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Viruses with anti-sense RNA genomes, such as the influenza virus carry three core polypeptides inside their viral capsids in order to successfully enter a host and initiate the [https://en.wikipedia.org/wiki/Influenza#Replication viral replication cycle]. Especially important for viral replication and coordination with the host cell's replication machinery is a protein able to bind single-strand RNA scripts (ssRNA), forming ribonucleoprotein complexes (RNPs). This protein is commonly referred to as nucleoprotein (NP). The NP binds and transports viral RNA scripts to and from the host cell nucleus for transcription, replication, and packaging into new virions. When NP binds RNA it is structure specific but not sequence specific, meaning NP will bind only ssRNA but will bind any ss-RNA script, viral or non-viral. Beyond the transport function, NP is an essential mediator between host and virus and coordinates complex processes during viral replication. Due to its important function, NP is intensively studied as a potential drug target for antiviral pharmaceuticals. <ref name ="[1]">[[http://jgv.microbiologyresearch.org/content/journal/jgv/10.1099/0022-1317-83-4-723#tab2]] A. Portela, P. Digard, The influenza virus nucleoprotein: a multifunctional RNA-binding protein pivotal to virus replication, Journal of General Virology. 83 (2002) 723–734. doi:10.1099/0022-1317-83-4-723.</ref> ref name="[2]">[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2957899/]] K. Das, J.M. Aramini, L.-C. Ma, R.M. Krug, E. Arnold, Structures of influenza A proteins and insights into antiviral drug targets, Nat Struct Mol Biol. 17 (2010) 530–538. doi:10.1038/nsmb.1779.</ref>

The globular NP protein is rich in arginine, serine, and glycine residues. The abundance of arginine residues gives the protein a net positive charge at pH 7. With a predicted pI of 9.3, NP is mainly composed of basic residues, except for a tail domain formed by the 30 C-terminal residues, which are acidic. The C-terminal domain has a pI of 3.7.<ref name = "[1]"/> The NP is a trimer of homomers with 498 amino acid residues encoded by the Influenza A RNA segment 5. Each homomer provides a binding site for the viral RNA script.<ref name = "[1]"/>

The homomers fold into a crescent shape with a head and a body domain, where the groove between the two domains hosts the ssRNA binding site on the outer surface of the homomer.<ref name = "[1]"/><ref name = "[2]"/> For the formation of functional RNPs, the NP needs to form a coordinated aggregate of three NPs through a process called homo-oligomerization.<ref name = "[2]"/>[3] The C-tail loop, marked in pink in the image below, is critical for this function. Residues 408-419, located at the back of the NP between head and tail domain, form a loop that is the basis of the interface between neighboring NPs. The C-tail loop interlocks with the loop binding cavity in the neighboring NP and forms a tight binding interaction, featuring both hydrophobic and hydrophilic residues, that keeps the trimer together. The NP oligomer conformation is especially stabilized by a salt bridge between R416 in the loop and E339 in the adjacent NP.<ref name = "[2]"/>

[[Image:Structural Features of NP.jpg]]<ref name = "[2]"/>

NP binds ssRNA in the RNA binding groove with an affinity of ~20 nM. The binding of ssRNA is not sequence specific and the approximate stoichiometry is 1 NP monomer:24 ssRNA nucleotides. Although the portions of ssRNA that are not directly bound in the groove wrap around NP, the RNA is not protected from digestion by RNase.<ref name = "[1]"/>

The RNA binding region (RNA binding groove) has been mapped (via deletion mutagenesis) to cluster around residues located in the N-terminal third of the nucleoprotein. This large region is aided in RNA binding by surrounding regions that affect the binding affinity. Thus, the RNA-binding mechanism of NP is characterized by co-operativity and allosteric regulation.<ref name = "[1]"/>

The influenza virus polymerase complex is essential for directing virus replication inside the host cell's nucleus. The viral polymerase complex consists of three sub units, PA, PB1, and PB2.[7] The polymerase complex directs both viral RNA replication and viral genome transcription. Additionally, specifically for viral genome transcription, the complex can steal 5' RNA primers from host mRNAs for proper transcription of viral RNA. [10] NP has been found to have substantial regulatory interaction with PB1 and PB2. The NP interaction with PB2 is host specific and depends on the amino acid sequence found in the C-terminal tail loop region. Thus, the direct interaction between NP and PB2 may be the switch between viral RNA replication (no 5' primer cap addition) and transcription (viral mRNA with added 5' primer caps).[6]<ref name = "[2]"/>

[[Image:NP functional-subunits.jpg]] <ref name = "[1]"/>

Importin α is a mediator protein that interacts with hundreds of different proteins in eukaryotic cells to ensure transport across the nuclear envelope through the nuclear pores. During early stages of infection, the NP is largely concentrated in the nucleus, importing viral RNA scripts for transcription and replication. The entrance of NP into the nucleus is guaranteed by the NP interaction with Importin α.<ref name = "[1]"/>

After the viral RNA scripts have been replicated, NP shuttles out of the nucleus by associating with CRM1 (chromosomal maintenance 1), also known as exportin.[12] In this later stage of infection, the concentration of NP in the cytoplasm outweighs the concentration of NP inside the nucleus. It is unknown if this is the case because the nuclear localization signals (NLS) are turned off or because the NLS are overwhelmed by the cytoplasmic accumulation signal (CAS), which describes the interaction between NP and the host cell cytoskeleton.<ref name = "[1]"/>

During packaging and assembly of new viral particles, the M1 protein (matrix protein 1 of influenza A) mediates the encapsidation of RNPs into the viral envelope. This interaction is critical for successful assembly of functional virus progeny and is characterized by direct protein-protein interaction between M1 and NP, which is stabilized by M1 simultaneously binding to the NP-bound RNA.<ref name = "[1]"/>

The loop binding cavity may constitute a viable drug target as disruption of the homo-oligomerization process prevents the formation of functional RNPs.<ref name = "[2]"/>[4] Special focus lies on the disruption of the salt bridge between R416 and E339.[4]