The highly conserved proline at position 438 in Pseudorabies Virus gH is important for regulation of membrane fusion

Membrane fusion in herpesviruses requires viral glycoproteins (g)B and gH/gL. While gB is considered the actual fusion protein but is non-fusogenic per se, the function of gH/gL remains enigmatic. Crystal structures for different gH homologs are strikingly similar despite only moderate amino acid sequence conservation. A highly conserved sequence motif comprises the residues serine-proline-cysteine corresponding to position 437-439 in Pseudorabies Virus (PrV) gH. The structures show that proline438 induces bending at the end of an alpha-helix, thereby juxtaposing cysteine404 and cysteine439 to allow formation of a strictly conserved disulfide bond. However, PrV vaccine strain Bartha unexpectedly carries a serine at this conserved position. To test the influence of this substitution we constructed different gH-chimeras carrying proline or serine at position 438 in gH either derived from PrV strain Kaplan or Bartha. Mutants expressing gH with serine438 showed reduced fusion activity in transient fusion assays and during infection, with delayed penetration kinetics and a small plaque phenotype which indicates that proline438 is important for efficient fusion. A more drastic effect was observed when disulfide bond formation was completely blocked by mutation of cysteine404 to serine. Although PrV expressing gHC404S was viable, plaque size and penetration kinetics were drastically reduced. Alteration of serine438 to proline in gH of strain Bartha enhanced cell-to cell spread and penetration kinetics but restoration of full activity required additional alteration of aspartic acid to valine at position 59.