Naturally occurring mutations in PB1 affect influenza A virus replication fidelity, virulence, and adaptability.
ABSTRACT: BACKGROUND:Mutations in the PB1 subunit of RNA-dependent RNA polymerase (RdRp) of influenza A virus can affect replication fidelity. Before the influenza A/H1N1 pandemic in 2009, most human influenza A/H1N1 viruses contained the avian-associated residue, serine, at position 216 in PB1. However, near the onset of the 2009 pandemic, human viruses began to acquire the mammalian-associated residue, glycine, at PB1-216, and PB1-216G became predominant in human viruses thereafter. METHODS:Using entropy-based analysis algorithm, we have previously identified several host-specific amino-acid signatures that separated avian and swine viruses from human influenza viruses. The presence of these host-specific signatures in human influenza A/H1N1 viruses suggested that these mutations were the result of adaptive genetic evolution that enabled these influenza viruses to circumvent host barriers, which resulted in cross-species transmission. We investigated the biological impact of this natural avian-to-mammalian signature substitution at PB1-216 in human influenza A/H1N1 viruses. RESULTS:We found that PB1-216G viruses had greater mutation potential, and were more sensitive to ribavirin than PB1-216S viruses. In oseltamivir-treated HEK293 cells, PB1-216G viruses generated mutations in viral neuraminidase at a higher rate than PB1-216S viruses. By contrast, PB1-216S viruses were more virulent in mice than PB1-216G viruses. These results suggest that the PB1-S216G substitution enhances viral epidemiological fitness by increasing the frequency of adaptive mutations in human influenza A/H1N1 viruses. CONCLUSIONS:Our results thus suggest that the increased adaptability and epidemiological fitness of naturally arising human PB1-216G viruses, which have a canonical low-fidelity replicase, were the biological mechanisms underlying the replacement of PB1-216S viruses with a high-fidelity replicase following the emergence of pdmH1N1. We think that continued surveillance of such naturally occurring PB1-216 variants among others is warranted to assess the potential impact of changes in RdRp fidelity on the adaptability and epidemiological fitness of human A/H1N1 influenza viruses.
Project description:Pigs are evidently more resistant to avian than swine influenza A viruses, mediated in part through frontline epithelial cells and alveolar macrophages (AM). Although porcine AM (PAM) are crucial in influenza virus control, their mode of control is unclear. To gain insight into the possible role of PAM in the mediation of avian influenza virus resistance, we compared the host effects and replication of two avian (H2N3 and H6N1) and three mammalian (swine H1N1, human H1N1 and pandemic H1N1) influenza viruses in PAM. We found that PAM were readily susceptible to initial infection with all five avian and mammalian influenza viruses but only avian viruses caused early and extensive apoptosis (by 6 h of infection) resulting in reduced virus progeny and moderated pro-inflammation. Full length viral PB1-F2 present only in avian influenza viruses is a virulence factor that targets AM for mitochondrial-associated apoptotic cell death. With the use of reverse genetics on an avian H5N1 virus, we found that full length PB1-F2 contributed to increased apoptosis and pro-inflammation but not to reduced virus replication. Taken together, we propose that early apoptosis of PAM limits the spread of avian influenza viruses and that PB1-F2 could play a contributory role in the process.
Project description:The pandemic of 1918 was caused by an H1N1 influenza A virus, which is a negative strand RNA virus; however, little is known about the nature of its direct ancestral strains. Here we applied a broad genetic and phylogenetic analysis of a wide range of influenza virus genes, in particular the PB1 gene, to gain information about the phylogenetic relatedness of the 1918 H1N1 virus. We compared the RNA genome of the 1918 strain to many other influenza strains of different origin by several means, including relative synonymous codon usage (RSCU), effective number of codons (ENC), and phylogenetic relationship. We found that the PB1 gene of the 1918 pandemic virus had ENC values similar to the H1N1 classical swine and human viruses, but different ENC values from avian as well as H2N2 and H3N2 human viruses. Also, according to the RSCU of the PB1 gene, the 1918 virus grouped with all human isolates and "classical" swine H1N1 viruses. The phylogenetic studies of all eight RNA gene segments of influenza A viruses may indicate that the 1918 pandemic strain originated from a H1N1 swine virus, which itself might be derived from a H1N1 avian precursor, which was separated from the bulk of other avian viruses in toto a long time ago. The high stability of the RSCU pattern of the PB1 gene indicated that the integrity of RNA structure is more important for influenza virus evolution than previously thought.
Project description:Highly pathogenic influenza A viruses (IAV) from avian hosts were first reported to directly infect humans 20 years ago. However, such infections are rare events, and our understanding of factors promoting or restricting zoonotic transmission is still limited. One accessory protein of IAV, PB1-F2, was associated with pathogenicity of pandemic and zoonotic IAV. This short (90-amino-acid) peptide does not harbor an enzymatic function. We thus identified host factors interacting with H5N1 PB1-F2, which could explain its importance for virulence. PB1-F2 binds to HCLS1-associated protein X1 (HAX-1), a recently identified host restriction factor of the PA subunit of IAV polymerase complexes. We demonstrate that the PA of a mammal-adapted H1N1 IAV is resistant to HAX-1 imposed restriction, while the PA of an avian-origin H5N1 IAV remains sensitive. We also showed HAX-1 sensitivity for PAs of A/Brevig Mission/1/1918 (H1N1) and A/Shanghai/1/2013 (H7N9), two avian-origin zoonotic IAV. Inhibition of H5N1 polymerase by HAX-1 can be alleviated by its PB1-F2 through direct competition. Accordingly, replication of PB1-F2-deficient H5N1 IAV is attenuated in the presence of large amounts of HAX-1. Mammal-adapted H1N1 and H3N2 viruses do not display this dependence on PB1-F2 for efficient replication in the presence of HAX-1. We propose that PB1-F2 plays a key role in zoonotic transmission of avian H5N1 IAV into humans.IMPORTANCE Aquatic and shore birds are the natural reservoir of influenza A viruses from which the virus can jump into a variety of bird and mammal host species, including humans. H5N1 influenza viruses are a good model for this process. They pose an ongoing threat to human and animal health due to their high mortality rates. However, it is currently unclear what restricts these interspecies jumps on the host side or what promotes them on the virus side. Here we show that a short viral peptide, PB1-F2, helps H5N1 bird influenza viruses to overcome a human restriction factor of the viral polymerase complex HAX-1. Interestingly, we found that human influenza A virus polymerase complexes are already adapted to HAX-1 and do not require this function of PB1-F2. We thus propose that a functional full-length PB1-F2 supports direct transmission of bird viruses into humans.
Project description:Amongst all the internal gene segments (PB2. PB1, PA, NP, M and NS), the avian PB1 segment is the only one which was reassorted into the human H2N2 and H3N2 pandemic strains. This suggests that the reassortment of polymerase subunit genes between mammalian and avian influenza viruses might play roles for interspecies transmission. To test this hypothesis, we tested the compatibility between PB2, PB1, PA and NP derived from a H5N1 virus and a mammalian H1N1 virus. All 16 possible combinations of avian-mammalian chimeric viral ribonucleoproteins (vRNPs) were characterized. We showed that recombinant vRNPs with a mammalian PB2 and an avian PB1 had the strongest polymerase activities in human cells at all studied temperature. In addition, viruses with this specific PB2-PB1 combination could grow efficiently in cell cultures, especially at a high incubation temperature. These viruses were potent inducers of proinflammatory cytokines and chemokines in primary human macrophages and pneumocytes. Viruses with this specific PB2-PB1 combination were also found to be more capable to generate adaptive mutations under a new selection pressure. These results suggested that the viral polymerase activity might be relevant for the genesis of influenza viruses of human health concern.
Project description:Gene mutations and reassortment are key mechanisms by which influenza A virus acquires virulence factors. To evaluate the role of the viral polymerase replication machinery in producing virulent pandemic (H1N1) 2009 influenza viruses, we generated various polymerase point mutants (PB2, 627K/701N; PB1, expression of PB1-F2 protein; and PA, 97I) and reassortant viruses with various sources of influenza viruses by reverse genetics. Although the point mutations produced no significant change in pathogenicity, reassortment between the pandemic A/California/04/09 (CA04, H1N1) and current human and animal influenza viruses produced variants possessing a broad spectrum of pathogenicity in the mouse model. Although most polymerase reassortants had attenuated pathogenicity (including those containing seasonal human H3N2 and high-pathogenicity H5N1 virus segments) compared to that of the parental CA04 (H1N1) virus, some recombinants had significantly enhanced virulence. Unexpectedly, one of the five highly virulent reassortants contained a A/Swine/Korea/JNS06/04(H3N2)-like PB2 gene with no known virulence factors; the other four had mammalian-passaged avian-like genes encoding PB2 featuring 627K, PA featuring 97I, or both. Overall, the reassorted polymerase complexes were only moderately compatible for virus rescue, probably because of disrupted molecular interactions involving viral or host proteins. Although we observed close cooperation between PB2 and PB1 from similar virus origins, we found that PA appears to be crucial in maintaining viral gene functions in the context of the CA04 (H1N1) virus. These observations provide helpful insights into the pathogenic potential of reassortant influenza viruses composed of the pandemic (H1N1) 2009 influenza virus and prevailing human or animal influenza viruses that could emerge in the future.
Project description:Vaccination is considered the most effective preventive means for influenza control. The development of a master virus with high growth and genetic stability, which may be used for the preparation of vaccine viruses by gene reassortment, is crucial for the enhancement of vaccine performance and efficiency of production. Here, we describe the generation of a high-fidelity and high-growth influenza vaccine master virus strain with a single V43I amino acid change in the PB1 polymerase of the high-growth A/Puerto Rico/8/1934 (PR8) master virus. The PB1-V43I mutation was introduced to increase replication fidelity in order to design an H1N1 vaccine strain with a low error rate. The PR8-PB1-V43I virus exhibited good replication compared with that of the parent PR8 virus. In order to compare the efficiency of egg adaptation and the occurrence of gene mutations leading to antigenic alterations, we constructed 6:2 genetic reassortant viruses between the A(H1N1)pdm09 and the PR8-PB1-V43I viruses; hemagglutinin (HA) and neuraminidase (NA) were from the A(H1N1)pdm09 virus, and the other genes were from the PR8 virus. Mutations responsible for egg adaptation mutations occurred in the HA of the PB1-V43I reassortant virus during serial egg passages; however, in contrast, antigenic mutations were introduced into the HA gene of the 6:2 reassortant virus possessing the wild-type PB1. This study shows that the mutant PR8 virus possessing the PB1 polymerase with the V43I substitution may be utilized as a master virus for the generation of high-growth vaccine viruses with high polymerase fidelity, low error rates of gene replication, and reduced antigenic diversity during virus propagation in eggs for vaccine production.IMPORTANCE Vaccination represents the most effective prophylactic option against influenza. The threat of emergence of influenza pandemics necessitates the ability to generate vaccine viruses rapidly. However, as the influenza virus exhibits a high mutation rate, vaccines must be updated to ensure a good match of the HA and NA antigens between the vaccine and the circulating strain. Here, we generated a genetically stable master virus of the A/Puerto Rico/8/1934 (H1N1) backbone encoding an engineered high-fidelity viral polymerase. Importantly, following the application of the high-fidelity PR8 backbone, no mutation resulting in antigenic change was introduced into the HA gene during propagation of the A(H1N1)pdm09 candidate vaccine virus. The low error rate of the present vaccine virus should decrease the risk of generating mutant viruses with increased virulence. Therefore, our findings are expected to be useful for the development of prepandemic vaccines and live attenuated vaccines with higher safety than that of the present candidate vaccines.
Project description:<h4>Background</h4>A new strain of human H1N1 influenza A viruses was broken out in the April 2009 and caused worldwide pandemic emergency. The present study is trying to estimate a temporal reassortment history of 2009 H1N1 viruses by phylogenetic analysis based on a total 394 sequences of H1N1viruses isolated from swine, human and avian.<h4>Results</h4>Phylogenetic trees of eight gene segments showed that viruses sampled from human formed a well-supported clade, whereas swine and avian lineages were intermixed together. A new divergence swine sublineage containing gene segments of 2009 H1N1 viruses was characterized, which were closely related with swine viruses collected from USA and South Korea during 2004 to 2007 in six segments (PB2, PB1, PA, HA, NP and NS), and to swine viruses isolated from Thailand during 2004 to 2005 in NA and M. Substitution rates were varied drastically among eight segments and the average substitution rate was generally higher in 2009 H1N1 than in swine and human viruses (F2,23 = 5.972, P < 0.01). Similarly, higher dN/dS substitution ratios were identified in 2009 H1N1 than in swine and human viruses except M2 gene (F2, 25 = 3.779, P < 0.05). The ages of 2009 H1N1 viruses were estimated around 0.1 to 0.5 year, while their common ancestors with closest related swine viruses existed between 9.3 and 17.37 years ago.<h4>Conclusion</h4>Our results implied that at least four reassortments or transmissions probably occurred before 2009 H1N1 viruses. Initial reassortment arose in 1976 and avian-like Eurasian swine viruses emerged. The second transmission happened in Asia and North America between 1988 and 1992, and mostly influenced six segments (PB2, PB1, PA, HA, NP and NS). The third reassortment occurred between North American swine and avian viruses during 1998 to 2000, which involved PB2 and PA segments. Recent reassortments occurred among avian-to-swine reassortant, Eurasian and classical swine viruses during 2004 to 2005. South Korea, Thailand and USA, were identified as locations where reassortments most likely happened. The co-circulation of multiple swine sublineages and special lifestyle in Asia might have facilitated mixing of diverse influenza viruses, leading to generate a novel virus strain.
Project description:The H1N1/pdm2009 virus is a new triple-reassortant virus. While Eurasian avian-like and triple-reassortant swine influenza viruses are the direct ancestors of H1N1/pdm2009, the classic swine influenza virus facilitate the spectrum of influenza A diversity in pig population when the reassortant events occurred during 1998 to April 2009. The factors that facilitate the final formation of this gene constellation for H1N1/pdm2009 virus from this complex gene pool remain unknown. Since a novel successful virus should efficiently replicate and transmit in their hosts, in this study, we estimated the adaptability of the codon usage patterns of the pool of genes from these lineages of swine influenza viruses to the human expression system. We found that the MP and NA genes of Eurasian avian-like swine influenza viruses, and the PB2, PB1 and PA genes of triple-reassortant swine influenza viruses were best adapted to the human codon usage pattern. As these genes participated in the development of H1N1/pdm2009, they might help in viral replication and strengthen its competitiveness during its emergence. After its emergence in the human population, a gradual optimization of codon usage patterns between 2009 and 2019 to the human codon usage for the H1N1/pdm2009 genes was detected. This reveals that ongoing adaptive evolution, after its original incursion, occurred to further increase the adaptability of overall gene cassette to human expression system.
Project description:Influenza A viruses cause an annual contagious respiratory disease in humans and are responsible for periodic high-mortality human pandemics. Pandemic influenza A viruses usually result from the reassortment of gene segments between human and avian influenza viruses. These avian influenza virus gene segments need to adapt to humans. Here we focus on the human adaptation of the synonymous codons of the avian influenza virus PB1 gene of the 1968 H3N2 pandemic virus. We generated recombinant H3N2 viruses differing only in codon usage of PB1 mRNA and demonstrated that codon usage of the PB1 mRNA of recent H3N2 virus isolates enhances replication in interferon (IFN)-treated human cells without affecting replication in untreated cells, thereby partially alleviating the interferon-induced antiviral state. High-throughput sequencing of tRNA pools explains the reduced inhibition of replication by interferon: the levels of some tRNAs differ between interferon-treated and untreated human cells, and evolution of the codon usage of H3N2 PB1 mRNA is skewed toward interferon-altered human tRNA pools. Consequently, the avian influenza virus-derived PB1 mRNAs of modern H3N2 viruses have acquired codon usages that better reflect tRNA availabilities in IFN-treated cells. Our results indicate that the change in tRNA availabilities resulting from interferon treatment is a previously unknown aspect of the antiviral action of interferon, which has been partially overcome by human-adapted H3N2 viruses.IMPORTANCE Pandemic influenza A viruses that cause high human mortality usually result from reassortment of gene segments between human and avian influenza viruses. These avian influenza virus gene segments need to adapt to humans. Here we focus on the human adaptation of the avian influenza virus PB1 gene that was incorporated into the 1968 H3N2 pandemic virus. We demonstrate that the coding sequence of the PB1 mRNA of modern H3N2 viruses enhances replication in human cells in which interferon has activated a potent antiviral state. Reduced interferon inhibition results from evolution of PB1 mRNA codons skewed toward the pools of tRNAs in interferon-treated human cells, which, as shown here, differ significantly from the tRNA pools in untreated human cells. Consequently, avian influenza virus-derived PB1 mRNAs of modern H3N2 viruses have acquired codon usages that better reflect tRNA availabilities in IFN-treated cells and are translated more efficiently.
Project description:The emergence of new pandemic influenza A viruses requires overcoming barriers to cross-species transmission as viruses move from animal reservoirs into humans. This complicated process is driven by both individual gene mutations and genome reassortments. The viral polymerase complex, composed of the proteins PB1, PB2, and PA, is a major factor controlling host adaptation, and reassortment events involving polymerase gene segments occurred with past pandemic viruses. Here we investigate the ability of polymerase reassortment to restore the activity of an avian influenza virus polymerase that is normally impaired in human cells. Our data show that the substitution of human-origin PA subunits into an avian influenza virus polymerase alleviates restriction in human cells and increases polymerase activity in vitro. Reassortants with 2009 pandemic H1N1 PA proteins were the most active. Mutational analyses demonstrated that the majority of the enhancing activity in human PA results from a threonine-to-serine change at residue 552. Reassortant viruses with avian polymerases and human PA subunits, or simply the T552S mutation, displayed faster replication kinetics in culture and increased pathogenicity in mice compared to those containing a wholly avian polymerase complex. Thus, the acquisition of a human PA subunit, or the signature T552S mutation, is a potential mechanism to overcome the species-specific restriction of avian polymerases and increase virus replication. Our data suggest that the human, avian, swine, and 2009 H1N1-like viruses that are currently cocirculating in pig populations set the stage for PA reassortments with the potential to generate novel viruses that could possess expanded tropism and enhanced pathogenicity.