The role of stearate attachment to the hemagglutinin‐esterase‐fusion glycoprotein HEF of influenza C virus

The only spike of influenza C virus, the hemagglutinin‐esterase‐fusion glycoprotein (HEF) combines receptor binding, receptor hydrolysis and membrane fusion activities. Like other hemagglutinating glycoproteins of influenza viruses HEF is S‐acylated, but only with stearic acid at a single cysteine located at the cytosol‐facing end of the transmembrane region. Previous studies established the essential role of S‐acylation of hemagglutinin for replication of influenza A and B virus by affecting budding and/or membrane fusion, but the function of acylation of HEF was hitherto not investigated. Using reverse genetics we rescued a virus containing non‐stearoylated HEF, which was stable during serial passage and showed no competitive fitness defect, but the growth rate of the mutant virus was reduced by one log. Deacylation of HEF does neither affect the kinetics of its plasma membrane transport nor the protein composition of virus particles. Cryo‐electron microscopy showed that the shape of viral particles and the hexagonal array of spikes typical for influenza C virus were not influenced by this mutation indicating that virus budding was not disturbed. However, the extent and kinetics of haemolysis were reduced in mutant virus at 37°C, but not at 33°C, the optimal temperature for virus growth, suggesting that non‐acylated HEF has a defect in membrane fusion under suboptimal conditions.     

Hemagglutinin-esterase-fusion (HEF) protein of influenza C virus

Influenza C virus, a member of the Orthomyxoviridae family, causes flu-like disease but typically only with mild symptoms. Humans are the main reservoir of the virus, but it also infects pigs and dogs. Very recently, influenza C-like viruses were isolated from pigs and cattle that differ from classical influenza C virus and might constitute a new influenza virus genus. Influenza C virus is unique since it contains only one spike protein, the hemagglutinin-esterase-fusion glycoprotein HEF that possesses receptor binding, receptor destroying and membrane fusion activities, thus combining the functions of Hemagglutinin (HA) and Neuraminidase (NA) of influenza A and B viruses. Here we briefly review the epidemiology and pathology of the virus and the morphology of virus particles and their genome. The main focus is on the structure of the HEF protein as well as on its co- and posttranslational modification, such as N-glycosylation,disulfide bond formation, S-acylation and proteolytic cleavage into HEF1 and HEF2 subunits. Finally, we describe the functions of HEF: receptor binding, esterase activity and membrane fusion.     

Structural and functional analysis of the roles of influenza C virus membrane proteins in assembly and budding

Assembly and budding of the influenza C virus is mediated by three membrane proteins: the hemagglutinin-esterase-fusion glycoprotein (HEF), the matrix protein (CM1), and the ion channel (CM2). Here we investigated whether the formation of the hexagonal HEF arrangement, a distinctive feature of influenza C virions is important for virus budding. We used super resolution microscopy and found 250-nm sized HEF clusters at the plasma membrane of transfected cells, which were insensitive to cholesterol extraction and cytochalasin treatment. Overexpression of either CM1, CM2, or HEF caused the release of membrane-enveloped particles. Cryo-electron microscopy of the latter revealed spherical vesicles exhibiting the hexagonal HEF clusters. We subsequently used reverse genetics to identify elements in HEF required for this clustering. We found that deletion of the short cytoplasmic tail of HEF reduced virus titer and hexagonal HEF arrays, suggesting that an interaction with CM1 stabilizes the HEF clusters. In addition, we substituted amino acids at the surface of the closed HEF conformation and identified specific mutations that prevented virus rescue, others reduced virus titers and the number of HEF clusters in virions. Finally, mutation of two regions that mediate contacts between trimers in the in-situ structure of HEF was shown to prevent rescue of infectious virus particles. Mutations at residues thought to mediate lateral interactions were revealed to promote intracellular trafficking defects. Taken together, we propose that lateral interactions between the ectodomains of HEF trimers are a driving force for virus budding, although CM2 and CM1 also play important roles in this process.     

Intrinsic Temperature Sensitivity of Influenza C Virus Hemagglutinin-Esterase-Fusion Protein

Influenza C virus replicates more efficiently at 33°C than at 37°C. To determine whether hemagglutinin-esterase-fusion protein (HEF), a surface glycoprotein of influenza C virus, is a restricting factor for this temperature sensitivity, we analyzed the biological and biochemical properties of HEF at 33°C and 37°C. We found that HEF exhibits intrinsic temperature sensitivities for surface expression and fusion activity.     

The role of proteolytic cleavage of viral glycoproteins in the pathogenesis of influenza virus infections

Infectivity, tropism, spread, and pathogenicity of influenza viruses are based on the interplay between the fusogenic glycoproteins and appropriate host endoproteases. The hemagglutinin (HA) of influenza A and B viruses and the HEF (hemagglutinating, esterase, fusion) glycoprotein of influenza C virus receive their full biological activity by proteolytic cleavage of a precursor molecule at a definite cleavage site. The amino acid motifs at the cleavage site and the availability of suitable proteases are critical for the clinical manifestation of the infection. Prototype cleavage proteases, including bacterial enzymes, are described.     

Analytical detection of 9(4)-O-acetylated sialoglycoproteins and gangliosides using influenza C virus.

The unique glycoprotein of influenza C virus, designated hemagglutinin (HEF), exhibits three functions: hemagglutination, esterase activity, and fusion factor. As the virus uses 9-O-acetylated sialic acid as a high-affinity receptor determinant for attachment to cells, its binding activity was used to reveal O-acetylated sialic acid residues after polyacrylamide gel electrophoresis and transfer onto nitrocellulose sheets of proteins and thin-layer chromatography of lipids. The specificity of the binding for O-acetylated sialoglycoconjugates was investigated. Our results showed that influenza C virus could detect the different forms of the two murine glycophorins which are known to be O-acetylated sialoglycoconjugates. The virus also bound to O-acetylated gangliosides isolated from embryonic chicken brain such as purified O-acetylated NeuAc alpha (2-8)NeuAc alpha (2-8)NeuAc alpha (2-3)Gal beta (1-4)Glc beta (1-1)ceramide (GT3). The esterase activity of the HEF protein of influenza C virus was used to unmask the sialic acid. After its deacetylation by the virus enzyme, the O-acetylated GT3 was recognized by a monoclonal antibody which binds only to the nonacetylated derivative. The results presented here show that influenza C virus is a discriminating analytical probe for identifying O-acetylated sialoglycoconjugates directly after Western blotting of proteins and thin-layer chromatography of lipids, thus providing a new analytical tool.     

The influenza virus hemagglutinin cytoplasmic tail is not essential for virus assembly or infectivity.

The influenza A virus hemagglutinin (HA) glycoprotein contains a cytoplasmic tail which consists of 10-11 amino acids, of which five residues re conserved in all subtypes of influenza A virus. As the cytoplasmic tail is not needed for intracellular transport to the plasma membrane, it has become virtually dogma that the role of the cytoplasmic tail is in forming protein-protein interactions necessary for creating an infectious budding virus. To investigate the role of the HA cytoplasmic tail in virus replication, reverse genetics was used to obtain an influenza virus that lacked an HA cytoplasmic tail. The rescued virus contained the HA of subtype A/Udorn/72 in a helper virus (subtype A/WSN/33) background. Biochemical analysis indicated that only the introduced tail- HA was incorporated into virions and these particles lacked a detectable fragment of the helper virus HA. The tail- HA rescued virus assembled and replicated almost as efficiently as virions containing wild-type HA, suggesting that the cytoplasmic tail is not essential for the virus assembly process. Nonetheless, a revertant virus was isolated, suggesting that possession of a cytoplasmic tail does confer an advantage.     

A seven-segmented influenza A virus expressing the influenza C virus glycoprotein HEF

Influenza viruses are classified into three types: A, B, and C. The genomes of A- and B-type influenza viruses consist of eight RNA segments, whereas influenza C viruses only have seven RNAs. Both A and B influenza viruses contain two major surface glycoproteins: the hemagglutinin (HA) and the neuraminidase (NA). Influenza C viruses have only one major surface glycoprotein, HEF (hemagglutinin-esterase fusion). By using reverse genetics, we generated two seven-segmented chimeric influenza viruses. Each possesses six RNA segments from influenza virus A/Puerto Rico/8/34 (PB2, PB1, PA, NP, M, and NS); the seventh RNA segment encodes either the influenza virus C/Johannesburg/1/66 HEF full-length protein or a chimeric protein HEF-Ecto, which consists of the HEF ectodomain and the HA transmembrane and cytoplasmic regions. To facilitate packaging of the heterologous segment, both the HEF and HEF-Ecto coding regions are flanked by HA packaging sequences. When introduced as an eighth segment with the NA packaging sequences, both viruses are able to stably express a green fluorescent protein (GFP) gene, indicating a potential use for these viruses as vaccine vectors to carry foreign antigens. Finally, we show that incorporation of a GFP RNA segment enhances the growth of seven-segmented viruses, indicating that efficient influenza A viral RNA packaging requires the presence of eight RNA segments. These results support a selective mechanism of viral RNA recruitment to the budding site.     

Importance of the cytoplasmic tails of the measles virus glycoproteins for fusogenic activity and the generation of recombinant measles viruses

The generation of replication-competent measles virus (MV) depends on the incorporation of biologically active, fusogenic glycoprotein complexes, which are required for attachment and penetration into susceptible host cells and for direct virus spread by cell-to-cell fusion. Whereas multiple studies have analyzed the importance of the ectodomains of the MV glycoproteins hemagglutinin (H) and fusion protein (F), we have investigated the role of the cytoplasmic tails of the F and H proteins for the formation of fusogenic complexes. Deletions in the cytoplasmic tails of transiently expressed MV glycoproteins were found to have varying effects on receptor binding, fusion, or fusion promotion activity. F tail truncation to only three amino acids did not affect fusion capacity. In contrast, truncation of the H cytoplasmic tail was limited. H protein mutants with cytoplasmic tails of <14 residues no longer supported F-mediated cell fusion, predominantly due to a decrease in surface expression and receptor binding. This indicates that a minimal length of the H protein tail of 14 amino acids is required to ensure a threshold local density to have sufficient accumulation of fusogenic H-F complexes. By using reverse genetics, a recombinant MV with an F tail of three amino acids (rMV-FcDelta30), as well as an MV with an H tail of 14 residues (rMV-HcDelta20), could be rescued, whereas generation of viruses with shorter H tails failed. Thus, glycoprotein truncation does not interfere with the successful generation of recombinant MV if fusion competence is maintained.     

The effect of the cytoplasmic tail of influenza C virus CM2 protein on its biochemical properties and intracellular processing

CM2 is an integral membrane protein encoded by the influenza C virus M gene. To examine the effects of the cytoplasmic tail of CM2 on its biochemical properties, deletion and substitution mutations were introduced into CM2 cytoplasmic tail at residues 47–115, and the expressed CM2 mutants were investigated. Although the cytoplasmic tail is not essential for the oligomerization of CM2, it may affect the degree of oligomerization. The residues 47–48, 67–69, 73–90 and 113–115 were all required for the proper expression of CM2. Pulse-chase experiments suggest that residues 47–48, 67–69, 73–75 and 79–87 stabilize CM2, thereby affecting CM2 expression. The C-terminal region at residues 61–115 is not essential for CM2 transport to the cell surface, and a 14-amino-acid, but not an 11-amino-acid, cytoplasmic tail is sufficient for the cell surface expression of CM2. These results suggest that either certain amino acid sequences or the length of the CM2 cytoplasmic tail are necessary for the proper conformational maturation, stability, expression level and intracellular transport of CM2.     

Shortened cytoplasmic domain affects intracellular transport but not nuclear localization of a viral glycoprotein

Herpes simplex virus (HSV) buds from the inner nuclear membrane of the infected cells. The glycoprotein gB-1 of HSV contains a stretch of 69 hydrophobic amino acids near the COOH terminus and a 109-amino acid cytoplasmic domain. By oligonucleotide-directed mutagenesis, five gB-1 mutants were constructed which either lack a cytoplasmic tail or contained 3, 6, 22, or 43 amino acids in the cytoplasmic tail. When expressed in COS cells all of the mutant glycoproteins were synthesized but the rate of intracellular transport and the appearance at the cell surface of the mutant gB-1 protein lacking the cytoplasmic tail or containing 3 and 6 amino acids in the cytoplasmic domain was drastically reduced. The wild-type gB-1 as well as all of the mutants in the cytoplasmic tail were, however, located on the nuclear envelope. These results suggest that the cytoplasmic domain of the glycoprotein gB may play a role in intracellular transport but not in the nuclear localization.     

Analysis of the cell fusion activities of chimeric simian immunodeficiency virus-murine leukemia virus envelope proteins: inhibitory effects of the R peptide.

It was previously reported that truncation or proteolytic removal of the C-terminal 16 amino acids (the R peptide) from the cytoplasmic tail of the murine leukemia virus (MuLV) envelope protein greatly increases its fusion activity. In this study, to investigate the specificity of the effect of the R peptide on the fusion activity of viral envelope proteins, we expressed simian immunodeficiency virus (SIV)-MuLV chimeric proteins in which the entire cytoplasmic tail of the SIV envelope protein was replaced by either the full-length MuLV cytoplasmic tail or a truncated MuLV cytoplasmic tail with the R peptide deleted. Extensive fusion of CD4-positive cells with the chimeric protein containing a truncated MuLV cytoplasmic tail was observed. In contrast, no cell fusion activity was found for the chimeric protein with a full-length MuLV cytoplasmic tail. We constructed another SIV-MuLV chimeric protein in which the MuLV R peptide was added to an SIV envelope protein cytoplasmic tail 17 amino acids from its membrane-spanning domain. No fusion activity was observed within this construct, while the corresponding truncated SIV envelope protein lacking the R peptide showed extensive fusion activity. No significant difference in the transport or surface expression was observed among the various SIV-MuLV chimeric proteins and the truncated SIV envelope protein. Our results thus demonstrate that the MuLV R peptide has profound inhibitory effects on virus-induced cell fusion, not only with MuLV but also in a distantly related retroviral envelope protein which utilizes a different receptor and fuses different cell types.     

An Open Receptor-Binding Cavity of Hemagglutinin-Esterase-Fusion Glycoprotein from Newly-Identified Influenza D Virus: Basis for Its Broad Cell Tropism

Influenza viruses cause seasonal flu each year and pandemics or epidemic sporadically, posing a major threat to public health. Recently, a new influenza D virus (IDV) was isolated from pigs and cattle. Here, we reveal that the IDV utilizes 9-O-acetylated sialic acids as its receptor for virus entry. Then, we determined the crystal structures of hemagglutinin-esterase-fusion glycoprotein (HEF) of IDV both in its free form and in complex with the receptor and enzymatic substrate analogs. The IDV HEF shows an extremely similar structural fold as the human-infecting influenza C virus (ICV) HEF. However, IDV HEF has an open receptor-binding cavity to accommodate diverse extended glycan moieties. This structural difference provides an explanation for the phenomenon that the IDV has a broad cell tropism. As IDV HEF is structurally and functionally similar to ICV HEF, our findings highlight the potential threat of the virus to public health.     

Cytoplasmic domains of cellular and viral integral membrane proteins substitute for the cytoplasmic domain of the vesicular stomatitis virus glycoprotein in transport to the plasma membrane

Oligonucleotide-directed mutagenesis was used to construct chimeric cDNAs that encode the extracellular and transmembrane domains of the vesicular stomatitis virus glycoprotein (G) linked to the cytoplasmic domain of either the immunoglobulin mu membrane heavy chain, the hemagglutinin glycoprotein of influenza virus, or the small glycoprotein (p23) of infectious bronchitis virus. Biochemical analyses and immunofluorescence microscopy demonstrated that these hybrid genes were correctly expressed in eukaryotic cells and that the hybrid proteins were transported to the plasma membrane. The rate of transport to the Golgi complex of G protein with an immunoglobulin mu membrane cytoplasmic domain was approximately sixfold slower than G protein with its normal cytoplasmic domain. However, this rate was virtually identical to the rate of transport of micron heavy chain molecules measured in the B cell line WEHI 231. The rate of transport of G protein with a hemagglutinin cytoplasmic domain was threefold slower than wild type G protein and G protein with a p23 cytoplasmic domain, which were transported at similar rates. The combined results underscore the importance of the amino acid sequence in the cytoplasmic domain for efficient transport of G protein to the cell surface. Also, normal cytoplasmic domains from other transmembrane glycoproteins can substitute for the G protein cytoplasmic domain in transport of G protein to the plasma membrane. The method of constructing precise hybrid proteins described here will be useful in defining functions of specific domains of viral and cellular integral membrane proteins.     

Multiple regulatory effects of varicella-zoster virus (VZV) gL on trafficking patterns and fusogenic properties of VZV gH.

Varicella-zoster virus (VZV) is an extremely cell-associated alphaherpesvirus; VZV infection is spread almost exclusively via cell membrane fusion. The envelope glycoprotein H (gH) is highly conserved among the herpesviruses. A virus-encoded chaperone, glycoprotein L (gL), associates with gH, and the gH:gL complex is required for gH maturation and membrane expression. We recently demonstrated that in the VZV system, the gH:gL complex facilitated cell membrane fusion and extensive polykaryon formation in transfected cells (K. M. Duus, C. Hatfield, and C. Grose, Virology 210:429-440, 1995). To further define the functions of the unusual VZV gL chaperone protein, we have performed a series of mutagenesis experiments with both gH and gL and analyzed the mutants by laser scanning confocal microscopy in a transfection-based fusion assay. We established the fact that immature gH exited the endoplasmic reticulum (ER) when coexpressed with either gE or gI and appeared on the cell surface in a patch pattern. A similar effect was observed on the cell surface with gH with a cytoplasmic tail mutagenized to closely resemble the vaccinia virus hemagglutinin cytoplasmic tail. Site-directed mutagenesis of the five gL cysteine residues demonstrated that four of five cysteines participated in the gL chaperone function required for proper maturation of gH. On the other hand, the same gL mutants facilitated transport of immature gH to the cell surface, where patching occurred. Studies of gL processing demonstrated that maturation did not require transport beyond the medial-Golgi; furthermore, gL was not detected in the outer cell membrane, nor was it secreted into the medium. Colocalization studies with 3,3'-dihexyloxa-cabocyanine iodide and N-(e-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)-D-erythro-sphingosine confirmed that gL was found primarily in the ER and cis/medial-Golgi when expressed alone. When all of these data were considered, they suggested a posttranslational gH:gL regulation model whereby the gL chaperone modulated gH expression via retrograde flow from the Golgi to the ER. In this schema, mature gL returns to the ER, where it escorts immature gH from the ER to the Golgi; thereafter, mature gH is transported from the trans-Golgi to the outer cell membrane, where it acts as a major fusogen.     

Differences in the role of the cytoplasmic domain of human parainfluenza virus fusion proteins.

We have investigated the roles of the cytoplasmic domains of the human parainfluenza virus type 2 (PI2) and type 3 (PI3) fusion (F) proteins in protein transport and cell fusion activity. By using the vaccinia virus-T7 transient expression system, a series of F protein cytoplasmic tail truncation mutants was studied with respect to intracellular and surface expression and the ability to induce cell fusion when coexpressed with the corresponding hemagglutinin-neuraminidase (HN) proteins. All of the cytoplasmic tail truncation mutants of PI2F were expressed at high levels intracellularly or on cell surfaces as measured by immunoprecipitation and cell surface biotinylation assays. In addition, when coexpressed with PI2HN, these truncation mutants of PI2F were all found to be essentially unimpaired in the ability to induce cell fusion as measured by a quantitative cell fusion assay. In contrast, surface expression and cell fusion activity were found to be eliminated by a mutant of PI3F in which the entire cytoplasmic tail was deleted, and the mutant protein appeared to be unable to assemble into a high-molecular-weight oligomeric structure. To further investigate whether there is a specific sequence requirement in the cytoplasmic tail of PI3F, a chimeric protein consisting of the PI3F extracellular and transmembrane domains and the PI2F cytoplasmic tail was constructed. This chimeric protein was detected on the surface, and it was capable of inducing cell fusion when expressed together with PI3HN, although the fusogenic activity was reduced compared with that of wild-type PI3F. These results demonstrate that although PI2 and PI3 viruses belong to the same parainfluenza virus genus, these viruses show marked differences with respect to functional requirements for the cytoplasmic tail of the F glycoprotein.     

Monoclonal antibodies detect different forms of influenza virus hemagglutinin during viral penetration and biosynthesis.

Monoclonal antibodies specific for the influenza virus A/PR/8/34 hemagglutinin (HA) were used to examine the structure of the HA glycoprotein by immunofluorescence techniques during infection of MDCK cells. One antibody (Y8-10C2), shown previously to detect conformational alterations in the HA coinciding with the acid induction of viral fusion activity, bound to internalized virus but not to virus adsorbed to the cell surface. The binding of Y8-10C2 was completely inhibited by ammonium chloride treatment of the cells. These findings are consistent with the idea that Y8-10C2 detects conformational changes in the HA which accompany the acid-induced fusion of viral and endosomal membranes. The same antibody also bound to newly synthesized HA but not to later forms of cytoplasmic HA or to HA incorporated into the cell membrane during virus maturation. A possible common denominator of the antigenic changes detected by antibody Y8-10C2 during virus entry and replication may be alterations in the structural relationship among the three HA monomers which form the mature HA molecule.     

Down-regulation of paramyxovirus hemagglutinin-neuraminidase glycoprotein surface expression by a mutant fusion protein containing a retention signal for the endoplasmic reticulum.

The human parainfluenza virus type 3 (HPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins are the principal components involved in virion receptor binding, membrane penetration, and ultimately, syncytium formation. While the requirement for both F and HN in this process has been determined from recombinant expression studies, stable physical association of these proteins in coimmunoprecipitation studies has not been observed. In addition, coexpression of other heterologous paramyxovirus F or HN glycoproteins with either HPIV3 F or HN does not result in the formation of syncytia, suggesting serotype-specific protein differences. In this study, we report that simian virus 5 and Sendai virus heterologous HN proteins and measles virus hemagglutinin (H) were found to be down-regulated when coexpressed with HPIV3 F. As an alternative to detecting physical associations of these proteins by coimmunoprecipitation, further studies were performed with a mutant HPIV3 F protein (F-KDEL) lacking a transmembrane anchor and cytoplasmic tail and containing a carboxyl-terminal retention signal for the endoplasmic reticulum (ER). F-KDEL was defective for transport to the cell surface and could down-regulate surface expression of HPIV3 HN and heterologous HN/H proteins from simian virus 5, Sendai virus, and measles virus in coexpression experiments. HN/H down-regulation appeared to result, in part, from an early block to HPIV3 HN synthesis, as well as an instability of the heterologous HN/H proteins within the ER. In contrast, coexpression of F-KDEL with HPIV3 wild-type F or the heterologous receptor-binding proteins, respiratory syncytial virus glycoprotein (G) and vesicular stomatitis virus glycoprotein (G), were not affected in transport to the cell surface. Together, these results support the notion that the reported serotype-specific restriction of syncytium formation may involve, in part, down-regulation of heterologous HN expression.     

Accumulation of membrane glycoproteins in lysosomes requires a tyrosine residue at a particular position in the cytoplasmic tail

Human lysosome membrane glycoprotein h-lamp-1 is a highly N-glycosylated protein found predominantly in lysosomes, with low levels present at the cell surface. The signal required for delivery of h-lamp-1 to lysosomes was investigated by analyzing the intracellular distribution of h-lamp-1 with altered amino acid sequences expressed from mutated cDNA clones. A cytoplasmic tail tyrosine residue found conserved in chicken, rodent, and human deduced amino acid sequences was discovered to be necessary for efficient lysosomal transport of h-lamp-1 in COS-1 cells. In addition, the position of the tyrosine residue relative to the membrane and carboxyl terminus also determined lysosomal expression. Supplanting the wild-type h-lamp-1 cytoplasmic tail onto a cell surface reporter glycoprotein was sufficient to cause redistribution of the chimera to lysosomes. A similar chimeric protein replacing the cytoplasmic tyrosine residue with an alanine was not expressed in lysosomes. Altered proteins that were not transported to lysosomes were found to accumulate at the cell surface, and unlike wild-type lysosomal membrane glycoproteins, were unable to undergo endocytosis. These data indicate that lysosomal membrane glycoproteins are sorted to lysosomes by a cytoplasmic signal containing tyrosine in a specific position, and the sorting signal may be recognized both in the trans-Golgi network and at the cell surface.