Polymerase basic protein 1 (PB1) as a molecular determinant of fitness and adaptation in influenza a virus

Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2017The World Health Organization and the National Institute of Allergy and Infectious Diseases reported growth deficits of influenza A(H1N1)pdm09 reverse genetic pandemic vaccine virus seeds. These have compromised the effective and timely distribution of vaccines in the 2009 pandemics and accentuated the need to improve the process of vaccine production. In pre-pandemic A(H5N1) research, seed viruses produced by reverse genetics have also been reported to present growth deficits. These deficits have been attributed to a putative sub-optimal protein interaction. The dynamics of the genetic evolution of influenza A viruses appears to suggest a gene segregation pattern between the Polymerase Basic protein 1 (PB1) and antigenic proteins Hemagglutinin (HA) and Neuraminidase (NA). In the reassortment events that lead to the emergence of the 1957 e 1968 pandemic viruses, the contemporary seasonal viruses acquired PB1 genomic segment together with antigenic glycoproteins originating from avian viruses. A similar pattern was identified in 1947, where a reassortment event between seasonal viruses, involving PB1 and antigenic proteins, has altered the epidemiology of the infection to a near-pandemic geographic dispersion. In both situations, viral fitness appears to have benefitted from acquiring a PB1 genomic segment homologous to antigenic proteins. Also, in retrospective studies on the genomic composition of high yield seasonal vaccine seeds produced by classical reassortment, PB1 is frequently co-incorporated with antigenic proteins HA and NA, further suggesting that the interaction between these proteins could have an impact in viral fitness. In this context, we proposed to address the question of PB1 genomic segment being a molecular determinant of fitness and adaptation in influenza A virus and, particularly, of the functional compatibility between PB1 and antigenic proteins being a driver of the overall viral fitness and putatively exploitable to improve seed virus production. The A(H1N1)pdm09 virus was used a model for this research because it is a product of viral reassortment with an unprecedented genomic composition of segments originating from avian, swine and human seasonal viruses. Additionally, the 2009 pandemic vaccine virus presented severe growth deficits and, since the A(H1N1)pdm09 persists in circulation with a seasonal epidemiologic profile, the demand for high yield A(H1N1)pdm09 vaccine seeds will be continuous and the need to adequate the immunogenic strain to the circulating viruses will be recurrent because of antigenic drifts. The objectives of this research were defined as 1) to evaluate the genetic evolution of PB1 in the zoonotic transmission of swine influenza virus and infer its putative contribution towards viral fitness and adaptation, and 2) to determine if the functional or structural compatibility between PB1 and antigenic proteins is a molecular determinant of the overall virus fitness in the reverse genetics A(H1N1)pdm09 vaccine seed model. The approach followed to accomplish objective 1 was to select a study sample of PB1 nucleotide sequences from swine virus that have infected the human host, to analyze phylogeny and mutation trends and to search for putative markers for viral adaptation on the basis of viral molecular epidemiology, genomic location of the polymorphisms and amino-acid properties. Our major findings were that the evolutionary history of PB1 is traceable in terms of lineage and host origin. Specific genomic markers in PB1 appear to putatively relate to the viral adaptation to mammalian hosts, 336I, 361R, 468K and 584Q, and to the viral adaptation to new genomic backgrounds possibly in the sequence of reassortment events, such as 638D and 618D. Residues 298I, 386K and 517V have been found to putatively relate to an enhanced compatibility between PB1 and HA of the H1 subtype, in the mammalian host. A subsequent in vitro investigation of the phenotypic impact of mutations L298I, R386K and I517V acquired by the A(H1N1)pdm09 during its evolutionary history, was performed by generating an A(H1N1)pdm09 recombinant virus and an A(H1N1)pdm09 reassortant in which the specific mutations have been reverted, by reverse genetics. This approach has resulted in two major findings. Acquiring these mutations has been found to putatively promote conformational changes in PB1 and enhance the span of complementary nucleotides possibly involved in PB1 interaction with HA at the RNA level and, on the other hand, has proven detrimental to viral growth kinetics in vitro. These findings have lead us to suggest that the interaction between genomic segments at the RNA level could be a determinant of co-segregation, concordant with a selective packaging model proposed by other authors, but that the mechanisms that drive this process are probably not dependent on a replicative advantage. Our approach to accomplishing objective 2) to determine if the functional or structural compatibility between PB1 and antigenic proteins is a molecular determinant of the overall virus fitness in the reverse genetic A(H1N1)pdm09 vaccine seed model, was to determine the genetic profile of A(H1N1)pdm09 strains circulating in Portugal during the pandemic period and select a prototype immunogenic strain, to generate reassortant viruses with the genomic composition of A(H1N1)pdm09 seed viruses prototypes bearing PB1 homologous and heterologous to antigenic proteins, and to evaluate viral growth and antigen yield in vitro. A sample of specimens collected from the pandemic period in Portugal were evaluated for genetic and phenotypic features and a strain similar to the consensus was selected as a prototype strain. Vaccine seed prototypes of the selected A(H1N1)pdm09 strain in an A/PuertoRico/08/34 backbone were generated by reverse genetics to present the genomic compositions of the 6:2 classical vaccine seed (PR8:HA,NA A(H1N1)pdm09) and a 5:3 seed prototype in which the PB1 segment from the immunogenic strain is co-incorporated with the antigenic proteins (PR8:HA,NA,PB1 A(H1N1)pdm09). Our major findings were that the presence of PB1 homologous to antigenic protein significantly increased viral replication, hemagglutination capacity and Neuraminidase activity. We have establishing proof of concept that, in the PR8:A(H1N1)pdm09 seed virus model, viral growth and antigen yield can be significantly improved by the inclusion of PB1 from the immunogenic strain when compared to the classical seed virus prototype. We consider that, additionally to the role of PB1 protein in viral replication, PB1 genomic segment may be a molecular determinant of the overall virus fitness and a determinant factor in the molecular epidemiology of the viruses by establishing interactions with other segments at the RNA level and by, apparently, being able to genetically change and adapt to improve these interactions. Further research is necessary to clarify the mechanisms of viral genome packaging, the role of interactions at the RNA level in establishing the co-segregation patterns and the specificities of this interactions at the subtype level. However, it becomes clear that the functional compatibility between PB1 and antigenic proteins is a driver of the overall viral fitness in the A(H1N1)pdm09 and is putatively exploitable to improve seed virus production. We also consider that exploring the concept of the compatibility between gene segments or proteins being a determinant factor in the overall viral fitness, can result in major improvements in the production of reverse genetics seed viruses of different influenza subtypes. Also, being aware of the fact that the genomic composition of influenza viruses can have a major phenotypic impact, and that consequently is a determinant of virulence even though the mechanisms that drive the selective packaging remain unclear, we consider that its inclusion in the risk assessment of influenza strains would be extremely relevant for seasonal and pandemic preparedness