Characterization of influenza A viruses with polymorphism in PB2 residues 701 and 702

Abstract

The 701 and 702 positions of influenza PB2 polymerase subunit are previously shown to have roles on host range. Limited polymorphisms at these two residues are identified in natural isolates, thereby limiting the study of their role in the polymerase. In this study, we generated 31 viable viruses by random mutagenesis at this region, indicating that these positions can tolerate a wide range of amino acids. These mutants demonstrated varying polymerase activities and viral replication rates in mammalian and avian cells. Notably, some mutants displayed enhanced polymerase activity, yet their replication kinetics were comparable to the wild-type virus. Surface electrostatic charge predication on the PB2 structural model revealed that the viral polymerase activity in mammalian cells generally increases as this region becomes more positively charged. One of the mutants (701A/702E) showed much reduced pathogenicity in mice while others had a pathogenicity similar to the wild-type level. Distinct tissue tropisms of the PB2-701/702 mutants were observed in infected chicken embryos. Overall, this study demonstrates that the PB2-701/702 region has a high degree of sequence plasticity and sequence changes in this region can alter virus phenotypes in vitro and in vivo.

Figure 1

Effect of PB2-701 and 702 mutations on polymerase activity in human and chicken cells. Viral RNP complexes with mutant PB2 genes were reconstituted in human 293T cells at (a) 33 °C, (b) 37 °C and (c) 39 °C, and avian DF-1 cells at (d) 33 °C, (e) 37 °C and (f) 39 °C. Mock represented a negative control without the transfection of PB2 gene. The polymerase activity was determined by the luciferase activity normalized with GFP expression. The relative polymerase activity was expressed in relative to the wild-type activity at the corresponding temperature, which was set as 100%. Data was presented as mean ± 1 s.d. (n = 3). (*p < 0.05 by Student’s t-test).

Figure 2

Correlation between surface electrostatic charge and polymerase activity. (a) Structural model of the PB2 C-terminal was predicted by the web-server Geno3D and visualized with Chimera. The protein surface was presented from PB2 residues 700 to 703 (left). Coulomb’s law was applied to estimate the electrostatic potential, in which blue and red color indicated positively and negatively charged surface respectively. A change in surface electrostatic charge were observed with PB2-701 or PB2-702 mutation (middle and right). Correlation analyses were performed between overall surface charge of the PB2-700 to 703 region and polymerase activities in (b) 293T cells and (c) DF-1 cells at 37 °C. Overall surface charge, in the unit of kT/e, was defined as the sum of the charges from residues 700 to 703 determined by Coulomb’s law.

Figure 3

Effect of PB2-701 and 702 mutations on viral growth kinetics in mammalian and avian cells. Mammalian MDCK cells were infected by wild-type and mutant viruses at an MOI of 0.01 at (a) 33 °C, (b) 37 °C, and (c) 39 °C. Avian DF-1 cells were infected by wild-type and mutant viruses at an MOI of 0.1 at (d) 33 °C, (e) 37 °C, and (f) 39 °C. Supernatant was harvested at 12 h, 24 h, 48 h and 72 h post-infection. The viral titre was determined by plaque assay in MDCK cells. Data was presented as mean ± 1 s.d. (n = 3).

Figure 4

Effect of PB2-701 and 702 mutations on virulence in mice. Female BALB/c mice (n = 6 per group) were intranasally infected by wild-type and PB2-701 and 702 mutant viruses at 1,000 PFU. Mice inoculated with PBS were used as negative control. (a) Body weight of the infected mice was measured daily for 14 days. The body weights were presented in relative to the initial body weight (set as 100%). Data was presented as mean ± 1 s.d. Mice with weight loss more than 25% of the initial body weight were humanely euthanized. (b) Survival rate of each group was also determined. (c) In lung viral titre determination, female BALB/c mice (n = 3 per group) were intranasally infected by wild-type and PB2-701 and 702 mutant viruses at 1,000 PFU as above. Mice were humanely euthanized on day 3 or day 6 post-infection. The lungs were collected and homogenized in ice-cold PBS. The viral titres of the lung homogenates were determined by plaque assay in MDCK cells. Data was presented as mean ± 1 s.d. (n = 3). (*p < 0.05, by Student’s t-test).

Figure 5

Differential tissue tropism of PB2-701 and 702 mutants in 12-day-old chicken embryos. Embryonic chicken eggs were inoculated with wild-type and PB2-701 and 702 mutant viruses at 100 PFU. PBS was inoculated as mock infection control. The chicken embryos were formalin-fixed at 48 h.p.i. Longitudinal sections of the chicken embryos were H&E stained and immunostained by anti-NP antibody. ^{^}Mutants with polymerase activity lower than wild-type. *Mutants with polymerase activity higher than wild-type. ^#Mutant with polymerase activity similar as wild-type.