Improvement of prediction of mutation positions in H5N1 hemagglutinins of influenza A virus using neural network with distinguishing of arginine, leucine and serine

In this study, we attempt to improve the prediction of mutation position in H5N1 hemagglutinin from influenza A virus using the neural network model with distinguishing of arginine, leucine and serine. In comparison with previous logistic regression prediction model, the 3-6-1 feedforward back propagation neural network model reduced independents from seven to three without compromising the predictability, and the prediction is made according to the most similar parameterised hemagglutinin rather than the population means.     

Mechanism of antigenic drift in influenza virus. Amino acid sequence changes in an antigenically active region of Hong Kong (H3N2) influenza virus hemagglutinin

During antigenic drift in influenza viruses, changes in antigenicity are associated with changes in amino acid sequence of the large hemagglutinin polypeptide, HA1. In ten variants of Hong Kong (H3N2) influenza virus selected with monoclonal antibodies, the proline residue at position 143 in HA1 changed to serine, threonine, leucine or histidine. In other variants, asparagine 133 changed to lysine, glycine 144 to aspartic acid and serine 145 to lysine. All these changes are possible by single base changes in the RNA except the last, which requires a double base change. Residues 142 to 146 also changed in field strains of Hong Kong influenza isolated between 1968 and 1977 (Laver et al., 1980). The single amino acid sequence changes in HA1 of the monoclonal variants were detected by comparing the compositions of the soluble tryptic peptides from the variants with the known sequences of these peptides from wild-type virus. Two insoluble tryptic peptides, comprising residues 110 to 140 and 230 to 255 in the HA1 molecule, were not examined and we do not know if additional changes occurred in these regions.In order to determine whether sequential changes at the same position occurred during antigenic drift, antibody prepared against the new antigenic site on the variants in which proline 143 changed to histidine or threonine was used to select second generation variants of these variants. In the first case, the glycine residue (144) next to the histidine changed to aspartic acid, and in the second, the threonine residue at position 143 reverted to proline and the virus regained the antigenicity of wild-type.Although monoclonal antibodies revealed dramatic antigenic differences between the variants and wild-type virus, only those variants with changes at position 144 of glycine to aspartic acid or at position 145 of serine to lysine could be distinguished from wild-type virus using heterogeneous rabbit or ferret antisera. The other variants, including those which showed sequence changes in widely separated positions of HA1, could not be distinguished from wild-type with heterogeneous antisera.These findings suggest that sequence changes in the region comprising residues 142 to 146 of HA1 affect an important antigenic site on the hemagglutinin molecule, but how these changes affect the antigenic properties, or whether this region actually forms part of the antigenic site is not known.     

A comparative study of phenylalanyl-tRNA synthetases from Escherichia coli and Thermus thermophilus by the tritium topography method

A comparative study of thermostability and amino acid composition of phenylalanyl-tRNA synthetases from E. coli and Thermus thermophilus HB8 has been carried out. In the thermophilic protein the proline, leucine, phenylalanine, arginine content was considerably increased, whereas that of asparagine, isoleucine, serine, threonine and lysine was decreased as compared to the mesophilic protein. Using tritium topography, Pro, (Leu + Ile) and Gly were found to be the most accessible on the surfaces of the both enzymes. In the E. coli enzyme the threonine residues were also easy to access, while on the surface of the thermophilic enzyme arginine residues were more abundant. A quantitative assay of the surface compositions revealed the increased exposure of (Leu + Ile) residues in the thermophilic protein as well as of the charged asparagine and arginine residues. A possible relationship of the observed effects to thermostability is discussed.     

Computational Identification of Antigenicity-Associated Sites in the Hemagglutinin Protein of A/H1N1 Seasonal Influenza Virus

The antigenic variability of influenza viruses has always made influenza vaccine development challenging. The punctuated nature of antigenic drift of influenza virus suggests that a relatively small number of genetic changes or combinations of genetic changes may drive changes in antigenic phenotype. The present study aimed to identify antigenicity-associated sites in the hemagglutinin protein of A/H1N1 seasonal influenza virus using computational approaches. Random Forest Regression (RFR) and Support Vector Regression based on Recursive Feature Elimination (SVR-RFE) were applied to H1N1 seasonal influenza viruses and used to analyze the associations between amino acid changes in the HA1 polypeptide and antigenic variation based on hemagglutination-inhibition (HI) assay data. Twenty-three and twenty antigenicity-associated sites were identified by RFR and SVR-RFE, respectively, by considering the joint effects of amino acid residues on antigenic drift. Our proposed approaches were further validated with the H3N2 dataset. The prediction models developed in this study can quantitatively predict antigenic differences with high prediction accuracy based only on HA1 sequences. Application of the study results can increase understanding of H1N1 seasonal influenza virus antigenic evolution and accelerate the selection of vaccine strains.     

Amino-terminal processing of mutant forms of yeast iso-1-cytochrome c. The specificities of methionine aminopeptidase and acetyltransferase

Amino-terminal processing in the yeast Saccharomyces cerevisiae has been investigated by examining numerous mutationally altered forms of iso-1-cytochrome c. Amino-terminal residues of methionine were retained in sequences having penultimate residues of arginine, asparagine, glutamine, isoleucine, leucine, lysine, and methionine; in contrast, the amino-terminal methionine residues were exercised from residues of alanine, glycine, and threonine and were partially excised from residues of valine. The results suggest the occurrence of a yeast aminopeptidase that removes amino-terminal residues of methionine when they precede certain amino acids. A systematic search of the literature for amino-terminal sequences formed at initiation sites suggests the hypothetical yeast aminopeptidase usually has the same specificity as the amino peptidase from bacteria and higher eukaryotes. Our results and the results from the literature search suggest that the aminopeptidase cleaves amino-terminal methionine when it precedes residues of alanine, glycine, proline, serine, threonine, and valine but not when it precedes residues of arginine, asparagine, aspartic acid, glutamine glutamic acid, isoleucine, leucine, lysine, or methionine. In contrast to the normal iso-1-cytochrome c and in contrast to the majority of the mutationally altered proteins, certain forms were acetylated including the following sequences: acetyl(Ac)-Met-Ile-Arg-, Ac-Met-Ile-Lys, Ac-Met-Met-Asn-, and Ac-Met-Asn-Asn-. We suggest yeast contains acetyltransferases that acetylates these mutant forms of iso-1-cytochromes c because their amino-terminal regions resemble the amino-terminal regions of natural occurring proteins which are normally acetylated. The lack of acetylation of closely related sequences suggest that the hypothetical acetyltransferases are specific for certain amino-terminal sequences and that the 3 amino-terminal residues may play a critical role in determining these specificities.     

Mutations at palmitylation sites of the influenza virus hemagglutinin affect virus formation.

The carboxy terminus of the hemagglutinin (HA) of influenza A viruses contains three cysteine residues which are highly conserved among HA subtypes. It has previously been shown for the H2, H3, and H7 subtypes of HA that these cysteine residues are modified by the covalent attachment of palmitic acid. In order to study the role of the acylated cysteines in the formation of infectious influenza viruses, we introduced mutations into the HA of influenza A/WSN/33 virus (H1 subtype) by reverse-genetics techniques. We found that the cysteine at position 563 of the cytoplasmic tail is required for infectious-particle formation. The cysteine at position 560 can be changed to alanine or tyrosine to yield virus strains that are attenuated in cell cultures. The change from cysteine at position 553 to serine or alanine does not significantly alter the phenotype of the virus. The requirement for a cysteine at position 563 suggests a functional role for palmitylation of the cytoplasmic tail. This interpretation is further supported by experiments in which two or more of the cysteine residues were mutated, eliminating potential palmitylation sites. None of these double or triple mutations resulted in infectious virus. Selection of revertants of the attenuated cysteine-to-tyrosine mutant (mutation at position 560) always resulted in reversion to cysteine rather than to other amino acids. Although our data indicate a biological role for the conserved cysteine residues in the cytoplasmic tail of the HA of influenza viruses, we cannot exclude the possibility that structural constraints in the cytoplasmic tail of the HA--rather than altered palmitylation--are the determining factors for infectious-particle formation.     

Influenza C virus hemagglutinin: comparison with influenza A and B virus hemagglutinins

The complete nucleotide sequence of the influenza C/California/78 virus RNA 4 was obtained by using cloned cDNA derived from the RNA segment. This gene is 2,071 nucleotides long and can code for a polypeptide of 654 amino acids. Although there are no convincing sequence homologies between RNA 4 and the hemagglutinin genes of influenza A and B viruses, we suggest, on the basis of structural features, that RNA 4 of the influenza C virus codes for the hemagglutinin. The structural features which are common to the hemagglutinins of influenza A, B, and C viruses include (i) a hydrophobic signal peptide, (ii) an arginine cleavage site between the hemagglutinin 1 and 2 subunits, (iii) hydrophobic regions at the amino and carboxyl termini of the hemagglutinin 2 subunit, and (iv) several conserved cysteine residues. Additional evidence that RNA 4 of influenza C virus codes for the hemagglutinin is that the tripeptide Ile-Phe-Gly, known to be present at the amino terminus of the hemagglutinin 2 subunit of influenza C virus, is encoded by RNA 4 at a point immediately adjacent to the presumptive arginine cleavage site. The lack of primary sequence homology between the influenza C virus hemagglutinin and the influenza A or B virus hemagglutinins, which all have similar functions, might be attributed to convergent rather than divergent evolution. However, the structural similarities among the influenza A, B, and C virus hemagglutinins strongly suggest that the three hemagglutinin genes have diverged from a common precursor.     

Two polar residues within C‐terminal domain of M1 are critical for the formation of influenza A Virions

The matrix protein 1 (M1) is the most abundant structural protein in influenza A virus particles. It oligomerizes to form the matrix layer under the lipid membrane, sustaining stabilization of the morphology of the virion. The present study indicates that M1 forms oligomers based on a fourfold symmetrical oligomerization pattern. Further analysis revealed that the oligomerization pattern of M1 was controlled by a highly conserved region within the C‐terminal domain. Two polar residues of this region, serine‐183 (S183) and threonine‐185 (T185), were identified to be critical for the oligomerization pattern of M1. M1 point mutants suggest that single S183A or T185A substitution could result in the production of morphologically filamentous particles, while double substitutions, M1‐S183A/T185A, totally disrupted the fourfold symmetry and resulted in the failure of virus production. These data indicate that the polar groups in these residues are essential to control the oligomerization pattern of M1. Thus, the present study will aid in determining the mechanisms of influenza A virus matrix layer formation during virus morphogenesis.     

Involvement of an Arginine Triplet in M1 Matrix Protein Interaction with Membranes and in M1 Recruitment into Virus-Like Particles of the Influenza A(H1N1)pdm09 Virus

The influenza A(H1N1)pdm09 virus caused the first influenza pandemic of the 21st century. In this study, we wanted to decipher the role of conserved basic residues of the viral M1 matrix protein in virus assembly and release. M1 plays many roles in the influenza virus replication cycle. Specifically, it participates in viral particle assembly, can associate with the viral ribonucleoprotein complexes and can bind to the cell plasma membrane and/or the cytoplasmic tail of viral transmembrane proteins. M1 contains an N-terminal domain of 164 amino acids with two basic domains: the nuclear localization signal on helix 6 and an arginine triplet (R76/77/78) on helix 5. To investigate the role of these two M1 basic domains in influenza A(H1N1)pdm09 virus molecular assembly, we analyzed M1 attachment to membranes, virus-like particle (VLP) production and virus infectivity. In vitro, M1 binding to large unilamellar vesicles (LUVs), which contain negatively charged lipids, decreased significantly when the M1 R76/77/78 motif was mutated. In cells, M1 alone was mainly observed in the nucleus (47%) and in the cytosol (42%). Conversely, when co-expressed with the viral proteins NS1/NEP and M2, M1 was relocated to the cell membranes (55%), as shown by subcellular fractionation experiments. This minimal system allowed the production of M1 containing-VLPs. However, M1 with mutations in the arginine triplet accumulated in intracellular clusters and its incorporation in VLPs was strongly diminished. M2 over-expression was essential for M1 membrane localization and VLP production, whereas the viral trans-membrane proteins HA and NA seemed dispensable. These results suggest that the M1 arginine triplet participates in M1 interaction with membranes. This R76/77/78 motif is essential for M1 incorporation in virus particles and the importance of this motif was confirmed by reverse genetic demonstrating that its mutation is lethal for the virus. These results highlight the molecular mechanism of M1-membrane interaction during the formation of influenza A(H1N1)pdm09 virus particles which is essential for infectivity.     

Studies on influenza haemagglutinin fusion peptide mutants generated by reverse genetics

Influenza haemagglutinin (HA) is responsible for fusing viral and endosomal membranes during virus entry. In this process, conformational changes in the HA relocate the HA(2) N-terminal 'fusion peptide' to interact with the target membrane. The highly conserved HA fusion peptide shares composition and sequence features with functionally analogous regions of other viral fusion proteins, including the presence and distribution of glycines and large side-chain hydrophobic residues. HAs with mutations in the fusion peptide were expressed using vaccinia virus recombinants to examine the requirement for fusion of specific hydrophobic residues and the significance of glycine spacing. Mutant HAs were also incorporated into infectious influenza viruses for analysis of their effects on infectivity and replication. In most cases alanine, but not glycine substitutions for the large hydrophobic residues, yielded fusion-competent HAs and infectious viruses, suggesting that the conserved spacing of glycines may be structurally significant. When viruses containing alanine substitutions for large hydrophobic residues were passaged, pseudoreversion to valine was observed, indicating a preference for large hydrophobic residues at specific positions. Viruses were also obtained with serine, leucine or phenylalanine as the N-terminal residue, but these replicated to significantly lower levels than wild-type virus with glycine at this position.     

Unique N-region determines low basal activity and limited inducibility of A-RAF kinase: the role of N-region in the evolutionary divergence of RAF kinase function in vertebrates

In mammals the RAF family of serine/threonine kinases consists of three members, A-, B-, and C-RAF. A prominent feature of RAF isoforms regards differences in basal and inducible kinase activities. To elucidate the nature of these differences, we studied the role of the nonconserved residues within the N-region (Negative-charge regulatory region). The nonconserved amino acids in positions -3 and +1 relative to the highly conserved serine 299 in A-RAF and serine 338 in C-RAF have so far not been considered as regulatory residues. Here we demonstrate the essential role of these residues in the RAF activation process. Substitution of tyrosine 296 in A-RAF to arginine led to a constitutively active kinase. In contrast, substitution of glycine 300 by serine (mimicking B- and C-RAF) acts in an inhibitory manner. Consistent with these data, the introduction of glycine in the analogous position of C-RAF (S339G mutant) led to a constitutively active C-RAF kinase. Based on the three-dimensional structure of the catalytic domain of B-RAF and using the sequences of the N-regions of A- and C-RAF, we searched by molecular modeling for the putative contact points between these two moieties. A tight interaction between the N-region residue serine 339 of C-RAF and arginine 398 of the catalytic domain was identified and proposed to inhibit the kinase activity of RAF proteins, because abrogation of this interaction contributes to RAF activation. Furthermore, tyrosine 296 in A-RAF favors a spatial orientation of the N-region segment, which enables a tighter contact to the catalytic domain, whereas a glutamine residue at this position in C-RAF abrogates this interaction. Considering this observation, we suggest that tyrosine 296, which is unique for A-RAF, is a major determinant of the low activating potency of this RAF isoform.     

Regulation of Influenza A Virus Nucleoprotein Oligomerization by Phosphorylation

In the influenza virus ribonucleoprotein complex, the oligomerization of the nucleoprotein is mediated by an interaction between the tail-loop of one molecule and the groove of the neighboring molecule. In this study, we show that phosphorylation of a serine residue (S165) within the groove of influenza A virus nucleoprotein inhibits oligomerization and, consequently, ribonucleoprotein activity and viral growth. We propose that nucleoprotein oligomerization in infected cells is regulated by reversible phosphorylation.     

Influence of acylation sites of influenza B virus hemagglutinin on fusion pore formation and dilation

The cytoplasmic tail (CT) of hemagglutinin (HA) of influenza B virus (BHA) contains at positions 578 and 581 two highly conserved cysteine residues (Cys578 and Cys581) that are modified with palmitic acid (PA) through a thioester linkage. To investigate the role of PA in the fusion activity of BHA, site-specific mutagenesis was performed with influenza B virus B/Kanagawa/73 HA cDNA. All of the HA mutants were expressed on Cos cells by an expression vector. The membrane fusion ability of the HA mutants at a low pH was quantitatively examined with lipid (octadecyl rhodamine B chloride) and aqueous (calcein) dye transfer assays and with the syncytium formation assay. Two deacylation mutants lacking a CT or carrying serine residues substituting for Cys578 and Cys581 promoted full fusion. However, one of the single-acylation-site mutants, C6, in which Cys581 is replaced with serine, promoted hemifusion but not pore formation. In contrast, four other single-acylation-site mutants that have a sole cysteine residue in the CT at position 575, 577, 579, or 581 promoted full fusion. The impaired pore-forming ability of C6 was improved by amino acid substitution between residues 578 and 582 or by deletion of the carboxy-terminal leucine at position 582. Syncytium-forming ability, however, was not adequately restored by these mutations. These facts indicated that the acylation was not significant in membrane fusion by BHA but that pore formation and pore dilation were appreciably affected by the particular amino acid sequence of the CT and the existence of a single acylation site in CT residue 578.     

Differential nucleocytoplasmic shuttling of the nucleoprotein of influenza a viruses and association with host tropism

The nucleoprotein (NP) of influenza A virus plays a crucial role in virus replication, infectivity, and host adaptation. As a major component of the viral ribonucleoprotein complexes (vRNP), NP initiates vRNP shuttling between the nucleus and cytoplasm in the host cell. However, the characteristics of the nucleocytoplasmic shuttling of NP from H1N1 influenza A virus still remain unclear. In the present study, the subcellular localization and the related key residues of the H1N1 influenza virus NP were identified and evaluated. The NP of influenza virus A/WSN/33 (H1N1; WSN) displayed a more obvious nuclear accumulation than A/Anhui/1/2013 (H7N9; AH) and A/chicken/Shandong/lx1023/2007 (H9N2; SD). NP residue K4, located in NLS1, and residue F253, located in NES3, from WSN NP are not conserved in H7N9 and H9N2, which instead encode Q4 and I253, respectively. Crucially, these residues are involved in the regulation of NP nucleocytoplasmic shuttling through interactions with CRM1 and importin‐α. Moreover, residues at position 253 also play important roles in the replication of the virus, resulting in an increase in vRNP polymerase activity and an alteration of the cell tropism and pathogenicity in mice. The present data revealed a pivotal role of the Q4 and I253 residues of NP from H7N9 in enhancing the cytoplasmic accumulation of NP and vRNP activity compared to the K4 and F253 residues in WSN‐NP. In addition, an F253I substitution in the NP of WSN altered the survival ratio of infected mice and the growth curve in infected avian‐origin cells (DF‐1). The current data indicate that the F253I mutation results in attenuated pathogenicity of the virus in mice and altered cell tropism. The present study demonstrated the dissimilarity in subcellular NP transport processes between H1N1 virus WSN and other influenza A virus strains, as well as uncovered the mechanism responsible for this difference.     

O-acetylation and de-O-acetylation of sialic acids. Sialic acid esterases of diverse evolutionary origins have serine active sites and essential arginine residues

We and others have recently described 9-O-acetyl-sialic acid esterase (9-O-Ac-SA esterase) activities that appear to be specific for removal of O-acetyl esters from the 9-position of naturally occurring sialic acids. We have now examined a variety of species for such enzymes and found them in vertebrates and higher invertebrates, but not in plants or in lower invertebrates. This evolutionary distribution correlates well with that of the sialic acids themselves. All of the 9-O-Ac-SA esterase activities tested were inhibited by diisopropyl fluorophosphate (DFP) in a dose-dependent fashion. This indicates that each of these enzymes has a serine active site similar to the well known serine esterases and serine proteases. Methyl esterification of the carboxyl group of 9-O-acetyl-N-acetylneuraminic acid significantly reduced the activity of all of the 9-O-Ac-SA esterases against the O-acetyl group. This indicates that each of these enzymes may recognize the negatively charged carboxyl group of the sialic acid. Enzymes that recognize anionic substrates frequently have an essential arginine residue (Riordan, J. F., McElvany, K. D., and Borders, C. L., Jr. (1977) Science 195, 884-886). We therefore studied the effects of the arginine-specific modifying reagents 2,3-butanedione and phenylglyoxal on 9-O-Ac-SA esterase activities from influenza C virus, human erythrocytes, rat liver, starfish gonads, and sea bass brain. All of these enzymes were inhibited in a dose-dependent fashion by both reagents, under conditions previously known to avoid nonspecific modification. In contrast, the typical serine proteases trypsin and kallikrein and the serine esterase acetylcholinesterase were not significantly affected, even by the highest concentrations of these reagents used. These data indicate that five 9-O-Ac-SA esterase activities from evolutionarily distinct origins all have serine active sites and essential arginine residues. We postulate that the arginine residue is involved in substrate recognition via the negatively charged carboxyl group of the sialic acids. Thus, these 9-O-Ac-SA esterase activities may be members of a previously undescribed class of serine esterase.     

Novel in-frame two codon translational hop during synthesis of bovine placental lactogen in a recombinant strain of Escherichia coli.

A recombinant Escherichia coli strain was constructed for the overexpression of bovine placental lactogen (bPL), using a bPL structural gene containing 9 of the rare arginine codons AGA and AGG. When high level bPL synthesis was induced in this strain, cell growth was inhibited and bPL accumulated to less than 10% of total cell protein. In addition, about 2% of the recombinant bPL produced from this strain exhibited an altered trypsin digestion pattern. Amino acid residues 74 through 109 normally produce 2 tryptic peptides, but the altered form of bPL lacked these two peptides and instead had a new peptide which was missing arginine residue 86 and one of the two flanking leucine residues. The codon for arginine residue 86 was AGG and the codons for the flanking leucine residues 85 and 87 were TTG. When 5 of the 9 AGA and AGG codons in the bPL structural gene were changed to more preferred arginine codons, cell growth was not inhibited and bPL accumulated to about 30% of total cell protein. When bPL was purified from this modified strain, which included changing the arginine codon at position 86 from AGG to CGT, none of the altered form of bPL was produced. These observations are consistent with a model in which translational pausing occurs at the arginine residue 86 AGG codon because the corresponding arginyl-tRNA species is reduced by the high level of bPL synthesis, and a translational hop occurs from the leucine residue 85 TTG codon to the leucine residue 87 TTG codon. This observation represents the first report of an error in protein synthesis due to an in-frame translational hop within an open reading frame.     

Nutrient uptake by Candida albicans: the influence of cell surface mannoproteins.

Numerous ultrastructural and biochemical analyses have been performed to characterize the cell wall composition and structure of Candida albicans. However, little investigation has focused on how subtle differences in cell wall structure influence the intracellular transport of amino acids and monosaccharides. In this study C. albicans 4918 and ATCC 10231 were grown in culture conditions capable of modifying surface mannoproteins and induced surface hydrophobic or hydrophilic yeast cell wall states. Subcultures of these hydrophobic and hydrophilic yeasts were subsequently incubated with one of seven L-[3H] amino acids: glycine, leucine, proline, serine, aspartic acid, lysine, or arginine. The transport of [3H] mannose and [3H] N-acetyl-D-glucosamine were also investigated. This study revealed significant strain differences (P < or = 0.05) between hydrophilic and hydrophobic yeast transport of these nutrients throughout a 2 h incubation. Hydrophilic cultures of 4918 and ATCC 10231 transported nearly two times more (pmol mg-1 dry weight) proline, mannose, and N-acetyl-D-glucosamine than hydrophobic yeast. Hydrophobic cultures preferentially incorporated serine and aspartic acid in both these strains. Strain variation was indicated with the transport of leucine, lysine, and arginine, as follows: experiments showed that hydrophilic 4918 cultures selectively transported leucine, lysine, and arginine, whereas, the hydrophobic ATCC 10231 cultures incorporated these amino acids.     

Influenza C virus esterase: analysis of catalytic site, inhibition, and possible function.

The active site serine of the acetylesterase of influenza C virus was localized to amino acid 71 of the hemagglutinin-esterase protein by affinity labeling with 3H-labeled diisopropylfluorophosphate. This serine and the adjacent amino acids (Phe-Gly-Asp-Ser) are part of a consensus sequence motif found in serine hydrolases. Since comparative analysis failed to reveal esterase sequence similarities with other serine hydrolases, we suggest that this viral enzyme is a serine hydrolase constituting a new family of serine esterases. Furthermore, we found that the influenza C virus esterase was inhibited by isocoumarin derivatives, with 3,4-dichloroisocoumarin being the most potent inhibitor. Addition of this compound prevented elution of influenza C virus from erythrocytes and inhibited virus infectivity, possibly through inhibition of virus entry into cells.     

Cleavage of influenza a virus hemagglutinin in human respiratory epithelium is cell associated and sensitive to exogenous antiproteases

Proteolytic cleavage of the hemagglutinin (HA) of human influenza viruses A/Aichi/2/68 (H3N2) and A/WSN/34 (H1N1) from HA0 to HA1/HA2 was studied in primary human adenoid epithelial cells (HAEC). HAEC contain a mixture of ciliated and nonciliated secretory cells and mimic the epithelium membrane of the human respiratory tract. Pulse-chase labeling with [(35)S]methionine and Western blot analysis with anti-HA antibodies of cellular and virion polypeptides showed that HAEC cleaved newly synthesized HA0 to HA1/HA2 ("cleavage from within") and significant amounts of cleaved HA accumulated within cells. It was also shown that HAEC was able to cleave HA0 of incoming virions ("cleavage from without"), whereas the HA0 of nonabsorbed virions free in extracellular fluid were not cleaved, supporting the conclusion that HA0 cleavage in HAEC is cell associated. Low-molecular-weight inhibitors of serine proteases, aprotinin and leupeptin, when added to influenza virus-infected HAEC suppressed HA0 cleavage and reduced the amount of cleaved HA1/HA2 both in cells and in progeny virions and thus diminished the infectivity of the virus. In contrast, the addition of fetal bovine serum, containing a number of high-molecular-weight antiproteases that compete for proteases in the extracellular environment, did not inhibit influenza virus growth in HAEC. These data suggest that in human respiratory epithelium the cleavage of influenza virus HA containing a single arginine in the proteolytic site (i) is a cell-associated process accomplished by serine-type protease(s) and (ii) is sensitive to low-molecular-weight exogenous inhibitors of serine proteases.