TI - The herpes simplex virus UL20 protein functions in glycoprotein K ( gK ) intracellular transport and virus-induced cell fusion are independent of UL20 functions in cytoplasmic virion envelopment . AB - The HSV-1 UL20 protein ( UL20p ) and glycoprotein K ( gK ) are both important determinants of cytoplasmic virion morphogenesis and virus-induced cell fusion . In this manuscript , we examined the effect of UL20 mutations on the coordinate transport and Trans Golgi Network ( TGN ) localization of UL20p and gK , virus-induced cell fusion and infectious virus production . Deletion of 18 amino acids from the UL20p carboxyl terminus (UL20 mutant 204t) inhibited intracellular transport and cell -surface expression of both gK and UL20 , resulting in accumulation of UL20p and gK in the endoplasmic reticulum ( ER ) in agreement with the inability of 204t to complement UL20-null virus replication and virus-induced cell fusion . In contrast , less severe carboxyl terminal deletions of either 11 or six amino acids ( UL20 mutants 211t and 216t , respectively ) allowed efficient UL20p and gK intracellular transport , cell -surface expression and TGN colocalization . However , while both 211t and 216t failed to complement for infectious virus production , 216t complemented for virus-induced cell fusion , but 211t did not . These results indicated that the carboxyl terminal six amino acids of UL20p were crucial for infectious virus production , but not involved in intracellular localization of UL20p/gK and concomitant virus-induced cell fusion . In the amino terminus of UL20 , UL20p mutants were produced changing one or both of the Tyr38 and Y49 residues found within putative PHOSphorylation sites . UL20p tyrosine-modified mutants with both tyrosine residues changed enabled efficient intracellular transport and TGN localization of UL20p and gK , but failed to complement for either infectious virus production , or virus-induced cell fusion . These results show that UL20p functions in cytoplasmic envelopment are separable from UL20 functions in UL20p intracellular transport , cell surface expression and virus-induced cell fusion .