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At least 19 recordsLinked to original sources
PubMed2006

Specific binding of heat shock protein 70 with HN-protein inhibits the HN-protein assembly in Sendai virus-infected Vero cells.

The production of hemagglutinating virus of Japan (HVJ; Sendai virus) was inhibited at 41 degrees C, whereas it was normal at 37 degrees C. In the infected Vero cells, viral specific proteins were synthesized even at 41 degrees C, but the synthesized HN protein was not integrated into the cell membrane, resulting in the inhibition of viral production. To investigate the relationship of HSP70 to the inhibition of HN-protein integration, the expression of HSP70 was induced by prostaglandin A1 (PGA1) at 37 degrees C, and the influence on viral infection was examined. The induction of HSP70 at 37 degrees C inhibited the viral production. Viral proteins were also synthesized, even in the presence of PGA1. However, HN protein was not as present on the cell membrane following PGA1-treatment as it was at 41 degrees C, whereas F protein was detected. An immunoprecipitation assay showed that HSP70 was coprecipitated with HN protein, but not with F protein. The results suggested that the specific interaction of HSP70 with HN protein prevented the protein from integrating into the cell membrane. In addition, the abnormal virus-like particles, of which HN protein and nucleocapsid were ablated, were released in the culture medium at 41 degrees C, although the size was smaller than the normal viral virions. The results suggest that HN protein is necessary for viral morphogenesis.

Animals↗
PubMed1989

Complete nucleotide sequence of the hemagglutinin-neuraminidase (HN) mRNA of mumps virus and comparison of paramyxovirus HN proteins.

The complete nucleotide sequence of the hemagglutinin-neuraminidase protein (HN) mRNA of the virulent SBL-1 strain of mumps virus has been determined. The mRNA contains 1887 nucleotides excluding the poly(A). The protein encoded by the mRNA has 582 amino acids and a membrane anchorage domain near the amino terminus. The calculated molecular mass (64 kDa) of the protein is in good agreement with that of the unglycosylated HN protein (63 kDa) identified in tunicamycin treated mumps virus infected cells (Herrler and Compans, 1983). The predicted sequence has nine potential N-glycosylation sites out of which two contain a cysteine residue and one has a proline residue as the variable amino acid X in the glycosylation site (Asn-X-Ser or Asn-X-Thr) and therefore, may not be utilized. One potential glycosylation site is in the cytoplasmic region which may not also be glycosylated. Comparison of the mumps HN protein sequence with the HN protein sequences of Sendai virus, simian-virus 5 (SV5), parainfluenza virus type 3 and Newcastle disease virus (NDV) shows two major homology regions, one region near the middle of the protein and the other in the second half of the molecule. In terms of percentage amino acid homology, the HN proteins of mumps virus, SV5 and NDV are closely related to each other but distinct from Sendai virus and parainfluenza virus type 3 HN proteins.

Amino Acid Sequence↗
PubMed1999

Fusion of sendai virus with liposome depends on only F protein, but not HN protein.

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.

Blotting, Western↗
PubMed2000

A single amino acid change in the Newcastle disease virus fusion protein alters the requirement for HN protein in fusion.

The role of a leucine heptad repeat motif between amino acids 268 and 289 in the structure and function of the Newcastle disease virus (NDV) F protein was explored by introducing single point mutations into the F gene cDNA. The mutations affected either folding of the protein or the fusion activity of the protein. Two mutations, L275A and L282A, likely interfered with folding of the molecule since these proteins were not proteolytically cleaved, were minimally expressed at the cell surface, and formed aggregates. L268A mutant protein was cleaved and expressed at the cell surface although the protein migrated slightly slower than wild type on polyacrylamide gels, suggesting an alteration in conformation or processing. L268A protein was fusion inactive in the presence or absence of HN protein expression. Mutant L289A protein was expressed at the cell surface and proteolytically cleaved at better than wild-type levels. Most importantly, this protein mediated syncytium formation in the absence of HN protein expression although HN protein enhanced fusion activity. These results show that a single amino acid change in the F(1) portion of the NDV F protein can alter the stringent requirement for HN protein expression in syncytium formation.

Alanine↗
PubMed2000

Assembly of Sendai virus: M protein interacts with F and HN proteins and with the cytoplasmic tail and transmembrane domain of F protein.

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.

Animals↗
PubMed1997

Paramyxovirus fusion (F) protein and hemagglutinin-neuraminidase (HN) protein interactions: intracellular retention of F and HN does not affect transport of the homotypic HN or F protein.

To investigate a possible intracellular coassociation of the paramyxovirus simian virus 5 (SV5) and human parainfluenza virus type 3 (HPIV-3) fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins in a living cell, without resorting to chemical crosslinking and antibody coimmunoprecipitation, we tagged the cytoplasmic N-terminus of SV5 HN with a RRRRR motif and HPIV-3 HN with a RRR motif for endoplasmic reticulum (ER) retention. In addition, we tagged the cytoplasmic C-terminus of SV5 and HPIV-3 F with a KK motif. The RRR- or RRRRR-tagged HN molecules were coexpressed in mammalian cells together with the homologous wt F proteins, and the KK-tagged F molecules were coexpressed with the homologous wt HN proteins, and in each case the transport of the wt F or HN molecules was investigated. The data suggest that an association of F and HN of sufficient affinity to alter the transport of the reporter molecule does not occur intracellularly in the ER or the Golgi apparatus.

Biological Transport↗
PubMed2003

Triggering of human parainfluenza virus 3 fusion protein (F) by the hemagglutinin-neuraminidase (HN) protein: an HN mutation diminishes the rate of F activation and fusion.

For human parainfluenza virus type 3 and many other paramyxoviruses, membrane fusion mediated by the fusion protein (F) has a stringent requirement for the presence of the homotypic hemagglutinin-neuraminidase protein (HN). With the goal of gaining further insight into the role of HN in the fusion process, we developed a simple method for quantitative comparison of the ability of wild-type and variant HNs to activate F. In this method, HN/F-coexpressing cells with red blood cells (RBC) bound to them at 4 degrees C are transferred to 22 degrees C, and at different times after transfer 4-guanidino-neu5Ac2en (4-GU-DANA) is added; this inhibitor of the HN-receptor interaction then releases all reversibly bound RBC but not those in which F insertion in the target membrane or fusion has occurred. Thus, the amount of irreversibly bound (nonreleased) RBC provides a measure of F activation, and the use of fluorescently labeled RBC permits microscopic assessment of the extent to which F insertion has progressed to fusion. We studied two neuraminidase-deficient HN variants, C28a, which has two mutations, P111S and D216N, and C28, which possesses the D216N mutation only. C28a but not C28 exhibits a slow fusion phenotype, although determination of the HNs' receptor-binding avidity (with our sensitive method, employing RBC with different degrees of receptor depletion) showed that the receptor-binding avidity of C28a or C28 HN was not lower than that of the wild type. The F activation assay, however, revealed fusion-triggering defects in C28a HN. After 10 and also 20 min at 22 degrees C, irreversible RBC binding was significantly less for cells coexpressing wild-type F with C28a HN than for cells coexpressing wild-type F with wild-type HN. In addition, F insertion progressed to fusion more slowly in the case of C28a HN-expressing cells than of wild-type HN-expressing cells. Identical defects were found for P111S HN, whereas for C28 HN, representing the 216 mutation of C28a, F activation and fusion were as rapid as for wild-type HN. The diminished fusion promotion capacity of C28a HN is therefore attributable to P111S, a mutation in the stalk region of the molecule that causes no decrease in receptor-binding avidity. C28a HN is the first parainfluenza virus variant found so far to be specifically defective in HN's F-triggering and fusion promotion functions and may contribute to our understanding of transmission of the activating signal from HN to F.

Animals↗
PubMed2003

Contribution of the length of the HN protein and the sequence of the F protein cleavage site to Newcastle disease virus pathogenicity.

Newcastle disease virus (NDV) possesses two envelope spike glycoproteins: the haemagglutinin-neuraminidase (HN) protein and the fusion (F) protein. The HN protein, which is responsible for virus attachment to sialic acid-containing receptors, varies in length due to differences in the sizes of the ORFs. An HN protein precursor of 616 aa has been found in avirulent but not in virulent NDV strains, whereas an HN protein of 571 aa can be detected in highly virulent strains only. An HN protein of 577 aa is present in virulent and avirulent strains. The F protein, which mediates virus-cell fusion, requires proteolytic activation at an internal cleavage site, whose amino acid composition determines cleavability by various proteases. Here, the functional significance of the length of the HN protein in combination with F protein cleavage sites typical for virulent (velogenic and mesogenic) or avirulent (lentogenic) strains was investigated. To this end, site-directed mutagenesis was used to construct recombinant NDV on the basis of an infectious clone of the lentogenic vaccine virus Clone-30. Only recombinant NDV expressing an F protein with a multibasic cleavage site typical of virulent strains was able to spread efficiently in cell culture, irrespective of the size of the HN protein. Moreover, as determined by the intracerebral pathogenicity index (ICPI) in 1-day-old, specific-pathogen-free chickens, pathogenicity was influenced by the cleavability of the F protein and not by the length of the HN protein. The maximum ICPI value obtained for these recombinants was 1.3, as compared to a possible maximum of 2. This demonstrates that the modifications introduced did not result in the conversion of the lentogenic Clone-30 to a velogenic strain with an ICPI value of >1.5 and suggests the involvement of additional virulence determinants that contribute to the pathogenicity of NDV.

Amino Acid Sequence↗
PubMed1985

Antigenic characterization of hemagglutinin-neuraminidase (HN) protein of avian paramyxoviruses by specific antisera to isolated HN subunits.

Specific antisera for the isolated HN proteins of eight reference strains of avian paramyxoviruses could be prepared in guinea pigs by intraperitoneal injection of guinea pig red blood cells (GRBC) coated with purified HN proteins. In the hemagglutination inhibition (HI) tests, all reference strains reacted strongly with each homologous antiserum to the isolated HN showing that a low level of cross-reactivity among the reference strains was greatly diminished by using specific antisera. Immuno-double-diffusion (IDD) tests showed that all antisera except those to turkey/Wisconsin/68 and duck/Hong Kong/D3/75 gave single well-defined lines only with the homologous viruses. The remaining two antisera developed a single definite precipitin line together with weak lines with homologous virus. Two isolates in Japan were clearly identified in HI and IDD tests with specific antisera to the HN subunits of the reference strains suggesting that the antisera were useful for identification of avian paramyxovirus isolates. Two isolates in Japan, H-70 from a munia-bird and Y-7 from a duck were found to have HN proteins related closely to those of finch/N. Ireland/Bangor/73 and duck/Hong Kong/199/77, respectively.

Animals↗
PubMed2002

Newcastle disease virus HN protein alters the conformation of the F protein at cell surfaces.

Conformational changes in the Newcastle disease virus (NDV) fusion (F) protein during activation of fusion and the role of HN protein in these changes were characterized with a polyclonal antibody. This antibody was raised against a peptide with the sequence of the amino-terminal half of the F protein HR1 domain. This antibody immunoprecipitated both F(0) and F(1) forms of the fusion protein from infected and transfected cell extracts solubilized with detergent, and precipitation was unaffected by expression of the HN protein. In marked contrast, this antibody detected significant conformational differences in the F protein at cell surfaces, differences that depended upon HN protein expression. The antibody minimally detected the F protein, either cleaved or uncleaved, in the absence of HN protein expression. However, when coexpressed with HN protein, an uncleaved mutant F protein bound the anti-HR1 antibody, and this binding depended upon the coexpression of specifically the NDV HN protein. When the cleaved wild-type F protein was coexpressed with HN protein, the F protein bound anti-HR1 antibody poorly although significantly more than F protein expressed alone. Anti-HR1 antibody inhibited the fusion of R18 (octadecyl rhodamine B chloride)-labeled red blood cells to syncytia expressing HN and wild-type F proteins. This inhibition showed that fusion-competent F proteins present on surfaces of syncytia were capable of binding anti-HR1. Furthermore, only antibody which was added prior to red blood cell binding could inhibit fusion. These results suggest that the conformation of uncleaved cell surface F protein is affected by HN protein expression. Furthermore, the cleaved F protein, when coexpressed with HN protein and in a prefusion conformation, can bind anti-HR1 antibody, and the anti-HR1-accessible conformation exists prior to HN protein attachment to receptors on red blood cells.

Amino Acid Sequence↗
PubMed2004

Amino acid substitutions in the F-specific domain in the stalk of the newcastle disease virus HN protein modulate fusion and interfere with its interaction with the F protein.

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus mediates attachment to sialic acid receptors, as well as cleavage of the same moiety. HN also interacts with the other viral glycoprotein, the fusion (F) protein, to promote membrane fusion. The ectodomain of the HN spike consists of a stalk and a terminal globular head. The most conserved part of the stalk consists of two heptad repeats separated by a nonhelical intervening region (residues 89 to 95). Several amino acid substitutions for a completely conserved proline residue in this region not only impair fusion and the HN-F interaction but also decrease neuraminidase activity in the globular domain, suggesting that the substitutions may alter HN structure. Substitutions for L94 also interfere with fusion and the HN-F interaction but have no significant effect on any other HN function. Amino acid substitutions at other positions in the intervening region also modulate only fusion. In all cases, diminished fusion correlates with a decreased ability of the mutated HN protein to interact with F at the cell surface. These findings indicate that the intervening region is critical to the role of HN in the promotion of fusion and may be directly involved in its interaction with the homologous F protein.

Amino Acid Sequence↗
PubMed1998

Membrane fusion promoted by increasing surface densities of the paramyxovirus F and HN proteins: comparison of fusion reactions mediated by simian virus 5 F, human parainfluenza virus type 3 F, and influenza virus HA.

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.

Animals↗
PubMed1998

Role of carbohydrate processing and calnexin binding in the folding and activity of the HN protein of Newcastle disease virus.

The role of carbohydrate processing and calnexin binding in the folding pathway and activity of the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) was explored in infected cells using the inhibitor castanospermine (CST). Calnexin-HN protein complexes were demonstrated by coimmunoprecipitation using antibody specific for calnexin or HN protein. As in other systems, this complex was not detected in CST treated cells. In cells incubated in CST, the synthesis and stability of the HN protein was unaffected. However, as monitored by the appearance of conformationally sensitive antigenic sites, the folding of the HN protein in CST treated cells was approximately twice as slow than in untreated cells. This folding was ultimately efficient since there was no evidence for significant amounts of irreversibly aggregated forms which never acquired a mature conformation. Most significantly, the folding sequence as measured by the order of appearance of conformationally sensitive antigenic sites (McGinnes and Morrison, Virology 199, 255) was unaffected by CST. Thus while calnexin functions to speed the folding of the HN protein, it is not required for the folding of this protein. In addition, the protein synthesized in the presence of CST had significant levels of neuraminidase and hemagglutination activity suggesting that processing of the carbohydrate has a minimal role in the activity of the protein.

Animals↗
PubMed1993

Mutations in the transmembrane domain of the HN protein of Newcastle disease virus affect the structure and activity of the protein.

To explore the role of the transmembrane domain of the HN protein in the structure and function of the molecule, three conserved leucine residues in this domain which occur in a heptad-repeat motif were changed to alanine singly or in combination by site-specific mutagenesis. None of the mutant proteins were defective in translocation and intracellular transport. All mutant proteins formed disulfide-linked dimers. However, tetrameric structures of proteins with mutations in the third or most carboxy-terminal leucine could not be detected by sucrose gradient analysis, and mutant proteins with changes in both the first and second leucine formed less-stable tetramers. These results suggest that the transmembrane domain plays a role in the tetrameric structure of the HN protein. These mutations also altered the biological activities of the protein. Mutant proteins with alterations in the third leucine were very defective in attachment activity and somewhat defective for neuraminidase activity while all other mutant proteins had wild-type levels of attachment and neuraminidase activity. While all mutant proteins showed diminished fusion-promotion activity, proteins with mutations in the third leucine and proteins with changes in both the first and second leucines were very defective in fusion promotion. These results suggest that elimination or destabilization of the tetrameric structure affects attachment activity and fusion-promotion activity of the HN protein.

Amino Acid Sequence↗
PubMed1993

The fusion promotion activity of the NDV HN protein does not correlate with neuraminidase activity.

Three activities, attachment, neuraminidase, and fusion promotion, have been associated with the hemagglutinin-neuraminidase (HN) protein encoded by paramyxoviruses such as Newcastle disease virus. The fusion promotion activity of the HN protein can be separated from its attachment activity by mutation (Sergel et al., 1993, Virology 193, 717-726). To determine if neuraminidase activity of the HN protein has any role in fusion promotion, two sets of mutants were characterized. First, a change of amino acid 193 from a serine to a proline and a change of amino acid 175 from isoleucine to a methionine diminished neuraminidase activity as previously reported. However, these mutant proteins retained fusion promotion activity. In addition, mutation of amino acid 200 from a histidine to a proline resulted in nearly twice the neuraminidase activity of wild-type as previously reported. This mutant also had wild-type levels of fusion promotion activity. Second, substitution of three leucine residues at amino acids 94, 96, and 97 with three alanines resulted in a mutant protein with full neuraminidase as well as full attachment activity but no fusion promotion activity. Thus, two sets of HN protein mutants demonstrate that the fusion promotion activity does not correlate with the level of neuraminidase activity.

Animals↗
PubMed1983

Thermostabilities of virion activities of Newcastle disease virus: evidence that the temperature-sensitive mutants in complementation groups B, BC, and C have altered HN proteins.

Four virion activities of Newcastle disease virus (hemagglutinating, neuraminidase, hemolytic, and infectious activities) were examined before and after heat stress in low-salt buffer and physiological salt buffer (phosphate-buffered saline). The hemagglutinating and neuraminidase activities of the Australia-Victoria wild-type (AV-WT) strain were thermostable at both salt concentrations tested, whereas the thermostabilities of the hemolytic and infectious activities were salt dependent (thermostable in phosphate-buffered saline but not in low-salt buffer). Virions of RNA(+) temperature-sensitive (ts) mutants of AV-WT were tested for the stabilities of the four activities. Some mutants in groups B, BC, and C were as stable as AV-WT in all functions, but others were much less stable in all functions. The unstable mutants in groups B, BC, and C affirmed the assignment of the ts lesions of these mutants to the hemagglutinin/neuraminidase (HN) protein gene because HN function(s) are required for all four activities. The instability of these ts mutants was not related to their decreased virion HN protein content and was not due to physical loss of the HN protein from the virions. Three of four ts(+) plaque-forming revertants of the least stable mutant, BC2, coreverted for stability, confirming that the unstable phenotype is indeed the result of the mutation responsible for the ts phenotype. Group D mutants were approximately as stable as AV-WT in hemagglutinating, neuraminidase, and hemolytic activities; this is consistent with this group representing a lesion in a gene other than the HN protein gene. However, the infectivities of two of the three group D mutants were less stable than the infectivity of AV-WT in low-salt buffer.

Genes, Viral↗
PubMed2006

Addition of N-glycans in the stalk of the Newcastle disease virus HN protein blocks its interaction with the F protein and prevents fusion.

Most paramyxovirus fusion (F) proteins require the coexpression of the homologous attachment (HN) protein to promote membrane fusion, consistent with the existence of a virus-specific interaction between the two proteins. Analysis of the fusion activities of chimeric HN proteins indicates that the stalk region of the HN spike determines its F protein specificity, and analysis of a panel of site-directed mutants indicates that the F-interactive site resides in this region. Here, we use the addition of oligosaccharides to further explore the role of the HN stalk in the interaction with F. N-glycans were individually added at several positions in the stalk to determine their effects on the activities of HN, as well as its structure. N-glycan addition at positions 69 and 77 in the stalk specifically blocks fusion and the HN-F interaction without affecting either HN structure or its other activities. N-glycans added at other positions in the stalk modulate activities that reside in the globular head of HN. This correlates with an alteration of the tetrameric structure of the protein, as indicated by sucrose gradient sedimentation analyses. Finally, N-glycan addition in another region of HN (residues 124 to 152), predicted by a peptide-based analysis to mediate the interaction with F, does not significantly reduce the level of fusion, arguing strongly against this site being part of the F-interactive domain in HN. Our data support the idea that the F-interactive site on HN is defined by the stalk region of the protein.

Amino Acid Sequence↗
PubMed1994

The role of the individual cysteine residues in the formation of the mature, antigenic HN protein of Newcastle disease virus.

The amino acid sequence of the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus (NDV) has 14 cysteine residues, two of which are variably present in the sequences of the HN proteins of different strains of NDV while the rest are absolutely conserved. The role of each residue in the formation of the mature, oligomeric structure of the HN protein was assessed by characterizing proteins with mutations in each of the cysteine residues by Western analysis, immunoprecipitation with conformationally sensitive antibodies, immunofluorescence, and sedimentation on sucrose gradients. Proteins with mutations in the first cysteine (amino acid 6) or the second cysteine (amino acid 123), the nonconserved cysteine residues, formed antigenically mature oligomers which were transported to the cell surface like wild type. Protein with a mutation at cysteine 2 did not, however, form covalently linked oligomers demonstrating that it is this residue that is responsible for intermolecular disulfide bonds in the mature oligomer. Proteins with mutations in cysteine 3 (amino acid 172) or cysteine 5 (amino acid 196) formed proteins with all antigenic sites except one, site 23. These mutant proteins formed disulfide-linked dimers and were efficiently transported to the cell surface. They did not, however, sediment on gradients like the wild-type protein. They were also defective in the biological activities associated with the wild-type protein. Proteins with mutations in cysteines 4 (amino acid 186), 6 (amino acid 238), 7 (amino acid 247), 8 (amino acid 251), 13 (amino acid 531), or 14 (amino acid 542) contained no mature antigenic sites but formed noncovalently linked oligomers. Proteins with mutations in cysteines 9 (amino acid 344), 10 (amino acid 455), 11 (amino acid 461), or 12 (amino acid 465) formed proteins with antigenic site 4 but no other mature antigenic sites. These mutant proteins also formed noncovalently linked oligomers. These results suggest that mutations in different cysteine residues block the maturation of the HN protein at different stages.

Animals↗