A functional sequence-specific interaction between influenza A virus genomic RNA segments

Influenza A viruses cause annual influenza epidemics and occasional severe pandemics. Their genome is segmented into eight fragments, which offers evolutionary advantages but complicates genomic packaging. The existence of a selective packaging mechanism, in which one copy of each viral RNA is specifically packaged into each virion, is suspected, but its molecular details remain unknown. Here, we identified a direct intermolecular interaction between two viral genomic RNA segments of an avian influenza A virus using in vitro experiments. Using silent trans-complementary mutants, we then demonstrated that this interaction takes place in infected cells and is required for optimal viral replication. Disruption of this interaction did not affect the HA titer of the mutant viruses, suggesting that the same amount of viral particles was produced. However, it nonspecifically decreased the amount of viral RNA in the viral particles, resulting in an eightfold increase in empty viral particles. Competition experiments indicated that this interaction favored copackaging of the interacting viral RNA segments. The interaction we identified involves regions not previously designated as packaging signals and is not widely conserved among influenza A virus. Combined with previous studies, our experiments indicate that viral RNA segments can promote the selective packaging of the influenza A virus genome by forming a sequence-dependent supramolecular network of interactions. The lack of conservation of these interactions might limit genetic reassortment between divergent influenza A viruses.Influenza A viruses (IAVs) belong to the Orthomyxoviridae family and cause annual influenza epidemics and occasional pandemics that represent a major threat for human health (1). The IAV genome consists of eight single-stranded negative-sense RNA segments (vRNAs), ranging from 890 to 2,341 nucleotides (nts) and packaged as viral ribonucleoproteins (vRNPs) containing multiple copies of nucleoprotein (NP) and a RNA-dependent RNA polymerase complex (2–4). The central coding region (in antisense orientation) of the vRNAs is flanked by short, segment-specific untranslated regions and conserved, partially complementary, terminal sequences that constitute the viral polymerase promoter and impose a panhandle structure to the vRNPs (4–9). The segmented nature of the IAV genome favors viral evolution by genetic reassortment. This process, which takes place when a single cell is coinfected by different IAVs, can generate pandemic viruses that represent a major threat for human health (1). However, segmentation complicates packaging of the viral genome into progeny virions.Although it had initially been proposed that the vRNAs are randomly packaged into budding viral particles, several lines of experiment suggest that IAVs specifically package one copy of each vRNA during viral assembly (7). First, electron microscopy and tomography revealed that the relative disposition of the eight vRNPs within viral particles is not random, even though some variability is tolerated, and they adopt a typical arrangement, with seven vRNPs surrounding a central one (10–12). Second, genetic and biochemical analysis revealed that the vast majority of IAV particles contain exactly one copy of each vRNA (7, 13, 14). Third, analysis of defective interfering RNAs (7, 15–17) and reverse genetic experiments (7, 18–25) identified specific bipartite packaging signals, most often located within the ends of the coding regions, in each segment. Of note, the terminal promoters are crucial for RNA packaging (8), but they cannot confer specificity to the packaging process (7).A selective packaging mechanism requires the existence of direct RNA–RNA or indirect RNA–protein interactions between vRNAs (7). Because all vRNAs associate with the same viral proteins to form vRNPs and no cellular protein has been identified that would specifically recognize an IAV packaging signal, we (10) and others (7, 12, 19) hypothesized that direct interactions between vRNAs might ensure selective packaging. However, these interactions remain elusive. We recently showed that the eight vRNAs of both a human H3N2 IAV (10) and an avian H5N2 IAV (26) form specific networks of intermolecular interactions in vitro, but the functional relevance of these interactions was not demonstrated. Here, we used a biochemical approach to identify, at the nt level, an interaction between two in vitro transcribed vRNAs. Unexpectedly, this interaction occurs between regions not previously identified as packaging signals. We then demonstrated that this interaction is important for infectivity and packaging of the viral genome.     

Critical role of segment-specific packaging signals in genetic reassortment of influenza A viruses

The fragmented nature of the influenza A genome allows the exchange of gene segments when two or more influenza viruses infect the same cell, but little is known about the rules underlying this process. Here, we studied genetic reassortment between the A/Moscow/10/99 (H3N2, MO) virus originally isolated from human and the avian A/Finch/England/2051/91 (H5N2, EN) virus and found that this process is strongly biased. Importantly, the avian HA segment never entered the MO genetic background alone but always was accompanied by the avian PA and M fragments. Introduction of the 5′ and 3′ packaging sequences of HA_MO into an otherwise HA_EN backbone allowed efficient incorporation of the chimerical viral RNA (vRNA) into the MO genetic background. Furthermore, forcing the incorporation of the avian M segment or introducing five silent mutations into the human M segment was sufficient to drive coincorporation of the avian HA segment into the MO genetic background. These silent mutations also strongly affected the genotype of reassortant viruses. Taken together, our results indicate that packaging signals are crucial for genetic reassortment and that suboptimal compatibility between the vRNA packaging signals, which are detected only when vRNAs compete for packaging, limit this process.The mechanisms by which animal viruses are introduced into and are disseminated through the human population remain to be addressed. In particular, emerging pathogenic influenza viruses, such as the highly pathogenic avian H5N1 virus and the 2009 “swine” H1N1 virus (H1N1pdm2009), pose major public health and scientific challenges (1, 2). Even though the natural reservoirs of influenza A viruses are wild aquatic birds, influenza A viruses exhibit a broad host range and a wide antigenic diversity, represented by combinations of 17 hemagglutinin (HA) and nine neuraminidase (NA) subtypes (3). Two subtypes of influenza A viruses, H1N1 and H3N2, currently are circulating in the human population.The genome of influenza A viruses is composed of eight single-stranded, negative-sense viral RNA (vRNA) segments. Each segment is associated with the heterotrimeric polymerase complex consisting of polymerase basic proteins 1 and 2 and polymeric acid (PB1/PB2/PA) and is covered by the viral nucleoprotein (NP) to form a viral ribonucleoparticle (vRNP). The fragmented nature of the genome allows the exchange of gene segments when two or more influenza viruses coinfect the same cell, in a process named “genetic reassortment” (4). Genetic reassortment is a major feature of influenza evolution and cross-species transmission and also is important for the generation of antigenically novel isolates by introducing novel HA segments in compatible genetic backgrounds (5–7). Future pandemic viruses most likely will carry different HA genes to which human populations are immunologically naive. The strains giving rise to the 1918 Spanish, 1957 Asian, and 1968 Hong Kong influenza pandemics all harbored an HA segment derived from an avian virus. The avian viruses circulating in the waterfowl are the source of the HA genes most likely to be introduced into the human population (8). Phylogenetic, epidemic, epizootic, and virology studies suggest that swine serve as “mixing vessels” for the generation of human–avian–swine reassortant viruses.When the reassortment process takes place between a human and an avian influenza virus, there are in theory 127 possible reassortant viruses harboring the avian HA segment. Two studies used forced reverse genetics (i.e., a minimal set of reverse genetic plasmids allowing no competition between segments) to generate all 127 reassortant viruses carrying the HA segment from an avian H5N1 virus in the genetic background of a human H3N2 or the HA segment from an avian H9N2 virus in the genetic background of the human 2009 pandemic H1N1 virus (9, 10). They showed that 49% (H5N1/H3N2) and 58% (H9N2/H1N1) of these reassortant viruses replicated efficiently in Madin–Darby canine kidney (MDCK) cells (9, 10). However, several reports indicated that the number of observed natural or experimental reassortant viruses is much smaller than 127, suggesting that reassortment is somehow restricted (4, 11, 12). When analyzing viruses from the nasal secretions of ferrets coinfected with human H3N2 and avian H5N1 viruses, Jackson et al. (13) observed that only 3.1% were reassortant viruses possessing the HA H5 gene, and they corresponded to only five distinct genotypes. Genetic reassortment between human H3N2 and an equine H7N7 virus has been studied using cotransfection (4). Only 1.6% of purified viruses, corresponding to two genotypes, were reassortant viruses possessing the HA H7 gene (4). In contrast, a high frequency of genetic reassortment was observed recently between swine-origin H1N1 and avian H5N1 viruses: 64% of purified viruses, corresponding to 20 different genotypes, were reassortant viruses possessing the HA H5 gene (14). In this case, the high reassortment rate was attributed to the triple reassortant internal gene cassette, consisting of the avian PA and PB2 genes, the nonstructural (NS), NP, and matrix (M) swine genes, and the human PB1 gene (15).The low number of reassortant genotypes usually generated from genetically diverse influenza viruses suggests incompatibilities at the protein and/or genomic level. Accumulating evidence indicates that protein incompatibility among the vRNP components is a limiting factor for reassortment between two viruses (4, 16–18), but little is known about genetic incompatibilities between the vRNA segments. Although incompatibility between proteins is expected to have similar effects in cotransfection or coinfection experiments and in forced reverse genetic experiments, genomic incompatibilities may have several possible effects, especially at the level of the vRNA-packaging signals. Some incompatibilities between packaging signals might reduce viral replication in the absence of competition (absolute incompatibility), whereas more subtle ones might be revealed only when vRNA segments from the two parental viruses compete for packaging (suboptimal compatibility). Reverse genetics-derived reassortant viruses (RGd-RV) that possess the H5N1 (H5) HA in an otherwise H3N2 genetic background show high replicative capacities in MDCK cells (10). Similarly, RGd-RV with the HA gene from H5N1 virus in the H1N1pdm2009 genetic background replicated efficiently in primary human respiratory epithelial cells and caused 100% mortality in mice (19). However, phylogenetic analyses of natural or experimental reassortant viruses have shown that the HA segment from avian, swine, or equine viruses was never incorporated alone in the genetic background of a human virus (13, 14, 20): The HA segment is packaged with additional groups of gene segments depending on the viral subtypes involved in the coinfection process (13, 14).The inability to obtain a virus containing a nonhuman HA gene in an otherwise human genetic background, in contrast with the ability to produce “7+1” RGd-RV with a high yield of replication, suggests that the reassortment process might be restricted by suboptimal compatibility between the vRNA-packaging signals (10).To predict how pandemic influenza viruses can emerge, the complex molecular mechanisms limiting or facilitating genetic reassortment must be deciphered. Using reverse genetics, cis-packaging signals of the human H1N1 WSN and PR8 strains were found to reside at both ends of each vRNA, including the UTRs, along with up to 80 bases of adjacent coding sequences (21–28). In this study, we generated reassortant viruses in vitro from avian H5N2 and human H3N2 viruses to identify incompatibilities between the two parental viruses arising at the vRNA level. Our experiments focusing on the generation of reassortant viruses containing the HA H5 gene segment in an H3N2 genetic background indicate that genomic suboptimal compatibility driven by the selective packaging mechanism limits the generation of HA H5 reassortant viruses in vitro.     

Replication protein of tobacco mosaic virus cotranslationally binds the 5′ untranslated region of genomic RNA to enable viral replication

Genomic RNA of positive-strand RNA viruses replicate via complementary (i.e., negative-strand) RNA in membrane-bound replication complexes. Before replication complex formation, virus-encoded replication proteins specifically recognize genomic RNA molecules and recruit them to sites of replication. Moreover, in many of these viruses, selection of replication templates by the replication proteins occurs preferentially in cis. This property is advantageous to the viruses in several aspects of viral replication and evolution, but the underlying molecular mechanisms have not been characterized. Here, we used an in vitro translation system to show that a 126-kDa replication protein of tobacco mosaic virus (TMV), a positive-strand RNA virus, binds a 5′-terminal ∼70-nucleotide region of TMV RNA cotranslationally, but not posttranslationally. TMV mutants that carried nucleotide changes in the 5′-terminal region and showed a defect in the binding were unable to synthesize negative-strand RNA, indicating that this binding is essential for template selection. A C-terminally truncated 126-kDa protein, but not the full-length 126-kDa protein, was able to posttranslationally bind TMV RNA in vitro, suggesting that binding of the 126-kDa protein to the 70-nucleotide region occurs during translation and before synthesis of the C-terminal inhibitory domain. We also show that binding of the 126-kDa protein prevents further translation of the bound TMV RNA. These data provide a mechanistic explanation of how the 126-kDa protein selects replication templates in cis and how fatal collision between translating ribosomes and negative-strand RNA-synthesizing polymerases on the genomic RNA is avoided.Virions of positive-strand RNA viruses contain genomic RNA of messenger sense. After infection, genomic RNA is released from the virions into the cytoplasm and translated to produce viral proteins, including viral RNA-dependent RNA polymerases and other replication-related proteins. These proteins are collectively called “replication proteins.” In eukaryotic positive-strand RNA viruses, replication proteins recruit genomic RNA to the cytoplasmic face of intracellular membranes to form replication complexes (1, 2). Negative-strand RNAs that are complementary to genomic RNAs are synthesized in the replication complexes, and then, using the negative-strand RNAs as templates, genomic RNA is copied and released into the cytoplasm. The recognition of template RNAs and their recruitment to the replication complexes are key processes in selective amplification of genomic RNA by positive-strand RNA viruses. In several positive-strand RNA viruses, cis-acting elements for replication-template selection have been identified, and, for some of them, it was demonstrated that replication proteins directly bind to these elements (3).Replication of tobacco mosaic virus (TMV), poliovirus, and many other positive-strand RNA viruses is cis-preferential: i.e., replication proteins recognize their own translation templates for replication (4–13). Because viral RNA replication is error-prone, it is important for viruses to selectively eliminate defective genomes. Template selection in cis is apparently advantageous in this regard because the genomes that encode replication proteins of lower performance are amplified less efficiently. Despite its importance in viral replication as well as evolution, little is known about how replication proteins select a template RNA in cis although it was proposed that requirement of nascent or newly synthesized replication proteins for replication and restricted diffusion or integrity of the proteins underlie the phenomenon (6).The genomic RNAs of positive-strand RNA viruses serve as templates for both translation and negative-strand RNA synthesis. During negative-strand RNA synthesis, viral RNA polymerases move along genomic RNA templates in a 3′-to-5′ direction. On the other hand, ribosomes synthesize viral proteins moving along the genomic RNA templates in a 5′-to-3′ direction. If these reactions take place on a single genomic RNA molecule at the same time, RNA polymerases and ribosomes collide, which results in the collapse of both reactions because these molecules cannot reverse direction or detach from the template RNA (14). Thus, positive-strand RNA viruses must clear ribosomes from the genomic RNA strands before negative-strand RNA synthesis occurs (15, 16).TMV belongs to the alpha-like virus superfamily of positive-strand RNA viruses. Its genome is a 5′-capped monopartite RNA and encodes at least four proteins, including the 5′ terminal 126-kDa protein, its translational read-through product of 183 kDa, a 30-kDa cell-to-cell movement protein, and a 17.5-kDa coat protein (17). The 126-kDa and 183-kDa proteins are replication proteins (18). The 126-kDa protein harbors a methyltransferase-like domain that is involved in RNA 5′ capping in its N-terminal region and a helicase-like domain in its C-terminal region. A region between these two domains is called the intervening region, or IR. The read-through part of the 183-kDa protein contains a polymerase-like domain (19). A deletion derivative of TMV RNA, named TMV126 RNA, that encodes the 126-kDa protein but not the 183-kDa protein can replicate when the 183-kDa protein is supplied in trans from a helper virus. However, TMV126 mutants that do not encode functional 126-kDa protein cannot replicate even if the wild-type 126-kDa and 183-kDa proteins are supplied in trans (8). This and other observations indicate that the 126-kDa protein functions primarily in cis (20, 21). The 5′ untranslated region (UTR) of TMV genomic RNA called Ω is ∼70 nucleotides (nt) in length, contains 12 CAA repeats, and is reported to have unusual tertiary structure with non-Watson–Crick base pairing (22, 23). The 5′ UTR of TMV RNA is a well-known translation enhancer (24, 25) and is essential for efficient virus multiplication (26). However, the role of the 5′ UTR in replication has been unclear, mainly due to the lack of experimental systems to separately evaluate translation of viral RNA and negative- and positive-stand RNA synthesis.To dissect the process that precedes the formation of the tobamovirus RNA replication complex on membranes, we previously developed an in vitro translation-replication system (27). Using an evacuolated tobacco protoplast extract (BYL) from which membranes were removed by centrifugation (membrane-depleted BYL, or mdBYL), we demonstrated that the replication proteins of tomato mosaic virus (ToMV), a close relative of TMV, bind ToMV RNA to form a ribonucleoprotein complex named premembrane-targeting complex (PMTC) in a translation-coupled manner (28). The PMTC is inactive in RNA synthesis but forms an active replication complex capable of synthesizing negative-strand and positive-strand RNA when it is mixed with membranes prepared from BYL. PMTC-like ribonucleoprotein (core-PMTC) is formed when a ToMV derivative that expresses the 126-kDa protein, but not the 183-kDa protein, is translated in mdBYL, which can form a replication complex when the 183-kDa protein and membranes are posttranslationally supplied (28). In the current study, we characterized tobamovirus PMTC and obtained results that provide insight into how the genomic RNA of TMV is selected as a template for replication preferentially in cis as well as how collisions between replication proteins and ribosomes are avoided.     

RNA virus replication depends on enrichment of phosphatidylethanolamine at replication sites in subcellular membranes

Intracellular membranes are critical for replication of positive-strand RNA viruses. To dissect the roles of various lipids, we have developed an artificial phosphatidylethanolamine (PE) vesicle-based Tomato bushy stunt virus (TBSV) replication assay. We demonstrate that the in vitro assembled viral replicase complexes (VRCs) in artificial PE vesicles can support a complete cycle of replication and asymmetrical RNA synthesis, which is a hallmark of (+)-strand RNA viruses. Vesicles containing ∼85% PE and ∼15% additional phospholipids are the most efficient, suggesting that TBSV replicates within membrane microdomains enriched for PE. Accordingly, lipidomics analyses show increased PE levels in yeast surrogate host and plant leaves replicating TBSV. In addition, efficient redistribution of PE leads to enrichment of PE at viral replication sites. Expression of the tombusvirus p33 replication protein in the absence of other viral compounds is sufficient to promote intracellular redistribution of PE. Increased PE level due to deletion of PE methyltransferase in yeast enhances replication of TBSV and other viruses, suggesting that abundant PE in subcellular membranes has a proviral function. In summary, various (+)RNA viruses might subvert PE to build membrane-bound VRCs for robust replication in PE-enriched membrane microdomains.Many steps in the infection cycles of positive-strand RNA viruses, including entry into the cell, replication, virion assembly, and egress, are associated with subcellular membranes (1–4). Therefore, viruses have to interact with different lipids, such as phospholipids and sterols, which affect the biophysical features of membranes, including the fluidity and curvature (5, 6). The subverted cellular membranes could protect the viral RNA from recognition by the host nucleic acid sensors or from destruction by the cellular innate immune system. In addition, membranes facilitate the sequestration of viral and coopted host proteins to increase their local concentrations and promote macromolecular assembly, including formation of the viral replicase complex (VRC) or virion assembly. To optimize viral processes, RNA viruses frequently manipulate lipid composition of various intracellular membranes (6–13). Overall, the interaction between cellular lipids and viral components is emerging as one of the possible targets for antiviral methods against a great number of viruses. Understanding the roles of various lipids in RNA virus infections is important to ultimately control harmful RNA viruses.Among the various lipids, the highly abundant phospholipids are especially targeted by RNA viruses (2). In general, phospholipids likely affect the replication of most RNA viruses, which takes place within membranous structures (1, 3, 4). Accordingly, lipidomics analyses of cells infected with Dengue virus and hepatitis C virus (HCV) (8, 9) revealed enhanced virus-induced lipid biosynthesis, resulting in changes in the global lipid profile of host cells. Also, the less abundant regulatory phosphatidylinositol-4-phosphate (PI4P) was shown to be enriched at sites of enterovirus and HCV replication due to recruitment of cellular lipid kinases (7, 14), suggesting that a microenvironment enriched for PI4P facilitates (+)RNA virus replication. However, our knowledge on the roles of various phospholipids in RNA virus replication is currently incomplete. By using tombusviruses, small model RNA viruses of plants that can replicate in a yeast surrogate host (15), which has the advantage of tolerating large changes in different phospholipid composition, a major role for global phospholipid and sterol biosynthesis, have been revealed (16–18). In this paper, a viral replicase reconstitution assay based on artificial phospholipid vesicles identified the essential role of phosphatidylethanolamine (PE) in replication of Tomato bushy stunt virus (TBSV). It has also been shown that TBSV could recruit and enrich PE to the sites of viral replication in yeast and plant cells. Moreover, genetic changes that either increase or decrease PE levels in yeast greatly stimulated or inhibited TBSV replication, confirming the key role of PE in the formation of TBSV replicase.     

Genome-wide mutagenesis of influenza virus reveals unique plasticity of the hemagglutinin and NS1 proteins

The molecular basis for the diversity across influenza strains is poorly understood. To gain insight into this question, we mutagenized the viral genome and sequenced recoverable viruses. Only two small regions in the genome were enriched for insertions, the hemagglutinin head and the immune-modulatory nonstructural protein 1. These proteins play a major role in host adaptation, and thus need to be able to evolve rapidly. We propose a model in which certain influenza A virus proteins (or protein domains) exist as highly plastic scaffolds, which will readily accept mutations yet retain their functionality. This model implies that the ability to rapidly acquire mutations is an inherent aspect of influenza HA and nonstructural protein 1 proteins; further, this may explain why rapid antigenic drift and a broad host range is observed with influenza A virus and not with some other RNA viruses.Influenza A virus (IAV) is a segmented negative-sense, single-stranded RNA virus (1). Every year in the United States alone, IAV is thought to infect 5–20% of the population, leading to more than 200,000 hospitalizations for IAV-related disease (2). Despite this major burden to human health, and significant research efforts into viral pathogenesis, there are still major aspects of IAV biology that remain poorly understood.Most RNA viruses, including IAV, encode an error-prone RNA-dependent RNA polymerase responsible for replicating the viral genome (1, 3). Despite the fact that all eight influenza viral segments have errors introduced during RNA replication (1), sequence analysis shows that the conservation of segments (and therefore proteins) range from highly variable to highly conserved (4–7). It is currently unclear as to whether this disparity arises purely from a lack of selective pressure (i.e., all proteins encoded by the virus tolerate a range of mutations, but only those that confer an evolutionary advantage become fixed in a population) or if there is an inherent difference in the ability of the viral proteins themselves to tolerate mutations. Currently, there is no direct experimental evidence to distinguish between these two possibilities.One approach to directly provide evidence of the potential mutability of the viral genome is genome-wide insertional mutagenesis. This technique is one in which relatively small insertions are randomly introduced at all (or at least the majority of) possible sites across a genome. By determining the locations at which the viral genome will tolerate insertions, one can evaluate the intrinsic flexibility of the genome. Viruses with small genomes are excellent candidates for this type of analysis. Genome-wide insertional mutagenesis of two positive-stranded RNA viruses has been reported (8, 9); however, no such mutagenesis has been performed for a negative-sense RNA virus.In this report, we performed near-saturating insertional mutagenesis of the IAV genome. Upon mapping tolerated insertion sites, we identified two regions of the viral genome that were highly enriched for mutations. The identified regions were in domains associated with host adaptation and immune response evasion (10, 11): the viral glycoprotein hemagglutinin (12, 13) and nonstructural protein 1 (NS1) (14, 15). These data suggest that although the majority of the viral genome is resistant to major mutation, two viral proteins contain extremely flexible proteins scaffolds. These flexible domains may allow for rapid adaptation to new or changing host environments.     

A common solution to group 2 influenza virus neutralization

The discovery and characterization of broadly neutralizing antibodies (bnAbs) against influenza viruses have raised hopes for the development of monoclonal antibody (mAb)-based immunotherapy and the design of universal influenza vaccines. Only one human bnAb (CR8020) specifically recognizing group 2 influenza A viruses has been previously characterized that binds to a highly conserved epitope at the base of the hemagglutinin (HA) stem and has neutralizing activity against H3, H7, and H10 viruses. Here, we report a second group 2 bnAb, CR8043, which was derived from a different germ-line gene encoding a highly divergent amino acid sequence. CR8043 has in vitro neutralizing activity against H3 and H10 viruses and protects mice against challenge with a lethal dose of H3N2 and H7N7 viruses. The crystal structure and EM reconstructions of the CR8043-H3 HA complex revealed that CR8043 binds to a site similar to the CR8020 epitope but uses an alternative angle of approach and a distinct set of interactions. The identification of another antibody against the group 2 stem epitope suggests that this conserved site of vulnerability has great potential for design of therapeutics and vaccines.Influenza viruses are a significant and persistent threat to human health worldwide. Annual epidemics cause 3–5 million cases of severe illness and up to 0.5 million deaths (1), and periodic influenza pandemics have the potential to kill millions (2). Inhibitors against the viral surface glycoprotein neuraminidase are widely used for the treatment of influenza infections, but their efficacy is being compromised by the emergence of drug-resistant viral strains (3). Vaccination remains the most effective strategy to prevent influenza virus infection. However, protective efficacy is suboptimal in the highest risk groups: infants, the elderly, and the immunocompromised (1). Furthermore, because immunity after vaccination is typically strain-specific and influenza viruses evolve rapidly, vaccines must be updated almost annually. The antigenic composition of the vaccine is based on a prediction of strains likely to circulate in the coming year, therefore, mismatches between vaccine strains and circulating strains occur that can render the vaccine less effective (4). Consequently, there is an urgent need for new prophylactic and therapeutic interventions that provide broad protection against influenza.Immunity against influenza viruses is largely mediated by neutralizing antibodies that target the major surface glycoprotein hemagglutinin (HA) (5, 6). Identification of antigenic sites on HA indicates that influenza antibodies are primarily directed against the immunodominant HA head region (7), which mediates endosomal uptake of the virus into host cells by binding to sialic acid receptors (8). Because of high mutation rates in the HA head region and its tolerance for antigenic changes, antibodies that target the HA head are typically only effective against strains closely related to the strain(s) by which they were elicited, although several receptor binding site-targeting antibodies with greater breadth have been structurally characterized (9–15). In contrast, antibodies that bind to the membrane-proximal HA stem region tend to exhibit much broader neutralizing activity and can target strains within entire subtypes and groups (16–25) as well as across influenza types (24). These stem-directed antibodies inhibit major structural rearrangements in HA that are required for the fusion of viral and host endosomal membranes and thus, prevent the release of viral contents into the cell (8). The stem region is less permissive for mutations than the head and relatively well-conserved across divergent influenza subtypes.Anti-stem antibodies are elicited in some, but not all, individuals during influenza infection or vaccination (20, 26) and thus, hold great promise as potential broad spectrum prophylactic or therapeutic agents and for the development of a universal influenza vaccine (27–29). The majority of the known heterosubtypic stem binding antibodies neutralize influenza A virus subtypes belonging to group 1 (17–20, 23, 25). Furthermore, two antibodies that target a similar epitope in the HA stem, like most heterosubtypic group 1 antibodies, are able to more broadly recognize both group 1 and 2 influenza A viruses (22) or influenza A and B viruses (24). Strikingly, group 2-specific broadly neutralizing Abs (bnAbs) seem to be rare, because only one has been reported to date (21). CR8020 uniquely targets a distinct epitope in the stem in close proximity to the viral membrane at the HA base and binds lower down the stem than any other influenza HA antibody (21).In the discovery process that led to the isolation of bnAb CR8020, we recovered additional group 2-specific bnAbs. Here, we describe one such bnAb, CR8043, which recognizes a similar but nonidentical footprint on the HA as CR8020 and approaches the HA from a different angle. Furthermore, these two bnAbs are derived from different germ-line genes and, consequently, use distinct sets of interactions for HA recognition. Thus, the human immune system is able to recognize this highly conserved epitope in different ways using different germ-line genes. Hence, this valuable information can be used for the design of therapeutics and vaccines targeting this site of vulnerability in group 2 influenza A viruses that include the pandemic H3N2 subtype.     

Relationship of the quaternary structure of human secretory IgA to neutralization of influenza virus

Secretory IgA (S-IgA) antibodies, the major contributors to humoral mucosal immunity to influenza virus infection, are polymeric Igs present in many external secretions. In the present study, the quaternary structures of human S-IgA induced in nasal mucosa after administration of intranasal inactivated influenza vaccines were characterized in relation to neutralization potency against influenza A viruses. Human nasal IgA antibodies have been shown to contain at least five quaternary structures. Direct and real-time visualization of S-IgA using high-speed atomic force microscopy (AFM) demonstrated that trimeric and tetrameric S-IgA had six and eight antigen-binding sites, respectively, and that these structures exhibited large-scale asynchronous conformational changes while capturing influenza HA antigens in solution. Furthermore, trimeric, tetrameric, and larger polymeric structures, which are minor fractions in human nasal IgA, displayed increased neutralizing potency against influenza A viruses compared with dimeric S-IgA, suggesting that the larger polymeric than dimeric forms of S-IgA play some important roles in protection against influenza A virus infection in the human upper respiratory tract.Antibodies in respiratory mucosa are primary mediators of protective immunity against influenza. Notably, preexisting secretory IgA (S-IgA) antibodies can provide immediate immunity by eliminating a pathogen before the virus passes the mucosal barrier (1–3). Parenteral vaccination induces serum IgG but not S-IgA, so vaccine efficacy is limited. In contrast, intranasal administration of an inactivated influenza vaccine elicits both S-IgA and IgG responses, thus improving the protective efficacy of current vaccination procedures (4–8).IgA in human serum exists predominantly in the form of monomers, whereas the majority of IgA in external secretions is present in the form of polymers. These polymeric IgA forms are associated with the extracellular portion of the polymeric Ig receptor, generating a complex (receptor + polymeric IgA) called S-IgA (9). S-IgA primarily corresponds to dimeric IgA, although low levels of some larger polymeric forms, particularly tetramers, are also present (9–15). Polymeric S-IgA has been shown (both in vitro and in experimental animal models) to be more effective than monomeric IgA or IgG for the neutralization of influenza viruses (16–19). However, little is known of the quaternary structures and neutralizing potencies in viral infection of the various forms of polymeric S-IgA in the human nasal mucosa. In this study, the quaternary structures and neutralizing potencies of nasal antibodies against influenza virus were examined using nasal wash samples from healthy adults who had received intranasally administered inactivated influenza vaccines. These nasal wash samples, containing variously sized Igs, were separated by gel filtration chromatography (GFC) and assessed for neutralization activity against influenza virus. The quaternary structures of the nasal IgA induced by intranasally administered inactivated influenza vaccines then were determined using biochemical techniques and high-speed atomic force microscopy (AFM). We found that human nasal IgA comprised at least five quaternary structures, including monomer, dimer, trimer, and tetramer structures, as well as a polymeric form larger than the tetramer structure. Among these forms, the polymeric structure demonstrated higher neutralizing potency against seasonal influenza viruses (H3N2) and highly pathogenic avian influenza virus (H5N1) compared with the dimeric form, suggesting that large polymeric S-IgA antibodies play crucial roles in protective immunity against influenza virus infection of the human upper respiratory tract.     

Pathogenesis and transmission of swine origin A(H3N2)v influenza viruses in ferrets

Recent isolation of a novel swine-origin influenza A H3N2 variant virus [A(H3N2)v] from humans in the United States has raised concern over the pandemic potential of these viruses. Here, we analyzed the virulence, transmissibility, and receptor-binding preference of four A(H3N2)v influenza viruses isolated from humans in 2009, 2010, and 2011. High titers of infectious virus were detected in nasal turbinates and nasal wash samples of A(H3N2)v-inoculated ferrets. All four A(H3N2)v viruses possessed the capacity to spread efficiently between cohoused ferrets, and the 2010 and 2011 A(H3N2)v isolates transmitted efficiently to naïve ferrets by respiratory droplets. A dose-dependent glycan array analysis of A(H3N2)v showed a predominant binding to α2-6–sialylated glycans, similar to human-adapted influenza A viruses. We further tested the viral replication efficiency of A(H3N2)v viruses in a relevant cell line, Calu-3, derived from human bronchial epithelium. The A(H3N2)v viruses replicated in Calu-3 cells to significantly higher titers compared with five common seasonal H3N2 influenza viruses. These findings suggest that A(H3N2)v viruses have the capacity for efficient replication and transmission in mammals and underscore the need for continued public health surveillance.Seasonal epidemics and periodic pandemics are an ever-present international public health burden. During seasonal epidemics, the risk for hospitalization and death are highest among persons at either end of the age spectrum as well as for individuals with underlying medical conditions (1, 2). Although the overall impact of the 2009 influenza pandemic, caused by an H1N1 virus [A(H1N1)pdm09] was more modest than those of prior pandemics, the disproportionate disease burden among children and younger adults distinguished this pandemic from seasonal influenza (3–5). The A(H1N1)pdm09 virus emerged from swine, with a unique constellation of genes from human, avian, and swine influenza viruses not previously observed in nature. Swine represent a unique host because of their ability to be infected by influenza viruses from multiple species and serve as a reservoir for specific subtypes of influenza capable of infecting humans (6–8). Triple-reassortant swine (TRS) H1N1 viruses, which share host gene-lineage origins with A(H1N1)pdm09 viruses, have been responsible for sporadic human cases since 2005 (9, 10). The emergence of the A(H1N1)pdm09 virus, to which the majority of children and younger adults had little preexisting immunity, highlights the public health threat posed by other swine-origin influenza virus subtypes.H3N2 viruses have circulated in humans since their pandemic emergence in 1968 and are generally associated with uncomplicated disease in young healthy adults. However, epidemics caused by H3N2 viruses have been more severe than those caused by seasonal H1N1 or influenza B viruses (11, 12). In 1997–1998, human H3N2 viruses infected swine and spread widely in North American swine (6–8). In particular, TRS H3N2 viruses with a human lineage polymerase subunit polymerase basic 1 (PB1) gene, avian lineage PB2 and polymerase acidic (PA) genes, and swine lineage nucleoprotein (NP), matrix (M), and nonstructural (NS) genes, referred to as the triple-reassortant internal gene (TRIG) constellation, have been isolated widely in pigs throughout the United States (6–8, 13). From the late 1990s to 2009, these novel variants of H3N2 viruses [A(H3N2)v] were limited to transmission among swine, with only occasional detections of transmission to humans (14). However, between September and November 2010, five cases of human infection with the novel swine-origin A(H3N2)v were reported (15). Although all five recovered fully from their illness, two of the five cases were hospitalized. In 2011, 12 additional human cases were documented in the United States, with limited human-to-human transmission in some cases (15–17). The 2011 A(H3N2)v viruses are similar to other A(H3N2)v viruses isolated from previous human infections over the past 2 y but are unique in that the M gene is derived from the A(H1N1)pdm09 virus. Antigenic characterization showed that the A(H3N2)v viruses are distinct from current seasonal H3N2 viruses but exhibit a low degree of serologic cross-reactivity with human H3N2 viruses that circulated in the early 1990s (6, 8, 13), suggesting that children born after this time period may be particularly susceptible to infection.The use of the ferret model has become indispensable for understanding the virulence and transmission of influenza viruses (18–20), partly because ferrets and humans share similar lung physiology as well as because human and avian influenza viruses exhibit similar patterns of binding to sialic acids, the receptor for influenza viruses distributed throughout the respiratory tract in both species (21, 22). In this study, we used glycan microarrays to determine the receptor-binding preference of the A(H3N2)v viruses isolated from humans. The culture model of bronchial epithelial Calu-3 cells was used to assess viral replication, and the ferret model was used to assess pathogenicity and transmissibility. Notably, the 2010 and 2011 swine-origin H3N2 viruses replicated even more efficiently than human seasonal influenza viruses in human airway Calu-3 cells and exhibited efficient respiratory-droplet (RD) transmission in ferrets. These findings suggest that swine-origin H3N2 viruses have the potential to cause additional human disease.     

Potential antigenic explanation for atypical H1N1 infections among middle-aged adults during the 2013–2014 influenza season

Influenza viruses typically cause the most severe disease in children and elderly individuals. However, H1N1 viruses disproportionately affected middle-aged adults during the 2013–2014 influenza season. Although H1N1 viruses recently acquired several mutations in the hemagglutinin (HA) glycoprotein, classic serological tests used by surveillance laboratories indicate that these mutations do not change antigenic properties of the virus. Here, we show that one of these mutations is located in a region of HA targeted by antibodies elicited in many middle-aged adults. We find that over 42% of individuals born between 1965 and 1979 possess antibodies that recognize this region of HA. Our findings offer a possible antigenic explanation of why middle-aged adults were highly susceptible to H1N1 viruses during the 2013–2014 influenza season. Our data further suggest that a drifted H1N1 strain should be included in future influenza vaccines to potentially reduce morbidity and mortality in this age group.Seasonal H1N1 (sH1N1) viruses circulated in the human population for much of the last century and, as of 2009, most humans had been exposed to sH1N1 strains. In 2009, an antigenically distinct H1N1 strain began infecting humans and caused a pandemic (1–3). Elderly individuals were less susceptible to 2009 pandemic H1N1 (pH1N1) viruses because of cross-reactive antibodies (Abs) elicited by infections with older sH1N1 strains (3–7). pH1N1 viruses have continued to circulate on a seasonal basis since 2009. Influenza viruses typically cause a higher disease burden in children and elderly individuals (8) but pH1N1 viruses caused unusually high levels of disease in middle-aged adults during the 2013–2014 influenza season (9–12). For example, a significantly higher proportion of individuals aged 30- to 59-y-old were hospitalized in Mexico with laboratory-confirmed pH1N1 cases in 2013–2014 relative to 2011–2012 (11).Most neutralizing influenza Abs are directed against the hemagglutinin (HA) glycoprotein. International surveillance laboratories rely primarily on ferret anti-influenza sera for detecting HA antigenic changes (13). For these assays, sera are isolated from ferrets recovering from primary influenza infections. Seasonal vaccine strains are typically updated when human influenza viruses acquire HA mutations that prevent the binding of primary ferret anti-influenza sera. Our laboratory and others have demonstrated that sera isolated from ferrets recovering from primary pH1N1 infections are dominated by Abs that recognize an epitope involving residues 156, 157, and 158 of the Sa HA antigenic site (14, 15). The pH1N1 component of the seasonal influenza vaccine has not been updated since 2009 because very few pH1N1 isolates possess mutations in residues 156, 157, and 158. The majority of isolates from the 2013–2014 season have been labeled as antigenically similar to the A/California/07/2009 vaccine strain (9).It is potentially problematic that major antigenic changes of influenza viruses are mainly determined using antisera isolated from ferrets recovering from primary influenza infections. Unlike experimental ferrets, humans are typically reinfected with antigenically distinct influenza strains throughout their life (16). In the 1950s, it was noted that the human immune system preferentially mounts Ab responses that cross-react to previously circulating influenza strains, as opposed to new Ab responses that exclusively target newer viral strains (17). This process, which Thomas Francis Jr. termed “original antigenic sin,” has been experimentally recapitulated in ferrets (14, 18), mice (19–21), and rabbits (22). Our group and others recently demonstrated that the specificity of pH1N1 Ab responses can be shaped by prior sH1N1 exposures (14, 23–26). We found that ferrets sequentially infected with sH1N1 and pH1N1 viruses mount Ab responses dominated against epitopes that are conserved between the viral strains (14). These studies indicate that primary ferret antisera may not be fully representative of human influenza immunity.It has been proposed that increased morbidity and mortality of middle-aged adults during the 2013–2014 influenza season is primarily a result of low vaccination rates within these populations (27). An alternative explanation is that recent pH1N1 strains have acquired a true antigenic mutation that has been mislabeled as “antigenically neutral” by assays that rely on primary ferret antisera. Here we complete a series of experiments to determine if recent pH1N1 strains possess a mutation that prevents binding of Abs in middle-aged humans who have been previously exposed to different H1N1 strains.     

Prevention of influenza by targeting host receptors using engineered proteins

There is a need for new approaches for the control of influenza given the burden caused by annual seasonal outbreaks, the emergence of viruses with pandemic potential, and the development of resistance to current antiviral drugs. We show that multivalent biologics, engineered using carbohydrate-binding modules specific for sialic acid, mask the cell-surface receptor recognized by the influenza virus and protect mice from a lethal challenge with 2009 pandemic H1N1 influenza virus. The most promising biologic protects mice when given as a single 1-μg intranasal dose 7 d in advance of viral challenge. There also is sufficient virus replication to establish an immune response, potentially protecting the animal from future exposure to the virus. Furthermore, the biologics appear to stimulate inflammatory mediators, and this stimulation may contribute to their protective ability. Our results suggest that this host-targeted approach could provide a front-line prophylactic that has the potential to protect against any current and future influenza virus and possibly against other respiratory pathogens that use sialic acid as a receptor.Influenza viruses continue to be a threat to human health and a burden on health services (1). The emergence of highly pathogenic H5N1 viruses and recent introductions of H7N9 viruses from avian sources (2), and their potential to acquire human transmissibility, increase the threat (3–5). Although vaccines remain a cornerstone of prevention, significant time is required to develop an effective vaccine against a new virus strain. Anti-influenza drugs approved by the Food and Drug Administration, such as the viral neuraminidase inhibitors oseltamivir (Tamiflu) and zanamivir (Relenza) and the M2 ion-channel blocker adamantanes (amantadine and rimantadine), are available, but their effectiveness can be compromised by the virus’s ability to mutate and become drug resistant (6, 7).The influenza virus binds to sialic acid receptors present on the respiratory tract epithelium via its surface HA glycoprotein, an event that triggers viral endocytosis (8). Other respiratory pathogens, such as parainfluenza viruses (9), some coronaviruses (10), and Streptococcus pneumoniae (11), also use sialic acid as a receptor. Human influenza viruses such as the 2009 pandemic H1N1 virus recognize α-2,6–linked sialic acid receptors present in the upper respiratory tract, whereas avian influenza viruses such as H5N1 predominantly recognize α-2,3–linked sialic acid receptors, which are present in the human lower respiratory tract as well (12, 13). The recently emerged human H7N9 influenza virus is unusual in recognizing both types of receptors and therefore has the possibility of sustained human-to-human transmission and pandemic potential (14, 15).We hypothesized that masking such receptors in the respiratory tract with proteins specific for sialic acid could provide a novel host-targeted therapeutic route to prevent infection. Numerous sialic acid-binding proteins are known, but most have low affinity for sialic acid (e.g., the HA monomer that has ∼2.5 mM affinity for its receptor but gains affinity by being present in high copy number on the virus surface) (16). We have shown previously that engineered multivalent polypeptides containing up to four tandemly linked copies of the sialic acid-recognizing carbohydrate-binding module (CBM) from Vibrio cholerae nanH sialidase display low (nanomolar) binding affinity compared with the 30-μM affinity of the single domain (17).Here we report the engineering and characterization of further sialic acid-recognizing multivalent CBMs (mCBMs) together with in vitro and in vivo evidence of their potential in preventing influenza infection. Significantly, our lead mCBM demonstrates protective in vivo efficacy when given to mice as a single 1-μg dose 7 d in advance of a lethal virus challenge with pandemic 2009 H1N1 influenza virus, indicating that these mCBMs show great promise as biologics for the prophylaxis of influenza and potentially other respiratory pathogens that recognize sialic acid receptors.     

Evolution of the H9N2 influenza genotype that facilitated the genesis of the novel H7N9 virus

The emergence of human infection with a novel H7N9 influenza virus in China raises a pandemic concern. Chicken H9N2 viruses provided all six of the novel reassortant’s internal genes. However, it is not fully understood how the prevalence and evolution of these H9N2 chicken viruses facilitated the genesis of the novel H7N9 viruses. Here we show that over more than 10 y of cocirculation of multiple H9N2 genotypes, a genotype (G57) emerged that had changed antigenicity and improved adaptability in chickens. It became predominant in vaccinated farm chickens in China, caused widespread outbreaks in 2010–2013 before the H7N9 viruses emerged in humans, and finally provided all of their internal genes to the novel H7N9 viruses. The prevalence and variation of H9N2 influenza virus in farmed poultry could provide an important early warning of the emergence of novel reassortants with pandemic potential.Human infection with a novel avian-origin H7N9 influenza A virus causing severe respiratory symptoms and mortality was first reported in eastern China in March 2013 (1). To date, the novel virus has caused two outbreaks of human infection, including 375 known cases and 115 deaths as of 11 March 2014 (2). Phylogenetic analysis suggests that the virus is a triple reassortant of H7, N9, and H9N2 avian influenza viruses (3, 4). The H7 and N9 genes may have been transferred from migratory birds to domestic ducks and then to chickens in the live poultry markets (3–5), after which reassortment with enzootic H9N2 viruses formed the H7N9 viruses identified in humans (3–5).H9N2 influenza virus has low pathogenicity for avians, replicating mainly in the upper respiratory tract and causing mild or no overt signs of illness in specific pathogen-free (SPF) chickens (6). In 1994, the H9N2 subtype was first identified in chicken farms in the Guangdong province of south China (7); it has since become widespread in chickens and has caused great economic loss from reduced egg production and highly lethal coinfections (8–11). To reduce the impact of H9N2 infection in chickens, the flocks have been vaccinated since 1998 with commercial inactivated vaccines, such as A/chicken/Guangdong/SS/1994 (Ck/GD/SS/94), A/chicken/Shandong/6/1996 (Ck/SD/6/96), and A/chicken/Shanghai/F/1998 (Ck/SH/F/98) (8, 12, 13). These H9N2 vaccines initially limited the outbreaks and virus spread. However, despite multiple doses, the H9N2 vaccines became less effective, especially after 2007, and H9N2 influenza virus continues to circulate in vaccinated chicken flocks and has caused sporadic disease outbreaks (8, 10, 12–20). However, the recent prevalence and molecular evolution of the H9N2 viruses in chickens especially in the flocks receiving large-scale vaccination, and their role in the emergence of human H7N9 virus, are not fully understood. In this study, we systematically investigated the prevalence and evolution of H9N2 viruses mainly focusing on farm chickens and their role in the genesis of the novel H7N9 viruses.