The non-structural (NS1) protein of influenza A viruses is a non-essential virulence factor that has multiple accessory functions during viral infection. In recent years, the major role ascribed to NS1 has been its inhibition of host immune responses, especially the limitation of both interferon (IFN) production and the antiviral effects of IFN-induced proteins, such as dsRNA-dependent protein kinase R (PKR) and 2'5'-oligoadenylate synthetase (OAS)/RNase L. However, it is clear that NS1 also acts directly to modulate other important aspects of the virus replication cycle, including viral RNA replication, viral protein synthesis, and general host-cell physiology. Here, we review the current literature on this remarkably multifunctional viral protein. In the first part of this article, we summarize the basic biochemistry of NS1, in particular its synthesis, structure, and intracellular localization. We then discuss the various roles NS1 has in regulating viral replication mechanisms, host innate/adaptive immune responses, and cellular signalling pathways. We focus on the NS1-RNA and NS1-protein interactions that are fundamental to these processes, and highlight apparent strain-specific ways in which different NS1 proteins may act. In this regard, the contributions of certain NS1 functions to the pathogenicity of human and animal influenza A viruses are also discussed. Finally, we outline practical applications that future studies on NS1 may lead to, including the rational design and manufacture of influenza vaccines, the development of novel antiviral drugs, and the use of oncolytic influenza A viruses as potential anti-cancer agents.

/pdf/the-multifunctional-ns1-protein-of-influenza-a-viruses-5dbqg8uwin.pdf

The multifunctional NS1 protein of influenza A viruses.

Influenza A Virus Lacking the NS1 Gene Replicates in Interferon-Deficient Systems

The hemagglutinin (HA) structure at 2.9 angstrom resolution, from a highly pathogenic Vietnamese H5N1 influenza virus, is more related to the 1918 and other human H1 HAs than to a 1997 duck H5 HA. Glycan microarray analysis of this Viet04 HA reveals an avian α2-3 sialic acid receptor binding preference. Introduction of mutations that can convert H1 serotype HAs to human α2-6 receptor specificity only enhanced or reduced affinity for avian-type receptors. However, mutations that can convert avian H2 and H3 HAs to human receptor specificity, when inserted onto the Viet04 H5 HA framework, permitted binding to a natural human α2-6 glycan, which suggests a path for this H5N1 virus to gain a foothold in the human population.

Structure and Receptor Specificity of the Hemagglutinin from an H5N1 Influenza Virus

Glycan Microarray Analysis of the Hemagglutinins from Modern and Pandemic Influenza Viruses Reveals Different Receptor Specificities

Influenza A and B viruses carry two surface glycoproteins, the haemagglutinin (HA) and the neuraminidase (NA). Both proteins have been found to recognise the same host cell molecule, sialic acid. HA binds to sialic acid-containing receptors on target cells to initiate virus infection, whereas NA cleaves sialic acids from cellular receptors and extracellular inhibitors to facilitate progeny virus release and to promote the spread of the infection to neighbouring cells. Numerous studies performed recently have revealed that an optimal interplay between these receptor-binding and receptor-destroying activities of the surface glycoproteins is required for efficient virus replication. An existing balance between the antagonistic HA and NA functions of individual viruses can be disturbed by various events, such as reassortment, virus transmission to a new host, or therapeutic inhibition of neuraminidase. The resulting decrease in the viral replicative fitness is usually overcome by restoration of the functional balance due to compensatory mutations in HA, NA or both proteins.

Functional balance between haemagglutinin and neuraminidase in influenza virus infections

The hemagglutinin (HA) of fowl plague virus A/FPV/Rostock/34 (H7N1) carries two N-linked oligosaccharides attached to Asn123 and Asn149 in close vicinity to the receptor-binding pocket. In previous studies in which HA mutants lacking either one (mutants G1 and G2) or both (mutant G1,2) glycosylation sites had been expressed from a simian virus 40 vector, we showed that these glycans regulate receptor binding affinity (M. Ohuchi, R. Ohuchi, A. Feldmann, and H. D. Klenk, J. Virol. 71:8377–8384, 1997). We have now investigated the effect of these mutations on virus growth using recombinant viruses generated by an RNA polymerase I-based reverse genetics system. Two reassortants of influenza virus strain A/WSN/33 were used as helper viruses to obtain two series of HA mutant viruses differing only in the neuraminidase (NA). Studies using N1 NA viruses revealed that loss of the oligosaccharide from Asn149 (mutant G2) or loss of both oligosaccharides (mutant G1,2) has a pronounced effect on virus growth in MDCK cells. Growth of virus lacking both oligosaccharides from infected cells was retarded, and virus yields in the medium were decreased about 20-fold. Likewise, there was a reduction in plaque size that was distinct with G1,2 and less pronounced with G2. These effects could be attributed to a highly impaired release of mutant progeny viruses from host cells. In contrast, with recombinant viruses containing N2 NA, these restrictions were much less apparent. N1 recombinants showed lower neuraminidase activity than N2 recombinants, indicating that N2 NA is able to partly overrule the high-affinity binding of mutant HA to the receptor. These results demonstrate that N-glycans flanking the receptor-binding site of the HA molecule are potent regulators of influenza virus growth, with the glycan at Asn149 being dominant and that at Asn123 being less effective. In addition, we show here that HA and NA activities need to be highly balanced in order to allow productive influenza virus infection.

Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics.

We used the yeast interaction trap system to identify a novel human 70-kDa protein, termed NS1-binding protein (NS1-BP), which interacts with the nonstructural NS1 protein of the influenza A virus. The genetic interaction was confirmed by the specific coprecipitation of the NS1 protein from solution by a glutathione S-transferase-NS1-BP fusion protein and glutathione-Sepharose. NS1-BP contains an N-terminal BTB/POZ domain and five kelch-like tandem repeat elements of approximately 50 amino acids. In noninfected cells, affinity-purified antibodies localized NS1-BP in nuclear regions enriched with the spliceosome assembly factor SC35, suggesting an association of NS1-BP with the cellular splicing apparatus. In influenza A virus-infected cells, NS1-BP relocalized throughout the nucleoplasm and appeared distinct from the SC35 domains, which suggests that NS1-BP function may be disturbed or altered. The addition of a truncated NS1-BP mutant protein to a HeLa cell nuclear extract efficiently inhibited pre-mRNA splicing but not spliceosome assembly. This result could be explained by a possible dominant-negative effect of the NS1-BP mutant protein and suggests a role of the wild-type NS1-BP in promoting pre-mRNA splicing. These data suggest that the inhibition of splicing by the NS1 protein may be mediated by binding to NS1-BP.

/pdf/ns1-binding-protein-ns1-bp-a-novel-human-protein-that-5faowwa3yh.pdf

NS1-Binding Protein (NS1-BP): a Novel Human Protein That Interacts with the Influenza A Virus Nonstructural NS1 Protein Is Relocalized in the Nuclei of Infected Cells

Importance of hemagglutinin glycosylation for the biological functions of influenza virus.

The influenza A virus M1 protein interacts with the cellular receptor of activated C kinase (RACK) 1 and can be phosphorylated by protein kinase C.

Borna disease virus (BDV) is unique among the non-segmented negative-strand RNA viruses of animals and man because it transcribes and replicates its genome in the nucleus of the infected cell. It has recently been discovered that BDV expresses a gene product of 87 amino acids, the p10 protein, from an open reading frame that overlaps with the gene encoding the viral p24 phosphoprotein. In addition, the p10 protein has been localized to intranuclear BDV-specific clusters containing viral antigens. Here, characterization of p10 interactions with the viral nucleoprotein p38/p39 and the p24 phosphoprotein is reported. Immunoaffinity chromatography demonstrated the presence of high-salt stable complexes of p10 containing the p24 and p38/p39 proteins in extracts of BDV-infected cells. Analyses in the yeast two-hybrid system and biochemical co-precipitation experiments suggested that the p10 protein binds directly to the p24 phosphoprotein and indirectly to the viral nucleoprotein. Mutational analysis demonstrated that a leucine-rich stretch of amino acids at positions 8–15 within the p10 protein is critical for interaction with p24. Furthermore, binding of p10 to the viral phosphoprotein was shown to be important for association with the BDV-specific intranuclear clusters that may represent the sites of virus replication and transcription in infected cells. These findings are discussed with respect to possible roles for the p10 protein in viral RNA synthesis or ribonucleoprotein transport.

Thorsten Wolff

Papers

Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics.

NS1-Binding Protein (NS1-BP): a Novel Human Protein That Interacts with the Influenza A Virus Nonstructural NS1 Protein Is Relocalized in the Nuclei of Infected Cells

Importance of hemagglutinin glycosylation for the biological functions of influenza virus.

The influenza A virus M1 protein interacts with the cellular receptor of activated C kinase (RACK) 1 and can be phosphorylated by protein kinase C.

A short leucine-rich sequence in the Borna disease virus p10 protein mediates association with the viral phospho- and nucleoproteins