The influenza A virus (IAV) HA protein must be activated by host cells proteases in order to prime the molecule for fusion. Consequently, the availability of activating proteases and the susceptibility of HA to protease activity represents key factors in facilitating virus infection. As such, understanding the intricacies of HA cleavage by various proteases is necessary to derive insights into the emergence of pandemic viruses. To examine these properties, we generated a panel of HAs that are representative of the 16 HA subtypes that circulate in aquatic birds, as well as HAs representative of the subtypes that have infected the human population over the last century. We examined the susceptibility of the panel of HA proteins to trypsin, as well as human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2). Additionally, we examined the pH at which these HAs mediated membrane fusion, as this property is related to the stability of the HA molecule and influences the capacity of influenza viruses to remain infectious in natural environments. Our results show that cleavage efficiency can vary significantly for individual HAs, depending on the protease, and that some HA subtypes display stringent selectivity for specific proteases as activators of fusion function. Additionally, we found that the pH of fusion varies by 0.7 pH units among the subtypes, and notably, we observed that the pH of fusion for most HAs from human isolates was lower than that observed from avian isolates of the same subtype. Overall, these data provide the first broad-spectrum analysis of cleavage-activation and membrane fusion characteristics for all of the IAV HA subtypes, and also show that there are substantial differences between the subtypes that may influence transmission among hosts and establishment in new species.

While the molecular mechanism of membrane fusion by the influenza virus hemagglutinin (HA) protein has been studied extensively in vitro, the role of acid-dependent HA protein activation in virus replication, pathogenesis, and transmission in vivo has not been characterized. To investigate the biological significance of the pH of activation of the HA protein, we compared the properties of four recombinant viruses with altered HA protein acid stability to those of wild-type influenza virus A/chicken/Vietnam/C58/04 (H5N1) in vitro and in mallards. Membrane fusion by wild-type virus was activated at pH 5.9. Wild-type virus had a calculated environmental persistence of 62 days and caused extensive morbidity, mortality, shedding, and transmission in mallards. An N114K mutation that increased the pH of HA activation by 0.5 unit resulted in decreased replication, genetic stability, and environmental stability. Changes of ؉0.4 and ؊0.5 unit in the pH of activation by Y23H and K58I mutations, respectively, reduced weight loss, mortality, shedding, and transmission in mallards. An H24Q mutation that decreased the pH of activation by 0.3 unit resulted in weight loss, mortality, clinical symptoms, and shedding similar to those of the wild type. However, the HA-H24 1 Q virus was shed more extensively into drinking water and persisted longer in the environment. The pH of activation of the H5 HA protein plays a key role in the propagation of H5N1 influenza viruses in ducks and may be a novel molecular factor in the ecology of influenza viruses. The data also demonstrate that H5N1 neuraminidase activity increases the pH of activation of the HA protein in vitro.

Currently available drugs for the prevention and treatment of seasonal influenza virus infections are the M2 ion channel blockers (amantadine and rimantadine) and the neuraminidase (NA) inhibitors (oseltamivir and zanamivir) (9). The clinical usefulness of amantadine and rimantadine is limited due to the increasing incidence of adamantane-resistant viruses in the population (3, 11). Moreover, the M2 ion channel blockers inhibit only influenza A virus replication and are associated with neurological side effects. NA inhibitors are favored clinically, since they are effective against all NA subtypes, are well tolerated, and have a higher barrier for resistance (27). However, drug-resistant isolates have been detected in A/H3N2-and A/H5N1-infected patients receiving oseltamivir treatment (10, 16). Even more reason for concern is the recent and worldwide isolation of oseltamivir-resistant A/H1N1 mutants, even among untreated patients (17, 46). Oseltamivir and, to a lesser extent, zanamivir have been stockpiled as part of pandemic preparedness plans and form the cornerstone of the response to the recent outbreak of the swine flu A/H1N1 virus (39, 40). However, it is unclear whether these antivirals will be sufficient to deal with larger influenza epidemics, so there is an urgent need to develop antivirals that act on a novel influenza virus target.An attractive antiviral strategy is to block influenza virus entry into the host cell, a process in which the viral hemagglutinin (HA) plays a key role (42). HA is a trimeric envelope glycoprotein that contains two disulfide-linked polypeptide chains, HA1 and HA2. After attachment of the receptor binding domain in the HA1 subunit to sialic acid-containing cell surface glycans, the virion is internalized by endocytosis. The acidic pH of the endosome leads to an extensive and irreversible conformational change of the HA protein, resulting in exposure of the fusion peptide, which inserts into the endosomal target membrane of the host cell (18). After fusion of the viral and endosomal membranes, the viral ribonucleoproteins are released into the cytosol and transported into the nucleus, where replication occurs (6). Crystallographic studies have provided detailed insight into the processes of HA refolding and extrusion of the fusion peptide (4). The latter is a sequence of hydrophobic amino acids located at the N terminus of the HA2 subunit, which is, in the prefusogenic conformation, sequestered in a pocket of ionizable residues at the monomer interface of the HA trimer (48). In order to exploit the HA protein as an antiviral target, several small-molecule inhibitors that block the acid-induced conformational change of HA have been identified (2,19,21,30,49). For many of these, development has been hindered by their subtype-dependent activities. On the other hand, these diverse fusion inhibitors represent excellent tools to identify the HA amino acid residues involved in the fusion process and/or delineate the structural differences among HA subtypes (36). We report here the i...

The receptor specificity and cleavability of the hemagglutinin (HA) protein have been shown to regulate influenza A virus transmissibility and pathogenicity, but little is known about how its pH of activation contributes to these important biological properties. To identify amino acid residues that regulate the acid stability of the HA protein of H5N1 influenza viruses, we performed a mutational analysis of the HA protein of the moderately pathogenic A/chicken/Vietnam/C58/04 (H5N1) virus. Nineteen HA proteins containing point mutations in the HA2 coiled-coil domain or in an HA1 histidine or basic patch were generated. Wild-type and mutant HA plasmids were transiently transfected in cell culture and analyzed for total protein expression, surface expression, cleavage efficiency, pH of fusion, and pH of conformational change. Four mutations to residues in the fusion peptide pocket, Y23H and H24Q in the HA1 subunit and E105K and N114K in the HA2 subunit, and a K58I mutation in the HA2 coiled-coil domain significantly altered the pH of activation of the H5 HA protein. In some cases, the magnitude and direction of changes of individual mutations in the H5 HA protein differed considerably from similar mutations in other influenza A virus HA subtypes. Introduction of Y23H, H24Q, K58I, and N114K mutations into recombinant viruses resulted in virus-expressed HA proteins with similar shifts in the pH of fusion. Overall, the data show that residues comprising the fusion peptide pocket are important in triggering pH-dependent activation of the H5 HA protein.

bInfluenza virus entry is mediated by the acidic-pH-induced activation of hemagglutinin (HA) protein. Here, we investigated how a decrease in the HA activation pH (an increase in acid stability) influences the properties of highly pathogenic H5N1 influenza virus in mammalian hosts. We generated isogenic A/Vietnam/1203/2004 (H5N1) (VN1203) viruses containing either wild-type HA protein (activation pH 6.0) or an HA2-K58I point mutation (K to I at position 58) (activation pH 5.5). The VN1203-HA2-K58I virus had replication kinetics similar to those of wild-type VN1203 in MDCK and normal human bronchial epithelial cells and yet had reduced growth in human alveolar A549 cells, which were found to have a higher endosomal pH than MDCK cells. Wild-type and HA2-K58I viruses promoted similar levels of morbidity and mortality in C57BL/6J mice and ferrets, and neither virus transmitted efficiently to naive contact cage-mate ferrets. The acid-stabilizing HA2-K58I mutation, which diminishes H5N1 replication and transmission in ducks, increased the virus load in the ferret nasal cavity early during infection while simultaneously reducing the virus load in the lungs. Overall, a single, acid-stabilizing mutation was found to enhance the growth of an H5N1 influenza virus in the mammalian upper respiratory tract, and yet it was insufficient to enable contact transmission in ferrets in the absence of additional mutations that confer ␣(2,6) receptor binding specificity and remove a critical N-linked glycosylation site. The information provided here on the contribution of HA acid stability to H5N1 influenza virus fitness and transmissibility in mammals in the background of a non-laboratory-adapted virus provides essential information for the surveillance and assessment of the pandemic potential of currently circulating H5N1 viruses.

Unlike nuclear localization signals, there is no obvious consensus sequence for the targeting of proteins to the nucleolus. The nucleolus is a dynamic subnuclear structure which is crucial to the normal operation of the eukaryotic cell. Studying nucleolar trafficking signals is problematic as many nucleolar retention signals (NoRSs) are part of classical nuclear localization signals (NLSs). In addition, there is no known consensus signal with which to inform a study. The avian infectious bronchitis virus (IBV), coronavirus nucleocapsid (N) protein, localizes to the cytoplasm and the nucleolus. Mutagenesis was used to delineate a novel eight amino acid motif that was necessary and sufficient for nucleolar retention of N protein and colocalize with nucleolin and fibrillarin. Additionally, a classical nuclear export signal (NES) functioned to direct N protein to the cytoplasm. Comparison of the coronavirus NoRSs with known cellular and other viral NoRSs revealed that these motifs have conserved arginine residues. Molecular modelling, using the solution structure of severe acute respiratory (SARS) coronavirus N-protein, revealed that this motif is available for interaction with cellular factors which may mediate nucleolar localization. We hypothesise that the N-protein uses these signals to traffic to and from the nucleolus and the cytoplasm.

The nucleolus is a dynamic sub-nuclear structure which is involved in ribosome subunit biogenesis, modulation of cell growth and response to cell stress. The nucleolar proteome varies particularly with regard to the cell cycle. Viral proteins can localise to the nucleolus and using the coronavirus nucleocapsid (N) protein as a model, the cell cycle dependent trafficking of viral proteins to the nucleolus was investigated. Cell synchronisation studies coupled to live cell confocal microscopy indicated that nucleolar localisation of N protein was greater in the G2/M phase of the cell cycle than at other stages. This result was reinforced when FRAP and FLIP analysis indicated that N protein was more mobile within the nucleoplasm and nucleolus in the G2/M phase of the cell cycle. The data suggested that viral nucleolar proteins can also localise to the nucleolus in a cell cycle dependent manner and this may be related to dynamic trafficking.

SummaryThe nucleolus is a dynamic subnuclear structure involved in ribosome subunit biogenesis, cell cycle control and mediating responses to cell stress, among other functions. While many different viruses target proteins to the nucleolus and recruit nucleolar proteins to facilitate virus replication, the effect of infection on the nucleolus in terms of morphology and protein content is unknown. Previously we have shown that the coronavirus nucleocapsid protein will localize to the nucleolus. In this study, using the avian infectious bronchitis coronavirus, we have shown that virus infection results in a number of changes to the nucleolus both in terms of gross morphology and protein content. Using confocal microscopy coupled with fluorescent labelled nucleolar marker proteins we observed changes in the morphology of the nucleolus including an enlarged fibrillar centre. We found that the tumour suppressor protein, p53, which localizes normally to the nucleus and nucleolus, was redistributed predominately to the cytoplasm.