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.

Influenza virus nucleoprotein (NP) is the major component of the viral ribonucleoprotein complex, which is crucial for the transcription and replication of the viral genome. We have determined the crystal structure of influenza B virus NP to a resolution of 3.2 Å. Influenza B NP contains a head, a body domain, and a tail loop. The electropositive groove between the head and body domains of influenza B NP is crucial for RNA binding. This groove also contains an extended flexible charged loop (amino acids [aa] 125 to 149), and two lysine clusters at the first half of this loop were shown to be crucial for binding RNA. Influenza B virus NP forms a crystallographic homotetramer by inserting the tail loop into the body domain of the neighboring NP molecule. A deeply buried salt bridge between R472 and E395 and a hydrophobic cluster at F468 are the major driving forces for the insertion. The analysis of the influenza B virus NP structure and function and comparisons with influenza A virus NP provide insights into the mechanisms of action and underpin efforts to design inhibitors for this class of proteins.

The ribonucleoprotein (RNP) complex is the essential transcription-replication machinery of the influenza virus. It is composed of the trimeric polymerase (PA, PB1 and PB2), nucleoprotein (NP) and RNA. Elucidating the molecular mechanisms of RNP assembly is central to our understanding of the control of viral transcription and replication and the dependence of these processes on the host cell. In this report, we show, by RNP reconstitution assays and co-immunoprecipitation, that the interaction between NP and polymerase is crucial for the function of the RNP. The functional association of NP and polymerase involves the C-terminal ‘627’ domain of PB2 and it requires NP arginine-150 and either lysine-627 or arginine-630 of PB2. Using surface plasmon resonance, we demonstrate that the interaction between NP and PB2 takes place without the involvement of RNA. At 33, 37 and 41°C in mammalian cells, more positive charges at aa. 627 and 630 of PB2 lead to stronger NP-polymerase interaction, which directly correlates with the higher RNP activity. In conclusion, our study provides new information on the NP-PB2 interaction and shows that the strength of NP-polymerase interaction and the resulting RNP activity are promoted by the positive charges at aa. 627 and 630 of PB2.

Influenza nucleoprotein (NP) is a major component of the ribonucleoprotein (vRNP) in influenza virus, which functions for the transcription and replication of viral genome. Compared to the nucleoprotein of influenza A (ANP), the N-terminal region of influenza B nucleoprotein (BNP) is much extended. By virus reconstitution, we found that the first 38 residues are essential for viral growth. We further illustrated the function of BNP by mini-genome reconstitution, fluorescence microscopy, electron microscopy, light scattering and gel shift. Results show that the N terminus is involved in the formation of both higher homo-oligomers of BNP and BNP-RNA complex.

Poster Sessions C410 chain forming the beta-helices are very different. Nevertheless, gpV, gp138 and PA0616 are water soluble and SDS-resistant proteins and show no obvious membrane affinity. They are unlikely to unfold upon interaction with the membrane during phage attachment.GpV, gp138 and PA0616 contain a conserved cluster of histidines at the tip of the beta-helical domain. These histidines bind a Fe atom in the octahedral configuration. In addition to Fe, gpV also contains Ca and Cl near the spike's apex.The peculiar topology and thermodynamic stability of gpV, gp138 and PA0616 suggests that these proteins are used as rigid and sharp needles to breach the outer membrane of the host cell using the energy of the contractile sheath. These spikes appear to create an opening in the host cell membrane into which the tail tube in inserted for subsequent DNA release into the host cell.[ The Influenza viruses are classified into three types: A, B and C. While influenza A virus is widely recognized as the most devastating one, influenza B virus also causes severe damages, in particular substantial mortality among patients younger than 18 years old. Influenza B virus is prevalent in Hong Kong. It accounts for 32% of the 554 laboratory-confirmed cases from February 27, 2011 to April 2, 2011. Among the influenza viral proteins, nucleoprotein (NP) is the major component of the ribonucleoprotein complex, which is crucial for the transcription and replication of the viral genome. We have recently determined the crystal structure of influenza B NP to a resolution of 3.2 Å. Two NP molecules, namely chains A and B, are found in an asymmetric unit. Homologous to the structure of influenza A NP [1,2], influenza B NP is composed of the head and body domains and a tail loop. Influenza B NP forms a tetramer in the crystal structure with two A chains and two B chains, in contrast to the trimer observed in influenza A NP. The homo-tetramer formation is the result of tail loop insertion from one NP molecule to its neighboring NP. Another major role of NP is to bind the genomic RNA of the virus. The putative RNA-binding regions are exposed in the influenza B NP tetramer. Residues involved in oligomerization and in RNA binding have been studied biochemically by static light scattering and surface plasmon resonance. The functional significance of these residues towards the ribonucleoprotein activities of the virus has also been investigated. The structure-function relationship of influenza B NP has enriched the current knowledge on influenza NP and provides valuable information for the design of anti-viral agents.This work was supported by a grant from the General Research Fund (Project no. 473810) from the Research Grants Council of Hong Kong SAR. Hepatitis A Virus (HAV) is a common cause of acute hepatitis worldwide, transmitted by fecal-oral route. HAV is classified within Picornaviridae family but it has some distinct biological characteristics like its slow viral replication and not inducing cellular lysis. Picornavirus genome en...