the PIV-5 life cycle was investigated by utilizing MCF-7 human breast cancer cells that stably express Cav-1 (MCF-7/Cav-1). PIV-5 entry into MCF-7 and MCF-7/Cav-1 was found to be Cav-1 independent. However, the interaction between HN and M proteins was dramatically reduced in the Cav-1 null MCF-7 cells, and PIV-5 grown in MCF-7 cells had a reduced infectivity. Similarly, when PIV-5 was grown in MDCK cells that stably expressed dominant negative Cav-1 (MDCK/P132LCav-1), the virus showed a reduced infectivity. Virions lacking Cav-1 were defective and contained high levels of host cellular proteins and reduced levels of HN and M. These data suggest that Cav-1 affects assembly and/or budding, and this is supported by the finding that Cav-1 is incorporated into mature viral particles.

A histidine switch in hemagglutinin-neuraminidase triggers paramyxovirus-cell membrane fusion.

Most paramyxovirus fusion proteins require coexpression of and activation by a homotypic attachment protein, hemagglutinin-neuraminidase (HN), to promote membrane fusion. However, the molecular mechanism of the activation remains unknown. We previously showed that the incorporation of a monohistidylated lipid into F-virosome (Sendai viral envelope containing only fusion protein) enhanced its fusion to hepatocytes, suggesting that the histidine residue in the lipid accelerated membrane fusion. Therefore, we explored whether a histidine moiety in HN could similarly direct activation of the fusion protein. In membrane fusion assays, the histidine substitution mutants of HN (H247A of Sendai virus and H245A of human parainfluenza virus 3) had impaired membrane fusion promotion activity without significant changes in other biological activities. Synthetic 30-mer peptides corresponding to regions of the two HN proteins containing these histidine residues rescued the fusion promoting activity of the mutants, whereas peptides with histidine residues substituted by alanine did not. These histidine-containing peptides also activated F-virosome fusion with hepatocytes both in the presence and in the absence of mutant HN in the virosome. We provide evidence that the HN-mimicking peptides promote membrane fusion, revealing a specific histidine "switch" in HN that triggers fusion.

Effects of hemagglutinin-neuraminidase protein mutations on cell-cell fusion mediated by human parainfluenza type 2 virus.

The monoclonal antibody M1-1A, specific for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 2 virus (HPIV2), blocks virus-induced cell-cell fusion without affecting the hemagglutinating and neuraminidase activities. F13 is a neutralization escape variant selected with M1-1A and contains amino acid mutations N83Y and M186I in the HN protein, with no mutation in the fusion protein. Intriguingly, F13 exhibits reduced ability to induce cell-cell fusion despite its multistep replication. To investigate the potential role of HPIV2 HN protein in the regulation of cell-cell fusion, we introduced these mutations individually or in combination to the HN protein in the context of recombinant HPIV2. Following infection at a low multiplicity, Vero cells infected with the mutant virus H-83/186, which carried both the N83Y and M186I mutations, remained as nonfused single cells at least for 24 h, whereas most of the cells infected with wild-type virus mediated prominent cell-cell fusion within 24 h. On the other hand, the cells infected with the mutant virus, carrying either the H-83 or H-186 mutation, mediated cell-cell fusion but less efficiently than those infected with wild-type virus. Irrespective of the ability to cause cell-cell fusion, however, every virus could infect all the cells in the culture within 48 h after the initial infection. These results indicated that both the N83Y and M186I mutations in the HN protein are involved in the regulation of cell-cell fusion. Notably, the limited cell-cell fusion by H-83/186 virus was greatly promoted by lysophosphatidic acid, a stimulator of the Ras and Rho family GTPases.

Insertion of the two cleavage sites of the respiratory syncytial virus fusion protein in Sendai virus fusion protein leads to enhanced cell-cell fusion and a decreased dependency on the HN attachment protein for activity.

Cell entry by paramyxoviruses requires fusion of the viral envelope with the target cell membrane. Fusion is mediated by the viral fusion (F) glycoprotein and usually requires the aid of the attachment glycoprotein (G, H or HN, depending on the virus). Human respiratory syncytial virus F protein (F(RSV)) is able to mediate membrane fusion in the absence of the attachment G protein and is unique in possessing two multibasic furin cleavage sites, separated by a region of 27 amino acids (pep27). Cleavage at both sites is required for cell-cell fusion. We have investigated the significance of the two cleavage sites and pep27 in the context of Sendai virus F protein (F(SeV)), which possesses a single monobasic cleavage site and requires both coexpression of the HN attachment protein and trypsin in order to fuse cells. Inclusion of both F(RSV) cleavage sites in F(SeV) resulted in a dramatic increase in cell-cell fusion activity in the presence of HN. Furthermore, chimeric F(SeV) mutants containing both F(RSV) cleavage sites demonstrated cell-cell fusion in the absence of HN. The presence of two multibasic cleavage sites may therefore represent a strategy to regulate activation of a paramyxovirus F protein for cell-cell fusion in the absence of an attachment protein.

Entry of parainfluenza virus into cells as a target for interrupting childhood respiratory disease.

Human parainfluenza viruses cause several serious respiratory diseases in children for which there is no effective prevention or therapy. Parainfluenza viruses initiate infection by binding to cell surface receptors and then, via coordinated action of the 2 viral surface glycoproteins, fuse directly with the cell membrane to release the viral replication machinery into the host cell's cytoplasm. During this process, the receptor-binding molecule must trigger the viral fusion protein to mediate fusion and entry of the virus into a cell. This review explores the binding and entry into cells of parainfluenza virus type 3, focusing on how the receptor-binding molecule triggers the fusion process. There are several steps during the process of binding, triggering, and fusion that are now understood at the molecular level, and each of these steps represents potential targets for interrupting infection.

Escaping from the cell: assembly and budding of negative-strand RNA viruses.

Negative-strand RNA virus particles are formed by a process that includes the assembly of viral components at the plasma membranes of infected cells and the subsequent release of particles by budding. Here, we review recent progress that has been made in understanding the mechanisms of negative-strand RNA virus assembly and bud- ding. Important topics for discussion include the key role played by the viral matrix proteins in assembly of viruses and viruslike particles, as well as roles played by additional viral components such as the viral glycoproteins. Various interactions that contribute to virus assembly are discussed, including interactions between matrix proteins and membranes, interactions between matrix proteins and glycoproteins, interactions between matrix proteins and nucleocapsids, and interactions that lead to matrix protein self-assembly. Selection of specific sites on plasma membranes to be used for virus assembly and budding is described, including the asymmetric budding of some viruses in polarized epithelial cells and assembly of viral components in lipid raft microdomains. Evidence for the involvement of cellular proteins in the late stages of rhabdovirus and filovirus budding is discussed as well as the possible involvement of similar host factors in the late stages of budding of other negative-strand RNA viruses.

Fusogenic activity of reconstituted newcastle disease virus envelopes: a role for the hemagglutinin-neuraminidase protein in the fusion process.

Enveloped viruses, such as newcastle disease virus (NDV), make their entry into the host cell by membrane fusion. In the case of NDV, the fusion step requires both transmembrane hemagglutinin-neuraminidase (HN) and fusion (F) viral envelope glycoproteins. The HN protein should show fusion promotion activity. To date, the nature of HN-F interactions is a controversial issue. In this work, we aim to clarify the role of the HN glycoprotein in the membrane fusion step. Four types of reconstituted detergent-free NDV envelopes were used, on differing in their envelope protein contents. Fusion of the different virosomes and erythrocyte ghosts was monitored using the octadecyl rhodamine B chloride assay. Only the reconstituted envelopes having the F protein, even in the absence of HN protein, displayed residual fusion activity. Treatment of such virosomes with denaturing agents affecting the F protein abolished fusion, indicating that the fusion detected was viral protein-dependent. Interestingly, the rate of fusion in the reconstituted systems was similar to that of intact viruses in the presence of the inhibitor of HN sialidase activity 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. The results show that the residual fusion activity detected in the reconstituted systems was exclusively due to F protein activity, with no contribution from the fusion promotion activity of HN protein.

Modulation of the activities of HN protein of Newcastle disease virus by nonconserved cysteine residues.

Comparisons of the sequences of the hemagglutinin-neuraminidase (HN) protein from thirteen different strains of Newcastle disease virus (NDV) show that while 12 cysteine residues are conserved in all strains, two cysteine residues are variably present (Sakaguchi et al. (1989) Virology 169, 260-272). One of these residues, at amino acid 6, is in the cytoplasmic domain. The other cysteine is at amino acid 123 in the ectodomain and is responsible for disulfide-linked HN dimers detected in some NDV strains (McGinnes and Morrison (1994) Virology 200, 470-483). To explore the role of these nonconserved residues in the structure and function of the protein, cysteine residues at amino acid 6 and 123 in the HN protein of the AV strain of NDV were mutated individually and in combination by site specific mutagenesis to serine and tryptophan, respectively. Proteins with mutations in either residue (C6S or C123W) or in both residues (C6S,123W) were transported to the cell surface. However, all three mutants had reduced attachment, neuraminidase, and fusion promotion activities. All three mutant proteins also showed an alteration in an antigenic site specific for oligomers of HN protein while all other antigenic sites were present at wild type levels. These results suggest that the nonconserved cysteine residues in the HN sequence may modulate the biological activities of the protein by affecting the oligomeric structure of the protein.

Role of a conserved sequence in the maturation and function of the NDV HN glycoprotein.

The hemagglutinin-neuraminidase (HN) proteins of viruses in the Paramyxovirus genus have a short conserved sequence, G(A, S)EGR(I, L, V). The role of this sequence in the intracellular processing and function of the Newcastle disease virus HN protein was explored by site directed mutagenesis. Mutations in this region fall into two categories. One set of mutants (G398A, E400D, R402K, and a deletion removing amino acids 400-403) was defective in folding. These mutant proteins formed little or no mature, disulfide linked oligomer. They had few or no antigenic sites found on the mature protein and they were transported to the cell surface poorly or not at all. The second class of mutants (A399G, G401A, G401L) was minimally affected in folding and intracellular transport. When normalized to surface expression, this group of mutant proteins had wild type levels of attachment activity, neuraminidase activity, and fusion promotion activity. Thus mutations in this region directly affect intracellular processing but not the biological activities of the protein. This sequence may, therefore, be conserved in the HN proteins of Paramyxoviruses because it is critical to the folding of the molecule.

An oligosaccharide at the C-terminus of the F-specific domain in the stalk of the human parainfluenza virus 3 hemagglutinin-neuraminidase modulates fusion.

The promotion of membrane fusion by the fusion (F) protein of human parainfluenza virus 3 (hPIV3) is dependent on a virus-specific contribution from the hemagglutinin-neuraminidase (HN) protein. By evaluation of chimeric hPIV3-Newcastle disease virus (NDV) HN proteins, we have previously shown that hPIV3-F-specificity is determined by a domain that extends from the middle of the membrane anchor to the 82nd residue in the ectodomain [Virology 209, (1995) 457; Arch. Virol. 13 (1997) 115]. If the corresponding NDV-derived residues replace the two C-terminal residues in this domain, no fusion is detected. However, these substitutions restore a glycosylation site present in NDV HN, but not in hPIV3 HN. Deletion of this site from a nested set of chimeras with hPIV3-derived N-terminal portions of decreasing length partially restores fusion, suggesting that an oligosaccharide near the top of hPIV3 HN stalk modulates fusion. In addition, further mutational analyses show that a chimera with only 125 N-terminal hPIV3-derived residues (72 in the stalk) actually promotes fusion more efficiently than the wt protein. These findings localize the C-terminus of the F-specific domain in hPIV3 HN a full 10 residues closer to the membrane than previously shown.

Function and immunogenicity of human parainfluenza virus 3 glycoproteins expressed by recombinant adenoviruses.

Human parainfluenza virus type 3 fusion (F) and hemagglutinin-neuraminidase (HN) cDNA sequences were inserted into the E3 region of the adenovirus type 5 genome. Cells infected with recombinant adenoviruses containing HPIV3 F (AdF) and HN (AdHN) sequences were shown to express HPIV3 F and HN proteins that were functional and immunogenic. The HN protein produced following AdHN infection was glycosylated, expressed on the surface of infected cells and exhibited both hemagglutinin and neuraminidase activities. AdF infection led to the synthesis of both the HPIV3 F0 precursor and its proteolytic cleavage product, F1. F proteins produced by AdF were glycosylated and expressed on the infected cell surface. Syncytium formation was observed in HeLa T4 cell monolayers upon coinfection with AdF and AdHN. The F and HN proteins expressed by recombinant adenoviruses were recognized by HPIV3 F- and HN-specific monoclonal antibodies. Mice injected intraperitoneally with AdF or AdHN produced antibodies that immunoprecipitated the appropriate HPIV3 glycoproteins and sera from immunized mice effectively neutralized HPIV3 virions. These results support future work using recombinant adenoviruses to study the immune response to individual HPIV3 glycoproteins as well as in protection studies using animal models.

Introduction of adhesive and costimulatory immune functions into tumor cells by infection with Newcastle Disease Virus.

We demonstrate in this study that infection of tumor cells by Newcastle Disease Virus (NDV) leads to changes in tumor cell surface adhesiveness and tumor immune costimulatory function. While adsorbtion of virions to the cell surface occurs after short-term (10 min) incubation and leads to cells expressing viral antigens at low antigen density (LAD), viral replication in the cytoplasm occurs within 5-24 h leading to tumor cells expressing viral antigens at high antigen density (HAD) as shown by quantitative FACS flow cytometry. Virus infected tumor cells showed an increased adhesiveness for erythrocytes and lymphocytes. When IL-2 preactivated human lymphocytes with cytotoxic potential were coincubated with 51Cr-labeled NDV-infected or non-infected human colon carcinoma cells increased lysis of the virus infected targets was observed. The virus mediated cell adhesion could be inhibited by monoclonal antibody (mAb) against the hemagglutinin-neuraminidase (HN) molecule but not by antibody against the fusion protein. HN cDNA transfectants also mediated increased lymphocyte adhesion in comparison to wild-type or neo-vector transfected control cells. Further experiments demonstrated that not only the adhesion domain of HN but also the neuraminidase plays a role in cell-cell interactions. A comparison of an NDV neuraminidase mutant of the strain Australian Victoria (AV-L1) with the parental AV strain revealed pronounced differences in their capacity to mediate lymphocyte binding and costimulatory activity. The mutant with highly decreased neuraminidase activity was very similar to NDV Ulster in adhesive and costimulatory activity while the parental line with high neuraminidase activity was negative for both functions. Costimulatory effects of NDV Ulster and AV-L1 were revealed when virions and suboptimal concentrations of anti-CD3 mAbs were coated to microtiter plates for induction of murine CD4 T cell proliferation. In human autologous mixed lymphocyte-tumor cell cultures up-regulation of T cell activation markers CD69 and CD25 was seen with NDV modified but not with non-modified tumor cells.

Paramyxovirus induced cell fusion. Hemagglutination and hemolysis is enhanced by methylphenidate.

The influence of methylphenidate (MP) upon cell fusion, hemagglutination and hemolysis induced by paramyxovirus was evaluated in vitro. A direct correlation between MP concentrations (range 40 mg/ml to 1.25 mg/ml) and hemagglutination and hemolysis induced by Newcastle disease virus (NDV) was found. Furthermore, MP (500 micrograms/ml) increased formation of the syncytia by LPM paramyxovirus in PK-15 cells. It is fair to speculate that MP leads to activation of the F and/or HN proteins in paramyxovirus by mechanisms other than proteolytic cleavage.

Single amino acid changes in the mumps virus haemagglutinin-neuraminidase and polymerase proteins are associated with neuroattenuation.

It has previously been shown that three amino acid changes, one each in the fusion (F; Ala/Thr-91-->Thr), haemagglutinin-neuraminidase (HN; Ser-466-->Asn) and polymerase (L; Ile-736-->Val) proteins, are associated with attenuation of a neurovirulent clinical isolate of mumps virus (88-1961) following serial passage in vitro. Here, using full-length cDNA plasmid clones and site-directed mutagenesis, it was shown that the single amino acid change in the HN protein and to a lesser extent, the change in the L protein, resulted in neuroattenuation, as assessed in rats. The combination of both amino acid changes caused neuroattenuation of the virus to levels previously reported for the clinical isolate following attenuation in vitro. The amino acid change in the F protein, despite having a dramatic effect on protein function in vitro, was previously shown to not be involved in the observed neuroattenuation, highlighting the importance of conducting confirmatory in vivo studies. This report provides additional supporting evidence for the role of the HN protein as a virulence factor and, as far as is known, is the first report to associate an amino acid change in the L protein with mumps virus neuroattenuation.