Amino acid changes in the influenza A virus PA protein that attenuate avian H5N1 viruses in mammals

Abstract

The influenza viral polymerase complex affects host tropism and pathogenicity. In particular, several amino acids in the PB2 polymerase subunit are essential for the efficient replication of avian influenza viruses in mammals. The PA polymerase subunit also contributes to host range and pathogenicity. Here, we report that the PA proteins of several highly pathogenic avian H5N1 viruses have attenuating properties in mammalian cells and that the attenuating phenotype is conferred by strain-specific amino acid changes. Specifically, lysine at position 185 of A/duck/Vietnam/TY165/2010 (TY165; H5N1) PA induced strongly attenuating effects in vitro and in vivo. More importantly, the introduction of the arginine residue commonly found at this position in PA significantly increased the viral polymerase activity of TY165 in mammalian cells and its virulence and pathogenicity in mice. These findings demonstrate that the PA protein plays an important role in influenza virulence and pathogenicity.

Importance: Highly pathogenic influenza viruses of the H5N1 subtype cause severe respiratory infections in humans, which have resulted in death in nearly two-thirds of the patients with laboratory-confirmed cases. We found that the viral PA polymerase subunit of several H5N1 viruses possesses amino acid changes that attenuate virus replication in mammalian cells (yet the H5N1 viruses possessing these mutations are highly pathogenic in mice). Specifically, we found that an arginine-to-lysine substitution at position 185 of an H5N1 virus PA protein significantly affected that virus's virulence and pathogenicity in mice. The PA protein thus plays a role in the pathogenicity of highly pathogenic H5N1 influenza viruses.

FIG 1

Attenuating properties of avian H5N1 PA proteins in human cells. 293T cells were transfected with PB2, PB1, PA, and NP protein expression plasmids, together with a minigenome encoding the luciferase gene [pPol(Hu)-VD5(50)M(50)-Luc] and an internal control plasmid encoding Renilla luciferase (pGL4.74[hRluc/TK]). Transfected cells were incubated at 37°C and 33°C for 48 h and then lysed. Luciferase activity was determined by using the dual-luciferase system kit. The relative polymerase activity was normalized to that of wild-type polymerase complexes. The data are presented as the averages of three independent experiments ± standard deviations. (A) Polymerase activities of TY165, LS1349, and QT517 PA proteins tested with VD5 or VD18 PB2, PB1, and NP proteins. (B) Polymerase activities of VD5 and VD18 PA proteins tested with TY165, LS1349, and QT517 PB2, PB1, and NP proteins.

FIG 2

Identification of attenuating amino acid changes in avian H5N1 PA proteins in human cells. Minireplicon experiments were carried out as described in the legend for Fig. 1. (A) Polymerase activities of TY165 PA proteins possessing a mutation shared with LS1349 (Table 1). (B) Polymerase activities of TY165, QT517, and LS1349 PA proteins possessing strain-specific mutations (Table 1). (C) Polymerase activities of VD18 and VN1203 PA proteins encoding the attenuating PA-R185K mutation.

FIG 3

Replicative ability of TY165 and TY165-PA-K185R viruses in human cells. Calu-3 cells were infected with viruses at a multiplicity of infection of 1 × 10⁻⁴ and incubated at 33°C and 37°C. Aliquots of the supernatants were collected at the indicated time points and titrated in MDCK cells by use of plaque assays. Values are presented as the averages of three wells ± standard deviations from one experiment.

FIG 4

Survival and body weight changes of mice infected with wild-type or mutant viruses. BALB/c mice (three/group) were inoculated intranasally with the indicated doses of TY165 and TY165-PA-K185R (A), VD18 and VD18-PA-R185K (B), or VN1203 and VN1203-PA-R185K (C) viruses. Infected animals were monitored daily for body weight changes and survival. The numbers of animals that survived infection are shown.

FIG 5

Replication of TY165 and TY165-PA-K185R viruses in mice. Nine mice per group were intranasally inoculated with 10² PFU of TY165 and TY165-PA-K185R viruses. Three mice were euthanized on days 2, 4, and 6 postinfection. Lungs, nasal turbinates, spleens, kidneys, brains, and nasal washes were collected and virus titers were determined by means of plaque assays in MDCK cells. Data shown are the mean virus titers with standard deviations. The P values were calculated by using Student's t test, comparing the virus titers between the wild-type and mutant viruses detected in the indicated organs. **, P < 0.001; *, P < 0.05.

FIG 6

Replication of VD18 and VD18-PA-K185R viruses in mice. Nine mice per group were intranasally inoculated with 10⁵ PFU of VD18 and VD18-PA-R185K viruses. Three mice were euthanized on days 2, 4, and 6 postinfection. Lungs, nasal turbinates, spleens, kidneys, brains, and nasal washes were collected and virus titers were determined by means of plaque assays in MDCK cells. Data shown are the mean virus titers with standard deviations. The P values were calculated by using Student's t test, comparing the virus titers between the wild-type and mutant viruses detected in the indicated organs. **, P < 0.001; *, P < 0.05.

FIG 7

Replicative abilities of TY165 and TY165-PA-K185R viruses in avian cells. (A) Minireplicon assays in avian DF-1 cells. DF-1 cells were transfected with PB2, PB1, PA, and NP protein expression plasmids, together with a minigenome encoding the luciferase gene [pPol(Ck)-VD5(50)M (50)-Luc] and an internal control plasmid encoding Renilla luciferase (pGL4.74[hRluc/TK]). Transfected cells were incubated at 37°C and 41°C for 48 h and then lysed. Luciferase activity was determined by using the dual-luciferase system kit. The relative polymerase activity was normalized to that of wild-type polymerase complexes. The values are presented as the averages of triplicate wells ± standard deviations from one experiment. (B) Viral growth curves in avian DF-1 cells. DF-1 cells were infected with viruses at an MOI of 2 × 10⁻⁵ and incubated at 37°C and 41°C. Aliquots of the supernatants were collected at the indicated time points and titrated in MDCK cells by use of plaque assays. Values are presented as the averages of three wells ± standard deviations from one experiment.