Effect on Virulence and Pathogenicity of H5N1 Influenza A Virus through Truncations of NS1 eIF4GI Binding Domain

Abstract

To study the effect of NS1 eIF4GI binding domain on virulence and pathogenicity of H5N1 influenza A virus, 5 recombinant H5N1 viruses encoding eIF4GI binding domain-truncated NS1 proteins and parental NS1 (NS1‐wt) were generated by an 8‐plasmid-based reverse genetics system. The results indicated that the recombinants with the addition of 5‐amino acid and the deletion position of 85-89 in NS1‐wt were attenuated in replication in vitro and in vivo, compared with the recombinant wild‐type virus rNS1‐wt, whereas the deletion position 85-94 or the entire eIF4GI binding domain in NS1‐wt displayed a significantly attenuated phenotype in chicken and mice. We also showed that the eIF4GI binding domain-truncated mutants were impaired in their ability to inhibit interferon production in vitro, and they did not replicate as efficiently as the parental recombinant strain in embryonated hen eggs, in Madin ‐Darby Canine Kidney cells, or in vivo in chickens and in a mouse model. Therefore, these attenuated NS1‐truncated viruses may have a great potential as live attenuated vaccine candidates against H5N1 influenza A virus.

Effects of the C-Terminal Truncation in NS1 Protein of the 2009 Pandemic H1N1 Influenza Virus on Host Gene Expression

“…After incubated at 37°C for 4 days, a second agar overlay containing 1∶10,000 neutral red was added [17].…”

“…The pre-transcriptional inhibition occurs by preventing dsRNA- and virus-mediated activation of the transcription factors (IRF-3, NF-κB and c-Jun/ATF-2), which are essential for IFN-β production [6]–[8], while the post-transcriptional inhibition is involved in limiting cellular mRNA maturation and nucleo-cytoplasmic transport through interaction of NS1 protein with the 30 KD cleavage and polyadenylation specificity factor (CPSF30) or PABII [9]–[10]. In addition, NS1 can block the function of two antiviral proteins including 2′-5′-oligoadenylate synthetase (OAS) [11] and serine/threonine protein kinase R (PKR) [12], stimulate phosphoinositide 3-kinase signaling (PI3K) by binding to p85β and/or CrkL [13]–[16], and limit host antiviral gene translation by binding to eIF4GI [17].…”

Effect of human activated NRAS on replication of delNS1 H5N1 influenza virus in MDCK cells

“…Previous studies suggested that binding of dsRNA by the NS1 protein blocked the action of the interferon (IFN)-inducible dsRNA-dependent protein kinase (PKR) [11,12]. The eIF4GI binding domain-truncated influenza virus did not efficiently replicate and was impaired in its ability to inhibit IFN production in vitro [13]. …”

Avian influenza virus NS1

“…The ED within amino acids 74 to 230 has specific regions to interact with several host factors and proteins (Table 1) including the cleavage and polyadenylation specificity factor 30 (CPSF30), eukaryotic translation initiation factor 4GI (eIF4GI), PABPII, p58b-subunit of phosphatidylinositol 3-kinase (P13K). In some viruses a second NLS and nucleolus localization signal (NoLS) exist 27 - 30 …”

“…As a non-structural protein, NS1 is not present in virions but it is abundant in the nucleus of influenza virus-infected cells early during infection and also in the cytoplasm at later stages of the viral replication cycle 30 . In their publication, Munir et al 8 showed that both NS1 proteins they studied localized primarily in the nucleus 24 h after transfection of human A549 cells.…”