The Study of Influenza A Virus Genomic Assembly

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Summary

The packaging of viral genome is an important step in the virus life cycle. Influenza A virus is an enveloped single-stranded negative-sense RNA virus contains eight segmented genome (vRNAs) (1). These eight vRNAs interact with polymerases and nucleoproteins to form ribonucleoprotein (RNP) complexes and have their segment-specific regions that extend from UTR to the open reading frame (ORF) (2). During virus assembly, all eight vRNAs need to be packaged into the new virion for future infection (3). The mechanism for influenza A virus assembly is only partially known. It has been proposed that all eight vRNAs form a genomic complex and are selectively packaged into the newborn virion. The study of assembly mechanism is challenging by the complicated interactions among different segmented RNAs and proteins.

By using biological and biochemical studies, we propose to investigate the interaction between segment 5 and segment 3, specific regions required for this interaction and the formation of segment 5 RNP in vitro. FRET study, in vivo fitness assay, electron microscopy and crystallography will be employed to for this purpose. These studies will not only case light on the mechanism of influenza A virus assembly, but also provide insight into potential target for the development of pharmaceuticals for influenza viruses. Moreover, these studies will contribute to the understanding of the assembly of other virus particles with segmented genome such as measles virus (MeV) (4).

Specific Aims

Influenza A virus is a pandemic threat to human beings. This virus is an enveloped single-stranded negative-sense RNA virus with eight segmented genome (vRNAs). All eight vRNAs share conservation and partial complementarily at both the 5' and the 3' untranslated regions (UTR), and have their segment-specific regions that extend from UTR to the open reading frame (ORF). A favorite assembly mechanism is that all eight vRNAs are specially selected for virus assembly and all vRNAs form a genomic complex before they are fully packaged into the newborn virion. Previous study implies segment 5 might interact with segment 3 (9). By using biophysical and biochemical methods, we aim to investigate the interaction between segment 5 and segment 3, as well as the regions required for this interaction.

Specific aim 1: Investigate the interaction between segment 3 and 5 that might take place during the influenza A virus assembly.

A synonymous mutation study of segment 5 packaging signal suggests an interaction between segment 3 and 5 during influenza A virus assembly. We hypothesize that segment 5 and 3 have a special interaction during viral assembly. In order to study the interaction between them, I propose to purify segment 3 and 5 in their ribonucleoprotein complexes from influenza A virus, using velocity sedimentation. These two segments will be studied by förster resonance energy transfer (FRET) experiment using Cy3-labeled segment 3 and Cy5-labeled segment 5. This experiment will allow us to evaluate the interaction between these two segments that might be involved in the virus assembly.

Specific aim 2: Identify nucleotides of segment 3 that are critical for the specific interaction.

It was found that mutations of segment 5 packaging signal affect the packaging of segment 3 but little is known about the interaction between them. In order to cast more light on the interaction, I propose to undertake site-directed point mutations or truncations in packaging signal regions of segment 3. These segment 3 mutants will be studies for their in vivo effects and purified to test their interactions with intact segment 5 by FRET as well. The identification of important regions will allow us to evaluate their importance and properties during the segment interaction.

Specific aim 3: Segment 3 reconstruction using biochemical methods and its structural study

The vRNA of influenza A form RNP complex with nucleoprotein NP and three RNA polymerases in intact virus. For further understanding of the RNP assembly, I propose to express and purify nucleoprotein and polymerases PB1, PB2 and PA, and also synthesize vRNA segment 3 to test the in vitro self-assembly. Electron microscopy and crystallization will be used to characterize the reconstructed RNP and also the interaction between it and segment 5 packaging signals.

Research Strategy

Influenza A virus can cause local and global pandemics with high fatality rate in the general population. The nearest catastrophe caused by influenza A was the 1918 Spanish flu which resulted in 40 million lives (5). Thus, deep understanding of the virus life cycle and its infection of host cells is needed for future prevention of such pandemics.

Influenza A is an enveloped single-stranded negative-sense RNA virus (1). The genome RNA (vRNA) contains eight segments which have their segment-specific regions that extend from untranslated regions (UTR) to the open reading frame (ORF) (6). The eight segments encode altogether 11 proteins including two non-structural proteins and nine structural proteins (1). Inside the virus, the segmented genome interacts with polymerases acidic polymerase protein (PA), basic polymerase protein 1 (PB1), basic polymerase protein 2 (PB2) and nucleoproteins (NP) to form eight ribonucleoprotein (RNP) complexes (1). Although the segmented genome provides advantages for virus infections and viabilities, it complicates the incorporation of all eight segmented vRNAs required for future infections (6).

During the late stage of viral life cycle, at least one copy of each of the eight vRNAs needs to be packaged into the new virion for future infection (1). Whether influenza A virus packages these eight segmented genome randomly or selectively is not clearly understood (2). A favorite hypothesis for this assembly mechanism is that all eight vRNAs interact with other segments nearby to form a genomic complex and are selectively packaged into the newborn virion (Figure 1). This model is supported by electron microscopy of influenza A virus particles which shows RNPs are organized as seven RNPs surrounding a central RNP (7). It has been proposed that vRNA segments interact via RNA-RNA interactions but the interaction array of different segments is unknown yet (8). The study of assembly mechanism is challenging by the formation of RNP complexes with tertiary structures and the complicated interactions among different genome segments and proteins. Most of the assembly studies were conducted in vivo using biological approach (8).

Figure 1: Proposed model for influenza A virus packaging. (2)

The assembly of Influenza A virus is an essential step in the virus life cycle. The study of Influenza A and its packaging mechanism is important for the further understanding of the viral life cycle, for prevention of further Influenza A virus pandemics, as well as the development of potential pharmaceuticals. Moreover, the study of Influenza A virus will provide insight into the assembly of other viruses with segmented genome such as measles virus (MeV) (4).

Specific aim 1: Investigate the interaction between segment 3 and 5 that might take place during the influenza A virus assembly.

Significance

The assembly mechanism of Influenza A virus is proposed that all eight virus RNA segments interact with neighboring segments to form a genomic complex before they are selectively packaged into the newborn virions. However, the details about the interactions between each of the eight segments are not clear. (2) A recent synonymous mutation study of segment 5 packaging signal reveals an interaction between segment 3 and segment 5 during virus assembly (9).

In order to investigate the potential interaction between intact segment 3 and segment 5, I aim to label segment 3 and segment 5 with fluorescent dyes and test the in vitro interaction between them with fluorescent dye-labeled binding assay. The result will not only improve our understanding of the specific interaction between these two segments in vivo, but also provide insight into the complicated interactions within other pairs of segments during virus assembly.

Approach

Human Embryonic Kidney 293 (HEK293) cells will be cultured in Dulbecco's modified Eagles' medium supplemented with fetal bovine serum to nearly 90% confluency as previously described (6). An eight plasmid reverse genetics system will be used to generate influenza A virus A/WSN/33 (H1N1). Segment 3 and segment 5 in their RNP complexes will be generated from the HEK293 cells as established previously (10). Naturally occurred cysteine residues in RNA polymerases and NP will be mutated by site-directed mutagenesis because Cy3 and Cy5 label free cysteine residues in proteins and this will interfere with the RNA labeling in the RNP in the following experiment. After the sonication of HEK293 by Bioruptor (Cosmo Bio), these two segments will then be separated from other viral components and cell debris using sucrose gradient velocity sedimentation. Velocity sedimentation is an analytical method that separates different macromolecules and provides information about their masses and shapes.

Segment 3 and 5 will then be labeled with Cy3 and Cy5 at the 5' end of RNA respectively (2). NP protein and polymerases have been shown not to affect the exposure of the RNA in RNP complex (2). These two labeled segments will be studied by förster resonance energy transfer (FRET) experiment to detect the proximity in vitro (12). FRET is useful in detecting molecular dynamics in biochemistry, such as protein-protein interactions and protein structural changes. It can also be used to study the dynamics of RNA -RNA interactions (13).

During FRET experiment, upon excitation at a specific wavelength, the Cy3 donor on segment 3 will transfer energy to Cy5 acceptor on segment 5 when the two dyes are brought in close proximity during the interaction of the two segments. If Cy3 and Cy5 are far away from each other, no energy transfer will take place. By measuring the energy transfer during the mix of labeled segments, we can infer the proximity of the two segments and thus interaction between them. RNP complex with random RNA is expected not to interact with segment 3 or segment 5. Thus, Cy5 or Cy3 labeled reconstituted RNP complex with random RNA will be generated (14) and used to test its interaction with segment 3 and 5 as a negative control in this experiment.

It is possible that segment 3 and 5 in their ribonucleoprotein complexes would not give appealing result from FRET experiment due to potential protein-protein interaction or protein-RNA interaction. In that case, segment 3 and 5 RNA will be purified from RNP complexes using RNA/Protein Purification Kit (QIAGEN) or the SV Total RNA isolation system (Promega) following purification from HEK293 cells. These segment3 and 5 RNA free from NP or polymerases will then be labeled and studied for their in vitro interaction using FRET experiment.

It is also possible that mutated RNA polymerases and NP will not maintain their in vivo properties in virus assembly. Circular dichroism spectroscopy will be employed to study the structures and stabilities. FRET experiment with only the RNAs of segment 3 and 5 would be conducted without proteins to avoid possible effect of mutant proteins if non-natural structures are indicated.

Although not likely, mutated RNA polymerases binding RNA at the termini and mutated NP may affect fluorescent labeling of RNA. If that is the case, labeled reconstituted RNP will be generated by transfection of pCDNA-PB2, pCDNA-PB and pCDNA-PA, and an NP-expressing reverse genetics plasmid (9).

All personals involved in this study will be properly trained to handle mammalian cells, RNA and virus particles. Courses on virus biology will benefit the study.

Specific aim 2: Identify nucleotides of segment 3 that are critical for the specific interaction.

Significance

Influenza A virus genome segment contains a coding region and a noncoding region at each end of the coding region (15). These noncoding regions of all eight vRNAs share conserved and partial complementarily sequences and are thought to harbor the packaging signals. Sequences specific for each segment are also located in the noncodoing regions. Recently, cis-acting elements that are important for the virus assembly are found in the coding region of influenza A virus (16).

The packaging signals of influenza A virus were identified by various studies (reference 2 for review, Figure 2 for segment 3). Synonymous mutational analysis is widely used in vivo biological study to investigate the assembly of influenza A virus. However, the exact functions and roles of these packaging signals are not fully elucidated. A recent finding shows that mutations of segment 5 packaging signal affect the incorporation of segment 3 in Madin-Darby canine kidney (MDCK) cells (9). We aim to identify nucleotides in the packaging signal of segment 3 that are critical for interaction between segment 5 and 3. The study will reveal the role of packaging signal in the interaction between segments and also highlight their significance in the formation of genomic complex to be packed into new virion.

Figure 2: Packaging signals in the influenza A virus genome segment 3 identifiedby different approaches (2). (a) ORF is shown in black with UTR in white. (b) Boundary of internal deletions of DI RNAs. (c) The shortest sequences to package reporter genes. (d) Codon variation shows conserved regions. (e) Mutated regions that reduces segment packaging.

Approach

In order to indentify critical nucleotides of segment 3 for the interaction between segment 3 and 5, I propose to undertake synonymous point mutations of the packaging signal at the terminal regions first. HEK 293 cells and MDCK cells will be prepared as previously described (9). Recombinant influenza A virus A/WSN/33 (H1N1) with point mutation in segment 3 packaging signal will be produced from HEK293 cells by site-directed mutagenesis using reverse genetic systems. The desired mutation in the sequence will be confirmed by sequencing PCR using terminal and internal primers, and native PFU polymerase (9). The transfected HEK 293 cells will be sonicated to harvest the influenza A virus with mutant segment 3. The mutant virus will then be used to infect MDCK cells at 37 oC for 48h. The plaque assay of MDCK cells will be done to provide growth fitness and characteristics of virus containing mutant segmen3. The intensities of different segments packaged in the new born virion will be analyzed by quantitative densitometry and normalized to wild type virus. Denaturing urea-PAGE and silver staining will be used to separate and visualize different RNA segments. Wide-type influenza A virus A/WSN/33 (H1N1) will be used as a positive control for above experiments.

The macromolecular synthesis of mutant virus will also be evaluated for the effect of point mutations. The infected MDCK cells will be lysated, and all components will be subjected to SDS-PAGE and stained by coomassie brilliant blue. Western blot study will also be employed to analyze protein components by using antibodies that recognize acidic polymerase protein (PA) encoded by segment 3.

One difficulty in study packaging signals is that they extend into the coding regions which might cripple the function of encoded protein. The reconstitution of RNP will be conducted as described in the method of specific aim 1 to evaluate the effect of mutations. The HEK293 cells will also be transfected with a reporter luciferase using Lipofectin (Invitrogen) for the RNP complex in vivo reconstitution. We expect the mutations will not affect the structure and function of RNP.

After the identification of essential nucleotides in segment 3 packing signal, the corresponding segment 3 mutants will be purified as described in the method in specific aim 1. Their interactions with intact segment 5 will be studied by FRET to test the effect of point mutations in packaging signal of segment 3 on the interaction between segment 3 and 5.

If biosynthesized segments 3 and 5 do not work for this study, the packing signal of segment 3 with mutation will be chemically synthesized and studied for the in vitro interaction with intact segment 5 with FRET.

It is possible that other regions of segment 3 are also involved in the interaction (Figure 2). Truncated segments 3 will be employed for FRET study with intact segments 5.

The identification of important nucleotides and regions in the packaging signal will allow us to evaluate their importance during the segment interaction, as well as the properties of the interaction. Also, the characterization of the packaging signal of segment 3 may also reveal interactions between segment 3 and other segments, thereby provides a direction for further study.

Specific aim 3: Segment 3 reconstruction using biochemical methods and its structural study

Significance

The segmented vRNAs of influenza A virus have to form ribonucleoprotein (RNP) complex with nucleoprotein (NP) and three RNA polymerases (PB1, PB2, PA) in intact virus to be functional in transcription and replication (14). The NP, which is encoded by segment 5, is a 55 kDa protein that binds RNA non-specifically (1, 17). PB1, which is a 83kDa protein encoded by segment 2, has RNA polymerase activity (1, 14, 18). The 83 kDa PB2 is encoded by segment 1 and has a cap-binding property which is important to initiate transcription (1, 14, 19). PA is a 78 kDa phosphoprotein encoded by segment 3 and has protease activity (1, 16, 20).

NP proteins interact with vRNA to form a super-coiled structure while PB1, PB2 and PA from a RNA polymerase complex and interact with the super-coiled structure at the one end to maintain the super-coiled structure (2). Previous study shows that NP can form coiled structure which is similar to RNP complex even without RNA (21). A deeper understanding of the formation of the RNP and its crystal structure in solution will reveal important residues involved in the segment formation and probably segment interaction. Moreover, the RNP complex and its components are important targets for developing vaccines and pharmaceuticals. Thus, the further characterization of RNP will also contribute to the prevention and preparation of potential influenza pandemics.

Approach

In order to further characterize the assembly of segment 3, I propose to express NP, PB1, PB2 and PA in E.coli separately along with a protease inhibitor. The NP protein might fail to be expressed solely because it has recently been found to have protease activity so it (1). In that case, NP will be coexpressed with RNA polymerases to prevent self-degradation (1) for the RNP self-assembly test.

The purification process will include 10% polyethyleneimine precipitation, ammonium sulfate precipitation and column chromatographies such as ion-exchange chromatography and gel filtration. Fractions contained interested proteins will be examined by SDS-PAGE and ESI-MS analysis. Segment 3 RNA will be fluorescein labeled at 3' end and synthesized in multiple steps with overlapping synthesized fragments. Then the proteins and RNA will be incubated at 37 oC for 36 h and tested by analytical ultracentrifugation for self-assembly. The in vitro self-assembled RNP will then be electroporated in to influenza A virus infected HEK297 cells (prepared as described in methods of specific aims 1), detected and evaluated its assembly into new intions by electron microscopy.

Crystallographic structural study will be employed to further evaluate the RNP reconstruction. This will be done by hanging-drop vapor diffusion method. Crystals will be screened and optimized based on the initial conditions and known crystal structures of NP and polymerases to obtain the crystal structure of RNP complex. Similarly, segment 3 RNP and the packaging signal of segment 5 will be co-crystallized to study important residues for the interaction of the two segments.

It is possible that the RNP needs components from influenza A virus or host cells for assembly. The crystallization study will be conducted using segment 3 RNP generated in vivo to ensure the formation of RNP (9).

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