Pattern of mutation in the genome of influenza A virus on adaptation to increased virulence in the mouse lung: identification of functional themes

Abstract

The genetic basis for virulence in influenza virus is largely unknown. To explore the mutational basis for increased virulence in the lung, the H3N2 prototype clinical isolate, A/HK/1/68, was adapted to the mouse. Genomic sequencing provided the first demonstration, to our knowledge, that a group of 11 mutations can convert an avirulent virus to a virulent variant that can kill at a minimal dose. Thirteen of the 14 amino acid substitutions (93%) detected among clonal isolates were likely instrumental in adaptation because of their positive selection, location in functional regions, and/or independent occurrence in other virulent influenza viruses. Mutations in virulent variants repeatedly involved nuclear localization signals and sites of protein and RNA interaction, implicating them as novel modulators of virulence. Mouse-adapted variants with the same hemagglutinin mutations possessed different pH optima of fusion, indicating that fusion activity of hemagglutinin can be modulated by other viral genes. Experimental adaptation resulted in the selection of three mutations that were in common with the virulent human H5N1 isolate A/HK/156/97 and that may be instrumental in its extreme virulence. Analysis of viral adaptation by serial passage appears to provide the identification of biologically relevant mutations.

Figure 1

SDS/PAGE of proteins of parental and MA clones of A/HK/1/68. (A) ³⁵[S]met-labeled infected MDCK cell proteins were immunoprecipitated to show the position of the HA0 precursor. (B) The unglycosylated form of HA0 protein synthesized in the presence of tunicamycin. (C) The position of the NP proteins is shifted for HKMA20B, -C, and -D. (D) The shift in mobility of the HA2 subunit generated by trypsin in variants.

Figure 2

The pH dependence of hemolysis by A/HK/1/68 and the HKMA12, HKMA20, HKMA20C virulent MA variants. The relative extent of hemolysis was measured in duplicate for each of two experiments at 0.1 pH increments. The hemolysis profiles of FM-MA and the J9 reassortant that possesses the same HA gene (17) are shown as controls.

Figure 3

Yield of HK and HKMA variants in different hosts. (A) Mouse lung; each point represents the average of three mice; values were not obtained for HKMA20C after 4 days because of lethality. (B) Yield in MDCK cells. (C) The yield of infectious virus in chicken allantoic cavity; values are the average plus standard deviation of two to six pools of three eggs.

Figure 4

Location of mutations selected on mouse adaptation on the functional genetic map of Influenza A virus. The location of mutations seen in FM-MA that have been confirmed to control virulence (9) are shown with black downward arrows, and those preexisting in FM virus are indicated in blue. Mutations observed in HKMA clones are shown with upward arrows. The functional regions and binding sites are described elsewhere (12). NLS, nuclear localization signal; POL, polymerase; NTP, nucleotide triphosphate; PKR, dsRNA-dependent protein kinase; effector indicates a multiple protein-binding region; NES, nuclear export signal; regions labeled with influenza virus protein names indicate sites of interaction.