All viruses with negative-sense RNA genomes encode a single-strand RNA-binding nucleoprotein (NP). The primary function of NP is to encapsidate the virus genome for the purposes of RNA transcription, replication and packaging. The purpose of this review is to illustrate using the influenza virus NP as a well-studied example that the molecule is much more than a structural RNAbinding protein, but also functions as a key adapter molecule between virus and host cell processes. It does so through the ability to interact with a wide variety of viral and cellular macromolecules, including RNA, itself, two subunits of the viral RNA-dependent RNA polymerase and the viral matrix protein. NP also interacts with cellular polypeptides, including actin, components of the nuclear import and export apparatus and a nuclear RNA helicase. The evidence for the existence of each of these activities and their possible roles in transcription, replication and intracellular trafficking of the virus genome is considered.

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To get insights into the role played by each of the influenza A virus polypeptides in morphogenesis and virus particle assembly, the generation of virus-like particles (VLPs) has been examined in COS-1 cell cultures expressing, from recombinant plasmids, different combinations of the viral structural proteins. The presence of VLPs was examined biochemically, following centrifugation of the supernatants collected from transfected cells through sucrose cushions and immunoblotting, and by electron-microscopic analysis. It is demonstrated that the matrix (M1) protein is the only viral component which is essential for VLP formation and that the viral ribonucleoproteins are not required for virus particle formation. It is also shown that the M1 protein, when expressed alone, assembles into virus-like budding particles, which are released in the culture medium, and that the recombinant M1 protein accumulates intracellularly, forming tubular structures. All these results are discussed with regard to the roles played by the virus polypeptides during virus assembly.The final step in the lytic cycle of enveloped viruses involves the budding of the newly formed particles from cellular membranes. Previous to this step, all viral structural components should have been transported, either individually or as preassembled complexes, to the cellular membrane, where viral proteins will drive the budding process.A number of studies have focused on the assembly and budding processes of viruses (arena-, alpha-, rhabdo-, paramyxo-, orthomyxo-, and retroviruses) that obtain their envelope from the plasma membrane (reviewed in references 2, 13, and 19). For the alphavirus Semliki Forest virus, it has been established that virus budding is strictly dependent on interactions between the transmembrane spike protein and the internal nucleocapsid (46). In retroviruses, however, interactions between the cytoplasmic tail of external virus proteins (Env) and the internal virus components (Gag polyprotein) are not a prerequisite for virus budding since expression of the Gag protein alone is sufficient to drive budding of virus-like particles (VLPs) (7,14). A different mechanism, which directs the assembly and release of coronavirus particles, which assemble at intracellular membranes, has been described (47). In this case, expression of viral membrane proteins alone is sufficient to drive the assembly and budding of VLPs (47).It is widely accepted that the matrix protein plays a pivotal role as an assembly organizer for RNA viruses containing a single negative-strand genomic RNA molecule (such as rhabdo-and paramyxoviruses) (reviewed in reference 25). In fact, rabies and measles viruses modified by reverse genetics technology to lack the matrix gene grow poorly, and the released matrix-less particles show drastically altered morphologies (3, 31). Moreover, it has been shown that the M1 proteins of vesicular stomatits virus (VSV) and human parainfluenza virus type 1 have intrinsic budding activity when expressed alone (5, 22, 26), an observation ...

Influenza A virus mutants expressing C-terminally deleted forms of the NS1 protein (NS1-81 and NS1-110) were generated by plasmid rescue. These viruses were temperature sensitive and showed a small plaque size at the permissive temperature. The accumulation of virion RNA in mutant virus-infected cells was reduced at the restrictive temperature, while the accumulation of cRNA or mRNA was not affected, indicating that the NS1 protein is involved in the control of transcription versus replication processes in the infection. The synthesis and accumulation of late virus proteins were reduced in NS1-81 mutant-infected cells at the permissive temperature and were essentially abolished for both viruses at the restrictive temperature, while synthesis and accumulation of nucleoprotein (NP) were unaffected. Probably as a consequence, the nucleocytoplasmic export of virus NP was strongly inhibited at the restrictive temperature. These results indicate that the NS1 protein is essential for nuclear and cytoplasmic steps during the virus cycle.The genome of influenza A virus consists of eight singlestranded RNA molecules of negative polarity associated with nucleoprotein (NP) molecules and the polymerase in the form of ribonucleoprotein (RNP) complexes (for reviews, see references 40, 43, and 66). The first step in viral gene expression is primary transcription from the incoming viral RNPs (28). The expression of virus proteins, at least NP, leads to the shift from transcription to the synthesis of complete positive-polarity RNAs (cRNAs) (29, 73), which serve as templates for the synthesis of virion RNAs (vRNAs). Transcription and replication of vRNA take place in the nucleus of the infected cell (30,34) and require at least the activity of the three subunits of the polymerase (PB1, PB2, and PA) and the NP (9,31,38,55,64). The syntheses of the various vRNAs are not simultaneous during the infection cycle. Thus, NS1 or NP vRNAs are replicated earlier than M1 or hemagglutinin (HA) vRNAs (72). Since transcription is coupled to replication at the beginning of vRNA synthesis, the NS1 protein and NP are expressed earlier than the M1 protein and HA (72). However, later in the process of vRNA synthesis, transcription is discontinued and viral protein synthesis rests on previously synthesized mRNAs (72). In the course of the infection, viral gene expression takes over the cell machinery, leading to the shutoff phenomenon. Several alterations induced by the virus in the infected cell may be connected to shutoff: nuclear retention and degradation of polymerase II transcripts in the nucleus (35), inhibition of cellular pre-mRNA cleavage and polyadenylation (56, 74), cytoplasmic degradation of preexisting cellular mRNAs (3, 32), and preferential utilization of the translation machinery by the virus-specific mRNAs (36).Influenza A virus encodes a nonstructural protein (NS1) that is translated from the unspliced transcript of segment 8 (33, 44). NS1 is a nuclear protein, both in the infected cell (5, 41) and when expressed from cDNA (23,45,6...