Functional Analysis of the Influenza Virus H5N1 Nucleoprotein Tail Loop Reveals Amino Acids That Are Crucial for Oligomerization and Ribonucleoprotein Activities

Abstract

Homo-oligomerization of the nucleoprotein (NP) of influenza A virus is crucial for providing a major structural framework for the assembly of viral ribonucleoprotein (RNP) particles. The nucleoprotein is also essential for transcription and replication during the virus life cycle. In the H5N1 NP structure, the tail loop region is important for NP to form oligomers. Here, by an RNP reconstitution assay, we identified eight NP mutants that had different degrees of defects in forming functional RNPs, with the RNP activities of four mutants being totally abolished (E339A, V408S P410S, R416A, and L418S P419S mutants) and the RNP activities of the other four mutants being more than 50% decreased (R267A, I406S, R422A, and E449A mutants). Further characterization by static light scattering showed that the totally defective protein variants existed as monomers in vitro, deviating from the trimeric/oligomeric form of wild-type NP. The I406S, R422A, and E449A variants existed as a mixture of unstable oligomers, thus resulting in a reduction of RNP activity. Although the R267A variant existed as a monomer in vitro, it resumed an oligomeric form upon the addition of RNA and retained a certain degree of RNP activity. Our data suggest that there are three factors that govern the NP oligomerization event: (i) interaction between the tail loop and the insertion groove, (ii) maintenance of the tail loop conformation, and (iii) stabilization of the NP homo-oligomer. The work presented here provides information for the design of NP inhibitors for combating influenza virus infection.

Characterization of Influenza H5N1 Nucleocapsid Protein for Potential Vaccine Design

“…These authors propose that homo-oligomerization is facilitated through the insertion of the tail loop domain of one NP protomer into the body domain of a neighboring NP protomer at the "insertion groove". This hypothesis is supported by their more recent work [20] demonstrating that mutations which disrupt the tail loop structure or abrogate the ability to form a salt bridge between R416 in the tail loop of one protomer and E339 in the insertion groove of another protomer were both deficient in ribonucleoprotein (RNP) activity (transcription-replication activity) and had lost the ability to oligomerize. Size exclusion chromatography experiments also indicate that, in the presence of RNA, the amount of wild type NP in the trimeric/tetrameric forms is greatly diminished, and largely replaced by higher oligomeric forms of NP complexed with RNA [20].…”

“…Further experiments using influenza-specific RNA as a titrant may resolve this issue. The conclusion from the RNase A and RNase-free experiments that the "anomalous" spectrum results from the presence of RNA is bolstered by literature indicating that recombinant NP from other negative stranded RNA viruses (e.g., Ebola, Vesicular Stomatitis Virus, Rabies) also have the propensity to bind RNA of their expression system [20][21][22][23]. The data we present here indicate that association of NP and RNA may well result in observable changes in influenza NP structure and such association must be tightly monitored by both vaccine manufacturers and regulators of NPbased vaccines for human use.…”

“…Unfortunately, attempts to reproduce the "anomalous" form of NP by adding total yeast RNA to RNA-free NP were unsuccessful. While it has been suggested that influenza NP binds RNA in a non-sequence-specific fashion [19], demonstrations of RNA binding to influenza NP by this group [20] and others [9] have been accomplished only when using a virus-specific oligonucleotide sequence. Further experiments using influenza-specific RNA as a titrant may resolve this issue.…”

Stabilization of Influenza Virus Replication Intermediates Is Dependent on the RNA-Binding but Not the Homo-Oligomerization Activity of the Viral Nucleoprotein

“…Homo-oligomerization of NP, in which the tail loop of one NP is inserted into a groove of the neighboring NP molecule, is an important factor in maintaining RNP structure. The amino acid residues involved in these interactions have been identified and well characterized (6,8,23), including in our laboratory (5,14). The expected inhibitory effects of replacing amino acid residues involved in the intra-and interchain interactions of the tail loop (E339A, V408S/P410S, R416A, and L418S/P419S) on viral replication and transcription (5) were confirmed by vRNP (Fig.…”

“…3D) is presumably due to the inability of the mutant NP to fully support replicative synthesis of vRNA and cRNA. As oligomerization NP mutants have previously been found to possess reduced RNA binding affinity compared to that of the wild type in vitro (5,9), probably due to their reduced oligomeric state, we analyzed the association of oligomerization NP mutants with cRNA. Consistent with the model that cRNA stabilization requires NP to bind RNA, the cRNA detected in the presence of inactive RdRp (Fig.…”