Genetic structure-function analysis of the essential glycoprotein gB in herpes simplex virus type 1-induced membrane fusion.

Abstract

A genetic structure-function analysis of the essential HSV-1 membrane glycoprotein gB transmembrane and cytoplasmic domains in virally-induced membrane fusion was performed. Membrane fusion is induced at two times during infection: (i) early, between the virion envelope and cellular plasma membrane to initiate infection and (ii) late, between adjacent cells. Certain mutant HSV strains induce extensive cell fusion, or syncytia (syn) during infection. Syn mutations have been identified within the gB cytoplasmic domain, which is essential for gB function in both viral penetration and cell fusion, presenting a paradox since this region of the molecule presumably does not interact with the adjacent cell plasma membrane. To test the ability of the large gB cytoplasmic domain (109 amino acids) to function in membrane fusion independently of the native gB molecule, recombinant viruses containing gene chimeras encoding hybrid proteins consisting of the extracellular and transmembrane domains of the non-essential HSV glycoprotein gC fused to a wild type or syn mutant gB cytoplasmic domain (gC:gB) were constructed. Both gC:gB hybrids, including one containing a missense mutation which induces extensive cell fusion when expressed within the context of the native gB molecule, failed to influence cell fusion, suggesting that the gB cytoplasmic domain must cooperate with other regions of the gB molecule in membrane fusion. In addition, post-translational processing of the gC:gB hybrids was profoundly altered relative to the native gC molecule. Using a generalized mutagenesis strategy targeted at the gB transmembrane and cytoplasmic domains, two discrete regions affecting cell fusion were identified within the cytoplasmic domain. Syn region I is located immediately proximal to the transmembrane domain, while syn region II is located distal from the transmembrane domain towards the C-terminus. A single rate of entry (roe) mutation was identified within or immediately proximal to syn region II. This roe mutation provides the first direct molecular link between viral penetration and cell fusion and demonstrates that the requirement for gB during viral penetration is related to its essential role in membrane fusion. The nature of individual mutations give clues to the function of gB during membrane fusion.