Influenza A viruses (IAVs) of the H1N1 and H3N2 serotypes are the major cause of seasonal influenza epidemics. IAVs are labeled based on the antigenic properties of the two surface glycoproteins and main antigens: hemagglutinin (HA/H) and neuraminidase (NA/N). HA and NA have opposing roles, with HA binding and NA enzymatically removing terminal acid residues from glycoconjugates. As HA is more abundant in the viral envelope and easily quantified, current vaccines are standardized only to HA amount. However, the seasonal vaccines have low efficacy, in part because they mainly elicit an immune response to the HA protein. Suggestions to actively include and standardize to NA amount as well has been made. Unfortunately, the active and most immunogenic form of NA is an unstable tetramer that easily disassociate during vaccine production. Knowledge of the stabilizing properties of NA is therefore needed. Additionally, due to the influenza error-prone polymerase and the many avian serotypes that could potentially make zoonotic jumps in the future, identifying residues and regions of the surface proteins that are conserved across HA and NA subtypes could help provide a more universal response to IAVs.

This thesis presents several studies aimed to increase the knowledge about influenza and the NA protein. First, a method is presented where labeled oligonucleotides (padlock probes) hybridize with the viral genome, allowing for identification of genomic segments during an infection. We show that using a handful of probes against highly conserved regions, the vast majority of IAV serotypes can be identified. Second, we show that the conserved central calcium site of NA is vital for enzymatic function and that the oligomeric structure allows for the rescue of inactive monomers by formation of enzymatically active heterotetramers. The final studies take aim at the conserved N-linked glycosylation sites and cysteines of NA ectodomains. We show that glycosylation of the head domain influences NA virion incorporation and provides stability to the protein. We also identify conserved NA residues, including a surface accessible tryptophan that is entirely conserved across a majority of IAV NA subtypes.

Together, these results provide a better knowledge of influenza neuraminidase and present several targets for next-generation vaccines.

N-linked glycans commonly contribute to secretory protein folding, sorting, and signaling. For enveloped viruses, such as the influenza A virus (IAV), large N-linked glycans can also be added to prevent access to epitopes on the surface antigens hemagglutinin (HA or H) and neuraminidase (NA or N). Sequence analysis showed that in the NA head domain of H1N1 IAVs, three N-linked glycosylation sites are conserved and that a fourth site is conserved in H3N2 IAVs. Variable sites are almost exclusive to H1N1 IAVs of human origin, where the number of head glycosylation sites first increased over time and then decreased with and after the introduction of the 2009 pandemic H1N1 IAV of Eurasian swine origin. In contrast, variable sites exist in H3N2 IAVs of human and swine origin, where the number of head glycosylation sites has mainly increased over time. Analysis of IAVs carrying N1 and N2 mutants demonstrated that the N-linked glycosylation sites on the NA head domain are required for efficient virion incorporation and replication in cells and eggs. It also revealed that N1 stability is more affected by the head domain glycans, suggesting N2 is more amenable to glycan additions. Together, these results indicate that in addition to antigenicity, N-linked glycosylation sites can alter NA enzymatic stability and the NA amount in virions.

Eukaryotic cells possess a dynamic network of membranes that vary in lipid composition. To perform numerous biological functions, cells modulate their shape and the lateral organization of proteins associated with membranes. The modulation is generally facilitated by physical cues that recruit proteins to specific regions of the membrane. Analyzing these cues is difficult due to the complexity of the membrane conformations that exist in cells. Here, we examine how different types of membrane proteins respond to changes in curvature and to lipid phases found in the plasma membrane. By using giant plasma membrane vesicles derived from transfected cells, the proteins were positioned in the correct orientation and the analysis was performed in plasma membranes with a biological composition. Nanoscale membrane curvatures were generated by extracting nanotubes from these vesicles with an optical trap. The viral membrane protein neuraminidase was not sensitive to curvature, but it did exhibit strong partitioning (coefficient of K = 0.16) disordered membrane regions. In contrast, the membrane repair protein annexin 5 showed a preference for nanotubes with a density up to 10-15 times higher than that on the more flat vesicle membrane. The investigation of nanoscale effects in isolated plasma membranes provides a quantitative platform for studying peripheral and integral membrane proteins in their natural environment.

Influenza neuraminidase (NA) is a sialidase that contributes to viral mobility by removing the extracellular receptors for the haemagglutinin (HA) glycoprotein. However, it remains unclear why influenza NAs evolved to function as Ca2+-dependent tetramers that display variable stability. Here, we show that the Ca2+ ion located at the centre of the NA tetramer is a major stability determinant, as this Ca2+ ion is required for catalysis and its binding affinity varies between NAs. By examining NAs from 2009 pandemic-like H1N1 viruses, we traced the affinity variation to local substitutions that cause residues in the central Ca2+-binding pocket to reposition. A temporal analysis revealed that these local substitutions predictably alter the stability of the 2009 pandemic-like NAs and contribute to the tendency for the stability to vary up and down over time. In addition to the changes in stability, the structural plasticity of NA was also shown to support the formation of heterotetramers, which creates a mechanism for NA to obtain hybrid properties and propagate suboptimal mutants. Together, these results demonstrate how the structural restrictions for activity provide influenza NA with several mechanisms for adaptation and diversification.

Influenza viruses replicate within the nucleus of the host cell. This uncommon RNA virus trait provides influenza with the advantage of access to the nuclear machinery during replication. However, it also increases the complexity of the intracellular trafficking that is required for the viral components to establish a productive infection. The segmentation of the influenza genome makes these additional trafficking requirements especially challenging, as each viral RNA (vRNA) gene segment must navigate the network of cellular membrane barriers during the processes of entry and assembly. To accomplish this goal, influenza A viruses (IAVs) utilize a combination of viral and cellular mechanisms to coordinate the transport of their proteins and the eight vRNA gene segments in and out of the cell. The aim of this review is to present the current mechanistic understanding for how IAVs facilitate cell entry, replication, virion assembly, and intercellular movement, in an effort to highlight some of the unanswered questions regarding the coordination of the IAV infection process.

Genome delivery to the proper cellular compartment for transcription and replication is a primary goal of viruses. However, methods for analyzing viral genome localization and differentiating genomes with high identity are lacking, making it difficult to investigate entry-related processes and co-examine heterogeneous RNA viral populations. Here, we present an RNA labeling approach for single-cell analysis of RNA viral replication and co-infection dynamics in situ, which uses the versatility of padlock probes. We applied this method to identify influenza A virus (IAV) infections in cells and lung tissue with single-nucleotide specificity and to classify entry and replication stages by gene segment localization. Extending the classification strategy to co-infections of IAVs with single-nucleotide variations, we found that the dependence on intracellular trafficking places a time restriction on secondary co-infections necessary for genome reassortment. Altogether, these data demonstrate how RNA viral genome labeling can help dissect entry and co-infections.

A primary function of 5' regions in many secretory protein mRNAs is to encode an endoplasmic reticulum (ER) targeting sequence. In this study, we show how the regions coding for the ER-targeting sequences of the influenza glycoproteins NA and HA also function as translational regulatory elements that are controlled by the viral RNA-binding protein (RBP) NS1. The translational increase depends on the nucleotide composition and 5' positioning of the ER-targeting sequence coding regions and is facilitated by the RNA-binding domain of NS1, which can associate with ER membranes. Inserting the ER-targeting sequence coding region of NA into different 5' UTRs confirmed that NS1 can promote the translation of secretory protein mRNAs based on the nucleotides within this region rather than the resulting amino acids. By analyzing human protein mRNA sequences, we found evidence that this mechanism of using 5' coding regions and particular RBPs to achieve gene-specific regulation may extend to human-secreted proteins.

We used the amphipathic styrene maleic acid (SMA) co-polymer to extract cytochrome c oxidase (CytcO) in its native lipid environment from S. cerevisiae mitochondria. Native nanodiscs containing one CytcO per disc were purified using affinity chromatography. The longest cross-sections of the native nanodiscs were 11 nm x 14 nm. Based on this size we estimated that each CytcO was surrounded by similar to 100 phospholipids. The native nanodiscs contained the same major phospholipids as those found in the mitochondrial inner membrane. Even though CytcO forms a supercomplex with cytochrome bc(1) in the mitochondria! membrane, cyt.bc(1) was not found in the native nanodiscs. Yet, the loosely-bound Respiratory SuperComplex factors were found to associate with the isolated CytcO. The native nanodiscs displayed an O-2-reduction activity of similar to 130 electrons CytcO(-1) s(-1) and the kinetics of the reaction of the fully reduced CytcO with 02 was essentially the same as that observed with CytcO in mitochondrial membranes. The kinetics of CO-ligand binding to the CytcO catalytic site was similar in the native nanodiscs and the mitochondrial membranes. We also found that excess SMA reversibly inhibited the catalytic activity of the mitochondrial CytcO, presumably by interfering with cyt. c binding. These data point to the importance of removing excess SMA after extraction of the membrane protein. Taken together, our data shows the high potential of using SMA-extracted CytcO for functional and structural studies.

N-linked glycans commonly contribute to secretory protein folding, sorting and signaling. For enveloped viruses such as the influenza A virus (IAV), the addition of large N-linked glycans can also alter epitopes displayed by the surface antigens HA and NA. Computational analysis shows that three N-linked glycosylation sites (Asn88, Asn146 and Asn235) are conserved in the NA head domain from H1N1 IAVs and that one additional site (Asn200) is conserved in H3N2 IAVs. In IAVs of human origin, the number of these sites has increased and then decreased over time in H1N1 strains, whereas the number has primarily increased over time in in H3N2 strains. Experimental analysis shows that the three conserved head glycosylation sites are not essential for H1N1 IAV replication in cells or eggs, but influences the final NA amount in the virion, and that the more efficiently recognized N-X-T sites located on top of the NA tetramer affect thermostability. These results imply that nucleotide changes which alter N-linked glycosylation sites in NA can change the enzymatic and virion properties in addition to antigenicity.