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The primary target of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is epithelial cells in the respiratory and intestinal tract. The cellular receptor for SARS-CoV, angiotensin-converting enzyme 2 (ACE2), has been shown to be localized on the apical plasma membrane of polarized respiratory epithelial cells and to mediate infection from the apical side of these cells. Here, these results were confirmed and extended by including a colon carcinoma cell line (Caco-2), a lung carcinoma cell line (Calu-3) and Vero E6 cells in our analysis. All three cell types expressed human ACE2 on the apical membrane domain and were infected via this route, as determined with vesicular stomatitis virus pseudotypes containing the S protein of SARS-CoV. In a histological analysis of the respiratory tract, ACE2 was detected in the trachea, main bronchus and alveoli, and occasionally also in the small bronchi. These data will help us to understand the pathogenesis of SARS-CoV infection.

The importance of sialic acid for infection by avian Infectious bronchitis virus (IBV) has been analysed. Neuraminidase treatment rendered Vero, baby hamster kidney and primary chicken kidney cells resistant to infection by the IBV-Beaudette strain. Sialic acid-dependent infection was also observed with strain M41 of IBV, which infects primary chicken kidney cells but not cells from other species. In comparison with Influenza A virus and Sendai virus, IBV was most sensitive to pre-treatment of cells with neuraminidase. This finding suggests that IBV requires a greater amount of sialic acid on the cell surface to initiate an infection compared with the other two viruses. In previous studies, with respect to the haemagglutinating activity of IBV, it has been shown that the virus preferentially recognizes α2,3-linked sialic acid. In agreement with this finding, susceptibility to infection by IBV was connected to the expression of α2,3-linked sialic acid as indicated by the reactivity with the lectin Maackia amurensis agglutinin. Here, it is discussed that binding to sialic acid may be used by IBV for primary attachment to the cell surface; tighter binding and subsequent fusion between the viral and the cellular membrane may require interaction with a second receptor.

Coronaviruses mature by a budding process at intracellular membranes. For two of the viral membrane proteins, M and E, it has been shown that they are intracellularly retained. Upon single expression, the M proteins of transmissible gastroenteritis virus (TGEV) and avian infectious bronchitis virus are localized in the cis-Golgi network or cis-Golgi complex. 2 The small membrane protein E transiently resides in a pre-Golgi compartment before it progresses to the Golgi apparatus. 5 The S protein of TGEV is retained intracellularly. Retention is mediated by a tyrosine-based signal within the cytoplasmic tail. In contrast, the S protein of SARS-CoV lacks a tyrosine-residue in the corresponding tail portion, and in fact, it is transported to the cell surface. 7 We analyzed the protein expression of TGEV S protein and SARS-CoV S protein in two different expression systems. In the pTM1 vector, gene expression is under the control of a T7 promoter. This expression system requires the use of cells expressing the T7 RNA-polymerase, e.g., BSR-T7/5 cells. To exclude the possibility that retention of the S protein of TGEV is affected by the expression system, we compared pTM1-driven expression with expression by a plasmid under the control of a CMV promoter. As coronavirus S proteins cannot be expressed by standard plasmid vectors containing a CMV promoter, we used the vector pCG1 (kindly provided by Dr. Cattaneo), which contains a rabbit β-globin intron. This plasmid vector allows the expression of the S protein in different cell lines independent of T7 expression.

The expression of heterologous proteins in microorganisms using genetic recombination is still the high point in the development and exploitation of modern biotechnology. People can produce bioactive proteins from relatively cheap culture medium instead of expensive extraction. Host cell systems for the expression of heterologous genes are generally prokaryotic or eukaryotic systems, both of which have inherent advantages and drawbacks. An optimal expression system can be selected only if the productivity, bioactivity, purpose, and physicochemical characteristics of the interest protein are taken into consideration, together with the cost, convenience and safety of the system itself. Here, we concisely review the most frequently used prokaryotic, yeast, insect and mammalian expression systems, as well as expression in eukaryote individuals. The merits and demerits of these systems are discussed.

Entry of most paramyxoviruses is accomplished by separate attachment and fusion proteins that function in a cooperative manner. Because of this close interdependence, it was not possible with most paramyxoviruses to replace either of the two protagonists by envelope glycoproteins from related paramyxoviruses. By using reverse genetics of Sendai virus (SeV), we demonstrate that chimeric respiratory syncytial virus (RSV) fusion proteins containing either the cytoplasmic domain of the SeV fusion protein or in addition the transmembrane domain were efficiently incorporated into SeV particles provided the homotypic SeV-F was deleted. In the presence of SeV-F, the chimeric glycoproteins were incorporated with significantly lower efficiency, indicating that determinants in the SeV-F ectodomain exist that contribute to glycoprotein uptake. Recombinant SeV in which the homotypic fusion protein was replaced with chimeric RSV fusion protein replicated in a trypsin-independent manner and was neutralized by antibodies directed to RSV-F. However, replication of this virus also relied on the hemagglutinin-neuraminidase (HN) as pretreatment of cells with neuraminidase significantly reduced the infection rate. Finally, recombinant SeV was generated with chimeric RSV-F as the only envelope glycoprotein. This virus was not neutralized by antibodies to SeV and did not use sialic acids for attachment. It replicated more slowly than hybrid virus containing HN and produced lower virus titers. Thus, on the one hand RSV-F can mediate infection in an autonomous way while on the other hand it accepts support by a heterologous attachment protein.

This chapter discusses the various routes of entry of viruses into the mucosal epithelial cells, which are primarily determined by the distribution of viral receptors on cell surfaces. The chapter also describes the process of viral release, which typically occurs in a polarized fashion in epithelial cells and tissues. The presence or absence of suitable surface receptors is a critical determinant for the sensitivity or resistance, respectively, of cells to virus infection. The plasma membrane of the epithelial cells is divided into an apical domain and a basolateral domain that differ from each other in their composition. If a virus receptor is restricted to the apical surface—virus infection is possible only through this membrane domain, and in the context of an organism—it is only via the lumen of the body cavity that is lined by the respective epithelium. An apical localization of CD46 is consistent with the initial stage of the measles virus infection, when the virus enters the organism via the respiratory tract. However, CD46 serves as a receptor only for vaccine strains that are applied by injection and do not enter the organism via respiratory infection.

Coronaviruses (CoV) mature by a budding process at intracellular membranes. Here we showed that the major surface protein S of a porcine CoV (transmissible gastroenteritis virus) is not transported to the cell surface but is retained intracellularly. Site-directed mutagenesis indicated that a tyrosine-dependent signal (YXXI) in the cytoplasmic tail is essential for intracellular localization of the S protein. Surface expression of mutant proteins was evident by immunofluorescence analysis and surface biotinylation. Intracellularly retained S proteins only contained endoglycosidase H-sensitive N-glycans, whereas mutant proteins that migrated to the plasma membrane acquired N-linked oligosaccharides of the complex type. Corresponding tyrosine residues are present in the cytoplasmic tails of the S proteins of other animal CoV but not in the tail portion of the S protein of severe acute respiratory syndrome (SARS)-CoV. Changing the SEPV tetrapeptide in the cytoplasmic tail to YEPI resulted in intracellular retention of the S protein of SARS-CoV. As the S proteins of CoV have receptor binding and fusion activities and are the main target of neutralizing antibodies, the differences in the transport behavior of the S proteins suggest different strategies in the virus host interactions between SARS-CoV and other coronaviruses.

Transmissible gastroenteritis coronavirus (TGEV) is a porcine pathogen causing enteric infections that are lethal for suckling piglets. The enterotropism of TGEV is connected with the sialic acid binding activity of the viral surface protein S. Here we show that, among porcine intestinal brush border membrane proteins, TGEV recognizes a mucin-type glycoprotein designated MGP in a sialic acid-dependent fashion. Virus binding assays with cryosections of the small intestine from a suckling piglet revealed the binding of TGEV to mucin-producing goblet cells. A nonenteropathogenic mutant virus that lacked a sialic acid binding activity was unable to bind to MGP and to attach to goblet cells. Our results suggest a role of MGP in the enteropathogenicity of TGEV.

Tachykinins, an evolutionary conserved family of peptide hormones in both invertebrates and vertebrates, are produced by neuronal cells as inactive preprotachykinins that are post-translationally processed into different neuropeptides such as substance P, neurokinin A, and neurokinin B. We show here that furin-mediated cleavage of the bovine respiratory syncytial virus fusion protein results in the release of a peptide that is converted into a biologically active tachykinin (virokinin) by additional post-translational modifications. An antibody directed to substance P cross-reacted with the C terminus of mature virokinin that contains a classical tachykinin motif. The cellular enzymes involved in the C-terminal maturation of virokinin were found to be present in many established cell lines. Virokinin is secreted by virus-infected cells and was found to act on the tachykinin receptor 1 (TACR1), leading to rapid desensitization of this G protein-coupled receptor as shown by TACR1-green fluorescent protein conjugate translocation from the cell surface to endosomes and by co-internalization of the receptor with beta-arrestin 1-green fluorescent protein conjugates. In vitro experiments with isolated circular muscle from guinea pig stomach indicated that virokinin is capable of inducing smooth muscle contraction by acting on the tachykinin receptor 3. Tachykinins and their cognate receptors are present in the mammalian respiratory tract, where they have potent effects on local inflammatory and immune processes. The viral tachykinin-like peptide represents a novel form of molecular mimicry, which may benefit the virus by affecting the host immune response.

International audience; Binding of influenza viruses to target cells is mediated by the viral surface protein hemagglutinin. To determine the presence of binding sites for influenza A viruses on cells and tissues, soluble hemagglutinins of the H7 and H9 subtype were generated by connecting the hemagglutinin ectodomain to the Fc portion of human immunoglobulin G (H7Fc and H9Fc). Both chimeric proteins bound to different cells and tissues in a sialic acid-dependent manner. Pronounced differences were observed between H7Fc and H9Fc, in the binding both to different mammalian and avian cultured cells and to cryosections of the respiratory epithelium of different virus host species (turkey, chicken and pig). Binding of the soluble hemagglutinins was similar to the binding of virus particles, but showed differences in the binding pattern when compared to two sialic acid-specific plant lectins. These findings were substantiated by a comparative glycan array analysis revealing a very narrow recognition of sialoglycoconjugates by the plant lectins that does not reflect the glycan structures preferentially recognized by H7Fc and H9Fc. Thus, soluble hemagglutinins may serve as sialic acid-specific lectins and are a more reliable indicator of the presence of binding sites for influenza virus HA than the commonly used plant lectins.

The human respiratory syncytial virus (Long strain) fusion protein contains six potential N-glycosylation sites: N27, N70, N116, N120, N126, and N500. Site-directed mutagenesis of these positions revealed that the mature fusion protein contains three N-linked oligosaccharides, attached to N27, N70, and N500. By introducing these mutations into the F gene in different combinations, four more mutants were generated. All mutants, including a triple mutant devoid of any N-linked oligosaccharide, were efficiently transported to the plasma membrane, as determined by flow cytometry and cell surface biotinylation. None of the glycosylation mutations interfered with proteolytic activation of the fusion protein. Despite similar levels of cell surface expression, the glycosylation mutants affected fusion activity in different ways. While the N27Q mutation did not have an effect on syncytium formation, loss of the N70-glycan caused a fusion activity increase of 40%. Elimination of both N-glycans (N27/70Q mutant) reduced the fusion activity by about 50%. A more pronounced reduction of the fusion activity of about 90% was observed with the mutants N500Q, N27/500Q, and N70/500Q. Almost no fusion activity was detected with the triple mutant N27/70/500Q. These data indicate that N-glycosylation of the F 2 subunit at N27 and N70 is of minor importance for the fusion activity of the F protein. The single N-glycan of the F 1 subunit attached to N500, however, is required for efficient syncytium formation.

Transmissible gastroenteritis virus (TGEV) is a prototype of enteropathogenic coronaviruses. The virus causes diarrhea in pigs of all ages. Infections are most severe in newborn piglets where letality can be as high as 100% (Pensaert et al 1993). The determinants of the enterotropism of TGEV are not known. A crucial factor appears to be the sialic acid binding activity of this virus (Schultze et al 1996). The ability of TGEV to attach to sialoglycoconjugates allows the virus to bind to erythrocytes. This interaction results in an agglutination reaction that probably has no physiological importance. However, hemagglutination provides a convenient assay for the sialic acid binding activity and allows quantitation of the virus. Mutants of TGEV that have lost their haemagglutinating activity because of a single point mutation in the S protein also had lost enteropathogenicity (Krempl et al 1997). Porcine respiratory Coronavirus (PRCV), a respiratory variant of TGEV also lacks hemagglutinating activity. Both PRCV and the hemagglutination-deficient mutants are still able to grow in cell culture. Therefore, the sialic acid binding activity appears to be essential for enteropathogenicity but dispensible for growth in cultured cells. The sialic acid binding activity is located on the viral surface protein S. Another binding activity of this glycoprotein is the ability to interact with aminopeptidase N, the cellular receptor for TGEV (Delmas et al 1992). PRCV and the hemagglutination-deficient mutants have retained the ability to bind to aminopeptidase N. Therefore, the interaction with aminopeptidase N — though essential for the infection of cells — does not explain the enteropathogenicity of TGEV.

A mucin-type glycoprotein has been described in murine, rat and canine tissues as a differentiation antigen and influenza-virus receptor. We have cloned a cDNA from human placenta RNA encoding the corresponding human protein, a type-I integral membrane protein of 162 amino acids. Madin-Darby canine kidney cells transfected with the cDNA clone directed the cell-surface expression of a 36-kDa O-glycosylated sialoglycoprotein, gp36, and two minor isoforms of 28 and 70 kDa. gp36 has a broad tissue distribution with strong expression in lung, placenta and skeletal muscle, as shown by PCR screening of different cDNA libraries. Immunohistochemical detection of gp36 in cryo-sections of human placenta, kidney, lung and nasal polyps showed that the glycoprotein is expressed at the apical plasma membrane of vascular endothelial cells. Expression of gp36 was not restricted to endothelial cells, as alveolar epithelial cells were found to express gp36 as well.

Bovine respiratory syncytial virus (BSRV) is a major cause of respiratory disease in calves and is closely related to human RSV. Bovine viral diarrhoea virus (BVDV) is responsible for disease that affects the respiratory, reproductive, immune and enteric systems of cattle. These diseases are a huge problem globally for the livestock industry and a mucosally targeted, bivalent vaccine which prevents infection and spread of these viruses would be valuable. We investigated the influence of the site of expression of the BVDV E2 protein on induction of immune responses using recombinant (r)BRSV expressing wild-type and mutant forms of the E2 protein targeted to different sites within polarised epithelial cells. Calves vaccinated intranasally with rBRSV expressing the E2 protein at the basolateral surface induced higher levels of E2-specific serum antibodies than animals vaccinated with rBRSV expressing E2 at the apical membrane, intracellularly or when secreted from cells. Targeting E2 to the basolateral surface also induced E2-specific IgA antibodies in respiratory secretions without compromising RSV-specific responses. Calves vaccinated intranasally with rBRSV expressing E2 targeted to the basolateral surface were completely protected against challenge with BRSV, and were also protected against nasopharyngeal excretion of BVDV. These studies highlight the importance of the site of expression of foreign proteins in polarised epithelial cells on priming of immune responses.

Infection of cells by influenza C virus is known to be initiated by virus attachment to cell surface glycoconjugates containing N-acetyl-9-O-acetylneuraminic acid. Using an in vitro virus binding assay, we have detected this carbohydrate on several glycoproteins of Madin-Darby canine kidney cells (type I), a polarized epithelial cell line permissive for infection with influenza C virus. Among these proteins, only one was found to be present to a significant extent on the cell surface. This protein, gp40, was characterized as an O-glycosylated (mucin-type) integral membrane protein of 40 kDa, which was predominantly localized on the apical plasma membrane of filter-grown cells. It is a major cell surface sialoglycoprotein in this cell line and was shown to be subject to constitutive and rapid endocytosis. Thus, this glycoprotein can mediate not only the binding of influenza C virus to the cell surface, but also its delivery to endosomes, where penetration occurs by membrane fusion. Other highly sialylated cell surface glycoproteins were also detected but did not mediate influenza C virus binding to a significant extent, indicating that only gp40 contains 9-O-acetylated sialic acids.

The post-translational processing of the influenza C virus glycoprotein HEF was analysed. In cells infected with influenza C virus, HEF protein is synthesized as a glycosylated 80K polypeptide. A post-translational conformational rearrangement involving the formation of intramolecular disulphide bonds results in a decrease in its electrophoretic mobility. Therefore, SDS-PAGE under non-reducing conditions suggests an M r of about 100K, whereas under reducing conditions an 80K protein is observed which is in accordance with the sequence data. The 100K form was detected 10 min after synthesis of HEF, and transport to the cell surface took about 60 min. This result indicates that the conformational change presumably occurs in the endoplasmic reticulum. A difference in post-translational processing was observed when the HEF gene was expressed in the absence of other influenza C virus genes. In cells infected with recombinant simian virus 40, the 80K precursor was synthesized, but this protein was neither converted to the 100K form nor transported to the cell surface. Deletion of the short cytoplasmic tail of HEF (Arg-Thr-Lys) or replacement of the two basic amino acids by hydrophobic (Ile) or acidic residues (Glu) resulted in HEF protein which was partially converted to the 100K form. Influenza C virus glycoprotein obtained after transient expression of the HEF gene using the vaccinia virus system was completely converted to the 100K form. However, in neither expression system was HEF transported to the cell surface. The possibility is discussed that the interaction of HEF with another viral protein is required for the post-translational folding and transport of this glycoprotein. The M protein of influenza C virus is suggested as a candidate for the chaperone which might interact with the cytoplasmic tail of HEF.

BACKGROUND: Swine are important hosts for influenza A viruses playing a crucial role in the epidemiology and interspecies transmission of these viruses. Respiratory epithelial cells are the primary target cells for influenza viruses. METHODOLOGY/PRINCIPAL FINDINGS: To analyze the infection of porcine airway epithelial cells by influenza viruses, we established precision-cut lung slices as a culture system for differentiated respiratory epithelial cells. Both ciliated and mucus-producing cells were found to be susceptible to infection by swine influenza A virus (H3N2 subtype) with high titers of infectious virus released into the supernatant already one day after infection. By comparison, growth of two avian influenza viruses (subtypes H9N2 and H7N7) was delayed by about 24 h. The two avian viruses differed both in the spectrum of susceptible cells and in the efficiency of replication. As the H9N2 virus grew to titers that were only tenfold lower than that of a porcine H3N2 virus this avian virus is an interesting candidate for interspecies transmission. Lectin staining indicated the presence of both α-2,3- and α-2,6-linked sialic acids on airway epithelial cells. However, their distribution did not correlate with pattern of virus infection indicating that staining by plant lectins is not a reliable indicator for the presence of cellular receptors for influenza viruses. CONCLUSIONS/SIGNIFICANCE: Differentiated respiratory epithelial cells significantly differ in their susceptibility to infection by avian influenza viruses. We expect that the newly described precision-cut lung slices from the swine lung are an interesting culture system to analyze the infection of differentiated respiratory epithelial cells by different pathogens (viral, bacterial and parasitic ones) of swine.

The importance of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) as a receptor determinant for bovine coronavirus (BCV) on cultured cells was analysed. Pretreatment of MDCK I (Madin Darby canine kidney) cells with neuraminidase or acetylesterase rendered the cells resistant to infection by BCV. The receptors on a human (CaCo-2) and a porcine (LLC-PK1) epithelial cell line were also found to be sensitive to neuraminidase treatment. The susceptibility to infection by BCV was restored after resialylation of asialo-MDCK I cells with Neu5,9Ac2. Transfer of sialic acid lacking a 9-O-acetyl group was ineffective in this respect. These results demonstrate that 9-O-acetylated sialic acid is used as a receptor determinant by BCV to infect cultured cells. The possibility is discussed that the initiation of a BCV infection involves the recognition of different types of receptors, a first receptor for primary attachment and a second receptor to mediate the fusion between the viral envelope and the cellular membrane.

Cholesterol‐enriched microdomains known as lipid rafts have been shown to be important for the life cycle of several viruses. Here, we investigated whether cholesterol is important during the initial steps of SARS‐CoV spike (S) glycoprotein‐mediated entry. Vero cells were treated with the cholesterol sequestering drug methyl‐β‐cyclodextrin (mβCD) and then exposed to SARS‐CoV and Vesicular stomatitis virus (VSV) pseudotyped with SARS‐CoV spike glycoprotein (VSV‐ΔG‐S). Furthermore, a cell‐based binding assay and a binding assay with soluble S protein demonstrated that the binding of S to its receptor angiotensin‐converting enzyme 2 (ACE2) is affected by cholesterol depletion and that multi‐ligand interactions might be important for the entry process of SARS‐CoV to target cells. Confocal laser microscopy studies and a membrane flotation assay in Vero cells show that the SARS‐CoV receptor is organized within lipid microdomains and cholesterol depletion results in a reduction of ACE2 in the buoyant detergent resistant membrane fraction after Triton‐X 100 solubilization. Further attempts are directed to understand the molecular role of cholesterol during the initial steps of SARS‐CoV life cycle.

Erythrocytes from chicken of different age were analysed for their agglutinability by influenza C virus, which has been shown recently to use N-acetyl-9-O-acetylneuraminic acid as a high-affinity receptor determinant for the attachment to cells. Only with birds not younger than six days complete agglutination of the erythrocytes was observed. The hemagglutination titer which was initially low reached its maximum value at the age of about 20 days. Sialic acid was isolated from erythrocytes, purified and analysed by colorimetry, thin-layer chromatography, high-performance liquid chromatography, and gas-liquid chromatography-mass spectrometry. The sialic acid content of erythrocytes from one-day old and adult chicken was 21 micrograms and 18 micrograms sialic acid/ml packed erythrocytes, respectively. While N-acetylneuraminic acid was the major type of sialic acid on erythrocytes from both one-day old and adult chicken, N-acetyl-9-O-acetylneuraminic acid was only detected on red blood cells from adult animals accounting for 30-40% of total sialic acid. These results indicate that N-acetyl-9-O-acetylneuraminic acid, in addition to serving as a receptor determinant for influenza C virus, represents a developmental marker on chicken erythrocytes.

Mumps virus was adapted to growth in Vero cells, which yielded virus of high infectivity titers. The structural polypeptides of purified virions grown in Vero cells were similar to those described previously for egg-grown mumps virus: L (200K), HN (79K), NP (72K), F1 (61K), P (45K), M (40K), and F2 (16K). We have analyzed the synthesis of viral polypeptides in Vero cells by pulse labeling with radioactive amino acid precursors. The nucleoprotein (NP) was the first to be detected intracellularly above the cellular protein background at 6 h.p.i. By 12 h.p.i., all viral polypeptides were observed except for the glycoproteins F1 and F2, which are derived from a precursor designated F0 (74K). Two low-molecular-weight polypeptides not present in purified virions were also detected in infected cells. They are designated pI (28K) and pII (19K). Peptide mapping revealed that these two polypeptides share regions of their amino acid sequence and that they are also related to the structural protein P. Polypeptides pI and pII were found in several cell types (Vero, CEF, MDBK cells) infected with mumps virus. Infection of Vero cells with other paramyxoviruses (SV5 and Sendai virus) did not induce the synthesis of proteins comparable to pI and pII, whereas in mumps virus-infected cells no counterpart to the nonstructural C protein of Sendai virus was detected. Pulse-chase experiments suggest that pI and pII may not be derived from P by proteolytic cleavage.

Posttranslational cleavage of the precursor glycoprotein gp88 of influenza C virus results in two subunit glycoproteins, gp65 and gp30, each of which exist in two molecular forms. Influenza C glycoproteins were isolated from purified influenza C virions by selective solubilization with Triton X-100 or octylglucoside. Only preparations obtained with octylglucoside showed hemagglutinating activity. Tryptic peptide analysis of the three species of viral glycoproteins, gp88, gp65 and gp30, revealed that gp30 and gp65 are distinct; when the peaks resolved for the two subunit glycoproteins are superimposed, the pattern corresponding to that of gp88 is obtained. The two molecular forms of gp65 have an identical polypeptide backbone as shown by tryptic peptide analysis. The N-termini of gp88 as well as gp65 were resistant to sequential Edman degradation. The gp30 terminal sequence contains a preponderance of hydrophobic residues and shows homology with the corresponding sequence of the HA^ subunit of influenza A viruses, except for an additional N-terminal glycine residue. The glycoprotein spikes on the surface of influenza C virions were found in regular hexagonal arrays, which appear to involve lateral interaction between the glycoprotein molecules, since the spikes sometimes maintain their arrangement in a network upon release from the viral membrane by limited proteolytic digestion, or upon spontaneous disruption of the viral membrane. In infected cells, closely packed surface projections were observed on crescent shaped outfoldings of the plasma membrane, where virus maturation is occurring. The fact that, in most cases, no nucleocapsids can be seen in such regions suggests that nucleocapsids may not be required to initiate outfolding of the plasma membrane in the budding process of influenza C virions.

Influenza C virions possess a single glycoprotein that is cleaved into two disulfide-linked subunits, gp65 and gp30. When analyzed under nonreducing conditions, the uncleaved (gpI) and cleaved (gpI) glycoproteins differ significantly in apparent molecular weight; however, we observed no difference in their tryptic peptide patterns. We have isolated the glycoproteins by selective solubilization with Triton X-100 or octylglucoside; only preparations obtained with the latter detergent showed hemagglutinating activity. In purified glycoprotein samples, gp65 was routinely observed as a doublet on SDS-polyacrylamide gels. Analysis of tryptic peptides by ion-exchange chromatography demonstrated that the two gp65 bands have indistinguishable polypeptide backbones; they appear to differ, however, in carbohydrate content. The uncleaved glycoprotein as well as gp65 were resistant to Edman degradation indicating the presence of blocked amino termini, whereas gp30 was observed to have the N-terminal tripeptide sequence NH2-Ile-Phe-Gly. This sequence is homologous to a sequence at the N termini of influenza A and B HA2 glycoproteins, except for the presence of an additional terminal glycine residue in these viruses. The influenza C glycoproteins form a regular hexagonal lattice on the viral envelope. This arrangement is sometimes maintained in disrupted virus preparations and in glycoprotein subunits released from the envelope by limited proteolysis, indicating that direct interactions between the glycoprotein molecules are responsible, at least in part, for the observed arrangement. Observations of clustered surface projections on plasma membranes of infected cells, and of released virus particles apparently devoid of internal nucleoproteins, are consistent with the suggestion that lateral interactions between the influenza C glycoproteins may be important in virus assembly.

Influenza C virions grown in chicken kidney cells contain two glycoprotein size classes when analyzed under nonreducing conditions. These are designated gpI and gpII, with molecular weights of ∼105,000 and ∼82,000, respectively. We have obtained evidence that these glycoproteins share common amino acid sequences, and that gpI is the primary viral gene product which may be converted into gpII by proteolytic cleavage. When analyzed under reducing conditions, gpI was observed as a single, uncleaved polypeptide chain, whereas gpII was found to consist of two subunits with molecular weights of ∼65,000 and 30,000 respectively. Influenza C virus preparations grown in different host cells varied in the extent of cleavage of gpI into gpII. Virions grown in chick embryo fibroblast (CEF) cells contained exclusively gpI, whereas egg-grown virions possessed predominantly gpII glycoproteins. Treatment of CEF-grown virions with trypsin converted gpI into gpII; the specific infectivity of such preparations was increased up to 50-fold by trypsin treatment. These results indicate that cleavage of gpI into gpII is essential for maximal viral infectivity.