Virulence Determinants of Avian H5N1 Influenza A Virus in Mammalian and Avian Hosts: Role of the C-Terminal ESEV Motif in the Viral NS1 Protein

Abstract

We assessed the prediction that access of the viral NS1 protein to cellular PDZ domain protein networks enhances the virulence of highly pathogenic avian influenza A viruses. The NS1 proteins of most avian influenza viruses bear the C-terminal ligand sequence Glu-Ser-Glu-Val (ESEV) for PDZ domains present in multiple host proteins, whereas no such motif is found in the NS1 homologues of seasonal human virus strains. Previous analysis showed that a C-terminal ESEV motif increases viral virulence when introduced into the NS1 protein of mouse-adapted H1N1 influenza virus. To examine the role of the PDZ domain ligand motif in avian influenza virus virulence, we generated three recombinants, derived from the prototypic H5N1 influenza A/Vietnam/1203/04 virus, expressing NS1 proteins that either have the C-terminal ESEV motif or the human influenza virus RSKV consensus or bear a natural truncation of this motif, respectively. Cell biological analyses showed strong control of NS1 nuclear migration in infected mammalian and avian cells, with only minor differences between the three variants. The ESEV sequence attenuated viral replication on cultured human, murine, and duck cells but not on chicken fibroblasts. However, all three viruses caused highly lethal infections in mice and chickens, with little difference in viral titers in organs, mean lethal dose, or intravenous pathogenicity index. These findings demonstrate that a PDZ domain ligand sequence in NS1 contributes little to the virulence of H5N1 viruses in these hosts, and they indicate that this motif modulates viral replication in a strain-and host-dependent manner.

Prevalence of the C-terminal truncations of NS1 in avian influenza A viruses and effect on virulence and replication of a highly pathogenic H7N1 virus in chickens

“…25 Likewise, truncation of NS1 from an Italian LPAIV H7N1 did not increase virulence of the virus in chickens 20 and extension of the C-terminal end of NS1 of an HPAIV H5N1 did not decrease virulence in chickens. 13 In our study, compared to the Hp virus, the NS1 mutants exhibited impaired virus excretion from infected birds at 4dpi particularly via the cloacal route. A reduced amount of virus excretion due to NS mutations may reduce bird-to-bird transmission 26 which, however, was not studied in our experiment.…”

“…In addition to the wellknown role as antiviral antagonist, it can affect virus replication, host-range, virulence and tropism mainly shown in mammal models or in-vitro, whereas only few studies have been performed in poultry. 3,12,13,21 Although truncation of the CTE of NS1 has been previously reported using a limited number of sequences, 9,14,22 little is known about the prevalence and distribution of NS1 DCTE in avian influenza viruses of all subtypes. Moreover, the impact of NS1 truncation on virulence particularly the HPAIV H7 viruses in poultry has not been adequately studied.…”

“…20 Another study showed that extension of NS1 of an HPAIV H5N1 did not attenuate viral replication on chicken fibroblasts. 13 Accordingly, the effect of NS1 DCTE on virus replication in-vitro is most likely to be cell-and strain-dependent.…”

“…10 In addition to its well-defined role as an antiviral antagonist, variations in the Postsynaptic density protein 95, Drosophila disc large tumor suppressor, and Zonula occludens 1 protein domain (PDZ) at the carboxyl terminal end (CTE) of NS1 resulted in expansion of the host range of some influenza viruses in different cell cultures and modulated virulence in mice and poultry. [11][12][13] Size variation of NS1 has been described, mainly due to a stop codon in the ED which results in truncation of the C-terminal end (DCTE). 9, 14 Nonetheless, prevalence and distribution of NS1 DCTE among AIV subtypes and their biological impact in poultry have not been well studied.…”

Improvement of H5N1 influenza vaccine viruses: Influence of internal gene segments of avian and human origin on production and hemagglutinin content

“…The H5N1-clade 1 vaccine strain NIBRG-14 and the A/Vietnam/1203/2004 WT virus were obtained from the National Institute for Biological Standards and Control (NIBSC, Hertfordshire, UK). Recombinant A/Vietnam/1203/04 wild type viruses with multi-(VN1203-hp) or monobasic (VN1203-lp) cleavage site were generated by reverse genetic methods in our laboratory [27]. Site-directed mutagenesis was used to replace the multibasic cleavage within in the corresponding HA plasmid from "RERRRKKRG" to the monobasic sequence "RETRG".…”