PB1-F2 Protein Research Articles

HOST-RESTRICTED RANGE OF H5N1 AVIAN INFLUENZA VIRUSES ASSOCIATED WITH CHARACTERS OF POLYMERASE COMPLEX OF PB2 AND PB1-F2 PROTEINS

Epidemiological studies on H5N1 avian influenza viruses indi-cated that the viruses do not transmit efficiently from human to human. Transmissibility of viruses among human population is very complex and polygenic. Studies on molecular determinants facilitating interspecies transmission of the viruses suggested that two polymerase complex proteins such as PB2 and PB1-F2 are important. PB2 is critical in determining the host specificity, whereas mutations in PB1-F2 increase the viral virulence. The study aimed to characterize the polymerase complex of PB2 and PB1-F2 proteins of H5N1 avian influenza viruses isolated from Indonesia. The DNA samples encoding the PB2 and PB1-F2 complex proteins of several H5N1 isolates were sequenced and analyzed. Pathogenicity of the viruses was studied in both avian and mammal models. The sequencing results showed that there was no mutation in both proteins of PB2 and PB1-F2 of the avian influenza virus isolates. The molecular character for host specificity was consistent with the animal experiment results. The H5N1 virus isolates were only infectious and pathogenic in chickens, but not in BALB/C mice as the mammal model. The study suggests that host range of H5N1 virus isolates of Indonesia is restricted to poultry and not transmisible to mammal model used in this study.

HOST-RESTRICTED RANGE OF H5N1 AVIAN INFLUENZA VIRUSES ASSOCIATED WITH CHARACTERS OF POLYMERASE COMPLEX OF PB2 AND PB1-F2 PROTEINS

Epidemiological studies on H5N1 avian influenza viruses indi-cated that the viruses do not transmit efficiently from human to human. Transmissibility of viruses among human population is very complex and polygenic. Studies on molecular determinants facilitating interspecies transmission of the viruses suggested that two polymerase complex proteins such as PB2 and PB1-F2 are important. PB2 is critical in determining the host specificity, whereas mutations in PB1-F2 increase the viral virulence. The study aimed to characterize the polymerase complex of PB2 and PB1-F2 proteins of H5N1 avian influenza viruses isolated from Indonesia. The DNA samples encoding the PB2 and PB1-F2 complex proteins of several H5N1 isolates were sequenced and analyzed. Pathogenicity of the viruses was studied in both avian and mammal models. The sequencing results showed that there was no mutation in both proteins of PB2 and PB1-F2 of the avian influenza virus isolates. The molecular character for host specificity was consistent with the animal experiment results. The H5N1 virus isolates were only infectious and pathogenic in chickens, but not in BALB/C mice as the mammal model. The study suggests that host range of H5N1 virus isolates of Indonesia is restricted to poultry and not transmisible to mammal model used in this study.

Induction of memory cytotoxic T cells to influenza A virus and subsequent viral clearance is not modulated by PB1‐F2‐dependent inflammasome activation

Expression of the viral virulence protein PB1-F2 during infection has been linked to NLRP3 inflammasome complex activation in macrophages and induction of early inflammatory events enhancing immunopathology during influenza disease. We sought to determine whether PB1-F2-specific NLRP3 inflammasome activation influenced the magnitude and/or robustness of the CD8+ T-cell responses specific for conserved viral antigens and subsequent virus elimination. Using murine heterosubtypic viral infection models, we showed that mice infected with virus unable to produce PB1-F2 protein showed no deficit in the overall magnitude and functional memory responses of CD8+ T cells established during the effector phase compared with those infected with wild-type PB1-F2-expressing virus and were equally capable of mounting robust recall responses. These data indicate that while expression of PB1-F2 protein can induce inflammatory events, the capacity to generate memory CD8+ T cells specific for immunodominant viral epitopes remains uncompromised.

PB1-F2 Protein Does Not Impact the Virulence of Triple-Reassortant H3N2 Swine Influenza Virus in Pigs but Alters Pathogenicity and Transmission in Turkeys

PB1-F2 protein, the 11th influenza A virus (IAV) protein, is considered to play an important role in primary influenza virus infection and postinfluenza secondary bacterial pneumonia in mice. The functional role of PB1-F2 has been reported to be a strain-specific and host-specific phenomenon. Its precise contribution to the pathogenicity and transmission of influenza virus in mammalian host, such as swine, and avian hosts, such as turkeys, remain largely unknown. In this study, we explored the role of PB1-F2 protein of triple-reassortant (TR) H3N2 swine influenza virus (SIV) in pigs and turkeys. Using the eight-plasmid reverse genetics system, we rescued wild-type SIV A/swine/Minnesota/1145/2007 (H3N2) (SIV 1145-WT), a PB1-F2 knockout mutant (SIV 1145-KO), and its N66S variant (SIV 1145-N66S). The ablation of PB1-F2 in SIV 1145 modulated early-stage apoptosis but did not affect the viral replication in swine alveolar macrophage cells. In pigs, PB1-F2 expression did not affect nasal shedding, lung viral load, immunophenotypes, and lung pathology. On the other hand, in turkeys, SIV 1145-KO infected poults, and its in-contacts developed clinical signs earlier than SIV 1145-WT groups and also displayed more extensive histopathological changes in intestine. Further, turkeys infected with SIV 1145-N66S displayed poor infectivity and transmissibility. The more extensive histopathologic changes in intestine and relative transmission advantage observed in turkeys infected with SIV 1145-KO need to be further explored. Taken together, these results emphasize the host-specific roles of PB1-F2 in the pathogenicity and transmission of IAV. Novel triple-reassortant H3N2 swine influenza virus emerged in 1998 and spread rapidly among the North American swine population. Subsequently, it showed an increased propensity to reassort, generating a range of reassortants. Unlike classical swine influenza virus, TR SIV produces a full-length PB1-F2 protein, which is considered an important virulence marker of IAV pathogenicity. Our study demonstrated that the expression of PB1-F2 does not impact the pathogenicity of TR H3N2 SIV in pigs. On the other hand, deletion of PB1-F2 caused TR H3N2 SIV to induce clinical disease early and resulted in effective transmission among the turkey poults. Our study emphasizes the continuing need to better understand the virulence determinants for IAV in intermediate hosts, such as swine and turkeys, and highlights the host-specific role of PB1-F2 protein.

The Ubiquitination of the Influenza A Virus PB1-F2 Protein Is Crucial for Its Biological Function

The aim of the present study was to identify what influences the short half-life of the influenza A virus PB1-F2 protein and whether a prolonged half-life affects the properties of this molecule. We hypothesized that the short half-life of PB1-F2 could conceal the phenotype of the protein. Because proteasome degradation might be involved in PB1-F2 degradation, we focused on ubiquitination, a common label for proteasome targeting. A cluster of lysine residues was demonstrated as an ubiquitination acceptor site in evolutionary and functionally distinct proteins. The PB1-F2 sequence alignment revealed a cluster of lysines on the carboxy terminal end of PB1-F2 in almost all of the GenBank sequences available to date. Using a proximity ligation assay, we identified ubiquitination as a novel posttranslational modification of PB1-F2. Changing the lysines at positions 73, 78, and 85 to arginines suppressed the ubiquitination of A/Puerto Rico/8/1934 (H1N1)-derived PB1-F2. The mutation of the C-terminal lysine residue cluster positively affected the overall expression levels of avian A/Honk Kong/156/1997 (H5N1)- and mammalian A/Puerto Rico/8/1934 (H1N1)-derived PB1-F2. Moreover, increased PB1-F2 copy numbers strengthened the functions of this virus in the infected cells. The results of a minigenome luciferase reporter assay revealed an enhancement of viral RNA-dependent RNA polymerase activity in the presence of stabilized PB1-F2, regardless of viral origin. IFNβ antagonism was enhanced in 293T cells transfected with a plasmid expressing stabilized K→R mutant variants of PB1-F2. Compared with PB1-F2 wt, the loss of ubiquitination enhanced the antibody response after DNA vaccination. In summary, we revealed that PB1-F2 is an ubiquitinated IAV protein, and this posttranslational modification plays a central role in the regulation of the biological functions of this protein.

Emergence of Highly Pathogenic Avian Influenza A(H5N1) Virus PB1-F2 Variants and Their Virulence in BALB/c Mice.

Influenza A viruses (IAVs) express the PB1-F2 protein from an alternate reading frame within the PB1 gene segment. The roles of PB1-F2 are not well understood but appear to involve modulation of host cell responses. As shown in previous studies, we find that PB1-F2 proteins of mammalian IAVs frequently have premature stop codons that are expected to cause truncations of the protein, whereas avian IAVs usually express a full-length 90-amino-acid PB1-F2. However, in contrast to other avian IAVs, recent isolates of highly pathogenic H5N1 influenza viruses had a high proportion of PB1-F2 truncations (15% since 2010; 61% of isolates in 2013) due to several independent mutations that have persisted and expanded in circulating viruses. One natural H5N1 IAV containing a mutated PB1-F2 start codon (i.e., lacking ATG) was 1,000-fold more virulent for BALB/c mice than a closely related H5N1 containing intact PB1-F2. In vitro, we detected expression of an in-frame protein (C-terminal PB1-F2) from downstream ATGs in PB1-F2 plasmids lacking the well-conserved ATG start codon. Transient expression of full-length PB1-F2, truncated (24-amino-acid) PB1-F2, and PB1-F2 lacking the initiating ATG in mammalian and avian cells had no effect on cell apoptosis or interferon expression in human lung epithelial cells. Full-length and C-terminal PB1-F2 mutants colocalized with mitochondria in A549 cells. Close monitoring of alterations of PB1-F2 and their frequency in contemporary avian H5N1 viruses should continue, as such changes may be markers for mammalian virulence. Although most avian influenza viruses are harmless for humans, some (such as highly pathogenic H5N1 avian influenza viruses) are capable of infecting humans and causing severe disease with a high mortality rate. A number of risk factors potentially associated with adaptation to mammalian infection have been noted. Here we demonstrate that the protein PB1-F2 is frequently truncated in recent isolates of highly pathogenic H5N1 viruses. Truncation of PB1-F2 has been proposed to act as an adaptation to mammalian infection. We show that some forms of truncation of PB1-F2 may be associated with increased virulence in mammals. Our data support the assessment of PB1-F2 truncations for genomic surveillance of influenza viruses.

Epidemiological and molecular characteristics of the PB1-F2 proteins in H7N9 influenza viruses, Jiangsu.

The recent sporadic infections of humans in China with previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern. The aim is to find out the epidemiological and molecular analysis of the PB1-F2 proteins in H7N9 influenza viruses, in Jiangsu province. Sequences were obtained from GISAID database. Data were analyzed by using Molecular Evolutionary Genetics Analysis software and Bayesian Markov chain Monte Carlo method. From March 1, 2013, to May 31, 2014, 53 patients were confirmed to be infected with the H7N9 virus; one was a retrospective case in Jiangsu province. 38 sequences of PB1 in H7N9 of Jiangsu were obtained from the GISAID online and were then divided into three lineages. Of these sequences, 4 sequences and 3 sequences encode an N-terminally truncated PB1-F2 (52aa)polypeptide and C-terminally truncated PB1-F2 (76aa) polypeptide, respectively. The remaining sequences encode a full-length PB1-F2 (90aa). We estimated a mean evolutionary rate of 3.053 × 10−3 subs/site/year (95% HPD: 2.021 × 10−3–4.051 × 10−3). The site-by-site analysis of selection pressure analysis revealed positively and negatively (12, 3), respectively, selected sites. Influenza A (H7N9) virus adapting into new host, PB1-F2 of H7N9, might be faced with higher selection pressures.

Computational analysis and mapping of novel open reading frames in influenza A viruses.

The influenza A virus contains 8 segmented genomic RNAs and was considered to encode 10 viral proteins until investigators identified the 11th viral protein, PB1-F2, which uses an alternative reading frame of the PB1 gene. The recently identified PB1-N40, PA-N155 and PA-N182 influenza A proteins have shown the potential for using a leaking ribosomal scanning mechanism to generate novel open reading frames (ORFs). These novel ORFs provide examples of the manner in which the influenza A virus expands its coding capacity by using overlapping reading frames. In this study, we performed a computational search, based on a ribosome scanning mechanism, on all influenza A coding sequences to identify possible forward-reading ORFs that could be translated into novel viral proteins. We specified that the translated products had a prevalence ≥5% to eliminate sporadic ORFs. A total of 1,982 ORFs were thus identified and presented in terms of their locations, lengths and Kozak sequence strengths. We further provided an abridged list of ORFs by requiring every candidate an upstream start codon (within the upstream third of the primary transcript), a strong Kozak consensus sequence and high prevalence (≥95% and ≥50% for in-frame and alternative-frame ORFs, respectively). The PB1-F2, PB1-N40, PA-N155 and PA-N182 proteins all fulfilled our filtering criteria. Subject to these three stringent settings, we additionally named 16 novel ORFs for all influenza A genomes except for HA and NA, for which 43 HA and 11 NA ORFs from their respective subtypes were also recognized.

Non-avian animal reservoirs present a source of influenza A PB1-F2 proteins with novel virulence-enhancing markers.

PB1-F2 protein, expressed from an alternative reading frame of most influenza A virus (IAV) PB1 segments, may possess specific residues associated with enhanced inflammation (L62, R75, R79, and L82) and cytotoxicity (I68, L69, and V70). These residues were shown to increase the pathogenicity of primary viral and secondary bacterial infections in a mouse model. In contrast to human seasonal influenza strains, virulence-associated residues are present in PB1-F2 proteins from pandemic H1N1 1918, H2N2 1957, and H3N2 1968, and highly pathogenic H5N1 strains, suggesting their contribution to viruses' pathogenic phenotypes. Non-human influenza strains may act as donors of virulent PB1-F2 proteins. Previously, avian influenza strains were identified as a potential source of inflammatory, but not cytotoxic, PB1-F2 residues. Here, we analyze the frequency of virulence-associated residues in PB1-F2 sequences from IAVs circulating in mammalian species in close contact with humans: pigs, horses, and dogs. All four inflammatory residues were found in PB1-F2 proteins from these viruses. Among cytotoxic residues, I68 was the most common and was especially prevalent in equine and canine IAVs. Historically, PB1-F2 from equine (about 75%) and canine (about 20%) IAVs were most likely to have combinations of the highest numbers of residues associated with inflammation and cytotoxicity, compared to about 7% of swine IAVs. Our analyses show that, in addition to birds, pigs, horses, and dogs are potentially important sources of pathogenic PB1-F2 variants. There is a need for surveillance of IAVs with genetic markers of virulence that may be emerging from these reservoirs in order to improve pandemic preparedness and response.

Influenza A virus protein PB1-F2 translocates into mitochondria via Tom40 channels and impairs innate immunity.

Mitochondria contribute to cellular innate immunity against RNA viruses. Mitochondrial-mediated innate immunity is regulated by signalling molecules that are recruited to the mitochondrial membrane, and depends on the mitochondrial inner membrane potential (Δψm). Here we examine the physiological relevance of Δψm and the mitochondrial-associating influenza A viral protein PB1-F2 in innate immunity. When expressed in host cells, PB1-F2 completely translocates into the mitochondrial inner membrane space via Tom40 channels, and its accumulation accelerates mitochondrial fragmentation due to reduced Δψm. By contrast, PB1-F2 variants lacking a C-terminal polypeptide, which is frequently found in low pathogenic subtypes, do not affect mitochondrial function. PB1-F2-mediated attenuation of Δψm suppresses the RIG-I signalling pathway and activation of NLRP3 inflammasomes. PB1-F2 translocation into mitochondria strongly correlates with impaired cellular innate immunity, making this translocation event a potential therapeutic target.

Identification of virulence determinants in influenza viruses.

To date there is no rapid method to screen for highly pathogenic avian influenza strains that may be indicators of future pandemics. We report here the first development of an oligonucleotide-based spectroscopic assay to rapidly and sensitively detect a N66S mutation in the gene coding for the PB1-F2 protein associated with increased virulence in highly pathogenic pandemic influenza viruses. 5′-Thiolated ssDNA oligonucleotides were employed as probes to capture RNA isolated from six influenza viruses, three having N66S mutations, two without the N66S mutation, and one deletion mutant not encoding the PB1-F2 protein. Hybridization was detected without amplification or labeling using the intrinsic surfaced-enhanced Raman spectrum of the DNA-RNA complex. Multivariate analysis identified target RNA binding from noncomplementary sequences with 100% sensitivity, 100% selectivity, and 100% correct classification in the test data set. These results establish that optical-based diagnostic methods are able to directly identify diagnostic indicators of virulence linked to highly pathogenic pandemic influenza viruses without amplification or labeling.

SERS molecular sentinel for the RNA genetic marker of PB1-F2 protein in highly pathogenic avian influenza (HPAI) virus

We have developed a simple and sensitive assay for the detection of the RNA genetic marker associated with high pathogenicity influenza (HPAI) virus. The assay constituted of an array of Raman label tagged hairpin-DNA immobilized on a surface-enhanced Raman scattering (SERS) active substrate as the molecular sentinel (MS) reporter. Upon incubation of the assay with the target RNA, the structure of the hairpin-DNA probe changed from stem-loop configuration (closed state) to DNA/RNA hybridization configuration (open state) so that the Raman label tag will be physically separated from the SERS substrate and induce a decrease of Raman scattering intensity. A metal film over nanosphere (MFON) substrate was developed with a SERS enhancement of about 1.7×105. Based on this MS-modified substrate, the SERS signal showed a linear relationship to the target RNA in the range of 0–60 attomoles and the limit of detect is 2.67 attomoles. The non-complementary RNA sequences control was also detected and no spectral response was observed. The sensing process only required a single hybridization step and post-hybridization washing could also be omitted. Given that this ultrasensitive biosensor assay is free of polymerase chain reaction (PCR) amplification, it would be a potential diagnostic tool for point-of-care HPAI virus detection.

Emergence of truncated PB1-F2 protein of H3N2 influenza virus during its epidemic period in Jiangsu Province, China

Background PB1-F2 protein has been proven to increase the pathogenicity of influenza A virus (IAV) strains in primary infection and in secondary bacterial infection. It can also regulate the activity of viral polymerase. However, it was shown in another retrospective study that a portion of IAVs do not express full-length PB1-F2 protein during virus development; different kinds of stop codons cause exits in the open reading frames and form PB1-F2 gene products with the corresponding genotypes. Truncated PB1-F2 in human H3N2 IAVs has long been detected in North America but its evolution in China is still unclear. Methods Influenza-like illnesses (ILIs) from the whole of Jiangsu Province were collected and inspected to determine the type and subtype of the viruses. A portion of isolates collected in the epidemic period were selected as samples for later whole-genome sequencing, and the exact sequences were determined and analyzed. Results H3N2 influenza virus was one of the epidemical strains which had been prevalent during 2009-2010, in Jiangsu. Five H3N2 isolates with truncated PB1-F2 protein (25aa) were detected in influenza samples from Nanjing and Xuzhou, while seven similar H3N2 isolates were also reported in Niigata, Japan. Conclusion This emergence indicates the possibility that there has been transmission of the H3N2 virus between the two countries.

Direct electrochemical detection of PB1-F2 protein of influenza A virus in infected cells

Influenza virus represents a major concern of human health and animal production. PB1-F2 is a small proapoptotic protein supposed to contribute to the virulence of influenza A virus (IAV). However, the molecular mechanism of action of PB1-F2 is still unclear.PB1-F2 expression and behavior during the viral cycle is difficult to follow with classical biochemical methods. In this work we have developed an electrochemical biosensor based on immuno-detection system for quantification of PB1-F2 protein in infected cell. The electrochemical immunosensor was based on conducting copolypyrrole integrating ferrocenyl group as redox marker for enhancing signal detection. A specific anti-PB1-F2 monoclonal antibody was immobilized on the copolypyrrole layer via biotin–streptavidin system. We demonstrate that this electrochemical system sensitively detect purified recombinant PB1-F2 over a wide range of concentrations from 5 nM to 1.5µM. The high sensor sensitivity allowed the detection of PB1-F2 in lysates of infected cells confirming that PB1-F2 is expressed in early stages of viral cycle. The immunosensor developed shows enhanced performances for the evaluation of PB1-F2 protein concentration in biological samples and could be applied for studying of PB1-F2 during influenza virus infection.

PB1-F2 Amino Acids Regulate Influenza A Viral Polymerase Activity

In 2001, the 11th influenza A viral protein PB1-F2 was detected and found to be encoded by an alternative open reading frame in the PB1 polymerase gene. PB1-F2 has several unique functions, including roles in promoting apoptosis, increasing inflammation, and regulating viral polymerase activity. This study focused on a single PB1-F2 function: regulation of polymerase activity. We constructed a minigenome system to determine the influence of PB1-F2 amino acid (aa) mutations on polymerase activity. We examined four types of aa mutations: three species-specific aa mutations and one mutation that alters pathogenicity in mice. We discovered that an arginine (R) residue at aa position 29 is highly conserved in avian-derived virus strains. Introducing this mutation into mammalian strain A/WSN/33 (H1N1) led to a marked increase in polymerase activity in mammalian cells.These findings suggest that as PB1-F2 in H5N1 viruses regulates viral polymerase activity, it could be targeted for control of avian influenza infection and drug discovery.

Transient expression of the influenza A virus PB1-F2 protein using a plum pox virus-based vector in Nicotiana benthamiana.

PB1-F2 protein of influenza A virus (IAV) was cloned in a plum pox virus (PPV) genome-based vector and attempts to express it in biolistically transfected Nicotiana benthamiana plants were performed. The vector-insert construct replicated in infected plants properly and was stable during repeated passage by mechanical inoculation, as demonstrated by disease symptoms and immunoblot detection of PPV capsid protein, while PB1-F2-specific band was more faint. We showed that it was due its low solubility. Modification of sample preparation (denaturation/solubilization preceding the centrifugation of cell debris) led to substantial signal enhancement. Maximal level of PB1-F2 expression in plants was observed 12 days post inoculation (dpi). Only 1% SDS properly solubilized the protein, other detergents were much less efficient. Solubilization with 8M urea released approximately 50% of PB1-F2 from the plant tissues, thus the treatment with this removable chaotropic agent may be a good starting point for the purification of the protein for eventual functional studies in the future.

Identification of Chicken Pulmonary miRNAs Targeting PB1, PB1-F2, and N40 Genes of Highly Pathogenic Avian Influenza Virus H5N1 In Silico.

Highly pathogenic Avian influenza (HPAI) is a notifiable viral disease caused by avian influenza type A viruses of the Orthomyxoviridae family. Type A influenza genome consists of eight segments of negative-sense RNA. RNA segment 2 encodes three proteins, PB1, PB1-F2, and N40, which are translated from the same mRNA by ribosomal leaky scanning and reinitiation. Since these proteins are critical for viral replication and pathogenesis, targeting their expression can be one of the approaches to control and resist HPAI. MicroRNAs are short noncoding RNAs that regulate a variety of biological processes such as cell growth, tissue differentiation, apoptosis, and viral infection. In this study, a set of 300 miRNAs expressed in chicken lungs were screened against the HPAI virus (H5N1) segment 2 with different screening parameter like thermodynamic stability of heteroduplex, seed sequence complementarity, conserved target sequence, and target-site accessibility for identifying miRNAs that can potentially target the transcript of segment 2 of H5N1. Chicken miRNAs gga-mir-133c, gga-mir-1710, and gga-mir-146c* are predicted to target the expression of PB1, PB1-F2, and N40 proteins. This indicates that chicken has genetic potential to resist/tolerate H5N1 infection and these can be suitably exploited in designing strategies for control of avian influenza in chicken.

Genomewide Analysis of Reassortment and Evolution of Human Influenza A(H3N2) Viruses Circulating between 1968 and 2011

Influenza A(H3N2) viruses became widespread in humans during the 1968 H3N2 virus pandemic and have been a major cause of influenza epidemics ever since. These viruses evolve continuously by reassortment and genomic evolution. Antigenic drift is the cause for the need to update influenza vaccines frequently. Using two data sets that span the entire period of circulation of human influenza A(H3N2) viruses, it was shown that influenza A(H3N2) virus evolution can be mapped to 13 antigenic clusters. Here we analyzed the full genomes of 286 influenza A(H3N2) viruses from these two data sets to investigate the genomic evolution and reassortment patterns. Numerous reassortment events were found, scattered over the entire period of virus circulation, but most prominently in viruses circulating between 1991 and 1998. Some of these reassortment events persisted over time, and one of these coincided with an antigenic cluster transition. Furthermore, selection pressures and nucleotide and amino acid substitution rates of all proteins were studied, including those of the recently discovered PB1-N40, PA-X, PA-N155, and PA-N182 proteins. Rates of nucleotide and amino acid substitutions were most pronounced for the hemagglutinin, neuraminidase, and PB1-F2 proteins. Selection pressures were highest in hemagglutinin, neuraminidase, matrix 1, and nonstructural protein 1. This study of genotype in relation to antigenic phenotype throughout the period of circulation of human influenza A(H3N2) viruses leads to a better understanding of the evolution of these viruses. Each winter, influenza virus infects approximately 5 to 15% of the world's population, resulting in significant morbidity and mortality. Influenza A(H3N2) viruses evolve continuously by reassortment and genomic evolution. This leads to changes in antigenic recognition (antigenic drift) which make it necessary to update vaccines against influenza A(H3N2) viruses frequently. In this study, the relationship of genetic evolution to antigenic change spanning the entire period of A(H3N2) virus circulation was studied for the first time. The results presented in this study contribute to a better understanding of genetic evolution in correlation with antigenic evolution of influenza A(H3N2) viruses.

A Novel Cytotoxic Sequence Contributes to Influenza A Viral Protein PB1-F2 Pathogenicity and Predisposition to Secondary Bacterial Infection

Enhancement of cell death is a distinguishing feature of H1N1 influenza virus A/Puerto Rico/8/34 protein PB1-F2. Comparing the sequences (amino acids [aa] 61 to 87 using PB1-F2 amino acid numbering) of the PB1-F2-derived C-terminal peptides from influenza A viruses inducing high or low levels of cell death, we identified a unique I68, L69, and V70 motif in A/Puerto Rico/8/34 PB1-F2 responsible for promotion of the peptide's cytotoxicity and permeabilization of the mitochondrial membrane. When administered to mice, a 27-mer PB1-F2-derived C-terminal peptide with this amino acid motif caused significantly greater weight loss and pulmonary inflammation than the peptide without it (due to I68T, L69Q, and V70G mutations). Similar to the wild-type peptide, A/Puerto Rico/8/34 elicited significantly higher levels of macrophages, neutrophils, and cytokines in the bronchoalveolar lavage fluid of mice than its mutant counterpart 7 days after infection. Additionally, infection of mice with A/Puerto Rico/8/34 significantly enhanced the levels of morphologically transformed epithelial and immune mononuclear cells recruited in the airways compared with the mutant virus. In the mouse bacterial superinfection model, both peptide and virus with the I68, L69, and V70 sequence accelerated development of pneumococcal pneumonia, as reflected by increased levels of viral and bacterial lung titers and by greater mortality. Here we provide evidence suggesting that the newly identified cytotoxic sequence I68, L69, and V70 of A/Puerto Rico/8/34 PB1-F2 contributes to the pathogenesis of both primary viral and secondary bacterial infections.

The multifaceted effect of PB1-F2 specific antibodies on influenza A virus infection

PB1-F2 is a small influenza A virus (IAV) protein encoded by an alternative reading frame of the PB1 gene. During IAV infection, antibodies to PB1-F2 proteins are induced. To determine their function and contribution to virus infection, three distinct approaches were employed: passive transfer of anti-PB1-F2 MAbs and polyclonal antibodies, active immunization with PB1-F2 peptides and DNA vaccination with plasmids expressing various parts of PB1-F2. Mostly N-terminal specific antibodies were detected in polyclonal sera raised to complete PB1-F2. Passive and active immunization revealed that antibodies recognizing the N-terminal part of the PB1-F2 molecule have no remarkable effect on the course of IAV infection. Interestingly antibodies against the C-terminal region of PB1-F2, obtained by immunization with KLH-PB1-F2 C-terminal peptide or DNA immunization with pC-ter.PB1-F2 plasmid, partially protected mice against virus infection. To our knowledge, this is the first report demonstrating the biological relevance of humoral immunity against PB1-F2 protein in vivo.