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

Second sialic acid-binding site of influenza A virus neuraminidase: Binding receptors for efficient release

Influenza A viruses (IAVs) are a major cause of human respiratory tract infections and cause significant disease and mortality. Human IAVs originate from animal viruses that breached the host species barrier. IAV particles contain sialoglycan receptor-binding haemagglutinin (HA) and receptor-destroying neuraminidase (NA) in their envelope. When IAV crosses the species barrier, the functional balance between HA and NA needs to be adjusted to the sialoglycan repertoire of the novel host species. Relatively little is known about the role of NA in host adaptation in contrast to the extensively studied HA. NA prevents virion aggregation and facilitates release of (newly assembled) virions from cell surfaces as well as from decoy receptors abundantly present in mucus and cell glycocalyx. In addition to a highly conserved catalytic site, NA carries a second sialic acid-binding site (2SBS). The 2SBS preferentially binds α2,3-linked sialic acids, and enhances activity of the neighboring catalytic site by bringing/keeping multivalent substrates in close contact with this site. In this way, the 2SBS contributes to the HA-NA balance of virus particles and affects virus replication. The 2SBS is highly conserved in all NA subtypes of avian IAVs, with some notable exceptions associated with changes in the receptor-binding specificity of HA and host tropism. Conservation of the 2SBS is invariably lost in human (pandemic) viruses as well as in several other viruses adapted to mammalian host species. Preservation or loss of the 2SBS is likely to be an important factor of the viral host range

Gradual adaptation of animal influenza A viruses to human-type sialic acid receptors

Influenza A viruses (IAVs) originating from animal reservoirs pose continuous threats to human health as demonstrated by the Spanish flu pandemic. Infection starts by attachment to host receptors, a crucial step that is targeted by immunological, prophylactic, and therapeutic intervention. Fine-tuning of virus hemagglutinin binding to host-specific receptor repertoires needs to remain balanced to receptor-destroying neuraminidase (NA) activity and is a key step in host adaptation. It determines NA-dependent virus motility, enabling IAVs to traverse the mucus layer and to bind to, and migrate over, the epithelial cell surface for reaching a location supporting endocytic uptake. Canonical adaptations in enzootic/zoonotic IAVs enhancing human-type receptor binding are well-known, but the context and timespan required for their selection pose many questions. We discuss recent developments, focusing on the dynamic nature of interactions of IAV with the heterogeneous receptor repertoires present in humans and potential intermediate hosts. Potential pre-adaption toward human-type receptor binding in intermediate hosts will be discussed

Mutations in influenza A virus neuraminidase and hemagglutinin confer resistance against a broadly neutralizing hemagglutinin stem antibody

Influenza A virus (IAV), a major cause of human morbidity and mortality, continuously evolves in response to selective pressures. Stem-directed, broadly neutralizing antibodies (sBnAbs) targeting influenza hemagglutinin (HA) are a promising therapeutic strategy, but neutralization escape mutants can develop. We used an integrated approach combining viral passaging, deep sequencing, and protein structural analyses to define escape mutations and mechanisms of neutralization escape in vitro for the F10 sBnAb. IAV was propagated with escalating concentrations of F10 over serial passages in cultured cells to select for escape mutations. Viral sequence analysis revealed three mutations in HA and one in neuraminidase (NA). Introduction of these specific mutations into IAV through reverse genetics confirmed their roles in resistance to F10. Structural analyses revealed that the selected HA mutations (S123G, N460S, and N203V) are away from the F10 epitope but may indirectly impact influenza receptor binding, endosomal fusion, or budding. The NA mutation E329K, which was previously identified to be associated with antibody escape, affects the active site of NA, highlighting the importance of the balance between HA and NA function for viral survival. Thus, whole genome population sequencing enables the identification of viral resistance mutations responding to antibody-induced selective pressure.IMPORTANCE Influenza A virus is a public health threat for which currently available vaccines are not always effective. Broadly neutralizing antibodies that bind to the highly-conserved stem region of influenza hemagglutinin (HA) can neutralize many influenza strains. To understand how influenza virus can become resistant or escape such antibodies, we propagated influenza A virus in vitro with escalating concentrations of antibody and analyzed viral populations with whole genome sequencing. We identified HA mutations near and distal to the antibody binding epitope that conferred resistance to antibody neutralization. Additionally, we identified a neuraminidase (NA) mutation that allowed the virus to grow in the presence of high concentrations of the antibody. Virus carrying dual mutations in HA and NA also grew under high antibody concentrations. We show that NA mutations mediate the escape of neutralization by antibodies against HA, highlighting the importance of a balance between HA and NA for optimal virus function

Avian influenza A virus transmission and the emergence of drug resistance

2011 Fall.Includes bibliographical references.As avian influenza A viruses (AIV) continue to circulate worldwide both naturally, within the reservoir host of wild waterfowl, and cross species barriers, eventually establishing itself in new host species, it is imperative to study the natural reservoir in respect to virus change and transmissibility. This dissertation will focus on the transmissibility of a mallard virus from mallards to other wild and domestic species as well as elucidate the possible outcomes of oseltamivir contamination in the environment and its effect on influenza A virus infected mallards. Low pathogenicity (LP) AIVs of the H5N2 and H7N3 subtypes were utilized to evaluate the ability of transmission of a mallard derived virus to other species present in a co-habitation (barnyard) scenario. Other species in contact with the mallards were chickens, blackbirds, rats, and pigeons. Viral replication was assessed directly from ducks in the barnyard with assessment of the other animals in the barnyard through sero-conversion. Additional animals of each species were directly inoculated with these two viruses and assessed for viral replication. The H5N2 virus was transmitted to other ducks and chickens in the barnyard through either direct or environmental contamination, but not to rats or blackbirds. The H7N3 virus was transmitted to other ducks, chickens, pigeons, and rats. Chickens and blackbirds directly inoculated with both virus strains shed significant amount of virus and seroconverted, but rats and pigeons (except for one pigeon) failed to shed virus but did develop antiviral antibodies. Knowing that both mallard viruses can directly transmit without adaptation, show the mallard to be a good model to further evaluate the outcome of oseltamivir contamination in the environment and its effect on AIV infected mallards. The environment has been shown to be contaminated with significant amounts of oseltamivir carboxylate (OC) in an area of high drug prescription use. We analyzed the outcomes of AIV in infected mallards when they have access to OC in their drinking water. Two separate LPAIV H5N2 viruses were tested for their ability to mutate under drug pressure. One H5N2 virus did not demonstrate any altered sequence after 7-10 days of drug access and infection. The other H5N2 virus did show mutations in the neuraminidase gene that led to an increase in resistance to oseltamivir caused by a specific mutation at E119V. This resistant virus was further evaluated for its ability to transmit between infected and naïve mallards. While the resistant virus did transmit duck to duck, the mutation at position 119 was not detected after challenge or transmission showing instability of this mutation. This could either be a reversion to wild-type or possibly the low level presence of wild-type present in the resistant strain stock that outcompeted with the mutant strain to succeed in the host. This shows, that in these duck experiments, the E119V mutation is not stable in the absence of drug pressure and unlikely to succeed in the host

Bench-to-bedside review: Vaccine protection strategies during pandemic flu outbreaks

Vaccination is the most effective means for the prevention of influenza, including pandemic strains. An ideal pandemic influenza vaccine should provide effective protection with the fewest number of doses in the shortest amount of time, and among the greatest proportion of the population. The current manufacturing processes required for embryonated chicken-egg-based influenza vaccines are limited in their ability to respond to pandemic situations - these limitations include problems with surge capacity, the need for egg-adapted strains, the possibility of contamination, and the presence of trace egg protein. Several vaccine strategies to circumvent the deficiencies intrinsic to an egg-based influenza vaccine are in various phases of development. These include the use of cell-culture-based growth systems, concomitant use of adjuvants, whole virus vaccines, recombinant protein vaccines, plasmid DNA vaccines, virus-like particle vaccines, and universal flu vaccines

Viral factors in influenza pandemic risk assessment

The threat of an influenza A virus pandemic stems from continual virus spillovers from reservoir species, a tiny fraction of which spark sustained transmission in humans. To date, no pandemic emergence of a new influenza strain has been preceded by detection of a closely related precursor in an animal or human. Nonetheless, influenza surveillance efforts are expanding, prompting a need for tools to assess the pandemic risk posed by a detected virus. The goal would be to use genetic sequence and/or biological assays of viral traits to identify those non-human influenza viruses with the greatest risk of evolving into pandemic threats, and/or to understand drivers of such evolution, to prioritize pandemic prevention or response measures. We describe such efforts, identify progress and ongoing challenges, and discuss three specific traits of influenza viruses (hemagglutinin receptor binding specificity, hemagglutinin pH of activation, and polymerase complex efficiency) that contribute to pandemic risk

Systematic Experimental Determination of Functional Constraints on Proteins and Adaptive Potential of Mutations: A Dissertation

Sequence-function relationship is a fundamental question for many branches of modern biomedical research. It connects the primary sequence of proteins to the function of proteins and fitness of organisms, holding answers for critical questions such as functional consequences of mutations identified in whole genome sequencing and adaptive potential of fast evolving pathogenic viruses and microbes. Many different approaches have been developed to delineate the genotype-phenotype map for different proteins, but are generally limited by their throughput or precision. To systematically quantify the fitness of large numbers of mutations, I modified a novel high throughput mutational scanning approach (EMPIRIC) to investigate the fitness landscape of mutations in important regions of essential proteins from the yeast or RNA viruses. Using EMPIRIC, I analyzed the interplay of the expression level and sequence of Hsp90 on the yeast growth and revealed latent effect of mutations at reduced expression levels of Hsp90. I also examined the functional constraint on the receptor binding site of the Env of Human Immunodeficiency Virus (HIV) and uncovered enhanced receptor binding capacity as a common pathway for adaptation of HIV to laboratory conditions. Moreover, I explored the adaptive potential of neuraminidase (NA) of influenza A virus to a NA inhibitor, oseltamivir, and identified novel oseltamivir resistance mutations with distinct molecular mechanisms. In summary, I applied a high throughput functional genomics approach to map the sequence-function relationship in various systems and examined the evolutionary constraints and adaptive potential of essential proteins ranging from molecular chaperones to drug-targetable viral proteins