Bovine Herpesvirus 1 U
L
49.5 Interacts with gM and VP22 To Ensure Virus Cell-to-Cell Spread and Virion Incorporation: Novel Role for VP22 in gM-Independent U
L
49.5 Virion Incorporation
Authors: Pannhorst Katrin , Wei Huiyong , Yezid Hocine , He Junyun , Chowdhury Shafiqul I. 
Published: April 2018

Alphaherpesvirus envelope glycoprotein N (gN) and gM form a covalently linked complex. Bovine herpesvirus type 1 (BHV-1) U
L
49.5 (a gN homolog) contains two predicted cysteine residues, C42 and C78. The C42 is highly conserved among the alphaherpesvirus gN homologs (e.g., herpes simplex virus 1 and pseudorabies virus). To identify which cysteine residue is required for the formation of the U
L
49.5/gM complex and to characterize the functional significance of the U
L
49.5/gM complex, we constructed and analyzed C42S and C78S substitution mutants in either a BHV-1 wild type (wt) or BHV-1 U
L
49.5 cytoplasmic tail-null (CT-null) virus background. The results demonstrated that BHV-1 U
L
49.5 residue C42 but not C78 was essential for the formation of the covalently linked functional U
L
49.5/gM complex, gM maturation in the Golgi compartment, and efficient cell-to-cell spread of the virus. Interestingly, the C42S and CT-null mutations separately did not affect mutant U
L
49.5 virion incorporation. However, when both of the mutations were introduced simultaneously, the U
L
49.5 C42S/CT-null protein virion incorporation was severely reduced. Incidentally, the anti-VP22 antibody coimmunoprecipitated the U
L
49.5 C42S/CT-null mutant protein at a noticeably reduced level compared to that of the individual U
L
49.5 C42S and CT-null mutant proteins. As expected, in a dual U
L
49.5 C42S/VP22Δ virus with deletion of VP22 (VP22Δ), the U
L
49.5 C42S virion incorporation was also severely reduced while in a gMΔ virus, U
L
49.5 virion incorporation was affected only slightly. Together, these results suggested that U
L
49.5 virion incorporation is mediated redundantly, by both U
L
49.5/gM functional complex and VP22, through a putative gM-independent novel U
L
49.5 and VP22 interaction.

IMPORTANCE
Bovine herpesvirus 1 (BHV-1) envelope protein U
L
49.5 is an important virulence determinant because it downregulates major histocompatibility complex class I (MHC-I). U
L
49.5 also forms a covalently linked complex with gM. The results of this study demonstrate that U
L
49.5 regulates gM maturation and virus cell-to-cell spread since gM maturation in the Golgi compartment depends on covalently linked U
L
49.5/gM complex. The results also show that the U
L
49.5 residue cysteine 42 (C42) mediates the formation of the covalently linked U
L
49.5-gM interaction. Furthermore, a C42S mutant virus in which U
L
49.5 cannot interact with gM has defective cell-to-cell spread. Interestingly, U
L
49.5 also interacts with the tegument protein VP22 via its cytoplasmic tail (CT). The putative U
L
49.5 CT-VP22 interaction is essential for a gM-independent U
L
49.5 virion incorporation and is revealed when U
L
49.5 and gM are not linked. Therefore, U
L
49.5 virion incorporation is mediated by U
L
49.5-gM complex interaction and through a gM-independent interaction between U
L
49.5 and VP22.