Structural determinants for nuclear envelope localization and function of Pseudorabies Virus pUL34

Herpesvirus proteins pUL34 and pUL31 form a complex at the inner nuclear membrane (INM) which is necessary for efficient nuclear egress. Pseudorabies Virus (PrV) pUL34 is a type II membrane protein of 262 amino acids, with the transmembrane region (TM) predicted between amino acids (aa) 245 and 261 leaving only one aa in the C-terminus probably extending into the perinuclear space. It is targeted to the nuclear envelope in the absence of other viral proteins pointing to intrinsic localization motifs, and shows structural similarity to cellular INM proteins like lamina-associated polypeptide (Lap) 2ß and Emerin. To investigate which domains of pUL34 are relevant for localization and function we constructed chimeric proteins by substituting parts of pUL34 by regions of cellular INM proteins. First, the 18 C-terminal aa encompassing the TM were exchanged with TM regions and C-terminal domains of Lap2ß and Emerin or with the first TM region of the polytopic Lamin B receptor (LBR) including nine following aa. All resulting chimeric proteins complemented the replication defect of PrV-?UL34 demonstrating that substitution of the TM and extension of the C-terminal domain does not interfere with function of pUL34. Complementation was reduced, but not abolished when the C-terminal 50 aa were substituted by corresponding LAP2ß sequences (pUL34-LapCT50). However, substituting the C-terminal 100 aa (pUL34-LapCT100) resulted in a non-functional protein, despite continuing pUL31 binding, pointing to an important functional role of this region. Substitution of 100 N-terminal aa (pUL34-LapNT100) had no effect on nuclear envelope localization but abrogated pUL31 binding and function.