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.

FIG. 1.

Crystal structure and tail loop interaction of NP. (A) NP consists of head and body domains and a long tail loop, which inserts into the neighboring NP to form homo-oligomers. (B) Intrachain interaction of the tail loop. Three regions of molecular contacts were identified from the NP crystal structure. T1 (dotted lines) involves hydrophilic interactions, while T2 and T3 (double solid lines) involve hydrophobic interactions. (C) Interchain interaction of the tail loop. Most of the interactions are hydrophilic in nature (I1, I2, I3, and I5), except for I4, which is hydrophobic. The thicknesses of the dotted lines indicate the strength of the hydrogen bond between the two residues. Amino acid residues of the tail loop are indicated by circles, while amino acid residues of the insertion groove are indicated by rectangles.

FIG. 2.

Identification of important amino acid residues involved in the tail loop insertion by an RNP reconstitution assay. (A) Western blotting of cell lysates expressing NP mutants. The amount of NP-expressing plasmids was adjusted to give similar expression levels. Beta-actin was used as an internal control. (B) In vivo RNP reconstitution followed by primer extension assay of NP mutants. Different pcDNA-NP plasmids were mixed with pcDNA-PB1, pcDNA-PB2, pcDNA-PA, and pPOLI-NA-RT and transfected into 293T cells. pcDNA3 instead of pcDNA-NP was used for the negative control. RNA was isolated from cells after 48 h of incubation and analyzed by a primer extension assay as described in Materials and Methods. Positions of vRNA, mRNA, cRNA, and 5S rRNA are indicated on the right. A representative result of three independent experiments is shown. (C) Quantitation of mRNA, cRNA, and vRNA. RNA levels for each NP mutant were compared to those of wild-type NP, which was set to 100%. 5S rRNA was used as an internal control to standardize RNA levels. The results represent the mean percentages ± standard deviations from three independent experiments (*, P < 0.02; **, P < 0.0005).

FIG. 3.

NP mutants defective in RNP activity possess the ability to interact with RNA and polymerase proteins. (A) Gel shift assay of the RNA binding activities of the defective NP variants. Purified NP was incubated with a 24-nt RNA, and the complexes were analyzed by agarose gel electrophoresis. Bovine serum albumin (BSA) instead of NP was used as a negative control. (B) Coimmunoprecipitation of NP using a Myc-tagged polymerase complex. Wild-type NP was used as a positive control. The Myc-tagged PA plasmid was left out in the negative control. Loading controls are shown at the top. For each variant, “+” refers to the presence of anti-Myc antibodies, while “−” refers to their absence.

FIG. 4.

Static light scattering analysis of purified NP variants. Wild-type or mutant NP proteins were subjected to static light scattering analysis using a miniDAWN triangle light scattering detector. The light scattering signal and refractive index were measured during the experiment. Curves are the light scattering signals. Horizontal lines are the calculated native molecular weights (MW) (in thousands) of proteins, which are proportional to the ratio of the light scattering signal to the refractive index. (A) NP variants totally defective in RNP activity are monomeric, while wild-type NP exists as a mixture of trimers and tetramers. (B) Partially defective NP variants (I406S, R422A, and E449A) gave a skewed and broad peak and higher native molecular weights than wild-type NP, indicating that they exist as a mixture of different types of oligomers. (C) R267A exists as monomer in the absence of RNA. The addition of an 1,850-nt RNA to both the wild type and the R267A variant led to a high-molecular-weight peak on the left, suggesting the formation of NP-RNA complexes. Errors of native molecular weight were calculated from the percent error of the experiments.

FIG. 5.

Inability of NP mutants defective in RNP activity to form homo-oligomers in vivo. (A) Plasmids expressing untagged NP and C-terminally Myc-tagged NP of the respective mutants were transfected into 293T cells. Cell lysates were analyzed by Western blotting (WB) using polyclonal anti-NP antibodies after 48 h of incubation. The negative control contained untagged wild-type NP alone. (B) Coimmunoprecipitation of untagged NP variants with the corresponding Myc-tagged NP in the presence or absence of RNA. RNase A was used to remove RNA during coimmunoprecipitation. For each variant, “+” refers to the presence of anti-Myc antibodies, while “−” refers to its absence.