A conserved region in the F₂ subunit of paramyxovirus fusion proteins is involved in fusion regulation

Paramyxoviruses utilize both an attachment protein and a fusion (F) protein to drive virus-cell and cell-cell fusion. F exists functionally as a trimer of two disulfide-linked subunits: F₁ and F₂. Alignment and analysis of a set of paramyxovirus F protein sequences identified three conserved blocks (CB): one in the fusion peptide/ heptad repeat A domain, known to play important roles in fusion promotion, one in the region between the heptad repeats of F₁ (CRF₁) (A. E. Gardner, K. L. Martin, and R. E. Dutch, Biochemistry 46:5094-5105, 2007), and one in the F₂ subunit (CBF₂). To analyze the functions of CBF₂, alanine substitutions at conserved positions were created in both the simian virus 5 (SV5) and Hendra virus F proteins. A number of the CBF₂ mutations resulted in folding and expression defects. However, the CBF₂ mutants that were properly expressed and trafficked had altered fusion promotion activity. The Hendra virus CBF₂ Y79A and P89A mutants showed significantly decreased levels of fusion, whereas the SV5 CBF₂ I49A mutant exhibited greatly increased cell-cell fusion relative to that for wild-type F. Additional substitutions at SV5 F I49 suggest that both side chain volume and hydrophobicity at this position are important in the folding of the metastable, prefusion state and the subsequent triggering of membrane fusion. The recently published prefusogenic structure of parainfluenza virus 5/SV5 F (H. S. Yin et al., Nature 439:38-44, 2006) places CBF₂ in direct contact with heptad repeat A. Our data therefore indicate that this conserved region plays a critical role in stabilizing the prefusion state, likely through interactions with heptad repeat A, and in triggering membrane fusion.